Abstract
A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40–60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8–9. Metal ions such as Ca2+, Mn2+, Na+, and K+ enhanced the lipase activity, but Mg2+, Zn2+, and Fe2+ inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
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Abbreviations
- DTT:
-
Dithiothreitol
- EDTA:
-
Ethylenediaminetetraacetic acid
- GST:
-
Glutathione S transferase
- IPTG:
-
Isopropyl β-D-1-thiogalactopyranoside
- PCMB:
-
p-Chloromercuribenzoic acid
- PMSF:
-
Phenylmethanesulfonylfluoride
- PBS:
-
Phosphate buffered saline
- SDS PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
References
Castro-Ochoa LD, Rodrı′guez-Go′mez C, Valerio-Alfaro G, Oliart- Ros RM (2005) Enzyme Microb Technol 37:648–654
Dutta S, Ray L (2009) Appl Biochem Biotechnol 159:142–154
Gutteridge A, Thornton J (2005) J Mol Biol 346:21–28
Jaeger KE, Dijkstra BW, Reetz MT (1999) Annu Rev Microbiol 53:315–351
Jensen RG (1983) Lipids 18:650–657
Kambourova M, Kirilova N, Mandeva R, Derekova A (2003) J Mol Catal B Enzymatic 22:307–313
Kim HK, Park SY, Lee JK, Oh TK (1998) Biosci Biotech Biochem 62(1):66–71
Kim MH, Kim HK, Lee JK, Park SY, Oh TK (2000) Biosci Biotechnol Biochem 64:280–286
Koshland D (1963) Science 142:1533–1541
Kumar S, Kikon K, Upadhyay A, Kanwar SS, Gupta R (2005) Protein Expr Purif 41(1):38–44
Kwon DY, Rhee JS (1986) J Am Oil Chem Soc 63(1):29–32
Lee DW, Koh YS, Kim KJ, Kim BC, Choi HJ, Kim DS (1999) FEMS Microbiol Lett 179:393–400
Leow TC, Rahman RN, Basri M, Salleh AB (2007) Extremophiles 11:527–535
Masomian M, Rahman RN, Salleh AB, Basri M (2010) World J Microb Biot 26:1693–1701
Matsumura H, Yamamoto T, Leow TC, Mori T, Salleh AB, Basri M, Inoue T, Kai Y, Rahman RNZRA (2007) Proteins 70(2):592–598
Nawani N, Kaur J (2000) Mol Cell Biochem 206:91–96
Nawani N, Dosanjh NS, Kaur J (1998) Biotechnol Lett 20(10):997–1000
Peng R, Lin JP, Wei DZ (2010) Appl Biochem Biotechnol 162:733–743
Rua ML, Schmidt-Dannert C, Wahl S, Sprauer A, Schmid RD (1997) J Biotechnol 56:89–102
Sinchaikul S, Sookkheo B, Phutrakul S, Pan FM, Chen ST (2001) Protein Expres Purif 22:388–398
Spafiu F, Mischie A, Ionita P, Beteringhe A, Constantinescu T, Balabanb AT (2009) ARKIVOC pp 174–194
Tayyab M, Rashid N, Akhtar M (2011) J Biosci Bioeng 111(3):272–278
Tsujii K (1998) Surface activity: principles, phenomena, and applications. Academic Press, San Diego
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This research was supported by the Ministry of Science, Technology and Innovation (MOSTI), Malaysia.
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Ali, M.S.M., Yun, C.C., Chor, A.L.T. et al. Purification and Characterisation of an F16L Mutant of a Thermostable Lipase. Protein J 31, 229–237 (2012). https://doi.org/10.1007/s10930-012-9395-8
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DOI: https://doi.org/10.1007/s10930-012-9395-8