Mutant penicillin G acylases

Details Mutant penicillin G acylases Mutant penicillin G acylases

1997-10-31

2002-06-11

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S252330, C435S320100, C435S471000, C435S045000, C536S023200

Reexamination Certificate

active

06403356

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to mutated genes encoding Type II penicillin G acylases, to penicillin G acylases encoded by these genes resulting in altered properties, and to methods for the synthesis of &bgr;-lactam antibiotics using these penicillin G acylases.
BACKGROUND OF THE INVENTION
Today, semisynthetic &bgr;-lactam derivatives such as ampicillin, amoxicillin, cephalexin, cefadroxil, and cefprozil are, on an industrial scale, prepared by chemical methods. The synthesis of these antibiotics catalyzed by enzymes constitutes a clear example of an enzymatic reaction of possible industrial importance. The enzymatic approach has several advantages as compared to conventional chemical methods: (1) avoidance of toxic reagents and solvents; (2) enzyme specificity renders protection of carboxyl groups in the antibiotic nucleus unnecessary; (3) avoidance of side reactions, including racemization.
In this context, penicillin G acylase offers a great advantage. Penicillin G acylase, also called penicillin G amidase or benzylpenicillin amidohydrolase [EC. 3.5.1.11], refers to a group of hydrolases from microorganisms, especially bacteria, capable of hydrolyzing the 6 acyl group of penicillins or the 7 acyl group of cephalosporins having the general structures of I and II to their corresponding free amine forms (6-APA and its derivatives, 3.5 III, and 7-ACA and its derivatives, IV).
wherein
R1=phenylacetyl, phenoxyacetyl, hydroxyphenylglycine, phenylglycine and their derivatives, acetyl, adipyl and their derivatives
R2, R3=aliphatic or aromatic entities with or without one or more O, S, N atoms
R4=aliphatic or aromatic alcohols and their derivatives with or without one or more O. S. N atoms
The preferred acyl group is phenylacetyl, although other aromatic and aliphatic (hydrophobic, or charged/polar) acyl groups can also be hydrolyzed to varying degrees (generally less). The preference for different acyl groups are not necessarily true for the reverse reaction, namely the formation of amide bonds between the acyl groups and 6-APA and 7-ACA (III and IV). For instance, the chloroacetyl group can be put on to 7-ACA much faster than most aromatic acyl groups (patent JP08000284-A). For many currently marketed &bgr;-lactam antibiotics, the acyl groups are aromatic functions with varying degrees of hydrophobicity. The wild type penicillin G amidase can catalyze the semisyntheses (amide bond formation) of these antibiotics, but the reactions rarely go to completion under suitable or economical conditions for the production of these antibiotics. Improvements in the production yield and efficiency of these reactions are highly desired.
There are many reports in the literature of penicillin G acylases which contain altered amino acid residues exhibiting altered substrate specificity or changes in catalytic activity. Prieto et al. (I. Prieto et al., Appl. Microbiol. Biotechnol.33 (1990) 553-559) replaced Met168 in penicillin G acylase from
K. citrophila
with Ala, Asp, Val, Asn, and Tyr resulting in modified kinetic parameters for penicillin G and penicillin V deacylation; the substitution of Asn with Lys375 or Tyr with His481 did not. Martin et al. (J. Martin & I. Prieto, Biochimica et Biophysica Acta 1037 (1990) 133-139) describe a mutant of penicillin G acylase with different substrate specificity and enhanced thermal stability when Met168 was changed to Ala. Wang Min et al. (Wang Min et al. Shiyan Shengwu Xuebao 24 (1991), 1, 51-54) reported the replacement of Ser177 in
E. coli
penicillin G acylase with Gly, Thr, Leu, Arg, all of which changes resulted in inactive enzymes. Kyeong Sook et al. (Kyeong Sook et al. Journal of Bacteriology 174 (1992) 6270-6276) and Slade et al. (Slade et al. Eur.J. Biochem. 197 (1991) 75-80) have demonstrated Ser290 to be an essential amino acid residue of penicillin G acylase from
E. coli
. Substitution of Ser290 with Cys completely inactivated the enzyme. Niersbach et al. (Niersbach et al. Biotechnology Letters 17, 1, (1995) 19-24) replaced Gly359 with aspartic acid in penicillin G acylase from
E. coli
. The mutant enzyme lost the ability to hydrolyze penicillin G but exhibited the novel ability to hydrolyze phthalyl-L-leucine and phthalyl-glycyl-L-proline. An enhanced stability at alkaline pH was demonstrated by a site-directed mutant of penicillin G acylase from
E. coli
when Trp431 was changed to Arg (Gabriel del Rio et al. Biotechnology and Bioengineering 48 (1995) 141-148).
The inventors herein present mutant penicillin G acylases having altered enzymatic activities when compared with the wild type enzyme.
SUMMARY OF THE INVENTION
In one aspect of the invention the DNA sequence of the Type II wild-type penicillin G acylase, preferably from prokaryotic organisms (the structure of the enzyme from
E. coli
is given in FIGS.
1
A through
1
D), is altered to encode mutant penicillin G acylases. Type II acylases all share a common molecular structure. Type II acylases are heterodimers composed of a small subunit (alpha; 16-26 kilodaltons (kDa)) and a large subunit (beta; 54-66 kDa). As used herein the term “penicillin G acylase” is intended to mean prokaryotic Type II acylase as well as its preenzyme and preproenzyme forms. The DNA sequence (SEQ.ID.NO.:1) and corresponding amino acid sequence (SEQ.ID.NO.:2) for the alpha subunit of the wild type penicillin G acylase from
E. coli
are shown in FIG.
1
A. The DNA sequence (SEQ.ID.NO.:3) and corresponding amino acid sequence (SEQ.ID.NO.:4) for the beta subunit of the wild type penicillin G acylase from
E. coli
are shown in
FIGS. 1B through 1D
. In accordance with the present invention, one or more selected amino acid residues are substituted with different amino acid residues from the group of natural amino acids. Of course, in the mutated DNA sequences of the invention corresponding changes in the DNA sequence are made in order to encode the desired amino acid(s) at the desired position(s). The structural changes were determined based on the X-ray crystallographic structure of the wild-type penicillin G acylase. The DNA and amino acid sequence changes for each substitution in accordance with the present invention are shown in FIG.
2
.
In accordance with the invention the following substitutions at one or more of the designated sites are provided:
1. On the Alpha Subunit:
DNA basepairs:A424-426 (MetA142—Ala)
DNA basepairs:A436-438 (PheA146—Ala)
2. On the Beta Subunit:
DNA basepairs:B70-72 (PheB24—Ala, Leu, Val,
Pro, Tyr, Met, Ser, Cys, Gly, Asp, Lys, Arg, Typ, Thr, Ile, Glu, Gln, Asn or His).
DNA basepairs:B166-168 (ValB56—Ser or Thr)
DNA basepairs: B529-531 (IleB177—Phe)
In the nomenclature used above, “A” represents the alpha subunit, “B” represents the beta subunit; the numbered positions are conventional amino terminus to carboxy terminus for amino acid sequences, and 5′ to 3′ for DNA sequences; the amino acid preceding the amino acid position number represents the wild type amino acid and the amino acid following the amino acid position number represents the substitute amino acid, for example, “ValB56—Ser or Thr” means that amino acid at position 56 in the wild type beta subunit is valine which is substituted with either serine or threonine to make a mutant acylase of the invention.
The altered acylases of the invention have altered enzymatic activities when compared with the corresponding wild-type penicillin G acylase.
The most preferred altered (mutant) penicillin G acylase has a single amino acid alteration (PheB24-Ala), and is capable of synthesizing &bgr;-lactam antibiotics with significantly higher yield and efficiency than the wild type enzyme.
In other aspects the present invention is also directed to vectors which comprise the altered nucleic acid sequences of the invention, and microorganism host cells transformed with said vectors. The invention also concerns processes for production of the altered acylases comprising culturing the host cells of the invention, preferably followed by isolation of the acylase.
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