Shinpei Yamamoto

Preparation and evaluation of an enzyme which degrades yeast cell walls

SummaryA method for the production and preparation of an enzyme which degrades yeast cell walls from a species of aRhizoctonia (tentatively identified asR. solani) was established on a commercial scale. The production of crude enzyme powder, having a lytic activity of 100 units/mg, in batches of 80 kg is feasible.The enzyme preparation was evaluated for industrial use. When yeast cells were treated with this enzyme, the digestibility of feed yeast was improved 1.4–2 fold in vitro; the efficiency of a mechanical disintegrator in extracting cellular substances was increased 35–50%; the release of soluble glucans having widely varying degrees of polymerization was induced; the extraction of cellular protein by alkali was facilitated 2–3 fold; an 80% release of cell-bound invertase was induced and 2–3 times more yeast extract could be prepared.

PURIFICATION AND PROPERTIES OF BACILLUS COAGULANS CYCLOMALTODEXTRIN GLUCANOTRANSFERASE

Abstract Cyclomaltodextrin glucanotransferase (CGTase), produced in a culture filtrate by Bacillus coagulans , was purified to a single, homogeneous protein. It has a monomeric structure with a molecular weight of 65,000, isoelectric point of 4.6, and contains 2 mol of Ca 2+ per mol of the enzyme. The enzyme was most active at pH 6.0 and at 70°C. It did not lose its activity by heat treatment at 70°C for 10 min in the presence of CaCl 2 in the pH range of 5.5∼9.5, and by incubation in the pH range of 5.0∼10.5 at 4°C for one month. The enzyme converted about 60% of potato starch to cyclodextrins for 20 h at 50°C, and the ratio of α-: β-: γ-cyclodextrin produced was 8.1:8.9:1.0 B. coagulans CGTase was compared with B. macerans CGTase which was purified by the same method.

Crystalline enzyme which degrades the cell wall of living yeast

Abstract An enzyme which degrades yeast glucan and the cell wall of living yeast was crystallized from the culture filtrate of a mold belonging to the genus Rhizopus . The enzyme appears to be homogeneous and has a molecular weight of 19,000. The exact bond split by the enzyme was not identified: however, from the studies on the enzymatic degradation products of yeast glucan and on the substrate specificity of the enzyme, it appears to act as a debranching enzyme cleaved glycosidic bond at the branch points in yeast glucan.

Cyclomaltodextrin glucanotransferase-producing moderate thermophile, Bacillus coagulans

Abstract A new CGTase-producing moderate thermophile was isolated from soil, and was identified as Bacillus coagulans which have not previously been listed as cyclodextrin producing bacteria. The culture filtrate of the isolate as the CGTase source converted about 60% of soluble starch to CDs in 20 h at 50°C, and the ratio of α-: β-: γ-CD produced was 1.0: 0.9: 0.3.

Amino acid residues involved in the action of endo-(1→3)-β-d-glucanase II from Flavobacterium dormitator var. glucanolyticae FA-5

Abstract The pH-dependence of the kinetic parameters for the hydrolysis of yeast glucan with endo-(1→3)-β- d -glucanase II from Flav. dormitator var. glucanolyticae FA-5 suggests that two residues (histidyl and carboxyl) are involved in the enzyme action. Chemical modification of the enzyme has been studied in order to identify the kinds and the number of amino acid residues involved in enzyme action. Photo-oxidation and carbethoxylation of the enzyme indicated that the decomposition of a histidine residue is responsible for the loss of activity. Modification of the enzyme with Woodwards reagent K indicated that ∼ 12 carboxyl residues in the enzyme are involved in the catalytic and/or substrate binding-site.

Hemolysate activation coefficient and its relationship to holoenzyme levels of aspartate aminotransefrase in rabbit erythrocytes

Abstract The relationship between the aspartate aminotransferase (AST) activation coefficient and the intracellular in situ proportion of the pyridoxal 5′-phosphate (PLP) form of AST (PLP-AST) in total AST was determined. Erythrocytes from Japanese-White strain rabbits had an activation coefficient of 1.85. This indicated that 54.1% of the AST in the hemolysates from these erythrocytes was the holoenzyme. Sodium borohydride reduction showed that 64.8% of the total erythrocyte AST was present as the PLP-AST holoenzyme. Increases in intracellular PLP and pyridoxamine 5′-phosphate (PMP) concentrations, generated by preincubation of the erythrocytes with pyridoxal (PL) and pyridoxamine (PM), significantly decreased the values of the activation coefficient. However, these increases did not significantly increase the intracellular proportion of PLP-AST in total AST. These results showed that the AST activation coefficient reflects the saturation level of AST in the hemolysate rather than the intracellular in situ level of the holo-form of AST (holoAST). Thus, establishment of standard conditions is required for use of the activation coefficient, which is inherently a valuable biochemical marker of the vitamin B-6 status, for accurate deduction of the nutritional status of vitamin B-6 in test organisms.