Site-directed Mutagenesis Of Klebsiella Variicola Pullulanase For Improvement Of Its Thermostability And Acid Resistance

Pullulanase is usually applied to specifically cleave the α-1,6-glucosidic linkages in amylaceous polysaccharides, belonging to a family of 13 glycosyl hydrolases. In the saccharification process,higher conversion rate can be reached with the application of pullulanase. However, pullulanase must have high optimum temperature and low optimum p H value to meet the requirements of saccharification process.In the previous work, Klebsiella variicola SHN-1 pullulanase(Kv Pul) was expressed in Escherichia coli. Yet the thermostability and acid resistance of Kv Pul was not suitable for the saccharification reaction and practical application. In this study, site-directed mutation was applied to improve the thermostability and acid resistance of pullulanase. Main contents of the study are as follows:(1) As reported, residue Phe562 was proposed to be the possible site relating to thermostability of K. pneumoniae pullulanase. Based on conserved sites and homology modeling analysis, the residue Phe581 in the K. variicola SHN-1 pullulanase was selected as the potential thermostability-related site and its role on thermostability and activity was investigated by site-saturated mutagenesis. By analyzing the active site of K. variicola SHN-1 pullulanase and alignment of the amino acid sequences of the pullulanases from K. pneumoniae, Bacillus acidopullulyticus and B. naganoensis, site-directed mutagenesis including H626 D, R756 E, H852 D, R908 E, I619 N, F723 Y, S850 T and K851 S were subjected to improve the acid resistance of K. variicola SHN-1 pullulanase.(2) Compared with the wild-type pullulanase, the optimum temperature of the mutants including F581 L, F581 Q, F581 R, F581 T, F581 V and F581 Y was increased from 53 °C to 56 °C, and correspondingly the half-lives of these mutants at 55°C were increased by 4.20, 3.70, 3.30, 7.16, 3.01 and 1.75 min, respectively. Most of the mutants had a positive shift of T50 except for F581 A and F581 C. And the mutation did not change the p H profile of the enzymes. Vmax and kcat/Km of F581 L and F581 V was increased which indicated that the catalytic efficiency of F581 L and F581 V was improved. By modeling the structure of the pullulanase, formation of more hydrogen bonds of F581 Q, F581 R and F581 T by single-site substitution was supposed to be responsible for the improvement of thermostability. As Leu and Val are more hydrophobic than Phe, therefore, the mutants F581 L and F581 V would be more stable due to hydrophobic interactions.(3) After activity assay of the pullulanases, the optimum p H of H852 D was shifted from 5.0 to 4.7, compared with the wild-type enzyme, and the stability of H852 D and K851 S was obviously improved when stored at p H 4.5. By modeling the structure of the pullulanase, formation of more hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of stability at low p H and the p Ka value of the active center of pullulanase may influence the p H profile.