摘要
用质位ｐＢＲ３２２作载体，大肠杆菌Ｅ．ｃｏｌｉＤＨ５αＦ＇作受体，克隆到耐热芽孢杆菌Ｅ２菌株的羧甲基纤维素酶（ＣＭＣａｓｅ）基因。重组质粒ｐＢＧ３从产生ＣＭＣａｓｅ的转化子中分离得到。克隆到的ＣＭＣａｒｅ基因位于４．０ｋｂＨｉｎｄＩＩＩ片段上。功能状态ＣＭＣａｓｅ基因被亚克隆到２．４ｋｂＤＮＡ片段上。同位素杂交结果显示出克隆到的ＣＭＣａｓｅ基因与供体菌染色体ＤＮＡ的亲合性。重组质粒ｐＢＧ３所表达的ＣＭＣａｓｅ的最适作用ｐＨ为６．５，最适作用温度为５５℃，不作用于任何实验过的天然纤维素。含重组质粒ｐＢＧ３的转化子产生的纤维素酶在细胞中的分布为：胞内占８２．９％，胞外占７．９％，质壁空间（ｐｅｒｉｐｌａｓｍｉｃｓｐａｃｅ）占９．２％。
The CMCase gene from the Bacillus sp. strain E2 was cloned into E,coli DH5αF'using plasmid pBR322 as a vector. Recombinant plasmid pBG3 was isolated from transformants producing CMCase,and the cloned CMCasc gene was found to be in a 4.0kb HindIII fragment. The CMCase gene was subcloned in a functional state on a 2.4kb DNA fragment.The optimum pH and temperature for activity of CMCase encoded by pBG3 were pH 6.5 and 55℃ respectively.The CMCase could not hydrolyze any natural cellulose examined.Eighty-three percent of the CMCase was detected in the inner cellular fraction,9%and 8%were in the periplasmic space and medium respectively.Result of Southern hybridization indicates that the cloned DNA fragment was derived from Bacillus sp.strain E2 chromosomal DNA. Received May 11,1994.This Project Supported by National Natural Science Foundation of China
基金
国家自然科学基金
