Mutants of penicillin G acylase from Achromobacter sp. CCM 4824 and uses thereof

A penicillin and acylase technology is applied to a penicillin G acylase mutant derived from Achromobacter CCM4824 and its application field, and can solve the problems of reducing the efficiency of acyl transfer reaction and the like

Active Publication Date: 2019-07-05
艾美科健株式会社
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This hydrolysis reaction results in the disappearance of the activated side chain, which reduces the efficiency of the acyl transfer reaction

Method used

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  • Mutants of penicillin G acylase from Achromobacter sp. CCM 4824 and uses thereof
  • Mutants of penicillin G acylase from Achromobacter sp. CCM 4824 and uses thereof
  • Mutants of penicillin G acylase from Achromobacter sp. CCM 4824 and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Preparation of wild-type Penicillium derived from Achromobacter sp. CCM 4824 Penicillin G acylase (APA)

[0108] Synthetic expression of wild-type penicillin G acylase gene derived from Achromobacter sp. CCM 4824

[0109] Wild-type APA (penicillin G acylase derived from Achromobacter sp. CCM 4824) is a precursor (precursor) type, through the α subunit shown in SEQ ID NO.1 and SEQ ID NO.2 The shown β subunit is expressed by a single-chain polypeptide (SEQ ID NO.4) connected and constituted by a spacer peptide shown in SEQID NO.3, and then through autocatalytic processing in cells to A mature active dimer form consisting of the α and β subunits is formed. The gene expressing wild-type APA precursor SEQ ID NO.4 (abbreviated as "apa gene") was synthesized by Bioneer Company (Daejeon, Korea).

[0110] Preparation of recombinant vector (pBC-APA) expressing wild-type APA

[0111] The apa gene obtained in was inserted into the XbaI and NotI restriction enzyme recogni...

Embodiment 2

[0113] Generation and screening of mutations with high levels of multiplex synthesis performance against multiple β-lactam antibiotics body

[0114] Prepare mutant library once by error prone PCR

[0115] The nucleotide sequence of the apa gene synthesized in the was artificially randomly mutagenized, for which error prone PCR was performed to prepare a mutant library. The procedure for preparing a library of specific mutations is as follows.

[0116] Specifically, error-prone polymerase chain reaction was performed using the Diversity PCR Random Mutagenesis Kit (Diversity PCR Random Mutagenesis kit, Clontech, USA) to generate one mutation per 1,000 bp. PCR reaction solution, by using the template DNA of the pBC-APA plasmid of 1ng / μL, each 10pmol of T3 primer (SEQ ID NO.13) and T7 primer (SEQ IDNO.14), 2mM dGTP, 50X diversity dNTP mixture, 10X Titanium Taq buffer solution and titanium Taq polymerase, the final volume is 50 μL. The PCR reaction conditions were that th...

Embodiment 3

[0134] Preparation and screening of highly efficient antibiotic multiplex synthesis mutants with reduced substrate-decomposing activity

[0135] In order to prepare mutants having a high level of synthetic performance against various β-lactam antibiotics and significantly reduced hydrolytic activity of their reaction substrates, the following procedure was performed.

[0136] Prepare 5 times mutant library by error prone PCR

[0137] In order to increase the antibiotic (represented by cephalosporin) synthetic activity of the CEP2-s4 mutant prepared in the embodiment , and reduce the hydrolysis activity to the reaction substrate (represented by PGM), use CEP2- The s4 plasmid was used as template DNA, and an error-induced mutation library was prepared in the same manner as in Example .

[0138] In order to study the synthetic ability to cephalosporins representatively, the test was carried out in the same manner as in Example . In addition, in order to study the reactivity...

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Abstract

The present invention relates to mutants of penicillin G acylase from Achromobacter sp. CCM 4824 and uses thereof, and more specifically relates to Mutants of penicillin G acylase in a wild type penicillin G acylase protein sequence including an Alpha subunit shown in SEQ ID NO.1 and a Beta subunit shown in SEQ ID NO.2 and a method of preparing (synthesizing) Beta-Lactam antibiotics by using theMutants of penicillin G acylase, wherein the Mutants of penicillin G acylase select at least one mutation from a population consisting of A65AlphaV, A102AlphaE, A106AlphaT, D74BetaQ, R120BetaC, T176BetaS, Y180BetaS, T193BetaI, Y248BetaF, F254BetaY, T292BetaS, F343BetaY, E366BetaV and T485BetaS as the feature. The Mutants of penicillin G acylase from Achromobacter sp. CCM 4824, having a specific mutation form, are characterized by multiple syntheses with high efficiency for multiple semi-synthesized Beta-Lactam antibiotics.

Description

technical field [0001] The present invention relates to a penicillin G acylase mutant derived from Achromobacter sp. (Achromobacter sp.) CCM 4824 and its application, more specifically to a α subunit comprising SEQ ID NO.1 and SEQ ID NO.1 The wild-type penicillin G acylase protein sequence of the β subunit shown in NO.2 includes A65αV, A102αE, A106αT, D74βQ, R120βC, T176βS, Y180βS, T193βI, Y248βF, F254βY, T292βS, F343βY, E366βV and A method for producing (synthesizing) a mutant penicillin G acylase characterized by at least one mutation in a group of T485βS combinations and a β-lactam antibiotic using the mutant penicillin G acylase. Background technique [0002] β-lactam antibiotics are the most important class of antibacterial compounds in clinical application. It is known that the antibacterial mechanism of β-lactam inhibits peptidoglycan by acting on transpeptidase and D-alanine carboxypeptidase that catalyze the synthesis of bacterial cell wall materials, namely peptid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/84C12N15/55C12N1/21C12P37/00C12P35/00C12R1/19
CPCC12N9/84C12P37/00C12P35/00C12Y305/01011C12P35/04
Inventor 慎镛喆朴哲尹荣晟王垠善李美京金昇乐金娜俐
Owner 艾美科健株式会社
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