| ?-amylase(?-1,4-D-glucan maltohydrolase,EC3.2.1.2)is an exo-amylase.When used for amylose,it mainly produces?-maltose,glucose and malt.When acting on amylopectin,the products are mainly maltose and limited dextrin.?-amylase has a wide range of applications in beer brewing,sugar-making and other production fields.Commercial?-amylase is mainly derived from plants and owns good thermal stability and p H tolerance,but the production process is complicated,the cost is higher,with unstable quality and low purity,Microbial-derived?-amylase cannot achieve industrial production due to its low expression level.Herein,we adopted the recombinant cloning expression technology of exogenous genes and selected single promoters to construct dual promoters,to enhance the intensity of gene expression regulation.And we synonymously mutated the highly conserved base sequence site of the?-amylase gene:carbon catabolite responsive element(CRE)to prevent catabolite control protein(catabolite control protein A,Ccp A)combine with the complex,thereby alleviating the carbon catabolite repression(carbon catabolite repression,CCR)to significantly increase the expression level of enzyme activity.Finally,fermentation process optimization was done in the shake flask fermentation,aiming to improve the heterologous expression of?-amylase at the laboratory level.The main research contents are as follows:(1)Construction of heterologous expression system for?-amylase gene:The?-amylase gene was derived from Bacillus megaterium DSM 319 was successfully amplified and His+tag was added.Promoters from Bacillus subtilis 168,Bacillus licheniformis,and Bacillus licheniformis were successfully amplified to constructed 22 strains of?-amylase mediated by a single promoter,The?-amylase activity of expressed recombinant strains were tested.After screening and comparison,the results showed that P43,Pamy Q,Psig W,Pspov G,Pgsi B,PB.liche-apr,and Pnpr Btransformants own higher enzyme activity level.Then recombinant strain with a pairwise combination of dual promoters was constructed based on these 7 strong single promoters.The results showed that?-amylase mediated by dual promoters P43-P43 owned,the highest enzyme activity of 2950.8 U/m L under shaking flask fermentation culture conditions.(2)Purification and enzymatic characteristics of recombinant?-amylase:After purification by nickel column affinity chromatography,the specific enzyme activity of recombinant?-amylase was 361.04 U/mg,with the recovery rate of 17.29%,and purification fold of 3.04,SDS-PAGE result showed that the molecular weight of the protein was about 50 k Da.The recombinant?-amylase Property analysis shows that the optimal p H value of recombinant?-amylase is 6.0,the optimal temperature is 50?,and the temperature is in the range of 35?45?.?-amylase has good stability,and the stability is better in the p H range of 5.0?6.0,and the activity of?-amylase decreases when p H is out of range.Adding different metalions affects the activity of?-amylase,Metalion Co2+has an obvious activating effect on the enzyme activity of recombinant?-amylase.Ca2+and Fe2+have an inhibitory effect on recombinase at low concentrations,but as the concentration increases,they have almost no effect on enzyme activity.When the concentration reaches 15 mmol/L,the inhibitory effect appears.Cu2+and Mn2+donot affect the enzyme activity of the recombinant enzyme in the low and middle concentration,but as the concentration increases,it shows an inhibitory effect.Zn2+,Mg2+,Fe3+,Ba2+always inhibit the enzyme activity as the concentration changes,While the inhibitory effect of Ba2+is the most obvious.(3)Mutation of carbon catabolite repression(CCR)element CRE to regulate the expression of?-amylase in Bacillus subtilis:When the recombinant?-amylase gene is expressed in Bacillus subtilis,the strain will be affected by carbon catabolite repression(CCR)when the strain is in an environment rich in fast-acting carbon sources such as glucose,thereby limiting the expression level of amylase enzyme activity in fermentation.Studies showed that there are highly conserved sequence base sites in the gene of?-amylase,called catabolite responsive element(CRE).CRE is the cis-regulatory element of the catabolite control protein(Ccp A).Site-directed mutation of the CRE site could repress the combination of the repressor complex with the CRE site,thereby alleviating the CCR effect.The results showed that the highest expression level of?-amylase activity of the strains subjected to site-directed mutation was4663.0 U/m L,which was 58.0%higher than the recombinant?-amylase activity of the unmutated strain.The number of bases could affect its role in enzyme activity.(4)Optimization of the fermentation process of the highly expressed mutant p BE05 and its application.After optimization,it is determined that the carbon source is 2.5%potato starch,and the nitrogen source(yeast powder,tryptone compound nitrogen source of(2:1)was added with a ratio of 3%,the initial p H of the culture medium is 7.0,and the temperature of the fermentation shake flask is 34?.After 27 hours of fermentation,the enzyme activity level of recombinant?-amylase in the supernatant reached 8616.1 U/m L.The enzyme activity increased by 84.8%before fermentation optimization,and the enzyme activity increased by 191.9%before the carbon repression effect was not alleviated.Recombinant?-amylase was used in the saccharification process,to investigate the influence of different amounts of recombinant?-amylase enzymes on the conversion rate of maltose.The conversion rate reached the highest level when the added amount was 200 U/g,and the highest maltose conversion ratereached57.6%.When the addition amount of the?-amylase enzyme continue to arise,the maltose conversion rate no longer increased. |
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