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Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

Do Bien-Cuong1, Dang Thi-Thu1, Jean-Guy Berrin2, Dietmar Haltrich3, To Kim-Anh1, Jean-Claude Sigoillot24 and Montarop Yamabhai5*

Author Affiliations

1 Institute of Biological and Food Technology, Hanoi University of Technology, Hanoi, Vietnam

2 INRA, UMR1163 de Biotechnologie des Champignons Filamenteux, ESIL, 163 avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France

3 BOKU - University of Natural Resources and Applied Life Sciences, Vienna, Austria

4 Université de Provence, UMR1163 de Biotechnologie des Champignons Filamenteux, ESIL, 163 Avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France

5 School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima, 30000, Thailand

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Microbial Cell Factories 2009, 8:59  doi:10.1186/1475-2859-8-59

Published: 13 November 2009



Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS).


A gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-D-mannan mannanohydrolase (E.C., commonly termed β-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-β-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330 s-1, 292 s-1 and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger β -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed.


This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-β-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant β-mannanase will be valuable in various biotechnological applications.