Molecular Engineering Of An Alkaline Xylanase XynHBN3

Xylan,which is the main component of almost all plant hemicelluloses and the second largest renewable resource in nature in present,is a kind of polypentose.Xylan can be hydrolyzed by xylanase to oligomeric xylose,some xylose,arabinose and mannose.Meanwhile,xylanase has considerable application prospect in the food,agriculture,functional XOS production paper making,textile,energy and other industries.However,the extreme environments prevalent in industrial production such as high temperature,high pressure,extreme pH and some other conditions limited the activity of commercial xylanase,significantly restricted the range of its applications.Hence,it's urgent to improve their properties by genetic engineering to meet the demands of industrial application.In our previous study,a 606-bp xylanase gene encoding 201 amino acids,named xynHB was cloned from Bacillus pumilus HBP8 by a shot-gun method and expressed in E.Coli.The enzymatic properties of XynHB have been characterized after it was purified,the studies showed that the optimal pH and temperature of XynHB was pH 6-9 and 40-60?,respectively.But the thermostability of the enzyme at 60? is unsatisfactory for the half-life of it only 12 min.Then,the amino acid N at position 188 was mutated into A via site-directed mutagenesis and the thermostability of the mutant significantly increased,the mutant xynHBN188A was named xynHBN3.Molecular modification was conducted based on xynHBN3 in the subsequent experiments.In the present study,we carry on the rational design of xynHBN3,use the PyMol,MODELLER,VMD of auxiliary software analyze the tertiary structure of the xylanase,and estimate the two plans:1)Mutation the 167th serine into arginine;2)Mutation the 41th asparagine into arginine;then design the primer for site-directed mutagenesis,study the effects of mutations in the protein of its structure and function.In the first embodiment,after the serine at position 167 mutated into arginine,respectively putting the original gene xynHBN3 and the mutation gene xynHBN3S167R to expressed in E.coli Rosetta(DE3),then purifying the expression product for nickel column affinity chromatography,finally comparing the enzymatic properties between XynHBN3 and XynHBN3S167R,the result showed that pH stability of XynHBN3S167R has been remarkable improved.In the second embodiment,the asparagine at position 41 mutated into arginine also purification after induced,a preliminary study found that the mutant protein had no big change compared with the original one.On the other hand,The xynHBN3 was mutated by random mutagenesis using error-prone PCR technique,the DNA fragment was cloned into vector pET-28a using the BamH I and Sal I restriction sites.The mutant library was transformed into E.coli Rosetta(DE3)according to the electroporation method.Using crosslinking-xylan substrate plate to observed the hydrolysis circle nearly 20,000 recombinants had been screened.After several rounds of screening 10 strains was selected,one positive clone XynHBN3217 whose specific activity of 2.8-fold increased was obtained by a shake flask fermentations selection.Two amino acid substitution of xynHBN3217 that would contribute to improving these function was identified by DNA sequencing;the 42th asparagine mutated into aspartic,149th threonine mutated into serine.In order to study how the two amino acid site of xynHBN3217 affect its protein structure and activity,we put the mutations in saturation mutagenesis and study the enzymatic properties.Meawhile.to obtain a higher pH stability of xylanase,we also combine xynHBN3217 with rational design,mutated the 167th amino acid serine into arginine,study of its enzymatic properties.