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雷氏普罗威登斯菌青霉素G酰化酶基因在大肠杆菌中的克隆与表达 被引量:2

Cloning and expression of penicillin G acylase gene from Providencia rettgeri in Escherichia coli
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摘要 利用PCR和分子克隆技术从雷氏普罗威登斯菌(Providencia rettgeri)(ATCC29944)的基因组DNA中获得一个青霉素G酰化酶(penicillin G acylase,PGA)基因并将其装入表达质粒pET24a。携带有重组质粒pETPGA的Escherichia coli基因工程菌BL21(DE3)/pETPGA实现了PGA的高效表达。对发酵条件的研究表明基因工程菌在24℃、添加5g/L甘油条件下以1.0mmol/L IPTG诱导1.5h酶活力即达到993.4U/L,比野生菌酶活力(15U/L)提高了66倍。 Based on PCR techniques, a gene encoding penicillin G acylase(PGA) was obtained from genomic DNA of Providencia rettgeri(A.TCC29944). The pET24a vector combined with the PGA gene was transformed into BL21(DE3) and the resulting recombinant Escherichia coli gave a high level expression of PGA. Several factors, including IPTG concentration, temperature and carbon source, were optimized for enzyme overproduction. The optimization significantly increased PGA expression by 66-fold compared to the native expression (15U/L) in P. rettgeri. After inducing with 1. 0mmol/L IPTG at 24! and fermentation in medium with additive of 5g/L glycerol for 1.5 hour, The enzyme expression of recombinant E. coli reached 993.4U/L.
出处 《工业微生物》 CAS CSCD 北大核心 2002年第3期1-5,共5页 Industrial Microbiology
基金 国家自然科学基金(No.30100029) "863"国家高科技资助项目(No.2001AA235081)
关键词 雷氏普罗威登斯菌 青霉素G 酰化酶 基因 大肠杆菌 克隆 表达 penicillin G acylase Providencia rettgeri cloning and expression
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  • 1[1]Kang J H, Hwang Y, Yoo OJ. Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis. J. Biotechnol., 1991, 17:99-108
  • 2[2]Ohashi H, Katsuta Y, et al. Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis. Appl. Environ. Microbiol. , 1989, 55: 1351-1356
  • 3[3]Alvaro G, Fernandez- Lafuente, et al. Penicillin G acylase from Kluyvera citrophila: New choice as industrial enzyme. Biotechnol. Lett., 1992, 14:285-290
  • 4[4]Daumy GO, Danley D, et al. Experimental evolution of penicillin G aeylases from Escherichia coli and Proteus rettgeri. J. Bacteriol., 1985, 163:925-932
  • 5[5]Klei HE, Daumy GO, Kelly JA. Purification and preliminary crystallographic studies of penicillin G acylase from Providencia rettgeri. Protein Sci. 1995, 4:433-441
  • 6[6]Chou CP, Wang WC, Lin MI, An approach for enhancing heterologous production of providencia rettgeri penicillin acylase in Escherichia coli. Biotechnol. Prog., 2000, 16: 315-318
  • 7[7]Kutzbach C, Rauenbusch E. Preparation and general properties of crystalline penicillin acylase from Escherichia coli ATCC11105.Hoppe - Seyler' s Z . Physiol. Chem. , 1974, 354: 45-53
  • 8[8]Duggleby, HJ, Tolley, SP, Hill, CP, Dodson, E J, Dodson, G, Moody, PC, Penicillin acylase has a single- amino- acid catalytic center. Nature, 1995, 373:264-268
  • 9[9]Yang S, Huang H, Zhang RA, Huang XD, Li SY, Yuan ZY. Expression and Purification of Extracellular Penicillin G Acylase in Bacillus subtilis. Protein Expression and Purification, 2001, 21:60-64

同被引文献20

  • 1Kutzbach C, Rauenbusch E. Preparation and general properties of crystalline penicillin acylase from Escherichia coli ATCClll05. Hoppe Seylers Z Physiol Chem, 1974, 355(1): 45 - 53.
  • 2Klei HE, Daumy GO, Kelly JA. Purification and preliminary crystallographic studies of penicillin G acylase from Providencia rettgeri. Protein Sci, 1995, 4(3): 433 -441.
  • 3Choi KS, Kim JA, Kang HS. Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105. J Bacteriol, 1992, 174 (19): 6270 -6276.
  • 4Martin J, Prieto I, Barbero JL, Perez-Gil J, Mancheno JM, Arche R. Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met-Ala168 mutant penicillin acylases from Kluyvera citrophila. Biochim Byiophys Acta, 1990, 1037(2): 133 - 139.
  • 5YangS HuangH LiSY YeYZ WanL ZhangFW YuanZY.Enhancing penicillin G acylase stability by site—directed mutagenesis[J].Acta Biochimica et Biophysica Sinica(生物化学与生物物理学报:英文版),2000,32(6):581-585.
  • 6Van Laan JM, Riemens AM, Quax WJ. Mutated penicillin G acylase genes. US Patent: 006033823A. 2000-03-07.
  • 7Alkema WB, Dijkhuis AJ, Vries E, Janssen DB. The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase. Eur J Biochem, 2002, 269(8):2093 -2100.
  • 8Chen RD. Enzyme engineering: Rational redesign versus directed evolution. Trends Biotechnol, 2001, 19(1): 13 - 14.
  • 9Crameri A, Raillard SA, Bermudez E, Stemmer WP. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature, 1998, 391(6664): 288 -291.
  • 10Stemmer WP. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994, 370(6488): 389 -391.

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