A kind of artificially designed pro-penicillin g acylase and its coding sequence and application

Abstract

The invention discloses an artificially designed pro-penicillin G acylase, its coding sequence and application. In the present invention, one or more of the large side chain amino acids near the substrate binding pocket of the wild-type penicillin G acylase is mutated into smaller side chain amino acids than the amino acid before the mutation, so as to reduce the substrate binding steric hindrance effect and promote substrate binding. Substances can quickly enter the active pocket; at the same time, more than one of the key hydrophilic amino acids or weakly hydrophobic amino acids that are closer to the substrate-active center in the active pocket is mutated into an amino acid with stronger hydrophobicity than the amino acid before the mutation, increasing The hydrophobicity around the substrate binding pocket reduces the binding of water molecules, reduces hydrolysis, increases S / H from the two aspects of reducing hydrolysis and increasing synthesis, and finally greatly improves the ability of penicillin G acylase to synthesize amoxicillin. The present invention provides the penicillin G acylase progen with the highest S / H so far, which has great potential in the synthesis of amoxicillin.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to an artificially designed pro-penicillin G acylase, its coding sequence and application. Background technique [0002] Penicillin acylase (E.C.3.5.1.11) is an important industrial enzyme widely used in industrial production of antibiotics. Due to the different substrate specificities of these enzymes, they can be divided into three classes: penicillin G acylase (PGA), penicillin V acylase (PVA) and ampicillin acylase (AEH). Penicillin G acylase can catalyze the hydrolysis of penicillin G to 6-aminopenicillanic acid (6-APA) or the hydrolysis of cephalosporin G to 7-aminodeacetoxycephalosporanic acid (7-ADCA). In addition, the enzyme can also catalyze the reverse reaction of the above hydrolysis reaction, that is, connect different side chains on the 6-APA and 7-ADCA cores to generate different types of semi-synthetic antibiotics, such as amoxicillin, etc. However, the properties of pen...

AI Technical Summary

Problems solved by technology

[0005] Existing schemes only mutate the large side chain amino acids near the mutated substrate binding pocket into small side chain amino acids. Although the synthesis activity is enhanced, the hydrolysis activity is also improved to a certain level, and finally the synthetase activity / hydrolase activity ( S / H) has a limited increase, and the currently known Escherichia coli penicillin G acylase synthetase activity / hydrolase activity ratio (S / H) is only 4.2 at the highest

The mutant penicillin G acylase patent [publication number: 103275960B] applied by our company in 2013 has a high hydrolysis activity, but the synthetase activity is obviously low, and the final synthesis Low enzyme activity / hydrolase activity (S / H)

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