Mixed Function Oxidase Inhibitors Protect Plants from Ozone Injury

κ-Casein and α_s1-κ-casein complex with a weight ratio of unity were dissolved in 50mm cacodylate-HCl-70 mm KC1 buffer containing 0.02% of sodium azide (pH 7.1), and their size and shape in the absence and/or presence of calcium ions were observed with the electron microscope. In the absence of calcium ions, both κ-casein and α_s1-κ-casein complex were spherical particles. However, the mean length of α_s1-κ-casein complex (12 nm) was smaller than that of κ-casein (17 nm), which suggested that complex formation led to dissociation of the κ-casein polymer. The addition of calcium ions to the complex led to the formation of bent chains, though micelle-like aggregates were not observed even at 20 nm calcium. Comparison of the frequency distributions of α_s1-κ-casein complex at 0, 5, 10, 15 and 20 mm of calcium with the calculated probability distributions suggested that most α_s1-κ-casein complexes had two binding sites above 10 mm of calcium, which seemed to be essential for the stability of casein micelle.     

Studies on κ-Casein of Bovine Milk

κ-Caseins were prepared by the calciurn-ethanol method, the Sephadex method and the urea-sulfuric acid method. Some important properties of κ-caseins were investigated using isoelectric focusing, starch gel electrophoresis, ultracentrifugation, chemical analysis, stabilization test of α_s-casein, and rennin treatment. Isoelectric focusing established that κ-casein had its isoelectric point near pH 6.0 in 6 m urea, usually accompanied by a second peak around pH 5.6. Ultracentrifugation, however, showed a single peak having a s_20,w value of 2.6 ~ 3.8 in the presence of 6 m urea and of 14.4 in the absence of such dispersing reagents. Normal contents of hexose, sialic acid, phosphorus, and nitrogen were about 1.5, 0.8, 0.2, and 14%, respectively. Relative patterns of amino acid composition were similar in all of the κ-caseins. In addition, amino acid composition in intact κ-casein and in the further purified κ-casein which formed the second peak in DEAE cellulose chromatography were almost identical, indicating that the κ-casein of the first peak is not an impurity but is one of the components which formed the original κ-casein complexes. The ability of κ-caseins to stabilize α_s-casein in the presence of calcium increased when purified by DEAE cellulose chromatography.     

Studies on the Interaction between αs1- and β-Caseins

The interaction of α_s1-casein with β-, dephosphorylated β-,γ- and R-caseins was studied. It was proved by the sedimentation velocity experiments that α_s1-casein formed a complex with each of these components at 25±C in the presence of 3 mm CaCl₂.

In the presence of 10 mm CaCl₂, β- and dephosphorylated β-casein prevented the precipitation of α_s1-casein and gave micelle-like turbid solutions. However, γ- and R-caseins, fragments of β-casein, did not stabilize α_s1-casein. It was concluded from these results that α-casein interacted with α_s1-casein through its hydropholic region corresponding to R-casein and that hydrophilic region of β-casein was responsible for the stabilization of α_s1-casein.     

Role of Tyrosine Residues in Interactions between Casein Components

It was indicated from ultraviolet difference spectra and ultracentrifugal experiments that associations occurred between two casein components (α_s- and κ-caseins, β- and κ-caseins and α_s- and β-caseins) at lower CaCl₂ concentrations (2~3 mm) and that aromatic amino acid residues participated in the associations. Chemical modification studies with 2-hydroxy-5-nitrobenzylbromide indicated that tryptophane residues of each casein component were not essential for these associations. It was also demonstrated by nitration of tyrosine residues with tetranitromethane that tyrosine residues of κ-casein were essential for α_s·κ-association and for β·κ-association and that tyrosine residues of α_s-casein were important to α_s·β-association.

Interactions between casein components were also studied at higher CaCl₂ concentration (10 mm) which is enough for micelle formation. It was found that tyrosine residues of κ- casein played an important role for the stabilization of α_s- and β-caseins. Properties of the nitrated-β-casein were almost the same as that of the native β-casein except the absorption spectrum. α_s·β-Interaction in the presence of 10 mm CaCl₂ was investigated by use of the nitrated-β-casein instead of the native β-casein. It was proved that α_s-casein was stabilized by the nitrated-β-casein and that precipitation of the nitrated-β-casein increased in the presence of α_s-casein.

The mechanism of interactions between casein components at higher CaCl₂ concentration (10 mm) are discussed in connection with the associations at lower CaCl₂ concentrations (2~3 mm).     

Flavonoids in Poncirus Hybrids

α_sl-Casein can be made either soluble or insoluble by adjusting the concentration of coexisting calcium ions. In this study, we tried to make a soluble-insoluble interconvertible enzyme through the formation of a conjugate of an enzyme and α_sl-casein using a heterobifunctional crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The conjugate of phosphoglyceromutase and native α_s1-casein did not exhibit sufficient calcium-dependent precipitation. However, conjugates of enzymes (phosphoglyceromutase, enolase or peroxidase) and α_sl-casein polymerized by transglutaminase precipitated almost completely in the presence of more than 50 mM CaCl₂. Most of the enzyme conjugates precipitated as calcium caseinates could be solubilized reversibly with EDTA, without a significant loss of activity. A mixture of the enzyme ? polymerized α_s1-casein conjugates prepared with phosphoglyceromutase, enolase and pyruvate kinase could catalyze sequential reactions which convert d-3-phosphoglycerate into pyruvate with the same efficiency as a mixture of free enzymes. These results indicate that conjugates of enzymes and polymerized α_s1-casein can be useful as soluble-insoluble interconvertible enzymes.     

Antigenic Reactivity of Dephosphorylated αs1-Casein,Phosphopeptide from β-Casein and O-Phospho-l-serine towards the Antibody to Native αs1 -Casein

To study whether the phosphoserine residue is associated with the antigenicity of bovine α_s1- casein, we examined the antigenic reactivity of dephosphorylated α_s1-casein, peptide 1~25 from bovine β-casein and three chemical reagents with IgG antibody specific to native α_s1-casein by an enzyme-linked immunosorbent assay.

The reaction between native α_s1-casein and its IgG antibody was inhibited more strongly by native α_s1-casein than by dephosphorylated α_s1-casein. Peptide 1~25, having a phosphoserine residue-concentrated region from bovine β-casein, noticeably inhibited the reaction between native α_s1 -casein and its antibody. Furthermore, the O-phospho-l-serine residue inhibited the reaction of peptide 61~123 with anti-native α_s1-casein antibody, although l-serine and sodium phosphate showed no measurable inhibition.

These results suggest that the phosphoserine residue associated with part of an antigenic site in bovine α_sl-casein.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

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.     

Effects of Calcium Ion on the Catalytic Activity of α-Chymotrypsin in Organic Solvents

Addition of small amounts of calcium ion markedly accelerated the transesterification of N-acetyl-l-tyrosine methyl ester to its ethyl ester by the catalysis of α-chymotrypsin in organic solvents. Maximum increase of the reaction rate was about 12-fold in the presence of 25 μm of calcium ion in ethanol. The rate increase was strongly dependent on calcium ion concentration and nature of organic solvents. Esterification of N-acetyl-l-tyrosine and hydrolysis of N-acetyl-l-tyrosine ethyl ester by α-chymotrypsin in organic solvents were also accelerated by calcium ion. The reactions obeyed Michaelis–Menten kinetics, and the acceleration of the reactions was due to the increase in k_cat.     

Gene Cloning of α-Methylserine Aldolase from Variovorax paradoxus and Purification and Characterization of the Recombinant Enzyme

The α-methylserine aldolase gene from Variovorax paradoxus strains AJ110406, NBRC15149, and NBRC15150 was cloned and expressed in Escherichia coli. Formaldehyde release activity from α-methyl-L-serine was detected in the cell-free extract of E.coli expressing the gene from three strains. The recombinant enzyme from V. paradoxus NBRC15150 was purified. The V _max and K _m of the enzyme for the formaldehyde release reaction from α-methyl-L-serine were 1.89 μmol min^?1 mg^?1 and 1.2 mM respectively. The enzyme was also capable of catalyzing the synthesis of α-methyl-L-serine and α-ethyl-L-serine from L-alanine and L-2-aminobutyric acid respectively, accompanied by hydroxymethyl transfer from formaldehyde. The purified enzyme also catalyzed alanine racemization. It contained 1 mole of pyridoxal 5′-phosphate per mol of the enzyme subunit, and exhibited a specific spectral peak at 429 nm. With L-alanine and L-2-aminobutyric acid as substrates, the specific peak, assumed to be a result of the formation of a quinonoid intermediate, increased at 498 nm and 500 nm respectively.     

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]_D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]_D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]_D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.     

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.     

Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme

ABSTRACT

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other α-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45°C, respectively. MalE was stable at a pH range of 4.5–10.4 and at ≤40°C. The phosphorolysis of maltose by MalE obeyed the sequential Bi–Bi mechanism. In reverse phosphorolysis, MalE utilized d-glucose, 1,5-anhydro-d-glucitol, methyl α-d-glucoside, 2-deoxy-d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, kojibiose, 3-deoxy-d-glucose, d-allose, 6-deoxy-d-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The k_cat(app)/K_m(app) value for d-glucosamine and 6-deoxy-d-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23–12% of that for d-glucose. MalE synthesized α-(1→3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized α-(1→4)-glucosides in the reaction with other tested acceptors.     

Formations of Oligopeptides from Dipeptides by the Protease from Streptomyces cellulosae

The protease from Streptomyces cellulosae formed more turbidity in a 16% soybean protein hydrolysate in the initial stage of the reaction than α-chymotrypsin did, when the proteolytic activity of the protease was same as that of α-chymotrypsin. In highly concentrated solutions (2.5%) of various dipeptides, oligopeptides were produced by condensation by the protease. The oligopeptides formed were (l-Leu-Gly)₂ and (l-Leu-Gly)₃ from l-Leu-Gly, (l-Phe-l-Val)₂ from l-Phe-l-Val, (l-Val-l-Phe)₂ and (l-Val-l-Phe)₃ from l-Val-l-Phe, and (l-Leu-l-Met)₂ and (l-Leu-l-Met)₃ from l-Leu-l-Met.     

Studies on the the Isoleucine Fermentation

Accumulation of L-isoleucine and L-valine was studied on 14 genera, 47 species and 110 strains of aerobic bacteria using bacterial type cultures. A large amount of L-isoleucine and a small amount of L-valine accumulated when 1% of DL-α-aminobutyric acid was added to the culture medium. As a rule, facultative aerobes such as Aerobacter, Erwinia, Serratia and Bacillus showed good accumulation. In the absence of α-aminobutyric acid, powerful L-isoleucine accumulators produced a large amount of L-valine, although the accumulation of L- isoleucine was scarcely observed under that condition. In the presence of α-aminobutyric acid, the accumulation of L-valine was generally suppressed, but in several strains, on the contrary, the accumulation increased as well as that of L-isoleucine. When DL-threonine was used instead of α-aminobutyric acid, the amount of L-isoleucine accumulated was not as high as that with α-aminobutyric acid in almost all strains except Serratia marcescens. It was concluded that a distinct relationship between bacterial genera or species and accumulation of L-isoleucine did not exist, that is, powerful accumulators were limited to special strains, and that the addition of α-aminobutyric acid was necessary for the accumulation of a large amount of L-isoleucine.     

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.     

New Pepsin Inhibitors (S-PI) from Streptomyces EF-44-201

Studies have been done on the inhibition and inactivation of the β-glucosidase and β-fucosidase enzyme from Thai Rosewood (Dalbergia cochinchinesis Pierre). The enzyme was inhibited by Tris with similar K_i of 11.7 mm and 14.3 mm for the hydrolysis of p/nitrophenyl β-d-glucoside (PNPG) and p/nitrophenyl β-d-fucoside (PNPF), respectively. Conduritol B epoxide inhibited both β-glucosidase and β/fucosidase activities to similar extents, with a pseudo-first-order rate constant (K_obs) of inactivation of 5.56 × 10^?3 s ^?1, and binding stoichiometry of 0.9 mol per subunit. Partially inactivated enzyme showed similar kinetics with PNPG and PNPF as substrates. Moreover, Tris at 300 mm protected both β-glucosidase and β-fucosidase activities from inactivation by 6mm CBE. The data support the idea that the Dalbergia cochinchinensis Pierre enzyme has a common active site for the hydrolysis of PNPG and PNPF.     

Purification and Properties of α-d-Galactosidase Produced by Corticium rolfsii

An α-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SE-Sephadex C-50 chromatography. The purified enzyme was demonstrated to be free of other possibly interfering glycosidases and glycanases. The maximum activity of the enzyme towards p-nitrophenyl α-d-galactopyrano-side was found to be at pH 2.5 to 4.5, and the enzyme was fairly active at pH 1.1 to 2.0. The enzyme was stable over a pH range 4.0 to 7.0 at 5°C for 72 hr and relatively unstable at pH 1.1 to 2.0 as compared with endo-polygalacturonase, α-l-arabinofuranosidase and β-d-galactosidase produced by C. rolfsii. The enzymic activity was completely inhibited by Hg²⁺ and Ag⁺ ions, respectively. Km values were determined to be 0.16 × 10^?3 m for p-nitrophenyl α-d-galactopyranoside and 0.26 × 10^?3m for o-nitrophenyl α-d-galactopyranoside. The values of V_max were also determined to be 26.6 μmoles and 28.6 μmoles per min per mg for p- and o-nitrophenyl α-d-galactopyranoside, respectively.     

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.     

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.