Kosuke Tomita

Substrate specificities in hydrolysis of polyhydroxyalkanoates by microbial esterases

SummaryEnzymatic degradations of 5 different polyhydroxyalkanoates (PHA) were investigated at 37°C in the aqueous solutions (pH 7.4) containing different microbial enzymes of 16 lipases and 5 PHA depolymerases. The substrate specificities of microbial PHA depolymerases on hydrolysis of polyhydroxyalkanoates were distinguished from those of microbial lipases.

Isolation of thermophiles degrading poly(L-lactic acid).

A thermophile, strain 93, which degrades poly(L-lactic acid) (PLA) film was isolated from 144 soil samples obtained from different locations by cultivation using an enrichment culture medium at 60 degrees C. Under this temperature condition, the strain grew on PLA and the dissolved total organic carbon (TOC) concentration in the medium changed according to the growth stage, i.e., after the TOC rapidly reached the minimum, it increased rapidly until it reached the peak and then decreased thereafter. For residual PLA, reduced viscosity decreased rapidly but weight decreased initially slowly with a gradually increasing rate. Gel permeation chromatograms showed a marked decrease in the main peak and the appearance of a new peak in the low molecular weight region. The strain was identified as Bacillus brevis, which has an optimum growth temperature of around 58 degrees C.

Isolation of a thermophile degrading poly(butylene succinate-co-butylene adipate).

A thermophile, strain 73, which degrades poly(butylene succinate-co-butylene adipate) (PBSA) film was isolated from 95 soil samples obtained from different locations by cultivation using an enrichment culture technique at 60 degrees C. At this temperature, the strain grew on PBSA and the dissolved total organic carbon (TOC) concentration in the medium changed according to the growth stage, i.e., after the TOC concentration rapidly reached the minimum, it increased rapidly until it reached a peak and then decreased thereafter. During cultivation, PBSA was almost eliminated and the solution viscosity of the residual PBSA decreased markedly. Gel permeation chromatograms of the residual PBSA showed a significant decrease in the main peak and the appearance of a new peak at the low molecular weight region. The strain was identified as Bacillus stearothermophilus, which has an optimum growth temperature of approximately 63 degrees C.

Thermostable Glucokinase from Bacillus stearothermophilus and Its Analytical Application

Enzymes have characteristics of substrate and reaction specificities and stereospecificities. Although enzymes are becoming popular in clinical chemical analysis, many of the enzymes from mesophiles tend to lose their activity on storage even a t r m m temperature. On the other hand, enzymes from thermophiles are remarkably superior to those from mesophiles in terms of thermal and storage stabilities. In this report, we shall describe the properties of glucokinase (GlcK) [EC 2.7.1.21 isolated from a moderate thermophilic bacterium Bacillus stearothermophilus and its application to the determination of some components in biological fluids.

Isolation of a thermophilic bacterium degrading some nylons

A thermophilic strain capable of degrading nylon 12 was isolated from 100 soil samples by enrichment culture technique at 60 °C. At this temperature, the strain grew on nylon 12, accompanied by a marked decrease in molecular weight of nylon 12. It was identified as a neighboring species to Bacillus pallidus, which has an optimum growth temperature of around 60 °C. The strain was also found to degrade nylon 6 as well as nylon 12, but not nylon 66.

Isolation of Thermophiles Assimilating Poly(Ethylene-co-Vinyl Alcohol)

A thermophile, strain 23, assimilating poly(ethylene-co-vinyl alcohol) (EVOH) was isolated from soil using an enrichment culture at 60°C. The strain grew on EVOH, decreased the solution viscosity of EVOH, and accumulated total organic carbon (TOC) in the medium. It was also capable of degrading poly(vinyl alcohol). The strain was identified as Bacillus stearothermophilus which has an optimum growth temperature around 58°C.

Stabilized enzymatic reagents for measuring glucose, creatine kinase and γ-glutamyltransferase with thermostable enzymes from a thermophile, Bacillus stearothermophilus

Stabilized enzymatic reagents for measuring some components in biological fluids have been successfully developed based on two kinds of thermostable enzymes derived from Bacillus stearothermophilus with separation of the reagent into two complementary solutions. The thermostable glucokinase produced was applied to the measurement of glucose and creatine kinase activity, while the alanine dehydrogenase produced was used for the measurement of gamma-glutamyltransferase activity. The enzymatic reagents were also stabilized by developing two separate reagents with an optimum pH for the main reagent components. The stability of the reagents in liquid form was examined at 10 degrees C. It was clearly shown that the reagents for measuring glucose and creatine kinase activity were stable and retained their full capability for accurate measurement in biological fluids for over one year. The alanine dehydrogenase product was stable for at least 40 days.

Enzymatic determination of acetylcarnitine for diagnostic applications

An enzymatic method was proposed for determination of acetylcarnitine (AcCar), even when carnitine (Car), non-acetylated form, co-exists. The method is consisted of four enzymatic reactions: First, AcCar is hydrolysed by acylcarnitine hydrolase to yield acetate; followed by the other three reactions coupled with three enzymes, respectively, acetate kinase, pyruvate kinase and lactate dehydrogenase; finally, the acetate formation causes a decrease in NADH. The amount of AcCar is then evaluated as the change in absorbance at 340 nm. The reagent composition of the reaction mixture was determined, and the characteristics of the method were investigated. The dilution test showed a good linearity over a wide range. The precision and accuracy tests produced satisfactory results. The co-existence of Car gave no effect on the measurement. The present method was found to be used easily, simply and rapidly for the selective determination of AcCar.

Usefulness of bacterial thermostable enzymes in clinical chemical analysis

Since enzymes have many excellent characteristics, e.g., high substrate and reaction specificities, they have become popular in clinical chemical analysis. However, many of the usual enzymes tend to lose their activities on storage, especially in a solution state, even at rather low temperature, which limits their uses remarkably. On the other hand, enzymes from thermophiles are known to be superior to usual enzymes in thermal and storage stabilities. The author et al. [1] have successfully prepared many thermostable enzymes from a thermophilic bacterium, Bacillus stearothermophilus (BS). Moreover, we have obtained several enzymes from a mesophilic bacterium, Zymomonas mobilis (ZM), and found they also have good thermal stability[2]. In this paper, we show their usefulness as tools in clinical chemical analysis, with focusing the application of two kinds of them, i.e., glucokinase (GlcK, EC 2.7.1.2) and alanine dehydrogenase (AlaDH, EC 1.4.1.1), to the determination of some components in a serum.

Enzymatic analysis of acetylpolyamine.

An enzymatic method was proposed for measuring acetylpolyamine (AcPA) alone, even when non-acetylated polyamine co-exists. The method consisted of four enzymatic reactions. First, AcPA was hydrolysed by acylpolyamine amidohydrolase to yield acetate; followed by the other three reactions coupled with three enzymes, respectively, acetate kinase, pyruvate kinase, and lactate dehydrogenase; the acetate formation caused a decrease in NADH. The quantity of AcPA was then evaluated as the change in absorbance at 340 nm. The reagent composition of the reaction mixture was determined, and characteristics of the method were investigated. The validation tests produced satisfactory results. The co-existence of non-acetylated polyamine gave no effect on the measurement. The present method was found to be used easily, rapidly and reliably for the selective determination of AcPA itself.