From the genomic library of Pseudomonas paucimohilis TMYl009 constructed with the cosmid pHC79, an 8-kb BamHI-KpnI fragment encoding Iignostilbene-α,β-dioxygenase (LSD) was cloned into pUC19 (designated pKSN2510). E. coli JM109 having pKSN2510 produced a small amount of LSD only when the lac promoter was induced by isopropyl-β-D-thio-galactopyranoside (IPTG). The 1.9-kb SalI fragment containing the LSD gene on pKSN2510 was subcloned into pUCl19 in opposite directions (designated pKHN2560 and pKHN2590). The LSD gene on pKHN2590 was expressed in E. coli MV1184 using the lac promoter.LSD produced by E. coli MV1184 transformed with pKHN2590 (cloned LSD) was purified and found to be identical with LSD-I, which was the major isozyme produced by P. paucimobilis TMY1009. Cloned 1.9-kb nucleotide was sequenced and one open reading frame composed to 486 codons was found.
从用cosmid pHC79构建的假单胞菌paucimohilis TMYl009
基因组文库中,将一个8kb的BamHI-KpnI片段克隆到pUC19(命名为p
KSN2510)中,该片段编码Iignostilbene-α,β-dioxygenase(L
SD)。只有当异丙基-β-D-
硫代-半
乳糖苷(
IPTG)诱导lac启动子时,携带p
KSN2510的E. coli JM109才会产生少量的L
SD。将p
KSN2510上包含L
SD基因的1.9kb SalI片段以相反方向亚克隆到pUCl19中(命名为pKHN2560和pKHN2590)。使用lac启动子在E. coli MV1184中表达pKHN2590上的L
SD基因。对转化了pKHN2590(克隆的L
SD)的E. coli MV1184产生的L
SD进行纯化,发现其与L
SD-I相同,后者是假单胞菌paucimohilis TMY1009产生的主要同工酶。对克隆的1.9kb核苷酸进行测序,发现一个由486个