Thermal treatment of a mixture of S-alkyl-L-cysteines (alkyl: methyl, MCS; allyl, ACS; propyl, PCS; cis-1-propenyl, PeCS) occurring in the Allium species and D-glucose (GIc) gave rise to volatile products which are responsible for characteristic food flavors. Flavor evaluations due to thermally treating mixtures MCS-Glc, ACS-Glc, PCS-Glc and PeCS-Glc indicated pickled radish-like, garlic and rice cracker-like, roasted onion-like, and rice cracker-like flavors, respectively. The major volatile products from MCS-Glc, ACS-Glc and PeCS-Glc were sulfur compounds generated from the thermal decomposition of MCS, ACS and PeCS, respectively. Interestingly, the compounds contributing to the good aroma of PCS-Glc were alkylpyrazines as well as sulfur compounds. Furthermore, novel compounds-2, 4-bis(propylthio)butanal and 2, 4-bis(propylthio)but-2-enal, which were thermally generated from an equimolar mixture of PCS and Glc, were isolated and identified by MS, IR, 1H-NMR and ¹³C-NMR.

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The glucoamylase enzyme (GAase) gene from Aspergillus shirousami was cloned and sequenced from genomic and cDNA libraries. The genomic gene was located in the 5.4 kb EcoRI fragment. The deduced amino acid sequence of GAase contained 639 amino acid residues with a relative molecular mass of approximately 68, 000 daltons (non-glycosylated form). The genomic gene of A. shirousami GAase was introduced into Aspergillus oryzae. These transformants had increased GAase and raw starch degradation (RSD) activity in culture media and in rice-koji extracts. Analysis by Southern, Northern, SDS-PAGE, and Western blot techniques confirmed the foreign gene was correctly transcribed, translated, and expressed in A. oryzae.

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(S)-4-Hydroxy-2, 2-dimethyl-l-cyclohexanone (1a) was prepared by two enzymatic methods. 1, 4-Cyclohexanediol was converted to monoacetal (11) via (±)-la. Enzymatic reduction of 11 by baker's yeast gave (S)-1 of almost 100 % e.e. Direct hydroxylation of 2, 2-dimethyl-l-cyclohexanone (14) by P-450 camphor monooxygenase of the cloned genes of Pseudomonas putida PpGl gave (S)-la of almost 100% e.e.) too. According to Mitsunobu's method, SN-2 inversion of (S)-1 gave (R)-1.

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Both chiral 4'-hydroxy and 1', 4'-dihydroxy-γ-ionylideneacetic acids (3, 4 and 5), biosynthetic intermediates of abscisic acid produced by Cercospora cruenta, were synthesized from a chiral starting material, (R)- or (S)-4-hydroxy-2, 2-dimethyl-1-cyclohexanone (7). [2.3]-Sigmatropic rearrangement of (S)-1-chloromethyl-3, 3-dimethyl-5-tetrahydropyranyl(THP)oxy-1-cyclohexene (8), followed by the Reformatsky reaction with 4-bromo-3-methyl-2-butenoate (10) gave (l'R, 4S)-4. The diastereomeric isomer, (l'R, 4'R)-3, was synthesized in the same manner. The reaction of (S)-2, 2-dimethyl-5-methylene-4-THPoxy-1-cyclohexanone (14) with a Grignard reagent prepared from (Z)-3-methyl-2-penten-4-ynyI THP ether (15) and subsequent conversion of the side chain gave (1'S, 4'S)-5. These synthetic compounds confirmed the stereochemistry of natural 3, 4 and 5.

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Four kinds of Carotenoid pigments were found in Rhodococcm rhodochrous RNMS1. Spectroscopic and chemical evidence showed two of them to be monocyclic carotenoids each containing a tertiary hydroxyl group at C₁, and their structures were determined as 1', 2'-dihydro-β, ψ-caroten-rol and 1'-hydroxy-r, 2'-dihydro-β, ψ-caroten-4-one. The other two were novel carotenoid derivatives, one being a monoglycoside of the latter carotenoid and the other monoesters of this carotenoid glycoside, which were esterified with nine major fatty acids at the C₆ hydroxyl group of the glucose moiety. The fatty acids consisted of four saturated (C16:0-C19:0), four mono-unsaturated (CIS: 1-C21:l) and one branched-chain (10-methyl C19:0) acid, including the odd-numbered ones. The structures were determined to be 1'-[(β-D-glucopyranosyl)oxy]-1', 2'-dihydro-β, ψ-caroten-4-one and 1'-[(6-O-acyl-β-D-glucopyranosyl)oxy]-1', 2'-dihydro-β, ψ-caroten-4-one.

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The inhibition of α-amylase from human saliva by polyphenolic components of tea and its specificity was investigated in vitro. Four kinds of green tea catechins, and their isomers and four kinds of their dimeric compounds (theaflavins) produced oxidatively during black tea production were isolated. They were (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), (-)-catechin (C), (-)-gallocatechin (GC), (-)-catechin gallate (Cg), (-)-gallocatechin gallate (GCg), theaflavin (TF1), theaflavin monogallates (TF2A and TF2B), and theaflavin digallate (TF3). Among the samples tested, EC, EGC, and their isomers did not have significant effects on the activity of a-amylase. All the other samples were potent inhibitors and the inhibitory effects were in the order of TF3>TF2A>TF2B>TFl>Cg> GCg > ECg > EGCg. The inhibitory patterns were noncompetitive except for TF3.

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An isozyme of acid phosphatase-1, acid phosphatase-1¹, was purified from the leaves of tomato (Lycopersicon esculentum) to homogeneity and characterized. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The gel filtration analysis showed that the native molecule had a relative molecular mass of about 61 kilodaltons (kDa). The relative molecular mass of the subunit on gel electrophoresis with sodium dodecyl sulfate was about 32 kDa, indicating that the native form of the enzyme was a homodimer. It was suggested by periodic acid-Schiff staining on the gel that the enzyme was a glycoprotein. The Km for p-nitrophenylphosphate was 2.9 × 10^-3 M. The enzyme had a pH optimum of 4.5 in 0.15 M potassium acetate buffer with p-nitrophenylphosphate as a substrate. This enzyme was activated by divalent metal ions, such as Zn²⁺, Mg²⁺, and Mn²⁺. The N-terminal amino acids were sequenced after the purified enzyme was treated with pyroglutamylpeptidase. It was suggested that the N-terminal amino acid was pyroglutamate.

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Antibodies against Schizophyllan (SPG) or SPG conjugated with bovine serum albumin (BSA) were raised in rabbits by multiple subcutaneous immunization. The titer of SPG-reactive antibodies in the antiserum to SPG-BSA conjugate was significantly higher than that in the antiserum to SPG as assessed by enzyme-linked immunosorbent assay (ELISA). The SPG-reactive antibodies purified by affinity chromatography using a Protein A-Sepharose CL-4B column interacted with the SPG-BSA conjugate spotted on a nitrocellulose membrane filter in a dose-dependent manner, but the anti-SPG antibodies did not interact with BSA.
Immunocytochemical staining has also shown that the anti-SPG antibodies reacted with SPG taken up by mouse peritoneal macrophages.

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Nitrogenase catalyzes not only the reduction of N₂ to NH₃ but also the reduction of C₂H₂ to C₂H₄ and H⁺ ion to H₂ gas, etc. The detailed mechanism of the nitrogenase reaction is not clear. We have prepared monoclonal antibodies against Component I nitrogenase of A. vinelandii and examined the effects of antibodies on the nitrogenase reactions. A monoclonal antibody designated MA-1 inhibited C₂H₂ reduction activity strongly but did not inhibit H₂ evolution activity. MA-2, on the contrary, inhibited only H₂ evolution activity. MA-8 inhibited both C₂H₂ reduction and H₂ evolution activity to the same extent.

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Hydrogen peroxide in methylotrophic yeasts can be metabolized in at least two distinct ways. Addition of exogenous hydrogen peroxide removes the dependance of catalase on endogenouslyproduced hydrogen peroxide resulting enhanced rates of alcohol oxidation. Exogenous hydrogen peroxide is also efficiently degraded by cytochrome c peroxidase (CCP), a competitive reaction which does not result in enhanced alcohol oxidation. To overcome the influence of cytochrome c peroxidase, artificial peroxisomes were prepared by coimmobilization of alcohol oxidase and catalase. These artificial peroxisomes mimic the peroxide-induced rate enhancement observed with whole cells.

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A method for predicting the enthalpy profile during extrusion cooking was developed. Wattmeters were connected to each barrel, and the heat loss from the barrels was measured under several temperature patterns without any material flow through the twin-screw extruder. A short section of some screws was replaced by a collar to make it possible to insert thermometers into the barrels during extrusion, and water was then pumped into the extruder while heating the barrels and the die. The measured temperature coincided with the temperature calculated from the consumed energy and the heat loss from each barrel, indicating that the enthalpy profile could be evaluated by this method. As an application of the method, the dependence of the enthalpy profile on the screw configuration was investigated. The enthalpy absorbed by the extruded materials was increased by using reverse screws and a needle valve at the outlet of the twin-screw extruder. These results suggest that the method developed in the present study will contribute to the design of extruder screws.

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The method for predicting the enthalpy profile that was developed in the preceding work was applied to the extrusion cooking of jelly. After selecting the enthalpy profile to produce jelly of good quality at a low pro duction rate, the production rate was increased. It was found necessary to obtain an enthalpy profile similar to that at the low production rate in order to maintain good product quality at the higher production rate. For that purpose, the heating capacity of the specified barrel had to be increased and the temperature patterns of the barrels had to be changed. It was ascertained that the method developed in the preceding work was effective for estimating the production capability and offers a strategy for modifying the twin-screw extruder.

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The interaction of ricin D with urea was studied spectroscopically. The fluorescence intensity of ricin D increased with increasing urea concentration up to 5 M, but it decreased greatly in 7 M urea accompanied by a red shift of the spectrum. The UV -difference spectra of ricin D induced by urea at concentrations below 6 M versus that in the absence of urea had maxima at 280 and 288 nm, and a trough around 300 nm, while those induced by urea at concentrations greater than 7 M had troughs at 284 and 292 nm. It is suggested that spectroscopic changes of ricin D induced by low concentrations of urea are ascribed to the perturbation by urea of the surface-localized chromophores, but those induced by high concentrations of urea are due to the exposure of chromophores on the surface of the protein molecule by conformational change. Together with the CD data, the critical concentration of urea to induce the conformational change of ricin D was estimated to be 6 M. The saccharide-binding ability of ricin D was retained in urea at concentrations up to 4 M, but it decreased greatly in 6 M urea. From the time-dependent changes in the UV-difference spectra of ricin D and its complex with lactose induced by 7 M urea, it is suggested that urea first interacts with ricin D in such a manner as to perturb the surface-localized chromophores and then induces a conformational change that is prevented by binding with lactose.

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The saccharide-binding properties of abrin-a, a toxic lectin from seeds of Abrus precatorius, were studied by equilibrium dialysis and spectroscopy. Equilibrium dialysis data suggest that abrin-a has two saccharide-binding sites with different affinities for galactopyranosides, a high affinity site (HAsite) and a low affinity site (LA-site). The binding of galactopyranosides to abrin-a induced a shift of the fluorescence spectrum to a shorter wavelength by 5nm and UV-difference spectra with maxima at 283 and 292 nm. Such spectroscopic changes, however, were not induced by binding with N-acetylgalactosamine and galactosamine, suggesting a difference in the binding mode or the site to bind between these saccharides and galactopyranosides. The association constants for binding of individual saccharides to abrin-a suggested that in the HA-site there may be a subsite that can interact with the glucopyranosyl moiety of the lactose molecule in addition to the galactose-recognition site, while the LA-site may not have such a subsite structure. Based on these results, we concluded that galactopyranosides bind to abrin-a in such a manner as to induce the change in the environment of the tryptophan residue or residues located at or near the respective binding sites.

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The presence of enzymes and substrates participating in H₂O₂-decomposing system (NADPH→glutathione→ascorbate→H₂O₂) found in chloroplasts, and superoxide dismutase (SOD) (EC 1.15.1.1) was confirmed in cultured tobacco cells. The specific activities of ascorbate peroxidase (AP), glutathione reductase (GR) (EC 1.6.4.2) and SOD were much higher in callus than in leaves, but catalase (EC 1.11.1.6) was not detected. Tobacco callus also had activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and isocitrate reductase (EC 1.1.1.42), which supplied NADPH. When tobacco callus was illuminated, they became green. The contents of GR, glutathione, and ascorbate were higher in the light than in the dark, but no difference in AP and SOD could be detected between in the light and dark. By the test of Ouchterlony double immunodiffusion using anti-spinach AP and anti-spinach GR, it was confirmed that callus AP and GR had different structures from the leaf enzymes. These results show that callus has developed an active oxygen-decomposing system for overcoming the oxidative stress brought about by becoming callus.

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The Saccharomycopsis fibuligera α-amylase (Sfamy) gene was expressed in Saccharomyces cerevisiae. The highest productivity of Sfamy was 70 mg per liter of culture broth. We purified Sfamy from the culture broth and identified the NH₂ terminal primary sequence. This sequence suggests that the Sfamy gene product is synthesized as a pre-pro-precursor, and the pro-sequence is cleaved after a Lys-Arg sequence with the calpain-like endopeptidase encoded by the KEX2 gene, resulting in mature Sfamy protein composed of 468 amino acids. Furthermore, the enzyme Sfamy is a glycoprotein in which one N-linked sugar chain containing mannose residues is attached to the Asn residue at the 198 position. The Km and k_cat values were 1.1×10^-4 M and 1.4×10² sec^-1, respectively, using amylose (the degree of polymerization n=18) as a substrate. Moreover, the secondary structure, the location of the secondary elements including α-helix, β-sheet, and loop, and tertiary structure were predicted theoretically on the basis of the molecular structure of Aspergillus oryzae α-amylase, Taka-amylase A (TAA). These results indicate that Sfamy protein is composed of main (M) and C-terminal (C) domains. The molecular structure of M domain closely resembles that of TAA, but the C domain appears to be more compact than that of TAA because of deletions at three regions forming turns and one region forming α-helix.

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The use of elemental sulfur (S°) as an energy source by T. ferrooxidans API9-3 was completely inhibited by above 108mM of Fe²⁺ added to S° (1%)-salts medium, in which about 0.6 mM Fe²⁺ remained after 30 days cultivation without being oxidized. In contrast, the use of Fe²⁺ by this strain was not inhibited by S°. It was found that specific activities of hydrogen sulfide: ferric ion oxidoreductase (SFORase) and sulfite: ferric ion oxidoreductase, which are crucial in sulfur and sulfite oxidations of this strain, were completely inhibited by 20mM and 1 mM Fe²⁺, suggesting that this may be a main cause for the cells not using sulfur as an energy source. The SFORase activity of the cells incubated with S°-Fe²⁺-salts medium decreased to 39% of that in control cells without Fe²⁺, suggesting that Fe²⁺ added to the medium inhibits a sulfite: ferric ion oxidoreductase, and as a results sulfite ions are accumulated in the cells and this damages the SFORase. Sodium sulfite completely inhibited cell growth on S°-salts medium at 0.1 mM, but not on iron-salts medium.

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Various conditions for obtaining hybrids of the auxotrophic mutants SH1509 and SH1512 of Saccharomyces cerevisiae by electrofusion were investigated. An AC field of 400 V_p/cm and a DC field of 2 square pulses (7kV/cm; 60μsec each) at an interval of 0.5 sec were effective. Treatment with 0.2 (SH1509) or 1.0mg/ml (SH1512) Zymolyase for 1 or 1.5 hr was essential. As to the molarity of the osmotic stabilizer (sorbitol), the hybrid yield peaked at 0.6 M. The presence of CaCl₂ (up to 0.4 mM) or 0.1 mM CaCl₂ with 0.1 mM MgCl₂ enhanced the yield. The temperature of the spheroplast suspension during pulsations also affected the yield, the most suitable temperature being 28°C.

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The effect was investigated of some polyphenol compounds on the growth and intracellular enzyme activity of human-derived cells and Chinese hamster ovary (CHO) cells. Quercetin, a mutagen, inhibited the growth of serum-free cultured human-human hybridomas (SI102 and HB4C5) and a human histiocytic lymphoma cell line (U-937), but did not affect the growth of CHO cells. Glycosides of quercetin such as quercetin-4'-glucoside (Q-4'-G), quercetin-3, 4'-glucoside (Q-3, 4'-G) and rutin, and other polyphenol compounds (catechin and epicatechin) had no significant inhibiting effect on the growth of human-derived cells or CHO cells. These compounds slightly promoted the growth of human-derived cells. Most of the polyphenols used increased the activity of a drug-metabolizing enzyme, NADPH-cytochrome C reductase, in the U-937 cells and CHO cells, this effect being more marked in the CHO cells than in the U-937 cells. Quercetin markedly reduced the activity of catalase in the human-derived cell lines, while it slightly activated catalase in the CHO cells. Rutin, Q-4'-G, Q-3, 4'-G, catechin and epicatechin produced no significant change in catalase activity. Quercetin also reduced the activity of glutamic oxaloacetic transaminase in the U-937 cells.

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A gene coding for a novel esterase of Pseudomonas fluorescens was cloned in this study. DNA sequencing showed that the open reading frame is comprised of 708 nucleotides. The coding sequence of the gene is preceded by a potential Shine-Dalgarno sequence and by a promoter-like structure. Following the stop codon a structure reminiscent of the E. coli rho-independent terminator is present. The enzyme expressed in an E. coli clone was mostly in the periplasmic space, released to the outside of the cell by osmotic shock and purified to homogeneity by QAE-Sephadex A-50 and DEAE-Sepharose columns. The native form of the enzyme consisted of two identical subunits, each with a molecular weight of 27, 000. By studying the properties and substrate specificity, the enzyme was classified as an arylesterase (EC 3.1.1.2).

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Bile pigments in the bile of yellowtail, Seriola quinqueradiata, red sea bream, Pagrus major, and flounder, Paralichthys olivaceus, were analyzed by HPLC and TLC.
In the biles of these three fish, ditaurobilirubin and biliverdin IXα were the predominant pigments. Bilirubin IXα glucuronides and bilirubin IXβ were present in the bile of red sea bream and yellowtail but scarcely present in the bile of flounder. Bilirubin IXα was detected only in the bile of red sea bream. These results are in accord with the previous reports, in which a diversity of bile pigment composition was demonstrated in some fish.

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This work proposes a new method for improving the functional properties of food proteins by intentional modification. Hen's egg white was used as an example of the food proteins to be improved. Commercially available egg white was freeze-concentrated using bacterial ice nuclei. The resulting increase in its solid content led to formation of a hard, but fragile gel by heat treatment at 90-120°C for 20 min. When modified with arginine, it was changed to a viscoelastic product. Scanning electron microscopic and pulsed NMR observations suggested that tight aggregation of constituent protein molecules occurred in the hard gel network by the heat treatment and that the aggregation partly became loose by the modification with arginine.

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We searched for a new cell aggregation factor for hepatoma AH109A cells, and found one we called HAF in the culture filtrate of Streptomyces sp. strain No. A-6143 isolated from a soil sample. HAF was purified by salting-out with ammonium sulfate, DEAE-cellulose column chromatography, gel filtration on Sephadex G-100, and hydroxylapatite column chromatography. HAF was a glycoprotein which had a molecular weight of about 73, 000. HAF was stable from pH 6 to 8 at 37°C and up to 40°C at pH 8.0, and the aggregation activity of HAF was maximum around pH 8 at 30°C. The activity was not influenced by some saccharides, but it was inhibited by EDTA and EGTA; moreover HAF activity was restored by the addition of calcium ions. HAF aggregated hepatoma AH136B and COS-7 cells as well as hepatoma AH109A cells, but it was inert to other cancer cells and human erythrocytes. These properties proved that HAF is completely different from other aggregation factors for cancer cells so far reported.

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S-Alkylcysteine α, β-lyase was found in a thermophile, Bacillus sp. 41A, which was newly isolated from soil, and purified to homogeneity from the cell extract. The enzyme has a molecular weight of about 76, 000, and is composed of two subunits identical in molecular weight (39, 000). The enzyme requires pyridoxal 5'-phosphate as a coenzyme, and catalyzes α, β-elimination of S-methyl- L-cysteine and its analogs such as S-ethyl-L-cysteine, L-djenkolate, L-cystine, Se-methyl-L-selenocysteine, and O-methyl-DL-serine. However, S-methyl-D-cysteine, D-cystine, L-methionine, and L-norleucine were inert. The enzyme also catalyzes the β-replacement reaction of S-methyl-L-cysteine with various thiols to yield the corresponding S-substituted cysteines. In addition to S-methyl-L-cysteine, Se-methyl-L-selenocysteine and O-methyl-DL-serine also serve as β-substituent acceptors in the β-replacement reaction. The enzyme is most active at 70°C and stable at high temperatures. Automated Edman degradation provided the N-terminal sequence of the first 44 amino acids. The amino acid sequence in the vicinity of the lysyl residue to which pyridoxal 5'-phosphate is bound, was -Lys-His-Gln-Arg- by Edman degradation of the pyridoxyl peptide obtained by digestion with trypsin after reduction with sodium borohydride.

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The inhibition of ascorbate-mediated protein damage by the histamine H₂-receptor antagonist, cimetidine, was chemically studied. In the presence of copper(II) ion, ascorbate gave rise to oxidative modification of both a protein and a histidine derivative under simulated physiological conditions (pH 7.2, 37°C), whereas the reactions were entirely inhibited by the addition of cimetidine. The inhibition of protein damage resulted in almost total disappearance of cimetidine within 24 hr and the appearance of two major products, a species (2) assigned as the 2-imidazolone derivative of cimetidine and cimetidine sulfoxide (3). The mechanism for the protective effect of cimetidine against copper(II)/ascorbate-mediated protein damage is discussed.

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Three new proteins which inhibit protein synthesis in rabbit reticulocyte lysates were isolated from an extract of sponge gourd (Luffa cylindrica) seeds by chromatography on a AF-Blue Toyopearl column followed by FPLC with a Mono S column. These three protein-synthesis inhibitory proteins (PSIs) have molecular masses of 19kDa, 15kDa, and 9kDa, and were designated 19K-PSI, 15K-PSI, and 9K-PSI, respectively. Although the 19K-PSI had no effect on protein synthesis in HeLa cells, its inhibitory activity on the cell-free protein synthesis was 340- and 83-fold stronger than those of ricin A-chain and luffin-a, respectively, probably due to hydrolyzing mRNA. The inhibitory activities of 15Kand 9K-PSIs on the cell-free protein synthesis were weaker than those of ricin A-chain and luffin-a. The 19K-PSI was a glycoprotein having an ordinary amino acid composition, three intramolecular disulfide bonds and a blocked N-terminal residue, while the 15K-PSI was extraordinarily rich in glycine and the 9K-PSI in arginine and glutamic acid (and/or glutamine). The amino acid composition of 19K-PSI was: Ser₂₇Glx₃Gly₁₆₄Tyr₇Lys₉His₆, and that of 9K-PSI was: Asx₃Glx₂₅Pro₂Gly₅Lys₂His₂Arg₂₅Trp₃.

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The metabolic fates of the carbon skeletons of L-[U-¹⁴C]methionine and L-[U-¹⁴C]cysteine were investigated in growing rats fed on diets containing different percentages of protein calories (0, 5, 10, 15, and 30 PC%) at 4100 kcal of metabolizable energy per kg of diet. The incorporation of ¹⁴C into the body protein at 12hr after the injection of labeled methionine or cysteine was extremely high in the dietary groups with 0 to 10 PC% (about 70% of the injected dose), and then it decreased gradually in the higher PC% groups. A linear increase in the expired ¹⁴CO₂ production from ¹⁴C-methionine was observed with increasing levels of the dietary protein, but the ¹⁴CO₂ production from ¹⁴C-cysteine was depressed in the lower PC% groups and it increased in the higher PC% groups, showing a break point at around 10 PC% in the diet. The ¹⁴CO₂ production from methionine was greater than that from cysteine in each dietary group. These results indicate that although cysteine is not an essential amino acid for the growth of rats, the carbon skeleton of cysteine is preferentially used for protein synthesis, and that the metabolic responses of both the amino acids to dietary protein level change at around 10 PC%, where the growth rate reached its approximate maximum.

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Microbial production of benzoylformic acid (BF), which can be used as a substrate of enzymatic synthesis of (R)-(-)-mandelic acid, was investigated. Among 145 strains of yeasts and actinomycetes, Saccharomycopsis lipolytica (IAM 4964) was the best producer of BF from DL-phenylglycine (DL-PG). Culture conditions for BF production by the organism were optimized. When 0.2% fructose as a carbon source and 0.7% Bacto-tryptone as a nitrogen source were used in the presence of 4% DL-PG, 14.5 mg/ml of BF was produced (about 37% molar yield) in 4 days of cultivation. BF was synthesized from the L-form of PG, but not from the D-form. The BF was isolated from culture broth in a crystalline form and physicochemically identified.

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A nonlytic endo-β-1, 3-glucanase I (1, 3-β-D-glucan glucanohydrolase, EC 3.2.1.6) was purified from the culture broth of Flavobacterium dormitator var. glucanolyticae, which produced several species of endo-β-1, 3-glucanases. The purified enzyme decomposed various β-1, 3-glucans such as laminarin, yeast glucan, and pachyman to yield glucose, laminaribiose, and laminaritriose as the main products; the enzyme is a smaller oligosaccharide-producing type of endo-β-1, 3-glucanase. The molecular weight and isoelectric point of the enzyme were 27, 000 and 5.8, respectively. The substrate specificity, the conditions for the maximum reactivity, stability, and immunochemical properties of the enzyme resembled those of the lytic endo-β-1, 3-glucanase II, but there were some differences in isoelectric points, molecular weights, and the lytic activity on viable yeast cells between β-1, 3-glucanases I and II. By addition of yeast cells to the culture medium, the secretion of nonlytic β-1, 3-glucanase I was induced with a lag period, following the secretion of lytic β-1, 3-glucanases II and IV. From the results of the enzymological, immunological, and limited proteolytic experiments, it is concluded that β-1, 3-glucanase I was formed by a proteolytic digestion of β-1, 3-glucanase II during the cultivation of the organism.

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The more potent inhibitory activity against angiotensin-converting enzyme (ACE) was excised from a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of Bacillus stearothermophilus by heating at 120°C in 1 M AcOH-20 mM HCI, as compared with GAPDH preparations of yeast and pig. Sufficient excision of B. stearothermophilus ACE inhibitors required a longer proteolysis time of 60 min. Two inhibitors were then purified by gel-permeation and reverse-phase chromatographies. One of the B. stearothermophilus ACE inhibitors, BG-1, was the GAPDH peptide 68-77 (Gly-Lys-Glu-Ile-Ile-Val-Lys-Ala-Glu-Arg, IC₅₀: 32 μM). Another inhibitor, BG-2 (Gly-Lys-Met-Val-Lys-Val-Val-Ser-Trp-Tyr, IC₅₀: 6 μM), corresponded to GAPDH peptide 304-313. These sequences were quite different from those of vertebrate GAPDH peptides and the venom peptide family with ACE inhibitory activity. BG-2 was found to be a non-competitive type inhibitor, differing from many natural peptide inhibitors. Thus, B. stearothermophilus GAPDH seemed to be a good source of new type ACE inhibitors, in addition to the advantages due to its thermophilic property.

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One of the three C-P bond formation steps, defined as step 5 in the bialaphos (BA) biosynthetic pathway, was analyzed using a new BA non-producing mutant NP71. The mutant was derived from a BA producer by gene replacement of an unidentified region next to the gene responsible for the step 5 deficiency of the mutant NP213, obtained by conventional mutation procedures. Biochemical analysis of these two mutants indicated that NP71 was defective in the formation of carboxyphosphonoenolpyruvate (CPEP), while NP213 lacked the enzyme CPEP phosphonomutase, which catalyzed the intramolecular rearrangement of CPEP.

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The effect of simulated weightlessness on the morphological and histochemical changes of skeletal muscles was investigated, using a suspension harness, for 10 days. The weights of the gastrocnemius and soleus muscles of the suspended rats decreased significantly, especially that of the soleus muscle. The mean fiber diameter and area of the gastrocnemius and soleus muscle from the suspended rats also decreased. The present results demonstrate that this model involving a suspension harness may be useful for studying the effects of muscle atrophy and simulated hypogravity on muscle metabolism.

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