Interaction Involving Disulfide Bridges between Subunits of Soybean Seed Globulin and between Subunits of Soybean and Sesame Seed Globulins

Native subunit proteins of glycinin, the acidic and the basic subunits designated as AS₁₊₂, AS₂₊₃, AS₄, AS₅, and AS₆ and BS, respectively, were isolated by DEAE-Sephadex A-50 column chromatography in the presence of 6 m urea and 0.2 m 2-mercaptoethanol.

Reconstitution of intermediary subunits involving a disulfide bridge from native acidic and basic subunits was investigated. Formation of the intermediary subunit was observed in combinations between BS and each acidic subunit except AS₆. The yields of the reconstituted intermediary subunits differed from one another.

Further, formation of the intermediary complexes was observed when native acidic and basic subunits of soybean glycinin and sesame 13 S globulin, respectively (or reverse combinations), were mixed under reductively denatured condition and subjected to the reconstitution procedure. Considerring the overall evidence, we may conclude that the complexes are probably a hybrid intermediary subunit.     

Isolation and Some Physico-chemical Properties of the Acidic Subunits of Soybean 11S Globulin

Four kinds of acidic subunits and three kinds of basic subunits of 11S globulin were separated by polyacryl amide gel electrophoresis in the urea system. The four acidic subunits designated as A₁, A₂, A₃ and A₄ (R_m=0.35, 0.40, 0.46 and 0.56 respectively) were isolated by stepwise elution followed by repeating gradient elution with DEAE-Sephadex A-50 in the presence of 6 m urea at 5°C.

Subsequently, some physico-chemical properties of the subunits were determined. For example, N-terminal amino acids were determined as phenylalanine for both A₁ and A₂ and as leucine (or isoleucine) for both A₃ and A₄ by the DNP-amino acid method. The molecular weights of A₁, A₂ and A₃ were shown as 37,000 and 45,000 for A₄ by SDS-gel electrophoresis. The amino acid compositions of the acidic subunits were roughly similar to each other, but some remarkable differences were observed in the content of basic amino acids (lysine, histidine and arginine), serine and proline.     

Subunit Structure of Sesame 13S Globulin

On SDS-polyacrylamide gel electrophoresis, sesame seed 13S globulin was separated into three intermediary subunits termed IS₁ IS₂ and IS₃. Following a treatment with 0.2M 2-mercaptoethanol, the globulin was separated into three acidic subunits termed AS₁ AS₂ and AS₃, and four basic subunits termed BS₁ BS₂, BS₃ and BS₄. Two dimensional SDS-gel electrophoresis before and after treatment with 0.2 M 2-mercaptoethanol revealed that IS₁ was composed of two combinations of acidic and basic subunits, these being S₁ and BS₂, and AS₂ and BS₂. IS₂ was found to be composed of AS₃ and BS₁, and IS₃ was composed of AS₂ and BS₃, and AS₂ and BS₄. These combinations were consistent with the reactivity of each subunit to a fluorescent thiol reagent. The amino acid compositions were similar among the three acidic subunits and also among the four basic subunits. However, between the acidic and basic subunits, there were great differences in the amino acid composition, especially in the amount of glutamic acid.     

Substrate Specificity of α-Glucosidase II in Rice Seed

The substrate specificity of rice α-glucosidase II was studied. The enzyme was active especially on nigerose, phenyl-α-maltoside and maltooligosaccharides. The actions on isomaltose and phenyl-α-glucoside were weak, and on sucrose and methyl-α-glucoside, negligible. The α-glucans, such as soluble starch, amylopectin, β-limit dextrin, glycogen and amylose, were also hydrolyzed.

The ratio of the maximum velocities for hydrolyses of maltose (G₂), nigerose (N), kojibiose (K), isomaltose (I), phenyl-α-maltoside (?M) and soluble starch (SS) was estimated to be 100: 94.4: 14.2: 7.1: 89.5: 103.1 in this order, and that for hydrolyses of malto-triose (G₃), -tetraose (G₄), -pentaose (G₅), -hexaose (G₆), -heptaose (G₇), -octaose (G₈), and amyloses ( and ), 113: 113: 113: 106: 113: 100: 106: 106. The Km values for N, K, I, ?M and SS were 2.4 mm, 0.58 mm, 20 mm, 1.6 mm and 5.0 mg/ml, respectively; those for G₂, G₃, G₄, G₅, G₆, G₇, G₈, and , 2.4 mm, 2.2 mm, 2.1 mm, 1.5 mm, 1.0 mm, 1.1 mm, 0.95 mm, 1.5 mm and 1.1 mm.

Rice α-glucosidase II is considered an enzyme with a preferential activity on maltooligosaccharides.     

Limited Tryptic Degradation of Urea Denatured Glycinin

Digestibilities of native, 5 m urea-denatured and 8 m urea-denatured glycinin were studied. Urea was removed by dialysis before digestion. The tryptic digestion of the proteins are influenced by ionic strength. Under low ionic strength condition (0 m NaCl), the proteins, even native glycinin, are well degraded. On the other hand, under high ionic strength condition (0.5 m NaCl), native glycinin resists the tryptic attack and 5 m urea-denatured glycinin is best degraded. The digestibility of 8 m urea-denatured glycinin is lower than that of 5 m urea-denatured one under the condition. The gel filtration and electrophoretic properties show that the digestion intermediate like glycinin-T (the intermediate from native glycinin) is contained in the digestion products. These suggest that the urea-denatured protein contains the almost renatured component after removal of urea. A larger amount of the glycinin-T-like protein was detected at 8 m urea denaturation than at 5 m urea. Therefore, glycinin renatures more readily from 8 m urea denaturation. Probably this is the cause of the decreased digestibility at 8 m urea denaturation.     

Continuous Production of l-Alanine with NADH Regeneration by a Nanofiltration Membrane Reactor

A conjugated enzyme system, alanine dehydrogenase (AIDH) for stereospecific reduction of pyruvate to l-alanine and glucose dehydrogenase (GDH) for regeneration of NADH, were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of l-alanine from pyruvate with NADH regeneration. Since pyruvate was proved to be unstable at neutral pH, it was kept under acidic conditions and supplied to NFMBR separately from the other substrates. As 0.2 m pyruvate in HCl solution (pH 4), 10 mm NAD, 0.2 m glucose, and 0.2 m NH₄Cl in 0.5 m Tris buffer (pH 8) were continuously supplied to NFMBR with immobilized AIDH (100 U/ml) and GDH (140 U/ml) at the retention time of 80 min, the maximum conversion, reactor productivity, and NAD regeneration number were 100%, 320 g/liter/d, and 20,000, respectively. To avoid the effect of pyruvate instability, a consecutive reaction system, lactate dehydrogenase (l-LDH) and AIDH, was also used. In this system, the l-LDH provides pyruvate, the substrate for the AIDH reaction, from l-lactate regenerating NADH simultaneously, so the pyruvate could be consumed as soon as it was produced. As 0.2 m l-lactate, 10 mm NAD, 0.2 m NH₄Cl in 0.5 m Tris buffer (pH 8) were continuously supplied to NFMBR with immobilized l-LDH (100 U/ml) and AIDH (100 U/ml) at the retention time of 160 min, the maximum conversion, reactor productivity, and the NAD regeneration number were 100%, 160 g/Iiter/d, and 20,000, respectively.     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

An Acidic Polysaccharide Isolated from the Midrib of Leaves of Nicotiana tabacum

An acidic polysaccharide (APS-H) purified from the hemicellulosic fraction of the midrib of Nicotiana tabacum was composed of d-galacturonic acid, l-rhamnose, l-arabinose and d-galactose in a molar ratio of 31.8: 15.4: 9.9: 42.9. Its molecular weight was estimated to be 90,000 by gel filtration chromatography. APS-H had a pectin-like structure in which the rhamnogalacturonan backbone was composed of (1 → 2)-linked l-rhamnopyranosyl and (1 → 4)-linked d-galacturonosyl residues in a ratio of approximately 1: 2.1. It also contained (1 → 4)-linked d-galactan and (1 → 5)-linked l-arabinofuranosyl moieties as the side chains. Branch points occurred mainly at C-4 of (1 → 2)-linked l-rhamnosyl residues in the backbone and at C-6 of (1 → 4)-linked d-galactosyl residues in the side chains.     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.     

Studies on the Autolysis of Aspergillus oryzae

In was found that an intracellular ribonuclease was present as an inactive form in the fresh mycelium of Asp. oryzae. It was about 3 times activated either by 3 m urea or by the autolysis of mycelium at 30°C for 20 hr. The optimum pH of the ribonuclease activity was 8.3. It was heat sensitive (60°C, 10 min), and completely inhibited by 5 mm EDTA. It was activated by 1 mm Mg²⁺ and inhibited by Zn²⁺, Ca²⁺, Cd²⁺, Co²⁺ and Cu²⁺.     

Preparation of Zeaxanthin from Berries of Boxthorn,Lycium chinensis

Purification and properties of a new alkaline protease of rat skeletal muscle have been reported. The purification procedure of the enzyme is as follows: skeletal muscle tissue was extracted successively with Hasselbach-Schneider solution, 5 m urea solution and 2% sodium deoxycholate solution. After then, the enzyme was extracted from the residue with 1.1 m potassium iodide solution. This enzyme solution was treated with n-butanol, and dialyzed against water. The enzyme precipitated during dialysis was collected and dissolved in 1.1 m potassium iodide solution. The enzyme solution was fractionated with acetone, and chromatographed on Sephadex G-200. The final preparation showed over 20,000 times of purity.

The optimum pH range of the enzyme activity is 9.5~10.5, and the maximum reaction rate occurs at 47~57°C. The enzyme is stable below 47°C at pH 7.3. At 37°C, the enzyme is stable during 30 min at least, in the pH range of 5.5~10.0. Below pH 5.0, it is relatively labile. Hg²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, and Zn²⁺ scarcely affect the enzyme activity at the concentration of 1 mm. Ethylenediaminetetraacetate shows little effect on the activity at the concentration of 10 mm, and iodoacetamide, 2,4-dinitrophenol, p-chloromercuribenzoate show the similar effect at the concentration of 1 mm. Diisopropyl-flurophosphate inhibits the enzyme activity. From the results obtained, this enzyme is presumed to be responsible for the activity of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range.     

Changes of α-Glycerophosphate Dehydrogenase Activity in Fatty Liver of Rats by Amino Acid Imbalance

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 = 1.34 μm, 0.113mm and 0.145 mm, 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 amino-peptidase [EC 3.4.11.14].     

Optimal Conditions for the Enzymatic Production of l-Aromatic Amino Acids from the Corresponding 5-Substituted Hydantoins

The reaction conditions for the production of l-tryptophan from dl-5-indolyl- methylhydantoin by Flavobacterium sp. AJ-3940, and the cultural conditions for the formation of the enzyme involved by this bacterium were investigated. The optimal pH of this reaction was around 8.5 and the optimal temperature was between 45 to 55°C. The amount of l-tryptophan produced was remarkably increased by the addition of inosine, which formed a water insoluble adduct with l-tryptophan, to the reaction mixture because of the release of end-product inhibition by l-tryptophan. This enzyme was inducibly and intracellularly produced by Flavobacterium sp. AJ-3940 in proportion to the increase in cell growth. Cells showing high activity were obtained using a medium containing 5 g glucose, 5 g (NH₄)₂SO₄, 1 g KH₂PO₄, 3 g K₂HPO₄, 0.1 g MgSO₄ · 7H₂O, 0.01 g CaCl₂ · 2H₂O, 50 ml corn steep liquor and 3.5 g dl-5-indolylmethylhydantoin in a total volume of 1 liter (pH 7.0). Under the best conditions, 43 mg/ml of l-tryptophan was produced from 50 mg/ml of dl-5-indolylmethylhydantoin with a molar yield of 97% in the presence of cells of Flavobacterium sp. AJ-3940. In addition, other l-aromatic amino acids such as l-phenylalanine, l-tyrosine, l-DOPA and related l-amino acids were also produced from the corresponding 5-substituted hydantoins by this bacterium containing the l-tryptophan-producing enzyme induced by dl-5-indolylmethylhydantoin.     

New Nematicidal Metabolites from a Fungus,Irpex lacteus

N-Benzoyl-l-alanine amidohydrolase was purified from a cell-free extract of Corynebacterium equi H-7 which was grown in a medium containing hippuric acid as the sole carbon source. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The molecular weight was 230,000 and the enzyme consisted of six subunits, identical in molecular weight (approximately 40,000). The isoelectric point of the enzyme was pH 4.6. The optimum pH of the enzyme reaction was 8.0 and the enzyme was stable from pH 7.0 to 8.0. The enzyme hydrolyzed N-benzoyl-l-alanine, N-benzoylglycine, and N-benzoyl-l-aminobutyric acid. The Km values for these substrates were 4.3 mm, 6.7 mm, and 4.3 mm, respectively. The enzyme was activated by Co²⁺.     

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

Sequential Oxidation and Reduction of Mono Methyl Ethers of Methyl 4,6-O-Benzylidene-α- and β-d-Glucopyranosides

(1) Both glutaminases A and B of Pseudomonas aeruginosa are inactivated by urea and guanidine hydrochloride, and the activities are partially restored by removal of the denaturants, while sodium lauryl sulfate denatured irreversibly the isozymes. (2) Glutaminase A consists of 4 identical subunits (mol. wt, 35,000) and B is composed of one polypeptide chain (mol. wt., 67,000). (3) Glutaminase A, which catalyzes the hydrolysis and also the hydroxylaminolysis of L and D isomers of glutamine and asparagine, does not act on γ-N-substituted glutamine e.g., γ-glutamylhydrazide. Some l- and d-γ-glutamyl derivatives, e.g., l- and d-γ-glutamyl-hydrazide, l- and d-γ-glutamylmethylester, and l-γ-glutamyl-l-alanine are substrates for glutaminase B, which does not catalyze the hydrolysis and hydroxylaminolysis of asparagine. α-Amino adipamic acid and α-amino substituted amino acids are inert for both the isozymes. (4) The acylation step is rate-limiting in the catalytic reactions by both the isozymes.     

Cooked Odor of Euphausia pacifica Hansen

Quinoxaline and benzimidazole derivatives obtained from L-rhamnose and L-fucose under deoxygenated, weakly acidic, heated conditions were studied using GLC, HPLC, and NMR.

Four quinoxalines and one benzimidazole were obtained from L-rhamnose (RHA-I, II, III, III′, and IV) and L-fucose (FUA-I, II, III, IV, and V) in an acidic solution (MeOH-AcOH-H₂I = 8 : 1 : 2) at 80°C. The total yield of the products as sugar was about 80% from either rhamnose or fucose.

The structure of RHA-I was (2′S)-2-methyl-3-(2′-hydroxypropyl)quinoxaline; RHA-II, (2′R,3′S)-2-(2′,3′-dihydroxybutyl)quinoxaline; RHA-III, (1′S,2′S,3′S)-2-(1′2′3′-trihydroxybutyl)quinoxaline[2-(L-arabino-1′,2′,3′-trihydroxybutyl)quinoxaline]; RHA-III′, 2-(L-ribo-1′,2′,3′-trihydroxybutyl)quinoxaline; and RHA-IV, 2-(L-manno-1′,2′,3′,4′-tetrahydroxypentyl)-benzimidazole, and the structure of FUA-I was the same as RHA-I; FUA-II, (2′S, 3′S)-2-(2′, 3′-dihydroxybutyl)quinoxaline; FUA-III, (1′R, 2′R, 3′S)-2-(1′,2′,3′-trihydroxybutyl)quinoxaline [2-(L-xylo-1′,2′,3′-trihydroxybutyl)quinoxaline; FUA-IV, 2-(L-lyxo-1′,2′,3′-trihydroxybutyl)-quinoxaline; and FUA-V, 2-(L-galacto-1′,2′,3′,4′-tetrahydroxypentyl)benzimidazole. These results suggest no significant difference for the pathways of quinoxaline and benzimidazole formation between L-rhamnose and L-fucose. Possible pathways are proposed for each sugar.     

Production of Xylanase by Streptomyces sp., Using Non-metabolizable Inducer

meso-Diaminopimelate dehydrogenase (EC 1.4.1.16) was purified to homogeneity from Corynebacterium glutamicum ATCC 13032. The enzyme had a molecular weight of about 70,000 and consisted of two subunits identical in molecular weight. The enzyme was highly specific for meso-2,6-diaminopimelate. The pH optima for deamination and amination were about 9.8 and 7.9, respectively. The Michaelis constants were 3.1mm for meso-2,6-diaminopimelate, 0.12mm for NADP⁺, 0.28 mm for l-2-amino-6-ketopimelate, 36 mm for ammonia, and 0.13 mm for NADPH. d and l isomers of 2,6-diaminopimelate competitively inhibited the oxidative deamination of meso-2,6-diaminopimelate. The enzyme was distributed in a wider range of bacterial species than reported previously [Misono et al., J. Bacteriol., 137, 22 (1979)] when assayed by a sensitive formazan formation method.     

A Novel Reaction of Sugars with Anion Radical of Carbon Dioxide Produced from Formate

Radiolysis of some monosaccharides (fructose, glucose and ribose) in air-free condition was markedly enhanced by the addition of formate at concentrations above 20 mm, while it was inhibited at concentrations below 20 mm. The following compounds were detected in the irradiated sugar solutions containing excess formate (100mm): 1-Deoxy-d-arabinohexulose (1, G=4.4) and 1,3- dideoxy-d-erythrohexulose (2, G= 1.3) from fructose; 2-deoxy-d-ribose (3, G=2.3) and 2-deoxyribitol (4, G =0.6) from ribose; and 2-deoxy-d-glucose (5, G=0.5) and 2-deoxy-d-glucitol (6, G=0.4) from glucose. A mechanism for radiolytic formation of the products was proposed, based on interaction of - formed from formate with sugars.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.