【摘要】 为实现利用生物酶转化法进行D 对羟基苯甘氨酸的工业化生产 ,构建了 3株海因酶基因工程菌 .利用PCR技术从恶臭假单胞菌 (Pseudomonasputida)CPU 980 1染色体DNA中扩增得到长约1.8kb的含编码区和自身启动子的海因酶全基因 .通过将海因酶全基因插入pMD18 T质粒、海因酶基因的编码区与pET 17 b质粒重组、海因酶基因编码区和T7强启动子一起插入pMD18 T质粒分别得到重组质粒pMD dht、pET dht和pMD T7 dht.将上述重组质粒分别转化大肠杆菌 (Escherichiacoli) ,通过地高辛标记菌落原位杂交和海因酶活力测定两种方法 ,筛选出具有海因酶活力的阳性转化子 .结果表明 ,大肠杆菌的RNA聚合酶能够识别和结合来自恶臭假单胞菌海因酶基因的自身启动子 ,该启动子在大肠杆菌中能够工作 .基因工程菌E .coliBL2 1 pMD dht、E .coliBL2 1 pET dht和E .coliBL2 1 pMD T7 dht的海因酶活力分别为 170 0U L、190 0U L和 2 5 0 0U L ,比野生菌P .putidaCPU 980 1的海因酶活力分别提高了 8倍、9倍和 12倍 .薄层扫描结果显示 ,这些工程菌的海因酶表达量分别约占菌体总可溶性蛋白质的 2 0 %、31%和 5 7%.SDS PAGE显示 ,海因酶的单体分子量约为 5 0kD .经工程菌E .coliBL2 1 pMD T7 dht催化 ,底物对羟基苯海因的转化率在 13h内可达到 9更多还原

【Abstract】 For the purpose of the industrial production of D-p-hydroxyphenylglycine (D-p-HPG), D-hydantoinase (dht) gene was amplified from Pseudomonas putida CPU 9801 by PCR technique and inserted into pMD 18-T plasmid and pET 17b plasmid, respectively. The recombinant plasmids were transformed into several strains of E.coli. The positive transformants with D-hydantoinase activity were obtained by the two steps screening, digoxigenin DNA labelling in situ hybridization and D-hydantoinase activity assay. The results showed that the D-hydantoinase activity of the genetic engineering strains E .coli BL21/pMD-dht, E. coli BL21/pET-dht and E. coli BL21/pMD-T7-dht were 1 700 U/L, 1 900 U/L and 2 500 U/L respectively, and increased as high as 8, 9 and 12 times compared with D-hydantoinase activity of wild strain P. putida CPU 9801. The amount of the recombinant D-hydantoinase were about 20, 31 and 57 percent of total bacterial soluble proteins, respectively. The molecular mass of the recombinant D-hydantoinase was about 50 kD on SDS-PAGE. The conversion yield of D,L-p-hydroxyphenylhydantoin (D,L-p-HPH) to N-carbamoyl-D-p-hydroxyphenyl-glycine (CpHPG) catalyzed by the genetic engineering strain E.coli BL21/pMD-T7-dht could reach 96% within 13 hours. These D-hydantoinase genetic engineering strains possess the initial industrial production prospects.更多还原