Reconstitution of Arachin from Isolated Subunits

Six subunits of arachin were isolated in urea solution. They were then reassociated by removing urea by co-dialysis against 20 mM sodium phosphate buffer (pH 7.9), containing 30% sucrose, 0.1 M> sodium chloride and 7 mM β-mercaptoethanol, without agitation at 25°C. The reconstitution yield was greater than 90%. The reconstituted molecule was indistinguishable from intact arachin in disc electrophoretic mobility, subunit composition, sedimentation behavior depending upon ionic strength, circular dichroism, ultraviolet absorption and fluorescence emission spectra, and stabilities against heating, proteases and guanidine hydrochloride. The reconstituted arachin was, therefore, suggested to be in native state.

On the other hand, we found that co-dialysis of four or five subunits of arachin formed hexamer which contained the corresponding four or five subunits. These hexamers were more labile than intact arachin against heating. These facts suggest that the assembly of all six subunits to a hexamer will most advantage the quaternary structure of arachin.     

Studies on the Pentose Isomerases of Lactic Acid Bacteria

Production of d-xylose and l-arabinose isomerases by lactic acid bacteria was greatly promoted by the addition of manganese ions in cultural medium. Effective concentration of the ions was 5 × 1O^-3 m. Ferrous ions were also effective for the production of d-xylose isomerase and cobaltous ions were somewhat effective for the production of l-arabinose isomerase. Zinc and cadmium ions inhibited bacterial growth. It was possible to increase the production of isomerase by changing MnSO₄ concentration to 5× 10^-3 m (0.l1 %) in place of 0.001 per cent in the normal medium.

Column chromatographic procedures for the purification of pentose isomerases were carried out. Cation and anion exchange resins were not suitable because of their low exchange capacities and instability of the enzyme at acidic pH range. But the isomerases were successfully purified by DEAE-cellulose column chromatography with high recovery (85~90%). Using a Tris buffer, KCl concentration was increased in gradient. d-Xylose isomerase was eluted at pH 7.0 at 0~0.2 m KCl, and l-arabinose isomerase at pH 8.0 at 0~0.4 m KCl. The purified isomerases, d-xylose isomerase and l-arabinose isomerase, both required manganese ions specifically for their activities.

D-Xylose isomerase and l-arabinose isomerase are different enzymes which can be separated from each other with acetone fractionation at pH 4.8~5.0, heat treatment or chromatography on a colnmn of DEAE-cellulose. In DEAE-cellulose chromatography with a linear gradient elution method, d-xylose isomerase is recovered in the first peak at pH 7.0 (Tris bnffer) with 0~0.2 m KCl, and l-arabinose isomerase is eluted in the second peak at pH 8.0 (Tris buffer) with a larger ionic strength.     

Purification and Crystallization of d-Arabinose (l-Fucose) Isomerase from Aerobacter aerogenes by Polyethylene Glycol

d-Arabinose(l-fucose) isomerase (d-arabinose ketol-isomerase, EC 5.3.1.3) was purified from the extracts of d-arabinose-grown cells of Aerobacter aerogenes, strain M-7 by the procedure of repeated fractional precipitation with polyethylene glycol 6000 and isolating the crystalline state. The crystalline enzyme was homogeneous in ultracentrifugal analysis and polyacrylamide gel electrophoresis. Sedimentation constant obtained was 15.4s and the molecular weight was estimated as being approximately 2.5 × 10⁵ by gel filtration on Sephadex G-200.

Optimum pH for isomerization of d-arabinose and of l-fucose was identical at pH 9.3, and the Michaelis constants were 51 mm for l-fucose and 160 mm for d-arabinose. Both of these activities decreased at the same rate with thermal inactivation at 45 and 50°C. All four pentitols inhibited two pentose isomerase activities competitively with same Ki values: 1.3–1.5 mm for d-arabitol, 2.2–2.7 mm for ribitol, 2.9–3.2 mm for l-arabitol, and 10–10.5 mm for xylitol. It is confirmed that the single enzyme is responsible for the isomerization of d-arabinose and l-fucose.     

Production,Purification, and Characterization of D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

The best inducers for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were a poor substrate, N-acetyl-;-methyl-D-leucine, and an inhibitor, N-acetyl-D-alloisoleucine. The enzyme has been homogeneously purified. The molecular weight of the native enzyme was estimated to be 58,000 by gel filtration. A subunit molecular weight of 52,000 was measured by SD8–PAGE, indicating that the native protein is a monomer. The isoelectric point was 5.2. The enzyme was specific to the D-isomer and hydrolyzed N-acetyl derivatives of D-leucine, D-phenylalanine, D-norleucine, D-methionine, and D-valine, and also N-formyl, N-butyryl, and N-propionyl derivatives of D-leucine. The K_m for N-acetyl-D-leucine was 9.8mM. The optimum pH and temperature were 7.0 and 50°C, respectively. The stabilities of pH and temperature were 8.1 and 40°C. D-Aminoacylases from three species of the genus Alcaligenes differ in inducer and substrate specificities, but are similar with respect to molecular weight and N-terminal amino acid sequence.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.     

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.     

Substrate Specificity of an α-Glucosidase in Sugar Beet Seed

The substrate specificity of sugar beet α-giucosidase was investigated. The enzyme showed a relatively wide specificity upon various substrates, having α-1,2-, α-1,3-, α-1,4- and α-l,6-glucosidic linkages.

The relative hydrolysis velocity for maltose (G2), nigerose (N), kojibiose (K), isomaltose (I), panose (P), phenyl-a-maltoside (?M) and soluble starch (SS) was estimated to be 100:130: 10.7: 22.6: 54.6: 55.8: 120 in this order; that for malto-triose (G3), -tetraose (G4), -pentaose (G5), -hexaose (G6), -heptaose (G7), -octaose (G8), amyloses (G13) and (G17), 91: 91: 91: 91: 80: 57: 75: 73. The Km values for N, K, I, P, and SS were 16.7 mM, 1.25 mM, 10.8 mM, 8.00 mM, 4.12 mM and 1.90 mg/ml, respectively; that for G2, G3, G4, G5, G6, G7, G8, G13 and G17 were 20.0 mM, 3.67 mM, 2.34 mM, 0,64 mM, 0.42 mM, 0.32 mM, 0.23 mM, 0.36 mM and 0.26 mM, respectively.

The enzyme, though showed higher affinity and activity toward soluble starch than toward maltose, was considered essentially to be an α-glucosidase.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

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.     

Studies on Utilization of Agar

Some chemical and physical properties of agarose (AG) and agaropectin (AP) isolated from agar of various red seaweeds were studied. The two components were isolated by acrinol. Methanol containing sodium iodide was superior to the mixed solvent of ethanol and acetone (1:1) as solvent for removing acrinol. Greater value of the ratio of intrinsic viscosity of both AG and AP in the solution of 0.1 m sodium chloride against that in the mixed solution of 4 m urea and 0.001 m sodium thiocyanate made water holding capacity greater except sample whose molecular weight is very small. Water holding capacity of AG was decreased with increasing ratio of d-galactose plus 6-O-methyl-d-galactose against 3,6-anhydro-l-galactose, and with lower liquefying temperature of gel. In the case of AP, however, these relations were not always distinct.     

Crystalline d-Mannitol:NAD Oxidoreductase from Leuconostoc mesenteroides

The crystalline d-mannitol dehyrogenase (d-mannitol:NAD oxidoreductase, EC 1.1.1.67) catalyzed the reversible reduction of d-fructose to d-mannitol. d-Sorbitol was oxidized only at the rate of 4% of the activity for d-mannitol. The enzyme was inactive for all of four pentitols and their corresponding 2-ketopentoses. The apparent optimal pH for the reduction of d-fructose or the oxidation of d-mannitol was 5.35 or 8.6, respectively. The Michaelis constants were 0.035 m for d-fructose and 0.020 m for d-mannitol. The enzyme was also found to be specific for NAD. The Michaelis constans were 1 × 10^?5 m for NADH₂ and 2.7 × 10^?4 m for NAD.     

Isolation and Identification of 6-Methylsalcylic Acid and Gentisyl Alcohol from Phyllosticta sp.

Bovine serum albumin was reduced by incubating with various concentrations (0–200 mM) of 2-mercaptoethanol, and its emulsifying properties were examined for an oil-in-water emulsion system. A particle size analysis revealed that albumin reduced at 30 mM of the thiol yielded smaller oil particles than either native protein, or the protein reduced at 70 or 200 mM of the thiol. Furthermore, the particle size was almost constant during 35 days of storage with albumin reduced at 30 mM of the thiol, while an emulsion prepared using the native protein, or the protein reduced at 70 or 200 mM of the thiol was unstable during the same storage period. Gel filtration chromatography and transmission electron micrography show that serum albumin made aggregates with high molecular size by its disulfide reduction with 70 or 200 mM, but not with 30 mM of 2-mercaptoethanol. It was, therefore, concluded that the emulsifying property of serum albumin can be improved by a mild disufide reduction.     

Comparative Analysis of Terpenoids in Roots of Ipomoea Species Induced by Inoculation of Ceratocystis fimbriata

A 7S protein in soybean globulins consisted of at least nine polypeptide chains (subunits). Complete dissociation into subunits, having a sedimentation coefficient of 1.1~1.4S and a molecular weight of 22,000~24,000, occurred in the presence of 8m urea and 4m guanidine hydrochloride. However, it was found by sedimentation, ultraviolet spectrophotometry and optical rotatory dispersion that the dissociation with various concentrations of urea accompanied simultaneously with the destruction of the internal structure of the protein. No disulfide bond appeared to participate in the binding between subunits from the results of sedimentation and disc electrophoresis. These results suggested that the subunits were very compactly and complicatedly folded on the formation of the gross structure, and hydrophobic bond with hydrogen bond also participated in the interaction between subunits.

The dissociation was interfered with the increase of ionic strength using sodium chloride, particularly in low concentration of urea. In other words, the gross structure of the 7S protein was stabilized with high ionic strength. This inclination was also recognized in alkali denaturation of the 7S protein.

Almost all tyrosine residues in the 7S protein ionized with a pK value (about 11), so they seemed to exist in the same state and to be burried in the interior of the molecule or to be participated in some combinations.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

Enzymatic Quantification of L-Tartrate in Wines and Grapes by Using the Secondary Activity of D-Malate Dehydrogenase

L-Tartrate in wines and grapes was enzymatically quantified by using the secondary activity of D-malate dehydrogenase (D-MDH). NADH formed by the D-MDH reaction was monitored spectrophotometrically. Under the optimal conditions, L-tartrate (a 1.0 mM sample solution) was fully oxidized by D-MDH in 30 min. A linear relationship was obtained between the absorbance difference and the L-tartrate concentration in the range of a 0.02-1.0 mM sample solution with a correlation coefficient of 0.9991. The relative standard deviation from ten measurements was 1.71% at the 1.0 mM sample solution level. The proposed method was compared with HPLC, and the values determined by both methods were in good agreement.     

Kinetic Studies on Xylanase Induction by β-Xyloside in Streptomyces sp.

Xylanase induction by β-xyloside was investigated in non-growing conditions using non-induced mycelia of Streptomyces sp. No. 3137 harvested from glucose medium. The mycelia started to produce xylanase without lag time when β-xyloside was added. The rate of xylanase synthesis was dependent on the concentration of β-xyloside added to the inducing culture medium. The induction constants of various β-xylosides were calculated from the Lineweaver-Burk plots; those of methyl-, isopropyl-, butyl- and ethylencyanohydrin-β-d-xylosides were 10.53 mm, 3.83 mm, 0.55mm and 0.25 mm, respectively. Some α-xylosides repressed xylanase synthesis. The rate of xylanase synthesis decreased suddenly after the addition of α-xyloside. The inhibition constants of methyl-, ethyl- and isopropyl-α-d-xylosides were 8.80 mm, 12.50 mm and 33.33 mm, respectively. The xylanase induction was also repressed by glucose. However, this repression was completely restored after consuming additional glucose.     

Protective Actions of l-Aspartate from Acid or Proteolytic Inactivation

1. l-Aspartate was found to replace l-asparagine in the protective action from acid inactivation of l-asparaginase (EC 3.5.1.1) produced by Escherichia coli A–1–3 and at the same time to inhibit the proteolytic inactivation by α-chymotrypsin.

2. l-Asparaginase changed in its chromatographic properties in the presence of l-aspartate and became to be absorbed on the CM Sephadex column.

3. The sedimentation patterns of l-asparaginase at pH 3.5 were identical either in the presence or absence of l-aspartate, showing partial dissociation. But the reversibility to the active state was observed only in the enzyme dissolved in the solution containing l-aspartate.

4. l-Aspartate did not prevent the enzyme either from the dissociation into subunits or from decrease in the activity by urea.

5. High concentration of l-aspartate was shown to inhibit the l-asparagine hydrolysis reaction.

6. l-Aspartate was suggested from ORD measurements to cause changes in the higher structure as well as the ionic properties or proteolytic inactivation.

     

Inhibition of l-Alanine Adding Enzyme by Glycine

l-Alanine adding enzymes from Bacillus subtilis and Bacillus cereus which catalyzed l-alanine incorporation into UDPMurNAc were partially purified and the properties of the enzymes were examined. The enzyme from B. subtilis was markedly stimulated by reducing agents including 2-mercaptoethanol, dithiothreitol, glutathione and cysteine. Mn²⁺ and Mg²⁺ activated l-alanine adding activity and their optimal concentrations were 2 to 5 mm and 10 mm, respectively. The optimum pH was 9.5 and the Km for l-alanine was 1.8×10^?4m. l-Alanine adding reaction was strongly inhibited by p-chloromercuribenzoate and N-ethyl-maleimide. Among glycine, l- and d-amino acids and glycine derivatives, glycine was the most effective inhibitor of the l-alanine adding reaction. The enzyme from B. cereus was more resistant to glycine than that from B. subtilis. Glycine was incorporated into UDPMurNAc in place of l-alanine, and the Ki for glycine was 4.2×l0^?3m with the enzyme from B. subtilis. From these data, the growth inhibition of bacteria by glycine is discussed.     

Pretreatment of Cells of Klebsiella pneumoniae with 50% (v/v) Dimethylsulfoxide Yields Purified Deoxyribonucleic Acid of Low Polysaccharide Content

The flow behavior and dynamic viscoelasticity of welan gum solutions were measured with a rheogoniometer. The welan gum showed shear-thinning behavior at a concentration of 0.1%, but plastic behavior above 0.3% at 25°C. The dynamic viscoelasticity increased with increasing concentration, and was scarcely changeable with increasing temperature even at 80°C. Gelation did not occur even in a polysaccharide concentration of 1.0% at low temperature (0°C). An increase of the dynamic modulus was not observed on the addition of CaCl₂ (6.8 mm). The dynamic viscoelasticity of welan gum solution was scarcely changeable in a wide range of pH from 2 to 12. The dynamic modulus was also scarcely changeable on addition of urea (4.0 m). Possible mode of intramolecular associations between the OH-4 of the d-glucosyl residue and the adjacent hemiacetal oxygen atom of the l-rhamnosyl residue, and between the methyl group of the l-rhamnosyl residue and the adjacent hemiacetal oxygen atom of the d-glucosyl residue were proposed.