Expression System Optimization Of Mannanase Man23 And Its Molecular Engineering

Mannanase Man23,produced by Bacillus subtilis B23,has high activity and good thermo-stability.In this paper,to elevate the expression level of gene man23 and to improve the stability and catalysis efficiency of mannanase Man23,semi-rational design methods, accompanying with skills in bioinformatics,biophysics,biochemistry and genetic engineering, were utilized to engineer gene man23 in vitro.At the first step,the gene man23 was ligated with pHY-p43 which carries a strong promoter p43.Then the recombinant plasmid pHY-p43-man23 was transferred into the protease-deficient bacillus WB600 and Brevis subtilis.Changing the host strain and the promoter is an efficient way to boost the gene expression and lower the hybridprotein.Next step,mannanase Man23 was purified and its chemical property was analyzed through chemical modifying methods.On the basis of knowledge about the relation of residual type and property,the bioinformatics software was used to predict the mannanase structure.The suspected sites were determined with alanine scanning and saturation mutation was detected at selected sites.At last,a stabile and efficient mannanase was obtained and its micro-environment was optimized,including metal ion,preservative and so on.The experiment results showed the promoter p43 greatly elevated the expression of gene man23.At the same time,the protease-deficient bacillus greatly prevented the target protein degrading in vitro.Both Bacillus subtilis B23 and Brevibacillus brevis,carrying the recombinant plasmid pHY-p43-man23,are successful expression systems.To detect the active center of mannanase Man23,the semi-rational strategy was set up as following:at first,chemical modifying methods were used to analyze the properties of mannanase Man23 and to determine the residues relevant to its active center.Secondly, alanine scanning was used to determine the sites relevant to its active center and then at the selected sites,saturation mutation was used to decide the direction of mannanase mutation.In this paper,absorption spectrum of mannanase Man23 was tryptophane spectrum because its absorption peak was at 336 nm.Cysteine,carboxy group and tryptophane were essential groups to catalytic activity.Moreover,the sites of D91 and E191 were active sites and the sites of H129,H190,W196,F197,W198 and W199 were substrate binding sites. Thinking about covalent bond,charge distribution and stereo-angle and so on,site mutation,accompanying with bioinformatics software,was used to determine the sites relevant to mannanase stability.The saturation mutation was used to decide the direction of mutation.At last,an mutative mannanase M0710 which was effectively mutated at 23 sites was obtain.The activity of this mutative mannanase M0710 rose up nearly by 10 times and its half-life at 80℃extended by 3 times.Polyalcohol,preservative,metal ion and carbohydrate were experimented through single-factor tests,potassium sorbate,Na⁺,K⁺,Ca²⁺,sucrose and lactose had great effects on the stability of mannanase M0710.Orthogonal experiments decided the optimal stabilizing agent were potassium sorbate 3%,sodium chloride 2%and sucrose 2%.