Increasing The Expression Level Of α-Amylase AmyM In Bacillus Subtilis By Random Mutagenesis

The α-amylase AmyM from Corallococus sp.EGB has the ability to hydrolyze starch to form maltooligosaccharides.Previous studies have shown that the enzyme has good stability in high salt solution or high temperature environment,and is the potential object of food processing enzyme preparation.However,AmyM has a low yield in the original host EGB,and it can not be expressed efficiently in industrial production.Bacillus subtilis is a common host of food enzyme expression,which has the advantages of good safety,strong protein secretion and mature fermentation methods.The previous researchers in this laboratory have expressed AmyM in Bacillus subtilis,but the expression is not up to the expected level.In this study,we intend to improve the expression of α-amylase AmyM in Bacillus subtilis by random mutagenesis.Firstly,the AmyM expression box was randomly mutated by error prone PCR,and then the host was modified by traditional mutagenesis(EMS chemical and UV mutagenesis).The specific results are summarized as follows.1.Increasing the expression of AmyM by random mutagenesis based on error prone PCRSignal peptide mutation,codon change or amino acid mutation may increase the expression of target protein.Therefore,we first used error prone PCR to randomly mutate the expression cassette of AmyM.Among about 16000 transformants,a mutant PDACa-3 with 5-fold increase in expression was screened.The highest enzyme activity of AmyM was 1194.6 U/m L in shake flask fermentation.DNA sequencing results showed that GAA at codon 436 of PDACa-3 was mutated into TAA,which resulted in the deletion of some region of AmyM.The predicted tertiary structure showed that most of the starch binding domains were missing in the mutant AmyM.We speculate that the deletion of starch binding domain will improve the secretion or folding efficiency of AmyM.We constructed the mutant PDACaQ3 with the deletion of starch binding domain,and found that its extracellular amylase activity was 2.6 times higher than that of the original strain,which was consistent with our conjecture.Based on this,we selected two mutants PDACa Y-13 and PDACa Y-21 by random mutagenesis on the basis of wild-type AmyM,and their expression levels were 1.3 and 2.2 times higher than those of the original strain,respectively,which further indicated that the starch binding domain had an important effect on the secretion and expression of AmyM.The enzyme analysis of truncated AmyM showed that the optimal p H and temperature had no significant change.These results provide a reference for optimizing the expression of multi domain protein in Bacillus subtilis.2.UV mutagenesis and EMS chemical mutagenesisIn order to further improve the expression of AmyM,we tried to modify the expression host by EMS chemical and UV mutagenesis.The expression of PDACa-U53 and PDACa-E11 were screened out by UV and EMS chemical mutagenesis.The expression of AmyM was 24.4% and 40.5% higher than PDACa-3,and the highest enzyme activity reached 1485.7 U/m L,1678.9 U/m L respectively.The highest activity of PDACa-E11 was 1932.3 U/m L after the initial fermentation in 5-L bioreactor.In conclusion,the study used error prone PCR to randomly mutate the expression box of AmyM,and a mutant PDACa-3 with significantly increased expression was screened.The results of three-dimensional structural prediction and protein expression showed that the starch binding domain of AmyM had an important influence on the secretion of AmyM.Deletion or mutation of the domain could significantly improve the expression of AmyM.Then we carried out EMS chemical and UV mutagenesis on PDACa-3,and screened two mutant PDACa-E11 and PDACa-U53 with further improvement of expression.The highest enzyme activity of PDACa-E11 with high expression in 5-L bioreactor was 1932.3 U/m L.The results provide a reference for further improving the production of AmyM and the recombinant expression of other amylase.