Kohji Ishihara

Biotransformation using plant cultured cells

This review outlines the recent progress during the last 25 years concerning the biotransformation of exogenous substrates by plant cultured cells. The plant cultured cells have abilities of the regio- and stereoselective hydroxylation, oxido-reduction, hydrogenation, glycosylation, and hydrolysis for various organic compounds as well as microorganisms. The reaction types and the stereochemistry of the products involved in the biotransformations are described. The development of techniques using immobilized plant cells are also delineated.

Structural aspects of acylated plant pigments: stabilization of flavonoid glucosides and interpretation of their functions

The enzymatic synthesis of acylated isoquercitrins was accomplished by the lipase-catalyzed transesterification with carboxylic acid vinyl esters as acyl donors in an organic solvent. The introduction of an acyl group into isoquercitrin improved its thermostability and light-resistivity. In particular, isoquercitrin p-coumarate was the most stable of all the acylated isoquercitrins tested.

Low catalase activity in blood is associated with the diabetes caused by alloxan

BACKGROUND Hydrogen peroxide is enzymatically processed by catalase, and catalase deficiency in blood is known as acatalasemia. We examined whether low catalase activity is a risk factor for diabetes mellitus. METHODS Blood glucose, insulin and glucose tolerance test were examined in acatalasemic and normal mice under non-stress and oxidative stress conditions. Alloxan administration was used as oxidative stress. RESULTS Alloxan, which was a drug that caused diabetes mellitus, mostly generated hydrogen peroxide by the reaction of alloxan and reduced glutathione, in vitro. Incidence of hyperglycemia in alloxan-untreated acatalasemic mice was as low as that in the normal mice. However, the incidence of acatalasemia mice treated with alloxan was higher than that in normal mice, and the number of pancreatic beta-cells in the acatalasemic mice was less than that in normal mice. CONCLUSION These results indicate that low catalase activity in the blood is associated with the diabetes mellitus caused by alloxan administration.

Stable earthworm serine proteases: Application of the protease function and usefulness of the earthworm autolysate

The fibrinolytic enzymes from Lumbricus rubellus [Nakajima, N. et al., Biosci. Biotechnol. Biochem., 57, 1726-1730 (1993), 60, 293-300 (1996), and 63, 2031-2033 (1999)] were further characterized to exploit their catalytic functions. These enzymes are stable in solution for long periods at room temperature and strongly resistant to organic solvents, even toluene and n-hexane. The serine proteases can act on various protein substrates such as elastin and hemoglobin as well as fibrin, and also catalyzed the hydrolysis of esters such as ethyl acetate and a bioplastic, poly[(R)-3-hydroxybutyrate] film. The enzymes, in the absence of microbial degradation, contributed to the production of the earthworm autolysate possessing antioxidant ability and protease activity, whose components were similar to those of soy sauce. The extract of the earthworm autolysate could be used as a peptone substitute in media for the cultivation of microorganisms.

Stereoselective reduction of keto esters: thermophilic bacteria and microalgae as new biocatalysts

This review covers the possibility of aerobic thermophilic bacteria (Bacillus strains and thermophilic actinomycetes) and microalgae (Chlorella strains and marine algae) as new biocatalysts for the stereoselective reduction of α- and β-keto esters. The mechanistic interpretation of the reduction by a thermophilic actinomycete is also delineated.

Purification and Characterization of α-Keto Amide Reductase from Saccharomyces cerevisiae

An NADPH-dependent α-keto amide reductase was purified from Saccharomyces cerevisiae. The molecular mass of the native enzyme was estimated to be 33 and 36 kDa by gel filtration chromatography and SDS–polyacrylamide gel electrophoresis, respectively. The purified enzyme showed a reducing activity not only for aromatic α-keto amides but also for aliphatic and aromatic α-keto esters. The internal sequence of the enzyme was identical with that of a hypothetical protein (ORF YDL 124w) coded by yeast chromosome IV.

Stereoselective biotransformation of limonene and limonene oxide by cyanobacterium, Synechococcus sp. PCC 7942

The biotransformation of limonene and limonene oxide by the cyanobacterium, Synechococcus sp. PCC 7942, was investigated. (S)-(+)-Limonene was hydroxylated stereo- and regioselectively at its allylic position of the endocyclic C=C double bond by the cyanobacterial cells to its corresponding alcohol. The cells also showed the ability for the enantio- and stereoselective cleavage of the epoxide group of (1S,2R,4R)-limonene oxide to give (1S,2S,4R)-limonene-1,2-diol. The repetitive production of carveol from limonene was achieved using Ca2+-alginate-immobilized cyanobacterial cells.

Stereocontrolled reduction of α-keto esters with thermophilic actinomycete, Streptomyces thermocyaneoviolaceus IFO 14271

Abstract The reduction of aliphatic and aromatic α-keto esters was carried out using a thermophilic actinomycete, Streptomyces thermocyaneoviolaceus IFO 14271, as a biocatalyst. Ethyl 3-methyl-2-oxobutanoate, methyl benzoylformate, and ethyl benzoylformate were reduced to the corresponding ( R )-alcohols with >98% enantiomeric excess (ee) at 37°C, while the reduction in the presence of glutamic acid gave the ( S )-hydroxy esters in excellent ee (>99%). Ethyl 2-oxopropanoate and ethyl 2-oxobutanoate were also reduced to the corresponding ( S )-alcohols with >99% ee in the presence of an amino acid such as asparagine or aspartic acid.

Enzyme-catalyzed enantiomeric resolution of N-Boc-proline as the key-step in an expeditious route towards RAMP

Abstract For the preparation of both enantiomers of N-carbamoyl-2-methoxymethylpyrrolidine, the precursors of Enders’ chiral auxiliaries, SAMP and RAMP, enzyme-catalyzed kinetic resolution of racemic N-carbamoyl, N-Boc, N-Cbz proline esters and prolinols were examined. B. licheniformis protease (subtilisin) preferentially hydrolyzed the (R)-carbamoylproline ester with an enantiomeric ratio (E) of 10. To a hydrophobic N-Cbz proline ester, subtilisin showed lower selectivity (E=2.8), and in contrast, a purified protease (isozyme A) from the earthworm showed the preference of (S)-enantiomer (E=13.6). In a practical sense, C. antarctica lipase B (Chirazyme L-2) was effective for the hydrolysis of both N-Boc and N-Cbz derivatives with E >100. The e.e. of (R)-N-carbamoyl-2-methoxymethylpyrrolidine was raised to be >99.9% by recrystallization at the N-Boc-prolinol stage, which was derived from the (R)-N-Boc-proline methyl ester (98.7% e.e.) through a preparative-scale enzyme-catalyzed resolution (49% yield) of the racemic substrate.

Synthesis of glycosides of resveratrol, pterostilbene, and piceatannol, and their anti-oxidant, anti-allergic, and neuroprotective activities.

Resveratrol was glucosylated to its 3- and 4′-β-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4′-β-glucoside. P. americana cells converted piceatannol into its 4′-β-glucoside. The 3- and 4′-β-glucosides of resveratrol were further glucosylated to 3- and 4′-β-maltosides of resveratrol, 4′-β-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-β-glucoside and 3-β-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4′-β-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4′-β-glucoside showed high phosphodiesterase inhibitory activity. Graphical Abstract Stilbenes were glucosylated to the corresponding β-glucosides by cultured cells of Phytolacca americana. Stilbene glucosides were converted into β-maltosides by cyclodextrin glucanotransferase.