Cloning And Expression Of An Endoxylanase Gene From Streptomyces Sp.WMN-3

Endoxylanase is the most important enzyme for xylanase system,which only randomly hydrolyzes internal ?-1,4-glycossidic bonds to produce xylose and xylo-oligosaccharides(XOs).According to the optimum pH for the enzymatic reaction,endoxylanase could be classified into three types: acidic endoxylanase,neutral endoxylanase and alkaline endoxylanase.In contrast to acidic endoxylanase,neutral endoxylanase and alkaline endoxylanase which are mainly produced by bacteria and actinomyces,have wider pH for catalysis,good thermostability and short fermentation time,etc.Therefore,it could be well applied in paper-making?food and feed industry.In this paper,we describe the screening of alkaline endoxylanase-producing bacteria,the purification and characterization of the alkaline endoxylanase from the cultrue supernatant of Streptomyces sp.WMN-3,and the cloning and express of the alkaline endoxylanase gene in E.coli BL21(DE3).The main contents of this paper are as follows:(1)A relatively high alkaline endoxylanase-producing strain Streptomyces sp.WMN-3 was isolated.Streptomyces sp.WMN-3 could produce high enzymatic acitivity.It was subsequently identified as Streptomyces sp.WMN-3 based on its morphological characters and 16 S rDNA sequence.Compared with the current industrial strains,Streptomyces sp.WMN-3 could be cultured in high alkaline condition(pH 9.0),in this way bacteria pollution could be effectively suppressed,and becoming more favorable for large-scale fermentation production.This paper focuses on the research of Streptomyces sp.WMN-3.(2)The alkaline endoxylanase was purified from the culture supernatant of Streptomyces sp.WMN-3 with cation-exchange chromatography.Purified enzyme showed an optimum activity at temperature 55? and pH 8.0.Furthermore,it was resistant to most metal ions and reagents(SDS?EDTA)examined,and had good thermostability and pH stability.This alkaline endoxylanase with superior stabilitycharacteristics coud be a suitable candidate in paper industry.(3)A new endoxylanase gene which had an open-reading frame(ORF)of 1476 bp and coded for 491 amino acids was successfully cloned from genome DNA of Streptomyces sp.WMN-3 through PCR method.BLAST analysis of the WMN-3sequence in NCBI database showed that it had 82% highest similarity to the?-1,4-endoxylanase from Jonesia quinghaiensis which belonged to family GH10.Based on the BLAST results and the differences in molecular mass,pI and enzymatic properties,it could be inferred that the gene WMN-3 was a novel endoxylanase gene.(4)The alkaline endoxylanase gene from Streptomyces sp.WMN-3 was successfully overexpressed in E.coli BL21(DE3).By the method of PCR,the endoxylanase gene was cloned from genome DNA of Streptomyces sp.WMN-3 and was ligated with the expressing vector pET-28a(+)through restriction endonuclease EcoR I and Xho I,generating a recombinant plasmid pET-28a-WMN-3 which was directly transformed into E.coli B121(DE3).The most efficient recombinant E.coli strain,which was designated as E.coli WMN-3,expressed large amount of endoxylanase when cultivated in IPTG containing medium.The recombinant endoxylanase was purified to homogeneity from the cultrue supernatant with a Ni-Sepharose column-chromatographic procedure,and the purified recombinant endoxylanase XYN-WMN-3 exhibited high specific activity of 131.2 U/mL.The properties of the recombinant endoxylanase XYN-WMN-3 was verified to be identical to the native endoxylanase in corresponding aspects,indicating that the neutral endoxylanase gene had been successfully expressed.