Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1998-08-13
2000-11-14
Achutamurthy, Ponnathapu
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435 691, 43525233, 4353201, 435471, 435 47, 435228, 530413, 536 232, 536 234, C12N 918, C12N 1500, C12N 1555, C12N 1570
Patent
active
061468711
DESCRIPTION:
BRIEF SUMMARY
SCOPE OF THE INVENTION
The present invention relates to a procedure for modifying a gene which codes for an enzyme with 7.beta.-(4-carboxybutanamide) cephalosporinacylase activity, by the use of recombinant DNA techniques. The modification made to the gene allows the purification of the said enzyme in a single chromatographic step. This modification consists in the fusion of a chain of six histidines to the aminoterminal end of the 7.beta.-(4-carboxybutanamide) cephalosporinacylase, which allows it to bind with great affinity to adsorption chromatography columns with immobilized metallic ions.
This procedure would facilitate the enzymatic preparation of 7-aminocephalosporanic acid from 7.beta.-(4-carboxybutanamide) cephalosporanic acid. 7-Aminocephalosporanic acid is a known intermediate for the manufacture of a wide range of antibacterial agents of the cephalosporin family.
STATE OF THE ART
For the production of 7.beta.-(4-carboxybutanamide) cephalosporinacylase (hereinafter referred to as GLA) by fermentation, use has been made of micro-organisms such as Pseudomonas and Acinetobacter sp. The production of these enzymes by the said micro-organisms entails many disadvantages. Firstly, the GLA activity level is very low, and secondly, apart from the GLA activity, other enzymes are produced such as .beta.-lactamases and acetylases which break down 7-aminocephalosporanic acid (hereinafter referred to as 7ACA), reducing the product yield and increasing the costs of the process for purification of the final product. In order to prevent this enzymatic contamination it is necessary to purify the GLA, which greatly increases the difficulties and costs of the enzymatic process for obtaining 7ACA acid from 7.beta.-(4-carboxybutanamide) cephalosporanic acid (hereinafter referred to as GLA-7ACA).
Various procedures have recently been described for the isolation of genes which code for GLA (hereinafter referred to as gla gene) from strains such as Pseudomonas GK-16 (Matsuda et al. (1985) J. Bacteriol. 163, 1222-1228), Pseudomonas SE-83 (Matsuda et al. (1987) J. Bacteriol. 169, 5821-5826) and Acinetobacter sp. ATCC 53891 (Croux et al. (1990) Span. Pat. P9002109, Eur. Pat. 0469919A2, U.S. Pat. No. 5,354,667) and have been expressed in strains of Escherichia coli, commonly used in genetic engineering, extremely high levels of enzyme production being obtained, in comparison to those obtained in the parental strains.
In the process for purification of the enzymes expressed in Escherichia coli, co-purification of the unwanted enzymes referred to earlier (.beta.-lactamases, esterases, etc.) is not entirely avoided, which means that the GLA obtained has to be purified in a number of chromatographic steps.
In the scientific literature no references have been found concerning any modification of the gla gene which would allow the enzyme GLA to be purified in a single chromatographic step.
GLA belongs to the group of beta-lactam acylases which, with the exception of penicillin V acylase, share a very similar molecular structure. These enzymes have a structure which is unusual among prokaryotic proteins; they are synthesized as an inactive precursor polypeptide which is secreted into the periplasmic space thanks to the presence of a signal peptide. This precursor is subsequently hydrolysed, releasing two polypeptides called the alpha subunit (16-25 kDa) and beta subunit (54-69 kDa); both join to form the active enzyme (Sudhakaran et al. (1992) Process Biochem. 27, 131-143). This type of processing common to all of them supports the idea that all of them belong to the same family of proteins and have evolved from a common ancestor.
With regard to substrate specificity, they can be divided into two groups. The first group comprises the beta-lactam acylases which are highly specific to penicillin G, but their specificity generally resides in the acyl residue of the substrate, which must be hydrophobic and preferably benzyl or alkyl. Examples of these enzymes are the penicillin G acylases of Escherichia coli, Kluyvera citrophila,
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Barredo Fuente José Luis
Collados de la Vieja Alfonso
Cortes Rubio Estrella
Diez Garcia Bruno
Garcia Lopez Jose Luis
Achutamurthy Ponnathapu
Moore William W.
Sntibioticos, S. A.
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