E.i. Dupont De Nemours and Company

Bristol-myers Squibb Pharma Company

Robert Dicosimo

Robert Donald Fallon

John Edward Gavagan

Mark Scott Payne

Sarita Chauhan

Lech Wilczek

Michael G Bramucci

Arie Ben-Bassat

Frank Edward Herkes

Leo Ernest Manzer

Daniel P O'Keefe

Anna Panova

Jeffery Scott Thompson

Philip Ma

Wonpyo Hong

Jaan A Pesti

Susan K Fager

This invention relates to a process for the preparation of a 3-hydroxycarboxylic acid from a 3-hydroxynitrile. More specifically, 3-hydroxyvaleronitrile is converted to 3-hydroxyvaleric acid in high yield at up to 100% conversion, using as an enzyme catalyst 1) nitrile hydratase activity and amidase activity or 2) nitrilase activity of a microbial cell. 3-Hydroxyvaleric acid is used as a substitute for &#x3b5;-caprolactone in the preparation of highly branched copolyester.

3-Hydroxycarboxylic acid production and use in branched polymers

3-hydroxycarboxylic acid production and use in branched polymers

Recombinant microbial strains are provided that express nitrilase enzyme and are useful as biocatalysts for the hydrolysis of nitrile-containing substrates. The recombinant cells are transformed with a foreign gene isolated from72W encoding a thermostable nitrilase enzyme that catalyzes the hydrolysis of nitrile-containing substrates to carboxylic acids under mild reaction conditions. The nucleotide sequence of the nitrilase gene and the deduced amino acid sequence encoded by the nitrilase gene are provided.

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Isolation and expression of a gene for a nitrilase from Acidovorax facilis 72W

A method for preserving immobilized or unimmobilized microbial cells having nitrilase activity and for stabilizing the nitrilase activity of unimmobilized or immobilized microbial cells has been developed. Aqueous suspensions containing at least 100 mM bicarbonate, carbonate, or carbamate salts limit microbial contamination of the stored enzyme catalyst, as well as stabilize the desired nitrilase activity of the unimmobilized or immobilized cells. Microorganisms which are characterized by an nitrilase activity and are stabilized and preserved by this method include 72-PF-15 (ATCC 55747), 72-PF-17 (ATCC 55745), 72W (ATCC 55746), and transformed microbial cells having nitrilase activity, the host cells transformed with 72W nitrilase activity. Especially preferred is an embodiment using ammonium carbamate as the inorganic salt.

Method for stabilizing nitrilase activity and preserving microbial cells with carbamate salts

The invention provides a process for the hydrolysis of acrylonitrile to acrylic acid, and for the hydrolysis of methacrylonitrile to methacrylic acid, in high yield and at high concentration with high specificity. Acrylonitrile or methacrylonitrile is hydrolyzed in a suitable aqueous reaction mixture by a catalyst characterized by a nitrile hydratase and amidase activity of Comamonas testosteroni 5-MGAM-4D, producing the corresponding acid. The acrylic acid or methacrylic acid is isolated as the acid or corresponding salt.

Method for producing methacrylic acid acrylic acid with a combination of enzyme catalysts

The present invention relates to a process for the enantioselective enzymatic aminolysis of racemic 2-&lsqb;3-(4-cyanophenyl)-4,5-dihydro-5-isoxazolyl&rsqb;acetate ((R/S)-I) by methyl N -Boc-L-&agr;,&bgr;-diaminoproprionate.p-toluenesulfonic acid. More specifically, this invention pertains to a process for the enantioselective enzymatic aminolysis of racemic isobutyl 2-&lsqb;3-(4-cyanophenyl)-4,5-dihydro-5-isoxazolyl&rsqb;-acetate by methyl N -Boc-L-&agr;,&bgr;-diaminoproprionate.p-toluene sulfonic acid to produce (R)-methyl-3-&lsqb;&lsqb;&lsqb;3-(4-cyanophenyl)-4,5-dihydro-5-isoxazolyl&rsqb;acetyl&rsqb;amino&rsqb;-N-(butoxycarbonyl)-L-alanine, an intermediate in the preparation of roxifiban, an isoxazoline-based platelet glycoprotein IIb/IIIa antagonist which has activity as an antithrombotic agent.

Enantioselective enzymatic aminolysis of a racemic 2-isoxazolylacetate alkyl ester

The present invention relates to a method for producing 2-hydroxyisobutyric acid where acetone cyanohydrin is converted to 2-hydroxyisobutyric acid using an enzyme catalyst having nitrilase activity, or having a combination of nitrile hydratase and amidase activities. The invention also encompasses production of methacrylic acid wherein the 2-hydroxyisobutyric acid produced with the catalyst described is dehydrated to produce methacrylic acid.

Method for producing 2-hydroxyisobutyric acid and methacrylic acid from acetone cyanohydrin

This invention relates to a process for the preparation of a 3-hydroxycarboxylic acid from a 3-hydroxynitrile. More specifically, 3-hydroxyvaleronitrile is converted to 3-hydroxyvaleric acid in high yield at up to 100% conversion, using as an enzyme catalyst 1) nitrile hydratase activity and amidase activity or 2) nitrilase activity of a microbial cell. 3-Hydroxyvaleric acid is used as a substitute for &egr;-caprolactone in the preparation of highly branched copolyester.

This invention relates to an improved process for preparing a carboxylic acid from the corresponding nitrile using a nitrilase catalyst. More particularly, the instant invention converts 2-methylglutaronitrile to 4-cyanopentanoic acid in aqueous solution using an enzyme catalyst having an nitrilase activity, immobilized in alginate, and crosslinked with glutaraldehyde and polyethylenimine.

Process for preparing 4-cyanopentanoic acid

This invention relates to a process for preparing dicarboxylic acid monoesters from cyanocarboxylic acid esters. More particularly, using the nitrilase from 72W (ATCC 55745) in various forms as an enzyme catalyst, an aliphatic or aromatic cyanocarboxylic acid ester is converted to the corresponding dicarboxylic acid monoester with high chemoselectivity at 100% conversion. An embodiment of the invention also provides a process for obtaining high chemoselectively and high regioselectivity.

Preparation of dicarboxylic acid monoesters from cyanocarboxylic acid esters

The present invention relates to a method for producing &agr;-hydroxy acids using an enzyme catalyst having nitrilase activity. More specifically, the invention pertains to use of 72W (ATCC 55746) nitrilase to hydrolyze glycolonitrile to glycolic acid. Glycolonitrile is reacted in an aqueous mixture with a catalyst having 72W nitrilase activity to give glycolic acid selectively, and at high concentration and high yield.

Method for producing glycolic acid from glycolonitrile using nitrilase

Method for producing &agr;-hydroxy acid from &agr;-hydroxy nitrile using nitrilase

A method for preserving immobilized or unimmobilized microbial cells having nitrilase activity and for stabilizing the nitrilase activity of unimmobilized or immobilized microbial cells has been developed. The unimmobilized or immobilized microbial cells are stored in an aqueous solution containing from about 0.10 M to the saturation concentration of an inorganic salt of bicarbonate or carbonate, including ammonium, sodium and potassium salts of bicarbonate or carbonate. Aqueous suspensions containing at least 100 mM bicarbonate or carbonate limit microbial contamination of the stored enzyme catalyst, as well as stabilize the desired nitrilase activity of the uninmmobilized or immobilized cells. Microorganisms which are characterized by an nitrilase activity and are stabilized and preserved by this method include 72-PF-15 (ATCC 55747), 72-PF-17 (ATCC 55745), 72W (ATCC 55746), and transformed microbial cells having nitrilase activity, preferably SS1001 (ATCC PTA-1177) which is transformed with 72W nitrilase activity.

Method for stabilizing nitrilase activity and preserving microbial cells

A method for the stabilization of nitrilase activity of unimmobilized or immobilized microbial cells has been developed. The unimmobilized or immobilized microbial cells are stored in an aqueous solution containing from 0.100 M to the saturation concentration of an inorganic salt of bicarbonate or carbonate, including ammonium, sodium and potassium salts of bicarbonate or carbonate. Aqueous suspensions containing at least 100 mM bicarbonate or carbonate limit microbial contamination of the stored enzyme catalyst, as well as stabilize the desired nitrilase activity of the unimmobilized or immobilized cells. Microorganisms which are characterized by an nitrilase activity and are stabilized and preserved by this method include,72-PF-15 (ATCC 55747),,72-PF-17 (ATCC 55745), and,72W (ATCC 55746).

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Rockland, DE, United States of America

Robert Dicosimo