High level expression of thermostable lipase from Geobacillus sp. strain T1
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...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Japan Society for Bioscience, Biotechnology, and Agrochemistry
2004
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| Online Access: | http://psasir.upm.edu.my/id/eprint/5635/1/High%20Level%20Expression%20of%20Thermostable%20Lipase%20from%20Geobacillus%20sp.pdf http://psasir.upm.edu.my/id/eprint/5635/ https://www.jstage.jst.go.jp/article/bbb/68/1/68_1_96/_article |
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| Institution: | Universiti Putra Malaysia |
| Language: | English |
| Summary: | 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°C and pH 9, respectively. It was stable up to 65°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. |
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