Hiroyuki Nakagawa

Purification and characterization of a novel β-agarase from an alkalophilic bacterium, Alteromonas sp. E-1

A novel beta-agarase (EC 3.2.1.81) was purified from an agar-degrading alkalophilic bacterium, Alteromonas sp. E-1 isolated from the soil. This enzyme was obtained from a cell-free extract after sonication and purified 40.9-fold through treatment with streptomycin, ammonium sulfate fractionation and successive chromatography on anion-exchange and gel filtration columns. The molecular weight was estimated to be 82 kDa by SDS-polyacrylamide gel electrophoresis and 180 kDa by Superdex 200 gel filtration. The enzyme was inhibited by Mn2+, Cu2+, Fe2+, Zn2+ and Hg2+, and activated by K+, Na+ and EDTA, and its optimum pH and temperature for agarose degradation were 7.5 and 40 degrees C, respectively. This beta-agarase hydrolyzed agarose with rapid reduction of viscosity, and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose] was detected from the early stage of the reaction. Neoagarobiose as the final product was selectively released from agarose, neoagarohexaose and neoagarotetraose by the reaction with this beta-agarase. This observation was different from that of other beta-agarases which produced mixtures of neoagarobiose and neoagarotetraose as the final hydrolysis products. The N-terminal amino acid sequence of this beta-agarase shows no homology to those of other beta-agarases that were so far reported.

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%.

Citric acid production from cellulose hydrolysate by a 2-deoxyglucose- resistant mutant strain of Aspergillus niger

Abstract Citric acid production from cellulose hydrolysate was done by a semi-solid culture of Aspergillus niger Yang no. 2, a parental strain, and C192, which is a 2-deoxy- d -glucose-resistant mutant strain. Cellulose powder was hydrolyzed to mainly glucose and a low concentration of cellobiose by a commercial cellulase in acetate or citrate buffer. The hydrolysate prepared in acetate buffer was concentrated to contain 150 g/l of reducing sugars and used as a medium for fermentation. Yang no. 2 and C192 produced 77·9 g/l and 98·7 g/l of citric acid in 9 days from the concentrated hydrolysate, respectively. Acetate buffer delayed the fermentation. When the hydrolysate was prepared in citrate buffer and used as a medium, Yang no. 2 and C192 produced 92·2 g/l and 102·3 g/l of citric acid within 3 days from the concentrated hydrolysate containing 150 g/l of reducing sugars, respectively. It is concluded that citric acid production from cellulose hydrolysate with high conversion yield, maximally 68·2% based on the reducing sugars supplied, was achieved first by C192.

Recycle Use of Sphingomonas sp. CDH-7 Cells for Continuous Degradation of Carbazole in the Presence of MgCl2

Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.660 3.3) were shaken in 50 mM K2HPO4-KH2PO4 buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 mM MgCl2. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl2. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells.

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.

Analysis of the Masked Metabolite of Deoxynivalenol and Fusarium Resistance in CIMMYT Wheat Germplasm

Fusarium head blight (FHB) causes significant grain loss and contamination of grains with harmful mycotoxins, especially deoxynivalenol (DON). Fusarium resistance and DON accumulation have been extensively investigated in various cultivars; however, the level of DON-3-O-glucoside (D3G) has not been as carefully studied. In this study, we measured accumulated DON and D3G levels in CIMMYT wheat elite germplasm using an analytical method validated in-house. Co-occurring nivalenol (NIV) and ergostrerol (ERG) were also analyzed. LC-MS/MS and LC-UV analyses were applied to the 50 CIMMYT elite wheat lines. D3G showed rather high correlation with DON (r = 0.82), while FHB symptoms showed slight correlation with DON and D3G (r = 0.36 and 0.32, respectively). D3G/DON ratio varied widely from 8.1 to 37.7%, and the ratio was not related with FHB resistance in this dataset.

Development of an LC-MS/MS Determination Method for T-2 Toxin and Its Glucoside and Acetyl Derivatives for Estimating the Contamination of Total T-2 Toxins in Staple Flours

A determination method previously validated for trichothecenes and zearalenone by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was adapted for the quantification of T-2 toxin (T-2) as well as its glucoside and acetyl derivatives, T-2-3-glucoside (T-2-3G) and 3-acetyl-T-2 (3A-T-2). HT-2 toxin (HT-2) and its acetyl derivative 3-acetyl-HT-2 (3A-HT-2) were also included as the target chemicals. Staple flours (56 samples collected from the Singapore market) were examined for contamination from T-2 and/or HT-2 and their derivatives. Among them, 16 flours were found to be contaminated with T-2 and/or HT-2, whereas none was contaminated with T-2-3G and 3A-HT-2, except for trace 3A-T-2 detected in 2 rye samples. Rye flour samples were frequently contaminated with both T-2 and HT-2. Some of the reference materials (RMs) were further analyzed, and T-2-3G and 3A-T-2 were quantitatively detected in corn and wheat RMs. The ratio of T-2-3G to T-2 in the RMs seemed to be much lower than the ratio of deoxynivalenol-3-glucoside to deoxynivalenol usually reported in former studies. To the best of our knowledge, the natural contamination of 3A-T-2 in staple flour is reported here for the first time.

Detection of N-(1-deoxy-D-fructos-1-yl) Fumonisin C1, C2 and C3 in Corn Powder by LC - Orbitrap MS

Detection of N-(1-deoxy-D-fructos-1-yl) fumonisin C1, C2 and C3 (NDfrc-FCs) in a reference material of corn powder were performed with LC-Orbitrap MS. The peaks of NDfrc-FCs were eluted 0.1 ~ 0.3 min earlier than those of fumonisin C1, C2 and C3 (FCs), from the C18 column, probably due to their hydrophilic structures having the carbohydrate residues. At negative ionization mode scan with LC-MS analysis, the fragment ions of the tricarballylic acid (TCA) and characteristic fumonisin ions lacking TCA were detected at the identical retention times with those of respective parent NDfrc-FCs. Mass fragmentation patterns of NDfrc-FCs were confirmed to be almost in consistent with those of FCs. This study is the first report of natural occurrence of NDfrc-FC1, FC2, and FC3 in corn powder.