Bovine trypsin was modified with glucose through the Maillard reaction at 50°C and 65% RH for various periods (1 to 8 days). Tryptic activity against both benzoyl-_L-arginine-p-nitroanilide and two protein substrates was enhanced with increases in the reaction period, and reached the maximum after a 4-day reaction. Although there were no big differences in pH dependency of trypsin activity between native and modified trypsins, the K_m of the modified trypsin decreased to about half the native one. The modified trypsin retained its original activity almost completely after incubation in a buffer solution of pH 8.0 at 37°C for 72h, while native trypsin was greatly inactivated. Furthermore, tryptic acitivity at high temperature, residueal activity after heating, and differential scanning calorimetric analysis showed that the modified trypsin was more heat-stable than native trypsin.

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Toughness and collagen content were measured for various muscle parts of the Japanese abalone, kuro-awabi (Haliotis discus), in relation to muscle structures. The dorsal surface of the foot was toughest, followed by the hard and soft part of the foot, then the upper and middle part of the adductor muscle, irrespective of being reared or wild specimens. When compared with other abalone species, kuro-awabi showed the highest toughness for all the muscle parts, followed by madaka (H. sieboldii) and megai-awabi (H. gigas), while ezo-awabi (H. discus hannai) was softest. Collagen content was parallel with muscle toughness: the higher the collagen content, the tougher the muscle. Light micrographs of kuro-awabi showed that foot and the dorsal surface of foot were dominated by connective tissues, while adductor muscle was mainly composed of myofibrils. Transmission electron micrographs demonstrated that myofibrils in the foot were surrounded by thick layers of collagen fibrils of about 1μm, confirming light microscopic observations.

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The effects of a single administration of ethanol after feeding a PCB-containing diet on tissue ascorbic acid, drug-metabolizing enzymes in the liver, and serum and liver lipids of rats were investigated. Male Donryu and Wistar rats that had been fed on a 20% casein diet with or without 0.03% PCB were given a 5g/kg of body weight ethanol solution (25%, w/v) via a stomach tube, and then killed 16h after the intubation. Intake of the PCB-containing diet accelerated the disappearance of blood ethanol. Dietary PCB and a single dose of ethanol independently affected the tissue levels of ascorbic acid. The combined effect on hepatic aniline hydroxylase activity was additive or synergistic. A single dose of ethanol did not significantly affect the hepatic aminopyrine N-demethylase activity. In Donryu rats, the effects from feeding on a PCB-containing diet and those from a single dose of ethanol on serum lipids were almost additive. In Wistar rats, the effects of ethanol alone on lipids were not necessarily apparent, but the effects of ethanol after feeding with the PCB-containing diet were strongly enhanced. Ethanol alone hardly affected the liver lipids. Most lipids that were increased by PCB alone were significantly decreased or tended to be decreased by a single dose of ethanol. The serum levels of GOT and GPT were markedly increased by a single large dose of ethanol concomitant with the PCB-containing diet in both strains of rats ; however, PCB alone or ethanol alone hardly changed the serum levels of GOT and GPT. These results indicate that the effects of a single large dose of ethanol on drug and lipid metabolism, and on the liver function were markedly modified by the intake of a PCB-containing diet.

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Methyl L-β-acetylthioisobutyrate was racemized with 1, 8-diazabicyclo-[5.4.0]- undecene-7 as a catalyst. Methyl methacrylate and thioacetic acid were identified as the intermediates of the reaction. Thioacetic acid was relatively unstable and susceptible to decomposition during the racemization process. The addition of excess methyl mechacrylate to the reaction mixture prevented a decrease of the racemate. The racemized ester was confirmed to be usable as a substrate for the enzymatic production of D-β-acetyl-thioisobutyric acid.

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To study the relationship between taste and structure of O-aminoacyl sugars, a number of O-aminoacyl sugars containing basic amino acids were prepared. Taste quality of O-aminoacyl sugars was dependent on the side chain length of basic amino acids that were introduced into sugars. O-Aminoacyl sugars had an excellent sodium ion diet effect that could reduce sodium ion intake to 10%.

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Tofu-curd is made by the flocculation of proteins in soybean milk with an addition of calcium. The proteins consist of soluble and particle fractions. The influences of calcium and pH on the protein solubility of these fractions were investigated. The protein particles precipitated at lower calcium concentrations than that of the soluble proteins. This precipitation of proteins took place at higher pH with calcium than that without calcium. Therefore, it is considered that the first step to tofu-curd is the formation of a network by the protein particles at low calcium concentration. The next step was seemed to be the binding of the soluble proteins to the network by further addition of calcium and decreasing pH.

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A 52-kDa metalloproteinase from Serratia marcescens (Serratia marcescens extracellular proteinase, EC 3.4. 24.4) was purified to homogeneity and studied for the roles of metal ions. There were 1 zinc and 7 calcium atoms per molecule of the enzyme. Although the enzyme activity was almost eliminated by treatments with EDTA and tetraethylenepentamine up to 10 mM at room temperature, the fluorescence emission properties of the enzyme were not affected appreciably by the same treatments. The guanidine hydrochloridemediated unfolding of the enzyme molecules, which was accompanied by changes in fluorescence emission properties and elimination of enzyme activity, was considerably accelerated by the presence of 4 mM EDTA. The thermal unfolding profiles varied according to the microenvironment of the enzyme molecules. The selective substitution of calcium for functional zinc atom did not change the stability of the enzyme appreciably below 55°C. The activity of the EDTA-inactivated enzyme was largely restored by the additions of Zn²⁺, Ca²⁺, Mn²⁺, Mg²⁺, and Fe²⁺ ions.

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A mutant strain of a methylotrophic yeast, Candida boidinii UV-16, which was obtained and characterized as a respiration-deficient mutant, showed an improved productivity of glycerol from methanol. Cultural conditions for glycerol production was studied. Feedings of methanol and a nitrogen source during cultivation promoted the glycerol production by strain UV-16. Biotin and thiamin exclusion from the culture medium repressed the growth but doubled the glycerol production. The presence of slight amounts of the vitamins from seed culture was necessary and stimulative for the glycerol production. Addition of antifoam stimulated the ascent of glycerol productivity. Under the best culture conditions, strain UV-16 produced 2. 18g/liters of glycerol in a methanol medium.

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In our previous work we cloned on λ Charon4A phage vector a 6. 4-kb EcoRI fragment, which had the transforming activity of inducing gene amplification with a 16-kb repeating unit on the Bacillus subtilis chromosome by competence transformation ; those transformants were selected as tunicamycin resistant (Tm^r) transformants. We found that this DNA fragment can induce another type of gene amplification with a 8. 7-kb repeating unit by transformation when we use an amyE07 mutant as a recipient cell and select both Tm^r and amyE⁺ transformants. In the latter (8.7-kb type) gene amplification, the copy number was increased up to 20 in contrast to about 10 copies in the former 16-kb type. We replaced a part of the 6.4 kb EcoRI fragment with a cat gene as a model of a foreign gene, and succeeded in the amplification of the cat gene on the chromosome by the latter type of transformation.

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The formation of transparent gels by 6% bovine serum albumin (BSA), pH 7. 5, was examined by one- and two-step heating methods. Heating of the BSA solutions at various NaCl concentrations produced transparent gels at 25-50m_M NaCl and transparent sols at 0-20m_M NaCl (one-step heating method). The transparent sol obtained by heating without NaCl was reheated after mixing with various amounts of NaCl (two-step heating method I). The result was almost identical to that obtained by the one-step heating method. However, when the first heating was done with 10m_M NaCl, transparent gels were obtained over a wide range of NaCl concentrations with a second heating (two-step heating method II). Analyses of sols obtained at various NaCl concentrations by gel permeation chromatography and transmission electron microscopy showed the presence of linear polymers in the heated BSA sol (10m_M NaCl) and gel networks formed by the linear polymers (20m_M NaCl). The mechanism of transparent gel formation in BSA may be similar to that in ovalbumin.

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The detailed action mechanism of dextrin dextranase (EC 2.4.1.2, DDase) from Acetobacter capsulatus ATCC 11894 was investigated. DDase catalyzed the transglucosylation action of the non-reducing terminal glucosyl residue of a donor substrate, and three transglucosylation modes were recognized ; (1) transfer of an α-1, 4 linked glucosyl residue forming an α-1, 6 linkage, (2) transfer of an α-1, 4 linked glucosyl residue forming an α-1, 4 linkage, (3) transfer of an α-1, 6 linked glucosyl residue forming an α-1, 6 linkage. From these results, the action of DDase was considered to transfer α-1, 4 or α-1, 6 linked glucosyl residues at non-reducing termini of oligosaccharides to glucose molecules or glucosyl residues at non-reducing termini of other oligosaccharides forming α-1, 4 or α-1, 6 linkages. As the α-1, 6 linked terminal glucosyl residues were less reactive than α-1, 4 linked terminal glucosyl residues, DDase accumulated the α-1, 6 linked glucosyl residues, which is dextran.

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The growth of Brevibacterium flavum FA1-30, a _L-lysine-producing mutant strain with an aspartokinase desensitized to feedback inhibition, was almost completely inhibited by α-ketobutyrate (αKB) at concentrations more than 4mg/ml. Aspartic acid hydroxamate (Asphx) did not inhibit the growth at a concentration up to 6mg/ml, but slightly stimulated the αKB inhibition. Mutants resistant to αKB in the presence and absence of Asphx (type-I and -II, respectively) were derived and further selceted by their lysine productivity. The best producers among the type-I and -II mutants produced 41. 9 and 29. 4g/liter of _L-lysine·HCl, 1. 9- and 1. 4-fold that by the parent, respectively. The type-II mutant was confirmed to be resistant to αKB alone, but was still sensitive to αKB in the presence of Asphx, similar to the parent. The type-I mutants were resistant to αKB in the presence and absence of Asphx. Pyruvate dehydrogenase (PD) activity or both PD and citrate synthase (CS) activities significantly decreased in the type-II or -I mutants, respectively. The apparent K_m of PD with respect to pyruvate and that of CS with respect to oxaloacetate increased 1. 7-fold higher than those of the parent, respectively.

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Cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus produced a series of oligosaccharides by the transglycosylation reaction with cyclomaltohexaose as a glycosyl donor and D-lactose as its acceptor. The content of oligosaccharide A as the main transfer product was 22. 1% of the total sugar. The content was increased to 34. 1% by hydrolysis with glucoamylase. Oligosaccharide A was isolated by column chromatography and crystallized. By chemical and enzymatic analyses, oligosaccharide A was found to be a novel non-reducing trisaccharide ; O-β-_D-galactopyranosyl-(1→4)-O-β-_D-glucopyranosyl α-_D-glucopyranoside.

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Delta 6-desaturase activity and fatty acid composition of liver microsomes were measured periodically in rats fed diets high in α-linolenic acid (perilla oil) containing either casein (CAS) or soybean protein (SOY) as a protein source. Delta 6-desaturase activity measured by using linoleic acid as a substrate (18:2n-6→18:3n-6) and the linoleic acid desaturation index as estimated by the (20 : 3 + 20 : 4)/18 : 2 ratio in microsomal phospholipids were significantly higher in rats fed the CAS diet than in those fed the SOY diet at days 4 and thereafter. The proportion of eicosapentaenoic acid (20 : 5n-3) was also significantly higher in the CAS group than in the SOY group. These results confirmed a differential impact of the dietary protein type on the metabolism of polyunsaturated fatty acids even when rats were fed fat rich in α-linolenic acid.

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The effects of palm oil, soybean oil, and perilla oil on the lipid profiles of liver and serum were compared in young (1 month old) and adult (8 months old) rats fed on hypercholesterolemic diets. In young rats, the concentration of serum cholesterol was highest in the order of palm oil (PAO), soybean oil (SBO), and perilla oil (PEO), whereas it was comparable among the groups of adult rats, resulting in an age-dependent increase in the rats fed on PEO. In young rats, but not in adult rats, the ratio of HDL-/total-cholesterol was significantly increased with an increase in the degree of unsaturation of dietary fats. Dietary PAO, when compared with SBO or PEO, prevented the accumulation of liver cholesterol in adult rats, when expressed as mg per liver. No fat-effect on liver cholesterol was seen in young rats. SBO amd PEO, when compared with PAO, markedly enhanced fecal output of bile acids in adult rats. The concentration of serum triacylglycerol showed a fat-dependent response, whereas that of liver triacylglycerol showed both an age- and fat-dependent response. Thus, PAO showed an effect similar to that of SBO on the serum cholesterol level in adult animals fed on a hypercholesterolemic diet.

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The effects of sugar acids on the kinetics and the protein conformation of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) were examined to differentiate the ligand-binding modes of the catalytic sites. D-Glucarate, L-glucarate, and mucate inhibited the carboxylase activity of RuBisCO competitively with respect to ribulose 1, 5-bisphosphate. Their inhibition constants were 2. 5 to 8. 5 m_M. The sugar acids induced such a change of the protein conformation of activated RuBisCO as is detected with a hydrophobic fluorescence probe. However, the change was not concerned with hysteresis of the enzyme. The sugar acids bound to the catalytic sites retarded the release of activator CO₂ from the sites in the absence of CO₂. These characteristics of the sugar acids were quite similar to those of 6-phosphogluconate, but not of fructose bisphosphate or 3-phosphoglycerate. It is proposed that sugar phosphates that bind to the catalytic sites of RuBisCO could be divided into two groups on the basis of the modes of the binding to the sites.

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A convenient apparatus for measuring the locomotor activity of caged rats was produced from thin metallic chest, video camera, personal computers, fluorescent lamp, infrared lamp, etc. at a cost not exceeding 200, 000 yen. This apparatus was large enough for a growing rat to move about at will, whose location and locomotion were memorized at intervals of a second with the connected personal computer. For this reason, the apparatus is more suitable for monitoring 'voluntary' or 'spontaneous' activity than a running wheel or so-called 'Animex' apparatus. The behavior of rats under nutritionally different conditions, as well as those accustomed to meal-feeding either with high-protein and protein-free diets or with diets containing 10% perilla or safflower oil, was successively measured. As a result, it was assumed that the relative locomotor activity would be affected by body mass rather than by the preference for a particular diet and that the time to search for food would not necessarily be shortened by ingesting perilla oil.

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Repeated column chromatography enabled the lactone fraction to be separated from the volatile condensate of pouchong tea. In this fraction, several unidentified lactones were found, and their structures were elucidated mainly by GC-MS and GC-FTIR. 2-Hexen-4-olide, 5-octen-4-olide, 5-octanolide, 2-nonen-4-olide, 7-decen-4-olide, and 3, 7-decadien-5-olide were finally identified by a comparison with respective standard compounds. These compounds seem to contribute to the characteristic pouchong tea aroma.

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L-Glutamic acid (Glu) hyper-producing mutants were isolated from Methylobacillus glycogenes ATCC 21276 and ATCC 21371 during the screening of amino acid auxotrophs. iA111, derived from ATCC 21276, and 1009 (Phe^-), derived from ATCC 21371, produced about 10 times as much Glu as the wild type strains. The highest producer, RV3, a phenylalanine auxotrophic revertant of 1009, produced 38.8g/liters of Glu in 84h in a 5-liter jar fermentor. iA111 and 1009 were mutagenized, and L-threonine (Thr) producing mutants were isolated among Thr and L-lysine (Lys) analog-resistant strains. The highest Thr producer derived from iA111, AL119 (AHV+Lys^R), produced 11.0g/liters of Thr in 72h in a 5-liter jar fermentor. AL119 was further mutagenized, and a Thr and Lys producer, DHL 122 (DHL^R), was isolated. DHL 122 co-produced 3.1g/liters of Lys and 5.6g/liters of Thr in 72h in a 5-liter jar fermentor.

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A gene of Lactococcus lactis ATCC19435, the product of which is homologous with the principal σ factors of Escherichia coli and Bacillus subtilis, was cloned and sequenced. The deduced amino acid sequence of the 340-residue protein and the upstream open reading frame of the cloned gene showed a homology to B. subtilis σ⁴³ factor (the rpoD product) and DNA primase (the dnaE product), respectively, suggesting that L. lactis also has the rpoD operon. Surprisingly, introduction of the cloned L. lactis rpoD gene into a rpoD temperature-sensitive mutant of E. coli caused partial complementation.

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A psychrotrophic bacterium that produces a thermolabile alanine racemase was isolated from raw milk, and identified as Pseudomonas fluorescens TM5-2. The enzyme was purified to homogeneity from the cell extract, and characterized to be compared with enzymes from mesophiles (Bacillus subtilis and Salmonella typhimurium) and a thermophile (Bacillus stearothermophilus). The enzyme has a molecular weight of about 76, 000 and consists of two subunits identical in molecular weight (38, 000). The enzyme contains two mol of pyridoxal 5'-phosphate per mol as a coenzyme. The amino acid composition was different from those of other alanine racemases in content of valine. The amino acid sequence of the amino terminal region (from ¹Met to ²⁵Gly) had 21-33% homology with those of other alanine racemases. Kinetic parameters of the enzyme were similar to those of other alanine racemases. The enzyme is extremely labile over 30°C, and shows the high catalytic activity even at 0°C; it is thermolabile and psychrotrophic.

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Two novel nematicidal cyclodepsipeptides, designated bursaphelocides A and B, were isolated from the culture filtrate of an imperfect fungus, strain D1084, belonging to Mycelia sterilia. Bursaphelocide A (1), containing 2-hydroxy-3-methylpentanoic acid, proline, isoleucine, N-methylalanine, N-methylvaline, and β-alanine in sequence, and bursaphelocide B (2), comprising 4-methylproline instead of proline in 1, are novel 2-hydroxy-3-methylpentanoic acid analogues of insecticidal destruxins.

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The amino acids of the chymotrypsin inhibitor (ECI) from the Erythrina variegata seeds have been sequenced. The sequence was solved by analysis of peptides derived from the protein by enzymatic digestions with trypsin and Staphylococcus aureus V8 proteinase, as well as by chemical cleavage with o-iodosobenzoic acid. The ECI consists of 179 amino acid residues with a pyroglutamic acid as the N-terminal residue and has a calculated molecular weight of 19, 791. Comparison of this sequence with the sequences of the two trypsin inhibitors, ETIa and ETIb, from the E. variegata seeds shows that about 60% of the residues of ECI are identical to those of ETIa and ETIb and that the reactive sites, Arg⁶³, in ETIa and ETIb change to Leu⁶⁴ in ECI.

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To clarify the relationship between the structure and inhibitory action toward β-D-galactosidase (EC 3.2.1.21) of isoflavones and isoflavone glycosides, a number of polyhydroxyisoflavones, and the α-L-rhamnosides and β-L-quinovosides of daidzein and genistein were synthesized. Among the polyhydroxyisoflavones, 2', 3', 4', 7-tetrahydroxyisoflavone showed the strongest inhibitory activity (K_i = 26 × 10^-6M). Among the glycosides, all the L-rhamnosides were strong inhibitors, of which genistein 4', 7-di-O-α-L-rhamnoside was the strongest (K_i = 4.44 × 10^-6M), while all the isoflavone β-L-quinovosides were considerably weak or possessed no inhibition.

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The amino acids of the B-chains of two abrins (designated as abrin-a and abrin-b) from the seeds of Abrus precatorius have been sequenced. The sequence of the B-chain of abrin-a was solved by analysis of peptides derived by enzymatic digestions with trypsin, lysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The sequence of the B-chain of abrin-b was analyzed by sequence analysis of tryptic peptides and comparing these sequences with those of corresponding peptides of the B-chain of abrin-a. The B-chains of abrin-a and abrin-b consist of 268 amino acid residues and share 256 identical residues. Comparison of their sequences with that of the ricin B-chain shows that 60% of the residues of both abrin B-chains are identical to those of the ricin B-chain and that two saccharide-binding sites in ricin B-chain identified by a crystallographic study are highly conserved in both abrin B-chains.

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