Two strains of killer yeasts, both identified as Hansenula anomala, were isolated from shoyu moromi. Both killer yeasts showed killer activity toward Zygosaccharomyces rouxii EA under high salt concentration conditions. The killer toxins produced by these strains were purified by ultrafiltration and ion-exchange chromatography followed by gel filtration. The molecular weights of the toxins were about 300 kd and both toxins were glycoproteins. The isoelectric point of the toxin, Kh-I, produced by one strain was pH 2.9 and that of the toxin, Kh-II, produced by the other strain was pH 3.6. The amino acid composition of Kh-I was different from that of Kh-II. Kh-II was more thermolabile than Kh-I. The killer activities of both toxins were not observed in the absence of NaCl in the medium but increased with increasing NaCl concentration. The killer spectra of Kh-I and Kh-II were different from those of the killer toxins known as K₁-K₁₁. The killer activities of these strains were not abolished by cycloheximide treatment and by cultivation at 37°C. No plasmid was detected in either killer yeast.

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A disaccharide produced from the polysaccharide of Fusarium sp. M7-1 by digestion with a bacterial enzyme preparation was isolated and identified as D-mannopyranosylβ1→2 mannopyranoside.

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To examine the conformation-toxicity relationship concerning PM-toxins, which are the fungus (Phyllosticta maydis)-produced host-specific corn pathotoxins having C₃₃- or C₃₅-linear poly-β-ketol structures, five mimics were synthesized as stereoisomeric mixtures in addition to twelve that had previously been synthesized. The synthetic mimic PM-B', which has a 1, 3-diene spacer instead of the central saturated spacer in natural PM-toxin B, was 30- to 100-fold less toxic than the natural toxin, while PM-777B, C and D (which are isomers of PM-777A), and PM-77777 (which has six β-ketol groups) were as active as the natural toxin. These results suggest that the PMtoxins should take conformations (e.g., helix or hairpin forms) that are more involved than the previously considered simple ring-like forms.

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The carbohydrate and alcohol contents of various types of vinegar, including traditional balsamic vinegar from Modena, "Modena Balsamic Vinegar, " wine vinegar and vinegar prepared from apple must, were studied. Assays were carried out using enzymatic techniques, which are particularly suited to this type of analysis. Determination of the components of the various vinegars, and of their interrelationship, is a crucial factor in the characterization of traditional balsamic vinegars.

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A low calcium requiring form of calcium activated protease (LCAP) and a high calcium requiring form of calcium activated protease (HCAP), purified from porcine skeletal muscle, were studied. LCAP was most active at pH 8.2. The HCAP was rather unstable at high temperature or at alkaline pHs: The antiserum to purified LCAP did not make a precipitin line with HCAP. Those proteases hydrolyzed myofibril proteins to disassemble the structure of myofibrils. The electronmicrogram of the myofibril treated by LCAP resembled that of the muscle obtained from a vitamin E deficient rat. Both LCAP and HCAP hydrolyzed purified α-actinin, liver actin, and some other proteins which are not assigned. The microsome proteins were resistant to protease treatment, and only the 180kd protein was hydrolyzed by LCAP.

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A new lipolytic enzyme (Lipase III), which specifically hydrolyzed p-nitrophenyl laurate (p-NPL), was found in the culture broth of Penicillium cyclopium Ml. The enzyme was purified with an overall yield of 27% and crystallized by the addition of ammonium sulfate. The crystalline preparation gave a single band on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was about 110, 000 by gel nitration. The enzyme consists of two subunits identical in molecular weight (54, 000), which was estimated by SDS-gel electorophoresis. The optimum pH and temperature for hydrolysis of p-NPL were 6.0 and 40°C, respectively. The enzyme released glycerol from olive oil more rapidly than lipases from Chromobacterium and Pseudomonas. The enzyme also rapidly hydrolyzed triglycerides in serum in the presence of surface active agents. A linear relationship was found between the absorbance at 555 nm and the amount of triglycerides in serum, when it was assayed by using lipase III, glycerol kinase, and α-glycerophosphate oxidase.

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In Euglena gracilis, the activity of pyruvate: NADP⁺ oxidoreductase, a novel pyruvate dehydrogenase, was lowered by a deficiency of thiamin in the cells, showing that the enzyme is dependent on thiamin pyrophosphate. In thiamin-sufficient cells, wax esters were rapidly synthesized from paramylon, the reserve polysaccharide, to generate ATP under anaerobiosis (wax ester fermentation), however, wax esters increased less under anaerobiosis in thiamin-deficient cells, demonstrating that pyruvate: NADP⁺ oxidoreductase participates in wax ester fermentation. In contrast, the paramylon content of deficient-cells rapidly decreased during the initial 12hr anaerobic incubation. Lactate, ethanol and propanol were produced on anaerobic incubation of deficient cells, but their amounts were significantly small compared with the amount of decomposed paramylon. It is suspected that unknown product(s) are synthesized from paramylon on anaerobic incubation in thiamin-deficient cells. During anaerobic incubation of thiamin-deficient cells the ATP level decreased rapidly and the number of living cells also decreased. These results indicate that the formation of wax esters is important for the generation of ATP in anaerobically incubated Euglena cells for survival and that thiamin is an essential factor in the mechanism.

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Gibberellin A₅ (GA₅), which is not known to be native to carrot (Daucus carota L.), was fed as either high specific activity [1-³H]GA₅ (HSA, 3.2 Ci/mmol) or low specific activity [1, 2-³H]GA₅ (LSA, 74 mCi/mmol) to cell suspension cultures of carrot. Gibberellins A₁, A₃, A₆, A₈ and their glucosyl conjugates were found as metabolites of HSA [³H]GA₅, as were GA₂₂, GA₂₉ and GA₅ glucoside and glucosyl ester, with all tentative identifications based on retention times on reversed phase C₁₈ HPLC with radiocounting. From LSA [1, 2-³H]GA₅ feeds, GA₁, GA₃, GA₆, GA₈ and GA₂₉ were identified by GC/SIM. For cells or medium, only 24 to 27%, respectively, of [³H]GA₅ was metabolized; major metabolites were [³H]GA₅ glucosyl conjugates (HSA and LSA feeds) and free [³H]GA_1/3 (LSA feeds). The system may thus be useful for production of labelled GAs and GA conjugates, and especially for conjugates of GA₅ and free GA₆ without dilution by endogenous GAs.

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Heat-induced gelling characteristics of myosin-actin from cardiac and skeletal muscle were investigated under various conditions in a model system. The interaction between actin and myosin at weight ratios of 1:15-1:9 from cardiac or skeletal muscle seemed to contribute to heat-induced gel strength at pH 6.0, but such interaction seemed to carry little significance in gel formation at pH 5.4.
A decrease in gel rigidity of chemically modified myosin (PCMB-treated) and actomyosin (TNBS-treated actin and PCMB-treated myosin) suggested that certain thiol (SH) group of cysteine residues in myosin and ε-NH₂ groups of lysine residues in actin were involved in the development of gel on heating. However, the magnitude of difference in gel strength at pH 6.0 (<l, 000dyn/cm²) and at pH 5.4 (9, 000 dyn/cm²) of SH-blocked myosin was indicative of the major contribution of electrostatic interactions in the gelation of myosin. The effects of ATP on the gelation of myosin and actomyosin were also pH dependent.
Total rod and LMM subfragments of cardiac myosin formed respectively 2.6 times and 2-times stronger gel than produced by HMM subfragments at identical conditions in the model system, but S-l subfragment formed extremely weak gels under those conditions, which suggested the importance of the "tail" segment of the myosin molecule in the heat-induced gelation process.

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In male rats of the Wistar strain that were fed with a 25% casein (25C) diet supplemented with cholesterol (CHO) and/or sodium cholate (SC), the effects of melanoidin (a mixture of low- and high-molecular weight components that was prepared from a glucose-lysine system) on the contents of lipids, CHO and bile acids were examined. Animals weighing 160- 180 g were given ad libitum the 25C meal containing either or both 1% CHO and 0.2% SC with or without 4% of melanoidin for 3 weeks. By adding melanoidin to the 25C diet supplemented with CHO or CHO and SC, the level of total plasma CHO was depressed, but the amounts of total liver CHO, total fecal CHO and total bile acids were enhanced in general. The 25C diet containing 0.2, 0.4 or 0.6% of SC with or without CHO and melanoidin was fed ad libitum to the rats having about 90 g of body weight the 3 days. The addition of melanoidin to the CHO meal containing 0.2% SC restrained the total plasma CHO concentration and, furthermore, elevated the amount of total fecal CHO. However, these effects were not observed by supplementing the 0.6% SC-added CHO diet with melanoidin.

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Derivatives of pisiferic acid (2), an antibiotic found in Chamaecyparis pisifera var. plumosa, were tested for antibacterial activities against Proteus vulgaris, Staphylococcus aureus and Bacillus subtilis. As structural factors for the activities, the C-10 COOH and/or C-12 OH were estimated to be the most important factors. In the mode of action of the pisiferic acid species on the bacterial macromolecule, peptidoglycan synthesis was predominantly inhibited in B. subtilis, while nonspecific inhibition was observed in P. vulgaris.

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The effects of pyridine and pyrazine carboxylic acids derivatives on growth, floral induction, and the contents of niacin and NAD in the short-day plant Lemna paucicostata 151 were investigated. Among the compounds which promoted plant growth, cinchomeronic acid and trigonelline were the most effective. Picolinic acid was the strongest inhibitor of plant growth among tested. These effects may be concerned with the biosynthesis and metabolism of NAD. The results obtained in this study suggested that the substituents on the third position of the pyridine ring require large volume and high electronegativity to promote plant growth. Floral induction was observed with nicotinamide, isonicotinamide, pyrazinamide, etc.

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In order to investigate the role of the side chain of amino acids in the bitterness in peptide, some oilgopeptides containing glycine, alanine, aminobutyric acid, valine, isoleucine, norvaline (n-Val) and norleucine (n-Leu) were synthesized and their tastes were evaluated. For the bitter taste to be exhibited, the side chain skeleton of the amino acid should consist of at least three carbons. The peptides consisting of amino acids having a side chain of less than three carbons did not exhibit bitterness, while the peptides consisting of amino acids having a side chain of more than three carbons did exhibit bitterness. Valine exhibited an intermediate feature, the taste of valine peptides varying dependent upon the structure. The difference of the branching structure in side chains was less concerned with peptide bitterness.

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In order to investigate the role of the proline residue in the bitter taste of peptides, some oligopeptides containing proline were synthesized and their tastes were evaluated. Proline peptides exhibited bitterness, this aspect being different from hydrophobic amino acids. The most significant role of proline residue in peptide bitterness was dependent on the conformational alternation of the peptide molecule by folding the peptide skeleton due to the imino ring of the proline molecule. It was demonstrated that 2 bitter taste determinant sites were essential for peptide bitterness and that they should be adjacent in the steric conformation of the peptides.

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Thermostable lipase from Humicola lanuginosa No. 3 was immobilized by entrapment in photo-crosslinkable resin prepolymers, urethane prepolymers or Ca-alginate. Adsorption to different types of resins or gels, and covalent binding methods were also employed. From all the methods examined, adsorption on Amberlite XAD-7 followed by crosslinking with glutaraldehyde was superior, as to several points. Firstly, the adsorption efficiency for lipase was relatively high, being about 70%. Secondly, the optimal temperature was about 10°C higher than that for free lipase. The resin immobilized lipase was not affected by pH changes, and finally it also exhibited excellent stability as to repeated batch hydrolysis of triglycerides, both in a shaking flask system and in a recycling column reactor for more than 2 and 3 weeks, respectively.

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We previously reported that the K₀ value, which we have proposed as an index of freshness for edible meat, would be a suitable index as far as the freshness of beef and rabbit muscles is concerned.
This time, we have investigated the applicability of the K₀ value to porcine and chicken muscles.
As a result, we found that inosine (HxR), hypoxanthine (Hx) and xanthine (x) accumulated in both muscles during cold storage. Of these three intermediates, HxR tended to accumulate faster in chicken muscle than in porcine muscle, and HxR accumulated in porcine muscle disappeared faster than in chicken muscle. The IMP content in the chicken muscle during cold storage was always less in quantity than the sum of the ATP, ADP and AMP contents.
Changes of ATP-related compounds in porcine and chicken muscles during cold storage showed distinct patterns different from those of beef and rabbit muscles, and could be categorized as falling midway between those of beef and rabbit muscles. From these results, we can safely conclude that the K₀ value can be used as an index of freshness for porcine and chicken muscles, as well.

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An aniline-assimilating bacterium, Rhodococcus erythropolis AN-13, grew on glucose markedly slowly as a sole carbon and energy source. When acetate was added to the glucose medium, this organism grew rapidly with the utilization of glucose as well as acetate. Several organic acids and amino acids, meat extract, peptone, aniline, etc. also promoted the glucose utilization and cell growth. R. erythropolis AN-13 may metabolize glucose by way of the Embden-Meyerhof-Parnas and pentose phosphate pathways. However, the enzyme activities of both the pathways in this organism were lower than those in Escherichia coli K12 and another aniline-assimilating bacterium, Frateuria sp. ANA-18. These activities in R. erythropolis AN-13 increased when it was incubated in a medium containing acetate. The relationship between the promotion of glucose utilization and the increases in the enzyme activities on the addition of acetate was discussed.

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Moranoline (1-deoxynojirimycin) isolated from mulberry root bark (Mori Cortex), is a potent intestinal α-glucosidase inhibitor. The IC₅₀ values for sucrase and maltase in various animals ranged around 10^-7M. Postprandial hyperglycemia in sucrose-, starch-, or maltose-loaded rats was significantly supressed by simultaneous administration of moranoline in doses over 6 mg/kg. In contrast to the potent inhibition of intestinal α-glucosidase, the inhibition of β-glucosidase, glucoamylase, and α-amylase was weak.
Among the N-substituted alkyl derivatives of moranoline, the methyl and ethyl derivatives had more potent hypoglycemic activity than moranoline in sucrose- or starch-loaded rat models. Nojirimycin, or 2, 5-dihydroxymethyl 3, 4-dihydroxypyrrolidine (DMDP), which structurally resembles moranoline, only weakly inhibited α-glucosidase but strongly inhibited β-glucosidase.

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(+)-Demethylcycloheximides (2-4) and (±)-4-[2-(3-isopropyl-6-methyl-2-oxocyclohexyl)-2-hydroxyethyl]-2, 6-piperidinedione (5) were synthesized by an aldol condensation of the respective ketones (6) with 4-(2-oxo-ethyl)-2, 6-piperidinedione (8). Their biological activities were examined against fungi, rice seedlings and lettuce seeds, but only some of them showed weak activity.

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Glutathione transferase was purified from Mucor javanicus and characterized as a dimer of identical subunits (M_r; 22, 000). Kinetics and Km values of M. javanicus glutathione transferase, 0.48 mM and 1.0 mM (at pH 6.5) for glutathione and 1-chloro-2, 4-dinitrobenzene, are close to those of rat liver enzyme, but occurrence of isoenzymes was not recognized in M. javanicus. The best substrate of M. javanicus glutathione transferase was 1-chloro-2, 4-dinitrobenzene. On the other hand, 2, 3-, 2, 4-, 2, 5-, and 3, 4-dichloronitrobenzenes were converted to the related conjugates by 0.17 to 0.35% of the velocity toward the best substrate. This is correlated to the previous result that 2, 3-and 2, 4-dichloronitrobenzenes were metabolized to 2-methylthio derivatives by the incubation with M. javanicus.

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[16-¹⁴C]Hexadecanoic acid in dimethyl sulfoxide was applied topically to a sex pheromone gland of the silkworm moth, Bombyx mori L. By the combination of normal-phase TLC and reversed-phase TLC or HPLC, bombykol (the sex pheromone of this species, (10E, 12Z)-10, 12-hexadecadien-1-ol) was purified, and its ¹⁴C-incorporation was successfully counted without any contamination by other alcohol components in the gland. The highest incorporation ratio (ca. 1.5%) of the ¹⁴C-acid into bombykol was observed when a 1-day-old virgin female was treated 4hr after lighting under a 16:8 light-dark cycle, the gland being excised for extraction 1 or 3 hr after the treatment. This reliable incorporation method is suggested to be useful for further investigations of the biosynthetic pathway. The ¹⁴C-acid was also incorporated into other components in the pheromone gland, which have been proposed as precursors of bombykol biosynthesis, namely the (10E, 12Z)-10, 12-hexadecadienoic and (Z)-ll-hexadecenoic acid moieties, hexadecan-1-ol, and (Z)-11-hexadecen-l-ol. The total recovery of radioactivity attained ca. 20%, including the unchanged ¹⁴C-acid.

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(+)-Catechin was isolated from immature seed of Phaseolus vulgaris as an inhibitor of the gibberellin biosynthesis enzyme. It inhibited the conversion of GA₁₂-aldehyde to GA₁₂ at a concentration of 1 × 10^-3 M, and was localized mainly in the testa and not in the embryo during the seed maturation.

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A new β-galactosidase inhibitor was found with a newly developed rapid and practical screening method for β-galactosidase inhibitors. Several strains of Actinomycetes that produced βgalactosidase inhibitors were isolated from soil samples using this method, and Streptomyces lydicus PA-5726 was found to produce a high concentration of new inhibitor in the culture medium. Improvement of the medium led to 20-25-fold augmentation (45 to 900-1, 100μg/ml) of the production. This new inhibitor, which was named galactostatin, was isolated by adsorption on Dowex-50W×8 (H⁺), crystallization from the bisulfite adduct, development on Dowe×-2 × 8 (OH^-) and precipitation with EtOH. Galactostatin displayed strong inhibitory activity toward several β-galactosidases in acidic and neutral media but low acute toxicity in mice (LD₅₀ 1016mg/kg, i.v.).

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The insecticide salithion (2-methoxy-4H-1, 3, 2-benzodioxaphosphorin 2-sulfide) and its 4-cyano derivatives were prepared in good yields by utilizing an intramolecular cyclization reaction as the key step. The structures of diastereomers of the 4-cyano derivatives were characterized on the bases of NMR spectroscopy and X-ray analysis.

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N-Methyl nucleoside hydrolase (TV-methyl nucleosidase, N-MeNase), which hydrolyzes 7-methylxanthosine to produce 7-methylxanthine, was detected in tea-leaf extracts and separated from adenosine nucleosidase (ANase, EC 3.2.2.7) by DEAE-cellulose column chromatography.
The optimum pH for the N-MeNase ranged from 8.0 to 8.5. The enzyme was strongly inhibited by EDTA. Inhibition by the hydrolysis products of 7-methylxanthosine and 7-methylinosine was also observed. The molecular weight was estimated to be about 55, 000 by gel-filtration.
Among purine and N-methylpurine nucleosides, 3- and 7-methylpurine nucleosides were hydrolyzed preferentially by N-MeNase. On the other hand, ANase could not hydrolyze 7-methylxanthosine, although the enzyme showed high activity toward 7-methyladenosine. As a result, it is suggested that N-MeNase catalyzes the hydrolysis reaction of 7-methylxanthosine in the pathway of caffeine biosynthesis, whereas ANase is not directly concerned with it.

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The L-methionine γ-lyase of Pseudomonas putida contains 16 cysteinyl residues per mol of enzyme, which is composed of four identical polypeptide chains. Eight of these residues are buried inside the enzyme structure; they reacted with 5, 5'-dithiobis(2-nitrobenzoic acid) only after the enzyme was denatured. In the native enzyme, two of them were cyanylated with 2-nitro-5-thiocyanobenzoic acid with concomitant inactivation of the enzyme. Both the cyanylation and the inactivation proceeded through biphasic pseudo-first order kinetics. Cyanylation of about 2 mol of cysteine residues per mol of enzyme practically abolished the α, γ-elimination activity: the enzyme shows a half-of-the-sites reactivity. L-Norleucine, a competitive inhibitor, which binds to the active site, strongly retarded the reaction of the essential cysteine residue with 2-nitro-5-thiocyanobenzoic acid, and protected the enzyme from inactivation. The cyanylated enzyme was reactivated with 2-mercaptoethanol formed in situ from S-(β-hydroxyethyl)-L-homocysteine by its remaining activity. Added 2-mercaptoethanol was much less effective than that formed in situ. These suggest that the essential cysteine residue is at or near the active site. The cyanylated enzyme was substantially inactive (residual activity, 5%) for the α, γ-elimination, but 40% active for the α, β-elimination. Kinetic analyses revealed that the affinity of enzyme for the substrates, particularly for long straight-chain amino acids, was decreased greatly by cyanylation. The cyanylated cysteine residue was found to be located at a position of approximately 20% of the total length of the polypeptide from its NH₂-terminus.

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Washed intact cells of iron-grown T. ferrooxidans AP19-3 could reduce manganese dioxide (MnO₂) enzymatically with elemental sulfur (S₀) and the mechanism of Mn⁴⁺ reduction was studied. The optimum pH for Mn⁴⁺ reduction was 2.5. The amount of Mn⁴⁺ reduced by the strain was proportional to the amount of S₀ added to the reaction mixture. An enzyme that directly catalyzes the reduction of Mn⁴⁺ to Mn²⁺ with S₀ was not found in the cell-free extract of this strain. T. ferrooxidans AP19-3 was found to possess sulfur: ferric ion oxidoreductase (SFORase), which catalyzes the reduction of Fe³⁺ with S₀ to give Fe²⁺ and sulfite. The reduction of Mn⁴⁺ was inhibited by a specific inhibitor of SFORase or diamide, strongly suggesting that SFORase is involved in the Mn⁴⁺ reduction in this strain. The involvement of SFORase in Mn⁴⁺ reduction was further supported by the following results. Both the amounts of Fe²⁺ and sulfite produced by SFORase were markedly reduced by MnO₂. The amount of Mn⁴⁺ reduced increased 5.4-fold with 1 mM ferric ion and 10-fold with 2.5 mM cyanide or an inhibitor of iron oxidase, respectively. The sulfite and Fe²⁺ were chemically oxidized by MnO₂ instantly. From the results, it is concluded that the Mn⁴⁺ reduction with S₀ in this strain occurs through two steps. In the first step S₀ is enzymatically oxidized by SFORase to give Fe²⁺ and sulfite, and in the second step these two reduced compounds are chemically reduced by MnO₂ to give Mn²⁺

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To produce bovine milk casein in Escherichia coli cells, we constructed expression plasmids for α_s1-casein cDNA, including the DNA region encoding the signal peptide. First, the production of casein was examined with the maxicell system, by which plasmid-coded proteins could be specifically detected with high sensitivity. The casein protein was produced in UV-irradiated CSR603 cells carrying the plasmids that contained casein cDNA preceded by the tac promoter. Then, plasmid pα_s1EK for higher production of casein was constructed; casein cDNA in this plasmid was preceded by two tac promoters connected in series and followed by the terminator (rrnBT₁T₂) of the rRNA gene. Casein production in the E. coli cells of strains C600 and JM103 carrying Pα_s1EK was detected immunologically with the blotting technique. Casein was constitutive ly produced in the C600 cells, and the JM103 cells also produced it when the inducer of the lac operator was present in the culture medium. Fractionation of the cellular compartments and observations by immunocolloidal gold electron microscopy indicated that the recombinant casein was present in an insoluble form diffusely in the cytoplasm and periplasm. The major product isolated from the induced JM103 cells had the complete signal peptide, which might have been responsible for the insolubility. The bacterial hosts producing casein elongated and, furthermore, they formed linear chains by linking one after another.

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A variety of chemical compounds were examined as to their abilities to inhibit the ice nucleating activity of Erwinia ananas IN-10 cells and their outer membrane fraction. The nucleating activity of the outer membrane fraction was inhibited by many surface-active species among the compounds examined, whereas that of cells was inhibited only by amines and ammonium salts having amphiphilic structures. Ammonium salts with both an n-alkyl group having a carbon number of more than 8 and a benzyl group were particularly effective in inhibiting the nucleating activity of the bacterial cells. The inhibitory ability of one of the amphiphilic ammonium salts was greater at 15°C than at 4°C. When a tea plant was sprayed with one of the effective ammonium salts prior to being kept at - 3°C overnight, it was possible to protect the plant from freeze-injury at the minimal concentration of 250 ppm.

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Glycyrrhizinic acid hydrolase produced by Aspergillus niger selectively hydrolyzed the 3-O-β-D-glucuronide linkage of glycyrrhizinic acid. The substrate specificity of this enzyme was investigated for synthetic glucuronides of aliphatic alcohols as well as natural glucuronide saponins. It was revealed that the glucuronide linkage with low molecular weight alcohols was not cleaved by this enzyme, while the 3-O-β-D-glucuronide linkage of saponins of oleanolic acid was selectively hydrolyzed. It was also disclosed that both the 4-hydroxyl and carboxyl groups of the glucuronide moiety must be unsubstituted for hydrolysis by this enzyme.

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Four novel triterpenoids, epoxyganoderiol A (1), B (2) and C (3) and ganoderal B (4), and one novel steroid (6) were isolated from the fungus Ganoderma lucidum, with the known ganoderol B (5) and 6β-hydroxy-ergosta-4, 7, 22-trien-3-one (7). Their structures were determined by spectroscopic methods. The absolute configuration of the α, β-epoxy alcohol moiety of 1-3 was elucidated by the lanthanide-induced Cotton effect in the CD spectrum.

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An arylamidase (aminopeptidase N) was purified from Escherichia coli HB101 about 300-fold by sequential chromatographies on DEAE-Sephadex, hydroxyapatite and Sephadex G-150, assaying the arylamidase and peptidase activities using L-leucyl-β-naphthylamide and Met-Ala-Ser as substrates, respectively. Both enzyme activities were inseparable throughout the purification. The purified enzyme appeared homogeneous by disc gel electrophoresis, and the two activities were detected at the same protein band. The enzyme was most active at pH 7.5 and had a molecular weight of 80, 000 and a pi 5.7. The enzyme was inhibited by o-phenanthroline, p-chloromercuribenzoate, puromycin, bestatin, and amastatin. Both the activities of arylamidase and peptidase were inhibited in parallel by o-phenanthroline and PCMB. These lost activities were restored by the addition of metal ion and 2-mercaptoethanol, respectively. By these experimental results, aminopeptidase N was concluded to possess not only arylamidase activity but also peptidase activity like aminopeptidase M from mammalian tissues, contrary to the results reported previously by several workers.

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It was found that azide bound to alcohol oxidase non-covalently and caused a color change from yellow to red. Alcohol oxidase was purified with a high yield from methanol-limited chemostat-grown cells of Candida boidinii S2 as an enzyme-azide complex by a simple procedure. That is, a cell-free extract was prepared from cells treated with a cationic detergent, Cation M2, and alcohol oxidase amounted to more than 80% of the soluble protein. Inactivation of the enzyme by H₂O₂ and aldehydes was decreased in the complex. The results were discussed as to the production of various aldehydes with the yeast cells.

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An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184, 000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki=1.34μM, 0.113mM and 0.145mM, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mM). The enzyme was also inhibited by L-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on L-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl aminopeptidase [EC 3.4.11.14].

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Phospholipids were isolated from tea pollen. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylglycerol accounted for 54, 34, 6, and 1% of total phospholipids, respectively. Major fatty acid constituents of these lipids were 16:0 and 18:3, Fatty acid compositions of PE and PI changed during pollen germination, with an increase in 16:0 and a decrease in 18:3. Major molecular species of PC isolated from ungerminated pollen were 1-16:0-2-18:3-PC (60%) and 1-18:3-2-18:3-PC (25%). PE had a molecular species quite similar to PC. These chemical compositions of phospholipids seem to be related to a feature of tea plant pollination at low temperatures.

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