通过克隆Bacillus clarkii 7364来源编码γ-环糊精葡萄糖基转移酶(γ-cyclodextrins glucanotransferase,γ-CGTase)的基因,构建并表达基因重组菌株E.coli BL21/pET28a(+)-γ-CGTase,对γ-CGTase酶学性质和产物特异性进行了研究。结果表明,所克隆表达的γ-CGTase分子质量为78 kDa,水解活力与环化活力在最适温度、最适pH、金属离子影响等方面均存在一定差异。以可溶性淀粉为底物,经HPLC测定催化产物中几乎无α-环糊精(α-cyclodextrin,α-CD),γ-CD/β-CD可达7.70,γ-CD转化率为15.83%,添加体积分数为10%的乙醇浓度γ-CD产量可提高89.91%,γ-CD/β-CD提高73.77%,转化率为33.44%。催化豌豆淀粉γ-CD/β-CD达12.92,转化率为19.1%,分别比可溶性淀粉提高了63.13%和22.90%。该研究为进一步提高γ-CGTase酶法制备γ-CD产量和专一性应用提供了重要的理论依据。
Cloning a gene encoded γ-cyclodextrins glucanotransferase (γ-CGTase) of Bacillus clarkii 7364, the recombinant strain Escherichia coli BL21/pET28a(+)-γ-CGTase was constructed and expressed,then the enzymatic properties and product specificity of γ-CGTase were studied. The molecular mass of γ-CGTase was 78 kDa, and there were some differences between hydrolysis activity and cyclization activity in the optimum temperature, the optimum pH and the influence of metal ions. Using soluble starch as substrate, there was hardly α-cyclodextrin (α-CD) in the catalytic product which was determined by HPLC, and the ratio of γ-cyclodextrin(γ-CD)/β-cyclodextrin(β-CD) was 7.70. the conversion rate of γ-CD was 15.83%. Through adding 10% ethanol, the yield of γ-CD and the ratio of γ-CD/β-CD was increased by 89.91% and 73.77%, respectively. And the conversion rate from starch into γ-CD was 33.44%. However, using pea starch in the production process, the ratio of γ-CD/ β-CD reached 12.92, and the conversion rate was 19.1%, which was 63.13% and 22.90% higher than that of soluble starch, respectively. This study provides an important theoretical basis for further improving the yield and specificity of enzymatic preparation of γ-CD by γ-CGTase.
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