Fermentation Optimization, Heterologous Expression And Enzymatic Properties Of Pullulanase From Klebsiella Oxytoca M5al

Pullulanase(EC 3.2.1.41), an important debranching enzyme, can specifically hydrolyze α-1,6-glucosidic bonds of starch and greatly promote the starch utilization. Pullulanase has been applied in food, textile, biofuel and detergent industry. In present study, fermentation optimization and enzymatic properties of pullulanase from Klebsiella oxytoca M5 al were studied.Firstly, carbon source, nitrogen source, amount of inoculation, initial p H, shaker speed, temperature and liquid volume were explored by single factor experiment. And the optimum medium was determined by response surface analysis. The production of pullulanase was 1.05 U×m L-1, and increased to 1.50 U×m L-1 after response surface analysis, increased by 44%. And the final production was further increased to 12.44 U×m L-1 in 7 L fermentor, which was the 8.29 times than that in 500 m L shake flask.In order to characterize enzymatic properties, pullulanase from K. oxytoca M5 al was cloned, expressed in E. coli BL21(DE3), and purified. The optimal temperature and p H was 60 oC and 5.5, the specific activity was 5.85 U×mg-1 with optimum condition. The study of stability indicated that the thermostability was poor when the temperture higher than 45 oC,and the p H stability was preferable. The effect of metal ions and organic solvents on recombinant pullulanase showed that the enzyme activity slightly enhanced by Fe3+, Mg2+, Fe2+ and greatly increased by Mn2+, while the activity was inhibited in the presence of Cu2+, and not effected by Ca2+. Moreover, low concentration of organic solvents such as SDS, methanol, urea, guanidine HCl, had slightly inhibitory effect on recombinant pullulanase. The results of thin layer chromatography(TLC) suggested the hydrolysis products of pullulan was maltotriose, and recombinant pullulanase had the wonderful α-1,6-glucanohydrolase activity. The study of substrate specificity showed pullulanase preferentially hydrolyzed pullulan, and its hydrolysis efficiency was more higher than soluble starch and amylopectin.In order to solve the questions of pullulanase instability and low catalytic efficiency, Phe655 was selected as targets for site-directed mutagenesis based on a structure-guided consensus approach in this study. Two mutants(F655G and F655C) were generated and characterized in details. Two mutants pullulanase were simlar with the wild in thermostability, but its catalytic efficiency improved significantly when soluble starch as substrate.Taken together, the pullulanase production was improved by the study of fermentation conditions; and some enzymatic properties were characterized, such as optimum reaction conditions, stability, tolerance and substrate specificity; Modified pullulanase was generated by gene engering with the purpose of getting good thermostability and catalytic efficiency. pullulanase from K. oxytoca M5 al was the first report in public, the study of enzymatic properties and site-directed mutagenesis laid a solid foundation for further application in industrial production.