Research On Co-expression Of ?-amylase And Screening Of Optimal Signal In Bacillus Subtilis

Nowadays,?-amylase is widely used in food and feed industry.This study attempts to improve the activity of amylase by optimizing the signal peptide in the expression system of Bacillus subtilis and break through the"bottleneck"in the heterogenous expression of amylase.In addition,with the diversification of the application of enzymes,it is hoped that a single protein can have a variety of enzymatic characteristics,so that production cost will be reduced.Therefore,the research and development of multi-functional combination enzymes will become a trend.Based on the study of amylase,the study on the co-expression of xylanase and?-amylase fusion proteins helped us understand the mechanism of co-expression system deeply,and provided some valuable references for the co-expression study of fusion proteins between different enzyme preparations in the future.1.AmyCBS 101883 of Aspergillus lacticoffeatus and XynCDBFV of Neocallimastix patriciarum were selected as the main research objects in this study.Three ligand peptides suitable for glycoside hydrolases were screened by Linker Database.The co-expression fusion protein of xylanase and?-amylase was constructed by means of splicing-by-overlap extension?SOE?.The fusion proteins were separately introduced into Escherichia coli and Pichia pastoris for induction expression.The enzymatic properties were studied with reference to the single enzymes Amy-E2 and Amy-pGAP.The conclusions were as follows:In terms of pH adaptability and stability,the optimum pH of both Amy-E2 and fusion protein Xyn-Amy-E2 was 7.5.When the pH was lower than 7.0,the activity of Amy-E2 decreased rapidly,while the relative residual activity of Xyn-Amy-E2remained above 70%at pH 4.5 to 7.5,and it had better adaptability under acidic conditions.Linker 1-linked fusion protein maintained relative residual enzyme activity of about 95%at pH 6.5 to 7.5,and had the best adaptability under alkaline conditions of pH 8.5.Moreover,the activity of?-amylase in the fusion protein was increased by23%.In addition,in the eukaryotic expression system,the pH adaptability of?-amylase was weakly alkaline,and the activity of fusion protein?-amylase was superior to that of the reference enzyme under acidic conditions.In terms of temperature adaptability and stability,Xyn-Amy-E2 had the best thermal stability and reached half-life after tolerant treatment of 50min.Both the fusion protein Xyn-L2-Amy-pGAP and Xyn-L3-Amy-pGAP had good adaptability in the temperature range of 55?and 70?,and their relative residual enzyme activities were more than 65%,which were better than that of the reference enzyme Amy-pGAP.The fusion expression showed that the activity of?-amylase expressed after fusion was lower than that of single?-amylase in the same expression system.The expression activity of Xyn-Amy,Xyn-L1-Amy,Xyn-L2-Amy and Xyn-L3-Amy decreased by36.9%,21.6%,59.0%and 39.0%respectively.When Linker 1 was introduced,the activity of fused?-amylase increased by 23%.The average activity of?-amylase in E.coli was 0.571 U/mL,and that in eukaryotic system was 4.208 U/mL.2.In this study,a mixed signal peptide screening library derived from Bacillus amyloliquefaciens?-amylase gene?DL-3-4-1?was constructed based on the expression system pBE-S of Bacillus subtilis.It was transformed into B.subtilis strain RIK1285by electro transformation.Based on 96-well plate microculture,the fermentation supernatants of 1920 strains were detected,and the strains whose enzyme activity ranked in the top 100 were sequenced.After screening,14 signal peptides were obtained:yomL,PhrA,PhrF,YvbX,YoqH,mpr,YuaB,YqxI,YjcM,ywoF,PhrK,peI,YceG,YddT and yobb.Among them,the signal peptide yomL,which guided the most efficient secretion of?-amylase,has an enzyme activity of 113.251 U/mL.Its enzymatic properties were further analyzed as follows:The optimum pH was 6.5,and the stability was better in alkalescence condition,and the optimum temperature was 65?.The half-lives were 80 min,70 min and 40min respectively,which indicated that the heat stability was strong.Triton x-100,Mn²⁺,Mg²⁺,EDTA,Ni²⁺,Co²⁺have an activation effect on pBE-SPyomL-DL-3-4-1;SDS,Al³⁺and Cu²⁺have certain inhibitory effects on the enzyme;Ag⁺and?-mercaptoethanol completely inhibit the enzyme.In addition,the enzyme had a good hydrolysis effect on crude starch from plant seeds and rhizomes,and had a high application value.