Modifications Of The Enzymatic Activity And Salt-Tolerance Of Salt-Tolerant Xylanase Xyn22 By Site-Directed Mutagenesis

Xylan is the most abundant,naturally occurring hemicellulose,endoxylanases play a key role in the bio-degradation of xylan. The xylanase with salt-tolerant properties has important application in high salt food processing, sewage treatment and bioethanol production from seeweed. In our previous study,a glycoside hydrolase (GH) family 11 xylanase Xyn22 with excellent salt-tolerance was found. This work aimed to improve the thermal stability and specific activity of Xyn22 and explore the key amino acid residues that affect the salt-tolerance of Xyn22 by site-directed mutagenesis, in order to lay a foundation for resolving the salt-tolerant mechanism of Xyn22.Based on the sequence alignment and protein structure simulation analysis, the amino acid residue T10 of Xyn22 was speculated to affect its enzymatic activity. The mutation T10Y was constructed by overlapping extension PCR and successfully expressed in Escherichia coli BL21 (DE3). The results suggested that the optimum reaction temperature of the mutation T10Y was 65?, which is higher than wild type Xyn22 (60?). The thermal stability of mutant T10Y was also significantly improved, the residual enzymatic activity was nearly 100% after treatment at 50? for 60 min. The specific activity of the mutant T10Y was 360.50 U/mg, which is higher than the wild type (220.22 U/mg). The Km of mutant T10was 11.11 mg·ml-1, which is lower than wild type (17.38 mg ml-1). In addition,the mutant T10Y still exhibited excellent salt-tolerance as wild type Xyn22.An increase in acidic amino acid on the surface of protein might lead to its excellent salt-tolerance. In this study, the surface acid amino acid residues D50, E137, E139, D167 and E169 of Xyn22 were speculated to relate to its salt-tolerance based on the .sequence alignment and protein structure simulation,. In order to explore the effects of these five amino acids on the salt-tolerance of Xyn22, mutants D50A, E137A, E139, D167A/E169,E137A/D167A/E169A and E139A/D167A/E169A were successfully constructed and expressed in Ecoli BL21 (DE3).The results showed the relative enzyme activity of mutant E137A, E137A/D167A/E169A and E139A/D167A/E169A was 40.42%, 19.39% and 36.76% in the presence of 5 M NaCl, respectively, which were significantly lower than that of wild Type (50.21%). Interestingly, the salt-tolerance of mutant D167A/E169A was further enhanced. The relative activity of mutant D167A/E169A was higher than that of wild type Xyn22 in the presence of 2 M to 5 M NaCI.In summary, the specific activity and thermal stability of muation T10Y were significantly improved. The surface acidic amino acid residues E137, D167 and E169 of Xyn22 were the key amino acid residues that leading to its high salt-tolerance. This study provide a theoretical basis for further analysis of the salt tolerance mechanism of GH11 family xylanase.