Bunterngsook Benjarat, Kanokratana Pattanop, Thongaram Taksawan, Tanapongpipat Sutipa, Uengwetwanit Tanaporn, Rachdawong Sansanalak, Vichitsoonthonkul Taweerat, Eurwilaichitr Lily
Division of Biotechnology, School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok, Thailand.
Biosci Biotechnol Biochem. 2010;74(9):1848-54. doi: 10.1271/bbb.100249. Epub 2010 Sep 7.
In this work, a metagenomic library was generated from peat-swamp forest soil obtained from Narathiwat Province, Thailand. From a fosmid library of approximately 15,000 clones, six independent clones were found to possess lipolytic activity at acidic pH. Analysis of pyrosequencing data revealed six ORFs, which exhibited 34-71% protein similarity to known lipases/esterases. A fosmid clone, designated LP8, which demonstrated the highest level of lipolytic activity under acidic conditions and demonstrated extracellular activity, was subsequently subcloned and sequenced. The full-length lipase/esterase gene, estPS2, was identified. Its deduced amino acid was closely related to a lipolytic enzyme of an uncultured bacterium, and contained the highly conserved motif of a hormone-sensitive family IV lipase. The EstPS2 enzyme exhibited highest activity toward p-nitrophenyl butyrate (C⁴) at 37 °C at pH 5, indicating that it was an esterase with activity and secretion characteristics suitable for commercial development.
在这项研究中，从泰国北大年府的泥炭沼泽森林土壤中构建了一个宏基因组文库。从一个约15000个克隆的fosmid文库中，发现有六个独立克隆在酸性pH条件下具有脂解活性。对焦磷酸测序数据的分析揭示了六个开放阅读框（ORF），它们与已知的脂肪酶/酯酶具有34%-71%的蛋白质相似性。一个名为LP8的fosmid克隆在酸性条件下表现出最高水平的脂解活性且具有胞外活性，随后对其进行亚克隆和测序。鉴定出了全长脂肪酶/酯酶基因estPS2。其推导的氨基酸序列与一种未培养细菌的脂解酶密切相关，并包含激素敏感的IV型脂肪酶家族的高度保守基序。EstPS2酶在pH 5、37℃时对丁酸对硝基苯酯（C⁴）表现出最高活性，表明它是一种具有适合商业开发的活性和分泌特性的酯酶。
