重组大肠杆菌生产极端耐热木聚糖酶

Production of Extremely Thermostable Xylanase in Recombinant Escherichia coli

将含有来自于嗜热网球菌(Dictyoglomusthermophilum)Rt46B.1编码极端耐热木聚糖酶基因xynB的表达载体pET?DBc转化大肠杆菌(Escherichiacoli)BL21(DE3),获得重组菌E.coliDB1,目的基因可表达出有活性且耐90oC的木聚糖酶.初步优化的E.coliDB1发酵培养基的组成为(g/L):葡萄糖50,NH4Cl3,MgSO40.5,CaCl20.6,Na2HPO4?7H2O12.8,KH2PO43.0,NaCl0.5.重组菌E.coliDB1木聚糖酶的耐热特性有利于木聚糖酶的下游回收和提取.

Xylanases have shown considerable potential in pulp bleaching, food and feed industries by partial hydrolysis of xylan. Cloning and expression in E. coli of thermostable xylanase gene from the extremely thermophilic anaerobe microbes were studied and the effects of medium composition and high temperature treatment on the production of the extremely thermostable xylanases by recombinant strains were investigated. Plasmids, pET-DBc and pET-TB, carrying the xynB gene from Dictyoglomus thermophilum Rt46B.1 and Thermotoga maritime MSB8, were transformed into Escherichia coli BL21(DE3), respectively, and recombined strains E. coli DB1 and E. coli TB were formed. Xylanase assay and SDS?PAGE analysis indicated that the xylanase gene was expressed. Four kinds of media were used for examination of E. coli DB1 culture in shaken flask. The results indicated that xylanase had a higher specific activity in LB and M9 media than in the other media. Maximal cell density was achieved in TB medium, while the specific activity of xylanase was low. Xylanase produced by E. coli DB1 exihibited high activity at 90°C. The preliminary optimized fermentation medium of E. coli DB1 consisted of (g/L): D-glucose 50, NH4Cl 3, MgSO4 0.5, CaCl2 0.6, Na2HPO4?7H2O 12.8, KH2PO4 3.0, NaCl 0.5. There is little difference between the maximum activity of the extremely thermostable xylanase induced with IPTG and that not induced. The thermostability of the xylanase produced by recombined E. coli DB1 can help to simplify its down-stream process for product recovery and extraction.