嗜冷杆菌EastSeaG5-1415脂肪酶基因的克隆和序列测定

从东海深海底泥中筛选到一株产低温碱性脂肪酶的海洋细菌EastSeaG5-1415，经鉴定为嗜冷杆菌Psychrobacter glacincola。将其染色体DNA用Sau3AI部分酶切后，低熔点琼脂糖回收2～10kb的。DNA片段，用Klenow大片段酶半补齐，与用Sal I酶切半补齐的质粒pUC19连接后，转化E．coli．JM109构建基因文库。用平板测活法从基因文库初步筛选到两株产碱性脂肪酶的阳性克隆，采用ELISA反应进一步鉴定。序列测定分析表明，两个重组质粒中都包含长度为951bp的碱性脂肪酶基因的完整开放阅读框架(ORF)和上游基因调控序列。此片段编码有317个氨基酸组成的酶，计算分子量为35000 Dal。通过Southem杂交证实了此片段来自于嗜冷杆菌Psychrobacter glacincola EastSeaG5-1415基因细。

Cloning and Sequencing of Lipase Gene from P. glacincola EastSeaG5-1415

Psychrobacter glacincola EastSeaG5-1415 which stably secreting low-temperature alkaline lipase was obtained. For the preparation of gene libraries,genomic DNA from P.glacincola EastSeaG5-1415 was digested partially with Sau3A I,and the fragment from 2kb to 10kb were recovered.The cohesive ends partial filled in by klenow fragment of DNA polymerase I(Pol I k).DNA of plasmid pUC19 was cleaved with SalI,and subsequently partially filled in with Pol I k.The plasmid and the genomic DNAs were mixed and ligated.Then the recombinant plasmid transformed E.coli.JM109.Two positive clones were obtained from the tributyrin flat and ELISA sieve method.The analysis of sequence detection show that two positive clones both include the Open Read Frame of low-temperature lipase gene. The fragment coded the enzyme of 317 amino acid,the molecular weight of which is 35 000Dal.Through Southern hybridization,the fragment is confirmed to come from P.glacincola EastSeaG5-1415 genomic DNA.