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Penicillin G acylase mutant and application thereof in enzymatic synthesis of cefamandole

A mutant, penicillin technology, applied in biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of not meeting the needs of actual industrial production, low synthetic hydrolysis, and only conversion rate, and achieve broad industrial application prospects, hydrolysis The effect of reducing the activity and increasing the ratio of synthesis to hydrolysis

Active Publication Date: 2020-08-07
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] There are more and more researches on biocatalysis at home and abroad. One of the key problems is that the enzymes used often cannot meet the needs of actual industrial production.
As the wild-type penicillin G acylase with the amino acid sequence involved in the present application as shown in SEQ ID NO: 2, although it can catalyze the condensation of 7-TMCA and methyl mandelate to synthesize cefamandole, the synthetic hydrolysis is relatively low, and the produced There are many by-products, so the conversion rate is only about 40%

Method used

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  • Penicillin G acylase mutant and application thereof in enzymatic synthesis of cefamandole
  • Penicillin G acylase mutant and application thereof in enzymatic synthesis of cefamandole
  • Penicillin G acylase mutant and application thereof in enzymatic synthesis of cefamandole

Examples

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Effect test

Embodiment 1

[0033] This example illustrates the site-directed mutagenesis of penicillin G acylase.

[0034] Carry out single-point mutation, two-point combined mutation and three-point combined mutation at the following three sites of penicillin G acylase: the 186th arginine (R) is mutated into another amino acid residue selected from the following C amino acid (A), aspartic acid (D), or lysine (L); the 187th phenylalanine (F) is mutated to any other amino acid residue (isoleucine (I), Valine (V)); the 329th phenylalanine (F) mutation is another amino acid residue selected from the following alanine (A), glycine (G), isoleucine (I) , Valine (V), Serine (S). Use oligo7 software to design blunt-end primers, and the gene sequence (SEQ ID NO: 1) encoded by the wild-type penicillin G acylase amino acid sequence (SEQ ID NO.2) derived from Achromobacter xylosoxidans PX02 is a template, and then Perform PCR site-directed mutagenesis.

[0035] The base sequence of the primer is:

[0036]

...

Embodiment 2

[0049] This example illustrates the induced expression and purification of penicillin G acylase mutants.

[0050] 1. Induced expression of penicillin G acylase mutant

[0051] (1) The recombinant transformants obtained in Example 1 were inoculated into LB medium containing kanamycin (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, pH 6.5, shake culture at 37°C for 12h, Obtain the seed solution.

[0052] (2) Insert the inoculum amount of 2% (v / v) into a 250mL Erlenmeyer flask equipped with 40mL LB medium, place it on a shaker at 37°C and 180rpm for culture, when the culture solution OD 600 When it reaches 0.6, add IPTG with a final concentration of 0.1mmol / L as an inducer, and induce at 16°C for 24h.

[0053] (3) Centrifuge the culture medium, collect the cells, and wash twice with saline to obtain resting cells. Suspend the resulting resting cells in a pH 8.0 buffer, ultrasonically break in an ice bath, and centrifuge to collect the supernatant, which is the crude enzyme solu...

Embodiment 3

[0057] This experiment illustrates the application of penicillin G acylase mutants in the synthesis of cefamandole.

[0058] The technical scheme adopted in this embodiment is as follows figure 1 shown.

[0059] Depend on figure 1 The reaction formula shows that penicillin G acylase can not only condense the mother nucleus 7-TMCA and the side chain methyl mandelate to produce the product cefamandole, but also decompose the product to form the mother nucleus and mandelic acid, and at the same time, the methyl mandelate itself can also be hydrolyzed into mandelic acid.

[0060] Reaction system: use pH 6.5 phosphate buffer as the reaction medium. The total reaction volume is 2mL, which contains 50mM 7-TMCA, 150mM R-MMA (R-methyl mandelate), add penicillin acylase mutant enzyme solution (prepared in Example 1-2), and the reaction temperature is 25 ° C, The reaction was carried out for 5 hours at a rotational speed of 200 rpm, and samples were taken regularly. The sample was d...

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Abstract

The invention relates to a penicillin G acylase mutant and application thereof in enzymatic synthesis of cefamandole. Penicillin G acylase derived from achromobacter xylosoxidans PX02 is mutated by virtue of a semi-rationally designed enzyme engineering modification technology, so that a PGA mutant with higher reaction activity, higher reaction rate and better conversion rate is obtained. The hydrolysis activity of the PGA mutant to a product is greatly reduced, and after the yield reaches the maximum value, the hydrolysis product is hardly generated, so that the PGA mutant has a wide industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a penicillin G acylase mutant derived from Achromobacter xylosoxidans and its application in the enzymatic synthesis of cefamandole. Background technique [0002] β-lactam antibiotics such as the second-generation cephalosporins of the genus Cefamandole are high-efficiency medical drugs with strong bactericidal power and broad antibacterial spectrum. The bacteria become filamentous or spherical, and finally dissolve, which has a strong killing effect on a variety of Gram-positive and Gram-negative bacteria. Clinically, it is mainly used to treat respiratory tract infection, biliary tract infection, abdominal cavity infection, pelvic cavity infection, urinary tract infection, skin and soft tissue infection, bone and joint infection and sepsis caused by sensitive bacteria. [0003] At present, domestic enterprises have achieved the localization of most cepha...

Claims

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Application Information

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IPC IPC(8): C12N9/84C12N15/70C12N1/21C12P35/04C12R1/025C12R1/19
CPCC12N9/84C12Y305/01011C12N15/70C12P35/04
Inventor 何冰芳李安妮储建林吴斌汲晓琪彭智熠
Owner NANJING UNIV OF TECH
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