一种产自出芽短梗霉的胞外脂肪酶的分离纯化和酶学性质研究（英文）
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摘要
目的:从出芽短梗霉所产的脂肪酶中筛选具有独特酶学性质的脂肪酶。创新点:发现了一种新的产自出芽短梗霉的脂肪酶,并对其酶学性质进行了研究。方法:通过超滤和DEAE-Sepharose Fast Flow阴离子层析柱方法对脂肪酶进行纯化,随后分别用对硝基酚邻酸盐(pN PP)法对纯化得到的脂肪酶进行了酶学性质研究,并用酸碱中和法检测了脂肪酶对可食用油脂的水解。结论:对分离纯化得到的脂肪酶的酶学性质研究表明,该酶的分子量为39.5 kDa,具有一个亚基,为胞外酶。最佳催化温度为40°C,最佳催化pH为7。该酶对一些有机溶剂、表面活性剂和离子具有优良的抗性。此外,它可以水解常见的食用油。这些良好的特性使该脂肪酶有可能被应用于洗涤剂生产、生物柴油合成和食品制造等一些工业领域。
An extracellular lipase from Aureobasidium pullulans was obtained and purified with a specific activity of 17.7 U/mg of protein using ultrafiltration and a DEAE-Sepharose Fast Flow column. Characterization of the lipase indicated that it is a novel finding from the species A. pullulans. The molecular weight of the lipase was 39.5 kDa, determined by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis(SDS-PAGE). The enzyme exhibited its optimum activity at 40 °C and pH of 7. It also showed a remarkable stability in some organic solutions(30%, v/v) including n-propanol, isopropanol, dimethyl sulfoxide(DMSO), and hexane. The catalytic activity of the lipase was enhanced by Ca~(2+) and was slightly inhibited by Mn~(2+) and Zn~(2+) at a concentration of 10 mmol/L. The lipase was activated by the anionic surfactant SDS and the non-ionic surfactants Tween 20, Tween 80, and Triton X-100, but it was drastically inhibited by the cationic surfactant cetyl trimethyl ammonium bromide(CTAB). Furthermore, the lipase was able to hydrolyze a wide variety of edible oils, such as peanut oil, corn oil, sunflower seed oil, sesame oil, and olive oil. Our study indicated that the lipase we obtained is a potential biocatalyst for industrial use.
An extracellular lipase from Aureobasidium pullulans was obtained and purified with a specific activity of 17.7 U/mg of protein using ultrafiltration and a DEAE-Sepharose Fast Flow column. Characterization of the lipase indicated that it is a novel finding from the species A. pullulans. The molecular weight of the lipase was 39.5 kDa, determined by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis(SDS-PAGE). The enzyme exhibited its optimum activity at 40 °C and pH of 7. It also showed a remarkable stability in some organic solutions(30%, v/v) including n-propanol, isopropanol, dimethyl sulfoxide(DMSO), and hexane. The catalytic activity of the lipase was enhanced by Ca~(2+) and was slightly inhibited by Mn~(2+) and Zn~(2+) at a concentration of 10 mmol/L. The lipase was activated by the anionic surfactant SDS and the non-ionic surfactants Tween 20, Tween 80, and Triton X-100, but it was drastically inhibited by the cationic surfactant cetyl trimethyl ammonium bromide(CTAB). Furthermore, the lipase was able to hydrolyze a wide variety of edible oils, such as peanut oil, corn oil, sunflower seed oil, sesame oil, and olive oil. Our study indicated that the lipase we obtained is a potential biocatalyst for industrial use.
引文
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Aref HL,Mosbah H,Fekih A,et al.,2014.Purification and biochemical characterization of lipase from Tunisian Euphorbia peplus latex.J Am Oil Chem Soc,91(6):943-951.https://doi.org/10.1007/s11746-014-2444-z
Bradford MM,Williams WL,1976.New,rapid,sensitive method for protein determination.Fed Proc,35(3):274.
Chi ZM,Wang F,Chi Z,et al.,2009.Bioproducts from Aureobasidium pullulans,a biotechnologically important yeast.Appl Microbiol Biotechnol,82(5):793-804.https://doi.org/10.1007/s00253-009-1882-2
de Hoog GS,1993.Evolution of black yeasts:possible adaptation to the human host.Antonie van Leeuwenhoek,63(2):105-109.https://doi.org/10.1007/BF00872386
Ebrahimpour A,Rahman RNZRA,Basri M,et al.,2011.High level expression and characterization of a novel thermostable,organic solvent tolerant,1,3-regioselective lipase from Geobacillus sp.strain ARM.Bioresour Technol,102(13):6972-6981.https://doi.org/10.1016/j.biortech.2011.03.083
Fauzi AHM,Amin NAS,2012.An overview of ionic liquids as solvents in biodiesel synthesis.Renewable Sustainable Energy Rev,16(8):5770-5786.https://doi.org/10.1016/j.rser.2012.06.022
Federici F,1982.Extracellular enzymatic activities in Aureobasidium pullulans.Mycologia,74(5):738-743.https://doi.org/10.1080/00275514.1982.12021580
Ghasemi Y,Rasoul-Amini S,Kazemi A,et al.,2011.Isolation and characterization of some moderately halophilic bacteria with lipase activity.Microbiology,80(4):483-487.https://doi.org/10.1134/S0026261711040060
Hamdy HS,Abo-Tahon MA,2012.Extracellular lipase of Aspergillus terreus var.africanus(CBS 130.55):production purification and characterisation.Ann Microbiol,62(4):1723-1736.https://doi.org/10.1007/s13213-012-0429-4
Hasan F,Shah AA,Hameed A,2006.Industrial applications of microbial lipases.Enzyme Microb Technol,39(2):235-251.https://doi.org/10.1016/j.enzmictec.2005.10.016
Hasan F,Shah AA,Hameed A,2009.Methods for detection and characterization of lipases:a comprehensive review.Biotechnol Adv,27(6):782-798.https://doi.org/10.1016/j.biotechadv.2009.06.001
Hiol A,Jonzo MD,Rugani N,et al.,2000.Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit.Enzyme Microb Technol,26(5-6):421-430.https://doi.org/10.1016/S0141-0229(99)00173-8
Ji QC,Xiao SJ,He BF,et al.,2010.Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production.J Mol Catal B Enzym,66(3-4):264-269.https://doi.org/10.1016/j.molcatb.2010.06.001
Joseph B,Ramteke PW,2013.Extracellular solvent stable cold-active lipase from psychrotrophic Bacillus sphaericus MTCC 7526:partial purification and characterization.Ann Microbiol,63(1):363-370.https://doi.org/10.1007/s13213-012-0483-y
Kanjanavas P,Khuchareontaworn S,Khawsak P,et al.,2010.Purification and characterization of organic solvent and detergent tolerant lipase from thermotolerant Bacillus sp.RN2.Int J Mol Sci,11(10):3783-3792.https://doi.org/10.3390/ijms11103783
Kudanga T,Mwenje E,Mandivenga F,et al.,2007.Esterases and putative lipases from tropical isolates of Aureobasidium pullulans.J Basic Microbiol,47(2):138-147.https://doi.org/10.1002/jobm.200610207
Laemmli UK,1970.Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature,227(5259):680-685.https://doi.org/10.1038/227680a0
Lailaja VP,Chandrasekaran M,2013.Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS11.World J Microbiol Biotechnol,29(8):1349-1360.https://doi.org/10.1007/s11274-013-1298-0
Leathers TD,Rich JO,Anderson AM,et al.,2013.Lipase production by diverse phylogenetic clades of Aureobasidium pullulans.Biotechnol Lett,35(10):1701-1706.https://doi.org/10.1007/s10529-013-1268-5
Li M,Yang LR,Xu G,et al.,2013.Screening,purification and characterization of a novel cold-active and organic solvent-tolerant lipase from Stenotrophomonas maltophilia CGMCC 4254.Bioresour Technol,148:114-120.https://doi.org/10.1016/j.biortech.2013.08.101
Li X,Qian P,Wu SG,et al.,2014.Characterization of an organic solvent-tolerant lipase from Idiomarina sp.W33and its application for biodiesel production using Jatropha oil.Extremophiles,18(1):171-178.https://doi.org/10.1007/s00792-013-0610-0
Li Y,Cai HY,Zhao MJ,et al.,2015.Screening and identification of high-yield thermostable lipase producing microorganisms.Biotechnol Bull,31(1):144-150(in Chinese).https://doi.org/10.13560/j.cnki.biotech.bull.1985.2015.01.022
Liu ZQ,Li XY,Chi ZM,et al.,2008a.Cloning,characterization and expression of the extracellular lipase gene from Aureobasidium pullulans HN2-3 isolated from sea saltern.Antonie Van Leeuwenhoek,94(2):245-255.https://doi.org/10.1007/s10482-008-9237-z
Liu ZQ,Chi ZM,Wang L,et al.,2008b.Production,purification and characterization of an extracellular lipase from Aureobasidium pullulans HN2.3 with potential application for the hydrolysis of edible oils.Biochem Eng J,40(3):445-451.https://doi.org/10.1016/j.bej.2008.01.014
Martinelle M,Holmquist M,Hult K,1995.On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase.Biochim Biophys Acta,1258(3):272-276.https://doi.org/10.1016/0005-2760(95)00131-U
Mogensen JE,Sehgal P,Otzen DE,2005.Activation,inhibition,and destabilization of Thermomyces lanuginosus lipase by detergents.Biochemistry,44(5):1719-1730.https://doi.org/10.1021/bi0479757
Peng Q,Wang X,Shang M,et al.,2014.Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production.Microb Cell Fact,13:1.https://doi.org/10.1186/1475-2859-13-1
Rahman RNZRA,Baharum SN,Basri M,et al.,2005.Highyield purification of an organic solvent-tolerant lipase from Pseudomonas sp.strain S5.Anal Biochem,341(2):267-274.https://doi.org/10.1016/j.ab.2005.03.006
Ramakrishnan V,Goveas LC,Suralikerimath N,et al.,2016.Extraction and purification of lipase from Enterococcus faecium MTCC5695 by PEG/phosphate aqueous-two phase system(ATPS)and its biochemical characterization.Biocatal Agric Biotechnol,6:19-27.https://doi.org/10.1016/j.bcab.2016.02.005
Ramani K,Kennedy LJ,Ramakrishnan M,et al.,2010.Purification,characterization and application of acidic lipase from Pseudomonas gessardii using beef tallow as a substrate for fats and oil hydrolysis.Process Biochem,45(10):1683-1691.https://doi.org/10.1016/j.procbio.2010.06.023
Ramírez-Zavala B,Mercado-Flores Y,Hernández-Rodríguez C,et al.,2004.Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus.FEMSMicrobiol Lett,235(2):369-375.https://doi.org/10.1111/j.1574-6968.2004.tb09612.x
Rúa ML,Díaz-Mauri?ob T,Fernández VM,et al.,1993.Purification and characterization of two distinct lipases from Candida cylindracea.Biochim Biophys Acta,1156(2)181-189.https://doi.org/10.1016/0304-4165(93)90134-T
Salihu A,Alam MZ,2015.Solvent tolerant lipases:a review.Process Biochem,50(1):86-96.https://doi.org/10.1016/j.procbio.2014.10.019
Saxena RK,Sheoran A,Giri B,et al.,2003.Purification strategies for microbial lipases.J Microbiol Methods,52(1):1-18.https://doi.org/10.1016/S0167-7012(02)00161-6
Sharma R,Chisti Y,Banerjee UC,2001.Production,purification,characterization,and applications of lipases.Biotechnol Adv,19(8):627-662.https://doi.org/10.1016/S0734-9750(01)00086-6
Shi H,Meng Y,Yang M,et al.,2014.Purification and characterization of a hydrolysis-resistant lipase from Aspergillus terreus.Biotechnol Appl Biochem,61(2):165-174.https://doi.org/10.1002/bab.1142
Singh RS,Saini GK,2008.Pullulan-hyperproducing color variant strain of Aureobasidium pullulans FB-1 newly isolated from phylloplane of Ficus sp.Bioresour Technol,99(9):3896-3899.https://doi.org/10.1016/j.biortech.2007.08.003
Sulong MR,Rahnian RNZRA,Salleh AB,et al.,2006.A novel organic solvent tolerant lipase from Bacillus sphaericus205y:extracellular expression of a novel OST-lipase gene.Protein Express Purif,49(2):190-195.https://doi.org/10.1016/j.pep.2006.04.015
Taher H,Al-Zuhair S,2017.The use of alternative solvents in enzymatic biodiesel production:a review.Biofuels Bioprod Biorefin,11(1):168-194.https://doi.org/10.1002/bbb.1727
Tang LL,Xia YL,Wu XL,et al.,2017.Screening and characterization of a novel thermostable lipase with detergentadditive potential from the metagenomic library of a mangrove soil.Gene,625:64-71.https://doi.org/10.1016/j.gene.2017.04.046
Tomke PD,Rathod VK,2015.Ultrasound assisted lipase catalyzed synthesis of cinnamyl acetate via transesterification reaction in a solvent free medium.Ultrason Sonochem,27:241-246.https://doi.org/10.1016/j.ultsonch.2015.04.022
Ugur A,Sarac N,Boran R,et al.,2014.New lipase for biodiesel production:partial purification and characterization of LipSB 25-4.ISRN Biochem,2014:289749.https://doi.org/10.1155/2014/289749
Wang L,Chi ZM,Wang XH,et al.,2007.Diversity of lipaseproducing yeasts from marine environments and oil hydrolysis by their crude enzymes.Ann Microbiol,57(4):495-501.https://doi.org/10.1007/BF03175345
Winkler UK,Stuckmann M,1979.Glycogen,hyaluronate,and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens.J Bacteriol,138(3):663-670.
Yang L,Dordick JS,Garde S,2004.Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity.Biophys J,87(2):812-821.https://doi.org/10.1529/biophysj.104.041269
Yuan DJ,Lan DM,Xin RP,et al.,2016.Screening and characterization of a thermostable lipase from marine Streptomyces sp.strain W007.Biotechnol Appl Biochem,63(1):41-50.https://doi.org/10.1002/bab.1338
Yuan L,Sadiq FA,Liu TJ,et al.,2018.Spoilage potential of psychrotrophic bacteria isolated from raw milk and the thermo-stability of their enzymes.J Zhejiang Univ-Sci B(Biomed&Biotechnol),19(8):630-642.https://doi.org/10.1631/jzus.B1700352
Aouf C,Durand E,Lecomte J,et al.,2014.The use of lipases as biocatalysts for the epoxidation of fatty acids and phenolic compounds.Green Chem,16(4):1740-1754.https://doi.org/10.1039/C3GC42143K
Aref HL,Mosbah H,Fekih A,et al.,2014.Purification and biochemical characterization of lipase from Tunisian Euphorbia peplus latex.J Am Oil Chem Soc,91(6):943-951.https://doi.org/10.1007/s11746-014-2444-z
Bradford MM,Williams WL,1976.New,rapid,sensitive method for protein determination.Fed Proc,35(3):274.
Chi ZM,Wang F,Chi Z,et al.,2009.Bioproducts from Aureobasidium pullulans,a biotechnologically important yeast.Appl Microbiol Biotechnol,82(5):793-804.https://doi.org/10.1007/s00253-009-1882-2
de Hoog GS,1993.Evolution of black yeasts:possible adaptation to the human host.Antonie van Leeuwenhoek,63(2):105-109.https://doi.org/10.1007/BF00872386
Ebrahimpour A,Rahman RNZRA,Basri M,et al.,2011.High level expression and characterization of a novel thermostable,organic solvent tolerant,1,3-regioselective lipase from Geobacillus sp.strain ARM.Bioresour Technol,102(13):6972-6981.https://doi.org/10.1016/j.biortech.2011.03.083
Fauzi AHM,Amin NAS,2012.An overview of ionic liquids as solvents in biodiesel synthesis.Renewable Sustainable Energy Rev,16(8):5770-5786.https://doi.org/10.1016/j.rser.2012.06.022
Federici F,1982.Extracellular enzymatic activities in Aureobasidium pullulans.Mycologia,74(5):738-743.https://doi.org/10.1080/00275514.1982.12021580
Ghasemi Y,Rasoul-Amini S,Kazemi A,et al.,2011.Isolation and characterization of some moderately halophilic bacteria with lipase activity.Microbiology,80(4):483-487.https://doi.org/10.1134/S0026261711040060
Hamdy HS,Abo-Tahon MA,2012.Extracellular lipase of Aspergillus terreus var.africanus(CBS 130.55):production purification and characterisation.Ann Microbiol,62(4):1723-1736.https://doi.org/10.1007/s13213-012-0429-4
Hasan F,Shah AA,Hameed A,2006.Industrial applications of microbial lipases.Enzyme Microb Technol,39(2):235-251.https://doi.org/10.1016/j.enzmictec.2005.10.016
Hasan F,Shah AA,Hameed A,2009.Methods for detection and characterization of lipases:a comprehensive review.Biotechnol Adv,27(6):782-798.https://doi.org/10.1016/j.biotechadv.2009.06.001
Hiol A,Jonzo MD,Rugani N,et al.,2000.Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit.Enzyme Microb Technol,26(5-6):421-430.https://doi.org/10.1016/S0141-0229(99)00173-8
Ji QC,Xiao SJ,He BF,et al.,2010.Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production.J Mol Catal B Enzym,66(3-4):264-269.https://doi.org/10.1016/j.molcatb.2010.06.001
Joseph B,Ramteke PW,2013.Extracellular solvent stable cold-active lipase from psychrotrophic Bacillus sphaericus MTCC 7526:partial purification and characterization.Ann Microbiol,63(1):363-370.https://doi.org/10.1007/s13213-012-0483-y
Kanjanavas P,Khuchareontaworn S,Khawsak P,et al.,2010.Purification and characterization of organic solvent and detergent tolerant lipase from thermotolerant Bacillus sp.RN2.Int J Mol Sci,11(10):3783-3792.https://doi.org/10.3390/ijms11103783
Kudanga T,Mwenje E,Mandivenga F,et al.,2007.Esterases and putative lipases from tropical isolates of Aureobasidium pullulans.J Basic Microbiol,47(2):138-147.https://doi.org/10.1002/jobm.200610207
Laemmli UK,1970.Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature,227(5259):680-685.https://doi.org/10.1038/227680a0
Lailaja VP,Chandrasekaran M,2013.Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS11.World J Microbiol Biotechnol,29(8):1349-1360.https://doi.org/10.1007/s11274-013-1298-0
Leathers TD,Rich JO,Anderson AM,et al.,2013.Lipase production by diverse phylogenetic clades of Aureobasidium pullulans.Biotechnol Lett,35(10):1701-1706.https://doi.org/10.1007/s10529-013-1268-5
Li M,Yang LR,Xu G,et al.,2013.Screening,purification and characterization of a novel cold-active and organic solvent-tolerant lipase from Stenotrophomonas maltophilia CGMCC 4254.Bioresour Technol,148:114-120.https://doi.org/10.1016/j.biortech.2013.08.101
Li X,Qian P,Wu SG,et al.,2014.Characterization of an organic solvent-tolerant lipase from Idiomarina sp.W33and its application for biodiesel production using Jatropha oil.Extremophiles,18(1):171-178.https://doi.org/10.1007/s00792-013-0610-0
Li Y,Cai HY,Zhao MJ,et al.,2015.Screening and identification of high-yield thermostable lipase producing microorganisms.Biotechnol Bull,31(1):144-150(in Chinese).https://doi.org/10.13560/j.cnki.biotech.bull.1985.2015.01.022
Liu ZQ,Li XY,Chi ZM,et al.,2008a.Cloning,characterization and expression of the extracellular lipase gene from Aureobasidium pullulans HN2-3 isolated from sea saltern.Antonie Van Leeuwenhoek,94(2):245-255.https://doi.org/10.1007/s10482-008-9237-z
Liu ZQ,Chi ZM,Wang L,et al.,2008b.Production,purification and characterization of an extracellular lipase from Aureobasidium pullulans HN2.3 with potential application for the hydrolysis of edible oils.Biochem Eng J,40(3):445-451.https://doi.org/10.1016/j.bej.2008.01.014
Martinelle M,Holmquist M,Hult K,1995.On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase.Biochim Biophys Acta,1258(3):272-276.https://doi.org/10.1016/0005-2760(95)00131-U
Mogensen JE,Sehgal P,Otzen DE,2005.Activation,inhibition,and destabilization of Thermomyces lanuginosus lipase by detergents.Biochemistry,44(5):1719-1730.https://doi.org/10.1021/bi0479757
Peng Q,Wang X,Shang M,et al.,2014.Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production.Microb Cell Fact,13:1.https://doi.org/10.1186/1475-2859-13-1
Rahman RNZRA,Baharum SN,Basri M,et al.,2005.Highyield purification of an organic solvent-tolerant lipase from Pseudomonas sp.strain S5.Anal Biochem,341(2):267-274.https://doi.org/10.1016/j.ab.2005.03.006
Ramakrishnan V,Goveas LC,Suralikerimath N,et al.,2016.Extraction and purification of lipase from Enterococcus faecium MTCC5695 by PEG/phosphate aqueous-two phase system(ATPS)and its biochemical characterization.Biocatal Agric Biotechnol,6:19-27.https://doi.org/10.1016/j.bcab.2016.02.005
Ramani K,Kennedy LJ,Ramakrishnan M,et al.,2010.Purification,characterization and application of acidic lipase from Pseudomonas gessardii using beef tallow as a substrate for fats and oil hydrolysis.Process Biochem,45(10):1683-1691.https://doi.org/10.1016/j.procbio.2010.06.023
Ramírez-Zavala B,Mercado-Flores Y,Hernández-Rodríguez C,et al.,2004.Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus.FEMSMicrobiol Lett,235(2):369-375.https://doi.org/10.1111/j.1574-6968.2004.tb09612.x
Rúa ML,Díaz-Mauri?ob T,Fernández VM,et al.,1993.Purification and characterization of two distinct lipases from Candida cylindracea.Biochim Biophys Acta,1156(2)181-189.https://doi.org/10.1016/0304-4165(93)90134-T
Salihu A,Alam MZ,2015.Solvent tolerant lipases:a review.Process Biochem,50(1):86-96.https://doi.org/10.1016/j.procbio.2014.10.019
Saxena RK,Sheoran A,Giri B,et al.,2003.Purification strategies for microbial lipases.J Microbiol Methods,52(1):1-18.https://doi.org/10.1016/S0167-7012(02)00161-6
Sharma R,Chisti Y,Banerjee UC,2001.Production,purification,characterization,and applications of lipases.Biotechnol Adv,19(8):627-662.https://doi.org/10.1016/S0734-9750(01)00086-6
Shi H,Meng Y,Yang M,et al.,2014.Purification and characterization of a hydrolysis-resistant lipase from Aspergillus terreus.Biotechnol Appl Biochem,61(2):165-174.https://doi.org/10.1002/bab.1142
Singh RS,Saini GK,2008.Pullulan-hyperproducing color variant strain of Aureobasidium pullulans FB-1 newly isolated from phylloplane of Ficus sp.Bioresour Technol,99(9):3896-3899.https://doi.org/10.1016/j.biortech.2007.08.003
Sulong MR,Rahnian RNZRA,Salleh AB,et al.,2006.A novel organic solvent tolerant lipase from Bacillus sphaericus205y:extracellular expression of a novel OST-lipase gene.Protein Express Purif,49(2):190-195.https://doi.org/10.1016/j.pep.2006.04.015
Taher H,Al-Zuhair S,2017.The use of alternative solvents in enzymatic biodiesel production:a review.Biofuels Bioprod Biorefin,11(1):168-194.https://doi.org/10.1002/bbb.1727
Tang LL,Xia YL,Wu XL,et al.,2017.Screening and characterization of a novel thermostable lipase with detergentadditive potential from the metagenomic library of a mangrove soil.Gene,625:64-71.https://doi.org/10.1016/j.gene.2017.04.046
Tomke PD,Rathod VK,2015.Ultrasound assisted lipase catalyzed synthesis of cinnamyl acetate via transesterification reaction in a solvent free medium.Ultrason Sonochem,27:241-246.https://doi.org/10.1016/j.ultsonch.2015.04.022
Ugur A,Sarac N,Boran R,et al.,2014.New lipase for biodiesel production:partial purification and characterization of LipSB 25-4.ISRN Biochem,2014:289749.https://doi.org/10.1155/2014/289749
Wang L,Chi ZM,Wang XH,et al.,2007.Diversity of lipaseproducing yeasts from marine environments and oil hydrolysis by their crude enzymes.Ann Microbiol,57(4):495-501.https://doi.org/10.1007/BF03175345
Winkler UK,Stuckmann M,1979.Glycogen,hyaluronate,and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens.J Bacteriol,138(3):663-670.
Yang L,Dordick JS,Garde S,2004.Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity.Biophys J,87(2):812-821.https://doi.org/10.1529/biophysj.104.041269
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