Enzyme and Microbial Technology Research Center, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia.
Protein J. 2013 Apr;32(4):317-25. doi: 10.1007/s10930-013-9488-z.
A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S(207), D(255) and H(313), based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.
从南极土壤中筛选到一株具有低温适应性的产胞外脂肪酶的细菌（命名为 AMS8 菌株），并采用分子生物学方法对其进行了进一步分析。16S rDNA 分析表明，AMS8 菌株与假单胞菌属（Pseudomonas）的细菌较为相似。从 AMS8 菌株中成功克隆、测序并在大肠杆菌中过表达了一种脂肪酶基因，命名为 lipAMS8。序列分析表明，lipAMS8 基因全长 1431bp，编码一个由 476 个氨基酸组成的多肽。该酶无 N 端信号肽，C 端含有一个甘氨酸和天冬氨酸丰富的九肽序列，这是重复序列毒素细菌脂肪酶的特征。此外，根据同源建模和多序列比对，确定 lipAMS8 的底物结合位点为 S(207)、D(255)和 H(313)。粗酶在 20°C 时表现出最大活性，在 10°C 时仍保留近 50%的活性。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定 lipAMS8 的分子量约为 50kDa。在大肠杆菌生长的最佳密度为 0.5 时，使用重组质粒 pET32b/BL21(DE3)在 15°C 下诱导 8 小时，异丙基-β-D-硫代半乳糖苷（IPTG）的最佳诱导浓度为 0.1mM 时，可获得最佳表达水平。
