南极耐冷Pseudomonas sp.7323低温脂肪酶的性质及基因克隆

Characterization and Gene Cloning of Cold-active Lipase from Antarctic Psychrotrophic Pseudomonas sp.7323

南极微生物是筛选低温酶的良好来源,但尚未得到充分的研究与开发.低温脂肪酶具有广阔的应用前景,其基因结构特征也具有重要的研究意义.本文对南极微生物开展了低温脂肪酶产生菌的筛选、基因克隆及特征分析.采用功能筛选的方法,从南极普里兹湾深海沉积物中获得一株产低温脂肪酶的菌株7323,其最适温度和最高生长温度分别为20℃和30℃,属于耐冷菌.16S rDNA序列分析表明,该菌属于假单胞菌属(Pseudomonas).通过设计引物扩增出的脂肪酶基因全长为1 854 bp,该基因编码一个由617氨基酸、分子量预计为64 466的蛋白质.氨基酸序列分析表明,该酶与Pseudomonassp.UB48的脂肪酶有89%的相似性,在催化区和C末端信号肽中存在高度保守的序列.纯化后的酶学性质研究表明,该脂肪酶的最适温度为35℃,最适pH值为9.0,为碱性低温酶.

Microorganisms in Antarctic were good resource for screening the cold active enzymes, and were still inadequately studied and exploited. The cold active lipase （CAL） had good potential in the industry, and its gene characteristic was worth of study. Therefore, the screening of CAL-producing bacteria from Antarctic sample, the CAL gene clone, and the characterization of CAL were reported in this study. By using medium containing lipase substrates, a bacterium 7323 with high cold active lipase activity was isolated from the deep sea sediment of Prydz Bay, Antarctic. The optimal and highest temperatures for its growth were 20 ℃ and 30℃, respectively, indicating that it was a psychrotrophic bacterium. It was affiliated with the genus Pseudomonas by the analysis of 16S rDNA sequences. The Pseudomonas sp. 7323 lipase gene with 1 854 bp was cloned by PCR. It encoded a protein of 617 amino acids, exhibiting 89 % of identity with the lipase amino acid sequences of Pseudomonas sp. UB48. Two conservative motifs were found in the catalytic site and C- terminal signal peptide domain. The optimal temperature and pH for the activity of purified lipase were 35 ℃ and pH 9.0, respectively, indicating that it belonged to alkaline cold active lipase.