Effects of Carbohydrate-binding Modules on the Enzymatic Properties of Xylanase
[Objective]This study is aimed to explore the binding ability of different sources of CBM to beech-xylanan,and to fuse exogenous CBM with high binding ability to the C-terminal and N-terminal of Streptomyces L10904 xylanase(XYN),to explore the effects of exogenous CBM on the enzymatic properties of xylanase.[Method]First,through the substrate adsorption method,the concentration of CBM in the solution before and after adsorption was detected by Coomassie Brilliant blue G250 method,and the substrate binding rate of CBM was calculated.CBM1 and CBM4 with better xylan binding ability were screened.In order to explore the effect of the fusion location of CBM with high substrate binding ability on the enzymatic properties of xylanase,CBM1 and CBM4 were fused with the C-terminal and N-terminal of XYN by flexible binding peptide,and four recombinant enzymes were obtained by expression in Escherichia coli BL21(DE3).They were named CBM1-XYN,XYN-CBM1,CBM4-XYN,and XYN-CBM4.[Result]The binding rates of CBM1 and CBM4 to xylan were 89%and 95%,respectively.The specific activities of XYN,CBM1-XYN,XYN-CBM1,CBM4-XYN and XYN-CBM4 were 32 274.81,49 342.21,602.48,230.42 and 2 362.24 U/mg,respectively,measured at 60℃and pH 7.0.The specific activities of CBM1-XYN were 1.5 times higher than the specific activities of XYN.The analysis of enzymatic properties showed that CBM1 improved the temperature stability and pH stability of XYN,XYN and CBM1-XYN were incubated at 60℃for 1 h,and the residual enzyme activity of CBM1-XYN and XYN was 81%and 28%,respectively.In the pH range of 3-11,CBM1-XYN maintained more than 90%enzyme activity after incubation at 4℃for 12 h.[Conclusion]Streptomyces derived xylanase was heterogeneically expressed in E.coli BL21(DE3),and CBM1 and CBM4 with high substrate binding rates were screened.CBM was successfully fused to XYN by protein fusion technology,and CBM1-XYN with improved enzymatic properties was obtained,which improved the temperature stability,pH tolerance and specific enzyme activity of xylanase.
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