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High external pH enables more efficient secretion of alkaline α-amylase AmyK38 by Bacillus subtilis

Kenji Manabe12, Yasushi Kageyama1, Masatoshi Tohata1, Katsutoshi Ara1, Katsuya Ozaki1* and Naotake Ogasawara2*

Author Affiliations

1 Biological Science Laboratories, Kao Corporation, 2606 Akabane Ichikai, Haga, Tochigi, 321-3497, Japan

2 Graduate School of Information Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101, Japan

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Microbial Cell Factories 2012, 11:74  doi:10.1186/1475-2859-11-74

Published: 8 June 2012



Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system.


Compared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l-1) of AmyK38 was obtained using strain MGB874.


We demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.

α-Amylase; AmyK38; Bacillus subtilis; CssRS; Genome reduction; HtrA; HtrB; MGB874; Secretion stress response