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《Acta Biologiae Experimentalis Sinica》 1989-01
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WHOLE NUCLEOTIDE SEQUENCE OF PENICILLIN G ACYLASE GENE AND ITS FLANKING REGION FROM E.COLI

Guo Li-he Ye Zheng-can Yang Jun Wang Min (Shanghai Institute of Cell Biology,Academia Sinica) Han Heng-xiang Zhang Qi-jiu (Institute of Biophysics,Academia Sinica)  
Some of microorganisms have been knownto possess penicillin G acylase activity.The E.coli derived penicillin G acylase(PGA) can catalyze the conversion of peni-cillin G into phenylacetic acid and 6-amino-penicillanic acid,the latter is used as thestarting compound for the industrial forma-tion of semi-synthetic penicillins.Apart fromits industrial importance,the enzyme PGAdisplays a number of interesting properties.Catalytically active enzyme is localized inthe periplasmic space of E.coli cells andcomposed of two dissimilar subunits.Thetwo subunits are apparently produced from aprecursor protein,via a processing pathwayhitherto unique in its features for a prokary-otic enzyme.The studies on processing ofthe precursor and on the relationship betweenstructure and function of the mature enzymeare important theoretically. Previously we cloned a 3.5 kb DNAfragment from a strain (E.coli AS 1.76),which displays PGA activity.In this paper,we report a nucleotide sequence of the 3.5kb DNA fragment containing PGA gene.After insertion of the DNA fragment intoEcoR Ⅰ and Hind Ⅲ sites in pWR 13,pPGA 20 had been obtained.We subclonedthe Hind Ⅲ and Bgl Ⅱ treated fragment of1.6 kb in length from pPGA 20 into HindⅢ and BamH Ⅰ sites of pWR 13 to get apPGA 1.6.and Bgl Ⅱ and EcoR Ⅰ treatedfragment of 1.9 kb in length into BamH Ⅰand EcoR Ⅰ sites of pWR 13 to get a pPGA1.9.The linearized pPGA 1.9 which weredigested with appropriate restriction enzymeswere progressively shortened from both endsrespectively by digestion with Bal 31 nu-clease,followed by cleavage of shortened tar-get DNA off vector DNA molcules with ap-proriate restriction enzymes.The series ofthe DNA fragments shortened from EcoR Ⅰend were then cloned into plasmid pWR 13which had previously digested with Hind Ⅲand Sma Ⅰ enzymes (Fig.1).The DNA frag-ment cloned in pWR 13 were directly se-quenced on the resulted plasmids by usingprimer Ⅰ and primer Ⅱ.Thus we have ob-tained the complete nucleotide sequence ofthe 3.5 kb DNA fragment. The 3.5 kb fragment contains an in-tact PGA gene which is 2.6 kb.The openreading frame in the gene consists of fourstructural domains:(i) nucleotide positionsNo.53—130 coding for a signal peptide of theprecusor enzyme,(ii) positions No.131—757coding for alpha-subunit of the mature en-zyme,(iii) positions No.758—919 coding forthe spacer peptide of the precusor enzyme,(iv) positions No.920—2590 coding for beta-subunit of the mature enzyme.Additionallyupstream from the starting codon ATG,thereis a promoter and a PBS (i,e.SD) sequen-ce,and downstream sequence from termina-tion codon TAA,there is a terminator.(Fig.3).
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【References】
Chinese Journal Full-text Database 5 Hits
1 Zhang,Qi-jiu Yan, Mao-gong Chen, Hong(Institute of Biophysics, Academia Sinica, Beijing) Zhao, Wu-ling(Department of Biochemistry, College of Biological Science, Beijing Agricultural University, Beijing);The Purification and Properties of Penicillin G Acylase From E.coli AE109[J];;1990-04
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【Co-references】
Chinese Journal Full-text Database 10 Hits
1 DAI Ming Hua 1, WANG En Duo 2, XIE Yong 3, JIANG Wei Hong 1* and ZHAO Guo Ping 1* ( 1 Laboratory of Molecular Regulation for Microbial Secondary Metabolism, Shanghai Institute of Plant Physiology the Chinese Academy of Sciences,;Site directed Mutagenesis of the Active Center of Penicillin Acylase from E. coli ATCC 11105[J];ACTA BIOCHIMICA ET BIOPHYSICA SINICA;1999-05
2 YANG Yun Gui 1, XU Jing Ning 2, HU Tai Shan 1, QIAN You Cun 1, YANG Sheng Li 1, GONG Yi 1* ( 1 Shanghai Research Center of Biotechnology, Institute of Biological Science, Chinese Academy of Sciences and Shanghai Center of Life;Increase in Hydrophobicity of Signal Peptide Enhances Secretion of Penicillin G Acylase[J];ACTA BIOCHIMICA ET BIOPHYSICA SINICA;2000-02
3 YANG Sheng, HUANG He, LI Shi Yun, YE Yu Zhen, WAN Lin, ZHANG Feng Wen, YUAN Zhong Yi * (Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, S hanghai 200031, China);Enhancing Penicillin G Acylase Stability by Site-directed Mutagenesis[J];Acta Biochimica Et Biophysica Sinica;2000-06
4 XU Jing\|Ning 1* \ YANG Yun\|Gui\+2\ GONG Yi\+2\ YANG Sheng\|Li\+2\ YU Jun\|Tang\+1 1(The State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China) 2(Shanghai Research Center of;The Bottleneck Steps Limiting Maturation of Penicillin G Acylase in Escherichia coli[J];Chinese Journal of Biotechnology;2001-05
5 YANG Zhi-Jian 1,2 CAI Jin 2 SUN Jian 3 YUAN Zhong-Yi 1* 1(Institute of Biochemistry and Cell Biology, Shanghai Institute of Life Science,Chinese Academy of Sciences,Shanghai 200031,China) 2(Department of Chemical and Biochemical Engineering,Zhejiang University, Hangzhou 310027,China) 3(Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China);Constitutive Expression and Purification of Alcaligenes faecalis Penicillin G Acylase in Escherichia coli[J];Chinese Journal of Biotechnology;2004-05
6 Li Min, Chen Changqing (Shanghai Research Center of Biotechnology,Chinese Academy of Sciences,Shanghai 200233);Progress Studies of High Cell-density Culture of Recombinant Escherichia coli[J];PROGRESS IN BIOTECHNOLOGY;2000-02
7 HUANG He,YANG Sheng,LI Ren\|bao 1) ,HUANG Xiao\|dong,YUAN Zhong\|yi * (Shanghai Institute of Biochemistry,the Chinese Academy of Sciences,Shanghai 200031,China);Optimization of Recombinant Penicillin G Acylase Expression in Bacillus subtilis[J];Chinese Journal of Biochemistry and Molecular Biology;2001-02
8 ZHOU Zheng 1) , CHEN Mei juan 2) , LI Jia da 1) , YANG Sheng 1) ,LIU Shu zhao 2) , YUAN Zhong yi 1)* ( 1) Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China;;Functional Display of Penicillin G Acylase from Bacillus megaterium on the Surface of Phage fd[J];Chinese Journal of Biochemistry and Molecular Biology;2002-03
9 HUANG Yanhong,YUAN Zhongyi,WANG Yinglai (Shanghai Institute of Biochemistry,Chinese Academy of Sciences,Shanghai 200031);Expression and Purification of the PGA gene of Bacillus megaterium in Bacillus subtilis[J];CHINESE JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY;1999-01
10 HUANG Hua~1,GOU Bin-quan~1,WANG Yong-hong~1,CHU Ju~1,ZHUANG Ying-ping~1,ZHANG Si-liang~(1*),YUAN Zhong-yi~2(1.State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China;2.Institute of Biochemistry and Cell Biology,Shanghai Institute of Science,Chinese Academy of Sciences,Shanghai 200031,China);Fermentation Conditions for High-level Constitutive Expression of Alcaligenes faecalis Penicillin G Acylase in Escherichia coli[J];Biotechnology;2006-04
【Secondary References】
Chinese Journal Full-text Database 3 Hits
1 Wang Min Fei Jian Guo Li-he (Shanghai Institute of Cell Biology, Academia Sinica) Zhang Qi-jiu (Institute ol Biophysics, Academia Sinica);EFFECT OF MUTAGENESIS AT Ser 177 RESIDURE IN PENICILLIN G ACYLASE ON ACTIVITY OF THE ENZYME[J];Acta Biologiae Experimentalis Sinica;1991-01
2 Kou Ruqin;Yuan Jinming(Institute of Molecular Science,Shanxi University);STUDY OF THE ACTIVATION OF PENICILLIN ACYLASEFROM B. MEGATERIUM BY Co(Ⅱ)[J];JOURNAL OF SHANXI UNIVERSITY (NATURAL SCIENCE EDITION);1995-01
3 MA Xiao-li,YAO Zi-hua,GAO Yuan (Health Science Center,Hebei University,Baoding 071002,China);X-ray Microanalysis of the Activity of Immobilized Glucose Oxidase on Chitosan Membrane[J];Journal of Instrumental Analysis;2006-04
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