| ?-amylase is an an important industrial enzyme.At present,the commonly used methods to determine the activity of?-amylase were 3-5 dinitrosalicylic acid?DNS?and p-nitrophenol maltopentoside?PNP?-G3?.DNS method is time-consuming and low sensitivity,the enzyme activity value of?-amylase will be affected by alpha-amylase,while barley seeds often contain a large number of alpha-amylase;PNP?-G3 method is simple,fast and sensitive,but the ability of amylase to hydrolyze starch can not be accurately evaluated according to its results.Therefore,the two methods can be linearly fitted to accurately and effectively determine the ability of amylase to hydrolyze starch in?-amylase preparations.High thermostability and catalytic activity are conducive to the application of barley?-amylase,and microbial fermentation production is easier to achieve automatic control.In this study,the?-amylase activity determination method was improved,the barley?-amylase gene was modified in Escherichia coli,Bacillus subtilis was used as host to express the modified barley?-amylase.The main results of this paper are as follows.1.Improvement and application of determination method of?-amylase activity:When the concentration of?-amylase was 12-60?g·mL-1,the linear correlation coefficient was high within this concentration range,the linear regression equation obtained by fitting the two methods was y=0.0079x+0.0089,R2=0.9886.The relative deviation was less than 10%when the method was applied to the determination of?-amylase products.The method can be used to quickly examine whether there are other amylases in the sample of?-amylase.2.Molecular modification of barley?-amylase:Through amino acid sequence alignment of 10 different barley varieties,R115 and T387 were inferred to have important effects on the catalytic properties and thermostability of barley?-amylase.By site-directed mutagenesis,R115C was found to have higher catalytic activity,T387A had better thermostability.Saturated mutation at T387 site shows that T387V has the highest thermostability.The optimum temperature?T50 value and half-life at 60°C of double mutant barley beta-amylase?amyBDM?obtained by combining mutation of R115C and T387V were increased by 5°C,3.6°C and 29.5 min,respectively,and the specific enzyme activity and Kcat/Km value were increased by 27.4%and 54.7%respectively compared with wild enzyme.Structure modeling analysis showed that the the main reason for the increase of thermal stability of enzymes was the increase of hydrophobicity of protein surface and the number of strong hydrogen bonds and salt bridges.The reason for the enhancement of catalytic ability of enzymes was the change of hydrogen bonds at the active center amino acid E378.3.Heterogenous expression of double mutant barley?-amylase in B.subtilis:The recombinant B.subtilis WB800/pP43NMK-amyBDM was successfully constructed.The activity of recombinant?-amylase reached 434 U·mL-1 at 80 h of fermentation.The specific activity of recombinant?-amylase was 864 U·mg-1.The optimum temperature was 60°C.The half-life of recombinant?-amylase at 60°C was 45.2%higher than that of barley?-amylase.The optimum pH of the recombinant enzyme was 5.0,and the activity of the recombinant enzyme remained 84%after 1 h of warm bath at pH 4.0.The effect of metal ions and inhibitors on the activity of recombinant enzymes was investigated,most metal ions and inhibitors inhibit the activity of recombinant enzymes,and the presence of Li2+?Na+and 1mM DTT has a slight activation effect on the activity of recombinant?-amylase.The conversion rates of maltose produced by hydrolysis of maltodextrin by barley?-amylase,commercial?-amylase and recombinant?-amylase were 62.4%,55.1%and 67.7%,respectively.The conversion rate of maltose could reach 85.5%when the recombinant?-amylase was combined with pullulanase. |
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