Molecular Evolution And Efficient Expression Of Thermophilic ?-amylase

Thermophilic ?-amylase is an important industrial enzyme widely used in starch,sugar,fermentation,brewing and textile industries.Breeding of strains capable of high-yield industrial production is the basis for the improvement of this enzyme preparation.Efficient preparation and industrial application of thermophilic ?-amylase have been conducted based on Bacillus licheniformis expression system in our preliminary work.In this paper,the directed evolution of the amylase gene was carried out through modern molecular biology techniques.The enzymatic properties of new enzyme molecules were analyzed and the high-expression strains constructed.Following results were obtained:(1)Construction of the mutant library of thermophilic ?-amylase.Based on Bacillus licheniformis thermostable ?-amylase mutant BLA(high expression has been achieved in the Bacillus licheniformis industrial expression system,but heat resistance and acid resistance performance needs to be improved)and thermophilic bacteria-derived thermostable ?-amylase GNA(Excellent heat resistance and acid resistance,but unable to perform high expression).The sequence and structure were analyzed,and crossover PCR which is similar to DNA shuffling technology is used to get fusion genes.The two amylase molecules were successfully fused and expressed.A mutant library containing 22 thermophilic ?-amylase mutants was obtained.(2)Biochemical properties of the mutants.Transparent circle comparison was used for assay of the mutant enzymes' activity and shake flask fermentation for expression,followed by the enzymatic properties and enzyme kinetic parameters analyzed.The biochemical characterization of the mutants changed to varying degrees.The varying range for optimum temperature and pH was 60?80? and 5.5?7.5,respectively.Heat resistance and acid-base resistance have also been elevated to varying degrees,but without regularity.(3)Comparative analysis of biochemical properties of representative mutants.The mutants V-2,V-12 and H-11 were analyzed in detail.Their heat and acid resistance were significantly improved,and were more efficient than BLA in starch hydrolysis.Their specific activity was 0.6,1.0 and 3.8 folds higher than that of BLA,respectively.The effects of metal ions,especially Ca2+,on the activity of mutants were significantly reduced.H-11 and V-12 had potential for further research.(4)High-level expression of the enzyme and construction of high-yield bacteria.Expression vector pBL-WZX was constructed by inserting the thermophilic ?-amylase gene VH11 fragment to the BamH ?/Sma ? site of pBL-WZX vector.A high-yield producer of thermophilic ?-amylase,BL-H-11,was obtained through electroporation transformation.Eventually,thermotolerant ?-amylase was overexpressed in B.licheniformis.