粘质沙雷氏菌脂肪酶的应用、固定化及突变体研究
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摘要
粘质沙雷氏菌脂肪酶属于α/β水解酶,可广泛应用于食品、制药以及生物能源等领域。本课题研究了重组粘质沙雷氏菌ECU1010(Serratia marcescens ECU1010)脂肪酶LipA合成生物柴油的生产工艺、活性包涵体的固定化,并利用基因工程方法构建了脂肪酶LipB的突变体,对突变体的有机溶剂耐受性及三维结构进行了研究。
通过单因素试验对生物柴油的酶法合成工艺进行了研究,重点考察了酶用量、醇油摩尔比、反应时间、反应温度等因素对脂肪酸甲酯(FAME)转化率的影响。以FAME为考察指标,通过响应面法(RSM)系统研究了酶用量、醇油摩尔比、反应时间、有机溶剂量等因素对转酯反应进程的影响,结果表明：以大豆油质量为基准,LipA用量10.8%(w/w),反应温度40℃,石油醚用量2.73mL/g,醇油摩尔比1.4,甲醇流加次数3次,每次流加间隔时间10.6h, FAME的转化率为84.2%,比优化前提高了10.7%。
采用Plackett-Burman设计和Box-Behnken设计对影响S. marcescens LipA活性包涵体固定化的诸多因素进行考察和评价,并对筛选出的重要因素进行统计学优化,经RSM优化后的固定化条件为：戊二醛浓度0.13%(w/v),酶浓度16.5mg/mL,戊二醛与酶用量比5.5：1。在此条件下,固定化酶的酶活回收率比优化前提高了45%。
通过实验研究了固定化后脂肪酶活性包涵体的部分酶学性质,对固定化酶的pH稳定性、热稳定性、金属离子和有机溶剂性耐受性、操作稳定性以及贮存稳定性进行了重点考察。通过与游离酶性质的对比,发现固定化酶在pH5.0～10.0范围内稳定性较游离酶高,且60℃温育1h后仍能保持48%以上的酶活。此外,固定化酶对亲水性有机溶剂、金属离子以及表面活性剂的耐受性增强,其操作稳定性和贮存稳定性也大有提高。
利用定点突变技术,以重叠延伸PCR的方法将脂肪酶LipB上Gly33、Alal87以及Thr270分别突变成脂肪酶LipA对应位置上的Asp33、Val187和Ala270,并对LipB三个突变体进行不同浓度的有机溶剂处理,以考察三个突变体对有机溶剂的耐受情况,再通过同源建模和结构分析软件对三个突变体的结构进行模拟和分析,发现第33位Gly对脂肪酶LipB的有机溶剂耐受性起了重要作用。
The extracellular lipases from Serratia marcescens ECU1010are a kind of α/β hydrolase fold protein; they are widely used in the food, medicine and bio-energy industries. In this paper, we investigated and optimized the production conditions of biodiesel using S. marcescens LipA as the catalyst. Through the single-factor experiment and response surface analysis, the optimal production conditions of biodiesel were obtained and the results were as follows:Based on the weight of soybean oil, enzyme dosage10.8%(w/w), reaction temperature40℃, petroleum ether2.73mL/g, substrate molar ratio1:4, adding methanol for3times, time interval10.6h. Under the optimal conditions, the conversion rate of FAME reached84.2%, which increased10.7%than prior to optimization.
The immobilization conditions of lipase present in active inclusion bodies were studied by response surface methodology. Plackett-Burman design and Box-Behnken design were used to optimize the immobilization conditions, respectively. Through the experiment, we obtained the optimal immobilized conditions. The details were as follows:glutaraldehyde0.13%(w/v), enzyme16.5mg/mL, ratio of glutaraldehyde and enzyme5.5:1. Under this condition, the enzymatic activity was increased45%than prior to optimization.
Comparative studies on the enzymatic properties of the free and immobilized enzymes were performed. The main properties studied in this work included the stability of pH and temperature, as well as in the presence of various metal ions and organic solvents. In addition, the operation stability and the storage stability were investigated. The immobilized lipase showed high stability in a broad pH range of5.0-10.0and remained over48%of its activity at60℃for1h. The tolerance to metal ions and water-miscible organic solvents was raised. The operation stability and the storage stability were also significantly improved.
The mutants (G33D, A187V and T270A) of S. marcescens LipB were obtained through the site-directed mutagenesis technology and the tolerance of LipB mutants to various organic solvents were investigated. Based on homology modeling and structural analysis, the33rd amino acid (Gly) of LipB played an important role in the tolerance against organic solvents.
通过单因素试验对生物柴油的酶法合成工艺进行了研究,重点考察了酶用量、醇油摩尔比、反应时间、反应温度等因素对脂肪酸甲酯(FAME)转化率的影响。以FAME为考察指标,通过响应面法(RSM)系统研究了酶用量、醇油摩尔比、反应时间、有机溶剂量等因素对转酯反应进程的影响,结果表明：以大豆油质量为基准,LipA用量10.8%(w/w),反应温度40℃,石油醚用量2.73mL/g,醇油摩尔比1.4,甲醇流加次数3次,每次流加间隔时间10.6h, FAME的转化率为84.2%,比优化前提高了10.7%。
采用Plackett-Burman设计和Box-Behnken设计对影响S. marcescens LipA活性包涵体固定化的诸多因素进行考察和评价,并对筛选出的重要因素进行统计学优化,经RSM优化后的固定化条件为：戊二醛浓度0.13%(w/v),酶浓度16.5mg/mL,戊二醛与酶用量比5.5：1。在此条件下,固定化酶的酶活回收率比优化前提高了45%。
通过实验研究了固定化后脂肪酶活性包涵体的部分酶学性质,对固定化酶的pH稳定性、热稳定性、金属离子和有机溶剂性耐受性、操作稳定性以及贮存稳定性进行了重点考察。通过与游离酶性质的对比,发现固定化酶在pH5.0～10.0范围内稳定性较游离酶高,且60℃温育1h后仍能保持48%以上的酶活。此外,固定化酶对亲水性有机溶剂、金属离子以及表面活性剂的耐受性增强,其操作稳定性和贮存稳定性也大有提高。
利用定点突变技术,以重叠延伸PCR的方法将脂肪酶LipB上Gly33、Alal87以及Thr270分别突变成脂肪酶LipA对应位置上的Asp33、Val187和Ala270,并对LipB三个突变体进行不同浓度的有机溶剂处理,以考察三个突变体对有机溶剂的耐受情况,再通过同源建模和结构分析软件对三个突变体的结构进行模拟和分析,发现第33位Gly对脂肪酶LipB的有机溶剂耐受性起了重要作用。
The extracellular lipases from Serratia marcescens ECU1010are a kind of α/β hydrolase fold protein; they are widely used in the food, medicine and bio-energy industries. In this paper, we investigated and optimized the production conditions of biodiesel using S. marcescens LipA as the catalyst. Through the single-factor experiment and response surface analysis, the optimal production conditions of biodiesel were obtained and the results were as follows:Based on the weight of soybean oil, enzyme dosage10.8%(w/w), reaction temperature40℃, petroleum ether2.73mL/g, substrate molar ratio1:4, adding methanol for3times, time interval10.6h. Under the optimal conditions, the conversion rate of FAME reached84.2%, which increased10.7%than prior to optimization.
The immobilization conditions of lipase present in active inclusion bodies were studied by response surface methodology. Plackett-Burman design and Box-Behnken design were used to optimize the immobilization conditions, respectively. Through the experiment, we obtained the optimal immobilized conditions. The details were as follows:glutaraldehyde0.13%(w/v), enzyme16.5mg/mL, ratio of glutaraldehyde and enzyme5.5:1. Under this condition, the enzymatic activity was increased45%than prior to optimization.
Comparative studies on the enzymatic properties of the free and immobilized enzymes were performed. The main properties studied in this work included the stability of pH and temperature, as well as in the presence of various metal ions and organic solvents. In addition, the operation stability and the storage stability were investigated. The immobilized lipase showed high stability in a broad pH range of5.0-10.0and remained over48%of its activity at60℃for1h. The tolerance to metal ions and water-miscible organic solvents was raised. The operation stability and the storage stability were also significantly improved.
The mutants (G33D, A187V and T270A) of S. marcescens LipB were obtained through the site-directed mutagenesis technology and the tolerance of LipB mutants to various organic solvents were investigated. Based on homology modeling and structural analysis, the33rd amino acid (Gly) of LipB played an important role in the tolerance against organic solvents.
引文
[1]盛梅,曹国民.蚕丝固定化脂肪酶催化猪油水解[J].中国油脂,1999,24(4)：36-38.
[2]王军.脂肪酶固定化及催化酯化反应[J].日用化学工业,1995,(3)：4-7.
[3]Alexander E I, Manfred P S. Methods for the immobilization of lipases and their use for ester synthesis[J]. J Mol Catal B:Enzymatic,1997,3(6):303-309.
[4]Bruins M E, Janssen A E M, Boom R M. Thermozymes and their applications[J]. Appl Biochem Biotechnol,2001,90(2):155-186.
[5]Wu H, Zong M H, Lou W Y. Transesterification of waste oil to biodiesel in solvent free system catalyzed by immobilized lipase[J]. Chin J Catal,2004, 25(11):903-908.
[6]Haki G D, Rakshit S K. Developments in industrially important thermostable enzymes:a review[J]. Bioresour Technol,2003,89(1):17-34.
[7]Kirk O, Borchert T V, Fuglsang C C. Industrial enzyme applications[J]. Curr Opin Biotechnol,2002,13(4):345-351.
[8]Bajpai P. Application of enzymes in the pulp and paper industry[J]. Biotechnol Prog,1999,15(2):147-157.
[9]Wong H, Schotz M C. The lipase gene family[J]. J Lipid Res,2002,43: 993-999.
[10]Sinchaikul S, Sookkheo B, Phutrakul S, et al. Optimization of a thermostable lipase from Bacillus stearothermophilus P1:overexpression, purification, and characterization[J]. Protein Expr Purif,2001,22:388-398.
[11]龙章德.粘质沙雷氏菌脂肪酶的制备及其在手性拆分中的应用[D].华东理工大学博士论文,2007：22.
[12]Fickers P, Fudalej F, Le-Dall M T, et al. Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica[J]. Fungal Genet Biol,2005,42:264-274.
[13]Uppenberg J, Hansen M T, Patkar S, Jones T A. The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica[J]. Structure,1994,2:293-308.
[14]Nardini M, Dijkstra B W. Alpha/bata hydrolase fold enzymes:the family keeps growing[J]. Curr Orin Struct Biol,1999,9:732-737.
[15]Reto M, Thomas D, Vera S, Karl E J, Ulrich B. A calcium-gated lid and a large-roll sandwich are revealed by the crystal structure of extracellular lipase from Serratia marcescens[J].J Biol Chem,2007,282:31477-31483.
[16]Holmquist M. Alpha/Beta-hydrolase fold enzymes:structures, functions and mechanisms[J]. Curr Protein Pept Sci,2000,1:209-235.
[17]Wei Y, Swenson L, Castro C, et al. Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases:Streptomyces exfoliatus lipase at 1.9 A resolution[J]. Structure,1998,6:511-519.
[18]Van P G, Eggert T, Jaeger K E, et al. The crystal structure of Bacillus subtilis lipase:a minimal alpha/beta hydrolase fold enzyme[J]. J Mol Biol,2001,309: 215-226.
[19]Arpigny J L, Jaeger K E. Bacterial lipolytic enzymes:classification and properties[J]. Biochem J.1999,343:177-183.
[20]Mosbah H, Sayari A, Mejdoub H, et al. Biochemical and molecular characterization of Staphylococcus xylosus lipase[J]. Biochim Biophys Acta, 2005,1723:282-291.
[21]Dimitris G H, John B M, Paul C, et al. Production and partial charaterisation of extracellular lipase from Aspergillus niger[J]. Biotech Lett,1996,18:547-552.
[22]Sayari A, Agrebi N, Jaoua S, et al. Biochemical and molecular characterization of Staphylococcus simulans lipase[J]. Biochimie,2001,83:863-871.
[23]Saleh A M, Heidi M Abdel-Mageed, Saadia A T, Mohamed A. Characterization of Mucor racemosus lipase with potential application for the treatment of cellulite[J]. Process Biochem,2011,46(3):642-648.
[24]Chen C S, Sih C J A. General aspects and optimization of enantioselective biocatalysis in organic solvents:The use of lipases[J]. Chem Int Ed Engl,1989, 28(6):695-707.
[25]祖元刚.生物柴油[M].北京：科学出版社,2007,54-61.
[26]汪永,欧仕益,温勇等.酶法催化合成生物柴油的研究进展[J].中国油脂,2006,31：65-68.
[27]Fangrui M. Biodiesel production:a review[J]. Bioresource Technol,1999,70: 1-15.
[28]忻耀年等.生物柴油的生产和应用[J].中国油脂,2001,26(5)：72-77.
[29]谭天伟等.生物柴油的生产和应用[J].现代化工,2002,22(2)：4-6.
[30]聂开立,王芳,谭天伟.固定酶法生产生物柴油[J].现代化工,2003,23(9)：35-38.
[31]Schafer A. Vegetable oil fatty acid methyl esters as alternative diesel fuels for commercial vehicles engines[J]. Plant Oil Fuel Proc Symp,1997,29-46.
[32]Kanmini N R, Iefuji H. Lipase catalyzed methanolysis of vegetable oils in aqueous medium by Oyptococcus spp. S-2[J]. Process Biochem,2001,37(4): 405-410.
[33]Mohamad I, Widyan A, Ali A. Experimental evaluation of the transesterification of waste palm oil into biodiesel[J]. Bioresource Technol,2002, (85):253-256.
[34]王学伟,常杰.固定化脂肪酶催化制备生物柴油[J].石油化工,2005,34(9)：855-858.
[35]Du W, Xu Y Y, Zeng J, et al. Comparative study on lipase-catalyzed transformation of soybean oil for biodiesel production with different acyl acceptors[J]. JMol Catal B:Enzymatic,2004,30:125-129.
[36]Marisa R, Verawat C, Navadol L. Biocatalytic ethanolysis of palm oil for biodiesel production using microcrystalline lipase in tert-butanol system[J]. Process Biochem,2010, (45):829-834.
[37]Soumanou M M, Bornschener U T. Improvement in lipase-catalyzed synthesis of fatty acid methyl esters from sunflower oil biodiesel using immobilized lipases[J]. Enzyme Microb Technol,2003,33(1):97-103.
[38]Yomi W, Praphan P, Toshihiro N, Asao Y, et al. Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase[J].J Mol Catal B:Enzymatic,2007,44:99-105.
[39]沅新,曾健青,张镜澄等.超临界二氧化碳中脂肪酶酯化酯交换反应的研究[J].化学通报,1998,(10)：34-35.
[40]林志勇,裘爱泳.超临界二氧化碳酶法酯交换反应研究进展[J].食品与发酵工业,1997,23(5)：78-82.
[41]Miller D A. Enzymic interesterification in supercritical carbon dioxide[J]. Acid Science,1990,13:534-537.
[42]陈惠晴,杨基础.超临界CO2的溶剂性质对脂肪酶催化反应的影响[J].清华大学学报(自然科学版),1999,39(6)：31-34.
[43]Jong H L, Sung B K, Seong W K, et al. Biodiesel production by a mixture of Candida rugosa and Rhizopus oryzae lipases using a supercritical carbon dioxide process[J]. Bioresource Technol,2011,102:2105-2108.
[44]杨继国,林炜铁,吴军林.酶法合成生物柴油的研究进展[J].化工环保,2004,24(2)：116-120.
[45]熊振平.酶工程[M].北京：化学工业出版社,1989,125.
[46]禹邦超,刘德立.应用酶学导论[M].武汉：华中师范大学出版社,1994,233.
[47]Wilhelm T, Volker K. Immobilizem enzymes:crystals of carriers[J]. Tibtech, 1999,17:326-334.
[48]Shan S W, Lee-Jun C W. Chemical crosslinking and the stabilization of protein and enzymes[J]. Enzyme Microb Technol,1992,14(11):866-874.
[49]Indu B, Rajinder P, Gulam N Q, Ganesh I. Lipase enzyme immobilization on synthetic beaded macroporous copolymers for kinetic resolution of chiral drugs intermediates[J]. Process Biochem,2008,43:321-330.
[50]Yasin Y. Biodiesel production from pomace oil by using lipase immobilized onto olive pomace[J]. Bioresource Technol,2011,102:3977-3980.
[51]寇秀芬,徐家立.在无溶剂系统中固定化脂肪酶合成聚乙二醇400月桂酸酯[J].生物工程学报,1997,13(3)：289-293.
[52]Chen B Q, Yin C H, Cheng Y Y, Li W N. Using silk woven fabric as support for lipase immobilization:The effect of surface hydrophilicity/hydrophobicity on enzymatic activity and stability[J]. Biomass Bioenerg,2010,10:1-8.
[53]Uswatun H Z, Mohd B A R, Mahiran B. Silylation of mica for lipase immobilization as biocatalysts in esterification[J]. Appl Clay Sci,2010,47: 276-282.
[54]Kuoa C H, Liub Y C, et al. Optimum conditions for lipase immobilization on chitosan-coated Fe3O4 nanoparticles[J]. Carbohyd Polym,2012,87:2538-2545.
[55]Zaks A, Klibabov A M. Enzymes catalysis in organic media at 100℃[J]. Science, 1984,224:1249-1253.
[56]Klibanov A M. Improving enzymes by using them in organic solvents[J]. Nature, 2001,409:241-246.
[57]Lee M Y, Dordick J S. Enzyme activation for nonaqueous media[J]. Curr Opin Biotech,2002,13:376-384.
[58]Khmelnitsky Y L, Rich J O. Biocatalysis in nonaqueous solvents[J]. Curr Opin Chem Biol,1999,3:47-53.
[59]Yang Z, Russell A J. Enzymatic reactions in organic media[M]. London:Blackie Academic& Professional,1996,43-69.
[60]Ye P, Xu Z K, Wang Z G, et al. Comparison of hydrolytic activities in aqueous and organic media for lipases immobilized on poly(acrylonitrile-co-maleic acid) ultrafiltration hollow fiber membrane[J]. J Mol Catal B:Enzymatic,2005,32: 115-121.
[61]Riva S, Chopineau J, Kieboom A P G, et al. Protease-catalyzed regioselective esterification of sugars and related compounds in anhydrous dimethylformamide[J]. J Am Chem Soc,1988,110:584-589.
[62]Li S X, Pang H Y, Lin K, et al. Refolding, purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010[J]. J Mol Catal B:Enzymatic,2011,71:171-176.
[63]Costantino H R, Griebenow K, Mishra P, et al. Fourier-transform infrared spectroscopic investigation of protein stability in the lyophilized form[J]. Biochimica et Biophysica Acta,1995,1253:69-74.
[64]Affleck R, Xu Z F, Suzawa V, et al. Enzymatic catalysis and dynamics in low-water environments[J]. Proc Natl Acad Sei,1992,89:1100-1104.
[65]Ghamgui H, Chaabouni M K, Gargouri Y.1-Butyl oleate synthesis by immobilized lipase from Rhizopus oryzae:a comparative study between n-hexane and solvent-free system[J]. Enzyme Microb Technol,2004,35: 355-363.
[66]Halling P J. Salt hydrates for water activity control with biocatalysts in organic media[J]. Biotechnol Tech,1992,6:271-278.
[67]曹淑桂.有机溶剂中酶催化研究的新进展——酶催化活性和选择性的控制与调节[J].化学通报,1995,(5)：5-12.
[68]Laane C, Boeren S, Vos K, et al. Rules for optimization of biocatalysis in organic solvent[J]. Biotechnol Bioeng,1987,30:81-87.
[69]Mestri S D, Pai J S. Effect of moisture on lipase-catalyzed esterification of geraniol of palmarosa oil in non-aqueous system[J]. Biotechnol Lett,1995,17: 459-461.
[70]罗师平,冷希冈.基于PCR的体外诱变技术[J].国外医学生物医学工程分册,2005,3(28)：188-192.
[71]Horton S N, Hunt H D. Site-directed mutagenesis by overlap extension using the polymerase chain reaction[J]. Gene,1989,77(1):51-59.
[72]Senanayake S D, Brian D A. Precise large deletions by the PCR-based overlap extension method[J]. Mol Biotechnol,1995,4(1):13-15.
[73]Urban A, Neukirchen S, Jaeger K E. A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR[J]. Nucleic Acids Res,1997,25(11):2227-2228.
[74]Warrens A N, Jones M D, Leehler R I. Splicing by overlap extension by PCR using asymmetric amplification:An improved technique for the generation ofhybrid proteins ofimmunological interest[J]. Gene,1997,186(1):29-35.
[75]Horton R M, Cai Z L, Horton S N. Gene splicing by overlap extension: Tailor-made genes using the polymerase chain reaction[J]. Biotechniques,1990, 8(5):528-535.
[76]徐圆圆,杜伟,刘德华.非水相脂肪酶催化大豆油合成生物柴油的研究[J].现代化工,2003,23：167-169.
[77]周泉城,吴某成.响应面法优化游离脂肪酸酯化条件[J].2006,21(1)：113-116.
[78]林康,李素霞,许建和等.戊二醛固定化脂肪酶活性包涵体的性质研究[J].中国药学杂志,2011,46(15)：1150-1153.
[79]林康,李素霞,许建和等.戊二醛交联法固定化脂肪酶活性包涵体的研究[J].食品与药品,2011,13(5)：153-156.
[80]林康,陈欣宁,李素霞,许建和等.响应面法优化脂肪酶活性包涵体固定化条件[J].食品与药品,2012,14(1)：1-5.
[81]钱军民,张兴,吕飞等.酶固定化载体材料研究进展[J].化工新型材料,2002,30(10)：21-24.
[82]Gao L, Xu J H, Zhao L L. Optimization of Serratia marcescens lipase production for enantioselective hydrolysis of 3-phenylglycidic acid ester[J]. J Ind Microbiol Biot,2004,31(11):525-530.
[2]王军.脂肪酶固定化及催化酯化反应[J].日用化学工业,1995,(3)：4-7.
[3]Alexander E I, Manfred P S. Methods for the immobilization of lipases and their use for ester synthesis[J]. J Mol Catal B:Enzymatic,1997,3(6):303-309.
[4]Bruins M E, Janssen A E M, Boom R M. Thermozymes and their applications[J]. Appl Biochem Biotechnol,2001,90(2):155-186.
[5]Wu H, Zong M H, Lou W Y. Transesterification of waste oil to biodiesel in solvent free system catalyzed by immobilized lipase[J]. Chin J Catal,2004, 25(11):903-908.
[6]Haki G D, Rakshit S K. Developments in industrially important thermostable enzymes:a review[J]. Bioresour Technol,2003,89(1):17-34.
[7]Kirk O, Borchert T V, Fuglsang C C. Industrial enzyme applications[J]. Curr Opin Biotechnol,2002,13(4):345-351.
[8]Bajpai P. Application of enzymes in the pulp and paper industry[J]. Biotechnol Prog,1999,15(2):147-157.
[9]Wong H, Schotz M C. The lipase gene family[J]. J Lipid Res,2002,43: 993-999.
[10]Sinchaikul S, Sookkheo B, Phutrakul S, et al. Optimization of a thermostable lipase from Bacillus stearothermophilus P1:overexpression, purification, and characterization[J]. Protein Expr Purif,2001,22:388-398.
[11]龙章德.粘质沙雷氏菌脂肪酶的制备及其在手性拆分中的应用[D].华东理工大学博士论文,2007：22.
[12]Fickers P, Fudalej F, Le-Dall M T, et al. Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica[J]. Fungal Genet Biol,2005,42:264-274.
[13]Uppenberg J, Hansen M T, Patkar S, Jones T A. The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica[J]. Structure,1994,2:293-308.
[14]Nardini M, Dijkstra B W. Alpha/bata hydrolase fold enzymes:the family keeps growing[J]. Curr Orin Struct Biol,1999,9:732-737.
[15]Reto M, Thomas D, Vera S, Karl E J, Ulrich B. A calcium-gated lid and a large-roll sandwich are revealed by the crystal structure of extracellular lipase from Serratia marcescens[J].J Biol Chem,2007,282:31477-31483.
[16]Holmquist M. Alpha/Beta-hydrolase fold enzymes:structures, functions and mechanisms[J]. Curr Protein Pept Sci,2000,1:209-235.
[17]Wei Y, Swenson L, Castro C, et al. Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases:Streptomyces exfoliatus lipase at 1.9 A resolution[J]. Structure,1998,6:511-519.
[18]Van P G, Eggert T, Jaeger K E, et al. The crystal structure of Bacillus subtilis lipase:a minimal alpha/beta hydrolase fold enzyme[J]. J Mol Biol,2001,309: 215-226.
[19]Arpigny J L, Jaeger K E. Bacterial lipolytic enzymes:classification and properties[J]. Biochem J.1999,343:177-183.
[20]Mosbah H, Sayari A, Mejdoub H, et al. Biochemical and molecular characterization of Staphylococcus xylosus lipase[J]. Biochim Biophys Acta, 2005,1723:282-291.
[21]Dimitris G H, John B M, Paul C, et al. Production and partial charaterisation of extracellular lipase from Aspergillus niger[J]. Biotech Lett,1996,18:547-552.
[22]Sayari A, Agrebi N, Jaoua S, et al. Biochemical and molecular characterization of Staphylococcus simulans lipase[J]. Biochimie,2001,83:863-871.
[23]Saleh A M, Heidi M Abdel-Mageed, Saadia A T, Mohamed A. Characterization of Mucor racemosus lipase with potential application for the treatment of cellulite[J]. Process Biochem,2011,46(3):642-648.
[24]Chen C S, Sih C J A. General aspects and optimization of enantioselective biocatalysis in organic solvents:The use of lipases[J]. Chem Int Ed Engl,1989, 28(6):695-707.
[25]祖元刚.生物柴油[M].北京：科学出版社,2007,54-61.
[26]汪永,欧仕益,温勇等.酶法催化合成生物柴油的研究进展[J].中国油脂,2006,31：65-68.
[27]Fangrui M. Biodiesel production:a review[J]. Bioresource Technol,1999,70: 1-15.
[28]忻耀年等.生物柴油的生产和应用[J].中国油脂,2001,26(5)：72-77.
[29]谭天伟等.生物柴油的生产和应用[J].现代化工,2002,22(2)：4-6.
[30]聂开立,王芳,谭天伟.固定酶法生产生物柴油[J].现代化工,2003,23(9)：35-38.
[31]Schafer A. Vegetable oil fatty acid methyl esters as alternative diesel fuels for commercial vehicles engines[J]. Plant Oil Fuel Proc Symp,1997,29-46.
[32]Kanmini N R, Iefuji H. Lipase catalyzed methanolysis of vegetable oils in aqueous medium by Oyptococcus spp. S-2[J]. Process Biochem,2001,37(4): 405-410.
[33]Mohamad I, Widyan A, Ali A. Experimental evaluation of the transesterification of waste palm oil into biodiesel[J]. Bioresource Technol,2002, (85):253-256.
[34]王学伟,常杰.固定化脂肪酶催化制备生物柴油[J].石油化工,2005,34(9)：855-858.
[35]Du W, Xu Y Y, Zeng J, et al. Comparative study on lipase-catalyzed transformation of soybean oil for biodiesel production with different acyl acceptors[J]. JMol Catal B:Enzymatic,2004,30:125-129.
[36]Marisa R, Verawat C, Navadol L. Biocatalytic ethanolysis of palm oil for biodiesel production using microcrystalline lipase in tert-butanol system[J]. Process Biochem,2010, (45):829-834.
[37]Soumanou M M, Bornschener U T. Improvement in lipase-catalyzed synthesis of fatty acid methyl esters from sunflower oil biodiesel using immobilized lipases[J]. Enzyme Microb Technol,2003,33(1):97-103.
[38]Yomi W, Praphan P, Toshihiro N, Asao Y, et al. Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase[J].J Mol Catal B:Enzymatic,2007,44:99-105.
[39]沅新,曾健青,张镜澄等.超临界二氧化碳中脂肪酶酯化酯交换反应的研究[J].化学通报,1998,(10)：34-35.
[40]林志勇,裘爱泳.超临界二氧化碳酶法酯交换反应研究进展[J].食品与发酵工业,1997,23(5)：78-82.
[41]Miller D A. Enzymic interesterification in supercritical carbon dioxide[J]. Acid Science,1990,13:534-537.
[42]陈惠晴,杨基础.超临界CO2的溶剂性质对脂肪酶催化反应的影响[J].清华大学学报(自然科学版),1999,39(6)：31-34.
[43]Jong H L, Sung B K, Seong W K, et al. Biodiesel production by a mixture of Candida rugosa and Rhizopus oryzae lipases using a supercritical carbon dioxide process[J]. Bioresource Technol,2011,102:2105-2108.
[44]杨继国,林炜铁,吴军林.酶法合成生物柴油的研究进展[J].化工环保,2004,24(2)：116-120.
[45]熊振平.酶工程[M].北京：化学工业出版社,1989,125.
[46]禹邦超,刘德立.应用酶学导论[M].武汉：华中师范大学出版社,1994,233.
[47]Wilhelm T, Volker K. Immobilizem enzymes:crystals of carriers[J]. Tibtech, 1999,17:326-334.
[48]Shan S W, Lee-Jun C W. Chemical crosslinking and the stabilization of protein and enzymes[J]. Enzyme Microb Technol,1992,14(11):866-874.
[49]Indu B, Rajinder P, Gulam N Q, Ganesh I. Lipase enzyme immobilization on synthetic beaded macroporous copolymers for kinetic resolution of chiral drugs intermediates[J]. Process Biochem,2008,43:321-330.
[50]Yasin Y. Biodiesel production from pomace oil by using lipase immobilized onto olive pomace[J]. Bioresource Technol,2011,102:3977-3980.
[51]寇秀芬,徐家立.在无溶剂系统中固定化脂肪酶合成聚乙二醇400月桂酸酯[J].生物工程学报,1997,13(3)：289-293.
[52]Chen B Q, Yin C H, Cheng Y Y, Li W N. Using silk woven fabric as support for lipase immobilization:The effect of surface hydrophilicity/hydrophobicity on enzymatic activity and stability[J]. Biomass Bioenerg,2010,10:1-8.
[53]Uswatun H Z, Mohd B A R, Mahiran B. Silylation of mica for lipase immobilization as biocatalysts in esterification[J]. Appl Clay Sci,2010,47: 276-282.
[54]Kuoa C H, Liub Y C, et al. Optimum conditions for lipase immobilization on chitosan-coated Fe3O4 nanoparticles[J]. Carbohyd Polym,2012,87:2538-2545.
[55]Zaks A, Klibabov A M. Enzymes catalysis in organic media at 100℃[J]. Science, 1984,224:1249-1253.
[56]Klibanov A M. Improving enzymes by using them in organic solvents[J]. Nature, 2001,409:241-246.
[57]Lee M Y, Dordick J S. Enzyme activation for nonaqueous media[J]. Curr Opin Biotech,2002,13:376-384.
[58]Khmelnitsky Y L, Rich J O. Biocatalysis in nonaqueous solvents[J]. Curr Opin Chem Biol,1999,3:47-53.
[59]Yang Z, Russell A J. Enzymatic reactions in organic media[M]. London:Blackie Academic& Professional,1996,43-69.
[60]Ye P, Xu Z K, Wang Z G, et al. Comparison of hydrolytic activities in aqueous and organic media for lipases immobilized on poly(acrylonitrile-co-maleic acid) ultrafiltration hollow fiber membrane[J]. J Mol Catal B:Enzymatic,2005,32: 115-121.
[61]Riva S, Chopineau J, Kieboom A P G, et al. Protease-catalyzed regioselective esterification of sugars and related compounds in anhydrous dimethylformamide[J]. J Am Chem Soc,1988,110:584-589.
[62]Li S X, Pang H Y, Lin K, et al. Refolding, purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010[J]. J Mol Catal B:Enzymatic,2011,71:171-176.
[63]Costantino H R, Griebenow K, Mishra P, et al. Fourier-transform infrared spectroscopic investigation of protein stability in the lyophilized form[J]. Biochimica et Biophysica Acta,1995,1253:69-74.
[64]Affleck R, Xu Z F, Suzawa V, et al. Enzymatic catalysis and dynamics in low-water environments[J]. Proc Natl Acad Sei,1992,89:1100-1104.
[65]Ghamgui H, Chaabouni M K, Gargouri Y.1-Butyl oleate synthesis by immobilized lipase from Rhizopus oryzae:a comparative study between n-hexane and solvent-free system[J]. Enzyme Microb Technol,2004,35: 355-363.
[66]Halling P J. Salt hydrates for water activity control with biocatalysts in organic media[J]. Biotechnol Tech,1992,6:271-278.
[67]曹淑桂.有机溶剂中酶催化研究的新进展——酶催化活性和选择性的控制与调节[J].化学通报,1995,(5)：5-12.
[68]Laane C, Boeren S, Vos K, et al. Rules for optimization of biocatalysis in organic solvent[J]. Biotechnol Bioeng,1987,30:81-87.
[69]Mestri S D, Pai J S. Effect of moisture on lipase-catalyzed esterification of geraniol of palmarosa oil in non-aqueous system[J]. Biotechnol Lett,1995,17: 459-461.
[70]罗师平,冷希冈.基于PCR的体外诱变技术[J].国外医学生物医学工程分册,2005,3(28)：188-192.
[71]Horton S N, Hunt H D. Site-directed mutagenesis by overlap extension using the polymerase chain reaction[J]. Gene,1989,77(1):51-59.
[72]Senanayake S D, Brian D A. Precise large deletions by the PCR-based overlap extension method[J]. Mol Biotechnol,1995,4(1):13-15.
[73]Urban A, Neukirchen S, Jaeger K E. A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR[J]. Nucleic Acids Res,1997,25(11):2227-2228.
[74]Warrens A N, Jones M D, Leehler R I. Splicing by overlap extension by PCR using asymmetric amplification:An improved technique for the generation ofhybrid proteins ofimmunological interest[J]. Gene,1997,186(1):29-35.
[75]Horton R M, Cai Z L, Horton S N. Gene splicing by overlap extension: Tailor-made genes using the polymerase chain reaction[J]. Biotechniques,1990, 8(5):528-535.
[76]徐圆圆,杜伟,刘德华.非水相脂肪酶催化大豆油合成生物柴油的研究[J].现代化工,2003,23：167-169.
[77]周泉城,吴某成.响应面法优化游离脂肪酸酯化条件[J].2006,21(1)：113-116.
[78]林康,李素霞,许建和等.戊二醛固定化脂肪酶活性包涵体的性质研究[J].中国药学杂志,2011,46(15)：1150-1153.
[79]林康,李素霞,许建和等.戊二醛交联法固定化脂肪酶活性包涵体的研究[J].食品与药品,2011,13(5)：153-156.
[80]林康,陈欣宁,李素霞,许建和等.响应面法优化脂肪酶活性包涵体固定化条件[J].食品与药品,2012,14(1)：1-5.
[81]钱军民,张兴,吕飞等.酶固定化载体材料研究进展[J].化工新型材料,2002,30(10)：21-24.
[82]Gao L, Xu J H, Zhao L L. Optimization of Serratia marcescens lipase production for enantioselective hydrolysis of 3-phenylglycidic acid ester[J]. J Ind Microbiol Biot,2004,31(11):525-530.