Enzymatic properties of dipeptidyl carboxypeptidase (DCP) from Bacillus pumilus were investigated. The enyme was more active on tri- and tetrapeptides than angiotensin-converting enzyme (ACE) from rabbit lung. The presence of chloride ion is essential for the hydrolysis. The Km value of angiotensin I for the enzyme was 0.119 × 10^-3 M. The enzyme was not inhibited by the mammalian ACE inhibitors lisinopril and enalaprilat. The enzyme is readily inhibited by EDTA but restored by Co²⁺, Mn²⁺, and Zn²⁺. Therefore, it seems to be a zinc-metallo protease.

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The amino acid sequence of kalij pheasant lysozyme has been analyzed. From the comparison of the tryptic peptide pattern of kalij pheasant lysozyme and maps from other bird lysozymes followed by the sequencing of tryptic peptides, the amino acid sequence of kalij pheasant was found to be: KVYGRCELAAAMKRLGLDNYRGYSLGNWVCAAKYESNFNTHATNRNTDGSTDYGIL-QINSRWWCNDGKTPGSRNLCHIPCSALLSSDITASVNCAKKIVSDGNGMNAW-VAWRNRCKGTDVSVWTRGCRL. This sequence had 9 amino acid substitutions compared with hen egg-white lysozyme. Two of these substitutions, positions 34 and 121, were newly detected in phasianid birds. The protein genealogy of phasianid bird lysozymes showed some discordance with the morphological classification of these birds.

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The amino acid sequence of reeves' pheasant lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and resulting peptides were analyzed using the DABITC/PITC double coupling manual Edman method. The established amino acid sequence had seven substitutions, Tyr3, Leul5, His41, His77, Ser79, Argl02, and Asnl21, compared with hen egg-white lysozyme. Ser79 was the first found in a bird lysozyme. A substitution in the active site was found in position 102 which has been considered to participate in the substrate binding at subsites A-C.

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A maltotetraose-producing amylase that hydrolyzes starch in maltotetraose units was first found in the culture filtrate of a strain of Bacillus circulans MG-4 newly isolated from soil. The enzyme was partially purified by ammonium sulfate fractionation, DEAE-Sepharose column chromatography, and Bio-Gel A-0.5 column chromatography. The basic enzymatic properties and saccharification conditions of starch for the use of this enzyme were examined with this partially purified enzyme. Its optimum pH and temperature were around 7.5 and around 50°C, respectively. The enzyme was protected from heat in the presence of calcium ion. This enzyme produced maltotetraose in a yield more than 60% from liquefied starch. A typical sugar composition of hydrolysis products from liquefied starch was about G₁ 1.5%, G₂ 8.2%, G₃ 10.9%, G₄ 64.9%, G₅ 0.0%, G₆ 1.0%, G₇ 1.2%, G₈ 1.3%, G₉ 1.0%, and G_10- 11.0%.

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Homogeneous quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 contains one Ca²⁺ ion per subunit of native enzyme. Treatment with trans-1, 2-diaminocyclohexane-N, N, N', N'-tetraacetic acid at 30°C led to an catalytically inactive apo-form. Upon incubation of the apo-form with Ca²⁺ and pyrroloquinoline quinone a fully active holo-enzyme was reconstituted. The absorption spectrum of this reconstituted enzyme is identical to that of the native enzyme. Incubation of apo-enzyme with Sr²⁺ and PQQ led to the formation of an active Sr²⁺-form. The Sr²⁺ and the Ca²⁺-forms of the enzyme differ in their absorption spectra. The Sr²⁺-form was inactivated by trans-1, 2-diaminocyclohexane-N, N, N', N'-traacetic acid twice as fast as the Ca²⁺-form. Ca²⁺ is necessary for pyrroloquinoline quinone to bind to the apo-form of quinoprotein ethanol dehydrogenase.

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Novel antioxidative phenylpropanoid-substituted tocopherol derivatives, prunusols A and B, were isolated from the leaf wax of Prunus grayana Maxim., and their structures were fully characterized by Spectroscopic and synthetic methods. Prunusols A and B were found to be the conjugates of γ-tocopherol and p-coumaric acid, which are diastereoisomers of each other. They showed almost the same antioxidative activity as α-tocopherol in a water/alcohol system measured by thiocyanate and TBA methods.

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The metabolism of salicylate in a strain of Pseudomonas sp. TA-2, which was grown on tobias acids as the sole source of carbon and nitrogen, was studied by tracer techniques with [7-¹⁴C]salicylate and [1(4, 5, 8)-¹⁴C]l-naphthalenesulfonate. The salicylate and gentisate oxidizing activities were induced, but the catechol oxidizing activity was not, when the cells grown on a nutrient medium were incubated with tobias acid (2-naphthylamine-1-sulfonate). [7-¹⁴C]Salicylate was metabolized to [7-¹⁴C]gentisate and [¹⁴C]pyruvate, and they were accumulated in the culture medium in the presence of excessive gentisate. The maximum sum of radioactivity of [7-¹⁴C]gentisate and [¹⁴C]pyruvate roughly corresponded to that of the degraded [7-¹⁴C]salicylate. [1(4, 5, 8)-¹⁴C]l-Naphthalenesulfonate was also degraded in the presence of gentisate and/or catechol, and [ring-¹⁴C]gentisate was accumulated in the medium, but [¹⁴C]catechol was not accumulated. These results indicate that salicylate is metabolized nearly quantitatively through the gentisate pathway by Pseudomonas sp. TA-2.

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A extreme thermophile that produces a heat-stable proteinase was isolated from hot-spring water and classified as Thermomicrobium sp. KN-22 (growth temperature, 50-83°C; and optimum growth temperature, 70°C). The proteinase was purified from the culture broth of this strain by fractionation with ammonium suit ate, chromatography on columns of DEAE-cellulose and CM-Sepharose CL-6B, and HPLC on TSKgel CM-5PW. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and a single peak after HPLC (yield 8.8%). The enzyme had maximum activity at pH 8.5 and at 75°C and it was stable up to 60°C. The molecular weight of the enzyme was 35, 000 by SDS-PAGE. Since the enzymatic activity was completely inhibited by EDTA, o-phenanthroline, and phosphoramidon, it appears that the enzyme is a metal proteinase.

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An endo-type semi-alkaline cellulase (CMCase I) produced by an alkalophilic Streptomyces strain has an extraordinarily long leader peptide of about 70 amino acids (aa), which can be grouped into four distinct regions, an NH₂-terminal region (13 aa), an Arg-cluster region (13 aa), a hydrophobic region (23 aa), and an Ala/Pro-repeat region (12 aa). For identification of the function of each part of the leader peptide for secretion of the enzyme, mutations in the leader peptide were generated by site-directed mutagenesis using the cloned gene, and the mutant genes were expressed in a cellulase-negative mutant strain, Streptomyces lividans HN1. Although all the alterations in the leader peptide, except for one case, decreased secretion to various extents, in S. lividans, the following conclusions were obtained. Comparison of the intra- and extra-cellular enzyme activities of the mutants suggested that the Arg-cluster region was essential in secretion of the cellulase. Deletion of 8 amino acids rich in threonine and proline in the NH₂-terminal region enhanced the secretion to a small extent. Deletion of the Ala/Pro repeat region had almost no effect on secretion.

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Cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19] was found to catalyze the transglycosylation from α-cyclodextrin (α-CD) to L-ascorbic acid (AA). A main product formed by this reaction was identified as 2-O-α-D-glucopyranosyl L-ascorbic acid (AA-2G) with the enzymatic hydrolysis and ultraviolet absorption spectra using standard AA-2G. A series of maltooligosaccharide substituted 2-O-derivatives of AA were also detected by HPLC. These were thoroughly hydrolized to AA-2G and glucose by treatment with glucoamylase [EC 3.2.1.3].
A large amount of AA-2G was prepared with 500 g of AA and 1000 g of α-CD. Two hundred and fifty grams of purified AA-2G, the content of which was 97%, was obtained through enzyme reactions and purification with HPLC using an ion-exchange resin.

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An n-alkane-assimilating yeast, Candida maltosa, has a diploid genome. Probed with the previously isolated gene of n-alkanc-inducible cytochrome P-450 (P-450alk), its allelic gene had been isolated, its nucleotides sequenced, and the interallelic divergence examined. Using one of the allelic genes, we disrupted the two alleles of the cytochrome P-450alk gene by a two-step gene disruption system. Surprisingly, the disruptant still assimilated n-alkane and contained n-alkane-inducible cytochrome P-450. This result indicates that, other than the disrupted two alleles, there is at least one other gene that encodes an n-alkane-inducible cytochrome P-450.

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Lipid peroxidation products in the diet induce oxidative stress. A hepatic enzyme inactivated in proportion to the oxidative stress was found. The oxidative stress was evaluated by a decrease in the tocopherol level and increases in contents of lipid peroxides and thiobarhituric acid-reactive substances in the serum and liver, when secondary peroxidation products of linoleic acid were fed to rats. The changes in activities of mitochondrial NAD-dependent aldehyde dehydrogenase, succinate dehydrogenase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase in the liver, the activities of which are decreased after the oral dose of lipid peroxidation products, were measured. A marked decrease in the phosphoglucomutase activity was closely related to the increase in oxidative stress.

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Aminopeptidase C was purified from fresh chicken skeletal muscle by ammonium sulfate fractionation, and by successive chromatography on DEAE-cellulose, Ultrogel AcA 34, DEAE-cellulose again, and an alanine AH-Sepharose 4B affinity column twice. The purified enzyme migrated as a single band by SDS-PAGE. Aminopeptidase C was purified about 300-fold over the crude extract with a yield of 0.6%. The molecular weight of this enzyme was found to be 185, 000 by gel filtration in a Sepharose 6B column and 92, 000 by SDS-PAGE. The optimum pH for the hydrolysis of L-leucine β-naphthylamide was 6.0-7.0, the enzyme being stable in the range of pH 6.5-8.0. The activity of this enzyme was strongly inhibited by EDTA and puromycin, and was high against the β-naphthylamide derivatives of Lys, Leu, Ala and Met. The enzyme was more active towards tri- and tetrapeptides than towards dipeptides.

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Aminopeptidase H was purified from fresh porcine muscle by ammonium sulfate fractionation and successive chromatographies of DEAE-cellulose, Ultrogel AcA 34, Phenyl-Sepharose CL-4B, hydroxylapatite, and a second DEAE-cellulose. The purified enzyme migrated as a single band on SDS-PAGE. Aminopeptidase H has activity against both L-leucine β-naphthylamide (LeuNap) and α-N-benzoyl-DL-arginine β-naphthylamide (BzArgNap). The molecular weight of the purified enzyme was 51, 000 on SDS-PAGE and 390, 000 on an Ultrogel AcA 34 column chromatography. The optimum pH for the hydrolysis of LeuNap and BzArgNap was 8.0. The Km values for the hydrolysis of LeuNap and BzArgNap were 0.37 and 1.25 HIM, respectively. These activities were strongly inhibited by monoiodoacetic acid and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, or bestatin. The enzyme had the large activities against Met-, Leu-, Lys-, Ala-, Glu-, and SerNap and no activity against ProNap.

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Two new pyrrolizidine alkaloids, ideamines A and B, together with other analogs (lycopsamine and parsonsine) were isolated in the N-oxide forms from adult bodies of the Apocynaceae-feeding danaine butterfly, Idea leuconoe. Ideamine A was characterized as a homolog of lycopsamine, in which the viridifloric acid moiety was replaced by a 2-ethyl-2, 3-dihydroxybutanoic moiety. Likewise, ideamine B was identified as a nor-derivative of parsonsine, in which the trachelanthic acid moiety was replaced by a 2-ethyl-2, 3-dihydroxybutanoic moiety diastereomeric to the necic acid from ideamine A.

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The effect of dietary oleate levels (18, 39, 57 and 74% of total fatty acids) on various tipid parameters was studied in rats given cholesterol-enriched diets containing fat with a constant P/S (3.1-3.2) and n-6/n-3 (5.4-6.2) ratio. High-oleic safflower oil was used as a source of oleic acid, and was replaced stepwise with a mixture of cotton seed and perilla seed oils. After three weeks of feeding, there were no significant differences in the concentrations of serum and liver cholesterol, although they tended to increase with an increasing dietary oleate level. A hypotriglyceridemic trend was observed toward an increasing proportion of oleic acid. The linoleate desaturation index, (dihonio-γ-linolenic acid + arachidonic acid)/linoleic acid, in tissue phosphatidylcholine tended to increase with an increasing proportion of oleate, whereas the production of prostacyctin by the aorta and thromboxane A2 by platelets was independent of the dietary oleate level. These results indicate that dietary oleate did not significantly modify the effect of polyunsaturated fatty acids on various lipid parameters under dietary conditions at which the P/S and n-6/n-3 ratios of the dietary fat were kept at an appropriate level to prevent ischemic heart disease.

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When incubated for 7hr with a rat liver homogenate, the prenylated isoflavone luteone, 5, 7, 2', 4'-tetrahydroxy-6-(3, 3-dimethylallyl)isoflavone (1), was converted to six derivatives, which were isolated and purified by thin-layer chromatography. Using physicochemical procedures, the metabolites were characterized as a dihydrofurano-isoflavone (2), four new isoflavones with variously oxygenated terminal methyl groups (3, 4, 6 and 7), and a coumaronochromone (5). The geometry of the oxygenated side-chain was unambiguously determined by NMR techniques (¹H-NMR, NOE and ¹³C-NMR).

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Sucrose phosphorylase (EC 2.4.1.7) was purified to homogeneity from Leuconostoc mesenteroides cells with a specific activity of 173.8 units per nig protein by ammonium sulfate fractionation, anion exchange HPLC on TSKgel DEAE-5PW, and hydrophobic HPLC on TSKgel Ether-5PW.
The purified enzyme was an acidic protein having an isoelectric point of pH 4.6 and s^O_{2O, W} of 4.34 S. The molecular weight of this enzyme was estimated to be 56, 400 by sedimentation equilibrium, 55, 000 by SDS-polyacrylamide gel electrophoresis, and HPLC gel filtration on TSKgel G3000SW, suggesting that the enzyme is a monomeric protein. With regard to molecular weight, amino acid composition, and N-terminal amino acid sequence of 30 residues, this enzyme is close to the glucosyltransferase A of Streptococcus mutans.

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Linalyl-β-glucoside (LBG), α-terpinyl-β-glucoside (TBG), neryl-β-glucoside (NBG), and geranyl-β-glucoside (GBG) in sweet potatoes were identified as TMS derivatives from their MS fragmentation patterns and retention times. Amounts of these monoterpene alcohol-β-glucosides were from 36.9 μg/kg sweet potato of LBG to 189.μg/kg sweet potato of TBG. On the other hand, 75.8μg/1 distillation residue of TBG and traces of other monoterpene alcohol-β-glucosides occurred in the distillation residue. β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) in shiro-koji were purified using p-nitrophenyl-β-glucoside (PNPG), and three active fractions, one major fraction and two very minor fractions, were found. The Km against GBB was 2.4 mM and the Ki against glucose was 7.6 mM. Relative activity in the 20% ethanol solution was 68%. Shiro-koji β-glucosidases were active on tested β-glucosides except for LBG and TBG.

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An efficient heterologous protein production system was developed in Trichoderma viride, a very efficient cellulase producer. An expression vector containing the Taka-amylase A gene from Aspergillus oryzae, which was fused to the strong promoter and signal peptide sequence of the cellobiohy drolase 1 gene (cbhl) of T. viride, and the hygromycin B resistance gene was used to transform protoplasts of T. viride. Using hygromycin B resistance, a frequency of 3 transformants per μg DNA on average was obtained. One transformant showed highly elevated α-amylase production, 1.0 g/1, which was shown to be under the control of the cbhl gene promoter. Analysis of the chromosomal DNA of the transformant showed the integration of more than one copy of the vector.

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The effect of hydrophobia particles on foam stability was quantitatively evaluated by the parameters in the capillary model. Polystyrene particles were dispersed into an ovalbumin solution, foam then being produced by introducing air into the ovalbumin solution with polystyrene particles (OSP) through a spinneret. The electric conductivity at selected positions in the foam and the drained liquid height below the foam were measured at constant intervals, the measured electric conductivity being converted to the OSP volume fraction in the foam. The capillary model described the OSP leakage rate from the foam and OSP volume fraction in the foam. As the particle concentration increased, the ratio of the OSP volume fraction in the foam to the volume fraction of the Plateau border, and time constant for drainage, Tincreased. This result indicates that the hydrophobic particles used stabilized the foam.

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The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Since blocking the free SH group of Cys⁴¹ in the B chain or treating the adjacent Met⁴² with CNBr removed its sweetness, this part of the molecule has been suggested to be essential for the sweetness. The [Ser⁴¹] B chain, an analogue of the B chain, was synthesized by the stepwise Fmoc solid-phase method in an overall yield of 1.9%. The synthetic B chain analogue was combined with the A chain, which was left over from a previous work, to give [Ser⁴¹] B-chain monellin in a yield of 31.0%. This synthetic monellin analogue was 2000 times as sweet as sucrose. Changing the Cys⁴¹ residue to the Ser residue significantly decreased the sweetness potency and stability of the molecule in solution. Crystallization was carried out by a vapor diffusion method.

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The volatile components of steamed, microbially fermented, dried (product), and stored Toyama kurocha were analyzed by GC and GC/MS. The amounts of terpene alcohols increased and methylether phenolic compounds were produced by natural fungal fermentation. Aliphatic alcohols, aldehydes, and ketones were greatly increased by solar drying. One hundred and twenty-five volatile components were identified in the stored sample. The formation of volatile phenolic compounds produced from ferulic acid and p-coumaric acid by Aspergillus niger and Leuconostoc mesenteroides was also investigated. Methylation of phenolic hydroxy group by Aspergillus niger was observed, similar to that of the fungal fermentation tea process. Leuconostoc mesenteroides could not methylate phenolic hydroxy groups.

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An orotidine-phosphorolyzing enzyme and a purine nucleoside phosphorylase (PNPase) of Erwinia carotovora AJ 2992, which is a potent producer of ribavirin (1-β-D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide), an antiviral agent, from orotidine and 1, 2, 4-triazole-3-carboxamide (TCA), were purified 23-fold and 103-fold, respectively. At each purification step, the orotidine-phosphorolyzing enzyme was always co-purified with an uridine phosphorylase (UPase) and its activity could not be separated from that of the UPase after it showed as a single band on SDS-polyacrylamide gel electrophoresis. These results suggest that this enzyme may be identical with UPase. The purified enzyme had a molecular weight of 68, 000 ± 2, 000, and seemed to be a dimer. The optimal temperatures and pH values were 60°C and 6.0 for orotidine phosphorolysis, and 70°C and 7.0 for uridine phosphorolysis. The Michaelis constants for uridine and orotidine were 0.75 mM and 10.87 mM, respectively, at 40°C. The PNPase of E. carotovora AJ 2992 had a molecular weight of 58, 000 ± 2, 000 and seemed to be a dimer. The Michaelis constants for inosine and guanosine were 1.92 mM and 1.85 mM, respectively, at 40°C. The PNPase was completely inactivated by p-chloromercuribenzoate.

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A human MDR1 cDNA was introduced into yeast cells. Immunoblot analysis and indirect immunostaining showed that some of the P-glycoprotein produced was situated in its native orientation in the yeast plasma membrane. Drug-binding activities of the recombinant P-glycoproteins were markedly decreased compared to that of the authentic P-glycoprotein. To identify the bases of decreased binding we studied the effects of membrane component sterols on the azidopine binding and found that ergosterol, which is the main sterol in the yeast membrane, and calciferol, which is produced from ergosterol by UV irradiation, inhibited azidopine binding. These sterols in yeast membrane probably inhibit the function of human P-glycoprotein as a multidrug transporter in yeast cells, because expression of P-glycoprotein in yeast cells did not confer resistance to doxorubicin.

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The metabolic fate of the carbon skeletons of L-[U-¹⁴C]asparagine and L-[U-¹⁴C]glutamine was investigated in growing rats fed with 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 12 hr after injecting ¹⁴C-labeled asparagine was high in the lower PC% groups, about 60% of the dose being recovered, but the value decreased in the higher PC% groups. Most of the radioactivity incorporated into the tissue protein was present in the aspartic acid and/or asparagine fraction. The pattern of expired ¹⁴CO₂ production was in inverse relation to that of ¹⁴C incorporation into the body protein. The incorporation of ¹⁴C-glutamine into the body protein was markedly less in all the dietary groups, but the expired ¹⁴CO₂ production was extremely high, more than 70% of the dose being oxidatively degraded. These results show that the metabolic response of glutamine to dietary protein well resembles that of glutamic acid, aspartic acid or alanine, but markedly differs from that of asparagine, which is preferentially used for body protein synthesis, especially during protein depletion.

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The preparation of 4-hydroxy-β-ionone of high enantiomeric excess was achieved via lipase-catalyzed transesterification of the corresponding racemate as the key-step. Starting from Its (R)-enantiomer, (S)-6-hydroxy-α-ionone, an important intermediate for synthetic abscisic acid analogs, was synthesized.

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Both enantiomers of aliphatic and aromatic cyanohydrins were hydrolyzed with the aid of Rhodococcus butanica ATCC 21197 to afford optically active α-hydroxy acids. The usefulness of this reaction is demonstrated by the synthesis of optically pure (R)-4-dodecanolide, a defensive secretion of rove beetles, starting from (R)-2-hydroxydecanenitrile.

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Desaturase activity that converts versicolorin B (VB) to versicolorin A (VA) was detected in the presence of NADPH in the microsomal fraction of Aspergillus parasiticus NIAH-26, which endogenously produced neither aflatoxins nor anthraquinone- or xanthone-precursors in a yeast-extract sucrose (YES) medium. A smaller amount of VA was also produced from versicolorin C (VC). When the same mold was cultured with VA in YES medium in feeding experiments, aflatoxins B₁ (AFB₁) and G₁ (AFG₁) were produced. When either VB or VC was fed to the mold, aflatoxins B₂ (AFB₂) and G₂ (AFG₂) as well as AFB₁ and AFG₁ were produced. These results indicate that the branching step between AFB₁•AFG₁ and AFB₂•AFG₂ corresponds to the desaturation reaction from VB to VA.

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Fusaric acid-resistance genes (fus) were isolated from Pseudomonas cepacia. The nucleotides of the 5437 base pairs containing the fus genes were sequenced.

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