霉菌脂肪酶基因的克隆与表达
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摘要
脂肪酶(Lipase，EC3．1．1．3)是一类催化脂肪酸甘油酯水解的酶类，它作为生物催化剂的重要组成部分有着广泛的用途。米根霉(Rhizopus oryzae)脂肪酶具有良好的1，3位置专一性，它能催化甘油三酯产生脂肪酸和单甘酯，单甘酯是一种重要的非离子型表面活性剂，已广泛地应用于食品、乳制品和化妆品等行业。因此，利用基因手段，构建产酶活力高的基因工程菌对生产单甘酯等具有重要的意义，是当前研究的热点，另外，脂肪酶活力至今仍然没有统一的测定方法，如何准确测定酶活力大小，对研究脂肪酶的特性及应用具有重要意义。
本论文利用本实验室保存的含米根霉(Rhizopus oryzae 3．2686)脂肪酶基因ROL的重组质粒pGMT-ROL，设计两对特异性引物定向克隆甘油三酰酯脂肪酶基因ROL，分别与与载体pET-28a和pET-11c连接，构建成表达重组载体pET-28a-ROL和pET-11c-ROL。采用氯化钙法，将重组表达载体分别转入受体菌组BL21(DE3)和Origami DE3中，分别通过Kana抗性平板和Amp抗性平板筛选初步筛选获得阳性克隆。通过菌落PCR和提取重组质粒做筛选，筛选到的重组子，用IPTG做诱导基因表达，表达产物经SDS-PAGE电泳分析，可鉴定重组菌表达了重组蛋白，约35KD。
另外，本文比较和改进了酸碱滴定法，对硝基苯酚比色法和铜皂法三种常用的脂肪酶活力测定方法。具体比较了其所耗时间，测出酶活大小的准确性，灵敏度。为对硝基苯酚比色法找到了有效的方法来终止反应，改进的对硝基苯酚比色法具有重复性好的特点。另外还对其中的铜皂法进行了改进，用甲苯代替了苯，取得了较好的结果，改进的铜皂法具有毒性小、快捷的优点。通过对几种测定方法的比较改进和优缺点的讨论，为以后测定脂肪酶活力提供选择依据。
Lipase is a typical enzyme which catalyzes the hydrolysis of long chain triglycerides. They constitute the most important group of biocatalysts for biotechnological applications. Lipase from Rhizopus oryzae has 1,3 positional specificity, it can hydrolyze triacylglycerol producing fatty acid and monoglyceride (MG), MG is an important non-ionic surfactants, widely used as emulsifier in the food stuff and other industries. As a result, today people focus on the construction of gene recombinant strain with high enzyme activity by gene measuses.Besides,. There is still no uniform method to measure the activity of lipase exactly , while it is important in reasearching of characters and amplication of lipases.
In This thesis , the gene which encoding lipase ROL from Rhizopus oryzae was amplified by PCR and cloned into vector pGM-T was saved in our lab.The Recombinant plasmid Is named pGMT-ROL, and I designed primer pair 4 and 4.2 to clone the lipase gene ROL from pGMT-ROL,separated connect with pET-28a and pET-11c. The recombinant plasmid are named pET-28a-ROL and pET-11c-ROL, and pET-28a-ROL was transferred into E.coli.BL21 and pET-11c-ROL was transferred into E.coli.Origami DE3. Expression Recombinant strains were obtained separated by Kana and Amp plates screening ,the Expression Recombinant strains were farther filtered by strain PCR, and The engineered strain was expressed the gene ROL by the induction of IPTG. The expression products of ROL gene was analysis by SDS-PAGE, the result indicated that ROL gene was functionally expressed in E.coliBL21 and Origami DE3. Recombinant lipase had a molecular mass of 35kD.
On the other side,This thesis discusses three assays in common use,including the spectrophotometry assay with the synthetic substrate p-NP palmitate, the spectrophotometric assay using the formation of copper soaps and the alkaly titrition assay.We compare the consuming time of reaction, vericity and repeatity of methods.We have found an effective method to terminate the reaction for the spectrophotometry assay with the substrate p-NP palmitate,this modified method shows good repeatity and takes less time relatively.In addition,we also improved the copper soaps assay by using toluene insead of benzene,it has merits of less toxicity and good repeatity. Through discussing merits and demerits of these assays and improvement of them ,This thesis provides chosen basises of assays for lipase activity measure.
本论文利用本实验室保存的含米根霉(Rhizopus oryzae 3．2686)脂肪酶基因ROL的重组质粒pGMT-ROL，设计两对特异性引物定向克隆甘油三酰酯脂肪酶基因ROL，分别与与载体pET-28a和pET-11c连接，构建成表达重组载体pET-28a-ROL和pET-11c-ROL。采用氯化钙法，将重组表达载体分别转入受体菌组BL21(DE3)和Origami DE3中，分别通过Kana抗性平板和Amp抗性平板筛选初步筛选获得阳性克隆。通过菌落PCR和提取重组质粒做筛选，筛选到的重组子，用IPTG做诱导基因表达，表达产物经SDS-PAGE电泳分析，可鉴定重组菌表达了重组蛋白，约35KD。
另外，本文比较和改进了酸碱滴定法，对硝基苯酚比色法和铜皂法三种常用的脂肪酶活力测定方法。具体比较了其所耗时间，测出酶活大小的准确性，灵敏度。为对硝基苯酚比色法找到了有效的方法来终止反应，改进的对硝基苯酚比色法具有重复性好的特点。另外还对其中的铜皂法进行了改进，用甲苯代替了苯，取得了较好的结果，改进的铜皂法具有毒性小、快捷的优点。通过对几种测定方法的比较改进和优缺点的讨论，为以后测定脂肪酶活力提供选择依据。
Lipase is a typical enzyme which catalyzes the hydrolysis of long chain triglycerides. They constitute the most important group of biocatalysts for biotechnological applications. Lipase from Rhizopus oryzae has 1,3 positional specificity, it can hydrolyze triacylglycerol producing fatty acid and monoglyceride (MG), MG is an important non-ionic surfactants, widely used as emulsifier in the food stuff and other industries. As a result, today people focus on the construction of gene recombinant strain with high enzyme activity by gene measuses.Besides,. There is still no uniform method to measure the activity of lipase exactly , while it is important in reasearching of characters and amplication of lipases.
In This thesis , the gene which encoding lipase ROL from Rhizopus oryzae was amplified by PCR and cloned into vector pGM-T was saved in our lab.The Recombinant plasmid Is named pGMT-ROL, and I designed primer pair 4 and 4.2 to clone the lipase gene ROL from pGMT-ROL,separated connect with pET-28a and pET-11c. The recombinant plasmid are named pET-28a-ROL and pET-11c-ROL, and pET-28a-ROL was transferred into E.coli.BL21 and pET-11c-ROL was transferred into E.coli.Origami DE3. Expression Recombinant strains were obtained separated by Kana and Amp plates screening ,the Expression Recombinant strains were farther filtered by strain PCR, and The engineered strain was expressed the gene ROL by the induction of IPTG. The expression products of ROL gene was analysis by SDS-PAGE, the result indicated that ROL gene was functionally expressed in E.coliBL21 and Origami DE3. Recombinant lipase had a molecular mass of 35kD.
On the other side,This thesis discusses three assays in common use,including the spectrophotometry assay with the synthetic substrate p-NP palmitate, the spectrophotometric assay using the formation of copper soaps and the alkaly titrition assay.We compare the consuming time of reaction, vericity and repeatity of methods.We have found an effective method to terminate the reaction for the spectrophotometry assay with the substrate p-NP palmitate,this modified method shows good repeatity and takes less time relatively.In addition,we also improved the copper soaps assay by using toluene insead of benzene,it has merits of less toxicity and good repeatity. Through discussing merits and demerits of these assays and improvement of them ,This thesis provides chosen basises of assays for lipase activity measure.
引文
[1] 张树政．酶制剂工业[M]．北京：科学出版社，1984：655-668
[2] G. Pauline, K. Martin , K. S. Trichur. Thermophilic bacterial isolates producing lipase[J]. FEMS Microbiology Letter. 1987,48:339-343
[3] 曲音波．脂肪酶菌种库的建立、酶学性质以及在有机合成中的应用[D]．山东：山东大学，1999
[4] Lee K T, Akoh C C. Structured Lipids:Synthesis and Application[J]. Fed Rev Int. 1998,14(1):17-34
[5] Xu X, Mu H, Skands A R H, et al. Parameters Affecting Diacylglycerol Formation During the Production of Specific Structured Lipids by Lipase-Catalyzed lnteresterification [J]. J. Am. Oil Chem.Soc., 1999,76(2): 175-181
[6] Balcao V M, Paiva A L, Malcata F X. Bioreactors with Immobilized Lipase: State of Art[J]. Enzyme Microb. Technil.,1996,18(6):392-416
[7] Yahya A R M, Anderson W S, Moo-Young M. Ester Synthesis in Lipase-Catalyzed Reactions[J]. Enzyme Microb. Technol., 1998,23(6):31-35
[8] 李香春，甄宗园．脂肪酶特性及其应用[J]．粮食与油脂，2003，3(1)：19-2
[9] Atsushi Tschiya,Hidekazu Nakazawa,Jinichi Toida.et al. Cloning and nucleotide sequence of the mono-and diacylglycerol lipase gene(mdlB) of Aspergillus oryzae[J]. FEMS Microbiol. Lett., 1996,143(7):63-67
[10] S. Yamaguchi, T. Mase. Purification and characterization of mono-and diacylglycerol lipase isolated from Pencillium camembertii U-150[J]. Appl. Microbiol. Biotechnol., 1991.34(3):720-725
[11] 曹淑桂．脂肪酶的底物特异性及其应用潜力[J]．生物化学与生物物理进展，1995，22(1)：9-13
[12] 刘书来，毕艳兰，杨天奎．单甘酯合成及其应用[J]．粮食与油脂，2001，11(5)：30-31
[13] Hess R. Lipase-catalyzed synthesis of monostearoylglycerol in organic solvents from 1,2-O-isopropylidene glycerol[J]. Enzyme and Microbial Technology, 1995,17(3): 725-727
[14] Julieta B. Lipase-catalyzed synthesis of monoacylglycerol in a homogeneous system[J]. Biotechnology Letters, 2003,25(6):641-643
[15] 傅四周，尹春华，徐家立等．根霉脂肪酶催化合成单甘酯的研究[J]．北京化工大学学报，2002，29(5)：30-33
[16] 邬敏辰．圆弧青霉PG37碱性脂肪酶的发酵生产、分离纯化及基因克隆和表达[D]．上海：复旦大学，2000
[17] K. Nikolcit. Comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase: Indication for a C-terminal phospholipase domain[J]. Eur. J. Biochem., 1995,228(8):732-738
[18] A. Sanchez, P. Ferrer, A Serrano, et al. A controlled fed-batch cultivation for the production of new crude lipase from Candida rugosa with improved properties in fine chemistry[J]. Biotechnol., 1999,69(4): 169-182
[19] K. G. Anderas, L. P. Annette, R. C. Leda, et al.Lipase production by Penicillium restrictum in solid-state fermentation using babassu oil cake as substrate[J].Process Biochem.,1999,35(4):85-90
[20] 邬敏辰，孙崇荣，李江华等．圆弧青霉碱性脂肪酶发酵和纯化的研究[J]．江苏食品与发酵，2000，1：1-4
[21] 孙宏丹，胡军，姚民昌．无根根霉脂肪酶的菌株筛选及产酶条件设置[J]．大连医科大学学报，2001，23(2)：150-152
[22] E. J. Gilbert, J. W. Drozd, C. W. Jones. Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2[J]. J. Gen. Microbiol., 1991,137(6):2215-2221
[23] H. Chander, et al. Factors affecting lipase production in Aspergillus wentii[J]. Food Science, 1980,45(4):598-600
[24] 杨学昊，谭天伟．固定化根霉发酵生产脂肪酶[J]．生物工程学报，2004，20(2)：284-286
[25] 郑毅，叶海梅，吴朝娟等．脂肪酶活力测定研究进展[J]．工业微生物，2005，35(4)：36-40
[26] 何耀强，王炳武，谭天伟．假丝酵母99-125脂肪酶的发酵工艺研究[J]．生物工程学报，2004，20(6)：918-921
[27] T. Vordewulbecke, K. Kieshich, H. Erdnann. Comparison of lipases by different assays[J]. Enzyme Microb. Technol,1992, 14(3):631-639
[28] F. Gotz, et al. Complete nucleotide sequence of the lipase gene from Staphylococcus hyicus clone in Staphylococcus carnou[J]. Nucleic Acids Res., 1985,13:5895-5906
[29] G. Gijs, W. J. Ronad and J. Q. Wim. Development of a lipase fermentation process that uses a recombinant Pseudomonas alcaligenes strain[J]. Appl. Enciron. Microbiol., 1998,64(3):2644-2651
[30] Takahashi S, Ueda M, Tanaka A. Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae[J]. Appl Microbiol Biotechnol, 2001,55(4):454-462
[31] Kohno M, Enatsu M, Yoshiizumi M, et al. High-Level Expression of Rhizopus niveus Lipase in the Yeast Saccharomyces cerevisiae and Structural Properties of the Expressed Enzyme[J]. Protein Expr Purif, I999,15(6):327-335
[32] Washida M, et al. Spacer-mediated display of active lipase on the yeast cell surface[J]. Appl Microbiol Biotechnol, 2001,56(3):681-686
[33] 林琳，谢必峰，杨冠珍，等．扩展青霉PF898碱性脂肪酶cDNA的克隆及序列分析[J]．中国生物化学与分子生物学报，2002，18(1)：32-37
[34] 袁彩，林琳，施巧琴，等．扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达[J]．生物工程学报，2003，19(2)：231-235
[35] 邬敏辰，朱吉，黄伟达，等．圆弧青霉脂肪酶基因的克隆和表达[J]．无锡轻工大学学报，2002，21(3)：218-223
[36] 邬敏辰．脂肪酶基因结构和氨基酸序列的比较[J]．江南学院学报，2001，16(2)：1-4
[37] Eli Reed J L, Palsson B Thirteen Years of Building Constraint—Based In Silico Models of Escherichia coli[J]. Journal of Bacteriology, 2003, 185(9): 2692
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[45] 何耀强，王炳武，谭天伟．假丝酵母99-125脂肪酶的发酵工艺研究[J]．生物工程学报，2004，20(6)：918-921
[46] T. Vordewulbecke, K. Kieshich, H. Erdnann. Comparison of lipases by different assays[J]. Enzyme Microbiology Technology, 1992,14(6):631-639
[47] Takeshi S, Toru N, Tatsuo K, Tokuzo N, Nobuyoshi E.A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: gene cloning, purification, and characterization[J]. J Mol Catal B Enzymatic, 2002;16(7):244-253
[48] W. G. Duncombe. The colorimetric determination of long chain fatty acids in the 0.05-0.5μmol range[J]. Biochem,1963,88(4):7-9
[49] 纪建业．脂肪酶活力测定方法的改进[J]．通化师范学院学报，2005，26(6)：51-53
[50] Schmidt DC, Sztajer H, Stocklein W, Menge U, Schmid RD. Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus. Biochim Biophys Acta,1994,1214(1):43-53
[2] G. Pauline, K. Martin , K. S. Trichur. Thermophilic bacterial isolates producing lipase[J]. FEMS Microbiology Letter. 1987,48:339-343
[3] 曲音波．脂肪酶菌种库的建立、酶学性质以及在有机合成中的应用[D]．山东：山东大学，1999
[4] Lee K T, Akoh C C. Structured Lipids:Synthesis and Application[J]. Fed Rev Int. 1998,14(1):17-34
[5] Xu X, Mu H, Skands A R H, et al. Parameters Affecting Diacylglycerol Formation During the Production of Specific Structured Lipids by Lipase-Catalyzed lnteresterification [J]. J. Am. Oil Chem.Soc., 1999,76(2): 175-181
[6] Balcao V M, Paiva A L, Malcata F X. Bioreactors with Immobilized Lipase: State of Art[J]. Enzyme Microb. Technil.,1996,18(6):392-416
[7] Yahya A R M, Anderson W S, Moo-Young M. Ester Synthesis in Lipase-Catalyzed Reactions[J]. Enzyme Microb. Technol., 1998,23(6):31-35
[8] 李香春，甄宗园．脂肪酶特性及其应用[J]．粮食与油脂，2003，3(1)：19-2
[9] Atsushi Tschiya,Hidekazu Nakazawa,Jinichi Toida.et al. Cloning and nucleotide sequence of the mono-and diacylglycerol lipase gene(mdlB) of Aspergillus oryzae[J]. FEMS Microbiol. Lett., 1996,143(7):63-67
[10] S. Yamaguchi, T. Mase. Purification and characterization of mono-and diacylglycerol lipase isolated from Pencillium camembertii U-150[J]. Appl. Microbiol. Biotechnol., 1991.34(3):720-725
[11] 曹淑桂．脂肪酶的底物特异性及其应用潜力[J]．生物化学与生物物理进展，1995，22(1)：9-13
[12] 刘书来，毕艳兰，杨天奎．单甘酯合成及其应用[J]．粮食与油脂，2001，11(5)：30-31
[13] Hess R. Lipase-catalyzed synthesis of monostearoylglycerol in organic solvents from 1,2-O-isopropylidene glycerol[J]. Enzyme and Microbial Technology, 1995,17(3): 725-727
[14] Julieta B. Lipase-catalyzed synthesis of monoacylglycerol in a homogeneous system[J]. Biotechnology Letters, 2003,25(6):641-643
[15] 傅四周，尹春华，徐家立等．根霉脂肪酶催化合成单甘酯的研究[J]．北京化工大学学报，2002，29(5)：30-33
[16] 邬敏辰．圆弧青霉PG37碱性脂肪酶的发酵生产、分离纯化及基因克隆和表达[D]．上海：复旦大学，2000
[17] K. Nikolcit. Comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase: Indication for a C-terminal phospholipase domain[J]. Eur. J. Biochem., 1995,228(8):732-738
[18] A. Sanchez, P. Ferrer, A Serrano, et al. A controlled fed-batch cultivation for the production of new crude lipase from Candida rugosa with improved properties in fine chemistry[J]. Biotechnol., 1999,69(4): 169-182
[19] K. G. Anderas, L. P. Annette, R. C. Leda, et al.Lipase production by Penicillium restrictum in solid-state fermentation using babassu oil cake as substrate[J].Process Biochem.,1999,35(4):85-90
[20] 邬敏辰，孙崇荣，李江华等．圆弧青霉碱性脂肪酶发酵和纯化的研究[J]．江苏食品与发酵，2000，1：1-4
[21] 孙宏丹，胡军，姚民昌．无根根霉脂肪酶的菌株筛选及产酶条件设置[J]．大连医科大学学报，2001，23(2)：150-152
[22] E. J. Gilbert, J. W. Drozd, C. W. Jones. Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2[J]. J. Gen. Microbiol., 1991,137(6):2215-2221
[23] H. Chander, et al. Factors affecting lipase production in Aspergillus wentii[J]. Food Science, 1980,45(4):598-600
[24] 杨学昊，谭天伟．固定化根霉发酵生产脂肪酶[J]．生物工程学报，2004，20(2)：284-286
[25] 郑毅，叶海梅，吴朝娟等．脂肪酶活力测定研究进展[J]．工业微生物，2005，35(4)：36-40
[26] 何耀强，王炳武，谭天伟．假丝酵母99-125脂肪酶的发酵工艺研究[J]．生物工程学报，2004，20(6)：918-921
[27] T. Vordewulbecke, K. Kieshich, H. Erdnann. Comparison of lipases by different assays[J]. Enzyme Microb. Technol,1992, 14(3):631-639
[28] F. Gotz, et al. Complete nucleotide sequence of the lipase gene from Staphylococcus hyicus clone in Staphylococcus carnou[J]. Nucleic Acids Res., 1985,13:5895-5906
[29] G. Gijs, W. J. Ronad and J. Q. Wim. Development of a lipase fermentation process that uses a recombinant Pseudomonas alcaligenes strain[J]. Appl. Enciron. Microbiol., 1998,64(3):2644-2651
[30] Takahashi S, Ueda M, Tanaka A. Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae[J]. Appl Microbiol Biotechnol, 2001,55(4):454-462
[31] Kohno M, Enatsu M, Yoshiizumi M, et al. High-Level Expression of Rhizopus niveus Lipase in the Yeast Saccharomyces cerevisiae and Structural Properties of the Expressed Enzyme[J]. Protein Expr Purif, I999,15(6):327-335
[32] Washida M, et al. Spacer-mediated display of active lipase on the yeast cell surface[J]. Appl Microbiol Biotechnol, 2001,56(3):681-686
[33] 林琳，谢必峰，杨冠珍，等．扩展青霉PF898碱性脂肪酶cDNA的克隆及序列分析[J]．中国生物化学与分子生物学报，2002，18(1)：32-37
[34] 袁彩，林琳，施巧琴，等．扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达[J]．生物工程学报，2003，19(2)：231-235
[35] 邬敏辰，朱吉，黄伟达，等．圆弧青霉脂肪酶基因的克隆和表达[J]．无锡轻工大学学报，2002，21(3)：218-223
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