【摘要】 对已克隆的环状芽孢杆菌（Ｂａｃｉｌｕｓｃｉｒｃｕｌａｎｓ）Ｃ－２的几丁酶基因（Ｃｈｉ１）片段所作的亚克隆分析表明，该几丁酶基因位于１．７ｋｂＰｓｔⅠ－ＳｔｙⅠ片段上。Ｃｈｉ１基因在大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）ＪＭ１０７，ＤＨ５α，ＸＬ１－ｂｌｕｅ，ＴＧ－１等菌株中均能表达，但表达水平不同，其中ＪＭ１０７的表达活性最高，其胞外几丁酶活性与供体菌Ｃ－２菌株的胞外酶活性几乎相当。经ＳＤＳ－聚丙烯酰胺凝胶电泳分析，重组质粒ｐＣＨＩ１所产生的胞外几丁酶分子量为６６ｋＤ，与环状芽孢杆菌Ｃ－２的胞外蛋白质中相应的几丁酶蛋白分子量相同。Ｃｈｉ１基因在大肠杆菌ＪＭ１０７中表达后的细胞定位测定表明，几丁酶不仅存在于胞周间质和胞内，而且大量存在于培养物上清液中。在高产酶时期，胞外、胞周间质和胞内的酶活性分布分别为３５．８％，３２．１％，３２．９％更多还原

【Abstract】 Subcloning analysis of a cloned DNA fragment from Bacillus circulans containing the chitinase gene Chi1 showed that the chitinase gene lies on an 1.7 kb PstIStyI fragment. The chitinase gene could be expressed in Escherichia coli strains JM107, DH5α, XL1blue and TG1 with various efficiencies. The expression level of chitinase gene was highest in JM107, which was almost the same as that in B. circulans C2. The molecular weight of extracellular chitinase was 66 kD by SDSPAGE analysis. Cell location determination of the expressed chitinase showed that the enzyme existed not only in cell periplasm and cytoplasm, but also in extracellular broth. When the expression of the enzyme was optimal, the distribution of enzyme activity in extracellular broth, periplasm and cytoplasm was 35.8%, 32.1% and 32.9%, respectively.更多还原