Rational Design Of Trypsin-resistant Bacillus Subtilis168β-1,4-endoxylanase

Objective: The protein project produced in the1980s and its implementationstrategies consist of two kinds: site-directed mutations representing the rational designand directed evolution representing the irrational design. Rational design was thebasic technology for modifying protein and the most important aspect was to find outthe relationship between molecular structure of protein and its function. Meanwhile, itneeds to select mutation site purposefully to modify the enzyme protein on the basisof understanding the advanced structure of protein molecule. The mutation site waschosen usually based on accumulating the related information of molecular structureand function. It was impossible to obtain the required mutation sites by rational designfor some protein modification without research accumulation. Therefore, it is indeed achallenged and valued work. The study combined the rational design of proteinmodification with resistance to protease based on the method of the biocatalysis andcomputational chemistry,which has been established to improve Bacillus subtilis1681,4-β-endoxylanase with resistance to protease and to get the excellent properties.Meanwhile, the rational design method of protein modification with resistance toprotease was proved.Methods: Firstly, the optimized three-dimensional structure of xylanase was obtainedby Discovery Studio3.0software and analyzed rationally to get hydrolysis sites oftrypsin. Secondly, the most possible candidate mutation site was determined, andmutation was simulated. According to the exposure and the mutation effect scores, themutated structures which were much more stable or eaqual to the wild type wereobtained. Thirdly, the three-dimensional structures before and after mutations wereconstructed through molecular dynamics simulations. The RMSD value ofconformation with the lowest energy was calculated and changes in whole structureafter mutation were determined. What's more, the lowest energy conformation ofthree mutants was evaluated on structure similarity to confirm the mutated combination types. Fourthly, we used site-directed mutagenesis technology with theoverlap-extension PCR method to induce mutations on Bacillus subtilis168endo-1,4-beta-xylanase gene. The PCR fragments were connected to@-@@a vectorand duplicated in E.coli DH5α. Then the correct recombinant plasmids weretransformed into E.coli BL21(DE3) to express the proteins. Then the properties ofpurified enzymes were analyzed. We treated them with simulated intestinal fluid (pH6.810mg/mL trypsin solution) and analysed the effect of trypsin treatment on stabilityof the XynA and mutants.Results: The two most external exposure extent**,**/**were chosen as themutation sites and the most internal exposure extent**was choosen as thecounterevidence site. We did single site mutation to**and**and double sitesmutations to**/**to get the fine properties of mutant enzymes XynA**, XynA**/**and XynA**. Fortunately, we finally got the excellent properties. The optima pH ofthe wild and mutant enzymes was6.0and the pH tolerance of XynA**, XynA**/**wasmuch wider than the wild type, ranging from pH4.0—9.0, While XynA**is similar tothe wild type. The optimal temperature of XynA, XynA**, XynA**/**was60℃, and itwas stable for them under50℃, which their half-life periods were40min under55℃.The optimal temperature of XynA**was40℃, and its temperature tolerance waslower than the wild type. When the temperature is higher than40℃,the deactivatingspeed of XynA**was quicken. XynA**was stable under40℃, and its half-life periodwas40min under45℃. During the treatment with simulated intestinal fluid (pH6.810mg/mL trypsin solution), the remained enzyme activity of mutants was muchhigher than the wild type. As for XynA**, its half-life period was193min,1.52timeshigherr than XynA. As for XynA**/**, its half-life period was257min,2.02timeshigher than XynA and1.33times higher than XynA**. As for XynA**, afterincubation with simulated intestinal fluid at40℃for different time, its half-life periodwas375min, only37min shorter than XynA.