The amount of CO₂ sorbed by microbial cells in a Saccharomyces cerevisiae-water system was measured by a gravimetric method with a quartz spring, and the correlation between CO₂ sorption and the sterilization effect of high-pressure CO₂ was investigated. The sterilization rate of Saccharomyces cerevisiae by high-pressure CO₂ was measured by varying the water content and CO₂ pressure, and analyzed by reaction kinetics. The sterilization rate could be described by a first-order reaction, and the dependence of the sterilization rate constant, k, on the water content and CO₂ pressure was evaluated. The amount of CO₂ sorbed by the microbial cells reached equilibrium at a constant CO₂ pressure within a few minutes and was correlated well with the value of k. In addition, the amount of unfreezable water was measured by DSC as an index of the state of water in the cell-water system, this being considered to be closely related to the amount of CO₂ sorbed by the microbial cells. The value of k increased with increasing water content; however, the increase wad only slight for a water content by which free water existed.

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Water sorption isotherms of superabsorbent polymers were measured, and their affinity for water was evaluated by solution thermodynamics. The results provide basic data for the functional packaging of food to control the water content of food during its transportation or storage. Water abtivity above 0.9 was measured by adding a specific amount of water to the samples, while that below 0.9 was measured with apparatus for evaluating water sorption isotherms. Thus, water sorption isotherms for superabsorbent polymers were obtained up to a water activity of approximately 0.98. The amount of water sorbed by the superabsorbent polymers was influenced by the type of functional groups in the polymers, and not by the degree of cross-linking in the polymers. The integral Gibbs free energy, which is the most suitable parameter for evaluating the affinity of a material for water, was evaluated from the water sorption isotherms by using solution thermodynamics.

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At least 15 allergenic proteins have been found in soybean using the sera of soybean-sensitive patients with atopic dermatitis [T. Ogawa et al., J. Nutr. Sci. Vitaminol., 35, 555-565 (1991)]. In the present study, a monoclonal antibody (mAb) against Gly m Bd 28K, one of the major allergens of soybean, was prepared, and Gly m Bd 28K was purified from defatted soybean tlakes by five purification steps, including immunoaffinity chromatography with the mAb as a ligand. The purified allergen was found to be a glycoprotein with a molecular mass of 26 kDa. During the purification process the allergen was converted to more acidic proteins with the same molecular mass, suggesting that the allergen is unstable. The sugar composition and amino acid sequence of Gly m Bd 28K suggest that the allergen is a new glycoprotein with an Asn-linked sugar moiety. The distribution of the allergen in soybean products was examined by an immunoblotting technique with the mAb.

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We have developed an efficient expression system for foreign genes in Acremonium chrysogenum. After inserting the foreign gene between the phosphoglycerate kinase (PGK) promoter and a terminator derived from A. chrysogenum, multiple copies of this expression unit are tandemly ligated into cosmids and the resultant cosmids are introduced into A. chrysogenum. We expressed Pseudomonas cephalosporin C acylase and a human thrombomodulin mutant protein containing the fourth, fifth, and sixth epidermal growth factor (EGF)-like structures (E456). The acylase activity in the transformants obtained using our system was several times higher than that in the transformants without the use of the system. The acylase proteins expressed had enzymatic and immunochemical properties identical to those of authentic acylase. The transformants with the expression plasmid for E456 secreted biologically active E456 protein into the culture medium. The amino terminal sequence of the purified E456 was identical to that of recombinant E456 obtained using mammalian cells.

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Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Corynebacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter (77.6 mM) from orotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY13505 for the enzymatic reaction using orotic acid as a substrate. CTP was produced at 8.95 g/liter (15.1 mM) after 23 h of this reaction. To produce CDP-choline, two additional enzyme activities were needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CKIase; encoded by the CKIgene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system. After 23 h of the reaction using orotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mM) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.

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A new method for enzymatic production of cytidine diphosphate choline (CDP-choline) from orotic acid and choline chloride was developed. To establish an industrial manufacturing process, we constructed a plasmid, pCKG55, which simultaneously expressed in Escherichia coli the three following enzymes; CTP synthetase (encoded by the pyrG gene from E. coli, cholinephosphate cytidylyltransferase (encoded by the CCT gene from Saccharomyces cerevisiae), and choline kinase (encoded by the CKI gene from S. cerevisiae). CCT and CKI genes on pCKG55 were designed to be expressed as a single CCT/CKI fused protein. This CCT/CKI fused protein retained both activities and the thermal stability of its cholinephosphate cytidylyltransferase activity was nearly the same as the native CCT enzyme. Corynebacterium ammoniagenes KY13505 and E. coli MM294/pCKG55 were cultured in 5-liter jar fermentor independently. Equal volumes of each broth were mixed in a 2-liter jar fermentor, and then the enzymatic reaction was done using 47 mM orotic acid and 60 mM choline chloride as substrates. After 23 h of the reaction at 32°C, 21.5 mM (11 g/liter)of CDP-choline was actumulated.

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An extracellular glucose-tolerant β-glucosidase was purified to homogeneity by alcohol fractionation and preparative isoelectric focusing from Aspergillus niger CCRC 31494. The enzyme was a dimeric protein with a subunit of 49, 000, and had its optimum activity at pH 5.0 and 55°C. The enzyme was completely inhibited by 5 mM Ag⁺. Thiol groups and serine residues were not essential for its activity. Low concentrations of alcohols (10%) except for methanol could activate the enzyme. It was very specific for para-nitrophenyl-β-D-glucoside (pNPG) and cellobiose. However, the enzyme also had some β-xylosidase activity, but showed no activity towards x-linked glycosidic substrates. The V_max of 124.4 U/mg and 21.6 U/mg were found for pNPG (K_m = 21.7 mM) and para-nitrophenyl-β-D-xyloside (pNPX) (K_m = 14.2 mM), respectively. The enzyme was tolerant to glucose inhibition with a K_i of 543 mM, while fructose, galactose, mannose, and xylose were not inhibitory.

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The interactions of glycosaminoglycans (GAGs) with collagen are thought to be important in cell adhesion and cell differentiation. To investigate whether the interactions of GAG or sulfated polysaccharide with collagen can maintain the functions of cultured primary rat hepatocytes, GAG- or sulfated polysaccharide-containing collagen gels were reconstituted in vitro and used for culture of hepatocytes. Among the GAGs and sulfated polysaccharides examined, heparin- and lambda carrageenan-containing collagen gels were found to be able to stimulate and sustain albumin synthesis, while the other GAG- or sulfated polysaccharide-containing collagen gels had almost no effect on maintenance of albumin synthesis. In the cultures using collagen gels that contained 400 μg/ml heparin or 100 μg/ml lambda carrageenan, albumin synthesis by rat hepatocytes was prolonged to about 4 and 5 weeks, respectively, but albumin synthesis was kept up for only one week in the cultures using conventional collagen gels. These results suggest that the interactions of heparin or lambda carrageenan with collagen might be of importance for long-term maintenance of hepatocyte functions.

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When a series of chitin oligosaccharides was added into a rice suspension culture, N-acetylchito-hexaose, N-acetylchitopentaose, and N-acetylchitotetraose caused an increase in extracellular chitinase activity, mainly due to induction of a class III chitinase. In the case of N-acetylchitohexaose, a substantial increase in the chitinase activity was observed at a concentration higher than 0.01μg/ml, and a maximum effect was reached at 1μg/ml. In contrast, N-acetylchitotriose, N-acetylchitobiose, N-acetyl-D-glucosamine, and chitohexaose (a chitosan oligosaccharide) were not very effective. Chitinase induction was also observed with laminarihexaose (a β-1, 3-glucan oligosaccharide), but about a 10-fold higher concentration, compared with N-acetylchitohexaose, was needed to get the maximum effect. β-1, 3-Glucanase activity was found in cells (but not in medium), and the activity was increased by neither n-acetylchitohexaose nor laminarihexaose. When cells were incubated with N-acetylchitohexaose, L-phenylalanine ammonia-lyase (PAL) activity increased promptly. A biphasic profile was obtained when a dose-dependent effect of the elicitor on the PAL induction was examined; the first phase was observed in a range from 0.01 to 1μg/ml and the second phase from 3 to 300μg/ml. Laminarihexaose also acted as an elicitor for PAL induction.

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A cDNA for a Na⁺/glucose cotransporter was cloned from a rat jejunum cDNA library. This transporter was expressed in Xenopus oocytes by injection of cRNA synthesized from the cDNA, and the transporter ability was electrophysiologically examined. The cotransporter had a very narrow sugar specificity. Only D-glucose, D-galactose, and some of their derivatives elicited significant electrical responses. These results of sugar specificity were compared with those of the H-+/hexose cotransporter of Chlorella. Dose-response relationships of several sugars followed a simple Michaelis-Menten type of kinetics. Both V_m and K_m were dependent on the sugars. Not only the affinity of sugars to the cotransporter but also the rate of conformational change of the cotransporter loaded with the sugar and Na⁺, which translocates them from outside to inside, possibly depends on the sugar structure. The rate-limiting step of the transportation may be the conformational change, i.e., isomerization, of the cotransporter that translocates both the sugar and Na⁺ from outside to inside.

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The amino acid sequence of 6.5k-arginine/glutamate rich polypeptide (6.5k-AGRP) from the seeds of sponge gourd (Luffa cylindrica) has been determined. The 6.5k-AGRP consists of a 47-residue polypeptide chain containing two disulfide bonds, and a molecular mass calculated to be 5695 Da, which fully coincides with a value of [M + H]⁺ = m/z 5693.39 obtained by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The mass spectrometric evidence indicated that 6.5k-AGRP is also present partially truncated at the C-terminus. In our preparations, approximately half of the polypeptide molecules have the C-terminal sequence Arg-Arg-Glu-Val-Asp; the other half lack Val-Asp and end with the glutamic acid, making a total of 45 residues in the polypeptide chain. The two disulfide bonds connect Cys¹² to Cys³³ and Cys¹⁶ to Cys²⁹. Comparison of the amino acid sequence of 6.5k-AGRP with those of the other known proteins included in the PIR protein sequence database showed that it is related to the amino acid sequencc of the N-terminal region encoded by the first exon of the cocoa (Theobroma cacao) and cotton seeds vicilin genes, sharing a characteristic two Cys-Xaa-Xaa-Xaa-Cys motif.

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Assimilation of DFA III by Arthrobacter sp. H65-7 was found to consist of two sequential enzyme steps, hydrolysis of DFA III to inulobiose (1-O-β-D-fructofuranosyl-D-fructopyranose) and inulobiose to fructose, that is, x-(2→3') and β-(2'→1) fructosidic linkages were split separately. The enzyme catalyzing the first step, named DFA III hydrolysis enzyme (DFA IIIase), has been purified from the cell-free extracts of Arthrobacter sp. H65-7 to an electrophoretically pure state by heat-treatment, ammonium sulfate fractionation, and chromatographies on DEAE-Toyopearl 650M, Butyl-Sepharose 4B and TSKgel G3000SW_XL, and Biophoresis III. The molecular weight of the purified enzyme was estimated to be 125, 000 by gel filtration, and 61, 000 by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity at pH 6.0 and 45°C, and was stable from pH 4.5 to 10.0 and up to 60°C. The K_m of this enzyme for DFA III was 12.5 mM. The enzyme also catalyzed the reverse reaction, inulobiose to DFA III.

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Pokeweed antiviral protein (PAP) belongs to a group of ribosome-inactivating proteins (RIPs) that inactivate ribosomes by depurinating rRNA at a specific site. To study the mechanism for the antiviral activity of PAP, the actions of PAP on TMV-infected and uninfected tobacco protoplasts were investigated. The addition of 0.33 μM PAP to TMV-inoculated protoplasts caused a complete inhibition of TMV production. The same concentration of PAP was found to inhibit protein synthesis in the virus-infected protoplasts and to kill the cells, but it had no effect on the uninfected protoplasts. The concentration dependence of protein synthesis-inhibition by PAP was related to that of inhibition of viral multiplication. Furthermore, two other RIPs (ricin A-chain and luffin-a), which showed 240 and 430-fold less activity on tobacco ribosomes than PAP in d cell-free system, did not inhibit viral multiplication even at a concentration of 3.3μM. The analysis of RNAs from the virus-infected and PAP-treated protoplasts demonstrated that 25S rRNA was depurinated by PAP in the infected cells. These results suggest that PAP, which is normally unable to penetrate the plasma membrane of uninfected protoplasts, gains entrance to the cytosol of infected cells and prevents viral multiplication by inactivating ribosomes.

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Proteasomes are the major cytosolic protease complexes responsible for energy-dependent and extra-lysosomal proteolysis. Ubiquitinated proteins are degraded by the 26S proteasome which is composed of a 20S proteasome and two regulatory complexes. Changes in the 20S and 26S proteasome activity levels and protein abundance in spinach seeds during imbibition and germination were examined by using glycerol density gradient centrifugation. The 26S proteasome activity level decreased transiently during imbibition, reached a minimum one day after starting imbibition, and then increased again. During the period of minimal accumulation, the protein being detected with an anti-26S proteasome antibody shifted to a lower sedimentation coefficient fraction. In contrast, the 20S proteasome activity level increased during imbibition, and remained high for two days. The change in the 20S proteasome level corresponded to the change in the 20S proteasome activity level.

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We have previously demonstrated the bile-pancreatic juice (BPJ)-independent stimulation of pancreatic enzyme secretion in chronic BPJ-diverted rats. Pancreatic and intestinal adaptation to 7-day BPJ diversion was next examined. Pancreatic enzyme mRNA and cholecystokinin mRNA in the jejunal mucosa were measured in rats with BPJ diverted into the ileum (PBD rats) in comparison with the figures for rats with BPJ returned to the duodenum (normal rats) or laparotomized (Intact) rats under well-nourished conditions. Amylase mRNA in the pancreas was lower and trypsinogen plus chymotrypsinogen mRNA was higher in the PBD rats than in the intact rats. The change in pancreatic mRNA was similar to that in the specific activities of the enzymes after a chronic BPJ diversion. This finding suggests that these pancreatic enzymes were regulated by the mRNA level. The portal concentration of cholecystokinin in the postabsorptive period (exogenously non-stimulated status) was 4-fold higher in the PBD group than in the normal and intact groups. Cholecystokinin mRNA in the jejunal mucosa of PBD rats was somewhat higher than that of intact rats. These results suggest that intestinal cholecystokinin was predominantly increased at the translational or later stage by chronic BPJ diversion.

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A strain of streptolmycete isolated from a soil sample was found to produce a novel amino acid metabolite. The compound was purified from the culture fluid by column chromatography, using cation exchange resin, a synthetic adsorbent, and finally by preparative HPLC with a reverse-phase column. The structure of the compound was established as N^σ-(5-methyl-4-oxo-2-imidazolin-2-yl)-L-ornithine on the basis of an analysis of the spectral data and chemical degradation. This was confirmed by comparing the NMR spectrum of the metabolite with that of the compound synthesized by treating methylglyoxal and N^x-acetyl-L-arginine. The substance did not show any antimicrobial activity against bacteria, fungi and yeasts by the agar plate method, but exhibited a weak preventive effect on cucumber powdery mildew disease in a pot test.

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Male weanling rats were fed on diets containing 10% of either oxidized or fresh (control) soybean oil for periods of 4 and 7 weeks. The ingestion of oxidized oil was found to reduce the content of free thiols in tbe erytbrocyte membrane and to increase the amount of membrane-extractable spectrin. The level of reactive carbonyl groups was higher in the proteins of the ghosts and of the muscle tissue derived from the experimental animals. These alterations, which are characteristic of peroxidized proteins, suggest that oxidative stress caused by oxidized dietary lipids may damage tissue proteins.

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A phosphate buffer was found to significantly promote tetramethylpyrazine (TMP) formation in an acetoin (3-hydroxy-2-butanone)/ammonium hydroxide system. The effect of the phosphate ion on TMP formation was additive in the range of 0.05-0.2 M. The change in pH value of the system reveals that a proton-coupled redox type of reaction occurred during TMP formation. Phosphate serves both as proton donor and acceptor to facilitate proton transfer during the Schiff base formation between ammonia and 3-hydroxy-2-butanone. Protic solvents, methanol, and ethanol, were found to attract the water released from the system. The combination of a phosphate buffer and protic solvent led to the completion of TMP formation. The TMP formation mechanism in a phosphate buffer (PH 7.2) is proposed.

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RAPD (randomly amplified polymorphic DNA) analysis was developed for rapid identification of the spoilage bacterium, Alicyclo-bacillus acidoterrestris. Three primers, Ba-10, F-61, and F-64, allowed adequate discrimination between strains of A. acidoterrestris and related bacteria in RAPD analyses. As compared with the conventional method and a RAPD assay against the isolates from various environmental and food samples, the results obtained from the RAPD pattern were identical to the identification by the conventional method. We suggest that the RAPD analyis is a rapid and reliable technique to distinguish A. acidoterrestris from other related bacteria.

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We report an investigation of electrotransformation by three different topological isomers, circular supercoiled (sc DNA), circular relaxed (cr DNA), and linearized (In DNA) forms of the plasmids pUB110 (4.5 kbp) and pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214. Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of sc DNA with restriction endonuclease linearized the DNA. The transformation with the sc DNA of pUB110 resulted in the maximum efficiency of (2.6±0.6)×10⁵ transformants per μg DNA higher than that ((2.0±0.3)×10⁴ transformants per μg DNA) for the cr DNA, using the DNA concentration of 20μg/ml at an electric field strength of 7 kV/cm and a capacitance of 10μF with a single decayed pulse. The transformation efficiency (TE) for the ln DNA was zero. The variations of TE for different topological forms of DNA reflected their relative stability in the host cells. The molecular efficiency (ME, transformants per molecule) for sc DNA was nearly one order of magnitude greater for the lower molecular size of pUB110 DNA than that for the higher molecular size of pBDR331T DNA.

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Bacillus spores were effectively inactivated by the supercritical (SC) CO₂ micro-bubble method. The micro-bubble SC CO₂ treatment of B. cereus, B. subtilis, B. megaterium, B. polymyxa, and B. coagulans at 40°C and 30 MPa for 30 min produced greater reduction (about 3log cycles of reduction) than a similar treatment without a filter. The SC CO₂ treatment of B. polymyxa, B. cereus, and B. subtilis spores at 45°C, 50°C and 55°C, respectively, and 30 MPa for 60 min resulted in a 6-log cycle reduction of survival. The SC CO₂ treatment under the foregoing conditions should offer higher efficiency than that of heat treatment at 100°C for 60 min. In addition, the SC CO₂ treatment (30 MPa, 60°C, 30 min) of B. polymyxa and B. cereus spores also produced a 6-log cycle reduction.

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Caffeine was separated from supercritical carbon dioxide with a zeolite membrane that had been tested for pervaporation. The pore size of the NaY zeolite membrane was evaluated to be of subnanometer scale from the result of a Dubinin-Astakhov analysis. The rejection of caffeine was 0.98 by the zeolite membrane which would make it applicable for SCCO₂ membrane separation.

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Humulone, a bone resorption inhibitor isolated from hop extract, induced apoptosis in the premyocytic leukemia cell line HL-60 between 1 and 100μg/ml. Our data suggested that there was a correlation between the apoptosis-inducing activity of humulone and its antioxidative activity.

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The effects of various types of alginic acid consisting of L-guluronic acids (G) and D-mannuronic acids (M) on hyaluronidase and mast cell degranulation were examined. Alginic acid with an M/G ratio of 1.0 exhibited the strongest inhibition of both activities, the higher molecular weight alginic acids of 150 to 370kDa being preferable in both cases. Esterification of the carboxyl residue enhanced the latter activity.

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Circulation of a solution of 1 M D-fucose and 1 M D-glucose through a reaction system consisting of serial columns of immobilized recombinant β-D-glucosidase of Bifidobacterium breve clb and activated charcoal gave two oligosaccharides. Structural analysis identified these oligosaccharides as D-fucosylglucose (6-O-β-D-Fucopyranosyl-D-glucose) and gentiobiose (6-O-β-D-Glucopyranosyl-D-glucose). The D-fucosylglucose obtained was well assimilated by many Bifidobacteria but not by the other intestinal bacteria tested.

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The oxidative rate of three kinds of synthetic triacylglycerols (TAGs) consisting of eicosapentaenoic acid (EPA) and palmitic acid was measured by the induction period method to understand the mechanism for the autoxidation of TAGs containing highly unsaturated fatty acids (HUFAs) such as marine oils. The propagation rate, oxidizability, and kinetic chain length increased with the number of moles of EPA in a single TAG molecule, although the initiation rate was almost the same for all the TAG samples. These results demonstrate that the oxidative rate of EPA in TAGs was affected by the TAG structure and that EPA highly concentrated in a TAG molecule was very susceptible to free-radical oxidation.

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An efficient method is described for synthesizing and four possible diastereomers of stereochemically defined monodeuterated glycerols by utilizing Sharpless asymmetric dihydroxylation.

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Erythrina variegata trypsin inhibitors designated ETIa and ETIb belong to the Kunitz family trypsin inhibitor, but ETIa is unique in its ability to inhibit tissue-type plasminogen activator, while ETIb is not. The cDNA clone encoding ETIb was isolated from the seed cDNA library constructed in the lambda phage λgt11. The ETIb cDNA insert consists of 765 bp, including an open reading frame of 606 pb from ATG to TGA codons. The deduced amino acid sequence consists of 202 amino acids, having the signal peptides of 22 amino acids in the N-terminus and 2 amino acids in C-terminus. The cDNA fragment encoding the mature form of ETIb was introduced into an expression vector, pET-22b, and expressed in Escherichia coli BL21 (ED3) in a functional form. Furthermore, the ETIb mutant bP61R/F62L, in which Pro⁶¹ and Phe⁶² in ETIb were changed to the corresponding amino acid residues Arg and Leu, respectively, as in ETIa, was constructed, and its inhibitory potency toward tPA was assayed. This mutant showed significant tPA inhibitory activity, albeit less than ETIa. The result demonstrates that the Arg⁶¹ and Leu⁶² residues in ETIa are important in inhibiting tPA, and also suggests that beside these two residues, the other amino acid(s) or other structural element may be involved in interaction of ETIa with tPA.

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A mouse hybrid-hybridoma, HH1-4-3, secreting IgG₁ class bispecific antibodies to bovine lactoferrin (bLF) and horseradish peroxidase (HRPO), was established previously. The competitive enzyme-linked immunosorbent assay (ELISA) of bLF using the HH1-4-3 culture supernatant was not sensitive enough to measure bLF concentration in biological fluids. To improve the sensitivity of the competitive ELISA, we fractionated the bispecific antibodies by antigen affinity column chromatography. A column immobilized with bLF adsorbed 60%o of the antibodies of the HH1-4-3 supernatant, and the amount of antibodies adsorbed on a column immobilized with HRPO was less than 5%. The competitive ELISA of bLF using the affinity purified bispecitio antibodies through an HRPO-immobilized column chromatography showed a good standard curve at bLF concentrations of 10 ng/ml to 100 μg/ml.

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A Streptomyces plasmid, pR4C2, with an inserted DNA fragment of R4 phage, was encapsidated into R4 phage particles in vivo and transduced to Streptomyces lividans at 3×10^-6CFU/PFU. Formation of transducing phage was dependent on the inserted R4 DNA, and some of the transducing phages had larger DNA than R4 phage. A possible transduction mechanism through plasmid-phage cointegrate formation in vivo is discussed.

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Pediococcus sp. ISK-1 isolated in our laboratory from well-aged Nukadoko, produces a bacteriocin which has a unique antimicrobial spectrum anlong pediocins. The bacteriocin was stable at acidic pH, and more than 60% of the antimicrobial activity still remained even after being autoclaved at 121°C for 20min in the pH range of 3 to 8. This is the first report dealing with a bacteriocin produced by lactic acid bacteria isolated from Nukadoko.

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An in vitro degradation test of angiotensin (ANG) II or III in normotensive supine human plasma from 9 healthy male subjects confirmed the production of smaller ANG metabolites with angio-tensin I-converting enzyme inhibitory activity. These metabolites were identified as ANG (3-8), ANG (4-8), ANG (5-8), and ANG (3-4), whose respective peptide concentrations were determined by our proposed naphthalene-2, 3-dialdehyde (NDA)-HPLC method to be 64±9, 39±5, 176±22, and 197±35 fmol/ml of plasma.

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A new enzyme, D-carnitine dehydrogenase from Agrobacterium sp. 525a, was purified by DEAE-Toyopearl, ammonium sulfate fractionation, Sephadex G-75, affinity chromatography, and Mono Q and TSK-gel filtration column chromatography. The enzyme had the molecular mass of 89 kDa and consisted of three identical subunits. The optimum pH for the oxidation reaction was 9.3. The Michaelis constants for D-carnitine and NAD⁺ were 3.1 and 0.07 mM, respectively. The N-terminal 20 amino acids were sequenced.

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