Susumu Shimura

Production of bioactive triterpenes by Eriobotrya japonica calli

Callus tissue cultures induced from an axenic leaf of Eriobotrya japonica (Rosaceae) produced triterpenes in large amounts (ca. 50 mg/g dry wt). Nine triterpenes were characterized as ursolic acid, oleanolic acid, 2alpha-hydoxyursolic acid, maslinic acid, tormentic acid, 2alpha, 19alpha-dihydroxy-3-oxo-urs-12-en-28-oic acid, hyptadienic acid and a mixture of 3-O-cis-p-coumaroyltormentic acid and 3-O-trans-p-coumaroyltormentic acid. The triterpene composition in the callus tissues was noticeably different from that in intact leaves. The contents of tormentic acid with antidiabetic action, and 2alpha, 19alpha-dihydroxy-3-oxo-urs-12-en-28-oic acid with anti-HIV activity, were much larger than those in the intact leaves. All of the triterpenes isolated from the callus tissues showed an inhibitory effect comparable to (-)-epigallocatechin gallate (EGCG) of green tea on the activation of Epstein-Barr virus early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). 2alpha, 19alpha-Dihydroxy-3-oxo-urs-12-en-28-oic acid was the most potent inhibitor among them and caused a significant delay of two-stage carcinogenesis on mouse skin.

Flavan dimers with lipase inhibitory activity from Cassia nomame

Five flavan dimers which showed lipase-inhibiting effects were isolated from fruits of Cassia nomame (Leguminosae). Structures of two new compounds among them were determined to be (2S)-3′,4′,7-trihydroxyflavan-(4β → 8)-catechin and (2S)-3′,4′,7-trihydroxyflavan-(4α → 8)-catechin. Fouf flavan dimers structurally related to these two compounds were also synthesized for spectral comparison. Among 10 flavan dimers tested for lipase-inhibitory activity, (2S)-3′,4′,7-trihydroxyflavan-(4α → 8)-catechin showed the most potent inhibitory effect. A partially purified fraction composed of oligomeric flavans with Mn 1020 also showed a noticeable inhibitory effect.

Lipase production by Trichosporon fermentans WU-C12, a newly isolated yeast

Abstract From the soil samples of various locations, 245 strains of microorganisms were isolated by the enrichment culture method using olive oil as a carbon source. Of these microorganisms one deuteromycotinous yeast was the best producer of extracellular lipase, and the strain WU-C12 was identified as Trichosporon fermentans from the morphological and taxonomical properties. When cultivated at 30°C for 4 d in the medium containing 8% (w/v) corn steep and 3% (v/v) olive oil as sources of nitrogen and carbon, T. fermentans WU-C12 produced 126 U/ml of extracellular lipase. When 3% (v/v) tung oil was used instead of 3% (v/v) olive oil, 146 U/ml of the lipase was produced. Although lipase production decreased to 40 U/ml by the addition of 2% (w/v) glucose to the corn steep-olive oil medium, the strain WU-C12 produced 34 U/ml of lipase in the medium containing 2% (w/v) glucose instead of 3% (v/v) olive oil. On the other hand, T. fermentans WU-C12 could grow and produce lipase in the medium containing n -paraffin as a carbon source.

Anomer-selective glucosylation of l-menthol using lyophilized cells of Saccharomyces cerevisiae

Abstract A novel glucoside of l -menthol was synthesized from l -menthol and maltose by reaction with the lyophilized cells of Saccharomyces cerevisiae . Conditions for the synthetic reaction were examined and optimized. When 50 mg of l -menthol and 1.4 M maltose in 10 mM citrate-10 mM phosphatebuffer (pH 7.0) were incubated with the lyophilized cells for 96 h at 40°C, l -menthyl α- d -glucopyranoside (α-MenG) was selectively obtained as a product, and the maximum molar conversion yield based on l -menthol supplied reached 19.8%.

Ester synthesis by a crude lipase of Rhizopus oligosporus in an aqueous system

Abstract Ester synthesis in an aqueous system using a crude lipase of Rhizopus oligosporus was investigated. Oleic acid was used as a substrate to evaluate the yield of esterification. Oleyl alcohol was esterified with the yield of 44.0%. In the primary alcohols (C 1 ∼C 10 ), 1-propanol was esterified with the maximum yield of 38.5%. The lipase was able to esterify secondary alcohols, and 2-propanol was esterified with a maximum yield of 36.5%.

Selective alpha-glucosylation of eugenol by alpha-glucosyl transfer enzyme of Xanthomonas campestris WU-9701

For one-step enzymatic synthesis of eugenyl alpha-glucoside as a promising pro-drug for a hair restorer and a derivative of spices, selective alpha-glucosylation of eugenol was carried out using the alpha-glucosyl transfer enzyme of Xanthomonas campestris WU-9701. When 130 micromol eugenol and crude enzyme showing 1.0 unit of alpha-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 8.0) containing 1.2 M maltose as a glucosyl donor at 40 degrees C, only one form of eugenyl glucoside was selectively obtained as a product and identified as eugenyl alpha-D-glucopyranoside (alpha-EG) by 13C-NMR, 1H-NMR, and two-dimensional heteronuclear multiple-bond coherence analyses. In the reaction, no other glucosylated products such as maltotriose or eugenyl maltoside were detected in the reaction mixture. The reaction at 40 degrees C for 48 h under the above conditions yielded 68 mumol alpha-EG in 2 ml suspension, and the maximum molar conversion yield based on the amount of eugenol supplied reached 52%.

Identification of a Methioninase Inhibitor, Myrsinoic Acid B, from Myrsine seguinii Lév., and Its Inhibitory Activities

A methioninase inhibitor from Myrsine seguinii was purified and identified as myrsinoic acid B. Its inhibitory activities as to crude methioninase from periodontal bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola were determined. The IC50 values were 10.5, 82.4, and 30.3 μM respectively.

Purification of extracellular lipases from Trichosporon fermentans WU-C12

Abstract Two types of extracellular lipases (I and II) from Trichosporon fermentans WU-C12 were purified by acetone precipitation and successive chromatographies on Butyl-Toyopearl 650 M, Toyopearl HW-55F and Q-Sepharose FF. The molecular weight of lipase I was 53 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and 160 kDa by gel filtration, while that of lipase II was 55 kDa by SDS-PAGE and 60 kDa by gel filtration. For the hydrolysis of olive oil, the optimum pH and temperature of both the lipases were 5.5 and 35°C, respectively. The lipases showed stable activities after incubation at 30°C for 24 h in a pH range from 4.0 to 8.0. The thermostability of lipase I for 30 min at a reaction pH of 5.5 was up to 40°C, while that of lipase II under the same conditions was up to 50°C. Both lipases could hydrolyze the 1-, 2-, and 3-positions of triolein, and cleave all three ester bonds, regardless of the position in the triglyceride.

Enhancement of lipase production from hydrocarbons by mutation of Trichosporon fermentans

Lipase high-producing mutants with petroleum products as carbon sources were successfully induced from Trichosporon fermentans WU-C12 by ultraviolet (UV) light irradiation. In the first mutation step, one mutant strain, PU-30, derived from strain WU-C12 was selected. The productivity of extracellular lipase of PU-30 reached 58 units (U)/ml in the medium containing kerosene, being approximately twice the productivity of the parental strain WU-C12. In the second mutation step, the mutant strain 2PU-18 was induced from strain PU-30. In medium containing kerosene, gas oil and liquid paraffin, the 2PU-18 produced 70 U/ml, 62 U/ml and 60 U/ml of extracellular lipase, respectively. When various n-alkanes (C8-C18) were used as carbon sources, the parental strain WU-C12 produced more than 20 U/ml of lipase only from C9-C12 alkanes, but 2PU-18 could produce more than 50 U/ml of lipase from C8-C18 alkanes. When cultivated for 3 days in medium containing liquid paraffin, the activity ratios of extracellular lipase to total lipase and the values of extracellular lipase activity per dry-cell weight were 0.44 and 0.65 U/mg for WU-C12, and 0.62 and 1.82 U/mg for 2PU-18, respectively. These results indicate that the mutant strain 2PU-18 is superior in both total lipase productivity and permeability of lipase to the parental strain WU-C12 when petroleum products are used as carbon sources.

Myrsinoic acid B inhibits the production of hydrogen sulfide by periodontal pathogens in vitro

Recently, we reported that myrsinoic acid B purified from Myrsine seguinii inhibited methyl mercaptan (CH(3)SH) production by Fusobacterium nucleatum JCM8532. Since hydrogen sulfide (H(2)S) is the main component of physiological halitosis, while CH(3)SH is involved in pathological oral halitosis, the objective of this study is to determine whether myrsinoic acid B inhibits H(2)S production by oral microorganisms. F. nucleatum, Porphyromonas gingivalis and Treponema denticola were incubated with myrsinoic acid B and a substrate such as l-cysteine or l-methionine. H(2)S or CH(3)SH concentration in the headspace air, was determined using a gas chromatograph. The concentration of myrsinoic acid B inhibiting 50% (IC(50)) of H(2)S production by F. nucleatum was 0.142 µg ml(-1), and the IC(50) of P. gingivalis and T. denticola were 2.71 µg ml(-1) and 28.9 µg ml(-1), respectively. The presence of pyruvate, a by-product of H(2)S production, was determined. The IC(50) values of myrsinoic acid B for pyruvate production were 22.9 µg ml(-1) for F. nucleatum, 87.7 µg ml(-1) for P. gingivalis and 165 µg ml(-1) for T. denticola. We concluded that myrsinoic acid B inhibited the production of both H(2)S and pyruvate by periodontal pathogens.