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Potential and utilization of thermophiles and thermostable enzymes in biorefining

Pernilla Turner, Gashaw Mamo and Eva Nordberg Karlsson*

Author Affiliations

Dept Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden

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Microbial Cell Factories 2007, 6:9  doi:10.1186/1475-2859-6-9


The electronic version of this article is the complete one and can be found online at: http://www.microbialcellfactories.com/content/6/1/9


Received:4 January 2007
Accepted:15 March 2007
Published:15 March 2007

© 2007 Turner et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts.

Background

Thermostable enzymes and microorganisms have been topics for much research during the last two decades, but the interest in thermophiles and how their proteins are able to function at elevated temperatures actually started as early as in the 1960's by the pioneering work of Brock and his colleagues [1]. Microorganisms are, based on their optimal growth temperatures, divided into three main groups, i.e. psychrophiles (below 20°C), mesophiles (moderate temperatures), and thermophiles (high temperatures, above 55°C) [2]. Only few eukaryotes are known to grow above this temperature, but some fungi grow in the temperature range 50 – 55°C [3]. Several years ago Kristjansson and Stetter [4], suggested a further division of the thermophiles and a hyperthermophile boundary (growth at and above 80°C) that has today reached general acceptance. Most thermophilic bacteria characterised today grow below the hyperthermophilic boundary (with some exceptions, such as Thermotoga and Aquifex [5]) while hyperthermophilic species are dominated by the Archaea.

Use and development of molecular biology techniques, permitting genetic analysis and gene transfer for recombinant production, led to dramatically increased activities in the field of thermostable enzymes during the 1990's. This also stimulated isolation of a number of microbes from thermal environments in order to access enzymes that could significantly increase the window for enzymatic bioprocess operations. One of the early successful commercialised examples was analytical use of a thermostable enzyme, Taq-polymerase, in polymerase chain reactions (PCR) for amplification of DNA, and a number of other DNA modifying enzymes from thermophilic sources have, since then, been commercialised in this area [6-8]. Another area of interest has been the prospecting for industrial enzymes for use in technical products and processes, often in a very large scale. Enzymes can be advantageous as industrial catalysts as they rarely require toxic metal ions for functionality, hence creating the possibility to use more environmentally friendly processing [9]. Thermostable enzymes offer robust catalyst alternatives, able to withstand the often relatively harsh conditions of industrial processing.

Conversion of biomass into sugars for e.g. energy utilization was a topic of concern about 30 years ago. Renewed interest in biocatalytic conversions has recently emerged, with the growing concern on the instability and possible depletion of fossil oil resources as well as growing environmental concern, and focus is again put on biorefining, and the biorefinery concept. In biorefining, renewable resources such as agricultural crops or wood are utilized for extraction of intermediates or for direct bioconversion into chemicals, commodities and fuels [10,11]. Thermostable enzymes have an obvious advantage as catalysts in these processes, as high temperatures often promote better enzyme penetration and cell-wall disorganisation of the raw materials [12]. By the parallel development in molecular biology, novel and developed stable enzymes also have a good chance to be produced at suitable levels. This review will discuss the potential and possibilities of thermostable enzymes, developed or isolated from thermophiles, including examples where whole cells are considered, in bioconversions of renewable raw materials with a biorefining perspective. Examples of commercial thermostable enzymes acting on renewable raw materials will be illustrated.

Stability and development of thermostable enzymes

In industrial applications with thermophiles and thermostable enzymes, isolated enzymes are today dominating over microorganisms. An enzyme or protein is called thermostable when a high defined unfolding (transition) temperature (Tm), or a long half-life at a selected high temperature, is observed. A high temperature should be a temperature above the thermophile boundary for growth [>55°C]. Most, but not all proteins from thermophiles are thermostable. Extracellular enzymes generally show high thermostability, as they cannot be stabilised by cell-specific factors like compatible solutes [13]. In addition, a few thermostable enzymes have also been identified from organisms growing at lower temperatures (see for example B. licheniformis amylase below). Fundamental reasons to choose thermostable enzymes in bioprocessing is of course the intrinsic thermostability, which implies possibilities for prolonged storage (at room temperature), increased tolerance to organic solvents [14], reduced risk of contamination, as well as low activity losses during processing (when staying below the Tm of the enzyme) even at the elevated temperatures often used in raw material pre-treatments.

Discovery and use of thermostable enzymes in combination with recombinant production and development using site-directed and enzyme evolution technologies, have erased some of the first identified hinders (e.g. limited access and substrate specificity) for use in industrial biocatalysis. Today, a number of biotechnology companies are continuously prospecting for new, and adapting existing enzymes to reactions of higher volumes and more severe process conditions [15]. Enzyme prospecting often focuses on gene retrieval directly from Nature by molecular probing techniques, followed by recombinant production in a selected host. Availability of genes encoding stable enzymes, and knowledge on structural features in the enzymes, can also be utilized in molecular development for enzyme improvement (Table 1).

Table 1. An overview of suggested features for internal thermostability, selected from structural studies of homologues, along with some development approaches to introduce thermostability, and development of thermostable proteins.

In vitro evolution strategies can utilize genes encoding thermostable proteins as stable scaffolds. When developing thermostable enzyme scaffolds, the starting material is an already stable backbone, thus creating a good possibility for evolution to optimize function at selected conditions for activity. An example where this type of development has been utilized is the diversification of the binding specificity of a carbohydrate binding module, CBM4-2 originating from a xylanase from the thermophilic bacterium Rhodothermus marinus [30]. Carbohydrate binding modules allow fine-tuned polysaccharide recognition [31] and have potential as affinity handles in different types of applications, as recently reviewed by Volkov and co-workers [32]. Using CBM4-2, which has both high thermostability and good productivity in E. coli expression systems, a single heat stable protein could be developed with specificity towards different carbohydrate polymers [27], as well as towards a glycoprotein [33], showing the potential of molecular biology for selective specificity development of a single protein with overall desirable properties.

In vitro evolution strategies are more commonly used to increase stability (Table 1), often using genes encoding non-thermostable enzymes with desired activities, for development of better thermostability, and using the temperature of the screening assay as selection pressure [34-36]. This could for instance include development of thermostable cellobiohydrolases, which are uncommon among thermophiles, but beneficial for lignocellulose conversions. In addition, such strategies can be used to optimise stability inside the host-cell during recombinant expression [37]. Alternatively, the identification of thermostabilising features in stable enzymes can be utilized to engineer stability into less stable enzymes, using site-directed mutagenesis (Table 1). Adaptations of biomolecules to extreme conditions involve a compromise of stability and flexibility in order to optimise the functional state of proteins rather than to maximize stability [38,39]. The free energy of stabilization (ΔGN→U) of unrelated globular proteins of mesophilic origin is marginal (in the range 30–65 kJ/mol), corresponding to a few weak interactions, and the difference between a thermostable protein and a protein of mesophilic origin (ΔΔGN→U), corresponds to only a few additional interactions. In addition, despite several statistical studies of primary sequences, no general strategies in terms of preferred amino acid exchanges are to be expected [38-43], and very small 3D-structural alterations may hence suffice to cope with the various extreme conditions [38,42]. To rationally identify the type of stabilising interactions used, several studies have been undertaken where 3D-structures of one unique enzyme isolated from a range of organisms growing at different temperatures have been investigated. These studies include a number of intracellular enzymes [17,19,20,42] and a few extracellular enzymes, e.g. endoglucanase [23] and lipase [44]. A number of features have been proposed from these studies (Table 1), and e.g. increase in ion-pairs and ion-pair networks has frequently been observed, especially in enzymes from hyperthermophilic species. Disulphide bonds is another protein stabilising feature, shown to be important for many enzymes and proteins, that has recently also been shown for intracellular hyperthermophilic proteins, seeming to be especially common in small proteins [18]. Stabilisation of less stable proteins using these strategies requires structural knowledge and it can be rather complicated to predict the effect of introducing novel interacting amino acid residues. Despite these difficulties, continued developments of stable enzymes with desired activities, using both site-directed and random techniques, pave the way for more efficient enzymes. It is thus expected that use of thermostable enzymes in industrial applications will increase with time, ultimately leading to wider availability and lower price, hence improving their potential in large scale applications like biorefining.

Biorefineries for renewable resource utilization

The biorefinery has lately become a key concept used in the strategies and visions of many industrial countries, being driven by a combination of environmental (encouraging renewable chemicals and fuels, and discouraging net greenhouse gas), political and economical concerns [45-49]. A biorefinery is defined as a system combining necessary technologies between renewable raw materials, industrial intermediates and final products [10,11] (Fig. 1). The goal is to produce both high value, low volume products and low value, high volume products (e.g. fuels) [10]. The feedstocks (or their rest products) can be used directly as raw materials for bioprocessing, or be used as cheap substrates for fermentation processes from which products can be extracted [50]. Depending on the feedstock available in different countries, biomass of different origins have been suggested as raw materials, and include for example corn [51], wheat [52], sugar cane [46,53], rape, cotton, sorgo, cassava [54] and lignocellulose [47]. The simplest biorefinery systems have in principal fixed processing of one type of feedstock (e.g. grains) to one main product, while the most flexible ones use a mix of biomass feedstock to produce an array of products. Different types of biomass feedstock can be used, such as whole crop (e.g. cereals and corn), or lignocellulose feedstock (e.g. biomass from wood or waste) [10,11]. In order to achieve efficient conversion of the raw material, a mixture of mechanical, biocatalytic and chemical treatments are expected to be combined. Our focus will be on the biocatalytic conversions, and examples using crops or lignocellulosics as raw materials will be given.

thumbnailFigure 1. Schematic overview of the basic principle of a biorefinery, along with some product examples.

Biocatalysis, involving enzymatic or microbial actions, undertake a dual task in the biorefinery systems, both generating metabolizable building blocks (generating sugars from polymers) for further conversions, and acting as specific catalysts in the conversion of building blocks into desired products (conversion specificity). A wide range of reaction types, e.g. oxidations, reductions, carbon-carbon bond formations, and hydrolysis, can be catalysed using enzymes. To give a few examples, monooxygenases can be used for hydroxylation and Baeyer-Villiger oxidation reactions [55]. Stereoselective reduction of carbonyl compounds to chiral alcohols can be made using alcohol dehydrogenases, among which some of thermophilic origin are reported [56]. As these enzymes are coenzyme dependent, regeneration strategies have to be considered (see below next section). Epoxide synthesis, using lipases or oxidoreductases, have great potential for the synthesis of a wide range of chemicals, and enzymatic reactions could replace some toxic chemicals [57]. C-C-bond formation can be carried out with lyases [58]. Glycoside hydrolases and transferases can catalyse glycoside synthesis (eventually via reverse hydrolysis), for production of glyco-oligosaccharides of defined lengths, as well as other glyco-conjugates as for example alkyl-glycosides, and thermostable enzymes have been utilized for this purpose [59,60]

These reactions may be performed using free or immobilised whole cells, crude, purified or immobilised enzymes, many of which are based on recombinant organisms [15]. To increase the substrate availability, polymer-hydrolysing enzymes give a significant contribution. For example, glycoside hydrolases (which are also used in food and feed processing) degrade the polymeric storage and building materials of plants and trees into oligo- and monosaccharide building blocks that are easier for microorganisms to take up and metabolize. This can be desirable if whole cell biocatalysts (i.e. native, recombinant protein producing or otherwise metabolically engineered microorganisms) are selected, which could be the case when metabolic pathway products are the target compounds. Enzymes acting on glycosidic bonds can also be utilized for modification of glycoside-containing natural products like flavonoid antioxidants [61]. The possibility to use whole cells, as well as isolated enzymes for further processing increases the diversity of potentially produced building blocks, and a number of metabolic products have already today been identified as interesting platform chemicals.

Platform chemicals

The US Department of Energy has published a list of top value chemical building blocks, i.e. platform chemicals that can be derived from biomass by biological or chemical conversion and subsequently converted to a number of high-value bio-based chemicals or materials [62]. The 12 top value building blocks are listed in Table 2. Each building block can be converted to numerous high-value chemicals or materials and the potential industrial applications are immense (some of which are listed in Table 2). All building blocks listed can be produced from biomass (cellulose, hemicellulose, starch or vegetable oils) either by fermentation or by in vitro enzymatic conversions via the intermediate sugars; glucose, fructose, xylose, arabinose, lactose, and sucrose, respectively (glycerol excepted). In the suggested biocatalytic routes, fermentations of mesophilic organisms are still dominating among the top 12, and in some cases the biotransformation route is not known and needs to be explored. In order to achieve a proficient utilization of biomass materials (e.g. to release as much sugars as possible from the raw material), it is believed that there is a need for efficient thermostable biocatalysts.

Table 2. Prioritized sugar-derived building blocks as listed by the US Department of Energy. Adapted from [62].

Catalysis at high temperature could for example be advantageous in bioconversion of the hemicellulose xylan from lignocellulosic materials into xylitol (Table 2, [63]). The difficulty of lignocellulose degradation has been reported by several authors [64-66], and a thermal pre-treatment is often included to enhance the degradability of these materials. Thermal treatment is also reported to improve the enzyme penetration for hemicellulase conversions [12], improving xylan availability. Three enzymes are needed for the xylan to xylitol conversion: xylanase (EC 3.2.1.8), xylosidase (EC 3.2.1.37), and xylose reductase (EC 1.1.1.21). Use of thermoactive and thermostable xylanase allow the enzymatic action to take place simultaneously with the heating step, without need to pre-cool the system, hence shortening processing time. By adding thermostable xylosidase (active on xylo-oligosaccharides), efficient hydrolysis into xylose monomers can be achieved. Conversion of xylose to xylitol is however catalysed by a NAD(P)H-dependent xylose reductase: therefore, to reduce the need of co-factor (and its costs), addition of a co-factor recycling enzyme, or whole cell catalysis utilizing intracellular co-factors should be considered. Today, xylose to xylitol conversions are often reported using different pentose utilizing yeast strains [67] but a problem with these strains is further conversion of xylitol into xylulose. In xylose fermenting yeasts, like Pichia and Candida, this step is catalysed by an NAD+-dependent xylose dehydrogenase, while in bacteria the corresponding step is catalysed by a xylose isomerase. Metabolically engineeed Saccharomyces cerevisiae transformed with xylose reductase (from P. stipidis) has xylitol as an end product, and this organism has been used for the conversion of xylose to xylitol with more than 95% conversion, but as a new co-factor dependent enzyme is introduced, co-factor recycling has to be considered [68].

Industrial enzymes and biorefining/related applications

To further illustrate the use of thermostable biocatalysts on renewable raw materials in large scale, we will focus on the potential and applications of hydrolytic enzymes (proteases, lipases and glycoside hydrolases), which are established in industrial scale. Protease and lipase applications will only be mentioned briefly (for reviews, see [69-72]) and special emphasis will be put on glycoside hydrolases.

According to a report from the Business Communications Company Inc, the global market for industrial enzymes was estimated to totally $2 billion in 2004 [73]. Furthermore, the annual growth rate of industrial enzymes is predicted to be between 4 and 5% and with this comes lower prices of enzymes due to an intensified competition on the market. The industrial enzyme market can be separated into application sections: (1) technical enzymes, (2) food enzymes, and (3) animal feed enzymes. The largest section is technical enzymes where enzymes used for detergents and pulp and paper constitute 52% of the total world market [73]. Leading enzymes in this section are hydrolytic enzymes, classified as proteases and amylases, which comprise 20 and 25% of the total market, respectively [73]. Hydrolases are generally easy to use in bioprocesses, as they normally do not require co-factors or complex substrates. Moreover, they can be used at an early stage on the readily available material found in the forest and agricultural sectors. Some available applications from biomass materials where thermostable variants have been considered are listed [see Additional file 1] together with the enzyme activities which can be used for their degradation or modification. Applications of selected examples with a biorefining perspective will be further discussed in the text in the respective sections below.

Additional File 1. Examples of possible and current applications of thermostable hydrolases, and sequence-related transferases. The table shows applications of thermostable enzymes; hydrolases and transferases, along with EC numbers.

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Crop biorefining

The initial step in crop biorefining is fractionation. This is achieved by both physical, chemical and biological processes [74]. After a starting physical step, often milling, the biological process employs different hydrolases, depending on what kind of crop is fractionated. Fractionation is often accompanied by elevated temperatures, which demands thermostable and thermoactive enzymes. Chemical processes may be used for some applications, but may generate toxic and unwanted side products, and we will not focus on those methods here. Instead enzymatic degradation of starch from grains and utilization of products gained from this will serve as an example of the potential of thermostable enzymes in this type of processing. The straw may also be processed to utilize the carbohydrates present in the lignocellulosic fraction (see below).

Starch degradation and modification

Starch from cultivated plants is one of the most abundant and accessible energy sources in the world. It consists of amylose and amylopectin, and an overview of the principal structure indicating sites of enzymatic attack is given in Fig. 2. Corn is the most used crop in starch processing in industries, but wheat, potato and tapioca are also important crops while rice, sorghum, sweet potato, arrowroot, sago and mung beans are used to a lesser extent [75].

thumbnailFigure 2. Enzymatic attack on part of an amylopectin molecule. Glucose molecules are indicated as circles and the reducing ends are marked by a line through the circle.

Hydrolases (and sequence-related transferases) acting on starch are members of the α-amylase superfamily, which consists of a large number of primary sequence-related enzymes with a retaining catalytic mechanism [76], liberating groups in the α-configuration. The superfamily belongs to glycoside hydrolase clan GH-H, and consists of 3 sequence-related families of glycoside hydrolases (GH13, 70 and 77 [77]) catalysing a range of reactions [see Additional file 1]. Specific consensus sequences, and a varying number of domains, are believed to be responsible for the specificity variations, leading to hydrolysis or transferase activity, as well as differing substrate specificity.

Processed starch is mainly used for glucose, maltose, and oligosaccharide production, but a number of products/intermediates can also be produced via cyclodextrins. Glucose can be further converted to high-fructose syrups, crystalline dextrose and dextrose syrups, which are used in food applications [78]. Glucose can of course also be fermented to produce ethanol (see Biofuel below), amino acids or organic acids [78]. Conversion to high-fructose syrup by glucose isomerase (EC 5.3.1.5) is usually run at 55–60°C and pH 7.0–8.5 [78], requiring a thermostable enzyme. Fructose is a popular sweetener, partly because of the availability of bulk quantities of corn starch at low cost.

Starch processing is usually performed in a two-step hydrolysis process of liquefaction and saccharification. Liquefaction is the conversion of granular starch into soluble, shorter-chain-length dextrins [DE (dextrose equivalents) 9–14]. In liquefaction, starch is gelatinized by thermal treatment requiring a temperature around 70–90°C (for corn) [78], but to assure the removal of all lipid-amylose complexes, a preferred process temperature is above 100°C [78]. When the starch-slurry is cooled down it forms a thermo-irreversible gel, by a process known as retrogradation, in which the amylose chains interact by hydrogen bonding [79]. The crystalline order is then lost and the starch granules swell as the amylose and amylopectin chains are hydrated [80]. A thermostable α-amylase [see Additional file 1] is added before the heat treatment, which takes place at 105–110°C for 5–7 min [81]. The starch-slurry is then flash-cooled to 95°C and kept at that temperature for 60–120 min to complete the enzymatic liquefaction [81,82]. Consequently, a highly thermostable enzyme is required which will be active during the whole procedure. Nowadays there are, in addition to the originally used enzymes from Bacillus stearothermophilus or B. licheniformis, numerous examples available and marketed e.g. the Valley "Ultra-thin™" from Valley Research/Diversa, Multifect AA 21L® from Genencor and Termamyl® and Liquozyme® from Novozymes [see Additional file 2]. Ideally, the enzyme should be active and stable at a low pH (~4.5) and not demand calcium for stability. Some engineered enzymes have been reported to fulfill these desired properties [see Additional file 2]. The water content in the starch-slurry is generally quite high (35%), as a high viscosity increases the melting temperature of starch [83]. Reduction of the moisture content could be more economical, and has shown to be possible when including a shearing treatment [82]. This was however accompanied by increased formation of isomaltose [82], and increased temperatures would also require enzymes with very high thermostability.

Additional File 2. Properties of some thermostable wild-type or engineered members of the α-amylase family acting on starch and related molecules. The table shows some properties of thermostable enzymes acting on starch and related molecules.

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Saccharification involves hydrolysis of remaining oligosaccharides (8–12 glucose units) into either maltose syrup by β-amylase or glucose/glucose syrups by glucoamylase [84]. The process is run at pH 4.2–4.5 and 60°C, at which temperature the currently used Aspergillus niger glucoamylase is stable. Still, the temperature has to be cooled down after liquefaction and the pH has to be adjusted, in order for the glucoamylase to act. More economically feasible would be to utilize an enzyme active in the same pH and temperature range as the liquefaction enzymes. Kim et al. have recently reported on a glucoamylase from Sulfolobus solfataricus, which is optimally active at 90°C and pH 5.5–6.0. This enzyme also formed less isomaltose, a common side reaction, than the commercially available fungal glucoamylase [85]. To increase the efficiency in saccharification, a debranching enzyme, such as pullulanase, can be added to the process. Thermostable enzyme mixes are today available on the market containing both glucoamylase and pullulanase, e.g. OPTIMAX® from Genencor.

Gelatinized starch (obtained from liquefaction) can also be modified by amylomaltases (EC 2.4.1.25, and members of GH 77) that are 4-α-glucanotransferases transferring α-1,4-linked glucan fragments from the starch to an acceptor, which may be the 4-OH group of another α-1,4-linked glucan or glucose [86]. In plants, this enzyme is also called disproportionating enzyme or D-enzyme [79]. Several industrially relevant thermostable and thermoactive amylomaltases are known to date (Thermus species, Thermococcus species, and Aquifex aeolicus, [see Additional file 2]), with optimal temperatures between 75 and 90°C. Amylomaltase catalysis results in conversion into a thermoreversible starch gel that consists of amylopectin with shortened and elongated side-chains, but free of amylose [79]. The obtained gel behaves similar to gelatin (and may substitute gelatin obtained from the bone marrow of cows) and has many uses in the food industry. Applications of amylomaltases on starch also include formation of cycloamyloses [87] and production of isomalto-oligosaccharides [88].

Cyclodextrins (CDs) are other starch-derived products with a range of possible applications, due to the apolar interior that can host "guest molecules" and solubilize and stabilize them [89]. There are CDs of different sizes, suitable for different applications. Examples of applications of CDs and derivatives thereof are: carriers for therapeutically important peptides, proteins and oligonucleotides [90], solubilization and stabilization of a range of pharmaceutical molecules [91], analytical separations [92], and various applications in foods and cosmetics, textiles, and adhesives [93]. There are also large cyclic dextrins, commonly known as cycloamyloses [94] or LR-CDs [95]. These products can be synthesized by CGTases [96] or amylomaltases [87,97]. Cycloamyloses can be used as a coating material, in adhesives, for biodegradable plastics, as a high energy additive to soft drinks, as a retrogradation retardant for bread improvement, for freeze resistant jellies and for production of non-sticky rice as described by Larsen, 2002, and references therein [98]. Cycloamyloses have also been proposed to aid in protein refolding by acting as an artificial chaperone [99] and for solubilization of larger compounds, e.g. Buckminster fullerene (C60, C70) [95].

Biodegradation and modification of lignocellulose

Lignocellulose is an important example of an abundant raw material, produced in large quantities for the production of forest products, often leaving a significant fraction of unutilized waste products. Agricultural waste, such as straw, also has significant lignocellulose content. Enzymes (including commercially available feed enzymes) that hydrolyze the polymeric lignocellulose into shorter metabolizable intermediates, or that reduce viscosity of non-starch polysaccharide in feed cereals (e.g. barley, rye, oats) [100] can be used to improve utilization of the lignocellulosic carbohydrate fraction. As the lignocellulosic materials often are subjected to thermal treatments to facilitate degradation, thermostable enzymes have a clear advantage. Feed enzymes have been on the market for 15 years and the estimated value of this market is around $US360 million [100]. Feed processing is normally performed at high temperatures [101], so use and development of stable and robust enzymes has been imperative.

Lignocelluloses of plant cell walls are composed of cellulose, hemicellulose, pectin, and lignin (the three former being polysaccharides). Cellulose is the major constituent of all plant material and the most abundant organic molecule on Earth [102], while hemicelluloses and pectins are the matrix polysaccharides of the plant cell wall. Many enzymes are involved in the degradation of this biomass resource [103], and they are often built up by discrete modules (the most common being catalytic or carbohydrate-binding modules), linked together by short linker peptides, sometimes connecting one catalytic module with specificity towards cellulose with a hemicellulose-specific module. Such multiple enzyme systems aid in creating efficient degradation of the lignocellulosic materials. In addition, several microorganisms produce multiple individual enzymes that can act synergistically. Fig. 3 shows an overview of some polymers present in lignocellulose, and the sites of attack for a number of enzymes acting on these substrates. More examples of the lignocellulose degrading enzymes of thermophilic origin with differing specificities are given [see Additional file 3].

Additional File 3. Properties of some thermostable hydrolases of both thermophilic and mesophilic origin acting on lignocellulosic materials. The table shows some properties of enzymes acting on lignocellulosics.

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thumbnailFigure 3. Simplified structures and sites of enzymatic attack on polymers from lignocellulose. A cellulose chain fragment (A) is shown, along with hypothetical fragments of the hemicelluloses xylan (B), glucomannan (C), and pectin (D). Sites of attack of some of the major enzymes acting on the respective material are indicated by arrows. The glycosidic bond type of the main-chain is indicated in brackets to the right of each polymer fragment. Carbohydrates are indicated as circles, and the reducing end of each main chain is marked by a line through the circle. White = glucose, green = xylose, yellow = glucuronic acid, red = arabinose, light blue = mannose, dark blue = galactose, grey = galacturonic acid, and pink = undefined sugar residues. Acetate groups are shown as triangles, phenolic groups as diagonals, and methyl groups as rombs.

Cellulose conversion by cellulases

Cellulose is a homopolysaccharide composed of β-D-glucopyranose units, linked by β-(1→4)-glycosidic bonds. The smallest repetitive unit is cellobiose, as the successive glucose residues are rotated 180° relative to each other [104-106]. The cellulose hydrolysing enzymes (i.e. cellulases) are divided into three major groups: endoglucanases, cellobiohydrolases (and exoglucanases), and β-glucosidases, all three attacking β-1,4-glycosidic bonds [107,108]. The endoglucanases ([EC 3.2.1.4], classified under 12 different GH families with both inverting and retaining reaction mechanisms, and with different folds) catalyse random cleavage of internal bonds in the cellulose chain, while cellobiohydrolases (EC 3.2.1.91, GH 5, 7 [retaining] and 6, 9 [inverting]) attack the chain ends, releasing cellobiose. β-glucosidases (EC 3.2.1.21, GH1, 3 [retaining] and 9 [inverting]) are only active on cello-oligosaccharides and cellobiose, releasing glucose (Fig. 3A).

A significant industrial importance for cellulases was reached during the 1990's [109], mainly within textile, detergent and paper and pulp industry (e.g. in deinking of recycled paper). Several thermostable enzymes have been characterized [see Additional file 3], and there has been many trials in these areas as thermostability is highly relevant for the performance of the enzymes.

Degradation of cellulose (Fig. 3A) into fermentable sugars for commodity product production is a biorefining area that has invested enormous research efforts as it is a prerequisite for the subsequent production of energy, see Biofuel below. It is likely to be performed at least partly at high temperatures to facilitate the degradation, thus making thermostable enzymes (or thermophilic microorganisms) desirable. Although cellulases cleave a single type of bond, the crystalline substrates with their extensive bonding pattern necessitate the action of a consortium of free enzymes or alternatively multi-component complexes called cellulosomes [110]. Carbohydrate-binding modules connected by linkers to the catalytic modules can also give significant contribution to the action of the enzymes, and improve the degradation efficiency, especially on complex lignocellulosic substrates [111-113]. Further improvements in the efficiency level in cellulose degradation (more rapid and less costly), would create both environmental and economic benefits, motivating trials using enzyme blends, as well as engineered cells, and is still a key challenge open for research [114].

Hemicellulose conversions

Hemicellulose is the second most abundant renewable biomass and accounts for 25–35% of lignocellulosic biomass [115]. Hemicelluloses are heterogeneous polymers built up by pentoses (D-xylose, D-arabinose), hexoses (D-mannose, D-glucose, D-galactose) and sugar acids [115]. Hemicelluloses in hardwood contain mainly xylans (Fig. 3B), while in softwood glucomannans (Fig. 3C) are most common [115]. There are various enzymes responsible for the degradation of hemicellulose. In xylan degradation, e.g. endo-1,4-β-xylanase (EC 3.2.1.8), β-xylosidase (EC 3.2.1.37), α-glucuronidase (EC 3.2.1.139), α-L-arabinofuranosidase (EC 3.2.1.55) and acetylxylan esterase (EC 3.1.1.72) (Fig. 3B) all act on the different heteropolymers available in Nature. In glucomannan degradation, β-mannanase (EC 3.2.1.78), and β-mannosidase (EC 3.2.1.25) are cleaving the polymer backbone (Fig. 3C). The main chain endo-cleaving enzymes (xylanases and mannanases) are among the most well-known. Most xylanase sequences are classified under GH family 10 and 11 (both retaining), and a few additional enzymes are found in other families (both inverting and retaining [77]). Mannanases are predominantly classified under GH family 5 and 26 (both with retaining mechanism), and only one bifunctional enzyme is to date classified in GH44 [inverting]. These families all have representatives of thermophilic origin.

Hemicellulose is, like cellulose, an important source of fermentable sugars for biorefining applications (see also Biofuel below), and efficient degradation is vital for its use. As exemplified above, we can also predict an application potential in the production of intermediates for green chemicals (e.g. xylitol). Other biotechnological applications are also established for these enzymes, many of which motivate the use of thermostable enzymes. A selection of enzymes is shown below [see Additional file 3]. Use of endo-1,4-β-xylanases (EC 3.2.1.8.) in the bleaching process of pulps for paper manufacturing is a concept introduced by Finnish researchers, which is of great environmental interest due to the possibility to decrease chemical bleaching consumption in subsequent steps [116,117]. Due to process conditions, enzymes functioning at high temperatures and high pH-values are desirable in the following bleaching process. Enzymes from thermophiles meet the temperature demand, as they display intrinsic thermostability, and maximum activity at high temperature, and e.g. the xylanase Xyn10A from R. marinus has been shown to improve brightness in bleaching sequences of hardwood and softwood kraft pulps prepared by Kraft processing, when introducing the enzyme treatment step at 80°C [118]. Several patents have been filed on thermostable xylanases in relation to use in pulping [119-121], including e.g. amino acid substituted GH11 enzymes for improved performance [122]. Xylanases are also produced in industrial scale as additives in feed for poultry [123] and as additives to wheat flour for improving the quality of baked products [63].

Mannanases have potential in pulp bleaching, especially in combination with xylanase [124], and applications in food and feed include viscosity decreasing action in coffee extracts for instant coffee production [125].

Conversion of pectins

Pectins are the third main structural polysaccharide group of plant cell walls, abundant in sugar beet pulp [126] and fruit, e.g. in citrus fruit and apple, where it can form up to half of the polymeric content of the cell wall [127]. The pectin backbone, which consists of homo-galacturonic acid regions (sometimes methylated), and regions of both rhamnose and galacturonic acid (Fig. 3D), has neutral sugar sidechains made up from L-rhamnose, arabinose, galactose and xylose [128]. L-rhamnose residues in the backbone carry sidechains containing arabinose and galactose. There are also single xylogalacturonan side chains [127]. Pectin has found widespread commercial use, especially in the textile industry [129] and in the food industry as thickener, texturizer, emulsifier, stabilizer, filler in confections, dairy products, and bakery products, etc [130]. It is also studied for its potential in drug delivery and in the pharmaceutical industry [131], and is interesting as a dietary supplementation to humans due to its possible cholesterol-lowering effect [132]. Pectin also has a potential in making biodegradable films [133]. Despite these applications, pectins are, similar to cellulose and hemicelluloses, common waste materials that can be converted to soluble sugars, ethanol [134], and biogas [135].

Microbial pectinases account for 25% of the global food enzymes sales [136], and are used extensively for fruit juice clarification, juice extraction, manufacture of pectin-free starch, refinement of vegetable fibers, degumming of natural fibers, waste-water treatment, curing of coffee, cocoa and tobacco and as an analytical tool in the assessment of plant products [136,137]. In some applications, it can be more proficient to use thermostable enzymes, particularly when using substrates (which can also be other naturally-occurring glycoside-containing molecules with similar linkages as in pectin) that are poorly soluble at ambient temperatures, such as naringin and rutin, present in fruits [138]. Many enzymes are involved in pectin degradation (some major examples shown in Fig 3D), but are referred to by several different names, which can be quite confusing. They may be acting either by hydrolysis or by trans-elimination; the latter performed by lyases [128]. Polymethylgalacturonase, (endo-)polygalacturonase (pectin depolymerase, pectinase, EC 3.2.1.15), exopolygalacturonase (EC 3.2.1.67), and exopolygalacturanosidase (EC 3.2.1.82) hydrolysing the polygalacturonic acid chain by addition of water, are all classified under GH28, and are the most abundant among all the pectinolytic enzymes [128,139]. α-L-rhamnosidases (EC 3.2.1.40, in GH family 28, 78 and 106) hydrolyze rhamnogalacturonan in the pectic backbone. α-L-Arabinofuranosidases (EC 3.2.1.55, α-L-AFases found in 5 different GH families) hydrolyze the L-arabinose side-chains, and endo-arabinase (EC 3.2.1.99, GH43) act on arabinan side-chains in pectin [140]. These two enzymes operate synergistically in degrading branched arabinan to yield L-arabinose [126]. Polysaccharide lyases (PL), which like GH have been classified under sequence-related families, cleave the galacturonic acid polymer by β-elimination and comprise e.g. polymethylgalacturonate lyase (pectin lyase, EC 4.2.2.10), polygalacturonate lyase (pectate lyase, EC 4.2.2.2), and exopolygalacturonate lyase (pectate disaccharide-lyase, EC 4.2.2.9) [77,139,141]. Pectinesterase (pectinmethyl esterase, pectinmethoxylase, EC 3.1.1.11) de-esterify the methyl ester linkages of the pectin backbone [139]. Thermostable pectinases are not so frequently described, but reports show a few thermostable α-L-rhamnosidases, e.g. from Clostridium stercorarium [142] and from a strain closely related to Thermomicrobium [138]. A thermostable polygalacturonase from a thermophilic mould, Sporotrichum thermophile, optimally active at 55°C has also been reported and may be relevant for the fruit juice industry [143] [see Additional file 3]. Several thermostable α-L-AFases (also involved in side-chain degradation of xylan) are described in the literature (listed under hemicellulases [see Additional file 3]).

Biofuel

During the world oil crisis in the 70's the interest in the use of cellulases to produce fermentable sugars from cellulosic wastes was awakened both in the United States and in Europe. The aim was then to become less dependent on oil and reduce the oil imports. At present, this need is even more outspoken, not only because of the increasing cost of oil, but also since there is a need to reduce greenhouse gas emissions and overall improve air quality. Today, there are special programs in a number of countries targeted towards developing biofuel production from renewable resources, examining the possibilities of for example biogas, bioethanol, biodiesel and fuel cells.

Bioethanol is the most common renewable fuel today, and e.g the "Biofuels Initiative" in the U.S. (US Department of Energy), strives to make cellulosic ethanol cost-competitive by 2012 and supposedly correspond to a third of the U.S. fuel consumption by 2030. The "Energy for the Future" in the EU, has the objective of having 12% renewable energy in the EU by 2010 [144]. Ethanol is commonly derived from corn grain (starch) or sugar cane (sucrose) [145]. Sucrose can be fermented directly to ethanol, but starch is hydrolyzed to glucose before it can be fermented, generally by Saccharomyces cerevisiae [146]. Ethanol fermentation from starch can be improved by utilizing better enzymes and strains and preferably hydrolyze the starch from whole grains without a chemical pre-treatment and with simultaneous liquefaction, saccharification and fermentation [147].

However, the starch biomass material, as well as sugar cane, is limited and for renewable biofuel to be able to compete with fossil fuel, a cost-efficient process of an even more abundant renewable resource is needed. Agricultural and forest biomass are available in large enough quantities to be considered for large-scale production of alcohol-based fuels [148]. Urban wastes are an additional source of biomass; it is estimated that cellulose accounts for 40% of municipal solid waste [148]. Cellulose-based products can be competitive with products derived from fossil resources provided processing costs are reduced [149]. Unfortunately, because of the complex and crystalline structure of lignocellulose, this material is much more difficult to hydrolyze than starch. Efficient conversion of lignocellulosic material to fermentable sugars is necessary, but requires better strains or enzyme systems which are able to convert both pentoses and hexoses and tolerate stress conditions [150]. Use of thermostable cellulases, hemicellulases, and thermophilic microorganisms in the degradation of the lignocellulosic material offers an advantage by minimizing the risk of contamination and could enable a single-step process of enzymatic hydrolysis, fermentation, and distillation of formed ethanol [151].

Today, the hydrolysis and fermentation steps are separate. The fermentation step is usually performed by Saccharomyces cerevisiae or Zymomonas mobilis, but this can be a disadvantage, since the temperature has to be reduced from the hydrolysis step, which is better performed at higher temperature, at least 50°C [152]. Thermoactive yeast, Kluyveromyces marxianus, active up to 50°C, performed equally well as S. cerevisiae [153], but even higher temperatures are desired. The fermentation can also be done by thermo-active anaerobic bacteria. For example, some thermophiles isolated from Icelandic hot springs performed quite well in ethanol production from lignocellulolytic hydrolysates, but need further testing [154].

Enzymatic cellulose hydrolysis to glucose is today predominantly carried out by fungi, e.g. Trichoderma, Penicillium and Aspergillus [155], but to compete with results from acid hydrolysis, more efficient degradation, presumably at higher temperature is needed, and some relevant enzymes have been described from thermophiles and hyperthermophiles [see Additional file 3]. The obstacle lies in expressing a range of proteins and assembling them in vitro [151], but it has been shown that cellulases from different origins, with different temperature optima ranging from mesophilic to thermophilic, can be matched together and still exhibit substantial synergism in the degradation of cellulosic material [156]. An endoglucanase from Acidothermus cellulolyticus, which was fused to T. reesei cellobiohydrolase and expressed in T. reesei was for example enhancing saccharification yields [157]. Endoglucanase and cellobiohydrolase activity is however not sufficient, as the degradation product (cellobiose) inhibits the former enzymes and blocks further depolymerization of the cellulose. To solve this product inhibition, β-glucosidases have to be added, or engineered into production strains that are able to ferment cellobiose and cellotriose to ethanol [158]. Thermophiles have not yet played any major role in metabolic engineering, due to the limited amount of vectors and tools available for their modification. Instead, well-known mesophiles like S. cerevisiae are used, and has recently been modified with genes from a fungal xylose pathway and from a bacterial arabinose pathway, which resulted in a strain able to grow on both pentose and hexose sugars with improved ethanol yields [159]. Better technologies for biomass pretreatment are also needed. Mechanical, chemical, biological or thermal pre-treatments enhance the cellulase accessibility by removing lignin and hemicelluloses and by partially disrupting the fiber structure. A recent review is given by Wyman et al. [160] and a comparison has been made between leading technologies [161].

Production possibilities of the biocatalysts

An important consideration when selecting a biocatalyst is the prospect of producing it in sufficient amounts. These considerations include the choice of either producing by the native host, or if the gene encoding an enzyme of interest should be transferred to a selected host for recombinant production. Generally, gene expression is not a problem related to the thermophilicity of the target protein and those originating from thermophilic resources meet the same production bottlenecks as their counterparts from mesophiles.

Another important consideration, crucial for the implementation of biocatalysts, is the production cost, and a few years ago e.g. Genencor International was working under a subcontract from the office of Biomass Program (USA), to reduce the cellulase costs in order to make degradation into fermentable sugars more cost-effective [162].

Cellulose degradation by cellulases in large scale is (as stated in the Biofuel-section) usually carried out by fungal strains [155], but to introduce more thermoactive enzymes there is a possibility for heterologous production in bacterial hosts, which generally have higher growth rates than fungi. The difficulty using bacterial cellulases is that they are larger, more complex enzymes and often part of a cellulosome with many different activities. Research has also been aimed towards improving presently used fermentation strains by metabolic engineering.

Enzyme production by thermophiles

Cultivation of thermophiles at high temperature is technically and economically interesting as it reduces the risk of contamination, reduces viscosity, thus making mixing easier, and leads to a high degree of substrate solubility. However, compared to their mesophilic counterparts, the biomass achieved by these organisms is usually disappointingly low. The low cell yield poses problems for both large and small scale production, which makes extensive studies of their enzymes very difficult. This has triggered considerable research aiming to improve thermophilic cell yield. To date, several reports on media compositions and culture optimization of different thermophiles are available [163]. Special equipments and specific processes have been developed to improve fermentation processes of thermophiles and hyperthermophiles [164]. However, due to factors such as requirement of complex and expensive media [163], low solubility of gas at high temperature, and low specific growth rates and product inhibition [164], large scale commercial cultivation of thermophiles for enzyme production remains an economical challenge. The high cost of large-scale fermentation processes to produce enzymes by thermophiles and hyperthermophiles is justifiable only for very few specific applications.

Recombinant enzyme production in mesophilic and thermophilic hosts

Reduction of the production cost of thermophilic enzymes is fundamental for their breakthrough in large scale. One alternative to reduce production costs and increase the yield of these processes is to use recombinant technology. A wide variety of thermostable enzymes have been cloned and successfully expressed in mesophilic organisms, such as Escherichia coli [165], Bacillus subtilis [166], Saccharomyces cervisae [167], Pichia pastoris [168], Aspergillus oryzae [169], Kluyveromyces lactis [170], and Trichoderma reesei [171].

However, differences in codon usage or improper folding of the proteins can result in reduced enzyme activity or low level of expression [172,173]. Moreover, many complex enzymes, like heterooligomers or those requiring covalently bound co-factors can be very difficult to produce in mesophilic hosts. This initiated the search of genetic tools for the overexpression of such enzymes in thermophilic host systems. So far, a number of vectors have been developed for expression of proteins in various thermophilic hosts (Table 3). Use of the novel thermophilic expression systems is, however, still at research level and more work remains before exploitation at large or industrial scale can be considered.

Table 3. Vectors constructed for thermophilic expression system

Isolated enzymes or whole cell applications?

Thermophilic enzymes are potentially applicable in a wide range of industrial processes mainly due to their extraordinary operational stability at high temperatures and denaturant tolerance. Such enzymes are used in the chemical, food, pharmaceutical, paper, textile and other industries [182-185]. Most of these applications utilize recombinant thermostable enzymes that have been expressed in mesophilic hosts. Depending on the type of application, the nature of reactions and product purity, the enzyme preparation can be cell-free (crude, partially purified or homogenous) or cell-associated. For example, the use of cell-free dehydrogenases is hampered by the need for expensive and sensitive co-factors [186] while transaminases suffer from unfavourable reaction equilibria [187]. In this regard, whole cell applications can be more attractive. Whole cell applications have also been reported in food processing, making use of recombinant thermophilic α-glucosidase expressed in Lactococcus lactis [188].

The usage of whole cells is of special interest for transformation of lignocellulosics. The bioconversion involves two major steps; saccharification and fermentation. Saccharification is the hydrolysis of carbohydrate polymers (cellulose and hemicellulose) into sugars, and this hydrolysate is then utilized as substrate in the fermentation step by microorganisms that transform it into metabolic products (e.g. ethanol, see Biofuel). Whole-cell microbial bioconversion offers an attractive possibility of a single step transformation, in which the microorganisms produce saccharolytic enzymes that degrade the lignocellulose and ferment the liberated sugars, which could lead to higher efficiency than in the common multistep lignocellulosic conversions [189,190].

The close association of cellulose and hemicellulose to lignin in the plant cell wall, however, make this substrate difficult to degrade into monomer sugars at high yields (compared to sugar- or starch-containing crops, e.g. sugar cane or maize). Pre-treatment (using steam, acid or alkali) is thus necessary to make the carbohydrate polymers available for enzymatic hydrolysis and fermentation [155,191]. Among pre-treatment methods, high temperature pre-treatment using liquid hot water is shown to make the biomass (specifically the cellulose part) more accessible to enzymatic attack. Development of fermentation systems for thermophiles is here appealing, as it allows energy savings by reducing the cooling cost after steam pre-treatment, lowering the risk of contamination, and improving saccharification and fermentation rates. Moreover, in production of ethanol, thermophilic conditions result in continuous ethanol evaporation allowing harvest during fermentation. Simultaneous fermentation and product recovery can decrease product inhibition of the fermentation process (by the ethanol), reduce the volume of water consumed for distillery cooling, and the time required for distillation, leading to a more efficient process. A problem associated with lignocellulose pre-treatment procedures is, however, liberation of degradation products that can inhibit microbial growth [191], but some thermophilic bacteria have shown promising results in fermenting lignocellulosic hydrolysates to ethanol, like the xylanolytic anaerobic thermophilic bacterium, Thermoanaerobacter mathranii, shown to ferment the xylose in the hemicellulose fraction from alkaline wet oxidized wheat straw to ethanol with no prior detoxification [191]. Still, growth on pre-treated lignocellulose may vary dependent on both organism and substrate origin [189]. Moreover, the insolubility of lignocellulosics creates problems in maintaining homogeneity in reactors making monitoring and control of process parameters difficult. Therefore, like for their mesophilic counterparts, efficient utilization of thermophiles in integrated bioprocesses needs thorough investigation. In the last few years, reports have been made on solid state cultivation of thermophiles on lignocellulosics [192,193]. In some cases, compared to the more traditional submerged liquid fermentation, better conversion has been reached under solid sate cultivation [194].

Use of naturally occurring microorganisms is, however, generally not efficient enough in transforming the substrate into higher value products. Thus, it is imperative to enhance the robustness of the microbes towards increased substrate hydrolysis and higher product yields through metabolic engineering. Metabolic engineering has been pursued in mesophilic hosts, resulting in strains of biorefinery interest that produce high yields of ethanol [195,196], propanediol [197,198], acetate [199], adipic acid [200], succinic acid [201] and lactic acid [202]. However, such metabolic engineering reports have been very rare for thermophiles [203], but may increase with the availability/development of genetic tools. Several thermophilic organisms such as Thermoanaerobium brockii [204], Clostridium thermohydrosulfuricum [205], and Moorella sp. HUC22-1 [206], have been studied for ethanol production. Metabolic engineering of such thermophiles to improve ethanol productivity and efficiency of utilizing different substrates like cellulose, hemicellulose and pectin can be very interesting.

Concluding remarks

Thermophiles and especially thermophilic enzymes have to date gained a great deal of interest both as analytical tools, and as biocatalysts for application in large scale. Utilization of these enzymes is however still today, despite many efforts, often limited by the cost of the enzymes. With an increasing market for the enzymes, leading to production in higher volumes, the cost is however predicted to decrease. Moreover, with a paradigm shift in industry moving from fossils towards renewable resource utilization, the need of microbial catalysts is predicted to increase, and certainly there will be a continued and increased need of thermostable selective biocatalysts in the future.

References

  1. Brock TD, Freeze H: Thermus aquaticus gen. n. and sp. n., a non-sporulating extreme thermophile.

    Journal of Bacteriology 1969, 98:289-297. PubMed Abstract | PubMed Central Full Text OpenURL

  2. Brock TD: Introduction, an overview of the thermophiles. In Thermophiles: General, Molecular and Applied Microbiology. Edited by Brock TD. New York: John Wiley & Sons; 1986:1-16. OpenURL

  3. Maheshwari R, Bharadwaj G, Bhat MK: Thermophilic fungi: Their physiology and enzymes.

    Microbiology and Molecular Biology Reviews 2000, 64:461-488. OpenURL

  4. Kristjansson JK, Stetter KO: Thermophilic bacteria. In Thermophilic bacteria. Edited by Kristjansson JK. London: CRC Press Inc; 1992:1-18. OpenURL

  5. Stetter KO: Hyperthermophilic prokaryotes.

    FEMS Microbiology Reviews 1996, 18:149-158. OpenURL

  6. Satyanarayana T, Raghukumar C, Shivaji S: Extremophilic microbes: Diversity and perspectives.

    Current Science 2005, 89:78-90. OpenURL

  7. Fujiwara S: Extremophiles: Developments of their special functions and potential resources.

    Journal of Bioscience and Bioengineering 2002, 94:518-525. OpenURL

  8. Podar M, Reysenbach AL: New opportunities revealed by biotechnological explorations of extremophiles.

    Current Opinion in Biotechnology 2006, 17:250-255. PubMed Abstract OpenURL

  9. Comfort DA, Chhabra SR, Conners SB, J CC, L EK, Johnson MR, Jones KL, Sehgal AC, Kelly RM: Strategic biocatalysis with hyperthermophilic enzymes.

    Green Chemistry 2004, 6:459-465. OpenURL

  10. Fernando S, Adhikari S, Chandrapal C, Murali N: Biorefineries: Current status, challenges and future direction.

    Energy and Fuels 2006, 20:1727-1737. OpenURL

  11. Kamm B, Kamm M: Principles of biorefineries.

    Applied Microbiology and Biotechnology 2004, 64:137-145. OpenURL

  12. Paes G, O'Donohue MJ: Engineering increased thermostability in the thermostable GH-11 xylanase from Thermobacillus xylanilyticus.

    Journal of Biotechnology 2006, 125:338-350. PubMed Abstract OpenURL

  13. Santos H, da Costa MS: Compatible solutes of organisms that live in hot saline environments.

    Environmental Microbiology 2002, 4:501-509. PubMed Abstract OpenURL

  14. Kristjansson JK: Thermophilic organisms as sources of thermostable enzymes.

    Trends in Biotechnology 1989, 7:349-353. OpenURL

  15. OECD: The application of biotechnology to industrial sustainability. Paris, France: OECD publications service; 2001.

  16. Bruins ME, Janssen AEM, Boom M: Thermozymes and their applications.

    Applied Biochemistry and Biotechnology 2001, 90:155-186. OpenURL

  17. Vieille C, Zeikus GJ: Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

    Microbiology and Molecular Biology Reviews 2001, 65:1-43. OpenURL

  18. Ladenstein R, Ren B: Protein disulfides and protein disulfide oxidoreductases in hyperthermophiles.

    FEBS Journal 2006, 273:4170-4185. PubMed Abstract OpenURL

  19. Danson MJ, Hough DW: Structure, function and stability of enzymes from the Archaea.

    Trends in Microbiology 1998, 6:307-314. PubMed Abstract OpenURL

  20. Yip KSP, Britton KL, Stillman TJ, Lebbink J, de Vos WM, Robb FT, Vetriani C, Maeder D, Rice DW: Insights into the molecular basis of the thermal stability from the analysis of ion-pair networks in the glutamate dehydrogenase family.

    European Journal of Biochemistry 1998, 255:336-346. PubMed Abstract OpenURL

  21. Karshikoff A, Ladenstein R: Ion pairs and the thermotolerance of proteins from hyperthermophiles: a "traffic rule" for hot roads.

    Trends in Biochemical Sciences 2001, 26:550-556. PubMed Abstract OpenURL

  22. Arnott MA, Michael RA, Thompson CR, Hough D, Danson MJ: Thermostability and thermoactivity of citrate synthases from the thermophilic and hyperthermophilic archaea, Thermoplasma acidophilum and Pyrococcus furiosus.

    Journal of Molecular Biology 2000, 304:657-668. PubMed Abstract OpenURL

  23. Crennell SJ, Hreggvidsson GO, Nordberg Karlsson E: The structure of Rhodothermus marinus Cel12A, a highly thermostable family 12 endoglucanase, at 1.8 Å resolution.

    Journal of Molecular Biology 2002, 320:883-897. PubMed Abstract OpenURL

  24. Yano JK, Poulos TL: New understandings of thermostable and peizostable enzymes.

    Current Opinion in Biotechnology 2003, 14:360-365. PubMed Abstract OpenURL

  25. Crennell SJ, Cook D, Minns A, Svergun D, Andersen RL, Nordberg Karlsson E: Dimerisation and an increase in active site aromatic groups as adaptations to high temperatures: X-ray solution scattering and substrate-bound crystal structures of Rhodothermus marinus endoglucanase Cel12A.

    Journal of Molecular Biology 2006, 356:57-71. PubMed Abstract OpenURL

  26. Johannes TW, Zhao H: Directed evolution of enzymes and biosynthetic pathways.

    Current Opinion in Microbiology 2006, 9:261-267. PubMed Abstract OpenURL

  27. Cicortas Gunnarsson L, Nordberg Karlsson E, Albrekt AS, Andersson M, Holst O, Ohlin M: A carbohydrate binding module as a diversity-carrying scaffold.

    Protein Engineering Design and Selection 2004, 17:213-221. OpenURL

  28. Hasan Z, Renirie R, Kerkman R, Ruijssenaars HJ, Hartog AF, Wever R: Laboratory-evolved vanadium chloroperoxidase exhibits 100-fold higher halogenating activity at alkaline pH – Catalytic effects from first and second coordination sphere mutations.

    The Journal of Biological Chemistry 2006, 281:9738-9744. PubMed Abstract OpenURL

  29. Short JM: Directed evolution of thermostable enzymes.

    Patent. US 5830696 1998. OpenURL

  30. Cicortas Gunnarsson L, Nordberg Karlsson E, Andersson M, Holst O, Ohlin M: Molecular engineering of a thermostable carbohydrate-binding module.

    Biocatalysis and Biotransformation 2006, 24:31-37. OpenURL

  31. Boraston AB, Bolam DN, Gilbert HJ, Davies GD: Carbohydrate binding modules: fine tuning polysaccharide recognition.

    Biochemical Journal 2004, 382:769-781. PubMed Abstract | PubMed Central Full Text OpenURL

  32. Volkov IY, Lunina NA, Velikodvorskaya GA: Prospects for the practical application of substrate-binding modules of glycosyl hydrolases.

    Applied Biochemistry and Microbiology 2004, 40:427-432. OpenURL

  33. Cicortas Gunnarsson L, Dexlin L, Nordberg Karlsson E, Holst O, Ohlin M: Evolution of a carbohydrate binding module into a human IgG4-specific protein binder.

    Biomolecular Engineering 2006, 23:111-117. PubMed Abstract OpenURL

  34. Kim Y-W, Choi J-H, Kim J-W, Park C, Kim J-W, Cha H, Lee S-B, Oh B-H, Moon T-W, Park K-H: Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance.

    Applied and Environmental Microbiology 2003, 69:4866-4874. OpenURL

  35. Turner NJ: Directed evolution of enzymes for applied biocatalysis.

    Trends in Biotechnology 2003, 21:474-478. PubMed Abstract OpenURL

  36. Tang S-Y, Le Q-T, Shim J-H, Yang S-J, Auh J-H, Park C, Park K-H: Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling.

    FEBS Journal 2006, 273:3335-3345. PubMed Abstract OpenURL

  37. Roodvelt C, Aharoni A, Tawfik DS: Directed evolution of proteins for heterologous expression and stability.

    Current Opinion in Structural Biology 2005, 15:50-56. PubMed Abstract OpenURL

  38. Jaenicke R: Protein stability and molecular adaptations to extreme conditions.

    European Journal of Biochemistry 1991, 202:715-728. PubMed Abstract OpenURL

  39. Jaenicke R: What ultrastable globularproteins teach us about protein stabilisation.

    Biochemistry (Moscow) 1998, 63:312-321. OpenURL

  40. Daniel RM: The upper limits of enzyme thermal stability.

    Enzyme and Microbial Technology 1996, 19:74-79. OpenURL

  41. Daniel RM, Dines M, Petach HH: The denaturation and degradation of stable enzymes at high temperatures.

    Biochemical Journal 1996, 317:1-11. PubMed Abstract | PubMed Central Full Text OpenURL

  42. Ladenstein R, Antranikian G: Proteins from hyperthermophiles: Stability and enzymatic catalysis close to the boiling point of water. In Advances in Biochemical Engineering/Biotechnology. Volume 61. Edited by Scheper T. Berlin: Springer; 1998::37-85. PubMed Abstract OpenURL

  43. Schäfer T, Böhnisch H, Kardinahl S, Schmidt C, Schäfer G: Three extremely thermostable proteins from Sulfolobus and a reappraisal of the 'Traffic rules'.

    Biological Chemistry 1996, 377:505-512. PubMed Abstract OpenURL

  44. Tyndall JD, Sinchaikul S, Fothergill-Gilmore LA, Taylor P, Walkinshaw MD: Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1.

    Journal of Molecular Biology 2002, 323:859-869. PubMed Abstract OpenURL

  45. Mabee WE, Gregg DJ, Saddler JN: Assessing the emerging biorefinery sector in Canada.

    Applied Biochemistry and Biotechnology 2005, 121:765-778. OpenURL

  46. Pye EK: Biorefining; a major opportunity for the sugar cane industry.

    International Sugar Journal 2005, 107:222. OpenURL

  47. Pan X, Kadla JF, Ehara K, Gilkes N, Saddler JN: Organosolv ethanol lignin from hybrid poplar as a radical scavenger: relationship between lignin structure, extraction conditions, and antioxidant activity.

    Journal of Agricultural and Food Chemistry 2006, 54:5806-5813. OpenURL

  48. U.S. Department of Energy. Biomass program [http://www.eere.energy.gov/biomass] webcite

  49. European union biofuels policy and agriculture: An overview [http://www.usembassy.it/pdf/other/RS22404.pdf] webcite

  50. Solaiman DKY, Ashby RD, Foglia TA, Marmer WN: Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates).

    Applied Microbiology and Biotechnology 2006, 71:783-789. OpenURL

  51. Gaspar M, Juhasz T, Szengyel Z, Reczey K: Fractionation and utilisation of corn fibre carbohydrates.

    Process Biochemistry 2005, 40:1183-1188. OpenURL

  52. Koutinas AA, Wang R, Webb C: Restructuring upstream bioprocessing: Technological and economical aspects for production of a generic microbial feedstock from wheat.

    Biotechnology and Bioengineering 2004, 85:524-538. OpenURL

  53. Edye LA, Doherty WOS, Blinco JA, Bullock GE: The sugarcane biorefinery: Energy crops and processes for the production of liquid fuels and renewable commodity chemicals.

    International Sugar Journal 2006, 108:19-27. OpenURL

  54. Enze M: Developing biorefinery by utilizing agriculture and forestry biomass resources: Striding forward the "carbohydrate" era.

    Progress in Chemistry 2006, 18:132-141. OpenURL

  55. Mihovilovic MD, Müller B, Stanetty P: Monooxygenase-mediated Baeyer-Villiger oxidations.

    European Journal of Organic Chemistry 2002, 2002:3711-3730. OpenURL

  56. Kroutil W, Mang H, Edegger K, Faber K: Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols.

    Current Opinion in Chemical Biology 2004, 8:120-126. PubMed Abstract OpenURL

  57. Hatti-Kaul R, Törnvall U, Gustafsson L, Börjesson P: Industrial biotechnology for production of bio-based chemicals – a cradle-to-grave perspective.

    Trends in Biotechnology, in press. PubMed Abstract OpenURL

  58. Fessner W-D: Enzyme mediated C-C bond formation.

    Current Opinion in Chemical Biology 1998, 2:85-97. PubMed Abstract OpenURL

  59. Hansson T, Adlercreutz P: Enzymatic synthesis of hexyl glycosides from lactose at low water activity and high temperature using hyperthermostable β-glycosidases.

    Biocatalysis and Biotransformation 2002, 20:167-178. OpenURL

  60. Turner P, Svensson D, Adlercreutz P, Nordberg Karlsson E: A novel variant of Thermotoga neapolitana β-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation.

    Journal of Biotechnology, in press. OpenURL

  61. Turner C, Turner P, Jacobson G, Waldebäck M, Sjöberg P, Nordberg Karlsson E, Markides K: Subcritical water extraction and β-glucosidase-catalyzed hydrolysis of quercetin in onion waste.

    Green Chemistry 2006, 8:949-959. OpenURL

  62. Werpy T, Petersen G (Eds): Top value added chemicals from biomass: Volume 1 – Results of screening for potential candidates from sugars and synthesis gas [http://www1.eere.energy.gov/biomass/pdfs/35523.pdf] webcite

    In U.S. Department of Energy; NREL/TP-510-35523; 2004.

  63. Wong KKY, Saddler JN: Applications of hemicellulases in the food, feed, and pulp and paper industries. In Hemicellulose and Hemicellulases. Edited by Coughlan MP, Hazlewood GP. London and Chapel Hill: Portland Press; 1993:127-143. OpenURL

  64. Puls J, Schuseil J: Chemistry of hemicelluloses: relation between hemicellulose structure and enzymes required for hydrolysis. In Hemicellulose and hemicellulases. Edited by Coughlan MP, Hazlewood GP. London and Chapel Hill: Portland Press; 1993:1-27. OpenURL

  65. Viikari L, Kantelinen A, Buchert J, Puls J: Enzymatic accessibility of xylans in lignocellulosic materials.

    Applied Microbiology and Biotechnology 1994, 41:124-129. OpenURL

  66. Das H, Singh SK: Useful byproducts from cellulosic wastes of agriculture and food Industry – a critical appraisal.

    Critical Reviews in Food Science and Nutrition 2004, 44:77-89. OpenURL

  67. Guo C, Zhao C, He P, Lu D, Shen A, Jiang N: Screening and characterization of yeasts for xylitol production.

    Journal of Applied Microbiology 2006, 101:1096-1104. PubMed Abstract OpenURL

  68. Hallborn J, Walfridsson M, Airaksinen U, Ojamo H, Hahn-Hägerdal B, Penttila M, Keranen S: Xylitol production by recombinant Saccharomyces cerevisiae.

    Bio/technology 1991, 9:1090-1095. OpenURL

  69. Gupta R, Beg QK, Lorenz P: Bacterial alkaline proteases: molecular approaches and industrial applications.

    Applied Microbiology and Biotechnology 2002, 59:15-32. OpenURL

  70. Kumar CG, Takagi H: Microbial alkaline proteases: From a bioindustrial viewpoint.

    Biotechnology Advances 1999, 17:561-594. PubMed Abstract OpenURL

  71. Hasan F, Shah AA, Hameed A: Industrial applications of microbial lipases.

    Enzyme and Microbial Technology 2006, 39:235-251. OpenURL

  72. Svendsen A: Lipase protein engineering.

    Biochimica et Biophysica Acta – Protein Structure and Molecular Enzymology 2000, 1543:223-238. OpenURL

  73. Enzymes for Industrial Applications [http://www.bccresearch.com] webcite

    Business Communications Company, Inc; RC-147U; 2004.

  74. Duchiron F, Legin E, Ladrat C, Gantelet H, Barbier G: New thermostable enzymes for crop fractionation.

    Industrial Crops and Products 1997, 6:265-270. OpenURL

  75. Wang TL, Bogracheva TY, Hedley CL: Starch: As simple as A, B, C?

    Journal of Experimental Botany 1998, 49:481-502. OpenURL

  76. Sinnott ML: Catalytic mechanisms of enzymic glycosyl transfer.

    Chemical Reviews 1990, 90:1171-1202. OpenURL

  77. Carbohydrate-active enzymes server [http://www.cazy.org/] webcite

  78. Crabb WD, Mitchinson C: Enzymes involved in the processing of starch to sugars.

    Trends in Biotechnology 1997, 15:349-352. OpenURL

  79. Kaper T, van der Maarel MJEC, Euverink G-JW, Dijkhuizen L: Exploring and exploiting starch-modifying amylomaltases from thermophiles.

    Biochemical Society Transactions 2004, 32:279-282. PubMed Abstract OpenURL

  80. Tester RF, Debon SJJ: Annealing of starch – a review.

    International Journal of Biological Macromolecules 2000, 27:1-12. PubMed Abstract OpenURL

  81. SPEZYME FRED. Low calcium, low pH, thermostable α-amylase. Product information

    Genencor International, Inc. SZFRED01, REV0306 2006. OpenURL

  82. Van der Veen ME, Veelaert S, Van der Goot AJ, Boom RM: Starch hydrolysis under low water conditions: A conceptual process design.

    Journal of Food Engineering 2006, 75:178-186. OpenURL

  83. Souza RCR, Andrade CT: Investigation of the gelatinization and extrusion processes of corn starch.

    Advances in Polymer Technology 2002, 21:17-24. OpenURL

  84. Pandey A: Glucoamylase research – an overview.

    Starch 1995, 47:439-445. OpenURL

  85. Kim M-S, Park J-T, Kim Y-W, Lee H-S, Nyawira R, Shin H-S, Park C-S, Yoo S-H, Kim Y-R, Moon T-W, et al.: Properties of a novel thermostable glucoamylase from the hyperthermophilic archaeon Sulfolobus solfataricus in relation to starch processing.

    Applied and Environmental Microbiology 2004, 70:3933-3940. OpenURL

  86. Kaper T, Talik B, Ettema TJ, Bos H, van der Maarel M, Dijkhuizen L: Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels.

    Applied and Environmental Microbiology 2005, 71:5098-5106. OpenURL

  87. Terada Y, Fujii K, Takaha T, Okada S: Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: Production of cycloamylose.

    Applied and Environmental Microbiology 1999, 65:910-915. OpenURL

  88. Lee H-S, Auh J-H, Yoon H-G, Kim M-J, Park J-H, Hong S-S, Kang M-H, Kim T-J, Moon T-W, Kim J-W, et al.: Cooperative action of alpha-glucanotransferase and maltogenic amylase for an improved process of isomaltooligosaccharide (IMO) production.

    Journal of Agricultural and Food Chemistry 2002, 50:2812-2817. OpenURL

  89. Szejtli J: Introduction and general overview of cyclodextrin chemistry.

    Chemical Reviews 1998, 98:1743-1754. PubMed Abstract OpenURL

  90. Irie T, Uekama K: Cyclodextrins in peptide and protein delivery.

    Advanced Drug Delivery Reviews 1999, 36:101-123. PubMed Abstract OpenURL

  91. Davis ME, Brewster ME: Cyclodextrin-based pharmaceutics: Past, present and future.

    Nature Reviews Drug Discovery 2004, 3:1023-1035. PubMed Abstract OpenURL

  92. Muderawan IW, Ong TT, Ng SC: Urea bonded cyclodextrin derivatives onto silica for chiral HPLC.

    Journal of Separation Science 2006, 29:1849-1871. PubMed Abstract OpenURL

  93. Szejtli J: Utilization of cyclodextrins in industrial products and processes.

    Journal of Materials Chemistry 1997, 7:575-587. OpenURL

  94. Sträter N, Przylas I, Saenger W, Terada Y, Fujii K, Takaha T: Structural basis of the synthesis of large cycloamyloses by amylomaltase.

    Biologia, Bratislava 2002, 57(Suppl 11):93-99. OpenURL

  95. Ueda H: Physicochemical properties and complex formation abilities of large-ring cyclodextrins.

    Journal of Inclusion Phenomena and Macrocyclic Chemistry 2002, 44:53-56. OpenURL

  96. Terada Y, Sanbe H, Takaha T, Kitahata S, Koizumi K, Okada S: Comparative study of the cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases.

    Applied and Environmental Microbiology 2001, 67:1453-1460. OpenURL

  97. Fujii K, Minagawa H, Terada Y, Takaha T, Kuriki T, Shimada J, Kaneko H: Use of random and saturation mutagenesis to improve the properties of Thermus aquaticus amylomaltase for efficient production of cycloamyloses.

    Applied and Environmental Microbiology 2005, 71:5823-5827. OpenURL

  98. Larsen KL: Large cyclodextrins.

    Journal of Inclusion Phenomena 2002, 43:1-13. OpenURL

  99. Machida S, Ogawa S, Xiaohua S, Takaha T, Fujii K, Hayashi K: Cycloamylose as an efficient artificial chaperone for protein refolding.

    FEBS Letters 2000, 486:131-135. PubMed Abstract OpenURL

  100. Brufau J, Francesch M, Pérez-Vendrell AM: The use of enzymes to improve cereal diets for animal feeding.

    Journal of the Science of Food and Agriculture 2006, 86:1705-1713. OpenURL

  101. Pasamontes L, Haiker M, Wyss M, Tessier M, van Loon APGM: Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus.

    Applied and Environmental Microbiology 1997, 63:1696-1700. OpenURL

  102. Bhat MK: Cellulases and related enzymes in biotechnology.

    Biotechnology Advances 2000, 18:355-383. PubMed Abstract OpenURL

  103. Ward OP, Mooyoung M: Enzymatic degradation of cell-wall and related plant polysaccharides.

    Critical Reviews in Biotechnology 1989, 8:237-274. PubMed Abstract OpenURL

  104. Kuhad RC, Singh A, Eriksson K-EL: Microorganisms and enzymes involved in the degradation of plant fiber cell walls. In Biotechnology in the pulp and paper industry. Edited by Eriksson K-EL. Berlin: Springer-Verlag; 1997:45-125. OpenURL

  105. Marchessault RH, Sundararajan PR: Cellulose. In The Polysaccharides. Volume 2. Edited by Aspinall GO. London: Academic Press Inc; 1983::11-95. OpenURL

  106. Sjöström E: Wood Chemistry: Fundamentals and applications. London: Academic Press Inc; 1993.

  107. Teeri TT: Crystalline cellulose degradation: new insight into the function of cellobiohydrolases.

    Trends in Biotechnology 1997, 15:160-167. OpenURL

  108. Wood TM, Garcia-Campayo V: Enzymology of cellulose degradation.

    Biodegradation 1990, 1:147-161. OpenURL

  109. Schulein M: Protein engineering of cellulases.

    Biochimica et Biophysica Acta – Protein Structure and Molecular Enzymology 2000, 1543:239-252. OpenURL

  110. Bayer EA, Chanzy H, Lamed R, Shoham Y: Cellulose, cellulases and cellulosomes.

    Current Opinion in Structural Biology 1998, 8:548-557. PubMed Abstract OpenURL

  111. Bolam DN, Ciruela A, McQueen-Mason S, Simpson P, Williamson MP, Rixon JE, Boraston A, Hazlewood GP, Gilbert HJ: Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity.

    Biochemical Journal 1998, 331:775-781. PubMed Abstract | PubMed Central Full Text OpenURL

  112. Kittur FS, Mangala SL, Abu Rus'd A, Kitaoka M, Tsujibo H, Hayashi K: Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan.

    FEBS Letters 2003, 549:147-151. PubMed Abstract OpenURL

  113. Zverlov VV, Volkov IY, Velikodvorskaya GA, Schwarz WH: The binding pattern of two carbohydrate-binding modules of laminarinase Lam16A from Thermotoga neapolitana: differences in beta-glucan binding within family CBM4.

    Microbiology 2001, 147:621-629. PubMed Abstract OpenURL

  114. Himmel ME, Ruth MF, Wyman CE: Cellulase for commodity products from cellulosic biomass.

    Current Opinion in Biotechnology 1999, 10:358-364. PubMed Abstract OpenURL

  115. Saha BC: α-L-Arabinofuranosidases – biochemistry, molecular biology and application in biotechnology.

    Biotechnology Advances 2000, 18:403-423. PubMed Abstract OpenURL

  116. Suurnäkki A, Tenkanen M, Buchert J, Viikari L: Hemicellulases in the bleaching of chemical pulps. In Biotechnology in the pulp and paper industry. Volume 57. Edited by Eriksson K-EL. Berlin: Springer-Verlag; 1997::261-287. OpenURL

  117. Viikari L, Kantelinen A, Sundquist J, Linko M: Xylanases in bleaching: from an idea to the industry.

    FEMS Microbiology Reviews 1994, 13:335-350. OpenURL

  118. Pfabigan N, Nordberg Karlsson E, Ditzelmueller G, Holst O: Prebleaching of kraft pulp with full-length and functional domains of a thermostable xylanase from Rhodothermus marinus.

    Biotechnology Letters 2002, 24:1191-1197. OpenURL

  119. Bergquist PL, Gibbs MD, Morris D: Xylanase from Dictyoglomus thermophilum and its use in bleaching of cellulose products.

    Patent. WO 9736995 1997. OpenURL

  120. Sandal T, Kofod LV, Kauppinen MS, Andersen LN, Dybdal L: Enzyme with xylanase activity.

    Patent. WO 9727292 1997. OpenURL

  121. Fagerstrom RB, Paloheimo M, Lantto R, Lahtinen T, Suominen P: Xylanases and their use.

    Patent. US 5922579 1999. OpenURL

  122. Sung WL, Yaguchi M, Ischikawa K: Modification of xylanases to improve thermophilicity, alkalophilicity, and thermostability for pulp bleaching.

    Patent. EP 828002 1998. OpenURL

  123. Niehaus F, Bertoldo C, Kähler M, Antranikian G: Extremophiles as a source of novel enzymes for industrial application.

    Applied Microbiology and Biotechnology 1999, 51:711-729. OpenURL

  124. Clarke JH, Davidson K, Rixon JE, Halstead JR, Fransen MP, Gilbert HJ, Hazlewood GP: A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and α-galactosidase.

    Applied Microbiology and Biotechnology 2000, 53:661-667. OpenURL

  125. Sachslehner A, Foidl G, Foidl N, Gubitz G, Haltrich D: Hydrolysis of isolated coffee mannan and coffee extract by mannanases of Sclerotium rolfsii.

    Journal of Biotechnology 2000, 80:127-134. PubMed Abstract OpenURL

  126. Spagnuolo M, Crecchio C, Pizzigallo MDR, Ruggiero P: Fractionation of sugar beet pulp into pectin, cellulose, and arabinose by arabinases combined with ultrafiltration.

    Biotechnology and Bioengineering 1999, 64:685-691. OpenURL

  127. Brummell DA: Cell wall disassembly in ripening fruit.

    Functional Plant Biology 2006, 33:103-119. OpenURL

  128. Kashyap DR, Vohra PK, Chopra S, Tewari R: Applications of pectinases in the commercial sector: a review.

    Bioresource Technology 2001, 77:215-227. PubMed Abstract OpenURL

  129. Henriksson G, Akin DE, Slomczynski D, Eriksson KEL: Production of highly efficient enzymes for flax retting by Rhizomucor pusillus.

    Journal of Biotechnology 1999, 68:115-123. OpenURL

  130. Liu Y, Shi J, Langrish TAG: Water-based extraction of pectin from flavedo and albedo of orange peels.

    Chemical Engineering Journal 2006, 120:203-209. OpenURL

  131. Chambin O, Dupuis G, Champion D, Voilley A, Pourcelot Y: Colon-specific drug delivery: Influence of solution reticulation properties upon pectin beads performance.

    International Journal of Pharmaceutics 2006, 321:86-93. PubMed Abstract OpenURL

  132. Fernandez ML, Sun DM, Tosca MA, McNamara DJ: Citrus pectin and cholesterol interact to regulate hepatic cholesterol homeostasis and lipoprotein metabolism: a dose-response study in guinea pigs.

    American Journal of Clinical Nutrition 1994, 59:869-878. PubMed Abstract OpenURL

  133. Hoagland PD, Parris N: Chitosan/pectin laminated films.

    Journal of Agricultural and Food Chemistry 1996, 44:1915-1919. OpenURL

  134. Doran JB, Cripe J, Sutton M, Foster B: Fermentations of pectin-rich biomass with recombinant bacteria to produce fuel ethanol.

    Applied Biochemistry and Biotechnology 2000, 84–6:141-152. OpenURL

  135. Hutnan M, Drtil M, Mrafkova L: Anaerobic biodegradation of sugar beet pulp.

    Biodegradation 2000, 11:203-211. PubMed Abstract OpenURL

  136. Singh SA, Ramakrishna M, Appu Rao AG: Optimisation of downstream processing parameters for the recovery of pectinase from the fermented bran of Aspergillus carbonarius.

    Process Biochemistry 1999, 35:411-417. OpenURL

  137. Alkorta I, Garbisu C, Llama MJ, Serra JL: Industrial applications of pectic enzymes: a review.

    Process Biochemistry 1998, 33:21-28. OpenURL

  138. Birgisson H, Hreggvidsson GO, Fridjonsson OH, Mort A, Kristjansson JK, Mattiasson B: Two new thermostable α-L-rhamnosidases from a novel thermophilic bacterium.

    Enzyme and Microbial Technology 2004, 34:561-571. OpenURL

  139. Jayani RS, Saxena S, Gupta R: Microbial pectinolytic enzymes: A review.

    Process Biochemistry 2005, 40:2931-2944. OpenURL

  140. Takao M, Akiyama K, Sakai T: Purification and characterization of thermostable endo-1,5-α-L-arabinase from a strain of Bacillus thermodenitrificans.

    Applied and Environmental Microbiology 2002, 68:1639-1646. OpenURL

  141. Marin-Rodriguez MC, Orchard J, Seymour GB: Pectate lyases, cell wall degradation and fruit softening.

    Journal of Experimental Botany 2002, 53:2115-2119. PubMed Abstract OpenURL

  142. Zverlov VV, Hertel C, Bronnenmeier K, Hroch A, Kellermann J, Schwarz WH: The thermostable α-L-rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial α-L-rhamnoside hydrolase, a new type of inverting glycoside hydrolase.

    Molecular Microbiology 2000, 35:173-179. PubMed Abstract OpenURL

  143. Kaur G, Kumar S, Satyanarayana T: Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis.

    Bioresource Technology 2004, 94:239-243. PubMed Abstract OpenURL

  144. Energy for the future: renewable sources of energy [http://ec.europa.eu/energy/res/index_en.htm] webcite

  145. Gray KA, Zhao L, Emptage M: Bioethanol.

    Current Opinion in Chemical Biology 2006, 10:141-146. PubMed Abstract OpenURL

  146. Sorensen HR, Pedersen S, Vikso-Nielsen A, Meyer AS: Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue.

    Enzyme and Microbial Technology 2005, 36:773-785. OpenURL

  147. Bhargava S, Frisner H, Bisgard-Frantzen H, Tams JW: A process of producing a fermentation product.

    Patent. WO2005113785 2005. OpenURL

  148. Tomme P, Warren RAJ, Gilkes NR: Cellulose hydrolysis by bacteria and fungi. In Advances in Microbial Physiology. Volume 37. Edited by Poole RK. London: Academic Press; 1995. PubMed Abstract OpenURL

  149. Wyman CE: Potential synergies and challenges in refining cellulosic biomass to fuels, chemicals, and power.

    Biotechnology Progress 2003, 19:254-262. PubMed Abstract OpenURL

  150. Zaldivar J, Nielsen J, Olsson L: Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration.

    Applied Microbiology and Biotechnology 2001, 56:17-34. OpenURL

  151. Rabinovich ML: Ethanol production from materials containing cellulose: The potential of Russian research and development.

    Applied Biochemistry and Microbiology 2006, 42:1-26. OpenURL

  152. Stenberg K, Galbe M, Zacchi G: The influence of lactic acid formation on the simultaneous saccharification and fermentation (SSF) of softwood to ethanol.

    Enzyme and Microbial Technology 2000, 26:71-79. OpenURL

  153. Singh D, Nigam P, Banat IM, Marchant R, McHale AP: Ethanol production at elevated temperatures and alcohol concentrations: Part II – Use of Kluyveromyces marxianus IMB3.

    World Journal of Microbiology & Biotechnology 1998, 14:823-834. OpenURL

  154. Sommer P, Georgieva T, Ahring BK: Potential for using thermophilic anaerobic bacteria for bioethanol production from hemicellulose.

    Biochemical Society Transactions 2004, 32:283-289. PubMed Abstract OpenURL

  155. Galbe M, Zacchi G: A review of the production of ethanol from softwood.

    Applied Microbiology and Biotechnology 2002, 59:618-628. OpenURL

  156. Sheehan J, Himmel M: Enzymes, energy, and the environment: A strategic perspective on the U.S. Department of Energy's research and development activities for bioethanol.

    Biotechnology Progress 1999, 15:817-827. PubMed Abstract OpenURL

  157. Bower B, Larenas E, Mitchinson C: Exo-endo cellulase fusion protein.

    Patent. WO2005093073 2005. OpenURL

  158. Wood BE, Ingram LO: Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

    Applied and Environmental Microbiology 1992, 58:2103-2110. OpenURL

  159. Karhumaa K, Wiedemann B, Hahn-Hagerdal B, Boles E, Gorwa-Grauslund MF: Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains.

    Microbial Cell Factories 2006., 5 PubMed Abstract | PubMed Central Full Text OpenURL

  160. Wyman CE, Dale BE, Elander RT: Coordinated development of leading biomass pretreatment technologies.

    Bioresource Technology 2005, 96:1959-1967. PubMed Abstract OpenURL

  161. Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY: Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover.

    Bioresource Technology 2005, 96:2026-2032. PubMed Abstract OpenURL

  162. Mitchinson C: Improved cellulose products for biomass conversion. In Abstract Book Groningen. Netherlands: 5th Carbohydrate Bioengineering Meeting; 2003. OpenURL

  163. Krahe M, Antranikian G, Mäirkl H: Fermentation of extremophilic microorganisms.

    FEMS Microbiology Reviews 1996, 18:271-285. OpenURL

  164. Schiraldi C, De Rosa M: The production of biocatalysts and biomolecules from extremophiles.

    Trends in Biotechnology 2002, 20:515-521. PubMed Abstract OpenURL

  165. Ramchuran SO, Nordberg Karlsson E, Velut S, Maré de L, Hagander P, Holst O: Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3).

    Applied Microbiology and Biotechnology 2002, 60:408-416. OpenURL

  166. Soutschek-Bauer E, Staudenbauer WL: Synthesis and secretion of a heat-stable carboxymethylcellulase from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus.

    Molecular and General Genetics 1987, 208:537-541. OpenURL

  167. Moracci M, La Volpe A, Pulitzer JF, Rossi M, Ciaramella M: Expression of the thermostable β-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression.

    Journal of Bacteriology 1992, 174:873-882. PubMed Abstract | PubMed Central Full Text OpenURL

  168. Ramchuran SO, Vargas V, Hatti-Kaul R, Nordberg Karlsson E: Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris.

    Applied Microbiology and Biotechnology 2006, 71:463-472. OpenURL

  169. Shinohara ML, Ihara M, Abo M, Hashida M, Takagi S, Beck TC: A novel thermostable branching enzyme from an extremely thermophilic bacterial species. Rhodothermus obamensis.

    Applied Microbiology and Biotechnology 2001, 57:653-659. OpenURL

  170. Walsh DJ, Gibbs MD, Bergquist PL: Expression and secretion of a xylanase from the extreme thermophile Thermotoga strain FjSS3-B1 in Kluveromyces lactis.

    Extremophiles 1998, 2:2-16. OpenURL

  171. Bergquist P, Te'o V, Gibbs M, Cziferszky A, de Faria FP, Azevedo M, Nevalainen H: Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts.

    Extremophiles 2002., 6 PubMed Abstract OpenURL

  172. Ciaramella M, Cannio R, Moracci M, Pisani FM, Rossi M: Molecular biology of extremophiles.

    World Journal of Microbiology & Biotechnology 1995, 11:71-64. OpenURL

  173. Duffner F, Bertoldo C, Andersen JT, Wagner K, Antranikian G: A new thermoactive pullulanase from Desulfurococcus mucosus: Cloning, sequencing, purification and characterization of the recombinant enzyme after expression in Bacillus subtilis.

    Journal of Bacteriology 2000, 182:6331-6338. PubMed Abstract | PubMed Central Full Text OpenURL

  174. Mather MW, Fee JA: Development of plasmid cloning vectors for Thermus thermophilus HB8: Expression of a heterologous, plasmid-borne kanamycin nucleotidyltransferase gene.

    Applied and Environmental Microbiology 1992, 58:421-425. OpenURL

  175. Moreno R, Zafra O, Cava F, Berenguer J: Development of a gene expression vector for Thermus thermophilus based on the promoter of the respiratory nitrate reductase.

    Plasmid 2003, 49:2-8. PubMed Abstract OpenURL

  176. Contursi P, Cannio R, Prato S, Fiorentino G, Rossi M, Bartolucci S: Development of a genetic system for hyperthermophilic Archaea: expression of a moderate thermophilic bacterial alcohol dehydrogenase gene in Sulfolobus solfataricus.

    FEMS Microbiology Letters 2003, 218:115-120. PubMed Abstract OpenURL

  177. Jain S, Durand H, Tiraby G: Development of a transformation system for the thermophilic fungus Talaromyces sp. CL240 based on the use of phleomycin resistance as a dominant selectable marker.

    Molecular and General Genetics 1992, 234:489-493. OpenURL

  178. Bjornsdottir SH, Thorbjarnardottir SH, Eggertsson G: Establishment of a gene transfer system for Rhodothermus marinus.

    Applied Microbiology and Biotechnology 2005, 66:675-682. OpenURL

  179. Lucas S, Toffin L, Zivanovic Y, Charlier D, Moussard H, Forterre P, Prieur D, Erauso G: Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi.

    Applied and Environmental Microbiology 2002, 68:5528-5536. OpenURL

  180. Mai V, Wiegel J: Advances in development of a genetic system for Thermoanaerobacterium spp.: Expression of genes encoding hydrolytic enzymes, development of a second shuttle vector, and integration of genes into the chromosome.

    Applied and Environmental Microbiology 2000, 66:4817-4821. OpenURL

  181. Aravalli RN, Garrett RA: Shuttle vectors for hyperthermophilic archaea.

    Extremophiles 1997, 1:183-191. PubMed Abstract OpenURL

  182. Zaks A: Industrial biocatalysis.

    Current Opinion in Chemical Biology 2001, 5:130-136. PubMed Abstract OpenURL

  183. van Beilen JB, Li Z: Enzyme technology: an overview.

    Current Opinion in Biotechnology 2002, 3:338-344. OpenURL

  184. Demirjian DC, Moris-Varas F, Cassidy CS: Enzymes from extremophiles.

    Current Opinion in Chemical Biology 2001, 5:144-151. PubMed Abstract OpenURL

  185. Gomes J, Steiner W: The biocatalytic potential of extremophiles and extremozymes.

    Food Technology and Biotechnology 2004, 42:223-235. OpenURL

  186. Stewart JD: Dehydrogenases and transaminases in asymmetric synthesis.

    Current Opinion in Chemical Biology 2001, 5:120-129. PubMed Abstract OpenURL

  187. Taylor PP, Pantaleone DP, Senkpeil RF, Fotheringham IG: Novel biosynthetic approaches to the production of unnatural amino acids using transaminases.

    Trends in Biotechnology 1998, 16:412-418. PubMed Abstract OpenURL

  188. Giuliano G, Schiraldi C, Marotta MR, Hugenholtz J, De Rosa M: Expression of Sulfolobus solfataricus α-glucosidase in Lactococcus lactis.

    Applied Microbiology and Biotechnology 2004, 64:829-832. OpenURL

  189. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial cellulose utilization: Fundamentals and biotechnology.

    Microbiology and Molecular Biology Reviews 2002, 66:506-577. OpenURL

  190. Lynd LR, Van Zyl WH, McBride JE, Laser M: Consolidated bioprocessing of cellulosic biomass: an update.

    Current Opinion in Biotechnology 2005, 16:577-583. PubMed Abstract OpenURL

  191. Klinke HB, Thomsen AB, Ahring BK: Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass.

    Applied Microbiology and Biotechnology 2004, 66:10-26. OpenURL

  192. Hatzinikolaou DG, Kalogeris E, Christakopoulos P, Kekos D, Macris BJ: Comparative growth studies of the extreme thermophile Sulfolobus acidocaldarius in submerged and solidified substrate cultures.

    World Journal of Microbiology & Biotechnology 2001, 17:229-234. OpenURL

  193. Patel MA, Ou MS, Harbrucker R, Aldrich HC, Buszko ML, Ingram LO, Shanmugam KT: Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid.

    Applied and Environmental Microbiology 2006, 72:3228-3235. OpenURL

  194. Chinn MS, Nokes SE, Strobel HJ: Screening of thermophilic anaerobic bacteria for solid substrate cultivation on lignocellulosic substrates.

    Biotechnology Progress 2006, 22:53-59. PubMed Abstract OpenURL

  195. Ingram LO, Aldrich HC, Borges ACC, Causey TB, Martinez A, Morales F, Saleh A, Underwood SA, Yomano LP, York SW, et al.: Enteric bacterial catalysts for fuel ethanol production.

    Biotechnology Progress 1999, 15:855-866. PubMed Abstract OpenURL

  196. Tao H, Gonzalez R, Martinez A, Rodriguez M, Ingram LO, Preston JF, Shanmugam KT: Engineering a homo-ethanol pathway in Escherichia coli: Increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation.

    Journal of Bacteriology 2001, 183:2979-2988. PubMed Abstract | PubMed Central Full Text OpenURL

  197. Altaras NE, Cameron DC: Metabolic engineering of a 1,2-propanediol pathway in Escherichia coli.

    Applied and Environmental Microbiology 1999, 65:1180-1185. OpenURL

  198. Nakamura CE, Gatenby AA, Hsu AK-H, La Reau RD, Haynie SL, Diaz-Torres M, Trimbur DE, Whited GM, Nagarajan V, Payne MS, et al.: Method for the production of 1,3-propanediol by recombinant microorganisms.

    Patent. US 6013494 2000. OpenURL

  199. Causey TB, Zhou S, Shanmugam KT, Ingram LO: Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: Homoacetate production.

    Proceedings of the National Academy of Sciences of the United States of America 2003, 100:825-832. PubMed Abstract | PubMed Central Full Text OpenURL

  200. Nie W, Draths KM, Frost JW: Benzene-free synthesis of adipic acid.

    Biotechnology Progress 2002, 18:201-211. PubMed Abstract OpenURL

  201. Vemuri GN, Eiteman MA, Altman E: Effects of growth mode and pyruvate decarboxylase on succinic acid production by metabolically engineered strains of Escherichia coli.

    Applied and Environmental Microbiology 2002, 68:1715-1727. OpenURL

  202. Zhou SD, Causey TB, Hasona A, Shanmugam KT, Ingram LO: Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110.

    Applied and Environmental Microbiology 2003, 69:399-407. OpenURL

  203. Desai SG, Guerinot ML, Lynd LR: Cloning of L-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SL-YS485.

    Applied Microbiology and Biotechnology 2004, 65:600-605. OpenURL

  204. Ben-Bassat A, Lamed R, Zeikus JG: Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium Brockii.

    Journal of Bacteriology 1981, 146:192-199. PubMed Abstract | PubMed Central Full Text OpenURL

  205. Lovitt RW, Longin R, Zeikus JG: Ethanol production by thermophilic bacteria: physiological comparison of solvent effects on parent and alcohol-tolerant strains of Clostridium thermohydrosulfuricum.

    Applied and Environmental Microbiology 1984, 48:171-177. OpenURL

  206. Sakai S, Nakashimada Y, Yoshimoto H, Watanabe S, Okada H, Nishio N: Ethanol production from H2 and CO2 by a newly isolated thermophilic bacterium, Moorella sp. HUC22-1.

    Biotechnology Letters 2004, 26:1607-1612. PubMed Abstract OpenURL

  207. van der Maarel MJEC, van der Veen B, Uitdehaag JCM, Leemhuis H, Dijkhuizen L: Properties and applications of starch-converting enzymes of the α-amylase family.

    Journal of Biotechnology 2002, 94:137-155. PubMed Abstract OpenURL

  208. Gaouar O, Aymard C, Zakhia N, Rios GM: Enzymatic hydrolysis of cassava starch into maltose syrup in a continuous membrane reactor.

    Journal of Chemical Technology and Biotechnology 1997, 69:367-375. OpenURL

  209. THOR Products. Textile specialities – Enzyme products [http://www.thor.com] webcite

  210. Fuel ethanol application sheet [http://www.novozymes.com] webcite

    Novozymes A/S OpenURL

  211. Termamyl SC. A novel α-amylase for liquefaction of whole grain

    Novozymes A/S 1999.

    Ethanol/2001-14846-01.pdf

    OpenURL

  212. Application sheet. Liquefaction of starch from dry-milled grains

    Novozymes A/S 2004.

    2004-04542-01

    OpenURL

  213. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R: Advances in microbial amylases.

    Biotechnology and Applied Biochemistry 2000, 31:135-152. OpenURL

  214. Ara K, Saeki K, Igarashi K, Takaiwa M, Uemura T, Hagihara H, Kawai S, Ito S: Purification and characterization of an alkaline amylopullulanase with both α-1,4 and α-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378.

    Biochimica et Biophysica Acta 1995, 1243:315-324. PubMed Abstract OpenURL

  215. Chiang C-M, Yeh F-S, Huang L-F, Tseng T-H, Chung M-C, Wang C-S, Lur H-S, Shaw J-F, Yu S-M: Expression of a bi-functional and thermostable amylopullulanase in transgenic rice seeds leads to autohydrolysis and altered composition of starch.

    Molecular Breeding 2005, 15:125-143. OpenURL

  216. Satyanarayana T, Noorwez SM, Kumar S, Rao JLUM, Ezhilvannan M, Kaur P: Development of an ideal starch saccharification process using amylolytic enzymes from thermophiles.

    Biochemical Society Transactions 2004, 32:276-278. PubMed Abstract OpenURL

  217. Biwer A, Antranikian G, Heinzle E: Enzymatic production of cyclodextrins.

    Applied Microbiology and Biotechnology 2002, 59:609-617. OpenURL

  218. Shim JH, Kim YW, Kim TJ, Chae HY, Park JH, Cha HJ, Kim JW, Kim YR, Schaefer T, Spendler T, et al.: Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR.

    Protein Engineering Design and Selection 2004, 17:205-211. OpenURL

  219. Tachibana Y, Takaha T, Fujiwara S, Takagi M, Imanaka T: Acceptor specificity of 4-α-glucanotransferase from Pyrococcus kodakaraensis KOD1, and synthesis of cycloamylose.

    Journal of Bioscience and Bioengineering 2000, 90:406-409. OpenURL

  220. Christophersen C, Otzen DE, Norman BE, Christensen S, Schafer T: Enzymatic characterisation of Novamyl (R), a thermostable α-amylase.

    Starch-Starke 1998, 50:39-45. OpenURL

  221. Application sheet. Novamyl for anti-staling

    Novozymes A/S 2004.

    Cereal food/2001-00403-04.pdf

    OpenURL

  222. Gantelet H, Duchiron F: A new pullulanase from a hyperthermophilic archaeon for starch hydrolysis.

    Biotechnology Letters 1999, 21:71-75. OpenURL

  223. Lea B, Yuval S, Eugene R: Characterization and delignification activity of a thermostable α-L-arabinofuranosidase from Bacillus stearothermophilus.

    Applied Microbiology and Biotechnology 1993, 40:57-62. OpenURL

  224. Numan MT, Bhosle NB: α-L-Arabinofuranosidases: the potential applications in biotechnology.

    Journal of Industrial Microbiology and Biotechnology 2006, 33:247-260. OpenURL

  225. Gusakov AV, Sinitsyn AP, Salanovich TN, Bukhtojarov FE, Markov AV, Ustinov BB, van Zeijl C, Punt P, Burlingame R: Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolase I (Cel7A) produced by the industrial strain of Chrysosporium lucknowense.

    Enzyme and Microbial Technology 2005, 36:57-69. OpenURL

  226. Muzzarelli RAA: Human enzymatic activities related to the therapeutic administration of chitin derivatives.

    Cellular and Molecular Life Sciences 1997, 53:131-140. OpenURL

  227. van Wyk JPH, Mohulatsi M: Biodegradation of wastepaper by cellulase from Trichoderma viride.

    Bioresource Technology 2003, 86:21-24. PubMed Abstract OpenURL

  228. Bornscheuer UT: Microbial carboxyl esterases: classification, properties and application in biocatalysis.

    Fems Microbiology Reviews 2002, 26:73-82. PubMed Abstract OpenURL

  229. Margolin AL: Enzymes in the synthesis of chiral drugs.

    Enzyme and Microbial Technology 1993, 15:266-280. OpenURL

  230. Singh P, Gill PK: Production of inulinases: Recent advances.

    Food Technology and Biotechnology 2006, 44:151-162. OpenURL

  231. Liebl W, Brem D, Gotschlich A: Analysis of the gene for beta-fructosidase (invertase, inulinase) of the hyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed in Escherichia coli.

    Applied Microbiology and Biotechnology 1998, 50:55-64. OpenURL

  232. Ganter C, Bock A, Buckel P, Mattes R: Production of thermostable, recombinant α-galactosidase suitable for raffinose elimination from sugar beet syrup.

    Journal of Biotechnology 1988, 8:301-310. OpenURL

  233. Comfort DA, Chhabra SR, Conners SB, Chou C-J, Epting KL, Johnson MR, Jones KL, Sehgal AC, Kelly RM: Strategic biocatalysis with hyperthermophilic enzymes.

    Green Chemistry 2004, 6:459-465. OpenURL

  234. Panesar PS, Panesar R, Singh RS, Kennedy JF, Kumar H: Microbial production, immobilization and applications of beta-D-galactosidase.

    Journal of Chemical Technology and Biotechnology 2006, 81:530-543. OpenURL

  235. Soares NFF, Hotchkiss JH: Naringinase immobilization in packaging films for reducing naringinin concentration in grapefruit juice.

    Journal of Food Science 1998, 63:61-65. OpenURL

  236. Lee SG, Lee DC, Hong SP, Sung MH, Kim HS: Thermostable D-hydantoinase from thermophilic Bacillus stearothermophilus SD-I: characteristics of purified enzyme.

    Applied Microbiology and Biotechnology 1995, 43:270-276. OpenURL

  237. Wu G, Liu Z, Bryant MM, Roland DAS: Comparison of Natuphos and Phyzyme as phytase sources for commercial layers fed corn-soy diets.

    Poultry Science 2006, 85:64-69. PubMed Abstract OpenURL

  238. Jalal MA, Scheideler SE: Effect of supplementation of two different sources of phytase on egg production parameters in laying hens and nutrient digestiblity.

    Poultry Science 2001, 80:1463-1471. PubMed Abstract OpenURL

  239. Protex 6L. Genencor bacterial alkaline protease. Product information

    Genencor International, Inc. PRO44AX, REV0704 2004. OpenURL

  240. Soria F, Ellenrieder G: Thermal inactivation and product inhibition of Aspergillus terreus CECT 2663 α-L-rhamnosidase and their role on hydrolysis of naringin solutions.

    Bioscience, Biotechnology and Biochemistry 2002, 66:1442-1449. OpenURL

  241. Spagna G, Barbagallo RN, Martino A, Pifferi PG: A simple method for purifying glycosidases: α-L-rhamnopyranosidase from Aspergillus niger to increase the aroma of Moscato wine.

    Enzyme and Microbial Technology 2000, 27:522-530. OpenURL

  242. Multifect Xylanase. Genencor xylanase. Product information

    Genencor International, Inc. MUL33CX, REV0704 2004. OpenURL

  243. Luminase PB-100. Enzyme for improved pulp bleaching [http://www.diversa.com] webcite

    Diversa corporation

    D-1018.5.

    OpenURL

  244. Saito N: A thermophilic extracellular α-amylase from Bacillus licheniformis.

    Archives of Biochemistry and Biophysics 1973, 155:290-298. OpenURL

  245. Declerck N, Machius M, Joyet P, Wiegand G, Huber R, Gaillardin C: Hyperthermostabilization of Bacillus licheniformis α-amylase and modulation of its stability over a 50°C temperature range.

    Protein Engineering 2003, 16:287-293. PubMed Abstract OpenURL

  246. Lee S, Oneda H, Minoda M, Tanaka A, Inouye K: Comparison of starch hydrolysis activity and thermal stability of two Bacillus licheniformis α-amylases and insights into engineering α-amylase variants active under acidic conditions.

    Journal of Biochemistry 2006, 139:997-1005. PubMed Abstract OpenURL

  247. Burhan A, Nisa U, Gökhan C, Ömer C, Ashabil A, Osman G: Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6.

    Process Biochemistry 2003, 38:1397-1403. OpenURL

  248. Malhotra R, Noorwez SM, Satyanarayana T: Production and partial characterization of thermostable and calcium-independent α-amylase of an extreme thermophile Bacillus thermooleovorans NP54.

    Letters in Applied Microbiology 2000, 31:378-384. PubMed Abstract OpenURL

  249. Dong G, Vieille C, Savchenko A, Zeikus JG: Cloning, sequencing, and expression of the gene encoding extracellular α-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

    Applied and Environmental Microbiology 1997, 63:3569-3576. OpenURL

  250. Koch R, Spreinat A, Lemke K, Antranikian G: Purification and properties of a hyperthermoactive α-amylase from the archaeobacterium Pyrococcus woesei.

    Archives of Microbiology 1991, 155:572-578. OpenURL

  251. Leveque E, Haye B, Belarbi A: Cloning and expression of an α-amylase encoding gene from the hyperthermophilic archaebacterium Thermococcus hydrothermalis and biochemical characterisation of the recombinant enzyme.

    FEMS Microbiology Letters 2000, 186:67-71. PubMed Abstract OpenURL

  252. Richardson TH, Tan X, Frey G, Callen W, Cabell M, Lam D, Macomber J, Short JM, Robertson DE, Miller C: A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable α-amylase.

    The Journal of Biological Chemistry 2002, 277:26501-26507. PubMed Abstract OpenURL

  253. Schumann J, Wrba A, Jaenicke R, Stetter KO: Topographical and enzymatic characterization of amylases from the extremely thermophilic eubacterium Thermotoga maritima.

    Febs Letters 1991, 282:122-126. PubMed Abstract OpenURL

  254. Schen G-J, Saha BC, Lee Y-E, Bhatnagar L, Zeikus JG: Purification and characterization of a novel thermostable b-amylase from Clostridium thermosulphurogenes.

    Biochemical Journal 1988, 254:835-840. PubMed Abstract | PubMed Central Full Text OpenURL

  255. Brown SH, Kelly RM: Characterization of amylolytic enzymes, having both α-1,4 and α-1,6 hydrolytic activity, from the thermophilic archaea Pyrococcus furiosus and Thermococcus litoralis.

    Applied and Environmental Microbiology 1993, 59:2614-2621. OpenURL

  256. Erra-Pujada M, Chang-Pi-Hin F, Debeire P, Duchiron F, O'Donohue MJ: Purification and properties of the catalytic domain of the thermostable pullulanase type II from Thermococcus hydrothermalis.

    Biotechnology Letters 2001, 23:1273-1277. OpenURL

  257. Van Der Maarel MJEC, Vos A, Sanders P, Dijkhuizen L: Properties of the glucan branching enzyme of the hyperthermophilic bacterium Aquifex aeolicus.

    Biocatalysis and Biotransformation 2003, 21:199-208. OpenURL

  258. Murakami T, Kanai T, Takata H, Kuriki T, Imanaka T: A novel branching enzyme of the GH-57 family in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.

    Journal of Bacteriology 2006, 188:5915-5924. PubMed Abstract | PubMed Central Full Text OpenURL

  259. Thiemann V, Donges C, Prowe SG, Sterner R, Antranikian G: Characterisation of a thermoalkali-stable cyclodextrin glycosyltransferase from the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii.

    Archives of Microbiology 2004, 182:226-235. PubMed Abstract OpenURL

  260. Leemhuis H, Rozeboom HJ, Dijkstra BW, Dijkhuizen L: Improved thermostability of Bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt bridge.

    Proteins-Structure Function and Genetics 2004, 54:128-134. OpenURL

  261. Chung HJ, Yoon SH, Lee MJ, Kim MJ, Kweon KS, Lee IW, Kim JW, Oh BH, Lee HS, Spiridonova VA, et al.: Characterization of a thermostable cyclodextrin glucanotransferase isolated from Bacillus stearothermophilus ET1.

    Journal of Agricultural and Food Chemistry 1998, 46:952-959. OpenURL

  262. Wind RD, Liebl W, Buitelaar RM, Penninga D, Spreinat A, Dijkhuizen L, Bahl H: Cyclodextrin formation by the thermostable α-amylase of Thermoanaerobacterium thermosulfurigenes EM1 and reclassification of the enzymes as a glycosyl transferase.

    Applied and Environmental Microbiology 1995, 61:1257-1265. OpenURL

  263. Rashid N, Cornista J, Ezaki S, Fukui T, Atomi H, Imanaka T: Characterization of an archaeal cyclodextrin glucanotransferase with a novel C-terminal domain.

    Journal of Bacteriology 2002, 184:777-784. PubMed Abstract | PubMed Central Full Text OpenURL

  264. Liebl W, Feil R, Gabelsberger J, Kellerman J, Schleifer K-H: Purification and characterization of a novel 4-a-glucanotransferase of Thermotoga maritima cloned in Escherichia coli.

    European Journal of Biochemistry 1992, 207:81-88. PubMed Abstract OpenURL

  265. Tachibana Y, Fujiwara S, Takagi M, Imanaka T: Cloning and expression of the 4-α-glucanotransferase gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1, and characterization of the enzyme.

    Journal of Fermentation and Bioengineering 1997, 83:540-548. OpenURL

  266. Specka U, Mayer F, Antranikian G: Purification and properties of a thermoactive glucoamylase from Clostridium thermosaccharolyticum.

    Applied and Environmental Microbiology 1991, 57:2317-2323. OpenURL

  267. Ganghofner D, Kellermann J, Staudenbauer WL, Bronnenmeier K: Purification and properties of an amylopullulanase, a glucoamylase, and an α-glucosidase in the amylolytic enzyme system of Thermoanaerobacterium thermosaccharolyticum.

    Bioscience, Biotechnology and Biochemistry 1998, 62:302-308. OpenURL

  268. Thorsen TS, Johnsen AH, Josefsen K, Jensen B: Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus.

    Biochimica Et Biophysica Acta-Proteins and Proteomics 2006, 1764:671-676. OpenURL

  269. Basaveswara Rao V, Sastri NVS, Subba Rao PV: Purificatin and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus.

    Biochemical Journal 1981, 193:379-387. PubMed Abstract | PubMed Central Full Text OpenURL

  270. Chang ST, Parker KN, Bauer MW, Kelly RM: α-Glucosidase from Pyrococcus furiosus. In Methods in Enzymology. Volume 330. Academic Press; 2001::260-269. PubMed Abstract OpenURL

  271. Bertoldo C, Antranikian G: Starch-hydrolyzing enzymes from thermophilic archaea and bacteria.

    Current Opinion in Chemical Biology 2002, 6:151-160. PubMed Abstract OpenURL

  272. Harald M, Wolfgang L: Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides.

    European Journal of Biochemistry 1998, 258:1050-1058. PubMed Abstract OpenURL

  273. Bertoldo C, Armbrecht M, Becker F, Schafer T, Antranikian G, Liebl W: Cloning, sequencing, and characterization of thermoalkalistable type I pullulanase from Anaerobranca gottschalkii.

    Applied and Environmental Microbiology 2004, 70:3407-3416. OpenURL

  274. Koch R, Hippe H, Jahnke KD, Antranikian G: Purification and properties of a thermostable pullulanase from a newly isolated thermophilic anaerobic bacterium, Fervidobacterium pennavorans Ven5.

    Applied and Environmental Microbiology 1997, 63:1088-1094. OpenURL

  275. Rudiger A, Jorgensen PL, Antranikian G: Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli.

    Applied and Environmental Microbiology 1995, 61:567-575. OpenURL

  276. Gomes I, Gomes J, Steiner W: Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium Rhodothermus marinus: production and partial characterization.

    Bioresource Technology 2003, 90:207-214. PubMed Abstract OpenURL

  277. Kriegshäuser G, Liebl W: Pullulanase from the hyperthermophilic bacterium Thermotoga maritima: purification by β-cyclodextrin affinity chromatography.

    Journal of Chromatography 2000, 737:245-251. PubMed Abstract OpenURL

  278. Tuohy MG, Walsh DJ, Murray PG, Claeyssens M, Cuffe MM, Savage AV, Coughlan MP: Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii.

    Biochimica et Biophysica Acta – Protein Structure and Molecular Enzymology 2002, 1596:366-380. OpenURL

  279. Bronnenmeier K, Kern A, Liebl W, Staudenbauer WL: Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials.

    Applied and Environmental Microbiology 1995, 61:1399-1407. OpenURL

  280. Bok JD, Yernool DA, Eveleigh DE: Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana.

    Applied and Environmental Microbiology 1998, 64:4774-4781. OpenURL

  281. Wicher KB, Abou-Hachem M, Halldorsdottir S, Thorbjarnadóttir SH, Eggertsson G, Hreggvidsson GO, Nordberg Karlsson E, Holst O: Deletion of cytotoxic N-terminal putative signalpeptide results in a significant increase in production yields in E. coli and improved specific activity of Cel12A from Rhodothermus marinus.

    Applied Microbiology and Biotechnology 2001, 55:578-584. OpenURL

  282. Turner P, Svensson D, Adlercreutz P, Nordberg Karlsson E: A novel variant of Thermotoga neapolitana β-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation.

    Journal of Biotechnology, in press. OpenURL

  283. Zverlov VV, Volkov IY, Velikodvorskaya TV, Schwarz WH: Thermotoga neapolitana bglB gene, upstream of lamA, encodes a highly thermostable β-glucosidase that is a laminaribiase.

    Microbiology 1997, 143:3537-3542. PubMed Abstract OpenURL

  284. Kengen SWM, Luesink EJ, Stams AIM, Zehnder AIB: Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus.

    European Journal of Biochemistry 1993, 213:305-312. PubMed Abstract OpenURL

  285. Degrassi G, Vindigni A, Venturi V: A thermostable α-arabinofuranosidase from xylanolytic Bacillus pumilus: purification and characterisation.

    Journal of Biotechnology 2003, 101:69-79. PubMed Abstract OpenURL

  286. Gomes J, Gomes I, Terler K, Gubala N, Ditzelmuller G, Steiner W: Optimisation of culture medium and conditions for α-L-arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus.

    Enzyme and Microbial Technology 2000, 27:414-422. OpenURL

  287. Debeche T, Cummings N, Connerton I, Debeire P, O'Donohue MJ: Genetic and biochemical characterization of a highly thermostable α-L-arabinofuranosidase from Thermobacillus xylanilyticus.

    Applied and Environmental Microbiology 2000, 66:1734-1736. OpenURL

  288. Parker KN, Chhabra SR, Lam D, Callen W, Duffaud GD, Snead MA, Short JM, Mathur EJ, Kelly RM: Galactomannanases Man2 and Man5 from Thermotoga species: Growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes.

    Biotechnology and Bioengineering 2001, 75:322-333. OpenURL

  289. Politz O, Krah M, Thomsen KK, Borriss R: A highly thermostable endo-(1,4)-β-mannanase from the marine bacterium Rhodothermus marinus.

    Applied Microbiology and Biotechnology 2000, 53:715-721. OpenURL

  290. Abou-Hachem M, Olsson F, Nordberg Karlsson E: Probing the stability of the modular family 10 xylanase from Rhodothermus marinus.

    Extremophiles 2003, 7:483-491. PubMed Abstract OpenURL

  291. Chen C-C, Adolphson R, Dean JFD, Eriksson K-EL, Adams MWW, Westpheling J: Release of lignin from kraft pulp by a hyperthermophilic xylanase from Thermotoga maritima.

    Enzyme and Microbial Technology 1997, 20:39-45. OpenURL

  292. Mamo G, Hatti-Kaul R, Mattiasson B: A thermostable alkaline active endo-beta-1-4-xylanase from Bacillus halodurans S7: Purification and characterization.

    Enzyme and Microbial Technology 2006, 39:1492-1498. OpenURL

  293. Duffaud GD, McCutchen CM, Leduc P, Parker KN, Kelly RM: Purification and characterization of extremely thermostable β-mannanase, β-mannosidase, and α-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068.

    Applied and Environmental Microbiology 1997, 63:169-177. OpenURL