葡萄糖氧化酶基因的克隆及高效表达
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摘要
本文从一株产葡萄糖氧化酶(Glucose Oxidase,简称GOD)的点青霉中克隆得到了葡萄糖氧化酶编码序列,构建了高效表达、有效分泌的葡萄糖氧化酶的重组毕赤酵母生物反应器,这为开发高效生产葡萄糖氧化酶的生物技术提供途径。序列分析表明,该葡萄糖氧化酶基因长1815bp,共编码604个氨基酸,前18个氨基酸为信号肽编码序列,氨基酸序列中共有4个潜在的糖基化位点。将克隆得到的葡萄糖氧化酶基因在大肠杆菌和毕赤酵母中进行表达,构建出GOD的高产酵母菌株。在酵母α-Factor及AOX1启动子和终止子(TT)的调控下,点青霉GOD在GS115中大量表达并分泌到胞外。在3L发酵罐水平检测条件下,甲醇诱导132小时,发酵液中的酶活达到111.3U/mL。
The gene for Penicillium notatum glucose oxidase(EC1.1.3.4)has been cloned from its genome. The gene, isolated by PCR consisted of 1815 bp encoding 604 amino acid residues, and is preceded by a 18-amino acid presequence. The enzyme contains 4 potential sites for N-linked glycosylation.
The DNA fragment encoding Penicillium notatum glucose oxidase, was cloned into vector of pPIC9 for expression in Pichia pastoris. When transformed into methylotrophic yeast Pichiapastoris GS115, The constructed plasmid pPIC9-sgod directed the synthesis and secretion of functionally active GOD. After induction in 3L fermentor for 132h, the GOD activity in the medium reached to 111.3U/mL.
The gene for Penicillium notatum glucose oxidase(EC1.1.3.4)has been cloned from its genome. The gene, isolated by PCR consisted of 1815 bp encoding 604 amino acid residues, and is preceded by a 18-amino acid presequence. The enzyme contains 4 potential sites for N-linked glycosylation.
The DNA fragment encoding Penicillium notatum glucose oxidase, was cloned into vector of pPIC9 for expression in Pichia pastoris. When transformed into methylotrophic yeast Pichiapastoris GS115, The constructed plasmid pPIC9-sgod directed the synthesis and secretion of functionally active GOD. After induction in 3L fermentor for 132h, the GOD activity in the medium reached to 111.3U/mL.
引文
[1]张树政.酶制剂工业.北京:科学出版社,1983
[2]罗九甫.酶与酶工程.上海:上海交通大学出版社,1996
[3]王志新,于宏伟,贾英民等.黑曲霉A9葡萄糖氧化酶的酶学性质研究.河北农业大学学报.2006,7(4):69～72
[4]李艳,李静.葡萄糖氧化酶及其应用.食品工程.2006,9(3):9～11
[5]周亚凤,张先恩,刘虹,等。黑曲霉葡萄糖氧化酶基因的克隆及其在酵母中的高效表达.生物工程学报.2001,17(4):400～405
[6]李晶,赵晓祥,沙长清,张淑梅,田洁萍,甲醇酵母基因表达系统的研究进展,生物工程进展,1999,19(2),17～20
[7]张伟,姚斌,王磊等,来源于Aspergillus candidus的乳糖酶基因的克隆及序列分析,生物工程学报,2002,18(5):566～571
[8]郭晓贤,刘峰.葡萄糖氧化酶产生菌的快速筛选及其产酶条件的研究.饲料工业.2007,28(8):17～20
[9] Frederick K R ,Tung J,et al. Glucose oxidase from Aspergillus niger. Biol Chem ,1990,265 :3793～3802
[10] Whittington H , Kerry2Williams S,et al.Expression of the Aspergillusniger glucose oxidase gene in A. nidulans and Saccharomyces cerevi2siae. Curr Genet,1991,18:531～536 [11 CereghinoGP, Cregg JM., Applications of yeast in biotechnology: protein production and genetic analysis., Curr Opin Biotechnol, 1999: 10(5), 422～427
[12]刘文,胡巍,酵母表达基因工程产物特性分析,生物工程进展,2001:21(2),74～76)
[13]马孟根,王红宁,巴斯德毕赤酵母表达外源蛋白研究进展,四川农业大学学报,2001:19(3),277～280
[14] Remacle JE, Albrecht G, Brys R, Braus GH, Huylebroeck D., Three classes of mammalian transcription activation domain stimμLate transcription in Schizosaccharomyces pombe, EMBO J, 1997: 16(18), 5722～5729
[15]Tohda H, Okada H, Giga-Hama Y, Okayama H, A copy-number-controlled expression vector for the fission yeast Schizosaccharomyces pombe, Gene, 1994:150(2), 275～280
[16] Braspenning J, Meschede W, Marchini A, MμLler M, Gissmann L, Tommasino M., Secretion of heterologous proteins from Schizosaccharomyces pombe using the homologous leader sequence of pho1+ acid phosphatase, Biochem Biophys Res Commun, 1998:245(1), 166～171
[17]李艳,王正祥,诸葛健,酵母作为外源基因表达系统的研究进展,生物工程进展,2001:21(2),10～14
[18]Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA., Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris, Gene, 1997:190(1), 55～62
[19]彭毅,步威,康良仪,甲醇酵母表达系统,生物技术通报,2000:1,38～41
[20]李晶,赵晓祥,沙长清,张淑梅,田洁萍,甲醇酵母基因表达系统的研究进展,生物工程进展,1999:19(2),17～20
[21]James M. Cregg, Kevin J. Barringer, Anita Y. Hessler, Knut R. Madden, Pichia pastoris as a host system for transformations, MolecμLar and CellμLar Biology, 1985: 5(12), 3376～3385
[22]章如安,杨晟,邱荣德,袁中一,巴斯德毕赤酵母表达体系研究及进展,微生物学报,2000:27(5),371～373
[23]马孟根,王红宁,巴斯德毕赤酵母表达外源蛋白研究进展,四川农业大学学报,2001:19(3),277～280
[24] Rank D, Majak, Stephan T, Use of the glyceraldehyde-3-phosphate dehydrogenase promoter for production of functional mammalian membrane transport proteins in the yeast Pichia pastoris, Biochem Bio-phy Res Commun, 1998:250, 531～536
[25]阳菁,龙綮新,杨林,王珣章,甲基营养型酵母表达载体系统研究进展,应用与环境微生物,2001:7(5),502～506
[26]Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM., Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regμLation and use of its promoter, Gene, 1997: 186(1), 37～44
[27]聂东宋,梁宋平,李敏,外源蛋白在巴氏毕赤酵母中高效表达的策略,吉首大学学报(自然科学版),2001:22(3),40～44
[28] Wegner G H, Biochemical conversions by yeast fermentation at high cell dendities,USA Patent, 1983:44(14)329
[29]Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA., Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris, Gene, 1997:190(1), 55～62
[30]刘文,胡巍,酵母表达基因工程产物特性分析,生物工程进展,2001:21(2)74～76
[31]李育阳,外源基因在酵母中表达产物的分泌,生物工程学报,1987:3(2),81～85
[32]姜勇,王忠,施蕴渝等.利用PCR方法鉴定甲醇酵母转化子的表型.激光生物学报,1998:7(3),188～191
[33]刘谨,王汉中,丝状真菌外源基因表达系统研究的现状及展望,微生物学通报,2000:27(6),453～457
[34] Barr KA, Hopkins SA, Sreekrishna K, Protocol for efficient secretion of HAS developed from Pichia pastoris, Pharm Eng, 1992:12, 48～51
[35] Mei M, Whittaker, James W, Expression of recombinant galactose oxidase by Pichia pastoris, Protein Expression and Purification, 2000:20, 105～111
[36]郑华学试论生物技术在我国食品工业中的应用。河西学院学报,2003:(5)99～102
[37] KESHSVARZ K. Nonphytase phosphorus requirement of laying hens with and without phytase on a phase feeding program[J ] . PoμLt Sci ,2000 , 79 (5) : 748～763
[38]ZHAND Z B , KORNEGAY E T , RADCL IFFE J S ,et al . Comparison of genetically engineered microbial and plant phytase for young broilers [ J ] . PoμLt Sci ,2000 ,79 (5) : 709～717
[39] STAHL C H , RONEKER K R , THORNTON J R ,et al . A new phytase expressed in yeast effectively im- proves the bioavailability of phytase phosphorus to weanling pigs [J ] . J Anim Sci , 2000 ,78 (3) : 668～674
[40]PEN J , VERWOERD T C , VAN PARIDON P A ,et al . Phytase2containing transgenic seeds as a novelfeed additive for improved phosphorus utilization [J ] .Research , 1993 , 11(6) : 811～814
[41] VERWOERD T C , VAN PARIDON P A , ALBERT J , et al . Stable accumμLation of Aspergillus niger phy-tase in transgenic tobacco leaves [ J ] . Plant Physiol ,1995 ,109 :1 199～1 205
[2]罗九甫.酶与酶工程.上海:上海交通大学出版社,1996
[3]王志新,于宏伟,贾英民等.黑曲霉A9葡萄糖氧化酶的酶学性质研究.河北农业大学学报.2006,7(4):69～72
[4]李艳,李静.葡萄糖氧化酶及其应用.食品工程.2006,9(3):9～11
[5]周亚凤,张先恩,刘虹,等。黑曲霉葡萄糖氧化酶基因的克隆及其在酵母中的高效表达.生物工程学报.2001,17(4):400～405
[6]李晶,赵晓祥,沙长清,张淑梅,田洁萍,甲醇酵母基因表达系统的研究进展,生物工程进展,1999,19(2),17～20
[7]张伟,姚斌,王磊等,来源于Aspergillus candidus的乳糖酶基因的克隆及序列分析,生物工程学报,2002,18(5):566～571
[8]郭晓贤,刘峰.葡萄糖氧化酶产生菌的快速筛选及其产酶条件的研究.饲料工业.2007,28(8):17～20
[9] Frederick K R ,Tung J,et al. Glucose oxidase from Aspergillus niger. Biol Chem ,1990,265 :3793～3802
[10] Whittington H , Kerry2Williams S,et al.Expression of the Aspergillusniger glucose oxidase gene in A. nidulans and Saccharomyces cerevi2siae. Curr Genet,1991,18:531～536 [11 CereghinoGP, Cregg JM., Applications of yeast in biotechnology: protein production and genetic analysis., Curr Opin Biotechnol, 1999: 10(5), 422～427
[12]刘文,胡巍,酵母表达基因工程产物特性分析,生物工程进展,2001:21(2),74～76)
[13]马孟根,王红宁,巴斯德毕赤酵母表达外源蛋白研究进展,四川农业大学学报,2001:19(3),277～280
[14] Remacle JE, Albrecht G, Brys R, Braus GH, Huylebroeck D., Three classes of mammalian transcription activation domain stimμLate transcription in Schizosaccharomyces pombe, EMBO J, 1997: 16(18), 5722～5729
[15]Tohda H, Okada H, Giga-Hama Y, Okayama H, A copy-number-controlled expression vector for the fission yeast Schizosaccharomyces pombe, Gene, 1994:150(2), 275～280
[16] Braspenning J, Meschede W, Marchini A, MμLler M, Gissmann L, Tommasino M., Secretion of heterologous proteins from Schizosaccharomyces pombe using the homologous leader sequence of pho1+ acid phosphatase, Biochem Biophys Res Commun, 1998:245(1), 166～171
[17]李艳,王正祥,诸葛健,酵母作为外源基因表达系统的研究进展,生物工程进展,2001:21(2),10～14
[18]Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA., Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris, Gene, 1997:190(1), 55～62
[19]彭毅,步威,康良仪,甲醇酵母表达系统,生物技术通报,2000:1,38～41
[20]李晶,赵晓祥,沙长清,张淑梅,田洁萍,甲醇酵母基因表达系统的研究进展,生物工程进展,1999:19(2),17～20
[21]James M. Cregg, Kevin J. Barringer, Anita Y. Hessler, Knut R. Madden, Pichia pastoris as a host system for transformations, MolecμLar and CellμLar Biology, 1985: 5(12), 3376～3385
[22]章如安,杨晟,邱荣德,袁中一,巴斯德毕赤酵母表达体系研究及进展,微生物学报,2000:27(5),371～373
[23]马孟根,王红宁,巴斯德毕赤酵母表达外源蛋白研究进展,四川农业大学学报,2001:19(3),277～280
[24] Rank D, Majak, Stephan T, Use of the glyceraldehyde-3-phosphate dehydrogenase promoter for production of functional mammalian membrane transport proteins in the yeast Pichia pastoris, Biochem Bio-phy Res Commun, 1998:250, 531～536
[25]阳菁,龙綮新,杨林,王珣章,甲基营养型酵母表达载体系统研究进展,应用与环境微生物,2001:7(5),502～506
[26]Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM., Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regμLation and use of its promoter, Gene, 1997: 186(1), 37～44
[27]聂东宋,梁宋平,李敏,外源蛋白在巴氏毕赤酵母中高效表达的策略,吉首大学学报(自然科学版),2001:22(3),40～44
[28] Wegner G H, Biochemical conversions by yeast fermentation at high cell dendities,USA Patent, 1983:44(14)329
[29]Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA., Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris, Gene, 1997:190(1), 55～62
[30]刘文,胡巍,酵母表达基因工程产物特性分析,生物工程进展,2001:21(2)74～76
[31]李育阳,外源基因在酵母中表达产物的分泌,生物工程学报,1987:3(2),81～85
[32]姜勇,王忠,施蕴渝等.利用PCR方法鉴定甲醇酵母转化子的表型.激光生物学报,1998:7(3),188～191
[33]刘谨,王汉中,丝状真菌外源基因表达系统研究的现状及展望,微生物学通报,2000:27(6),453～457
[34] Barr KA, Hopkins SA, Sreekrishna K, Protocol for efficient secretion of HAS developed from Pichia pastoris, Pharm Eng, 1992:12, 48～51
[35] Mei M, Whittaker, James W, Expression of recombinant galactose oxidase by Pichia pastoris, Protein Expression and Purification, 2000:20, 105～111
[36]郑华学试论生物技术在我国食品工业中的应用。河西学院学报,2003:(5)99～102
[37] KESHSVARZ K. Nonphytase phosphorus requirement of laying hens with and without phytase on a phase feeding program[J ] . PoμLt Sci ,2000 , 79 (5) : 748～763
[38]ZHAND Z B , KORNEGAY E T , RADCL IFFE J S ,et al . Comparison of genetically engineered microbial and plant phytase for young broilers [ J ] . PoμLt Sci ,2000 ,79 (5) : 709～717
[39] STAHL C H , RONEKER K R , THORNTON J R ,et al . A new phytase expressed in yeast effectively im- proves the bioavailability of phytase phosphorus to weanling pigs [J ] . J Anim Sci , 2000 ,78 (3) : 668～674
[40]PEN J , VERWOERD T C , VAN PARIDON P A ,et al . Phytase2containing transgenic seeds as a novelfeed additive for improved phosphorus utilization [J ] .Research , 1993 , 11(6) : 811～814
[41] VERWOERD T C , VAN PARIDON P A , ALBERT J , et al . Stable accumμLation of Aspergillus niger phy-tase in transgenic tobacco leaves [ J ] . Plant Physiol ,1995 ,109 :1 199～1 205
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