Fermatation And Preparation Of Xylanase By Bacillus Methylotrophicus YH158 And Cloning Of Its Related Genes

In this paper,225 strains of xylan degradation bacteria that can formation transparent hydrolysis circle on select media,xylan as sole carbon source,were isolated from soils sampled from Shanxi Province,Heilongjiang province and around Hunan province preliminarily,etc.Then,by secondly screening,according to the xylanase activity and hydrolysates after shaking flask fermentation,the strain YH158 was deloveped as more optimal strain,with xylanase activity of 12.53 IU/mL.finally,strain YH158 was identified as Bacillus methylotrophicus by morphological observation and homology analysis of sequence of 16 S rRNA.And the products of hydrolysis xylan and corncob were rich in 2-5 xylose molecules.It's xylanase was able to tolerate high alkali and had high temperature stability.In the pH range of 6.0 ~ 10.0,the relative enzyme activity were over 76%,in the pH range of 6.0 ~ 9.0,processing after 1 h,relative residual enzyme activity in more than 88%,processing after 2 h,still can keep more than 80%;When the temperature reached 80 ?,the processing after 1 h,the residual enzyme activity and 40%,dealing with residual enzyme activity was 26% after 2 h.The liquid fermentation conditions for xylanase by Bacillus methylotrophicus YH158 was optimized by single factor experiment and orthogonal experiment.The optimization conditions were as follows: the bran 5.0% as carbon source,soyabean meal 0.7% as nitrogen source,MnSO4 0.1%,initial pH 8.0,and cultured at ratation speed of 180 r/min at 37? for 36 h.Under optimal liquid-state fermentation conditions.the xylanase activity of Bacillus methylotrophicus YH158 reached 28.18 IU/mL.According to the gene sequences of XynA,XynB,XynC and XynD in the NCBI database,four corresponding primers were designed for cloning xylanase gene of Bacillus methylotrophicus YH158 by method of seamless cloning.Xylanase genes Bacillus methylotrophicus YH158 were cloned in expression vector of E.coli BL21 and expressed successfully.By gene sequencing and BLAST alignment consistency and sequence analysis four genes of Bacillus methylotrophicus YH158,the sequences of nucleotide sequences consistency was 98.0~99.55%,for coding sequence of amino acids,the consistency was 100%.After the expression by E.coli BL21,The xylanase activities of xynA-pBAD-HisD,xynB-pBAD-HisD,xynC-pBAD-HisD and xynD-pBAD-HisD reached 12.23 IU/mL,10.34 IU/mL,10.85 IU/mL,and 9.56 IU/mL,respectively.The product of hydrolysates of xylan were xylose and xylooligosaccharides(contains 2~5 xylose molecules),the results of this study laid a foundation for further research on Bacillus methylotrophicus YH158.