Kamoda S, Saburi Y
Department of Forest Products, Faculty of Agriculture, University of Tokyo, Japan.
Biosci Biotechnol Biochem. 1993 Jun;57(6):926-30. doi: 10.1271/bbb.57.926.
From the genomic library of Pseudomonas paucimobilis TMY1009 constructed with the cosmid pHC79, an 8-kb BamHI-KpnI fragment encoding lignostilbene-alpha,beta-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-beta-D-thio-galactopyranoside (IPTG). The 1.9-kb SalI fragment containing the LSD gene on pKSN2510 was subcloned into pUC119 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.
从用黏粒pHC79构建的少动假单胞菌TMY1009基因组文库中，将一个编码木质芪α,β -双加氧酶（LSD）的8 kb BamHI - KpnI片段克隆到pUC19中（命名为pKSN2510）。携带pKSN2510的大肠杆菌JM109仅在异丙基 -β - D -硫代半乳糖苷（IPTG）诱导lac启动子时才产生少量LSD。将pKSN2510上包含LSD基因的1.9 kb SalI片段以相反方向亚克隆到pUC119中（命名为pKHN2560和pKHN2590）。pKHN2590上的LSD基因利用lac启动子在大肠杆菌MV1184中表达。用pKHN2590转化的大肠杆菌MV1184产生的LSD（克隆的LSD）经纯化后发现与LSD - I相同，LSD - I是少动假单胞菌TMY1009产生的主要同工酶。对克隆的1.9 kb核苷酸进行测序，发现一个由486个密码子组成的开放阅读框。
