bglS基因的亚克隆及功能分析

Subcloning and Functional Analysis of the bglS Gene

带有枯草杆菌β-1,3-1,4葡聚糖酶基因(bglS)7.1kb的EcoRI片段,经亚克隆,将1.5kb的EcoR I-Pst I酶切片段插入pUC19的polylinker上,转化E.coli JM101后合成出β-1,3-1,4葡聚糖酶,它能专一性地降解大麦β-1,3-1,4葡聚糖和地衣多糖,酶的大部分(>50%)留在胞内,其中25%分泌到胞外。根据酶特性分析,大肠杆菌中合成的β-1,3-1,4葡聚糖酶完全与出发菌株枯草杆菌相同。

This article describes a 7.1kb EcoRI DNA fragment carrying a Baci-llus subtilis β-1,3-1,4-glucanase gene (bg1S). By subcloning, a 1.5kbEcoRI-PstI DNA fragment has been inserted into the polylinker cloningsite of the plasmid pUC19 and transferred to Escherichia coli JM101.The fragment directs the synthesis in E. coli of a β-1,3-1,4-glucanasewhich specifically degrades barley glucan and lichenin. The largest pro-portion (50%)of the total enzyme activity is intracellular, 25% of theβ-1,3-1,4-glucanase activity is present in the extracellular fractions. Bycomparison of the substrate specificity and optimal pH, the enzmespurified from E.coli and from Bacillus subtilis 1.88 are proved to beidentical.