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Recombinant escherichia coli strain expressing cephalosporin deacetylase and construction method thereof
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A technology of recombinant Escherichia coli and deacetylase, applied in the field of genetic engineering and microbial fermentation, to achieve the effect of stable enzyme activity
Active Publication Date: 2009-10-14
中国科学院上海生命科学研究院湖州工业生物技术中心 +1
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[0004] The present invention creatively adopts gene recombination technology to construct two Escherichia coli strains capable of highly expressing cephalosporin deacetylase. The enzyme activity produced by
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Embodiment 1
[0027] Example 1: Construction and identification of recombinant Escherichia coli expressing cephalosporin deacetylase
[0028] 1. Plasmid construction
[0029] 1) Plasmid pET-24a(+): Product of Novagen, 5.31kb in size, containing kanamycin resistance gene, lactose repressor lac I gene, T7 lac promoter, and multiple restriction endonucleases location.
[0030] Plasmid pMD18-T: a product of TaKaRa Company, with a size of 2.7kb, containing an ampicillin resistance gene, the promoter is the lac Z gene, and has multiple restriction endonuclease sites.
[0031] Bacillus subtilis 168 was purchased from the Bacillus subtilis strain collection center, and Escherichia coli host strain BL21 (DE3) was purchased from Novagen.
[0032] 2) Using the Bacillus subtilis 168 genome as a template, select primers CEF, CER1 and CEF, CER2 to amplify cephalosporin deacetylase genes CE1 and CE2 respectively. The polymerase for PCR amplification is KOD, and the amplification conditions of the two g...
[0038] The recombinant bacteria MM1384 and MM1385 expressing cephalosporin deacetylase were carried out in shake flask fermentation, and the formulas of the two culture media were as follows:
[0039] TB medium: prepared with 2.4% yeast extract, 1.2% peptone, 0.4% glycerol, and phosphate buffer;
[0040] LB medium: prepared with 1% peptone, 0.5% yeast extract, 1% sodium chloride, and phosphate buffer.
[0041] To compare the fermentation effects of recombinant bacteria in different media, pick a single colony of MM1384 and MM1385 and inoculate it into 4 mL of fresh LB liquid medium. The inoculum amount was transferred to 30ml of TB and LB liquid medium, cultivated to OD at 37°C and 220rpm 600 At about 1.0, add lactose with a final concentration of 1% for induction, induce at 30° C. and 220 rpm for 6 hours and 18 hours, collect bacteria respectively, and measure the activity of cephalos...
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Abstract
The invention provides two recombinant escherichia coli strains expressing cephalosporin deacetylase. Two recombinant plasmids for constructing the recombinant strains both take T7 1ac as promoters and contain cephalosporin deacetylase genes and kanamycin genes. The method for constructing the recombinant strains comprises the following steps: 1) two pairs of primers PCR are designed for amplifying the cephalosporin deacetylase genes; 2) positive clones are selected; 3) recombinant plasmids of pMM1384 and pMM1385 are constructed; and 4) two inducible type escherichia coli recombinant strains expressing cephalosporin deacetylase are constructed. The recombinant strains has wide application prospect in the antibiotic preparation industry.
Description
technical field [0001] The invention relates to the fields of genetic engineering and microbial fermentation, in particular to a recombinant bacterial strain expressing cephalosporin deacetylase and a construction method thereof. Background technique [0002] Cephalosporin deacetylase (EC, cephalosporin C deacetylase, CE) can catalyze the deacetylation reaction of cephalosporin C, 7-ACA and its derivatives, and the products generated are used to prepare semi-synthetic cephalosporin antibiotics, such as cephalosporins Fuxin z (cefuroxime), S-1108, etc., are widely used in the antibiotic industry. [0003] Studies have shown that many organisms contain cephalosporin deacetylases. For example, citrus peel, mammalian liver, Bacillus subtilis, Streptomyces clavulatus, Cephalosporium acremonium, Nocardia, Rhodotorula and Aspergillus niger (Takimoto A, Takakura T, Tani H, Yagi S, Mitsushima K .2004. Applied Microbiology and Biotechnology 65:263-7). Japan's Shionogi Pharmaceutica...
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