Cloning,Expression And Application Of A Recombinant ?-xylosidase From Cellulosimicrobium Cellulans Sp.21

Xylan,one of the most abundant polysaccharides in nature,is the major components in plant hemicellulose.Xylan is the most important renewable resource in nature,which has been widely applicated in paper,food,medicine and other industries.The complete degradation of xylan requires a number of hydrolytic enzymes including xylanases and ?-xylosidases.Among them,?-xylosidases is a key enzyme,which synergistically acted with xylanase todegrade xylan into xylose.Therefore,the screening and preparation of high active ?-xylosidase is one of the research hotspot in the xylan degradation field.In this study,one ?-xylosidase encoding gene was cloned from Cellulosimicrobium cellulans and expressed in Escherichia coli BL21(DE3).The main research results are as follows:1.A multifunctional ?-xylosidase/?-L-arabinofuranosidase/?-glucosidase gene(ccxyl3a)belonging to glycoside hydrolase family 3(GH3)was cloned from C.cellulans sp.21,the gene was ligated with pET-28a(+)to form the recombinant plasmid pET-28a/ccxyl3a,and transformed into E.coli BL21(DE3)component cells,to get recombinant strain BL21/pET-28a/ccxyl3a.2.The recombinant strain BL21/pET-28a/ccxyl3 awas induced at 16? with 0.5 mM IPTG for 20 h.After that,the cells were harvest and lyzed and the recombinant enzyme CcXyl3A was purified from the supernatant of lysis solution by Ni sepharose fastflow column.3.The recombinant CcXyl3A was characterized.The molecular mass of recombinant CcXyl3A was estimated to be approximately 95 kDa.With p-nitrophenyl-?-D-xyloside(pNP?Xyl)as a substrate,the purified recombinant CcXyl3A presented an optimal pH of 8.5 and an optimal temperature of 45 ?.K~+ and Na~+ were found to be activitor for CcXyl3A,the enzyme activity of which was increased 132.2% and 132.6% in the presence of K~+ and Na~+ at concentration of 50 mM.Purified CcXyl3A demonstrated multifunctional activities on pNP?Xyl,p-nitrophenyl-?-D-glucoside(pNP?Glc),and p-nitrophenyl-?-L-arabinofuranoside(pNP?Araf).The greatest catalytic activity were found on pNP?Xyl,followed by pNP?Araf and pNP?Glc,respectively.Using xylooligosaccharides as substrate,CcXyl3A completely hydrolyzed xylobiose,xylotriose,xylotetraose and xylohexaose,xylose was the sole product.4.CcXyl3A synergistically acted with Thermomyces lanuginosus xylanase in the degradation of beechwood xylan,released xyloses from intermediate xylooligosaccharides produced by T.lanuginosus xylanase.These results would be useful for hemicellulose degradation.