A screening test was undertaken to isolate microorganisms that produced ascorbate oxidase. The enzyme activity was found in a culture filtrate of a fungal strain (HI-25), newly isolated from a soil sample. Based on the morphological characteristics, this isolate was identified as Acremonium sp. From the examinations of cultural conditions, optimum conditions for enzyme production were found ; strain HI-25 was aerobically cultured by a jar fermenter at 25°C in a medium containing 5% glycerol, 2% defatted soybeans, 0.1% monosodium L-glutamate, 0.1% KH₂PO₄, 0.02% MgSO₄·7H₂O, and 0.01% KCl, pH 6.0. After cultivation, an ascorbate oxidase was purified from the culture filtrate by an ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and Butyl-Toyopearl, and gel filtration twice on Sephadex G-100. The purification was 850-fold with an activity yield of 8.8%. The purified enzyme gave a single band on SDS polyacrylamide gel electrophoresis, and had a molecular weight of 80, 000 by SDS polyacrylamide gel electrophoresis and 76, 000 by native gel filtration. This enzyme was most active at pH 4.0, 45°C, and was most stable between pH 6.0-10.0 and at temperatures below 60°C.

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A bacterium that was isolated from soil and identified as Bacillus circulans was found to produce a highly viscous extracellular polysaccharide when it was grown aerobically in a medium containing glucose as a sole source of carbon. The product was characterized by TLC and GC analyses as a novel heteropolysaccharide consisted of rhamnose, mannose, galactose, and mannuronic acid as sugar components. A maximal yield of polysaccharide reached about 2g/liter by jar-fermentor culture at 30°C for 48 hr with a medium containing 1% glucose, 0.05% asparagine, 0.005% yeast extract, and small amounts of inorganic salts. Some culture conditions for the production of polysaccharide were investigated with flask culture ; an optimal production was attained with a medium containing 0.1-1% glucose and 0.01-0.05% asparagine, pH 7-8, at 30°C under aerobic conditions.

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Differeces in the polymorphism and crystallinity of chitosan were found in membranes prepared by different procedures when examined by X-ray diffraction measurements for four samples of chitosan differing in the degree of polymerization. When an acetic acid solution of chitosan was dried in air and then soaked in an alkaline solution (method A), both hydrated and anhydrous polymorphs of chitosan were present in the resulting memebranes ; the latter polymorph made chitosan insoluble in common solvents of chitosan, and its crystallinity increased with decreasing chitosan molecular weight. When a highly concentrated chitosan solution in aqueous acetic acid was neutralized with an alkaline solution (method B), no anhydrous polymorphs were detected in the membrane because of incomplete drying. When aqueous formic acid was used as the solvent, behavior basically similar to that in aqueous acetic acid was observed. In contrast, even with method A, aqueous hydrochloric acid gave a chitosan membrane having very little anhydrous crystallinity. The crystalline polymorph called"1-2", which has been proposed to be one of four chitosan polymorphs, is considered to be a mixture of hydrated and anhydrous crystals.

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To investigate the usefulness of ornithine and urea as freshness indices for marine invertebrates, the muscle tissue from freshly caught samples was stored in ice for 15 days and the free amino acid concentration was measured. The total amount of free amino acids decreased slightly after 1 day in most of the samples. The arginine concentration also decreased in most of the samples, especially in kuro shrimp during ice storage, while the ornithine and urea levels increased markedly in arrow squid and kuro shrimp, and increased considerably in kuruma prawn. Based on these results, it is proposed to use urea and ornithine as freshness indices for kuro shrimp and arrow squid, while only urea is proposed for kuruma prawn.

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An artificially inserted extra peptide (21 amino acid peptide) between the B.subtilis α-amylase signal peptide and the mature thermostable α-amylase was completely cleaved by B.subtilis alkaline protease in vitro. The cleavage to form a mature enzyme was observed between pH 7.5 and 10, but not between pH 6.0 and 6.5, although a similar protease activity toward Azocall was observed between pH 6.0 and 7.5. To analyze the effects of pH on the cleavage, CD spectra at pH 6, 8, and 11 of the NH₂-terminally extended thermostable α-amylase were analyzed and the results were compared with those of the mature form of the α-amylase. It is suggesteded that the cleavage of the NH₂-terminally extended peptide is controlled by the secondary and tertiary structure of the precursor enzyme. Similar cleavage of different NH₂-terminally extended peptides by the alkaline protease was also found in other hybrid thermostable α-amylases obtained.

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The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M_r of 27, 865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M_r of 91, 040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like seauence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E.coli and B.subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expresson in both host organisms.

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Endopeptidase was partially purified from the globulin fraction of 4-hr-imbibed soybean seeds. The protease fraction obtained had proteolytic activity on the glycinin A₄A₅ subunit at both pH 4 and 8. A suitable peptidic substrate for the endopeptidase was isolated from the tryptic digest of the carboxymethylated A₄A₅ subunit. Using the tryptic peptide of glycinin A₅ subunit, a simple assay system for the soybean endopeptidase activity has been established. The activity was significantly inhibited by phenylmethylsulfonyl fluoride, indicating the endopeptidase is a serine protease.

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A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the α-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145kDa) having both α-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original α-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.

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Alkaliphilic Bacillus penicillinase produced by Escherichia coli is distributed in several subcellular compartments according to cultivation conditions. The penicillinase that accumulated in particular subcellular fractions of E.coli grown under different conditions was purified and characterized. Periplasmic or extracellular penicillinase (24kDa) was mature protein, indicating that the putative precursor (27kDa) was processed at the correct amino acid residue, probably by signal peptidase I. Cytoplasmic penicillinase contained two unusual proteins(25kDa) that are produced by proteolytic cleavage of the precursor within its signal sequence.

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The hydroxyl radical scavenging action of green tea tannin given orally to rats with experimental renal failure was examined by using the urinary methylguanidine excretion as an index. In rats given 2mg of a green tea tannin mixture, the urinary methylguanidine excretion was significantly decreased or had a tendency to decrease. (-)-Epigallocatechin 3-O-gallate, the predominant component of the green tea tannin mixture, effected a decrease in the urinary methylguanidine excretion in rats at a dose as low as 0.25mg, suggesting a hydroxyl radical scavenging action.

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Two calcium-stimulated ATPases at optimal pH values of 5.0 and 7.0, which are designated as acid and neutral Ca²⁺-ATPase, respectively, were found in the membrane fraction of human milk, and their enzymatic properties were studied. For maximal activity, neutral Ca²⁺-ATPase required 0.45mM Ca ion, while acid Ca²⁺-ATPase required 207mM Ca ion. Neutral Ca²⁺-ATPase activity was not enhanced by adding the Mg ion at more than 0.1 mM. Among the nucleotides, neutral Ca²⁺-ATPase showed a higher substrate specificity to GTP, CTP, ITP, and UTP than to ATP, while ATP was the best substrate for acid Ca²⁺-ATPases. Neutral and acid Ca²⁺-ATPases had apparent K_m values of 0.361 and 0.192 mM, and V_max of 186 and 178 μmol ATP hydroyzed/mg of protein per min, respectively. Both Ca²⁺-ATPases were potently inhibited by fluoride, lanthanide, vanadate, and p-chloromercuribenzoate, and inactivated by EDTA, EGTA, and CDTA, but were unaffected by N-ethylmaleimide, NaN₃, ouabain, oxidized glutathione, or oligomycin, and were inactivated by heating at 60°C for 10 min. These enzmes were concentrated in the membrane fraction of the cream and skim milk membrane.

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We have cloned the xynA gene coding for xylanase A, a major commponent of the xylanase family, from Aspergillus kawachii. The cDNA was isolated from an A.kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein. Nucleotide sequence analysis of the cDNA showed a 981-bp open reading frame that encoded a protein of 327 amino acid residues. The signal peptide was composed of 25 amino acid residues and the N-terminus of the mature protein was pyroglutamic acid. The transformed yeast with a cloned cDNA prodcued xylanase. The genomic DNA was arranged as ten exons and nine introns.

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In a study of genetic lesions responsible for amino acid requirements in multiple auxotrophic lactic acid bacteria, a systematic attempt was made to isolate mutants that could synthesize each of the amino acids required by the parental strains of Lactococcus lactis, Enterococcus faecium, and Pediococcus acidilactici. After treatment with appropriate mutagens or, in some cases, spontaneously, such mutants could indeed be obtained with respect to many but not all essential amino acids. Successful isolation of mutants for a given amino acid means that a minor genetic lesion reparable by single-step mutations affects its biosynthesis ; a failure to isolate mutants suggests the involvement of more extensive lesions. Analysis of the results obtained showed certain regularities : some of the biosynthetic pathways for individual amino acids were virtually unaffected or affected by minor lesions in all the strains tested, while others were affected to varying extents among the different strains. Further studies showed that the ability to synthesize a number of amino acids had been acquired simultaneously in several of the amino acid-synthesizing mutants obtained after a single-step mutagenesis in E.faecium and P.acidilactici. Some detailed analysis with one of such mutants from E.faecium showed that a structural alteration of RNA polymerase caused by a single-step mutation is to some extent associated with simultaneous acquisition of the synthetic ability for a number of amino acids.

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The respiration inhibitory effects of the inhibitory uncouplers, 2, diiodo-4-(2, 2-dicyanovinyl)phenol and 2, 6-dimethoxy-4-(2, 2-dicyanovinyl)phenol, the binding site of which is cytochrome bc1 (cyt.bc1) complex, were studied with rat-liver mitochondria. The inhibitory potency of 2, 6-diiodo-4-(2, 2-dicyanovinyl) phenol and of 2, 6-dimethoxy-4-(2, 2-dicyanovinyl) phenol was increased and decreased, respectively, by steep dissination of the transmembrane electrical potential after adding the potent uncoupler, fluazinam, the uncoupling activity of which disappears with time. Changes in the inhibitory potency may have been due to variation of the binding affinity of these compounds to cyt.bc1 complex. Furthermore, the enhancement to the binding affinity of 2, 6-diiodo-4-(2, 2-dicyanovinyl) phenol was governed by the degree of reduction in the transmembrane electrical potential. These results suggest that the extent of conformational changes of cyt.bc1 complex, which resulted in an enhanced interaction between 2, 6-diiodo-4-(2, 2-dicyanovinyl) phenol and its binding niche, increased with decreasing transmembrane electrical potential. From examinations of the reduction of cyt.b, it is suggest that the action site of 2, 6-diiodo-4-(2, 2-dicyanovinyl) phenol may be close to or partially overlapping that of antimycin A.

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The protease I from Streptomyces griseus var.alcalophilus strongly catalyzed the transfer reaction forming hydroxamic acids of various amino acids, especially aromatic and hydrophobic amino acids. Dimethyl sulfoxide (DMSO), 1, 4-butane diol, and glycerol protected this enzyme from heat deaturation. Moreover, only DMSO strongly activated the transfer action of protease I and the velocity of reaction was accelerated three times in the presence of 50% DMSO. The yield of transfer product strongly depended on reaction time and the concentrations of enzyme and substrates in the presence of DMSO. The velocity of the transfer reaction was faster than that of the hydrolysis reaction under the conditions of 0.1 _M Phe-OEt, 0.75_M NH₂OH, and 50% DMSO.

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Scavenging of active oxygen species by glycated proteins was investigated. Glycated proteins were prepared from bovine serum albumin (BSA), insulin, and lysozyme incubated with glucose. Glyated BSA at concentration of 0.5% scavenged 34% of hydroxyl radicals by ESR experiments using DMPO as a spin-trapping reagent. The ability to scavenge hydroxyl radicals by glycated BSA was higher than that by BSA. Hydrogen peroxides also were largely scavenged with an increase in the concentration of glycated proteins. However, the ability to scavenge superoxides by glycated BSA was lower than that by BSA because glycated proteins produced superoxides. Experiments using model compounds such as Amadori compound and caproyl pyrraline suggested that the scavenging ability of glycated proteins against hydroxyl radicals depends on Maillard reaction products in the advanced stage, while the ability against hydrogen peroxides is dependent upon Maillard reaction products in the early stage and brown pigments.

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We have developed apparatus to assess spinnability that uses an impedance to prevent of electrolysis. The spinning distance and the stress confirmed by measurement of dial gauge. A total of 10 starch pastes was prepared from corn, sago, katakuri, sweet potato, kuzu, edible canna, cassava, Indian lotus root, bracken, and potato, at 2% and 4% concentrations. The spinning distance of each starch paste showed a dependence on the concentration and the tensile velocity. The strain at the point of maximum stress increased in all samples with increases in the tensile velocity. The spinning energy increased with increase in the tensile velocity, with a particularly large dependence on the tensile velocity of potato. Cluster analysis was done using the spinning property values of 4% starch pastes as the parameters, which showed a division at a distance of 20, yielding four clusters, with each cluster showing a distintive spinning pattern.

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Isomaltooligosaccharides (IMO), sweeteners derived from corn-starch, selectively promote the growth of bifidobacteria in the human intestine. The metabolic fate of IMO in healthy men was investigated. The expiration rates of excess ¹³CO₂ and hydrogen of six men were measured while sedentary and while taking physical exercise after the ¹³C-labeled IMO intakes. The breath H₂ excretion kept at a constant state after IMO ingestion in the sedentary test and increased in the exercise test. The serum glucose and serum insulin increased 30min after IMO ingestion. The ¹³CO₂ recoveries were 28.7% in the sedentary test and 60.9% in the exercise test. These recoveries were 70-80% compared those of maltose. These results indicated that a part of IMO was digested and the residual IMO was fermented by intestinal flora. The energy value of IMO might be about 75% of that of maltose.

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An erythrose reductase was obtained from the cells of an Aureobasidium sp. mutant having high erythritol-producing activity. This enzyme was purified 600-fold over the cell-free extract by ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, and hydrophobic chromatography. It gave a single band on polyacrylamide gel electrophoresis, and had a molecular weight of 37, 000 and an isoelectric point of 4.8. This enzyme had maximum reductive activity at 45°C and pH 6.5. The optimum pH of the oxidative reaction was 9.5. It was stable at pH 6.0-8.0 and below 40°C. The enzyme showed the maximum activity to D-erythrose. D-Glyceraldehyde was reduced at a rate 66% of that for D-erythrose. p-Nitrobenzaldehyde, L-erythrulose, dihydroxyacetone, and D-glucuronate were also reduced, although at shower rates. The oxidative activity was less than 0.1% of the reductive one.

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Five capsaicinoids, the pungent ingredients of hot peppers, have been efficiently synthesized by a method involving the Wittig reaction between isobutyl triphenylphosphorane and the appropriate lactols (3), followed by the nitrous acid-induced isomerization of the resultant (Z)-olefins (4) to (E)-olefins (5).

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An isolate of Cladosporium herbarum obtained from leaves of sugar beet produced a phytotoxic red pigment which was identified as ent-isophleichrome (1). Production of the pigment was dependent on composition of the culture medium. The toxicity of ent-isophleichrome to sugar beet was strongly influenced by light. The polyketide biosynthetic origin of ent-isophleichrome in C.herbarum was demonstrated by ¹³C acetate labelling/¹³C NMR studies.

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Ice structure was photographically analyzed for frozen soy protein curd and egg albumin gel frozen under various conditions. Dendritic ice structure was observed growing from the cooling plate parallel to the direction of the heat flux. The change in the ice structure size was analyzed at different locations from the cooling plate in the plane perpendicular to the direction of heat flux. In accordance with the theoretical relationship proposed by us before, the mean ice structure size was inversely proportional to the moving speed of the freezing front and the proportionality constant was not very much different from the diffusion coefficient of water, showing the important role of the molcular diffusion mechanism in the process of ice crystal growth. For the freezing accompanied with supercooling, the ice structure became very small, reflecting the very rapid moving speed of the freezing front when supercooling ceased. The theoretical model by us had advantages over the models proposed in the literature for its simple theoretical basis and wider applicability.

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Eight strains of Aspergillus species isolated from katsuobushi (dried bonito) were grown in a liquid medium containing acetophenone, to examine the possibility of asymmetric reduction of acetophenone during the molding process in katsuobushi production. Seven strains produced predominantly the R-stereoisomer of α-phenethylalcohol. However, one strain gave the S-isomer predominantly.

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