Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis

• Published in: BMC biotechnology Vol. 16; no. 1; p. 77
• Main Authors: Bai, Wenqin, Cao, Yufan, Liu, Jun, Wang, Qinhong, Jia, Zhenhu
• Format: Journal Article
• Language: English
• Published: England BioMed Central 08-11-2016
• Subjects: Alkalies - chemistry; Bacillus - classification; Bacillus - enzymology; Bacillus - genetics; Cellulose; Endo-1,4-beta Xylanases - chemistry; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - metabolism; Enzyme Activation; Enzymes; Genetic Enhancement - methods; Glutamine - genetics; High temperature; Hydrogen-Ion Concentration; Mutagenesis; Mutagenesis, Site-Directed - methods; Mutation; Mutation - genetics; Protein Engineering; Proteins; Random Allocation; Site-directed mutation; Catalytic efficiency; Error-prone PCR; Xylanase; Alkalophilicity; Site-saturation mutagenesis
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/s12896-016-0310-9

Family 11 alkaline xylanases have great potential economic applications in the pulp and paper industry. In this study, we would improve the alkalophilicity of family 11 alkaline xylanase Xyn11A-LC from Bacillus sp. SN5, for the better application in this field.
A random mutation library of Xyn11A-LC with about 10,000 clones was constructed by error-prone PCR. One mutant, M52-C10 (V116A and E135V), with improved alkalophilicity was obtained from the library. Site-directed mutation showed that the mutation E135V was responsible for the alkalophilicity of the mutant. The variant E135V shifted the optimum pH of the wild-type enzyme from 7.5 to 8.0. Compared to the relative activities of the wild type enzyme, those of the mutant E135V increased by 17.5, 18.9, 14.3 and 9.5 % at pH 8.5, 9.0, 9.5 and 10.0, respectively. Furthermore, Glu135 saturation mutagenesis showed that the only mutant to have better alkalophilicity than E135V was E135R. The optimal pH of the mutant E135R was 8.5, 1.0 pH units higher than that of the wild-type. In addition, compared to the wild-type enzyme, the mutations E135V and E135R increased the catalytic efficiency (k
/K
) by 57 and 37 %, respectively. Structural analysis showed that the residue at position 135, located in the eight-residue loop on the protein surface, might improve the alkalophilicity and catalytic activity by the elimination of the negative charge and the formation of salt-bridge.
Mutants E135V and E135R with improved alkalophilicity were obtained by directed evolution and site saturation mutagenesis. The residue at position 135 in the eight-residue loop on the protein surface was found to play an important role in the pH activity profile of family 11 xylanases. This study provided alkalophilic mutants for application in bleaching process, and it was also helpful to understand the alkaline adaptation mechanism of family 11 xylanases.