title: [Recombinant cephalosporin-acid synthesase: optimisation of expression in E.coli cells, immobilisation and application for biocatalytic cefazolin synthesis].
abstract: Cephalosporin acid synthetase (CASA) is responsible for specific to synthesis of cephalosporin-acids, its expression in Escherichia coli cells is accompanied by accumulation of unprocessed insoluble precursor. In order to optimize conditions of recombinant CASA production we have studied the effects of several parameters of strain cultivation, including growth media composition, temperature, and inoculation dose. Also plasmids for production of CASA variants with the signal sequence of Erwinia carotovora L-asparaginase (ansCASA) and "leaderless" CASA were created in search of more efficient expression constructs. Removal of the N-terminal secretion signal sequence reduced the production of functionally active CASA more than 10-fold and inhibited strain growth. Insertion of the L-asparaginase signal sequence increased the specific enzyme activity in the resultant recombinant strain. The ansCASA producing strain was used to develop the method of immobilization of the recombinant enzyme on an epoxy-activated macroporous acrylic support. The resultant biocatalyst performed effective synthesis of cefazolin from 3-[(5-methyl-1,3,4-thiadiazol-2-il)-thiomethyl]-7- aminocephalosporanic acid (MMTD-7-ACA) and methyl ester of 1(H)-tetrazolilacetic acid (МETzAA), under mild conditions a transformation level of MMTD-7-ACA to cefazolin of 95% is reached.
authors.affiliation: Centre "Bioengineering", Russian Academy of Sciences, Moscow, Russia.
authors.forename: M A
authors.initials: MA
authors.lastname: Eldarov
authors.affiliation: State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
authors.forename: A V
authors.initials: AV
authors.lastname: Sklyarenko
authors.affiliation: Centre "Bioengineering", Russian Academy of Sciences, Moscow, Russia.
authors.forename: M V
authors.initials: MV
authors.lastname: Dumina
authors.affiliation: State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
authors.forename: N V
authors.initials: NV
authors.lastname: Medvedeva
authors.affiliation: Centre "Bioengineering", Russian Academy of Sciences, Moscow, Russia.
authors.forename: A A
authors.initials: AA
authors.lastname: Jgoun
authors.affiliation: State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
authors.forename: J E
authors.initials: JE
authors.lastname: Satarova
authors.affiliation: State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
authors.forename: A I
authors.initials: AI
authors.lastname: Sidorenko
authors.affiliation: Research and Manufacturing Centre "Armbiotechnology"of Armenian National Academy of Science, Armenia.
authors.forename: A S
authors.initials: AS
authors.lastname: Emperian
authors.affiliation: State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
authors.forename: S V
authors.initials: SV
authors.lastname: Yarotsky
mesh_terms: D000179:Acrylates / Q000737:chemistry; D001215:Asparaginase / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002437:Cefazolin / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering; D055767:Immobilized Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D016973:Pectobacterium carotovorum / Q000737:chemistry / Q000201:enzymology; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000179:Acrylates; D001426:Bacterial Proteins; D003470:Culture Media; D055767:Immobilized Proteins; D009097:Multienzyme Complexes; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C038760:cephalosporin C synthetase system; D001215:Asparaginase; D002437:Cefazolin
keywords: antibiotics; biocatalyc synthesis; cefazolin; cephalosporin-acids synthetase; expression optimization; recombinant enzymes