Check patentability & draft patents in minutes with Patsnap Eureka AI!

Penicillin g acylase mutant and its application in enzymatic synthesis of cefamandole

A mutant and penicillin technology, applied in the direction of biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems that cannot meet the needs of actual industrial production, the conversion rate is only low, and the synthetic hydrolysis is low, so as to achieve broad industrial application prospects and improve Synthetic hydrolysis ratio and effect of hydrolysis activity reduction

Active Publication Date: 2022-01-18
NANJING TECH UNIV
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Penicillin g acylase mutant and its application in enzymatic synthesis of cefamandole
  • Penicillin g acylase mutant and its application in enzymatic synthesis of cefamandole
  • Penicillin g acylase mutant and its application in enzymatic synthesis of cefamandole

Examples

Experimental program
Comparison scheme
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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a penicillin G acylase mutant and its application in enzymatically synthesizing cefamandole. The penicillin G acylase derived from Achromobacter xylosoxidans (Achromobacter xylosoxidans) PX02 was mutated by semi-rational design enzyme engineering technology, and a higher reactivity, faster reaction rate and better conversion rate were obtained. PGA mutants. The hydrolysis activity of the PGA mutant to the product is greatly reduced, and the product is almost not hydrolyzed after the yield reaches the maximum value, and has broad industrial application prospects.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
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 TECH UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com