D-海因酶和水解酶在中华根瘤菌中的增强表达

Enhanced Expression of D-Hydantoinase and N-Carbamoyl-D-Amino Acid Amidohydrolase in Sinorhizobium

以质粒pBBR1MCS-5为载体,将D-Case和D-Hase基因以多顺反子形式置于其lac启动子控制下,转入中华根瘤菌,经30℃发酵培养24 h,在不添加诱导剂的情况下,总酶活比野生菌提高了2倍.为减少葡萄糖效应,将lac启动子替换成lacUV5,对核糖体结合位点进行优化,工程菌的酶活比野生菌提高了4倍,达0.5 U/mL.

A recombinant plasmid pBBR1MCS-5-1ac-CH was constructed based on pBBR1MCS-5 vector by combining the D-Hase and D-Case genes with a polycistronic structure under the control of lac promoter. After it was successfully transformed into the Sinorhizobium （Sino 416） and fermented for 24 h at 30 ℃, its total enzyme activity was increased about 2 times over the wild strains without induction. In order to reduce the glucose effect in Sino 416, the lac promoter was replaced by lacUV5, and the ribosome bind site optimized as well. The results show that the total enzyme activity could reach 0.5 U/mL by using of the recombinant plasmid, which is almost 4 times of that of the wild strains.