皱褶假丝酵母（Candidarugosa）脂肪酶基因的克隆与表达
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摘要
近年来,脂肪酶的工业化应用领域越来越宽,获取更多具有不同特性的脂肪酶及其基因资源,并实现规模化生产,成为生物技术的研发热点。皱褶假丝酵母(Candida rugosa,原名Candida cylindracea)脂肪酶(CRL)是脂肪酶中较为重要的品种之一,可以催化脂水解、酯交换、酯合成等反应,广泛应用于油脂加工、食品、医药、日化等工业领域中,其含有多个同工酶,性质特异,并均能在异源宿主中实现一定程度的表达、分泌,具有较好的发展前景。
本文从皱褶假丝酵母(ATCC 14830)菌株出发,以总DNA基因组为模板扩增出六个脂肪酶基因(lip1-lip5和lipJ08)。其中,基因lipJ08的全长序列为首次报道。同时,通过置换17个非常规丝氨酸密码子,实现了lipJ08基因在巴斯德毕赤酵母(GS115)中的异源表达。主要工作如下:
(1)借助生物信息学,对已克隆的皱褶假丝酵母脂肪酶全长基因序列进行同源比对,根据保守序列设计引物,在基因组DNA水平上,从皱褶假丝酵母(ATCC 14830)菌株中克隆得到六个脂肪酶基因(lip1-lip5和lipJ08),其中lip1-lip5序列与已报道的皱褶假丝酵母脂肪酶同工酶相同, lipJ08基因为皱褶假丝酵母脂肪酶新的同工酶基因。序列分析表明,lipJ08基因全长为1650 bp,编码549个氨基酸,包括一个含15个氨基酸残基的信号肽,成熟脂肪酶由534个氨基酸组成。
(2)由于C.rugosa脂肪酶基因中存在非通用密码子CTG,编码Ser,而在毕赤酵母中则编码Leu。因此,使得以Ser为脂肪酶活性中心的皱褶假丝酵母脂肪酶异源表达时得到的是没有催化活性的表达产物。利用重叠延伸PCR(Over-lap extension PCR)技术对lipJ08中的17个CTG位点进行了突变改造,转换为在毕赤酵母中编码Ser的TCT,突变基因命名为MlipJ08。
(3)将lipJ08和MlipJ08分别亚克隆到表达载体pPIC9K中,成功构建了表达质粒pPIC9K-lipJ08和pPIC9K-MlipJ08,并将表达载体电转导入巴斯德毕赤酵母(GS115)中,成功地得到分泌表达。SDS-PAGE结果显示,表达的LIPJ08和MLIPJ08的分子量大小约为60 kDa。酶学特性研究表明,经密码子改造后获得的重组脂肪酶MLIPJ08对橄榄油的水解活力达4.65 U/mL,而LIPJ08活力几乎检测不到;MLIPJ08的最适温度为37℃,最适pH为7.0,对长碳链(C16)和短碳链(C4)pNp酯的水解活力高于对中碳链C8、C10和C12 pNp酯。
In recent years, the industrialized application of lipases has attracted an increasing attention, it is becoming a hot spot in biotechnological field to get more and more the versatile lipases and their gene resources. C. rugosa (formerly C. cylindracea) lipase (CRL) is an important industrial enzyme capable of catalyzing lipolytic, transesterification, esterification reaction, has been applied in many fields such as oil processing, food, pharmaceuticals, and refined chemical manufacture. The CRL family includes several lipase isozymes, and they have different properties from each other, some of them have been successfully expressed in heterologous hosts. The lipase exhibits a good prospect in industrialized application.
In this paper, six lipase genes (lip1-lip5 and lipJ08) were amplified from the total genomic DNA of the C. rugosa ATCC 14830, among which the full sequence of lipJ08 was reported for the first time. Moreover, lipJ08 gene was functionally expressed in Pichia pastoris GS115 system after substituting 17 CTGser triple codons with TCTser universal codons. The main work was as follows:
(1) By means of bioinformatics, we aligned nucleotide sequences of reported lipase isoforms from C. rugosa. Primers were designed based on the conservative nucleotide sequences, and six lipase genes (lip1-lip5 and lipJ08) of C.rugosa ATCC 14830 were cloned. The nucleotide sequencing revealed that the lipJ08 gene with an ORF of 1650 bp without any intron, was a novel gene of CRL isoform, encoding a protein of 534 amino acid residues, including a potential signal sequence of 15 amino acid residues. The deduced amino acid sequence of the lipase showed an overall identity of 66 % to lip1-lip5 from C.rugosa. The nucleotide sequences of the other 5 isoforms of lipase genes cloned from C. rugosa ATCC 14830 were identical to CRL isoforms, lip1-lip5, as previously reported, respectively.
(2) The deviations from the universal genetic code for the asporogenic yeast C.rugosa, in which the universal codon for leucine, CUG, is used to code for serine, including the catalytic Ser-209, has hindered the use of recombinant CRL isoenzymes (isoforms). The conversion of most or all CTG codons is required for the expression of functional lipase proteins in heterologous system. To overcome the difficulty, in this work, 17 of the non-nuniversal serine codons (CTG) of lipJ08 were converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis, and the optimized gene was termed as MlipJ08.
(3) The native and codon-optimized MlipJ08 genes were subcloned into pPIC9K vector, and expressed in P.pastoris GS115, respectively. SDS-PAGE analysis of LipJ08 produced by P. pastoris harboring the plasmid pPIC9K-lipJ08 or pPIC9K-MlipJ08 respectively showed a single protein band about 60 kDa. The assays of emzyme characteristics demonstrated that the hydrolysis active of MLIPJ08 toward olive oil from the supernatant was 4.65 U/mL, whereas the native lipase LIPJ08 has no hydrolysis activity after methanol induction for 120 hours in shake flasks. Furthermore, the observation of the MLIPJ08 hydrolyzing pNp esters with variety of carbon chain length showed that it exhibited a preference for both long-chain (C16 acyl group) and short-chain (C4 acyl group) pNp esters to medium-chain cholesteryl esters (C8, C10 and C12 acyl groups), and its optimal reaction temperature and pH were 37℃and pH 7.0, respectively.
本文从皱褶假丝酵母(ATCC 14830)菌株出发,以总DNA基因组为模板扩增出六个脂肪酶基因(lip1-lip5和lipJ08)。其中,基因lipJ08的全长序列为首次报道。同时,通过置换17个非常规丝氨酸密码子,实现了lipJ08基因在巴斯德毕赤酵母(GS115)中的异源表达。主要工作如下:
(1)借助生物信息学,对已克隆的皱褶假丝酵母脂肪酶全长基因序列进行同源比对,根据保守序列设计引物,在基因组DNA水平上,从皱褶假丝酵母(ATCC 14830)菌株中克隆得到六个脂肪酶基因(lip1-lip5和lipJ08),其中lip1-lip5序列与已报道的皱褶假丝酵母脂肪酶同工酶相同, lipJ08基因为皱褶假丝酵母脂肪酶新的同工酶基因。序列分析表明,lipJ08基因全长为1650 bp,编码549个氨基酸,包括一个含15个氨基酸残基的信号肽,成熟脂肪酶由534个氨基酸组成。
(2)由于C.rugosa脂肪酶基因中存在非通用密码子CTG,编码Ser,而在毕赤酵母中则编码Leu。因此,使得以Ser为脂肪酶活性中心的皱褶假丝酵母脂肪酶异源表达时得到的是没有催化活性的表达产物。利用重叠延伸PCR(Over-lap extension PCR)技术对lipJ08中的17个CTG位点进行了突变改造,转换为在毕赤酵母中编码Ser的TCT,突变基因命名为MlipJ08。
(3)将lipJ08和MlipJ08分别亚克隆到表达载体pPIC9K中,成功构建了表达质粒pPIC9K-lipJ08和pPIC9K-MlipJ08,并将表达载体电转导入巴斯德毕赤酵母(GS115)中,成功地得到分泌表达。SDS-PAGE结果显示,表达的LIPJ08和MLIPJ08的分子量大小约为60 kDa。酶学特性研究表明,经密码子改造后获得的重组脂肪酶MLIPJ08对橄榄油的水解活力达4.65 U/mL,而LIPJ08活力几乎检测不到;MLIPJ08的最适温度为37℃,最适pH为7.0,对长碳链(C16)和短碳链(C4)pNp酯的水解活力高于对中碳链C8、C10和C12 pNp酯。
In recent years, the industrialized application of lipases has attracted an increasing attention, it is becoming a hot spot in biotechnological field to get more and more the versatile lipases and their gene resources. C. rugosa (formerly C. cylindracea) lipase (CRL) is an important industrial enzyme capable of catalyzing lipolytic, transesterification, esterification reaction, has been applied in many fields such as oil processing, food, pharmaceuticals, and refined chemical manufacture. The CRL family includes several lipase isozymes, and they have different properties from each other, some of them have been successfully expressed in heterologous hosts. The lipase exhibits a good prospect in industrialized application.
In this paper, six lipase genes (lip1-lip5 and lipJ08) were amplified from the total genomic DNA of the C. rugosa ATCC 14830, among which the full sequence of lipJ08 was reported for the first time. Moreover, lipJ08 gene was functionally expressed in Pichia pastoris GS115 system after substituting 17 CTGser triple codons with TCTser universal codons. The main work was as follows:
(1) By means of bioinformatics, we aligned nucleotide sequences of reported lipase isoforms from C. rugosa. Primers were designed based on the conservative nucleotide sequences, and six lipase genes (lip1-lip5 and lipJ08) of C.rugosa ATCC 14830 were cloned. The nucleotide sequencing revealed that the lipJ08 gene with an ORF of 1650 bp without any intron, was a novel gene of CRL isoform, encoding a protein of 534 amino acid residues, including a potential signal sequence of 15 amino acid residues. The deduced amino acid sequence of the lipase showed an overall identity of 66 % to lip1-lip5 from C.rugosa. The nucleotide sequences of the other 5 isoforms of lipase genes cloned from C. rugosa ATCC 14830 were identical to CRL isoforms, lip1-lip5, as previously reported, respectively.
(2) The deviations from the universal genetic code for the asporogenic yeast C.rugosa, in which the universal codon for leucine, CUG, is used to code for serine, including the catalytic Ser-209, has hindered the use of recombinant CRL isoenzymes (isoforms). The conversion of most or all CTG codons is required for the expression of functional lipase proteins in heterologous system. To overcome the difficulty, in this work, 17 of the non-nuniversal serine codons (CTG) of lipJ08 were converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis, and the optimized gene was termed as MlipJ08.
(3) The native and codon-optimized MlipJ08 genes were subcloned into pPIC9K vector, and expressed in P.pastoris GS115, respectively. SDS-PAGE analysis of LipJ08 produced by P. pastoris harboring the plasmid pPIC9K-lipJ08 or pPIC9K-MlipJ08 respectively showed a single protein band about 60 kDa. The assays of emzyme characteristics demonstrated that the hydrolysis active of MLIPJ08 toward olive oil from the supernatant was 4.65 U/mL, whereas the native lipase LIPJ08 has no hydrolysis activity after methanol induction for 120 hours in shake flasks. Furthermore, the observation of the MLIPJ08 hydrolyzing pNp esters with variety of carbon chain length showed that it exhibited a preference for both long-chain (C16 acyl group) and short-chain (C4 acyl group) pNp esters to medium-chain cholesteryl esters (C8, C10 and C12 acyl groups), and its optimal reaction temperature and pH were 37℃and pH 7.0, respectively.
引文
[1]谈重芳,王雁萍,陈林海等.微生物脂肪酶在工业中的应用及研究进展.食品工业科技, 2006, 27(7): 193-195
[2]阎金勇,杨江科,徐莉等.白地霉Y162脂肪酶基因克隆及其在毕赤酵母中的高效表达.微生物学报, 2008, 48(2): 184-190
[3] Kawaguchi Y, Honda H, Taniguchi-Morimura J, et al. The codon CUG is read as serine in an asporogenic yeast Candida cylindracea. Nature, 1989, 341: 164-166
[4]郭诤,张根旺.脂肪酶的结构特征和化学修饰.中国油脂, 2003, 28(7): 6-8
[5] Jaeger K E, Reetz M T. Microbial lipases form versatile tools for biotechnology. Trends in Biotechnology, 1998, 16: 396-403
[6] Brockerhoff B, Brockman H. L. Lipolytic Enzymes. New York. Academic Press, 1974, 1-340
[7]林志勇,裘爱泳.无溶剂状态下乌桕皮油酶催化酯交换改性制取类可可酯的研究.中国油脂, 1998, 3(1): 9-13
[8] Chang M K, Abraham G, John V T, et al. Production of cocoa butter-like fat from interesterfication of vegetable oils. Journal of the American Oil Chemists’Society, 1990, 67(11): 832-834
[9] RGandolfi R, Converti A, Pirozzi D, et al. Efficient and selective microbial esterification with dry mycelium of Rhizopus oryzae. Journal of Biotechnology, 2001, 92(1): 21-26
[10]黄玉丽,王宪迎,王树兰.羊毛生物防毡缩技术的研究.印染, 2004, 30(6): 1- 3
[11] Gulrajani M L, Agarwal R, Grover A, et al. Degumming of silk with lipase and protease. Indian Journal of Fibre and Textile Research, 2000, 25(1): 69-74
[12] Kathiervelu S S. Enzymatic preparatory processe. Textile Trends, 2002, 45(9): 93-96
[13] Al-Bahrani A Z, Ammori B J. Clinical laboratory assessment of acute pancreatitis. Clinica Chimica Acta, 2005, 362 (1-2): 26-48
[14]丰鲜明.血清淀粉酶_脂肪酶的联合检测在急性胰腺炎诊断中作用初探.医疗卫生, 1997, 12: 48-49
[15] Hasan F, Aamer A S, Abdul H. Industrial applications of microbial lipases. Enzyme Microbial Technology, 2006, 39(2): 235-251
[16] Vicente G F, Rosario B, Vicente G. Lipases: useful biocatalysts for the preparation of pharmaceuticals. Journal of Molecular Catalysis: B, Enzymatic, 2006, 40(3-4): 111-120
[17] Patel R N, Banerjee A, Ko R Y, et al. Enzymic preparation of (3R-cis)-3- (acetyloxy)-4-phenyl-2-azetidinone: a taxol side-chain synthon. Biotechnology and Applied Biochemistry, 1994, 20(1): 23-33
[18]黄瑛,黎洁,闫云君.固定化脂肪酶催化棉籽油酯交换制备生物柴油新工艺.中国油脂, 2008, 33(2): 40-43
[19]纪锡和,王孝英,黄月桂等.生物柴油专用脂肪酶的开发及其应用研究.广西轻工业, 2007, 108(11): 1-3
[20]倪婉星,张晓鸣.非水相脂肪酶催化合成生物柴油.食品与机械, 2007, 23(1): 25-28
[21] Kazuhiro B , Masaru K, Takeshi M, et al. Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochemical Engineering Journal, 2001, 8 : 39-43
[22] Hama S, Yamai H, Kaieda M, et al. Effect of fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel production. Biochemical Engineering Journal, 2004, 21(2): 155-160
[23]秦梦华,陈嘉翔.制浆造纸厂树脂沉积的机理及其控制.中国造纸, 1995(4): 45-50
[24]陈建平,黄儒珠,吴松刚.碱性脂肪酶水解纸浆中的甘油三酸酯的研究.福建师范大学学报, 1998, 14(3): 72-76
[25]顾琪萍,勇强,尤纪雪等.脂肪酶用于废报纸脱墨的研究.中国造纸, 2003, 22(4): 7-9
[26] Benjiaming S, Pandey A. Candida rugosa lipases: Molecular biology and versatility in biotechnology. Yeast, 1998, 14: 1069-1087
[27] Yamada K, Machida H. Studies on the production of lipase by microorganisms II.Quantitative determination of lipase. Journal of Agriculture Chemistry and Society, Japan, 1962, 86: 858
[28] Tomizuka N, Ota Y, Yaminaga Y. Studies on lipase from Candida cylindracea. Agriculture Biology Chemistry, 1966, 30: 576-584
[29] Longhi S, Fusetti F, Grandori R, et al. Cloning and nucleotide sequences of two lipase genes from Candida cylindracea. Biochimica Biophysica Acta, 1992, 1131: 227-232
[30] Lotti M, Grandori R, Fusetti F, et al. Cloning and analysis of Candida cylindracea lipase sequences. Gene, 1993, 124: 45-55
[31] Brocca S, Grandori R, Breviario D, et al. Localization of lipase genes on Candida rugosa chromosomes. Current Genetics, 1995, 28: 454-457
[32] Lotti M, Grandori R, Fusetti F, et al. Cloning and analysis of Candida cylindracea lipase sequences. Gene, 1993, 124: 45-55
[33] Ohama T, Suzeki T, Mori M, et al. Non-universal decoding of the leucine codon CUG in several Candida species. Nucleic Acids Research, 1993, 21(17): 4039-4045
[34] Fusetti F, Brocca S, Porro D, et al. Effect of the leader sequence on the expression of recombinant C. rugosa lipase by S. cerevisiae cells. Biotechnology Letter, 1996, 18(3): 281-286
[35] Secundo F, Carrea G, Tarabiono C, et al. Activity and enantioselectivity of wide-type and lid mutated Candida rugosa lipase isoform 1 in organic solvent, Biotechology and Bioengineering, 2004, 86: 236-240
[36] Brocca S, Schmidt-Dannert C, Lotti M, et al. Total synthesis and functional expression of Candida rugosa lip1 gene coding for a major industrial lipase. Protein Science, 1998, 7: 1415-1422
[37] Chang S W, Shieh C J, Lee G C, et al. Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris. Applied Microbiology Biotechnology, 2005, 67: 215-224
[38] Lee G C, LEE L C, Sava V, et al. Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris. Biochemistry. 2002, 366: 603-611
[39] Chang S W, Lee G C, SHAW J F. Efficient Production of Active Recombinant Candida rugosa LIP3 Lipase in Pichia Pastoris and Biochemical Characterization of the Purified Enzyme. Agricultural and Food Chemistry, 2006, 54: 5831-5838
[40] Tang S J, Shaw J F, Sun K H, et al. Recombinant Expression and Characterization of the Candida rugosa lip4 Lipase in Pichia pastoris: Comparison of Glycosylation Activity and Stability. Archives of Biochemistry and Biophysics, 2001, 387(1): 93-98
[41] Tang S J, Shaw J F, Sun G H, et al. Recombinant expression of the Candida rugosa lip4 in Escherichia coli. Protein Expression Purification, 2000, 20: 308-313
[42] Grochulski P, Li Y, Schrag J D, et al. Insights into Interfacial Activation from an Open Structure of Candida rugosa Lipase. Biochemistry, 1993, 268(17): 12843-12847
[43] Grochulski P, Li Y, Schrag J D, et al. Two Conformational States of Candida rugosa Lipase. Protein Science, 1994, 3: 82-91
[44] Ghosh D, Wawrzak Z, Pletnev V Z, et al. Sructure of Uncomplexed and Linoleate-Bound Candida cylindracea Cholesterol Esterase. Structure, 1995, 3(3): 279-288
[45] Manche?o J M , Pernas M A, Martínez M J, et al. Structural Insights into the Lipase/Esterase Behavior in the Candida rugosa Lipases Family: Crystal Structure of the Lipase 2 Isoenzyme at 1. 97 ? Resolution. Moleculaer Biology, 2003, 332(5): 1059-1069
[46] Grochulski P, Bouthillier F, Kazlauskas R J, et al. Analogs of Reaction Intermediates Identify a Unique Substrate Binding Site in Candida rugosa Lipase. Biochemistry, 1994, 33(12): 3494-3500
[47] Cygler M, Grochulski P, Kazlauskas R J, et al. A Structural Basis for the Chiral Preferences of Lipases. Journal of the American Chemical Society, 1994, 116(8), 3180-3186
[48] Pletnev V, Addlagatta A, Wawrzak Z, et al. Three-Dimensional Structure of Homodimeric Cholesterol Esterase-Ligand Complex at 1. 4 ? Resolution. Acta crystallographica. Section D, Biological Crystallography, 2003, 59(1): 50-56
[49] Ollis, D L, Cheah, E, Cygler, M, et al. The Alpha/Beta Hydrolase Fold, ProteinEngineering, 1992, 5(3): 197-211
[50] Colton I J, Ahmed S, Kazlauskas R J. A 2-propanol treatment increases the enantioselectivity of Candida rugosa lipase toward esters of chiral carboxylic acids. The Journal of Organic Chemistry, 1995, 60(11): 212-217
[51] Chamorro S, Sanchez-Montero J M, Alcantara A R, et al. Treatment of Candida rugosa lipase with short-chain polar organic solvents enhances its hydrolytic and synthetic activities. Biotechnology Letter, 1998, 20(5): 499-505
[52] Graupner M, Haalck L, Spener F, et al. Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence. Biophysica, 1999, 77(1): 493-504
[53] Overbeeke P L A, Govardhan C, Khalaf N, et al. Influence of the lid conformation on lipase enantioselectivity. Journal of Molecular Catalysis. B, Enzymatic, 2000, 10(4): 385-393
[54] Turner N A, Needs E C, Khan JA, et al. Analysis of conformational states of Candida rugosa lipase in solution: implications for mechanism of interfacial activation and separation of open and closed forms. Biotechnology and Bioengineering, 2001, 72: 114-118
[55] Zhu K, Jutila A, Tuominen E K J, et al. Effects of i-propanol on the structural dynamics of Thermomyces lanuginose lipase revealed by tryptophan fluorescence. Protein Science, 2001, 10: 339-351
[56] James J, Lakshmi B, Raviprasad V, et al. Insights from molecular dynamics simulations into pH-dependent enantioselective hydrolysis of ibuprofen esters by Candida rugosa lipase. Protein Engineering, 2003, 16(12): 1017-1024
[57] Secundo F, Carrea G, Tarabiono C, et al. Activity and enantioselectivity of wildtype and lid mutated Candida rugosa lipase isoform 1 in organic solvents. Biotechnol- ogy and Bioengineering, 2004, 86(2): 236-240
[58] Tejo B A, Salleh A B, Pleiss J. Structure and dynamics of Candida rugosa lipase: the role of organic solvent. Journal of Moleculer Model, 2004: 10: 358-366
[59] Foresti M L, Ferreira M L. Computational approach to solvent-free synthesis of ethyl oleate using Candida rugosa and Candida antarctica B lipases I Interfacial activation and substrate (ethanol, oleic acid) adsorption. Biomacromolecules, 2004,5(23): 66-75
[60] Hockney R C. Recent developments in heterologous protein production in Escherichia coli. Trends in Biotechnology, 1994, 12(11): 456-463
[61] Bruel C, Cha K, Reeves P J, et a1. Rhodopsin kinase: expressionin mammalian cells and a two-step purification. Proceedings of the National Academy Sciences of United States of American, 2000, 97(7): 3004-3009
[62] Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Reviews, 2000, 24: 45-66
[63] Hancock R E, Lehrer R. Cationic peptides: a new source of antibiotic. Trends in Biotechnology, 1998, 16(2): 82-88
[64] Cregg J M, Vedvick T S, Raschke W C. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology, 1993, 11(8): 905-910
[65] Peng Y C, Acheson N H. Production of active polyomavirus large T antigen in yeast Pichia pastoris. Virus Research, 1997, 49(1): 41-47
[66]胡世界,罗素兰,张吉贞等.巴斯德毕赤酵母表达系统及其高水平表达策略.生物技术, 2007, 17(6): 78-83
[67] Scorer C A, Buckholz R G, Clare J J, et al. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene, 1993, 136: 111-119
[68] Romanos M A, Scorer C A, Clare J J. Foreign gene expression in yeast: A review Yeast, 1992, 8: 423-488
[69] Steffan N H, Hery D H, Robot M H, et al. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 1989, 77: 51-59
[70] Cregg J M, Cereghino J L, Shi J, et al. Recombinant Protein Expression in Pichia pastoris. Moleculer Biotechnology, 2000, 16(1): 23-25
[71] Shu Z Y, Yan Y J, Yang J K, et al. Aspergillus niger lipase: gene cloning, over-expression in Escherichia coli and in vitro refolding. Biotechnology Letter, 2007, 29: 1875-1879
[72]郭尧君.蛋白质电泳实验技术.科学出版社, 1998, 76-78
[2]阎金勇,杨江科,徐莉等.白地霉Y162脂肪酶基因克隆及其在毕赤酵母中的高效表达.微生物学报, 2008, 48(2): 184-190
[3] Kawaguchi Y, Honda H, Taniguchi-Morimura J, et al. The codon CUG is read as serine in an asporogenic yeast Candida cylindracea. Nature, 1989, 341: 164-166
[4]郭诤,张根旺.脂肪酶的结构特征和化学修饰.中国油脂, 2003, 28(7): 6-8
[5] Jaeger K E, Reetz M T. Microbial lipases form versatile tools for biotechnology. Trends in Biotechnology, 1998, 16: 396-403
[6] Brockerhoff B, Brockman H. L. Lipolytic Enzymes. New York. Academic Press, 1974, 1-340
[7]林志勇,裘爱泳.无溶剂状态下乌桕皮油酶催化酯交换改性制取类可可酯的研究.中国油脂, 1998, 3(1): 9-13
[8] Chang M K, Abraham G, John V T, et al. Production of cocoa butter-like fat from interesterfication of vegetable oils. Journal of the American Oil Chemists’Society, 1990, 67(11): 832-834
[9] RGandolfi R, Converti A, Pirozzi D, et al. Efficient and selective microbial esterification with dry mycelium of Rhizopus oryzae. Journal of Biotechnology, 2001, 92(1): 21-26
[10]黄玉丽,王宪迎,王树兰.羊毛生物防毡缩技术的研究.印染, 2004, 30(6): 1- 3
[11] Gulrajani M L, Agarwal R, Grover A, et al. Degumming of silk with lipase and protease. Indian Journal of Fibre and Textile Research, 2000, 25(1): 69-74
[12] Kathiervelu S S. Enzymatic preparatory processe. Textile Trends, 2002, 45(9): 93-96
[13] Al-Bahrani A Z, Ammori B J. Clinical laboratory assessment of acute pancreatitis. Clinica Chimica Acta, 2005, 362 (1-2): 26-48
[14]丰鲜明.血清淀粉酶_脂肪酶的联合检测在急性胰腺炎诊断中作用初探.医疗卫生, 1997, 12: 48-49
[15] Hasan F, Aamer A S, Abdul H. Industrial applications of microbial lipases. Enzyme Microbial Technology, 2006, 39(2): 235-251
[16] Vicente G F, Rosario B, Vicente G. Lipases: useful biocatalysts for the preparation of pharmaceuticals. Journal of Molecular Catalysis: B, Enzymatic, 2006, 40(3-4): 111-120
[17] Patel R N, Banerjee A, Ko R Y, et al. Enzymic preparation of (3R-cis)-3- (acetyloxy)-4-phenyl-2-azetidinone: a taxol side-chain synthon. Biotechnology and Applied Biochemistry, 1994, 20(1): 23-33
[18]黄瑛,黎洁,闫云君.固定化脂肪酶催化棉籽油酯交换制备生物柴油新工艺.中国油脂, 2008, 33(2): 40-43
[19]纪锡和,王孝英,黄月桂等.生物柴油专用脂肪酶的开发及其应用研究.广西轻工业, 2007, 108(11): 1-3
[20]倪婉星,张晓鸣.非水相脂肪酶催化合成生物柴油.食品与机械, 2007, 23(1): 25-28
[21] Kazuhiro B , Masaru K, Takeshi M, et al. Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochemical Engineering Journal, 2001, 8 : 39-43
[22] Hama S, Yamai H, Kaieda M, et al. Effect of fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel production. Biochemical Engineering Journal, 2004, 21(2): 155-160
[23]秦梦华,陈嘉翔.制浆造纸厂树脂沉积的机理及其控制.中国造纸, 1995(4): 45-50
[24]陈建平,黄儒珠,吴松刚.碱性脂肪酶水解纸浆中的甘油三酸酯的研究.福建师范大学学报, 1998, 14(3): 72-76
[25]顾琪萍,勇强,尤纪雪等.脂肪酶用于废报纸脱墨的研究.中国造纸, 2003, 22(4): 7-9
[26] Benjiaming S, Pandey A. Candida rugosa lipases: Molecular biology and versatility in biotechnology. Yeast, 1998, 14: 1069-1087
[27] Yamada K, Machida H. Studies on the production of lipase by microorganisms II.Quantitative determination of lipase. Journal of Agriculture Chemistry and Society, Japan, 1962, 86: 858
[28] Tomizuka N, Ota Y, Yaminaga Y. Studies on lipase from Candida cylindracea. Agriculture Biology Chemistry, 1966, 30: 576-584
[29] Longhi S, Fusetti F, Grandori R, et al. Cloning and nucleotide sequences of two lipase genes from Candida cylindracea. Biochimica Biophysica Acta, 1992, 1131: 227-232
[30] Lotti M, Grandori R, Fusetti F, et al. Cloning and analysis of Candida cylindracea lipase sequences. Gene, 1993, 124: 45-55
[31] Brocca S, Grandori R, Breviario D, et al. Localization of lipase genes on Candida rugosa chromosomes. Current Genetics, 1995, 28: 454-457
[32] Lotti M, Grandori R, Fusetti F, et al. Cloning and analysis of Candida cylindracea lipase sequences. Gene, 1993, 124: 45-55
[33] Ohama T, Suzeki T, Mori M, et al. Non-universal decoding of the leucine codon CUG in several Candida species. Nucleic Acids Research, 1993, 21(17): 4039-4045
[34] Fusetti F, Brocca S, Porro D, et al. Effect of the leader sequence on the expression of recombinant C. rugosa lipase by S. cerevisiae cells. Biotechnology Letter, 1996, 18(3): 281-286
[35] Secundo F, Carrea G, Tarabiono C, et al. Activity and enantioselectivity of wide-type and lid mutated Candida rugosa lipase isoform 1 in organic solvent, Biotechology and Bioengineering, 2004, 86: 236-240
[36] Brocca S, Schmidt-Dannert C, Lotti M, et al. Total synthesis and functional expression of Candida rugosa lip1 gene coding for a major industrial lipase. Protein Science, 1998, 7: 1415-1422
[37] Chang S W, Shieh C J, Lee G C, et al. Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris. Applied Microbiology Biotechnology, 2005, 67: 215-224
[38] Lee G C, LEE L C, Sava V, et al. Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris. Biochemistry. 2002, 366: 603-611
[39] Chang S W, Lee G C, SHAW J F. Efficient Production of Active Recombinant Candida rugosa LIP3 Lipase in Pichia Pastoris and Biochemical Characterization of the Purified Enzyme. Agricultural and Food Chemistry, 2006, 54: 5831-5838
[40] Tang S J, Shaw J F, Sun K H, et al. Recombinant Expression and Characterization of the Candida rugosa lip4 Lipase in Pichia pastoris: Comparison of Glycosylation Activity and Stability. Archives of Biochemistry and Biophysics, 2001, 387(1): 93-98
[41] Tang S J, Shaw J F, Sun G H, et al. Recombinant expression of the Candida rugosa lip4 in Escherichia coli. Protein Expression Purification, 2000, 20: 308-313
[42] Grochulski P, Li Y, Schrag J D, et al. Insights into Interfacial Activation from an Open Structure of Candida rugosa Lipase. Biochemistry, 1993, 268(17): 12843-12847
[43] Grochulski P, Li Y, Schrag J D, et al. Two Conformational States of Candida rugosa Lipase. Protein Science, 1994, 3: 82-91
[44] Ghosh D, Wawrzak Z, Pletnev V Z, et al. Sructure of Uncomplexed and Linoleate-Bound Candida cylindracea Cholesterol Esterase. Structure, 1995, 3(3): 279-288
[45] Manche?o J M , Pernas M A, Martínez M J, et al. Structural Insights into the Lipase/Esterase Behavior in the Candida rugosa Lipases Family: Crystal Structure of the Lipase 2 Isoenzyme at 1. 97 ? Resolution. Moleculaer Biology, 2003, 332(5): 1059-1069
[46] Grochulski P, Bouthillier F, Kazlauskas R J, et al. Analogs of Reaction Intermediates Identify a Unique Substrate Binding Site in Candida rugosa Lipase. Biochemistry, 1994, 33(12): 3494-3500
[47] Cygler M, Grochulski P, Kazlauskas R J, et al. A Structural Basis for the Chiral Preferences of Lipases. Journal of the American Chemical Society, 1994, 116(8), 3180-3186
[48] Pletnev V, Addlagatta A, Wawrzak Z, et al. Three-Dimensional Structure of Homodimeric Cholesterol Esterase-Ligand Complex at 1. 4 ? Resolution. Acta crystallographica. Section D, Biological Crystallography, 2003, 59(1): 50-56
[49] Ollis, D L, Cheah, E, Cygler, M, et al. The Alpha/Beta Hydrolase Fold, ProteinEngineering, 1992, 5(3): 197-211
[50] Colton I J, Ahmed S, Kazlauskas R J. A 2-propanol treatment increases the enantioselectivity of Candida rugosa lipase toward esters of chiral carboxylic acids. The Journal of Organic Chemistry, 1995, 60(11): 212-217
[51] Chamorro S, Sanchez-Montero J M, Alcantara A R, et al. Treatment of Candida rugosa lipase with short-chain polar organic solvents enhances its hydrolytic and synthetic activities. Biotechnology Letter, 1998, 20(5): 499-505
[52] Graupner M, Haalck L, Spener F, et al. Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence. Biophysica, 1999, 77(1): 493-504
[53] Overbeeke P L A, Govardhan C, Khalaf N, et al. Influence of the lid conformation on lipase enantioselectivity. Journal of Molecular Catalysis. B, Enzymatic, 2000, 10(4): 385-393
[54] Turner N A, Needs E C, Khan JA, et al. Analysis of conformational states of Candida rugosa lipase in solution: implications for mechanism of interfacial activation and separation of open and closed forms. Biotechnology and Bioengineering, 2001, 72: 114-118
[55] Zhu K, Jutila A, Tuominen E K J, et al. Effects of i-propanol on the structural dynamics of Thermomyces lanuginose lipase revealed by tryptophan fluorescence. Protein Science, 2001, 10: 339-351
[56] James J, Lakshmi B, Raviprasad V, et al. Insights from molecular dynamics simulations into pH-dependent enantioselective hydrolysis of ibuprofen esters by Candida rugosa lipase. Protein Engineering, 2003, 16(12): 1017-1024
[57] Secundo F, Carrea G, Tarabiono C, et al. Activity and enantioselectivity of wildtype and lid mutated Candida rugosa lipase isoform 1 in organic solvents. Biotechnol- ogy and Bioengineering, 2004, 86(2): 236-240
[58] Tejo B A, Salleh A B, Pleiss J. Structure and dynamics of Candida rugosa lipase: the role of organic solvent. Journal of Moleculer Model, 2004: 10: 358-366
[59] Foresti M L, Ferreira M L. Computational approach to solvent-free synthesis of ethyl oleate using Candida rugosa and Candida antarctica B lipases I Interfacial activation and substrate (ethanol, oleic acid) adsorption. Biomacromolecules, 2004,5(23): 66-75
[60] Hockney R C. Recent developments in heterologous protein production in Escherichia coli. Trends in Biotechnology, 1994, 12(11): 456-463
[61] Bruel C, Cha K, Reeves P J, et a1. Rhodopsin kinase: expressionin mammalian cells and a two-step purification. Proceedings of the National Academy Sciences of United States of American, 2000, 97(7): 3004-3009
[62] Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Reviews, 2000, 24: 45-66
[63] Hancock R E, Lehrer R. Cationic peptides: a new source of antibiotic. Trends in Biotechnology, 1998, 16(2): 82-88
[64] Cregg J M, Vedvick T S, Raschke W C. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology, 1993, 11(8): 905-910
[65] Peng Y C, Acheson N H. Production of active polyomavirus large T antigen in yeast Pichia pastoris. Virus Research, 1997, 49(1): 41-47
[66]胡世界,罗素兰,张吉贞等.巴斯德毕赤酵母表达系统及其高水平表达策略.生物技术, 2007, 17(6): 78-83
[67] Scorer C A, Buckholz R G, Clare J J, et al. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene, 1993, 136: 111-119
[68] Romanos M A, Scorer C A, Clare J J. Foreign gene expression in yeast: A review Yeast, 1992, 8: 423-488
[69] Steffan N H, Hery D H, Robot M H, et al. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 1989, 77: 51-59
[70] Cregg J M, Cereghino J L, Shi J, et al. Recombinant Protein Expression in Pichia pastoris. Moleculer Biotechnology, 2000, 16(1): 23-25
[71] Shu Z Y, Yan Y J, Yang J K, et al. Aspergillus niger lipase: gene cloning, over-expression in Escherichia coli and in vitro refolding. Biotechnology Letter, 2007, 29: 1875-1879
[72]郭尧君.蛋白质电泳实验技术.科学出版社, 1998, 76-78