Leow Thean Chor, Rahman Raja Noor Zaliha Raja Abdul, Basri Mahiran, Salleh Abu Bakar
Enzyme and Microbial Technology Research Group, Faculty of Science and Environmental Studies, Universiti Putra Malaysia, Serdang, Malaysia.
Biosci Biotechnol Biochem. 2004 Jan;68(1):96-103. doi: 10.1271/bbb.68.96.
A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg(-1) which corresponds to 2927.15 Ug(-1) of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65 degrees C and pH 9, respectively. It was stable up to 65 degrees C at pH 7 and active over a wide pH range (pH 6-11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.
嗜热栖热放线菌T1菌株的一种热稳定胞外脂肪酶在原核系统中被克隆。序列分析显示一个长度为1251 bp的开放阅读框，其编码一个由416个氨基酸残基组成的多肽。该多肽由一个信号肽（28个氨基酸）和一个388个氨基酸的成熟蛋白组成。保守五肽Gly-X-Ser-X-Gly的第一个残基由丙氨酸取代了甘氨酸。分别在araBAD、T7、T7 lac和tac启动子的控制下，利用pBAD、pRSET、pET和pGEX成功实现了基因表达。其中，pGEX经优化后比活性为30.19 Umg(-1)，相当于每克湿细胞2927.15 Ug(-1)。重组脂肪酶的最适温度和pH分别为65℃和pH 9。在pH 7时，它在高达65℃的温度下稳定，并且在较宽的pH范围（pH 6-11）内具有活性。本研究提出了一种快速克隆和过表达方法，旨在提高酶产量以成功应用于工业生产。
