Lipases are important enzymes for various biotechnological applications. By using functional expression screening, lipZ03, a novel lipase gene, was isolated from a soil-derived metagenomic library. The gene was supposed to encode a protein of 617 amino acids with a C-terminal targeting signal region and four potential N-linked glycosylation sites. The protein sequence shared a conserved GXSXG motif (X represents any amino acid residue) with other microbial lipases. Gene lipZ03 was expressed in Pichia pastoris and the molecular weight was estimated to be approximately 65 kDa by electrophoresis. The optimum reaction temperature and pH value for LipZ03 was 50°C and 9.0, respectively. The enzyme was highly stable in the temperature range of 40–60°C and under alkaline conditions (pH 8–10). Lipolytic activity was significantly enhanced by Ca2+ and Mg2+ ions, but dramatically inhibited by Cu2+, Ni2+ and Hg2+ ions and EDTA. The purified enzyme preferentially hydrolyzed relatively long-chain triacylglycerols and was a true lipase rather than an esterase. Using a multi-stepwise methanol supply, the purified LipZ03 achieved a conversion yield of biodiesel production up to 74% after 36 h. Some interesting characteristics described here showed that the recombinant lipase may have potential to be a useful enzyme in industrial applications.

1.
Bunterngsook B, Kanokratana P, Thongaram T, Tanapongpipat S, Uengwetwanit T, Rachdawong S, Vichitsoonthonkul T, Eurwilaichitr L: Identification and characterization of lipolytic enzymes from a peat-swamp forest soil metagenome. Biosci Biotechnol Biochem 2010;74:1848–1854.
2.
Cereghino GP, Cereghino JL, Ilgen C, Cregg JM: Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 2002;13:329–332.
3.
Cieslinski H, Bialkowska A, Tkaczuk K, Dlugolecka A, Kur J, Turkiewicz M: Identification and molecular modeling of a novel lipase from an antarctic soil metagenomic library. Pol J Microbiol 2009;58:199–204.
4.
Cote A, Shareck F: Expression and characterization of a novel heterologous moderately thermostable lipase derived from metagenomics in Streptomyces lividans. J Ind Microbiol Biotechnol 2010;37:883–891.
5.
Couto GH, Glogauer A, Faoro H, Chubatsu LS, Souza EM, Pedrosa FO: Isolation of a novel lipase from a metagenomic library derived from mangrove sediment from the south Brazilian coast. Genet Mol Res 2010;9:514–523.
6.
Cregg JM, Cereghino JL, Shi J, Higgins DR: Recombinant protein expression in Pichia pastoris. Mol Biotechnol 2000;16:23–52.
[PubMed]
7.
Duong F, Soscia C, Lazdunski A, Murgier M: The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system. Mol Microbiol 1994;11:1117–1126.
8.
Glogauer A, Martini VP, Faoro H, Couto GH, Muller-Santos M, Monteiro RA, Mitchell DA, de Souza EM, Pedrosa FO, Krieger N: Identification and characterization of a new true lipase isolated through metagenomic approach. Microb Cell Fact 2011;10:54.
9.
Han JY, Kim HK: Transesterification using the cross-linked enzyme aggregate of Photobacterium lipolyticum lipase M37. J Microbiol Biotechnol 2011;21:1159–1165.
10.
Henne A, Schmitz RA, Bomeke M, Gottschalk G, Daniel R: Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl Environ Microbiol 2000;66:3113–3116.
[PubMed]
11.
Hide WA, Chan L, Li WH: Structure and evolution of the lipase superfamily. J Lipid Res 1992;33:167–178.
12.
Jaeger KE, Eggert T: Lipases for biotechnology. Curr Opin Biotechnol 2002;13:390–397.
[PubMed]
13.
Jeon JH, Kim JT, Kim YJ, Kim HK, Lee HS, Kang SG, Kim SJ, Lee JH: Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome. Appl Microbiol Biotechnol 2009;81:865–874.
14.
Jin H, Liu G, Dai K, Wang H, Li Z, Shi Z: Improvement of porcine interferon-alpha production by recombinant Pichia pastoris via induction at low methanol concentration and low temperature. Appl Biochem Biotechnol 2011;165:559–571.
15.
Kim HK, Park SY, Lee JK, Oh TK: Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1. Biosci Biotechnol Biochem 1998;62:66–71.
16.
Kojima Y, Kobayashi M, Shimizu S: A novel lipase from Pseudomonas fluorescens HU380: gene cloning, overproduction, renaturation-activation, two-step purification, and characterization. J Biosci Bioeng 2003;96:242–249.
17.
Kojima Y, Shimizu S: Purification and characterization of the lipase from Pseudomonas fluorescens HU380. J Biosci Bioeng 2003;96:219–226.
18.
Kouker G, Jaeger KE: Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 1987;53:211–213.
19.
Krsek M, Wellington EM: Comparison of different methods for the isolation and purification of total community DNA from soil. J Microbiol Methods 1999;39:1–16.
20.
Lan DM, Yang N, Wang WK, Shen YF, Yang B, Wang YH: A novel cold-active lipase from Candida albicans: cloning, expression and characterization of the recombinant enzyme. Int J Mol Sci 2011;12:3950–3965.
21.
Lee MH, Lee CH, Oh TK, Song JK, Yoon JH: Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases. Appl Environ Microbiol 2006;72:7406–7409.
[PubMed]
22.
Li G, Wang K, Liu YH: Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the metagenome. Microb Cell Fact 2008;7:38.
23.
Liao H-K, Long J, Li J, Yang J-J, Feng Y-Q: Distribution characteristics of soil carbon and nitrogen under different vegetation in micro-habitats of karst dry-hot valley region of south western China. Soils 2012;44:421–428.
24.
Liaw RB, Cheng MP, Wu MC, Lee CY: Use of metagenomic approaches to isolate lipolytic genes from activated sludge. Bioresour Technol 2010;101:8323–8329.
25.
Lorenz P, Liebeton K, Niehaus F, Eck J: Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space. Curr Opin Biotechnol 2002;13:572–577.
26.
Luo Y, Zheng Y, Jiang Z, Ma Y, Wei D: A novel psychrophilic lipase from Pseudomonas fluorescens with unique property in chiral resolution and biodiesel production via transesterification. Appl Microbiol Biotechnol 2006;73:349–355.
27.
Meilleur C, Hupe JF, Juteau P, Shareck F: Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library. J Ind Microbiol Biotechnol 2009;36:853–861.
28.
Minning S, Serrano A, Ferrer P, Sola C, Schmid RD, Valero F: Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol 2001;86:59–70.
[PubMed]
29.
Natalello A, Ami D, Brocca S, Lotti M, Doglia SM: Secondary structure, conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fourier-transform infrared spectroscopy. Biochem J 2005;385:511–517.
30.
Ogawa J, Shimizu S: Microbial enzymes: new industrial applications from traditional screening methods. Trends Biotechnol 1999;17:13–21.
31.
Rogalska E, Douchet I, Verger R: Microbial lipases: structures, function and industrial applications. Biochem Soc Trans 1997;25:161–164.
32.
Roh C, Villatte F: Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organism. J Appl Microbiol 2008;105:116–123.
33.
San Clemente CL, Vadehra DV: Instrumental assay of microbial lipase at constant pH. Appl Microbiol 1967;15:110–113.
34.
Shi B, Zeng L, Song H, Shi Q, Wu S: Cloning and expression of Aspergillus tamarii FS132 lipase gene in Pichia pastoris. Int J Mol Sci 2010;11:2373–2382.
35.
Stratton J, Chiruvolu V, Meagher M: High cell-density fermentation. Methods Mol Biol 1998;103:107–120.
36.
Tang SJ, Shaw JF, Sun KH, Sun GH, Chang TY, Lin CK, Lo YC, Lee GC: Recombinant expression and characterization of the Candida rugosalip4 lipase in Pichia pastoris: comparison of glycosylation, activity, and stability. Arch Biochem Biophys 2001;387:93–98.
37.
Tripathi MK, Roy U, Jinwal UK, Jain SK, Roy PK: Cloning, sequencing and structural features of a novel Streptococcus lipase. Enzyme Microb Technol 2004;34:437–445.
38.
Yang J, Zhang B, Yan Y: Cloning and expression of Pseudomonas fluorescens 26-2 lipase gene in Pichia pastoris and characterizing for transesterification. Appl Biochem Biotechnol 2009;159:355–365.
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