Purification and characterization of 1,3-ß-D-glucan phosphorylase from Ochromonas danica.

1,3-ß-D-glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-ß-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C-30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-ß-glucans.

A wild and tolerant yeast suitable for ethanol fermentation from lignocellulose.

Ethanol fermentation from food wastes containing mainly starch without carrying out sterilization was investigated by using wild and tolerant yeast, Issatchenkia orientalis MF-121. The MF-121 strain is not a suitable choice for ethanol fermentation from lignocellulosic biomass because it is only capable of fermenting hexoses of glucose, mannose, and fructose to ethanol. Therefore, we first isolated acid- and salt-tolerant yeast that are capable of fermenting various monosaccharides to ethanol, and the isolated yeast that showed the ability to ferment ethanol from glucose, mannose, galactose, fructose, and xylose, was identified as Zygoascus hellenicus LK-5G on the basis of the 26S rRNA sequence analysis.

A comparative study of ethanol production by Issatchenkia orientalis strains under stress conditions.

The ability of 13 strains of multi-stress-tolerant Issatchenkia orientalis yeast to produce ethanol was examined under different stress conditions, including conditions of elevated Na2SO4 and Na2SO4 concentrations and increased heat. The MF-121 strain produced a significant amount of ethanol after the incubation in acidic media containing high concentrations of salt, e.g., 50 g/l Na2SO4 at pH 2.0, or at high temperatures, e.g., 43°C, when compared with other strains.

Construction of a beta-glucosidase expression system using the multistress-tolerant yeast Issatchenkia orientalis.

We demonstrate the value of the thermotolerant yeast Issatchenkia orientalis as a candidate microorganism for bioethanol production from lignocellulosic biomass with the goal of consolidated bioprocessing. The I. orientalis MF-121 strain is acid tolerant, ethanol tolerant, and thermotolerant, and is thus a multistress-tolerant yeast. To express heterologous proteins in I. orientalis, we constructed a transformation system for the MF-121 strain and then isolated the promoters of TDH1 and PGK1, two genes that were found to be strongly expressed during ethanol fermentation. As a result, expression of beta-glucosidase from Aspergillus aculeatus could be achieved with I. orientalis, demonstrating successful heterologous gene expression in I. orientalis for the first time. The transformant could convert cellobiose to ethanol under acidic conditions and at high temperature. Simultaneous saccharification and fermentation (SSF) was performed with the transformant, which produced 29 g l(-1) of ethanol in 72 h at 40 degrees C even without addition of beta-glucosidase when SSF was carried out in medium containing 100 g l(-1) of microcrystalline cellulose and a commercial cellulase preparation. These results suggest that using a genetically engineered thermotolerant yeast such as I. orientalis in SSF could lead to cost reduction because less saccharification enzymes are required.

Structure of lintnerized starch is related to X-ray diffraction pattern and susceptibility to acid and enzyme hydrolysis of starch granules.

Acid-resistant residues (lintnerized starches, Ls) were prepared from starches showing A-, B- and C- X-ray diffraction patterns. Ls retained the same X-ray crystalline type as their native counterparts with an improvement in diffraction intensity. Fluorophore-assisted capillary electrophoresis (FACE) study indicated that structural characteristics of Ls were associated with X-ray diffraction patterns. Double helices originated from linear chains with an approximate average degree of polymerisation (DP) 14, 16, and 15 would span the entire length of crystalline lamellae of A-, B-, and C-type starches, respectively. The proportion of singly branched materials (SB) with DP 25 protected in Ls was higher for A-type Ls (10-17%) than for B-type Ls (4-6%) and C-type Ls (8%). The structures of SB were similar in which branched chain (DP 13-15) was longer than main chain (DP 10-12). The structural characteristics of Ls are discussed in relation to acid and enzymatic degradations of starch granules.

Effect of Hatakeshimeji (Lyophyllum decastes Sing.) Mushroom on serum lipid levels in rats.

The effect of Hatakeshimeji (Lyophyllum decastes Sing.) mushroom on serum lipid levels was investigated in rats. When the mushroom (fruit body) powder or its hotwater extract was added at a level of 10% to a cholesterol-containing diet, the serum total cholesterol levels of rats fed the fruit body or the hot-water extract were markedly lower than that of controls, though there was no significant difference in serum HDL-cholesterol among the three groups. On a cholesterol-free diet, the addition of fruit body powder at a level of 5% significantly decreased serum total cholesterol. Serum triglycerides and phospholipids were significantly decreased in both the fruit body and hot-water extract groups. Furthermore, Hatakeshimeji in the diet significantly increased the activity of cholesterol 7a-hydroxylase, which converts cellular cholesterol to bile acids, as well as the fecal excretion of bile acids.