Changes in water-soluble arabinogalactan-proteins (AGPs) in cabbages at five stages (I, seedlings with cotyledon ; II, young seedlings with five to six leaves ; III, initiation of head formation ; IV, completion of head formation ; V, flower stalk development) during the course of development were investigated. The results indicate that only the white head portion contained the higher molecular weight AGP (A-I) together with the lower molecular weight AGP (A-II) with the largest at stage IV. On the other hand, green leaves contained only A-II and its content decreased rapidly with progress of growth from stage I to stage IV. The chemical composition analyses also showed that the hydroxyproline content decreased significantly with growth. Based on these results, we concluded that cabbage leaves contained water-soluble AGPs throughout the growth of cabbage and AGP A-I was specific for head formation of cabbage.

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The Forsythia koreana plant produces such lignans as (-)-secoisolariciresinol, and (+)-pinoresinol. Cell-free extracts from the plant catalyzed the enantioselective formation of (-)-[²H₁₀]secoisolariciresinol from [9, 9-²H₂, OC²H₃]coniferyl alcohol in the presence of NADPH and H₂O₂. On the other hand, [²H₁0]lariciresinol isolated from the enzymatic reaction products was found to be predominantly composed of the unnatural (-)-enantiomer [88% enantiomer excess (e. e. )]. The stereoselectivity for the formation of these lignans can be explained, at least in part, by the finding that the enzyme system also catalyzed the stereoselective reduction of (+)-lariciresinol, but not its (-)-enantiomer, to (-)-secoisolariciresinol.

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Alcaligenes latus was used to produce a new poly-sacchardide bioabsorbent that can absorb water at more than 1, 000 times its own weight, approximately 5 times greater than that of currently used commercially available synthetic high polymer absorbents. In addition, its water absorption capacity in the presence of NaCl was high when compared to synthetic polymers, with its moisture retention capacity in dry environments also found to be superior. This bioabsorbent is a polysaccharide.

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The structures of hetero-oligosaccharides obtained by the action of transglycosylation of Arthrobacter sp. K-1 β-fructofuranosidase, using sucrose as the fructosyl donor, and several mono- and di-sacchrides as the acceptors were investigated. The main transfer products to most reducing mono- and di-sacchrides were non-reducing oligosaccharides with a fructosyl residue linked to a hemiacetal hydroxyl group. In the presence of L-sorbose, the enzyme produced 2-O-β-D-fructofuranosyl-α-L-sorbopyranoside as the major product. With D-galactose or L-arabinose, the enzyme produced not only non-reducing oligosaccharides, but also reducing oligosaccharides, which were identified as 3-O-β-D-fructofuranosyl-D-galactopyranose and 4-O-β-D-fructofuranosyl-L-arabinopyranose, respectively. When a non-reducing sugar such as methyl α-glucoside was used as an acceptor, the product formed had a fructosyl residue linked at the C6 hydroxyl group.

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Nocardioides simplex IFO 12069 (Arthrobacter simplex ATCC 6946) was examined for the degradation of L-ornithine, L-citrulline, L-arginine, D-arginine, agmatine, carbamoylputrescine, and putrescine, and the results were compared with those obtained in a previous study with A. globiformis and Brevibacterium helvolum. D-Arginine as well as L-arginine was degraded via the arginine oxygenase pathway. A part of L-citrulline was probably degraded via the pathway. The L-ornithine degradation via the pathway was obscure. Guanidinobutyrase was induced by agmatine, which was shown to be degraded via a unique pathway. The first step of the pathway converted agmatine to 4-guanidinobutyraldehyde by transferring the amino group to pyruvate. The second step was the NAD-linked dehydrogenation of the aldehyde to 4-guanidinobutyrate, which was then degraded to 4-aminobutyrate. The enzymes for the first and second steps were identified as diaminopropane aminotransferase (EC 2. 6. 1. -) and aminobutyraldehyde dehydrogenase (EC 1. 2. 1. 19), respectively. These enzymes also degraded putrescine to 4-aminobutyrate. Carbamoylputrescine was converted by the aminotransferase to the corresponding aldehyde, which was accumulated in the medium.

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A 3', 5'-cyclicribonucleotide was enzymatically converted into a corresponding 2', 3'-cyclic-ribo-nucleotide by a culture extract of 5'-phosphodiesterase (5'-PDase ; EC 3. 1. 4. 1) hyperproductive mutant of Aspergillus niger, FS-44. Cyclic-ribonucleotide phosphomutase (cRNPMase) activity catalyzing the preceding reaction was not separated from the 5'-PDase activity during the purification steps. The enzyme was purified to the homogeneity of a single polypeptide with a molecular weight of 62, 000. The purified enzyme specifically catalyzed the conversion of 3', 5'-cyclic-ribonucleotides to corresponding 2', 3'-cyclic-ribonucleotides. The decreasing order of reaction rate was as follows : adenosine 3', 5'-cyclic monophosphate > inosine 3', 5'-cyclic monophosphate > uridine 3', 5'-cyclic monophosphate > guanosine 3', 5'-cyclic monophosphate > cytidine 3', 5'-cyclic monophosphate. The enzyme did not act on 2'-AMP, 3'-AMP, or 5'-AMP. The optimum pH of cRNPMase activity was pH 3.0, while that of 5'-PDase activity was pH 5. 6. Both activities was inhibited by diisopropyl flurophosphate, Fe²⁺, and Fe³⁺. While 5'-PDase activity was significantly inhibited by Zn²⁺ and Cu²⁺, cRNPMase activity was not at all. The Michaelis constant of cRNPMase was estimated to be 3. 72mM for adenosine 3', 5'-cyclic monophosphate and that of 5'-PDase was 0.96mM for adenosine 5'-p-nitrophenyl phosphate. The enzyme was a glycoprotein containing N-linked sugar chains. The isoelectric point was 4.8. This bifunctional enzyme was named cyclic-ribonucleotide phosphomutase-5'-phosphodiesterase (cRNPMase-5'-PDase).

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The effects of amino acid medium (AA medium) on the dissociation of rice callus tissues were examined in suspension-cultured cells, because a finely dispersed cell suspension had been obtained previously from rice callus tissues in this medium. The level of extracellular polysaccharides formed in cultured AA medium was much higher than that of those formed in cultured B5 medium. The polysaccharides were mainly composed of higher levels of arabinose, xylose, and galactose, suggesting the solubilization of arabinoxylan and (arabino)galactan. Nevertheless, wall polysaccharides in cells cultured in AA medium contained the same levels of arabinosyl-, xylosel-, and galactosyl-linkages as those in B5 medium. Based on amino acid analysis, rice cells in AA medium incorporated the amino acids in 3 days and formed ornithine and urea during the early stages of cultivation, and secreted urea into the culture medium. Transfer of rice tissue cultured in AA medium to B5 medium composed of inorganic nitrogen source caused most of the tissue aggregates to become larger than 1000 μm. However, after transfer to B5 medium containing 1 mM arginine, they were apparently maintained as a more-finely dispersed cell suspension at a size below 1000 μm. These findings suggest that arginine is metabolized in rice cells to form urea, which is then secreted and may solubilize the arabinoxylan and (arabino)galactan between cells. The test of binding capacity confirmed that arabinoxylan was bound to both insoluble xyloglucan and cellulose, and that urea dissociated arabinoxylan from the complex.

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Though estrogen prolongs the half-life of chicken ovalbumin (OVA) mRNA in vivo, little has been described about the molecular mechanism by which estrogen stabilizes the OVA mRNA. As a first trial to discover this molecular mechanism, the organ culture of estrogen-induced chick oviduct was introduced to know whether estrogen could stabilize OVA mRNA in vitro, as observed in vivo. In this culture system, the expression of egg white genes were induced and maintained by estrogen. Moreover, using a pulse-chase labeling method, we showed that estrogen stabilized the OVA mRNA, and that the half-lives of the OVA mRNA in the presence and the absence of estrogen were almost identical with those calculated from in vivo study. Thus, from this study, we concluded that this culture system had a faithful feature in egg white gene expression for the response to estrogen, and it will be useful to investigate the mechanism of estrogen-induced stabilization upon the OVA mRNA.

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A moderate thermophilic bacterium that converts DL-5-(2-methylthioethyl)hydantoin stereospecifically to L-methionine was isolated from soil and designated NS1122A. The bacterial strain NS1122A was identified as Bacillus stearothermophilus. The optimal temperature and pH of the reaction with the resting cells were 60-70°C and around 8, respectively. The addition of Co²⁺ or Mn²⁺ stimulated the production of L-methionine, while Cu²⁺ and Zn²⁺ strongly inhibited it. Under adequate conditions, 7. 6 mg/ml of L-methionine was produced from 10 mg/ml of DL-5-(2-methylthioethyl)hydantoin with a molar yield of 89%. The enzymes responsible for the conversion were purified from the cells of B. stearothermophilus NS1122A. The purified hydantoinase was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was approximately 200 kDa by gel filtration. The hydantoinase was capable of hydrolyzing L- and D-5-substituted hydantoins, and the D-form of 5-substituted hydantoins were more effectively hydrolyzed than the L-form. The partially purified N-carbamyl-amino acid amidohydrolase showed a broad substrate specificity with strict L-stereospecificities. The N-carbamyl-L-amino acid amidohydrolase required Co²⁺ (0. 5 mM), Mn²⁺ (0. 5 mM), or Ni²⁺ (5. 0 mM) ion for the activity.

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Based on the amide linkage formation catalyzed by water-soluble carbodiimide, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), various acidic polysaccharides could be labeled by the fluorescent probe of EDAN (N-1-ethylenediaminonaphthalene). The pectinase reaction was evaluated by the EDC-OPA (o-phthalaldehyde) method [Anal. Biochem., 189, 122-125 (1990)]. Moreover, paper chromatographic purification of the glucuronic acid-EDAN adduct allowed a 57-fold increase in the sensitivity of de-tection limit, i. e., about 0. 19 nmol of glucuronate could be measured. Pectin-EDAN adduct worked as the substrate for pectinase and changes in the molecular weight distribution during the course of pectinase digestion were monitored by gel filtration with a Sepharose 6B column. In the case of hyaluronic acid-EDAN adduct, the fluorescent substrate was unfavorable to the hyaluronidase action and 2 to 5-fold higher K_ms were obtained with the hyaluronic acids of lower and higher contents of EDAN, respectively.

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Dextran-dialdehyde was attached to trypsin molecules by incubation at 30°C and pH 8. 5. The cross-linking reaction was completed within a few hours, and the dextran-modified trypsin was isolated by gel filtration chromatography. The modified trypsin was resistant to auto-hydrolysis and a significant increases in thermal and pH stabilities was observed. Storage of modified trypsin at 4°C under the optimum pH caused little loss of activity. The modified trypsin was resistant to organic solvents such as 10% ethanol and 0. 2% sodium dodecylsulfate. Moreover, inhibition by soybean trypsin inhibitor was eliminated for the modified trypsin. Kinetic analysis indicated that access of a high-molecular-weight substrate (casein) to the modified enzyme was restricted compared with a low-molecular-weight substrate.

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A novel tryptophan metabolite colored with reddish orange was isolated by addition of diethyl-dithiocarbamate to washed cells of Chromobacterium violaceum. The molecular formula was identical to that of deoxyviolacein. The chemical structure was found to be 5-(3-oxoindolylidene)-3-(indol-3-yl)-3-pyrroline-2-one by means of 2D NMR in exhaustive comparisons with the structure of deoxyviolacein. The novel metabolite was named pseudodeoxyviolacein. The five cross peaks in HMBC spectra and the conformation analysis in NOESY spectra were helpful in proposing the chemical structure of the novel metabolite. Electronic spectra calculated by using a CAChe system (ZINDO) showed that the maximal absorption of pseudodeoxyviolacein is shorter than that of dexoyviolacein, giving additional evidence for the structure of pseudodeoxyviolacein. The structure differs from that of deoxyviolacein only with regard to the oxygenation for two indole rings.

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Alanine aminotransferase (AlaAT ; EC 2. 6. 1. 2) was purified to homogeneity from Candida maltosa that was grown on L-alanine as the sole source of carbon and nitrogen. The enzyme has a molecular mass of 99 kDa and consists of two subunits of equal molecular mass (52 kDa). Each subunit binds one mole of PLP. The enzyme has an isoelectric point of 5. 3 and an optimum pH of 6. 0-7. 5. The spectroscopic profile and an inhibition experiment showed that both PLP and free-SH groups are directly involved in the enzymatic catalysis. In the N-terminal region of the enzyme, the consensus sequence (five amino acids) that commonly appears in mammalian and higher plant enzymes is present. Compared with mammalian enzyme, the Candida AlaAT is heat-sensitive and a little looser in substrate specificity, and its affinity towards L-alanine is high.

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A novel arabinoxylooligosaccharide was prepared from wheat bran and its structure was analyzed. Wheat bran hemicellulose was digested with a commercial Aspergillus japonicus hemicellulase preparation and an oligosaccharide was purified by carbon column and gel filtration chromatographies to be a single component. The oligosaccharide has D. P. of 6 and contains arabinose and xylose with a molar ratio of 2 : 1. Xylotetraose was formed as an intermediate hydrolyzate on acid hydrolysis of this oligosaccharide. Methylation analysis indicated that both reducing and non-reducing end xylose residues were free from arabinose residues and that there is only one xylose residue attached by two arabinose residues. The structure of this saccharide was finally identified by ¹3C NMR as β-D-Xylp-(1→4)[α-L-Araf-/(1→2)][α-L-Araf-(1→3)]-β-D-Xylp-(1→4)-β-D-Xylp-(1→4)-D-Xylp.

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An active antimicrobial substance contained in coffee residue was purified by HPLC. As result of the identification using MS, ¹H-, and ¹³C-NMR, etc., 3', 4'-dihydroxyacetophenone was found to be the antimicrobial substance in coffee residue. Comparison of the antimicrobial spectra of 3', 4'-dihydroxy-acetophenone isomers and catechol analogs suggested that the antimicrobial activity of 3', 4'-dihydroxy-acetophenone isomers vary depending on the binding sites of hydroxyl groups, and the antimicrobial activity of catechol analogs was inversely proportional to the polarity of groups binding thereto.

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The Agrobacterium rhizogenes strain 1724 isolated from diseased melon plants in Japan [Shiomi et al., Ann. Phytopath. Soc. Jpn., 53, 454-459 (1987)] carries a mikimopine-type root-inducing plasmid with 215-kb size (pRi1724). This plasmid DNA has been partially digested with the restriction endonuclease BamHI, and the resulting DNA fragments cloned in a plasmid vector (pBR329) and a cosmid vector (pHC79)for a pRi1724 gene library. DNA inserted in each clone was identified by complete cutting with BamHI and subsequent agarose gel electrophoresis. The physical order of BamHI fragments was deduced from a series of overlapping clones, creating a circular map. A virulence region was located on the map by Southern blot hybridization toward those of the agropine-type plasmid pRiA4b.

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A fluorescent Edman-type reagent, fluorescein isothiocyanate (FITC), was adapted to a gas-phase sequencer fitted with a miniature reaction chamber. Fluorescein thiohydantoin amino acids were identified by on-line gradient HPLC with fluorescence monitoring. The optimized protocol for the coupling reaction using FITC and phenylisothiocyanate, and the degree of washing to remove the excess reagents were established. Myoglobin (5 pmol) and lysozyme (5 pmol) were analyzed by the sequencer to obtain a 35-51% initial yield (I. Y. ) and 82-88% repetitive yield (R. Y. ) for the former, and 51-66% I. Y. and 91-92% R. Y. for the latter. These figures permitted 20-25 amino acid residues to be identified from the N-terminus of 5 pmol samples applied to the sequencer.

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As a part of investigations on the biosynthesis and induction mechanisms of capsidiol, a green pepper phytoalexin, we focussed our attention on the enzymatic oxidation of 5-epi-aristolochene, a non-oxidized elemophilane biosynthetic intermediate, to capsidiol. We examined the optimum conditions for capsidiol production and the direct incorporation of 5-epi-aristolochene into capsidiol for enzymatic study. We found that green pepper seedlings were easily prepared and produced a large amount of capsidiol, and that it directly converted ¹³C-labelled 5-epi-aristolochene to capsidiol in 42%.

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The addition of a thermolabile variant of β-lactoglobulin A (BLG A) R40C/F82C to raw skim milk reduced whey syneresis in a set-type yogurt which was manufactured using a reduced processing temperature of 70°C. R40C/F82C BLG is a site-directed variant of BLG A that contains two additional free thiol groups. These cysteine residues have been positioned in a hydrophobic region distal to the free thiol at cysteine-121. The additional cysteine residues appear to confer a more open, less compact structure as compared to normal BLG A. The R40C/F82C BLG has been shown to form a much stronger gel network and its polymerization can be started at ≥ 70°C as compared to BLG A, which does not gel ≤ 85°C. Native and SDS polyacrylamide gel electrophoretic analyses showed that the aggregation of R40C/F82C BLG when heated at ≥ 70°C in a BLG-free skim milk background was due to both hydrophobic interactions and thiol / disulfide interchange. Yogurt was manufactured from raw skim milk containing 2% skim milk powder that was heated at 70°C or 85°C and then inoculated with a combination of Lactobacillus bulgaricus and Streptococcus thermophilus. The addition of 0.075% BLG A to the milk before heating at 70°C did not significantly reduce the whey separation. In contrast, the addition of increasing concentrations (up to 0. 075%) of R40C/F82C BLG decreased whey separation as much as 83%. Furthermore, the addition of R40C/F82C BLG decreased the time for curd formation as compared to when equivalent amounts of BLG A were added.

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The effects of palm oil diets on tissue lipids of rats were studied. Four groups of male Sprague-Dawley rats (4wk old) were maintained on diets containing (10% by weight) physically refined palm oil (PRPO), chemically refined palm oil (CRPO), soybean oil (SO), or canola oil (CO) for two months. Both PRPO and CRPO diets resulted in the appearance of 4, 7, 10, 13, 16-docosapentaenoic acid (22 : 5n-6) in blood plasma, red cells, and liver and muscle lipids, while SO and CO diets did not. This fatty acid was principally found in the phospholipid fraction, such as phosphatidylcholine and phosphatidylethanolamine. The increase of 22 : 5(n-6) compensated for the decrease of docosahexaenoic acid (DHA ; 22 : 6n-3). The results suggest that diet containing only palm oil as a fat source has a high potential of causing α-linolenic acid deficiency in rats.

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We isolated bacterial strains tolerant to high concentrations of oleic acid from soil samples, and studied biotransformation of fatty acids with the bacteria. We found that a strain BL0-3 identified as Micrococcus luteus metabolized oleic acid to 10-hydroxystearic acid, 10-oxostearic acid, and 4-oxolauric acid. Palmitoleic acid and myristoleic acid also served as substrates in the biotransformation, and 1O-oxopalmitic acid and 10-oxomyristic acid were produced, respectively. The formation of 4-oxolauric acid from oleic acid was inhibited by compounds which inhibit β-oxidation of fatty acids.

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The complete amino acid sequence of rye seed chitinase-a (RSC-a) has been analyzed. RSC-a was cleaved with cyanogen bromide and the resulting three fragments, CB1, CB2, and CB3, were separated by gel filtration. The amino acids of the N-terminal fragment CB1 were sequenced by analyzing the peptides produced by digestion with trypsin, lysylendopeptidase, or pepsin of reduced S-carboxymethylated or S-aminoethylated CB1. The sequences of fragments CB2 and CB3 were established by sequencing the tryptic peptides from reduced S-carboxymethylated CB2 and CB3, and by aligning them with the sequence of rye seed chitinase-c (RSC-c) to maximize sequence homology. The complete amino acid sequence of RSC-a was established by connecting these three fragments. RSC-a consists of 302 amino acid residues including hydroxyproline residues, and has a molecular mass of 31, 722 Da. RSC-a is basic protein with a cysteine-rich amino terminal domain, indicating that this enzyme belongs to class I chitinases. The amino acid sequence of RSC-a showed that the sequence from Gly60 to C-terminal Ala302 in this enzyme corresponds to that of RSC-c belonging to class II chitinases with 92% identity, and that RSC-a has high similarity to other plant class I chitinases but a longer hinge region and an extra disulfide bond.

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The substrate specificity of dextrin dextranase (EC 2.4.1.2; DDase) was investigated. This enzyme acted on maltose and isomaltose in addition to starch and dextrin, but did not act on other gluco-disaccharides. When various saccharides were allowed to react with salicin as a glucosyl acceptor, glucosyl residues were transferred to salicin on the reaction with maltose, isomaltose, starch, and dextran as glucosyl donors. On the other hand, when starch as a glucosyl donor was allowed to react with various saccharides, glucosyl residues of starch were transferred to D-glucose, D-xylose, and oligosaccharides that had glucosyl or xylosyl residues at non-reducing termini. Methyl α- and β-D-glucosides also acted as acceptors. Furthermore DDase transferred glucosyl residues from starch to glucose derivatives such as 2-deoxy-, 2-acetamido-2-deoxy-, 3-O-methyl-, and 6-deoxy-D-glucoses. When starch was used as a glucosyl donor, two products formed by transglucosylation to D-glucose as an acceptor were found to be maltose and isomaltose, and a product formed by transglucosylation to D-xylose as an acceptor was found to be glucosyl-α-1, 4-xylose.

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Compounds from a soil isolate of Penicillium sp. MG-11 showed remarkable convulsive activity against silkworms. Activity-based fractionation of an acetone extract of the fermentation products resulted in the isolation of four compounds. One was identified as verruculogen, which was an active principle. Another compound, acetoxydehydroaustin, was new and was structurally characterized by spectral data and a single-crystal X-ray analysis. The two other compounds were identified as dehydroaustin and austin on the basis of the spectral data. Although the latter three compounds were not active themselves, they enhanced the convulsive activity of verruculogen in a silkworm bioassay. Three additional compounds with no identifiable activity were also isolated. Two of them were identified as austinol and dehydroaustinol, and the third was determined to be a new related compound named neoaustin by an X-ray analysis of crystals.

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Hepatic α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase [EC 4. 1. 1. 45] (ACMSD), a key enzyme for tryptophan-niacin metabolism, is known to be suppressed by dietary polyunsaturated fatty acids. This enzyme is induced by glucocorticoid or dietary protein. We investigated the effect of linoleic acids on the induction of ACMSD activity by glucocorticoid at 40% or 10% casein levels and the effect of dietary linoleic acid on ACMSD activity in puromycin-treated rats to investigate the action of dietary linoleic acid. Dietary linoleic acid suppressed the hepatic ACMSD activity in the groups treated with and without glucocorticoid. However, in the case of the 10% casein diet, the ACMSD elevation ratio caused by glucocorticoid in the linoleic acid diet group was as large as that in the fat-free diet group. Puromycin suppressed ACMSD activity, but no additional suppression of hepatic ACMSD was observed by dietary linoleic acid.

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The existence of glycosylated DNA-binding proteins was demonstrated in a whole cell extract from a filamentous fungus, Aspergillus oryzae. The proteins were specifically eluted from a DNA-cellulose column by the eluate containing shared double-stranded DNA and were detected by wheat germ agglutinin (WGA)-probing. The apparent molecular masses of these proteins on SDS-PAGE were 140 kDa, 115 kDa, 105 kDa, 68 kDa, and 60 kDa. The labeling of the proteins by uridine 5'-diphosphate(UDP)-[¹⁴C]galactose using galactosyltransferase showed the same electrophoretic pattern with the WGA-probing. The [¹⁴C]-galactose-labeled saccharides were released from the proteins by mild-base treatment but not by N-glyco-peptidase F digestion, indicating the O-glycosidic linkage of the saccharide chain attachment to proteins. The [¹⁴C]galactose-labeled saccharides co-migrated with galactose-(β1→4)-N-acetylglucosaminitol on a silica gel plate. Thus, it was seen that several proteins which had the DNA-binding activity were modified by N-acetylglucosamine monosaccharide through an O-glycosidic linkage in A. oryzae.

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A new simple reactor of the tubing type was developed for polymerase chain reaction (PCR). A thin Teflon capillary tube was used as a tubing reactor in which the reaction mixture of PCR was driven by a pump at a constant flow rate. The sample was treated with three successive thermal stages for denaturation, annealing, and elongation of DNA and primers as a function of the position in the tube. The amplification yield was about a half of that obtained by a commercial thermocycler. Moreover, the total reaction time from 12 to 18 min, which was one-tenth of the time generally required by conventional thermocyclers using metal blocks, assured substantial amplification of a DNA fragment. In addition, this reactor could be also used for rapid cycle-sequences. This new device will be easily incorporated into automated and rapid DNA analysis systems for DNA sequencing.

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We found two enzymes that solubilize pectin from protopectin, tentatively named protopectinase-N (PPase-N) and protopectinase-R (PPase-R), in a culture filtrate of Bacillus subtilis IFO 3134. These enzymes were purified to homogeneity by hydrophobic, cation exchange and size exclusion chromatographies. The molecular weights of PPase-N and PPase-R were estimated to be 43, 000 and 35, 000, respectively, by SDS-PAGE. Their pIs were 9. 4 and 8. 2, respectively. These enzymes were stable in a wide range of pH and temperature. PPase-N and -R released water-soluble pectin by transeliminative cleavage of protopectin. According to their substrate specificities and modes of action, PPase-N and PPase-R could be classified as endo-pectate transeliminase (pectate lyase ; EC 4. 2. 2. 2) and endo-pectin transeliminase (pectin lyase ; EC 4. 2. 2. 10), respectively. Both enzymes were produced in a simple medium containing defatted soybean flour and phosphates. Production of PPase-N was repressed by addition of glucose while that of PPase-R was enhanced by phosphate.

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A new method for measuring the freezing denaturation of ovalbumin with 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is described. One ml of an ovalbumin solution (0. 1%) containing 50μM DTNB at pH 7.0 was subjected to 5 freeze-thaw (FT) cycles. After centrifugation of the solution, the absorption at 412nm was measured as an index of denaturation. This method was applied to study the cryoprotective effect of sucrose. The method offers a simple and accurate means for measuring the denaturation of ovalbumin produced by FT cycles and for measuring the effect of cryoprotectants on this denaturation.

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The effects of pyridoxal 5'-phosphate on the refolding of aspartate aminotransferase were studied. The refolding of the cytoplasmic isozyme of pig heart aspartate aminotransferase denatured with 6 M guanidine hydrochloride depends critically on the presence of pyridoxal 5'-phosphate. Pyridoxal 5'-phosphate affects a refolding intermediate at guanidine hydrochloride concentrations between 3.5 and 2.0 M to bring out the correct folding. In contrast, thermostable aspartate aminotransferase of Bacillus sp. YM-2 can be refolded independently of pyridoxal 5'-phosphate.

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A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for nattokinase, a subtilisin-like fibrinolytic enzyme in the fermented soybean food, natto. The assay method was developed using a combination of a murine anti-nattokinase monoclonal antibody coated on the microtiter well as a catching antibody and rabbit anti-nattokinase polyclonal antibody as a peroxidase-conjugated detecting antibody. The assay sensitivity was 0. 1 ng/ml and cross-reactivity with subtilisin BPN' and subtilisin Carlsberg was 0. 0002% and 0. 00002%, respectively. The ELISA value reflected the fibrinolytic activity of nattokinase. The correlation coefficient between nattokinase measured by a fibrinolytic assay and by the ELISA in 14 commercially available natto extracts was 0. 967, suggesting that nattokinase is the only fibrinolytic enzyme in natto.

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Three proteinase inhibitors (CLTI-I, -II, and -III) were purified from the seeds of Canavalia lineata by DEAE-Toyopearl, hydroxyapatite, and anhydrotrypsin-Sepharose column chromatographies. All the inhibitors bound to trypsin at a 1 : 1 molar ratio and inhibited the enzyme with dissociation constants of 3-7×10^-9M. They also showed the inhibitory activities on chymotrypsin. CLTI-I and -II had an identical M_r of 8000 and very close isoelectric points (4. 57 and 4. 50), and existed mainly as trimers under physiological conditions. The high content of half-cystine residues and the high stability to pH and heat have suggested that these are Bowman-Birk type inhibitors. On the other hand, CLTI-III, with an M_r of 20, 500 was classified as a Kunitz (soybean) family inhibitor on the basis of the amino acid composition as well as the homology of its N-terminal 17 residues to other Kunitz inhibitors.

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The amino acids of two Bowman-Birk type proteinase inhibitors (CLTI-I and -II) from the seeds of Canavalia lineata were sequenced by a manual Edman degradation using the DABITC/PITC double coupling method after enzymatic digestions with Achromobacter lyticus lysyl endopeptidase, Staphylococcus aureus V8 protease, and chymotrypsin. CLTI-I contains 75 amino acid residues. CLTI-II has an identical sequence to CLTI-I except an extra Asp residue attached at the C-terminus. The inhibitors showed a homology (40-70%) to other Bowman-Birk inhibitors. The reactive-site peptide bonds were estimated to be Lys²1-Ser²2 and Leu⁴8-Ser⁴9 against trypsin and chymotrypsin, respectively. An inhibitory active fragment containing only the chymotrypsin-reactive site was also described.

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Nine compounds isolated from finger millet as antifeedants for brown planthopper have been identified as known compounds, L-malic acid, isocitric acid, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxy-benzaldehyde, and vitexin, and new constituents, 2-O-[4-hydroxy-(Z and E)-cinnamoyl] glyceric acid and 8-C-β-D-[6"-O-(3-hydroxy-3-methyl) glutaroyl] glucopyranosylapigenin.

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The incorporation into tissue phospholipids and the effects on eicosanoid production of trans-fatty acid were studied in rats fed different proteins, casein or soybean protein. After feeding partially hydrogenated corn oil containing 32% t-18 : 1 at the 10% level for 3 weeks, the incorporation of trans-fatty acid into liver phospholipids was species-specific. More trans-acid was incorporated into phosphatidylinositol than into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and there was apparently no incorporation into cardiolipin. The incorporation of trans-acid tended to be higher in the rats fed soybean protein than in those fed casein. In general the proportion of linoleic acid increased, but that of arachidonic acid tended to be decreased by trans-fat as compared with cis-fat, suggesting an interference with linoleic acid desaturation, but this was not the case for the spleen of rats fed vegetable protein. The concentration of plasma prostaglanidin E₂ was not influenced by the type of dietary fat, but trans-fat reduced the production of leukotriene C₄ by the spleen on the basis of unit weight when the dietary protein source was casein, but not soybean protein. Thus, the influence of the trans-monoene fatty acid was quite diverse, and there was an interaction between dietary protein and trans-fatty acid.

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Both salt-soluble and salt-insoluble fractions of wheat flour have allergenicity, but their treatment with actinase, collagenase, and transglutaminase produced hypoallergenic flour. In detail, hard flour and soft flour were mixed with water containing either of these enzymes and then incubated at 20°C to obtain hypoallergenic flour products. Minimum amounts of the enzymes necessary to minimize the allergenicity were lower in soft flour than in hard flour, and in this sense the former was the best material. The product resulting from the treatment with collagenase or transglutaminase retained some high-molecular-weight proteinaceous components, suggesting that for food processing these may be preferable to the product resulting from the treatment with actinase.

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We isolated a Saccharomyces cerevisiae mutant, CH108, which shows hypersensitivity to cycloheximide, bleomycin, actinomycin D, 5-fluorouracil, and several other antibiotics. The mutant cells showed irregular shapes with swelling and were also hypersensitive to Zymolyase digestion. The mutant cell wall was swelled and twice as thick as the wild type cell wall. These defects were completely reversed by introduction of a genomic clone on a single-copy vector. Restriction analysis and sequencing showed that the responsible gene, DHS1, encodes a novel hydrophilic polypeptide of 483 amino acids.

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The cholesterol ester (CE) hydroperoxides (HPO) were measured in high density lipoprotein (HDL) and combined low and very low density lipoproteins (LDL + VLDL) by a high pressure liquid chromatography with a post column detection system using diphenyl-1-pyrenylphosphine. Human plasma lipoproteins were fractionated from 2 ml of plasma with dextran sulfate and Mg²⁺ to each fraction from which CE-HPO was extracted with n-hexane. The fractionation had no influence on the CE-HPO values in a sample. The relative standard deviations of the CE-HPO were 5. 4% (n = 6) from the HDL fraction and 4. 8% (n = 5) from the LDL + VLDL fraction. The CE-HPO were 6. 8 ± 2. 6 nM (in HDL) and 20. 0 ± 5. 6 nM (in LDL + VLDL) from 10 fresh human plasma samples. The method was used to follow the increase and decrease of CE-HPO in each lipoprotein fraction by the incubation of fresh plasma spiked with dilinoleoyl phosphatidylcholine hydroperoxide.

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A peptide model (called PαPβ) of bovine pancreatic trypsin inhibitor (BPTI) for studying the peptide folding structure was designed as a crucial folding intermediate. The PαPβ model folded but it was unstable at low pH. Four acidic groups in PαPβ : Glu-49, Asp-50, and C-termini at Phe-33 and Ala-58, were replaced individually with an Ala (in case of Glu and Asp) and an amide group (for C-terminal carboxyl groups) to study the effects of these negative charged groups on folding structure in terms of thermal stability and pH dependence. Substituting the Glu-49 or Asp-50 with Ala and blocking the C-terminus carboxyl group of Phe-33 in Pβ destabilized the structure, but blocking the negative charge of C-terminus in Ala-58 of Pαstabilized the structure at neutral pH. These results can be interpreted in terms of helix dipole momentum effects and/or salt bridge formation between Asp-50 and Arg-53, and of electrostatic interaction between positive and negative charges, which stabilized the structure. The melting temperature of model peptides (T_m) in low ionic strength buffer were lower than that in high ionic strength buffer except the C-terminus blocking of Pα.

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Sulfated schizophyllans with lower anticoagulant activity and higher anti-HIV activity were prepared. Those with sulfur contents above 6% showed anticoagulant activity irrespective of molecular weight. The activity was correlated with sulfur content. The triple helical structure of sulfated schizophyllans did not affect their anticoagulant activity. A sulfated schizophyllan with a sulfur content of 5.0% showed low anticoagulant activity and good anti-HIV activity. These results indicate that the latter sulfated schizophyllan (sulfur content, 5%) would be useful as an anti-HIV agent for treatment of HIV-infected hemophiliacs.

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Fresh and conditioned pork were incubated with and without lactic acid bacteria, and the meat protein degradation during incubation was investigated. The combination of meat-conditioning and addition of lactic acid bacteria considerably accelerated the protein degradation of meat mixtures. It was considered that the protein degradation in the lactic fermented pork was caused by proteases originated in meats, and both meat-conditioning and lactic acid fermentation acted as enhancing effectors.

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Two Enterococcus faecium isolated from silages produced antibacterial substances. Based on their narrow antibacterial spectrum, proteinaceous nature, and bactericidal mode of action, the antibacterial substances were concluded to be bacteriocins. Tricine-SDS-polyacrylamide gel electrophoresis and an electro-blotting-activity staining analysis indicated that both bacteriocins were peptides.

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This study outlines a simple alternative technique for the purification of the 11S soybean globulin from crude globulin using a single Sephacryl S-300 gel filtration chromatographic technique. All supporting data demonstrated that the 11S globulin was purified to homogeneity with no contamination from the 7S soybean globulin.

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An E. coli cell extract containing thermostable α-amylase and maltogenic amylase expressed from the clone pTMA322 was used to produce an anomalously linked oligosaccharides (Alo) mixture from starch. The liquefaction and saccharification of starch were done by one-step procedure using the above cell extract. The resulted Alo mixture contained over 40% oligosaccharides having DP 3 or more including branched forms.

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Transglycosylation from sucrose to kojic acid by sucrose phosphorylase from Leuconostoc mesenteroides was studied. Two transfer products were obtained. These structures were identified as kojic acid 5-O-α-D-glucopyranoside and kojic acid 7-O-α-D-glucopyran-oside on the bases of secondary ion mass spectrum analyses, the component analyses of their enzymatic hydrolyzates, and the carbon-13 nuclear magnetic resonance spectrum analyses.

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Hydroperoxide produced by the reaction of lipoxygenase (LOX) could be detected using a chemiluminescence (CL) analyzer. The minute activities of LOX were measured by an improved emission system under luminol and cytochrome c with a CL assay method.

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An N-acetylgalactosamine-specific lectin was isolated from Galactia tashiroi seeds by affinity chromatography on Galactose-Sepharose 6B. It was a glycoprotein with molecular weight of 90, 000-95, 000 and composed of four identical subunits of molecular weight of 24, 000. The purified lectin was specific for N-acetyl-D-galactosamine, while it was non-specific for human type ABO erythrocytes.

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Both folimycin and bafilomycin A₁, specific inhibitors of V-ATPase, blocked cell surface expression of viral envelope glyco-proteins without affecting their synthesis, but secretion of invertase was insensitive to both inhibitors. Invertase secreted in the presence of folimycin or bafilomycin A₁ migrated faster than the control on electrophoresis. A significant difference was also detected among the two treatments, suggesting that the sites of their action are different.

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Rhodococcus erythropolis strain S-1, which was isolated from soil, produces a bioflocculant. We have found that alcohols are useful carbon sources for its flocculant production. Ethanol was best for flocculant production and culture time. The bioflocculant produced on ethanol medium flocculated a wide range of suspended soils, alkaline and acid.

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A new antimicrobial compound was isolated from Scorodocarpus borneensis Becc. The chemical structure was determined to be methylthiomethyl (methylsulfonyl) methyl disulfide on the basis of its spectroscopic data. This compound exhibited considerably strong antimicrobial activities against all bacteria and fungi tested, except for Pseudomonas aeruginosa, with 12. 5-25 μg/ml of MIC.

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