Purification Of Alkaline Xylanase From Bacillus Sp. WMN1 And Cloning & Expression Of Its Enzyme Gene

Endo-xylanase is a key enzyme that degrading xylan.It can hydrolyze ?-1,4-glycosidic bonds of xylan.Xylanase can be divided into acid xylanase,neutral xylanase and alkaline xylanase according to the optimum pH value of the catalytic reaction.Alkaline xylanase shows good value in papermaking and other industries due to its wide pH adaptability and good thermal stability.In this paper,an alkaline endoxylanase-producing strain,Bacillus sp.WMN1,was screened.The alkaline endo-xylanase was purified and its enzymatic properties were studied.A new endo-xylanase gene was cloned,which was efficiently expressed in the E.coli.The main contents of this paper are as follows:(1)A strain producing alkaline endo-xylanase was isolated.It was initially identified as Bacillus sp.by 16 S rDNA gene sequence analysis,and named Bacillus sp.WMN1.The xylanase activity in liquid fermentation was 360 U / mL.(2)The alkaline endo-xylanase protein produced by Bacillus sp.WMN1 was isolated and purified.Through anion exchange chromatography(DEAE Sepharose Fast Flow)and gel filtration chromatography(Superdex 75),an endo-xylanase protein was purified,whose molecular weight was about 36 k Da.The enzymatic properties showed that the optimum pH and temperature of the xylanase were 8.5 and 50 ?,respectively.It had excellent temperature stability and pH stability,and also had resistance to various metal ions,surfactants and metal chelates.In the range of pH 5-11,the xylanase was stable and had a wide pH adaptability.That is,it had excellent tolerance of acid and alkali,which indicated its great potential for industrial applications in paper-making pulping and washing.(3)The endo-xylanase gene of Bacillus sp.WMN1 was cloned.The complete open reading frame(ORF)of the WMN1 xylanase gene with a length of 1308 bp was amplified from the genomic DNA of Bacillus sp.WMN1,encoding 435 amino acids.The amino acid sequence of the enzyme gene was searched for homology identity with NCBI Blast program.It was found that the amino acid sequence had the highest similarity of 83% to Jonesia quinghaiensis,which belonged to the glycoside hydrolase families 10.Therefore,it can be concluded that the gene encoding the amino acid sequence was a new endo-xylanase gene.(4)An endo-xylanase gene cloned from Bacillus sp.WMN1 was successfully expressed in E.coli BL21 Star(DE3).The full-length xylanase gene was cloned from the genomic DNA of Bacillus sp.WMN1 by PCR,and it was inserted into the Nde I and Xho I restriction sites of the expression vector pET-28a(+).The resulting recombinant plasmid,rWMN1,was transformed into E.coli BL21 Star(DE3)strain.A recombinant Escherichia coli strain with efficient expression was obtained.(5)A recombinant alkaline endo-xylanase of rWMN1 with better enzymatic properties was isolated.The molecular weight was about 42 kDa of the recombinant enzyme protein isolated by anion exchange chromatography.Enzymatic property analysis revealed that the recombinant alkaline endo-xylanase of rWMN1 was basically consistent with the wild enzyme in terms of optimal pH,optimal temperature,and tolerance to metal ions.Nevertheless,the recombinant alkaline xylanase had good tolerance to SDS,while compared with the wild enzyme,which was inhibited partially by SDS.The stability of the recombinant alkaline endo-xylanase in Liby detergent with a final concentration of 1.5% was significantly better than its natural enzyme protein,showing the good application value of the recombinant enzyme preparation in detergent and other industries.