Takizawa N, Murooka Y
Appl Environ Microbiol. 1985 Feb;49(2):294-8. doi: 10.1128/aem.49.2.294-298.1985.
The pullulanase gene (pul) of Klebsiella aerogenes was cloned into a pBR322 vector in Escherichia coli. Deletion analysis of the recombinant plasmid showed that the pul coding sequence, probably with the regulator gene, was located entirely within a 4.2-kilobase segment derived from the chromosomal DNA of K. aerogenes. E. coli cells carrying the recombinant plasmids produced about three- to sevenfold more pullulanase than did the wild-type strain of K. aerogenes W70. When the cloned cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Transfer of the plasmid containing the pul gene into K. aerogenes W70 resulted in about a 20- to 40-fold increase in total production of pullulanase, and the intracellular enzyme level was about 100- to 150-fold higher than that of the parent strain W70. The high level of pullulanase activity in K. aerogenes cells carrying the recombinant plasmid was maintained for at least 2 weeks.
产气克雷伯菌的支链淀粉酶基因（pul）被克隆到大肠杆菌的pBR322载体中。对重组质粒的缺失分析表明，pul编码序列可能与调节基因一起，完全位于源自产气克雷伯菌染色体DNA的一个4.2千碱基片段内。携带重组质粒的大肠杆菌细胞产生的支链淀粉酶比产气克雷伯菌W70野生型菌株多大约三到七倍。当携带克隆基因的大肠杆菌细胞用支链淀粉或麦芽糖培养时，大部分支链淀粉酶在细胞内产生，而产气克雷伯菌则在细胞外产生支链淀粉酶。将含有pul基因的质粒转入产气克雷伯菌W70后，支链淀粉酶的总产量增加了约20到40倍，细胞内酶水平比亲本菌株W70高约100到150倍。携带重组质粒的产气克雷伯菌细胞中高水平的支链淀粉酶活性至少维持了2周。
