Artificially designed penicillin G acylase proenzyme and coding sequence and applications thereof

Abstract

The invention discloses artificially designed penicillin G acylase proenzyme and a coding sequence and applications thereof. Substrate combining steric effects can be reduced and the substrate can beurged to rapidly enter an active pocket by mutating more than one of large side chain amino acids near a substrate binding pocket of wild penicillin G acylase proenzyme into side chain amino acids which are smaller than the amino acids before mutation; and at the same time, through the mutation of more than one of key hydrophilic amino acids or weakly hydrophobic amino acids in the active pocket near a substrate-active center into the amino acids whose hydrophobicity is stronger than that of amino acids before the mutation, the hydrophobicity around the substrate binding pocket can be increased, the combination of water molecules can be reduced, hydrolysis can be reduced, S/H can be increased from reducing hydrolysis and increasing synthesis, and the capability of synthesizing penicillin Gacylase into amoxicillin can be greatly enhanced. The penicillin G acylase proenzyme with the highest S/H so far is provided, and the penicillin G acylase proenzyme has great potential in amoxicillinsynthesis.

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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