Improving Thermostability Of Alkaline Amylase Through Molecular Modification

Alkaline amylases can hydrolyze starch, and have high catalytic efficiency and stability atthe alkaline conditions (pH9.0~11.0), they have been widely used in the textile and detergentsindustries. Thermostability is one of the most important parameters for alkaline amylases,especially for desizing in the textile industry, in which higher enzyme thermostability benefitscatalytic efficiency and reduces the cost of the textile treatment.In our early study, Alkalimonas amylolytica alkaline amylase (AmyK) was heterologouslyexpressed in Escherichia coli. On the basis, the thermostability of AmyK was significantlyimproved through molecular modification in this thesis. The main contents and results are asfollows:1．The online server SWISS-MODEL was used to predict the three-dimensional structure ofAmyK using the crystal structure of Halothermothrix orenii amylase AmyB (3bc9) as thetemplate, and based on the rational design and computer aided design to improve thethermostability of AmyK. The key residues (His, Gly, and Pro, et al.) related to the stability ofdomain A, B, and the whole AmyK structure, were selected as targets for site-directedmutagenesis. Among15constructed single-site mutants, four mutants—H209L, Q226V,N302W, and P477V—showed enhanced thermostability. Combinational mutations weresubsequently introduced, and the best mutant was H209L/Q226V/P477V. The half-life of thismutant at60℃improved by2.8-fold as compared with wild-type AmyK, the optimumtemperature increased from50℃to55℃, the optimum pH shifted from9.5to10.0, the stablepH range expanded from8.0~11.0to7.0~11.0, and the catalytic efficiency increased from1.8×104L·g-1·min-1to3.5×104L·g-1·min-1.2．Based on the computer simulation design, introduction of disul de bridges in AmyK,7residue pairs were chosen as targets for site-directed mutagenesis. Three single disul debridge mutants—P35C-G426C, G116C-Q120C, and R436C-M480C—of the7showedsigni cantly enhanced thermostability. Combinational mutations were subsequently assessed,and the best mutant was P35C-G426C/G116C-Q120C/R436C-M480C, its half-life at60℃improved by6.0-fold as compared with wild-type AmyK. The optimum temperature,optimum pH, pH stability, and catalytic ef ciency of this mutant also improved. The optimumtemperature increased from50℃to55℃, the optimum pH shifted from9.5to10.0, the stablepH range expanded from8.0~11.0to7.0~11.0, and the catalytic efficiency increased from1.8×104L·g-1·min-1to2.4×104L·g-1·min-1.3．Based on the computer simulation design, introduction of arginines on the surface ofAmyK, three series residues (Lys, Asn/Gln, and Ser) were selected as targets for site-directedmutagenesis. Five single-site mutants—S270R, K315R, Q327R, N346R, andN423R—showed enhanced thermostability. Five arginines were subsequently introduced onthe protein surface, and the best mutant was S270R/K315R/Q327R/N346R/N423R. Itshalf-life at60℃improved by6.4-fold as compared with wild-type AmyK, and its optimal pHshifted from9.5to11.0. In addition, the optimum temperature increased from50℃to55℃, and the catalytic efficiency increased from1.8×104L·g-1·min-1to3.6×104L·g-1·min-1.4．Based on the synergistic effect, the most thermostable mutations obtained from the aboveseparate strategies were combined to improve the thermostability of AmyK further. The bestmutant was S270R/K315R/Q327R/N346R/N423R, and its half-life at60℃improved by6.6-fold as compared with wild-type AmyK. In addition, the optimum temperature increasedfrom50℃to55℃, the optimal pH shifted from9.5to10.5, and the catalytic efficiencyincreased from1.8×104L·g-1·min-1to2.8×104L·g-1·min-1.