Ji Xiuling, Li Shan, Lin Lianbing, Zhang Qi, Wei Yunlin
Technol Health Care. 2015;23 Suppl 1:S109-17. doi: 10.3233/thc-150941.
A pychrotrophic bacterium secreing an extracellular alkaline cold-active lipase was isolated from refrigeratory. Based on morphology and 16S rRNA gene sequence analysis, the isolate which was named as PF16 was identified as a member of Pseudomonas. A novel lip-PF16 gene was obtained from the genomic DNA of strain PF16. Nucleotide sequence analysis showed that open reading frame (ORF) was consisting of 1,431 nucleotides encoding 476 amino acids long protein. Lip-PF16 showed 89.5% and 88% identity with the cold-adapted lipases from Pseudomonas fluorescens and Pseudomonas sp. KB700A, respectively. It was found to be a member of conserved subfamily I.3 bacterial lipase, which contained a lipase consensus sequence GXSXG. The lip-PF16 gene was expressed in Escherichia coli BL21 (DE3) and functional lipase was obtained successfully. The molecular weight was estimated to be 50 KDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The cloned lipase was active over broad range of temperature range with optimum activity at 4^°C, and found to be alkaline preferring with optimum activity at pH 9.0. This recombination lipase show high catalytic activity at low temperature.
从冷藏室中分离出一株分泌胞外碱性冷活性脂肪酶的嗜冷细菌。基于形态学和16S rRNA基因序列分析，将该分离株命名为PF16，鉴定为假单胞菌属成员。从菌株PF16的基因组DNA中获得了一个新的lip - PF16基因。核苷酸序列分析表明，开放阅读框（ORF）由1431个核苷酸组成，编码一个476个氨基酸的蛋白质。Lip - PF16与荧光假单胞菌和假单胞菌属KB700A的冷适应脂肪酶分别具有89.5%和88%的同源性。它被发现是保守的I.3亚家族细菌脂肪酶的成员，包含脂肪酶共有序列GXSXG。lip - PF16基因在大肠杆菌BL21(DE3)中表达，成功获得了具有功能的脂肪酶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS - PAGE）分析，其分子量估计为50 kDa。克隆的脂肪酶在较宽的温度范围内具有活性，最适活性温度为4℃，并且发现其偏好碱性，最适活性pH为9.0。这种重组脂肪酶在低温下表现出高催化活性。
