Journal
PROCESS BIOCHEMISTRY
Volume 48, Issue 8, Pages 1166-1173Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2013.06.014
Keywords
beta-Mannanase; Site-directed mutagenesis; Acid stability; Bacillus subtilis
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Funding
- Program for New Century Excellent Talents in University [NCET-10-0459]
- National Basic Research Program of China (973 Program) [2012CB720806]
- High-tech Research and Development Programs of China [2012AA022102]
- National Natural Science Foundation of China [21276110]
- Fundamental Research Funds for the Central Universities [JUSRP51306A, JUSRP1009]
- 'Five-twelfth' National Science and Technology Support Program [2012BAD33B06]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
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beta-Mannanase can randomly hydrolyze the (I -> 4)-beta-D-mannosidic linkages in mannans, galactomannans and glucomannans, yielding manno-oligosaccharides. In this study, the beta-mannanase (MAN) from Bacillus subtilis B10-02 was overexpressed successfully in B. subtilis 168 as a hexa-histidine tagged, secreted protein. The recombinant enzyme BsMAN6H was not stable under acidic conditions, which restricts its use in food and feed industry. We aimed to improve the acid stability of BsMAN6H by changing several surface-exposed amino acid residues to acidic or neutral ones. Among the mutations, the His54Asp resulted in a shift in the optimal pH from 6.5 to 5.5. Accordingly, the acid stability was improved by a factor of a negative potential on the structure surface around the mutated site. Furthermore, the H54D variant showed the enzyme activity up to 3207.82 U/mL in bioreactors using the cheap Kojac powder as substrate. As a result, a bacterial beta-mannanase was produced efficiently with increased acid stability, improving its applicability in the animal feed industry. (C) 2013 Elsevier Ltd. All rights reserved.
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