Nobuyoshi Nakajima

Enantioselective synthesis of various D-amino acids by a multi-enzyme system

Abstract We have developed an effective method for the synthesis of various D-amino acids from the corresponding α-keto acids and ammonia by coupling four enzyme reactions catalyzed by D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase. In this system, D-glutamate is continuously regenerated from α-ketoglutarate, ammonia and NADH by the coupled reaction of glutamate dehydrogenase and glutamate racemase, and used as an amino donor for the enantioselective D-amino acid synthesis by the D-amino acid aminotransferase reaction. The unidirectional formate dehydrogenase reaction is also coupled to regenerate NADH consumed. Under the optimum conditions, D-enantiomers of valine, alanine, α-keto analogues with a molar yield higher than 80%.

Enzymatic conversion of racemic methionine to the L-enantiomer

We have developed an enzymatic method for conversion of racemic methionine to the L-enantiomer with a yield of >95% and >99% enantiomeric excess; the method has been applied to the conversion of DL-alanine and DL-leucine to the L-enantiomers.

Stereoselective Reduction of α-Keto Ester and α-Keto Amide with Marine Actinomycetes, Salinispora Strains, as Novel Biocatalysts

To clarify the potential ability of marine actinomycetes as biocatalysts, the stereoselective reduction of α-keto esters and α-keto amide using Salinispora arenicola and Salinispora tropica was tested. The reduction of ethyl pyruvate and ethyl 2-oxobutanoate by S. tropica gave corresponding alcohol with high conversion ratio and in high e.e. (96% e.e. (S) and 99% e.e. (S), respectively). In the presence of l-glutamate as an additive, the reduction of ethyl pyruvate by S. tropica afforded the corresponding (S)-ethyl lactate with >99% e.e. Furthermore, 2-chlorobenzoylformamide was reduced by S. tropica to the corresponding (R)-2-chloromandelamide with high conversion ratio and excellent enantioselectivity (>99% e.e.). Thus, it was found that marine actinomycetes, Salinispora strains, had high ability for the stereoselective reduction of carbonyl compounds as useful biocatalysts.

Plant-based Paste Fermented by Lactic Acid Bacteria and Yeast: Functional Analysis and Possibility of Application to Functional Foods

A plant-based paste fermented by lactic acid bacteria and yeast (fermented paste) was made from various plant materials. The paste was made of fermented food by applying traditional food-preservation techniques, that is, fermentation and sugaring. The fermented paste contained major nutrients (carbohydrates, proteins, and lipids), 18 kinds of amino acids, and vitamins (vitamin A, B1 B2, B6, B12, E, K, niacin, biotin, pantothenic acid, and folic acid). It contained five kinds of organic acids, and a large amount of dietary fiber and plant phytochemicals. Sucrose from brown sugar, used as a material, was completely resolved into glucose and fructose. Some physiological functions of the fermented paste were examined in vitro. It was demonstrated that the paste possessed antioxidant, antihypertensive, antibacterial, anti-inflammatory, anti-allergy and anti-tyrosinase activities in vitro. It was thought that the fermented paste would be a helpful functional food with various nutrients to help prevent lifestyle diseases.

preparation of chiral 2-chloromandelamide: stereoselective Reduction of an Aromatic α-keto Amide with Actinomycete strains

The stereoselective reduction of an aromatic α-keto amide with actinomycete strains was investigated. It was found that 2-chlorobenzoylformamide was reduced to the corresponding 2-chloromandelamide by mesophilic and thermophilic strains of actinomycetes. Among the strains tested, the reduction of 2-chlorobenzoylformamide by Streptomyces thermocyaneoviolaceus (one of thermophilic strains) in the presence of glycerol as an additive produced only (S)-2-chloromandelamide in >99% conversion with >99% enantiomeric excess (e.e.). On the other hand, the reduction by Streptomyces thermocarboxydovorans NBRC16324 at 45 °C or Thermoactinomyces vulgaris NBRC15851 cultivated in a soluble starch-based medium gave the corresponding (R)-hydroxy amide (conversion, 99%; >99% e.e.). Mesophilic and other thermophilic actinomycete strains also catalyzed the reduction to the corresponding (R)-hydroxy amide with 85%–>99% e.e. Thus, the syntheses of both enantiomers of 2-chloromandelamide was achieved though the reduction of 2-chlorobenzoylformamide with different actinomycete strains.

A Simple Enzymatic Method for Microdetermination of D-Glutamate

Abstract A simple and sensitive spectrophotometric method for the determination of D-glutamate has been developed. The method is based on the racemization of D-glutamate with glutamate racemase (EC 5.1.1.3) and the simultaneous dehydrogenation of L-glutamate with L-glutamate dehydrogenase (EC 1.4.1.3). NADH produced was determined either by the measurement of absorbance at 340 nm or by the colorimetric measurement of a formazan formed at 515 nm. A linear relationship has been obtained between the absorbance and the amount of D-glutamate (0.01-0.2 μmol). Enantioselective determination was also possible by measuring L-glutamate with L-glutamate dehydrogenase before the addition of glutamate racemase.