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Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same

An expandase, penicillin technology, applied in the field of penicillin expandase, can solve problems such as low individual activity

Inactive Publication Date: 2003-10-15
骏翰生化股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this patent, several amino acid positions are mentioned, and the mutated expandase produced by changing one or more of the mentioned amino acids shows a higher ratio of penicillin G to penicillin N in the mixture of penicillin G and penicillin N activity ratio of N, but it has lower individual activities towards penicillin G and penicillin N than wild-type expandase

Method used

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  • Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same
  • Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same
  • Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 random mutagenesis

[0033] The cefE gene fragment was excised from pYB4, pYB4 was cloned with the Zero Blunt TOPOPCR Cloning kit from the main chain of pET24a with BamHI-Hind IIIcefE insertion from Streptomyces clavulus, and the cefE gene fragment was treated with 0.8M hydroxylamine at 65°C After 2 hours, it was purified with PCR Clean up-M kit, and then ligated back to the main chain vector. This mutated cefE pool was transformed into BL21(DE3) by electroporation and transformants were selected on plates containing 50 μl / mL kanamycin. Mutated transformants were subjected to activity improvement selection. Example 2 Activity improvement screening

Embodiment 2

[0033] The cefE gene fragment was excised from pYB4, pYB4 was cloned with the Zero Blunt TOPOPCR Cloning kit from the main chain of pET24a with BamHI-Hind IIIcefE insertion from Streptomyces clavulus, and the cefE gene fragment was treated with 0.8M hydroxylamine at 65°C After 2 hours, it was purified with PCR Clean up-M kit, and then ligated back to the main chain vector. This mutated cefE pool was transformed into BL21(DE3) by electroporation and transformants were selected on plates containing 50 μl / mL kanamycin. Mutated transformants were subjected to activity improvement selection. Example 2 Activity improvement screening

[0034] Mutant transformants were grown in 96-well plates with 56.7 μl of LB medium containing kanamycin in each well. 0.1 mM IPTG was then added to each well to indirectly induce DAOCA with shaking for 2 hours at 30°C, followed by an additional 1 hour of shaking after addition of 7 μl of 100 mg / ml lysozyme. The activity of DAOCA was determined by ad...

Embodiment 3

[0036] The wild-type expandase gene (ie, cefE) was cloned by PCR from Streptomyces clavulatus and inserted into the NdeI-HindIII site of pET30a, and the resulting plasmid was named pYS16. The Quick Change Mutagenesis Kit was used to generate site-directed mutagenesis of pYS16. Mutation sites were selected based on the crystal structure of DAOCS (Valegard K. et al., Nature, 394:805-809 (1998)) by selecting residues within 10 Å around the active center using the Swiss-Pdb Viewer (V3.7b2) program. The sites were converted first to Ala residues, then to positively charged residues, hydrophobic residues and sulfur-containing residues. Primers were designed according to the manufacturer's instructions. The resulting mutant was confirmed by DNA sequencing of the plasmid containing the gene, and crude cell extracts were prepared for DAOCS activity assay. Example 4 DAOCS Activity Determination

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Abstract

The present invention is mutant expandase with even higher activity to benzyl penicillin used for preparing phenylacetyl-7-aminodeacetoxy cephalosporanic acid (7-ADCA). The mutant expandase has one or several amino acid substitutes selected from M73T, S79E, V275I, L277K, C281Y, G300V, N304K, I305L and I305M.

Description

technical field [0001] The present invention relates to a mutant penicillin expandase (expandase) with high substrate specificity to penicillin G, a recombinant cell expressing the mutant expandase, and the preparation of 7-amino deacetoxycephalosporin with the mutant expandase 7-aminodeacetylcephalosporanic acid (7-ACDA) method. Background technique [0002] 7-aminodeacetoxy cephalosporin acid (7-ADCA) is one of the important intermediates for the preparation of antibiotic cephalosporins, cephalexin, cephradine and cefadroxil, which are frequently and long-term used in humans and animals. The industrial synthesis method of 7-ADCA mainly includes two steps: chemical ring expansion of penicillin G to phenylacetyl-7-ADCA, and enzymatic side chain cleavage of phenylacetyl-7-ADCA. However, the chemistry of ring expansion is complex and expensive, and by-products and organic solvents such as pyridine and hydrogen bromide are environmentally toxic. Therefore, there is an urgent ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/00C12N15/52C12N15/63C12P35/00C12P37/00
Inventor 杨运博魏佳俐蔡英杰许志行
Owner 骏翰生化股份有限公司
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