短杆菌胆固醇氧化酶基因的表达研究
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摘要
胆固醇氧化酶(EC1.1.3.6 COD)在食品加工、医疗检测、生物抗虫等方面的作用日益受到人们的重视,并且显示出巨大的应用潜力。本文主要研究短杆菌Brevibacterium sp. DGCDC-82胆固醇氧化酶在大肠杆菌和在巴斯德毕赤酵母中的表达。
用PCR方法从Brevibacterium sp. DGCDC-82染色体DNA中扩增出含信号肽序列的胆固醇氧化酶结构基因,插入大肠杆菌表达载体pET28a(+)中,构建成重组质粒pET28a-COD(s+)。以pET28a-COD(s+)为底物,扩增出不含信号肽的胆固醇氧化酶结构基因,构建成重组质粒pET28a-COD(s-)。两种重组载体转化大肠杆菌BL21(DE3)均获得活性表达,不含信号肽基因的转化子酶活较高。在转化子中,两种结构基因与pET28a(+)携带的His-tag融合表达,经IPTG诱导后均表达出分子量约为55kD的蛋白质,目的蛋白大都为没有活性的包涵体,占细胞总蛋白的50%以上。
将不含信号肽的胆固醇氧化酶基因克隆至含trp/lac双启动子的表达载体pTrc99a中,并在大肠杆菌JM109中进行了IPTG诱导表达,并分别考察了诱导温度、时间、IPTG浓度等因素对重组菌表达的胆固醇氧化酶的影响。在优化条件下,该胆固醇氧化酶的酶活可以达到700 U/L。酶学特性分析表明其反应的最适pH为7.5,最适温度为40℃。
将不含信号肽的胆固醇氧化酶结构基因插入巴斯德毕赤酵母分泌型表达载体pPIC9K的EcoR I和Not I位点,构建成pPIC9K-COD,重组质粒以Sac I线性化后电转化巴斯德毕赤酵母GS115。甲醇诱导条件下,重组胆固醇氧化酶仅有少量存在于细胞破碎液的上清液中,并没有在毕赤酵母中获得分泌表达。
The significant functions of cholesterol oxidase (EC 1.1.3.6, COD) in food processing, medical assay and biological pest-resistance were recognized gradually and showed potentially use. This research is mainly focused on expression of Cholesterol oxidase gene from Brevibacterium sp. DGCDC-82 in Escherichia coli and expression of the gene in eukaryotes.
The structure gene of Brevibacterium sp. DGCDC-82 Cholesterol oxidase with signal sequence was amplified from genomic DNA of Brevibacterium sp. DGCDC-82 by PCR technique. The recombinant vector, pET28a-COD(s+), was constructed by inserting the amplified fragment(about 1.5kb)into the expression vector pET28a(+). The DNA fragment encoding mature peptide of COD was amplified from pET28a-COD(s+) and was then inserted into pET28a(+) to construct pET28a-COD(s-).Both recombinant plasmids were transformed into E.coli BL21(DE3) respectively. Promoted and controlled by T7lac promoter, the fusion protein containing His-tag and the peptide of COD with or without signal peptide could be expressed within the cell of recombinant E.coli. The active recombinant enzyme was tested after the two recombinants induced by IPTG.. The expression products of full gene and mature enzyme gene were analyzed by SDS-PAGE indicating that about 55kD protein was obtained, which accounted for about 50% of the cell total protein.
The mature enzyme gene of COD from Brevibacterium sp. DGCDC-82 was inserted into a prokaryotic expression vector pTrc99a. The vector was controlled by trp/lac promoter. A recombinant plasmid pTrc99a-COD was obtained and then transformed into the expression host JM109. The influences of induction conditions such as IPTG concentration, the induction temperature and time of induction on the expression of the recombinant protein were investigated. Under optimal condition, the enzyme activity reached 700U/L.The enzymatic analysis of the cholesterol oxidase revealed that its optimal pH and temperature was 7.5 and 40℃, respectively.
The structure gene of COD without signal sequence was into the site EcoR I、Not I of pPIC9K, a secreting expression vector of Pichia pastoris. The plasmid pPIC9K-COD was linear with Sal I and transform P.pastoris GS115. Inducting with methanol, The active COD enzyme expressed from the recombinant P.pastoris was found only exsisting in the supernatant of the crude cell extract, but not secreted into culture medium.
用PCR方法从Brevibacterium sp. DGCDC-82染色体DNA中扩增出含信号肽序列的胆固醇氧化酶结构基因,插入大肠杆菌表达载体pET28a(+)中,构建成重组质粒pET28a-COD(s+)。以pET28a-COD(s+)为底物,扩增出不含信号肽的胆固醇氧化酶结构基因,构建成重组质粒pET28a-COD(s-)。两种重组载体转化大肠杆菌BL21(DE3)均获得活性表达,不含信号肽基因的转化子酶活较高。在转化子中,两种结构基因与pET28a(+)携带的His-tag融合表达,经IPTG诱导后均表达出分子量约为55kD的蛋白质,目的蛋白大都为没有活性的包涵体,占细胞总蛋白的50%以上。
将不含信号肽的胆固醇氧化酶基因克隆至含trp/lac双启动子的表达载体pTrc99a中,并在大肠杆菌JM109中进行了IPTG诱导表达,并分别考察了诱导温度、时间、IPTG浓度等因素对重组菌表达的胆固醇氧化酶的影响。在优化条件下,该胆固醇氧化酶的酶活可以达到700 U/L。酶学特性分析表明其反应的最适pH为7.5,最适温度为40℃。
将不含信号肽的胆固醇氧化酶结构基因插入巴斯德毕赤酵母分泌型表达载体pPIC9K的EcoR I和Not I位点,构建成pPIC9K-COD,重组质粒以Sac I线性化后电转化巴斯德毕赤酵母GS115。甲醇诱导条件下,重组胆固醇氧化酶仅有少量存在于细胞破碎液的上清液中,并没有在毕赤酵母中获得分泌表达。
The significant functions of cholesterol oxidase (EC 1.1.3.6, COD) in food processing, medical assay and biological pest-resistance were recognized gradually and showed potentially use. This research is mainly focused on expression of Cholesterol oxidase gene from Brevibacterium sp. DGCDC-82 in Escherichia coli and expression of the gene in eukaryotes.
The structure gene of Brevibacterium sp. DGCDC-82 Cholesterol oxidase with signal sequence was amplified from genomic DNA of Brevibacterium sp. DGCDC-82 by PCR technique. The recombinant vector, pET28a-COD(s+), was constructed by inserting the amplified fragment(about 1.5kb)into the expression vector pET28a(+). The DNA fragment encoding mature peptide of COD was amplified from pET28a-COD(s+) and was then inserted into pET28a(+) to construct pET28a-COD(s-).Both recombinant plasmids were transformed into E.coli BL21(DE3) respectively. Promoted and controlled by T7lac promoter, the fusion protein containing His-tag and the peptide of COD with or without signal peptide could be expressed within the cell of recombinant E.coli. The active recombinant enzyme was tested after the two recombinants induced by IPTG.. The expression products of full gene and mature enzyme gene were analyzed by SDS-PAGE indicating that about 55kD protein was obtained, which accounted for about 50% of the cell total protein.
The mature enzyme gene of COD from Brevibacterium sp. DGCDC-82 was inserted into a prokaryotic expression vector pTrc99a. The vector was controlled by trp/lac promoter. A recombinant plasmid pTrc99a-COD was obtained and then transformed into the expression host JM109. The influences of induction conditions such as IPTG concentration, the induction temperature and time of induction on the expression of the recombinant protein were investigated. Under optimal condition, the enzyme activity reached 700U/L.The enzymatic analysis of the cholesterol oxidase revealed that its optimal pH and temperature was 7.5 and 40℃, respectively.
The structure gene of COD without signal sequence was into the site EcoR I、Not I of pPIC9K, a secreting expression vector of Pichia pastoris. The plasmid pPIC9K-COD was linear with Sal I and transform P.pastoris GS115. Inducting with methanol, The active COD enzyme expressed from the recombinant P.pastoris was found only exsisting in the supernatant of the crude cell extract, but not secreted into culture medium.
引文
1. Inouye Y, Taguchi K, Fujii A, et al. Purification and Characterization of Extracellular 3β-H roxysteroid oxidase produced by Streptoverticillium cholesterolicum[J]. Chem Charm Bull, 1982, 30(3):951-958
2. Stadtman T C, Cherkes A, Anfinsen C B. Studies on the microbiological degradation of cholesterol[J]. J Biol Chem, 1954, 206:551—523
3. Uwajima T, Yagi H, Nakamura S, et al. Isolation and crystallization of Extraccllular 3β-Hydroxysteroid oxidase of Brevibacterium sterolicum sp.[J]. Agr Biol Chem,1973,37(10):2354—2350
4. Cheelham P S J, Dunnill P, Lilly M D. The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous[J]. J Biochem, 1982, 201: 515—52
5. Sampson N S, Vrielink A. Cholesterol oxidases: A study of nature’s approach to protein design[J]. Chem Res, 2003, 36: 713—722
6. Gadda G, Wels G, Pollegioni L, et al. Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum[J]. Eur J Biochem. 1997, 250: 369—376
7. Kreit J, Lefebvre G, Germain P. Membrane-bound cholesterol oxidase from Rhodococcus sp. cells. Production and extraction[J]. J Biotechnol. 1994, 33 (3): 271—282
8. Doudyu N, Aono R. Purification of extracellular cholesterol oxidase with high activity in the presence of organic solvents from Pseudomons sp. Strain S T-200[J]. Appl Environ Microbial, 1998, 64(5): 1929—1932
9. Inouye Y, Taguchi K, Fujii A, et al. Purification and characterization of extracellular 3-Hydroxysteroid oxidase produced by Streptoverticillum cholesterolicum[J]. Chem Pharm Bull, 1982, 30(3): 951—958
10.吕莹.胆固醇氧化酶的分离和提纯[D]:[硕士学位论文].北京:中国农业大学, 2001
11. Vrielink A, Lloyd L F, Blow D M. Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A°rerolutin[J]. J. Mol. Biol, 1991, 219(3):533—554
12. Kass I J, Sampson N S. The isomerization catalyzed by Brevibacterium sterolicum cholesterol oxidase proceeds stereospecifically with one base[J]. Biochem Biophy Research Comm, 1995, 206(2):688—693
13. Sampson N S, Kass I J. Isomerization, but not oxidation, is suppressed by a single point mutation, E361Q, in the reaction catalyzed by cholesterol oxidase[J]. J American Chemical Society, 1997, 119(5):856—862
14. Kamei T, Takiguchi Y, Suzuki H, et al. Purification of 3β-Hydroxysteroid oxidase of streptomyces violascens origin by affinity chromatography on cholesterol[J]. Chem Charm Bul, 1978, 26:2799—2804
15.季文明,陈毅力,张和春.比色法测定胆固醇氧化酶酶活[J].无锡轻工大学学报,2000,19(3):251—254
16. Murooka Y, Ishizaki T, Nimi O, et al. Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptonyces lividans with plasmid pIJ702[J]. Appl Environ Microbial, 1986, 52(6):1382—1385
17. Fajishiro K, Ohta T, Hasegawa M, et al. Isolation and identification of gene of cholesterol oxidasefrom Brevibacterium sterolicum ATCC21387, a widely used enzyme in clinical analysis[J]. Biochem Biophys Res Commun, 1990, 172(2):721—727
18.季文明,张和春,王武.胆固醇氧化酶基因的克隆和表达[J].微生物学通报,2000,27(3): 224—227
19. Molmir I, Choi K P , Hayashi N, et al . Secretory overproduction of Streptomyces cholesterol oxidase by Streptomycm lividuns with a multi-copy shuttle vector[J]. J Ferment Bioeng, 1991, 72(5):368—372
20. Ohta T, Fujishiro K, Yamaguchi K, et al. Sequence of gene choB encoding cholesterol oxidase of Brevibacterium sterlicum: comparisonwith choA of Streptomyces sp. SA-COO. Gene, 1991, 103(1) :93—96
21. Sampson N S, Xiaoyu Chen. Increased expression of Brevibacterium sterolicum cholesterol oxidase in Escherichia coli by Genetic modification[J]. Protein Expression and Purification, 1998, 12: 347—352
22. Yen-Rong Chen, Hsin-Ho Huang, Yi-Fang Cheng, et al. Expression of a cholesterol oxidase gene from Arthrobacter simplex in Escherichia coli and Pichia pastoris[J]. Enzyme and Microbial Technology, 2006, 39(4):854—856
23. Nishiya Y, Harada N, Teshima S, et al . Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation[J]. Progress in Biotechnology, 1998, 15:295—320
24. Nishiya Y, Murooka Y. Development of improved cholesterol oxidase and its application[J]. Seibutsu-kogaku, 1999, 77:429—432
25. Black M, Guiomar A J, Guthrie J T, et al. Immobilization of cholesterol oxidase on a hydroxypropyl cellulose-Acrylic acid support system: Relevance to Biosensor Development[J]. Celluosis, 1993, 28:171—181
26.吕陈峰,王龙刚,杨胜利等.胆固醇氧化酶转化蛋黄胆固醇工艺的优化[J].无锡轻工大学学报,2001, 20(6):555—559
27. Chenfeng Lv, Yixin Tang, Longang Wang, et a1. Bioconversion of yolk cholesterol by extracellular cholesterol oxidase from Brevibacterium sp[J]. Food Chemistry, 2002, 77(4): 457—463
28. Somokuti G A, Daniel K Y, Soliman D K, et al. Expression of Streptomyces sp. cholesterol oxidase in Lactobacillus casei[J]. Appl. Microbial Biotechnol, 1992, 37: 330—334
29. Somokuti G A, Soliman D K, Steinberg D H. Native promoter-plasmid vector for heterologous cholesterol oxidase synthesis in Streptococcus thermophilus[J]. Plasmid, 1995, 33(1):7—14
30.吕陈峰,陈毅力,王龙刚等.利用胆固醇氧化酶转化胆固醇制备胆甾一4一烯一3一酮[J].无锡轻工大学学报,2001,20(5):485—488
31. Marta V M, Eliseo R, Nuria A, et al. Cholesterol oxidases act as signaling proteins for the biosynthesis of the polyene macrolide pimaricin[J]. Chemistry & Biology, 2007, 14(3):279—290
32. Yandouzi E H, Grimellec C. Effect of cholesterol oxidase treatment on physical state of renal brush border membranes: evidence for a cholesterol pool interacting weakly with membrane lipids[J].Biochemistry, 1993, 32 :2047—2052
33. Stevens V L , Lambeth J D. Cholesterol pools in rat adrenal mitochondria: use of cholesterol oxidase to infer a complex pool structure[J]. Endocrinology, 1992, 130:1557—1563
34. Corbin D R, Greenplate J T, Wong E Y, et al. Cloning of an insecticidal cholesterol oxidase gene and its expression in bacteria and in plant protoplasts[J]. Applied and Environmental Microbiology, 1994, 60(12): 4239—4244
35.桑吉,王龙刚,李忠琴.胆固醇氧化酶高产菌株的复合诱变选育[J].食品工业科技, 2006, 27(6):87—89
36.季文明,陈毅力,吕陈峰.双水相非离子表面活性剂萃取法分离胆固醇氧化酶[J].江南学院学报,2000, 15(2):3—6
37.季文明,陈毅力,张春和.亲和层析法分离胆固醇氧化酶[J].生物技术,2000, 10(5):16—19
38.王龙刚,梁爽,王武.三相分离甾短杆菌胆固醇氧化酶及其性质[J].华东理工大学学报,2007, 33(2):190—194
39.季文明,张和春,王武.构建胆固醇氧化酶的短杆菌基因文库[J].生物技术,1999,9(6):4—8
40.王龙刚,邬敏辰,王武.胆固醇氧化酶基因的克隆及在E.coli中的表达[J].微生物学通报,2006,33(6):39—42
41. Longgang Wang ,Wu Wang. Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli[J]. Biotechnol Lett ,2007, 29:761—766
42.银国利,张锐,郭三堆等.胆固醇氧化酶基因的克隆及其在大肠杆菌中的表达[J].西北农业学报,2003,12(3):12—16,35
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45. Sreekrishna K, Brankamp RC, Kropp K E,et al. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris[J]. Gene, 1997,190(1) :55—58
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2. Stadtman T C, Cherkes A, Anfinsen C B. Studies on the microbiological degradation of cholesterol[J]. J Biol Chem, 1954, 206:551—523
3. Uwajima T, Yagi H, Nakamura S, et al. Isolation and crystallization of Extraccllular 3β-Hydroxysteroid oxidase of Brevibacterium sterolicum sp.[J]. Agr Biol Chem,1973,37(10):2354—2350
4. Cheelham P S J, Dunnill P, Lilly M D. The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous[J]. J Biochem, 1982, 201: 515—52
5. Sampson N S, Vrielink A. Cholesterol oxidases: A study of nature’s approach to protein design[J]. Chem Res, 2003, 36: 713—722
6. Gadda G, Wels G, Pollegioni L, et al. Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum[J]. Eur J Biochem. 1997, 250: 369—376
7. Kreit J, Lefebvre G, Germain P. Membrane-bound cholesterol oxidase from Rhodococcus sp. cells. Production and extraction[J]. J Biotechnol. 1994, 33 (3): 271—282
8. Doudyu N, Aono R. Purification of extracellular cholesterol oxidase with high activity in the presence of organic solvents from Pseudomons sp. Strain S T-200[J]. Appl Environ Microbial, 1998, 64(5): 1929—1932
9. Inouye Y, Taguchi K, Fujii A, et al. Purification and characterization of extracellular 3-Hydroxysteroid oxidase produced by Streptoverticillum cholesterolicum[J]. Chem Pharm Bull, 1982, 30(3): 951—958
10.吕莹.胆固醇氧化酶的分离和提纯[D]:[硕士学位论文].北京:中国农业大学, 2001
11. Vrielink A, Lloyd L F, Blow D M. Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A°rerolutin[J]. J. Mol. Biol, 1991, 219(3):533—554
12. Kass I J, Sampson N S. The isomerization catalyzed by Brevibacterium sterolicum cholesterol oxidase proceeds stereospecifically with one base[J]. Biochem Biophy Research Comm, 1995, 206(2):688—693
13. Sampson N S, Kass I J. Isomerization, but not oxidation, is suppressed by a single point mutation, E361Q, in the reaction catalyzed by cholesterol oxidase[J]. J American Chemical Society, 1997, 119(5):856—862
14. Kamei T, Takiguchi Y, Suzuki H, et al. Purification of 3β-Hydroxysteroid oxidase of streptomyces violascens origin by affinity chromatography on cholesterol[J]. Chem Charm Bul, 1978, 26:2799—2804
15.季文明,陈毅力,张和春.比色法测定胆固醇氧化酶酶活[J].无锡轻工大学学报,2000,19(3):251—254
16. Murooka Y, Ishizaki T, Nimi O, et al. Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptonyces lividans with plasmid pIJ702[J]. Appl Environ Microbial, 1986, 52(6):1382—1385
17. Fajishiro K, Ohta T, Hasegawa M, et al. Isolation and identification of gene of cholesterol oxidasefrom Brevibacterium sterolicum ATCC21387, a widely used enzyme in clinical analysis[J]. Biochem Biophys Res Commun, 1990, 172(2):721—727
18.季文明,张和春,王武.胆固醇氧化酶基因的克隆和表达[J].微生物学通报,2000,27(3): 224—227
19. Molmir I, Choi K P , Hayashi N, et al . Secretory overproduction of Streptomyces cholesterol oxidase by Streptomycm lividuns with a multi-copy shuttle vector[J]. J Ferment Bioeng, 1991, 72(5):368—372
20. Ohta T, Fujishiro K, Yamaguchi K, et al. Sequence of gene choB encoding cholesterol oxidase of Brevibacterium sterlicum: comparisonwith choA of Streptomyces sp. SA-COO. Gene, 1991, 103(1) :93—96
21. Sampson N S, Xiaoyu Chen. Increased expression of Brevibacterium sterolicum cholesterol oxidase in Escherichia coli by Genetic modification[J]. Protein Expression and Purification, 1998, 12: 347—352
22. Yen-Rong Chen, Hsin-Ho Huang, Yi-Fang Cheng, et al. Expression of a cholesterol oxidase gene from Arthrobacter simplex in Escherichia coli and Pichia pastoris[J]. Enzyme and Microbial Technology, 2006, 39(4):854—856
23. Nishiya Y, Harada N, Teshima S, et al . Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation[J]. Progress in Biotechnology, 1998, 15:295—320
24. Nishiya Y, Murooka Y. Development of improved cholesterol oxidase and its application[J]. Seibutsu-kogaku, 1999, 77:429—432
25. Black M, Guiomar A J, Guthrie J T, et al. Immobilization of cholesterol oxidase on a hydroxypropyl cellulose-Acrylic acid support system: Relevance to Biosensor Development[J]. Celluosis, 1993, 28:171—181
26.吕陈峰,王龙刚,杨胜利等.胆固醇氧化酶转化蛋黄胆固醇工艺的优化[J].无锡轻工大学学报,2001, 20(6):555—559
27. Chenfeng Lv, Yixin Tang, Longang Wang, et a1. Bioconversion of yolk cholesterol by extracellular cholesterol oxidase from Brevibacterium sp[J]. Food Chemistry, 2002, 77(4): 457—463
28. Somokuti G A, Daniel K Y, Soliman D K, et al. Expression of Streptomyces sp. cholesterol oxidase in Lactobacillus casei[J]. Appl. Microbial Biotechnol, 1992, 37: 330—334
29. Somokuti G A, Soliman D K, Steinberg D H. Native promoter-plasmid vector for heterologous cholesterol oxidase synthesis in Streptococcus thermophilus[J]. Plasmid, 1995, 33(1):7—14
30.吕陈峰,陈毅力,王龙刚等.利用胆固醇氧化酶转化胆固醇制备胆甾一4一烯一3一酮[J].无锡轻工大学学报,2001,20(5):485—488
31. Marta V M, Eliseo R, Nuria A, et al. Cholesterol oxidases act as signaling proteins for the biosynthesis of the polyene macrolide pimaricin[J]. Chemistry & Biology, 2007, 14(3):279—290
32. Yandouzi E H, Grimellec C. Effect of cholesterol oxidase treatment on physical state of renal brush border membranes: evidence for a cholesterol pool interacting weakly with membrane lipids[J].Biochemistry, 1993, 32 :2047—2052
33. Stevens V L , Lambeth J D. Cholesterol pools in rat adrenal mitochondria: use of cholesterol oxidase to infer a complex pool structure[J]. Endocrinology, 1992, 130:1557—1563
34. Corbin D R, Greenplate J T, Wong E Y, et al. Cloning of an insecticidal cholesterol oxidase gene and its expression in bacteria and in plant protoplasts[J]. Applied and Environmental Microbiology, 1994, 60(12): 4239—4244
35.桑吉,王龙刚,李忠琴.胆固醇氧化酶高产菌株的复合诱变选育[J].食品工业科技, 2006, 27(6):87—89
36.季文明,陈毅力,吕陈峰.双水相非离子表面活性剂萃取法分离胆固醇氧化酶[J].江南学院学报,2000, 15(2):3—6
37.季文明,陈毅力,张春和.亲和层析法分离胆固醇氧化酶[J].生物技术,2000, 10(5):16—19
38.王龙刚,梁爽,王武.三相分离甾短杆菌胆固醇氧化酶及其性质[J].华东理工大学学报,2007, 33(2):190—194
39.季文明,张和春,王武.构建胆固醇氧化酶的短杆菌基因文库[J].生物技术,1999,9(6):4—8
40.王龙刚,邬敏辰,王武.胆固醇氧化酶基因的克隆及在E.coli中的表达[J].微生物学通报,2006,33(6):39—42
41. Longgang Wang ,Wu Wang. Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli[J]. Biotechnol Lett ,2007, 29:761—766
42.银国利,张锐,郭三堆等.胆固醇氧化酶基因的克隆及其在大肠杆菌中的表达[J].西北农业学报,2003,12(3):12—16,35
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