Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1994-08-11
1996-06-04
Naff, David M.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
435135, 435148, 435190, 4352523, 4352554, 435280, 435921, C12N 902, C12N 904, C12P 762, C12P 726
Patent
active
055232230
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
1. Field of the Invention
The subject of the invention is a new ketoester reductase suitable for NADH dependent enzymatic conversion of .beta.-, .gamma.- and .delta.-ketoesters to the corresponding optically active .beta.-, .gamma.- and .delta.-hydroxy acid esters and which is isolatable from strains of Candida parapsilosis, Yarrowinia cellobiosa or Rhodococcus erythropolis and Pseudomonas acidovorans.
2. Background of the Invention
Optically active .beta.-, .gamma.- and .delta.- hydroxycarboxylic acid esters are valuable chiral intermediates with wide application in the synthesis of pharmaceuticals, aromatic substances, pheromones, agrochemicals and enzyme inhibitors. They can only be obtained with difficulty in conventional chemical ways since the separation of the enantiomer mixtures resulting from chemical reduction is difficult and cost intensive.
The fermentative production of .beta.-, .gamma.- and .delta.-hydroxy carboxylic acids and esters with microorganisms is known. In these works the microbial cells are always introduced in excess and the yield and enantiomer excess of products vary over a wide range depending upon the source of the cells.
Primarily the bakers yeast Saccharomyces cerevisiae has been used.
There are already known oxidoreductases which are isolated from Saccharomyces cerevisiae which catalyze the enzymatic reduction of the .beta.-, .gamma.-and .beta.-ketogroups from the corresponding ketoesters and the formation of the corresponding optically active hydroxy compounds (Heidlas et al (1988) Eur. J. Biochem, 172: 633-639). These known oxidoreductases require NADPH as coenzyme, which is difficult to regenerate so that this known enzymatic synthesis technique has not received commercial acceptance.
OBJECT OF THE INVENTION
The object of the invention is an enzymatic conversion of .beta.-, .gamma.- and .delta.- ketoesters catalyzed by an NADH dependent enzyme suitable therefor.
SUMMARY OF THE INVENTION
Such an enzyme has been surprisingly found in certain yeasts and bacteria and indeed in Candida parapsilosis and Yarrowinia cellobiosa as well as in Rhodococcus erythropolis and Pseudomonas acidovorans which are able to utilize n-alkane and n-alkanoic acids.
The ketoester reductase isolated from Candida parapsilosis and Rhodococcus erythropolis have been widely investigated.
Especially high activity of the new ketoester reductase (KERed) is obtained when Candida parapsilosis and Rhodococcus erythropolis are cultured on a nutrient medium which contains long-chain alkanoic acids or alkanes, especially dodecanoic acid and tetradecane as carbon sources.
The formation of the KERed with Candida parapsilosis DSM 70 125 and the isolation and purification of the enzyme from this strain has been especially intensively studied.
The purified KERed is characterized by the following parameters:
A pH optimum for the ketoester reduction between 7.8 and 8.0 and for the back reaction of pH 9.5.
A temperature optimum for the ketoester reduction between 36.degree. C. and 40.degree. C. and for the reverse reaction of 50.degree. C. to 56.degree. C.
Rapid deactivation by Hg.sup.2+ -, Pb.sup.2+ -, Ag.sup.+ -, Cu.sup.2+ -, Ni.sup.2+, Sn.sup.2+ and Co.sup.2+ ions. Strong inhibition by p-hydroxymercuribenzoate, 5,5'-dithio-bis(2-nitrobenzoate) and iodoacetamide as well as by the chelators 2,2'-bipryridyl and o-phenanthroline. Stabilization with SH-protective reagents like dithiothreitol.
In addition the reduction of aliphatic allcyclic and aromatic ketones, diketones, ketals and aldehydes as well as the oxidation of primary and secondary alcohols are catalyzed.
The reductive enzymatic conversion of .beta.-, .gamma.-and .delta.-ketoesters to the corresponding optically active hydroxy carboxylic acid esters is effected according to the following equation: ##STR1##
R and R' can be various residues as will be apparent from Table 4. n can assume values of 1 to 3. Beyond that the model of the substrate binding site (FIG. 5, Table 5, Example 3.G) reaches the limits of the substrate acceptance.
Apart fro
REFERENCES:
patent: 5169758 (1992-12-01), Fischer et al.
Utting et al., J. Biol. Chem., vol. 250, No. 13, 5233-42, 1975.
Ward et al., Enzyme Microb. Technol., 1990, vol. 12.
Peters et al., Appl. Microbiol. Biotechnol., 38, 334-40, Dec. 1992.
Kula Maria-Regina
Peters Jorg
Dubno Herbert
Forschungszentrum Julich GmbH
Meller Mike
Myers Jonathan
Naff David M.
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