Abstract
By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15 °C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9 kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40 °C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10–90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (eep 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.
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Abbreviations
- pNPA:
-
p-Nitrophenyl acetate
- pNPB:
-
p-Nitrophenyl butyrate
- pNPH:
-
p-Nitrophenyl hexanoate
- pNPC:
-
p-Nitrophenyl caprylate
- pNPL:
-
p-Nitrophenyl laurate
- pNPP:
-
p-Nitrophenyl palmitate
- DMSO:
-
Dimethyl sulfoxide
- GC:
-
Gas chromatography
- CD:
-
Circular dichroism
- NCBI:
-
National Center for Biotechnology Information
- ees :
-
Enantiomeric excess value of the substrate
- eep :
-
Enantiomeric excess value of the product
- C :
-
Conversion ratio
- E :
-
Enantioselectivity
References
Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177–183. https://doi.org/10.1042/0264-6021:3430177
Arpigny JL, Feller G, Gerday C (1993) Cloning, sequence and structural features of a lipase from the antarctic facultative psychrophile Psychorobacter-immobilis B10. Biochem Biophys Acta 1171:331–333. https://doi.org/10.1016/0167-4781(93)90078-r
Berglund P, Hutt K (2000) Biocatalytic synthesis of enantiopure compounds using lipases. In: Patel RN (ed) Stereoselective biocatalysis. Marcel Dekker, New York, pp 633–657
Brady L et al (1990) A serine protease triad forms the catalytic center of a triacylglycerol lipase. Nature 343:767–770. https://doi.org/10.1038/343767a0
Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR (2002) Low-temperature extremophiles and their applications. Curr Opin Biotechnol 13:253–261. https://doi.org/10.1016/s0958-1669(02)00317-8
Chakravorty D, Parameswaran S, Dubey VK, Patra S (2012) Unraveling the rationale behind organic solvent stability of lipases. Appl Biochem Biotechnol 167:439–461. https://doi.org/10.1007/s12010-012-9669-9
Chen CS, Fujimoto Y, Girdaukas G, Sih CJ (1982) Quantitative-analyses of biochemical kinetic resolutions of enantiomers. J Am Chem Soc 104:7294–7299. https://doi.org/10.1021/ja00389a064
Chen R, Guo L, Dang H (2011) Gene cloning, expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18. World J Microbiol Biotechnol 27:431–441. https://doi.org/10.1007/s11274-010-0475-7
de los Rios AP, van Rantwijk F, Sheldon RA (2012) Effective resolution of 1-phenyl ethanol by Candida antarctica lipase B catalysed acylation with vinyl acetate in protic ionic liquids (PILs). Green Chem 14:1584–1588. https://doi.org/10.1039/c2gc35196j
Doukyu N, Ogino H (2010) Organic solvent-tolerant enzymes. Biochem Eng J 48:270–282. https://doi.org/10.1016/j.bej.2009.09.009
Drouin EE et al (2013) A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease. Arthritis Rheum 65:186–196. https://doi.org/10.1002/art.37732
Fan Y, Duncan NW, de Greck M, Northoff G (2011a) Is there a core neural network in empathy? An fMRI based quantitative meta-analysis. Neurosci Biobehav Rev 35:903–911. https://doi.org/10.1016/j.neubiorev.2010.10.009
Fan Y, Zhang S, Kruer N, Keyhani NO (2011b) High-throughput insertion mutagenesis and functional screening in the entomopathogenic fungus Beauveria bassiana. J Invertebr Pathol 106:274–279. https://doi.org/10.1016/j.jip.2010.11.003
Feller G, Thiry M, Arpigny JL, Gerday C (1991) Cloning and expression in Escherichia coli of 3 lipase-encoding genes from the psychrotrophic antarctic strain Moraxella TA144. Gene 102:111–115. https://doi.org/10.1016/0378-1119(91)90548-p
Feller G, Zekhnini Z, LamotteBrasseur J, Gerday C (1997) Enzymes from cold-adapted microorganisms—the class C beta-lactamase from the Antarctic psychrophile Psychrobacter immobilis A5. Eur J Biochem 244:186–191. https://doi.org/10.1111/j.1432-1033.1997.00186.x
Feller G, Le Bussy O, Gerday C (1998) Expression of psychrophilic genes in mesophilic hosts: assessment of the folding state of a recombinant alpha-amylase. Appl Environ Microbiol 64:1163–1165 (WOS:000072292700057)
Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1:2876–2890. https://doi.org/10.1038/nprot.2006.202
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30:1229–1235. https://doi.org/10.1093/molbev/mst012
Invernizzi G, Papaleo E, Grandori R, De Gioia L, Lotti M (2009) Relevance of metal ions for lipase stability: structural rearrangements induced in the Burkholderia glumae lipase by calcium depletion. J Struct Biol 168:562–570. https://doi.org/10.1016/j.jsb.2009.07.021
Kasana RC, Yadav SK (2007) Isolation of a psychrotrophic Exiguobacterium sp SKPB5 (MTCC 7803) and characterization of its alkaline protease. Curr Microbiol 54:224–229. https://doi.org/10.1007/s00284-006-0402-1
Kimura T, Horikoshi K (1990) Characterization of pullulan-hydrolyzing enzyme from an alkalopsychrotrophic micrococcus sp. Appl Microbiol Biotechnol 34:52–56 (WOS:A1990EG04800011)
Klibanov AM (2001) Improving enzymes by using them in organic solvents. Nature 409:241–246. https://doi.org/10.1038/35051719
Korman TP, Bowie JU (2012) Crystal structure of proteus mirabilis lipase, a novel lipase from the proteus/psychrophilic subfamily of lipase family I.1. PLoS One 7(12):e52890. https://doi.org/10.1371/journal.pone.0052890
Kumar R, Singh R, Kaur J (2013) Characterization and molecular modelling of an engineered organic solvent tolerant, thermostable lipase with enhanced enzyme activity. J Mol Catal B: Enzym 97:243–251. https://doi.org/10.1016/j.molcatb.2013.09.001
Lee HK, Ahn MJ, Kwak SH, Song WH, Jeong BC (2003) Purification and characterization of cold active lipase from psychrotrophic Aeromonas sp. LPB 4. Indian J Mar Sci 42:510–515 (WOS:000182068000004)
Lima VMG, Krieger N, Mitchell DA, Baratti JC, de Filippis I, Fontana JD (2004a) Evaluation of the potential for use in biocatalysis of a lipase from a wild strain of Bacillus megaterium. J Mol Catal B Enzym 31:53–61. https://doi.org/10.1016/j.molcatb.2004.07.005
Lima VMG, Krieger N, Mitchell DA, Fontana JD (2004b) Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents. Biochem Eng J 18:65–71. https://doi.org/10.1016/s1369-703x(03)00165-7
Lu M, Wang S, Fang Y, Li H, Liu S, Liu H (2010) Cloning, expression, purification, and characterization of cold-adapted alpha-amylase from Pseudoalteromonas arctica GS230. Protein J 29:591–597. https://doi.org/10.1007/s10930-010-9290-0
Ma JS, Zhang ZM, Wang BJ, Kong XJ, Wang YG, Cao SG, Feng Y (2006) Overexpression and characterization of a lipase from Bacillus subtilia. Protein Expr Purif 45:22–29. https://doi.org/10.1016/j.pep.2005.06.004
Mancheno JM, Pernas MA, Martinez MJ, Ochoa B, Rua ML, Hermoso JA (2003) Structural insights into the lipase/esterase behavior in the Candida rugosa lipases family: crystal structure of the lipase 2 isoenzyme at 1.97 angstrom resolution. J Mol Biol 332:1059–1069. https://doi.org/10.1016/j.jmb.2003.08.005
Mayordomo I, Randez-Gil F, Prieto JA (2000) Isolation, purification, and characterization of a cold-active lipase from Aspergillus nidulans. J Agric Food Chem 48:105–109. https://doi.org/10.1021/jf9903354
Ogino H, Ishikawa H (2001) Enzymes which are stable in the presence of organic solvents. J Biosci Bioeng 91:109–116. https://doi.org/10.1263/jbb.91.109
Rabbani G, Ahmad E, Zaidi N, Fatima S, Khan RH (2012) pH-Induced molten globule state of Rhizopus niveus lipase is more resistant against thermal and chemical denaturation than its native state. Cell Biochem Biophys 62:487–499. https://doi.org/10.1007/s12013-011-9335-9
Secundo F, Spadaro S, Carrea G, Overbeeke PLA (1999) Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents. Biotechnol Bioeng 62:554–561. https://doi.org/10.1002/(sici)1097-0290(19990305)62:5<554::aid-bit7>3.0.co;2-2
Sharma R, Chisti Y, Banerjee UC (2001) Production, purification, characterization, and applications of lipases. Biotechnol Adv 19:627–662. https://doi.org/10.1016/s0734-9750(01)00086-6
Sinchaikul S, Sookkheo B, Phutrakul S, Pan FM, Chen ST (2001) Optimization of a thermostable lipase from Bacillus stearothermophilus P1: overexpression, purification, and characterization. Protein Expr Purif 22:388–398. https://doi.org/10.1006/prep.2001.1456
Vorderwulbecke T, Kieslich K, Erdmann H (1992) Comparison of lipases by different assays. Enzyme Microb Technol 14:631–639. https://doi.org/10.1016/0141-0229(92)90038-p
Wang Q et al (2016) Characterization of a salt-activated protease with temperature-dependent secretion in Stenotrophomonas maltophilia FF11 isolated from frozen Antarctic krill. J Ind Microbiol Biotechnol 43:829–840. https://doi.org/10.1007/s10295-016-1749-3
Winkler UK, Stuckmann M (1979) Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by serratia-marcescens. J Bacteriol 138:663–670 (WOS:A1979HA45300002)
Wu G, Zhang X, Wei L, Wu G, Kumar A, Mao T, Liu Z (2015) A cold-adapted, solvent and salt tolerant esterase from marine bacterium Psychrobacter pacificensis. Int J Biol Macromol 81:180–187. https://doi.org/10.1016/j.ijbiomac.2015.07.045
Yu X-W, Wang R, Zhang M, Xu Y, Xiao R (2012) Enhanced thermostability of a Rhizopus chinensis lipase by in vivo recombination in Pichia pastoris. Microb Cell Fact 11:102. https://doi.org/10.1186/1475-2859-11-102
Yumoto I, Hirota K, Sogabe Y, Nodasaka Y, Yokota Y, Hoshino T (2003) Psychrobacter okhotskensis sp. nov., a lipase-producing facultative psychrophile isolated from the coast of the Okhotsk Sea. Int J Syst Evol Microbiol 53:1985–1989. https://doi.org/10.1099/ijs.0.02686-0
Zhang J-w, Zeng R-y (2008) Molecular cloning and expression of a cold-adapted lipase gene from an Antarctic deep sea psychrotrophic bacterium Pseudomonas sp. 7323. Mar Biotechnol 10:612–621. https://doi.org/10.1007/s10126-008-9099-4
Zhang J, Lin S, Zeng R (2007) Cloning, expression, and characterization of a cold-adapted lipase gene from an Antarctic deep-sea psychrotrophic bacterium, Psychrobacter sp. 7195. J Microbio Biotechnol 17:604–610 (WOS:000246096700008)
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This work was supported by Panjin Campus for Food Science and Technology Research Initiative, Dalian University of Technology.
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Zhang, Y., Ji, F., Wang, J. et al. Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124. Extremophiles 22, 287–300 (2018). https://doi.org/10.1007/s00792-018-0997-8
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DOI: https://doi.org/10.1007/s00792-018-0997-8