Application of biocatalysis and biotransformations to the synthesis of pharmaceuticals

Preparation and characterization of Pseudomonas putida esterase immobilized on magnetic nanoparticles

A novel lactonohydrolase, an enzyme that catalyzes the hydrolysis of 3,4-dihydrocoumarin, was purified 375-fold to apparent homogeneity, with a 22.7% overall recovery, from Acinetobacter calcoaceticus F46, which was isolated as a fluorene-assimilating micro-organism. The molecular mass of the native enzyme, as estimated by high-performance gel-permeation chromatography, is 56 kDa, and the subunit molecular mass is 30 kDa. The enzyme specifically hydrolyzes 3,4-dihydrocoumarin, and the Km and Vmax for 3,4-dihydrocoumarin are 0.806 mm and 4760 U·mg−1, respectively. The N-terminal and internal amino acid sequences of the enzyme show high similarity to those of bacterial non-heme haloperoxidases. The enzyme exhibits brominating activity with monochlorodimedon in the presence of H2O2 and 3,4-dihydrocoumarin or an organic acid, such as acetate and n-butyrate.

3,4‐Dihydrocoumarin hydrolase with haloperoxidase activity from Acinetobacter calcoaceticus F46

Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-β-acetylthioisobutyrate (dl-MATI) to produce d-β-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed dl-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67°C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 A, confirming that EST is a member of the α/β hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket ∼12 A deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in dl-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.

Stereoselective Esterase from Pseudomonas putida IFO12996 Reveals α/β Hydrolase Folds for d-β-Acetylthioisobutyric Acid Synthesis

Lactone compounds are widely distributed in nature and play important roles in organisms. These compounds are synthesized and metabolized enzymatically in vivo; however, detailed investigation of these enzymes lags behind that of other common enzymes. In this paper, recent work on the enzymes involved in the metabolism of lactone compounds will be reviewed. In particular, fundamental and application studies on lactonases and Baeyer-Villiger monooxgenases of microbial origin are described.

Microbial enzymes involved in lactone compound metabolism and their biotechnological applications.

Microorganisms producing D-β-acetylthioisobutyric acid from methyl D-β-acetylthioisobutyrate were screened from stock cultures. The D-β-acetylthioisobutyric acid-producing ability was found in 15 strains belonging to the genera Pseudomonas, Agrobacterium, Enterobacter, Cellulomonas, Rhodococcus, Brevibacterium, and Torulopsis. A strain of Pseudomonas fluorescens, IFO 3081, was selected as the best microorganism. The cells having activity (558 units/g of dry cells) could be easily prepared by cultivation at 25°C at pH 6.6 for 24 hr in a glucose-containing medium. The D-form of methyl DL-β-acetylthioisobutyrate was selectively hydrolyzed with the cells so that D-β-acetylthioisobutyric acid (97.2% enantiomeric excess) was produced in a high yield.

Screening of Microorganisms Producing D-β-Acetylthioisobutyric Acid from Methyl DL-β-Acetylthioisobutyrate

PURPOSE:To efficiently separate the subject compound from a reactional system and purify the compound because of a low elusion of proteins from a catalyst into the reactional system by asymmetrically hydrolyzing a carboxylate ester with a specific immobilized vital catalyst. CONSTITUTION:A carboxylate ester of formula I (R1 is alkyl, aryl, etc.; R2, R3 are alkyl; (n) is 1, 2) (e.g. beta-acetylthio-alpha-methylpropionic acid methyl ester) is treated with an immobilized vital catalyst to provide the objective compound of formula II, the immobilized vital catalyst being produced by treating bacterial cells with a multifunctional crosslinking agent, and the bacterial cells having an ability to asymmetrically hydrolyze the ester bond. The bacterial cells asymmetrically hydrolyzing the ester bond is preferably Pseudomonas putida MR-2068 strain (FERN P-3846), etc. The multifunctional agent includes glutar aldehyde. The hydrolysis reaction is preferably performed at >=45 deg.C for 4-15hrs.

Production of optically active carboxylic acid and its antipode ester

PURPOSE:To efficiently obtain the titled ester useful for synthesizing various physiologically active substances, by reacting a specific ester with a microorganism of the genus Enterobacter, etc., capable of asymmetrically hydrolyzing ester bonds of the ester. CONSTITUTION:Enterobacter.cloacae IAM 1624 having the ability to asymmetrically hydrolyze ester bonds is inoculated into a culture medium containing a carbon source, nitrogen source, etc., and cultivated at 5-50 deg.C and pH2-11 by spinner culture with aeration to provide cultivated microbial cells, which are then suspended in a phosphoric acid buffer solution, etc. 0.01-50wt.% ester expressed by formula I (R1 is alkyl, aralkyl or aryl; R2 and R3 are alkyl; n is 1 or 2) (e.g. methyl S-acetyl-beta-mercaptoisobutyrate) is added and reacted at pH2-11 and 5-50 deg.C. An optically active carboxylic acid expressed by formula II (R1, R2 and n are same as those described above) and antipodal ester thereof produced in the reaction solution are subsequently subjected to extraction treatment, separated and purified by column chromatography, etc.

Production of optically active carboxylic acid and antipodal ester thereof

PURPOSE:To improve production efficiency by reacting a specific ester with a culture solution, etc., of a microorganism, belonging to the genus Corynebacterium and having the ability to asymmetrically hydrolyze ester bonds. CONSTITUTION:(A) Corynebacterium sp. N-6004 strain having the following properties is obtained by searching and separation from the natural world. Shape; polymorphic bacillus. Gram staining property; +. No recognized spore. Starch decomposition) incapable. Asymmetrically hydrolyzing ester bonds of an ester expressed by formula I (R1 is alkyl, aralkyl or aryl; R2 and R3 are alkyl; n is 1 or 2). The resultant strain is then cultured in a liquid culture medium containing a carbon source, nitrogen source, etc., at 5-50 deg.C and pH2-11 by spinner culture with aeration to provide (B) a culture solution. The ester expressed by formula I, such as methyl S-acetyl-beta-mercaptoisobutyrate, in an amount of 0.01-50wt.% is added to the culture solution (B) and reached therewith at 5-50 deg.C and pH2-11 to afford (C) a reaction solution, which is subsequently extracted and purified to produce an optically active carboxylic acid expressed by formula II and in antipodal ester thereof.

Hosoi Akihiko

Papers

Screening of Microorganisms Producing D-β-Acetylthioisobutyric Acid from Methyl DL-β-Acetylthioisobutyrate

Production of optically active carboxylic acid and its antipode ester

Production of optically active carboxylic acid and antipodal ester thereof

Production of optically active carboxylic and antipodal ester thereof