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1.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2720-2726
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. 相似文献
2.
《Bioscience, biotechnology, and biochemistry》2013,77(6):1299-1302
We identified two compounds that demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity from cultures of Lactobacillus plantarum. Spectroscopic analyses proved these compounds to be L-3-(4-hydroxyphenyl) lactic acid (HPLA) and L-indole-3-lactic acid (ILA). The respective EC50 values for HPLA and ILA were 36.6 ± 4.3 mM and 13.4 ± 1.0 mM. 相似文献
3.
《Bioscience, biotechnology, and biochemistry》2013,77(11):1764-1767
An NADP-specific glutamate dehydrogenase [L-glutamate: NADP+ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (Km 44 μM), 2-oxoglutarate (Km 3.13 mM), NADP+ (Km 29 μM), and L-glutamate (Km 6.06 mM). The Km for NH4Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C. 相似文献
4.
《Bioscience, biotechnology, and biochemistry》2013,77(4):612-615
An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn2+ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids. 相似文献
5.
《Bioscience, biotechnology, and biochemistry》2013,77(2):400-403
We produced a monoclonal antibody (mAb) against N G,N G-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM–100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively. 相似文献
6.
《Bioscience, biotechnology, and biochemistry》2013,77(12):2578-2586
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. 相似文献
7.
《Bioscience, biotechnology, and biochemistry》2013,77(7):1145-1148
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 (Km=12.5 mM), N-acetyl (Km=2.52 mM), N-propionyl (Km=0.194 mM), N-butyryl (Km=0.033 mM), and N-glycyl (Km =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. 相似文献
8.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2142-2147
In the course of screening for antioxidative carotenoids from bacteria, we isolated and identified a novel carotenoid, OH-chlorobactene glucoside hexadecanoate (4), and rare carotenoids, OH-chlorobactene glucoside (1), OH-γ-carotene glucoside (2) and OH-4-keto-γ-carotene glucoside hexadecanoate (3) from Rhodococcus sp. CIP. The singlet oxygen (1O2) quenching model of these carotenoids showed potent antioxidative activities IC50 14.6 μM for OH-chlorobactene glucoside hexadecanoate (4), 6.5 μM for OH-chlorobactene glucoside (1), 9.9 μM for OH-γ-carotene glucoside (2) and 7.3 μM for OH-4-keto-γ-carotene glucoside hexadecanoate (3). 相似文献
9.
Seven optical active 2-benzylamino alcohols were synthesized by reduction of N-benzoyl derivatives of L-alanine, L-valine, L-leucine, L-phenylalanine, L-aspartic acid, L-glutamic acid and L-lysine and applied for the resolution of (±)-trans-chrysanthemic acid. d-trans-Chrys-anthemic acid was obtained by resolution via the salts of 2-benzylamino alcohols derived from L-valine and L-leucine, while (?)-trans-chrysanthemic acid was prepared through the salts of the amino alcohols derived from L-alanine and L-phenylalanine. 相似文献
10.
《Bioscience, biotechnology, and biochemistry》2013,77(12):2145-2151
Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (Mr, 1–2 × 105) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (Mr, 3.5–4.5 × 105) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages. 相似文献
11.
12.
《Bioscience, biotechnology, and biochemistry》2013,77(5):1030-1034
Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a K m of 165 μM and a V max of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a K d of 0.52 μM by BiP, lower than that for RCMLA (K d=1.1 μM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (K d=21 μM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK. 相似文献
13.
Seiya Chiba Tokuji Shimomura Keiko Hatakeyama 《Bioscience, biotechnology, and biochemistry》2013,77(3):591-596
The transglucosylation reaction of buckwheat α-glucosidase was examined under the coexistence of 2-deoxy-d-glucose and maltose. As the transglucosylation products, two kinds of new disaccharide were chromatographically isolated in a crystalline form (hemihydrate). It was confirmed that these disaccharides were 3-O-α-d-glucopyranosyl-2-deoxy-d-glucose ([α]d + 132°, mp 130 ~ 132°C, mp of ±-heptaacetate 151 ~ 152°C) and 4-O-±-d-glucopyranosyl-2-deoxy-d-glucose ([±]d + 136°, mp 168 ~ 170°C), respectively. The principal product formed in the enzyme reaction was 3-O-±-d-glucopyranosyl-2-deoxy-d-glucose. 相似文献
14.
Hirokazu Matsui Seiya Chiba Tokuji Shimomura 《Bioscience, biotechnology, and biochemistry》2013,77(10):1855-1860
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. 相似文献
15.
《Bioscience, biotechnology, and biochemistry》2013,77(12):1959-1961
Single cells were prepared from mesocarp tissue of ripe persimmon (Diospyros kaki cv. Fuyu) fruits, and inter- or intracellular localization of acid invertase (AI, EC 3.2.1.26) was studied. AI was localized in the intercellular fraction (cell wall fraction). AI was isolated and purified from the cell wall fraction of ripe persimmon fruits by column chromatography on SE-53 cellulose and Toyopearl HW 55F. The specific activity of purified AI was 570 units per mg protein at 30°C. The molecular mass of AI was estimated to be 44 kDa by gel filtration over Sephacryl S-200 and 70 kDa by SDS–PAGE. The optimum pH of the activity for sucrose was 4.25. The purified enzyme hydrolyzed sucrose and raffinose but not melibiose. The enzyme had a Km of 3.2 mM for sucrose and a Km of 2.6 mM for raffinose. Silver nitrate (5 μM), HgCI2 (2 μM), p-chloromercuribenzoate (100mM), pyridoxamine (10mM), and pyridoxine (2.5mM) inhibited AI activity by 95, 85, 100, 41, and 300%, respectively. 相似文献
16.
Florentinus Suhadi Shigeru Kitayama Yoshishige Okazawa Akira Matsuyama 《Bioscience, biotechnology, and biochemistry》2013,77(10):1644-1647
Sulfated polysaccharides (SP) isolated from freshwater green algae, Spirogyra neglecta (Hassall) Kützing, and fractionated SPs were examined to investigate their molecular characteristics and immunomodulatory activity. The crude and fractionated SPs (F1, F2, and F3) consisted mostly of carbohydrates (68.5–85.3%), uronic acids (3.2–4.9%), and sulfates (2.2–12.2%) with various amounts of proteins (2.6–17.1%). d-galactose (23.5–27.3%), d-glucose (11.5–24.8%), l-fucose (19.0–26.7%), and l-rhamnose (16.4–18.3%) were the major monosaccharide units of these SPs with different levels of l-arabinose (3.0–9.4%), d-xylose (4.6–9.8%), and d-mannose (0.4–2.3%). The SPs contained two sub-fractions with molecular weights (Mw) ranging from 164 × 103 to 1460 × 103 g/mol. The crude and fractionated SPs strongly stimulated murine macrophages, producing considerable amounts of nitric oxide and various cytokines via up-regulation of their mRNA expression by activation of nuclear factor-kappa B and mitogen-activated protein kinases pathways. The main backbone of the most immunoenhancing SP was (1→3)-l-Fucopyranoside, (1→4,6)-d-Glucopyranoside, and (1→4)-d-Galactopyranoside. 相似文献
17.
《Bioscience, biotechnology, and biochemistry》2013,77(4):920-923
A simple and sensitive specrophotometric method combined with solid-phase extraction (SPE) for the simultaneous determination of sodium linear-dodecylbenzenesulfonate (DBS) and sodium dodecyl sulfate (SDS) is described. The C2 (ethyl group bonded silicagel) cartridge could be repeatedly used more than 500 times for SPE, and it enabled the anionic surfactants to be concentrated by 50-fold. The calibration graph for DBS was linear in the range from 1.6×10?8 M to 5.0×10?7 M and for SDS from 2.0×10?9 M to 3.0×10?7 M. The relative standard deviation (n=5) for 5.0×10?7 M DBS was 3.1% and for 2.5×10?7 M SDS was 1.7%. The proposed method was applied to the simultaneous determination of DBS and SDS in river-water samples. 相似文献
18.
Sonoe Ochiai Yanagi Masako Kito Maria A. M. Galeazzi 《Bioscience, biotechnology, and biochemistry》2013,77(10):2561-2563
The acylated, amidated and esterified derivatives of N-acetylglucosaminyl-α(1 → 4)-N-acetylmuramyl tri- and tetrapeptide were synthesized and examined as to their protective effect on pseudomonal infection in the mouse and pyrogenicity in the rabbit. Modifications of the terminal end function of the peptide moieties in their molecules caused enhancement of resistance to pseudomonal infection and reduction of pyrogenicity. Among the compounds tested, sodium N-acetylglucosaminyl-β(1 → 4)-N-acetylmuramyl-l-alanyl-d-isoglutaminyl-(l)-stearoyl-(d)-meso-2,6-diaminopimelic acid-(d)-amide and sodium N-acetylglucosaminyl-β(1 → 4)-N-acetylmuramyl-l-alanyl-d-isoglutaminyl-(l)-stearoyl-(d)-meso-2,6-diaminopimelic acid-(d)-amide-(l)-d-alanine were found to be advantageous and conceivably worthwhile for further investigation as immunobiologically active compounds. 相似文献
19.
β-N-Acetyl-D-hexosaminidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purifted by making an acetone-dried preparation of the mid-gut gland, extracting with 50 mM citrate-phosphate buffer (pH 4.0) (about 13% of the extracted proteins was β-N-acetyl-D-hexosaminidase), ammonium sulfate fractionation, and column chromatographies on CM-Sepharose and DEAE-Sepharose. The purifted β-N-acetyl-D-hexosaminidase was homogeneous on SDS–PAGE, and sufficiently free from other exo-type glycosidases. The molecular weight was 56,000 by SDS–PAGE. The enzyme hydrolyzed both p-nitrophenyl β-N-acetyl-D-glucosaminide and p-nitrophenyl β-N-acetyl-D-galactosaminide. For p-nitrophenyl β-N-acetyl-D-glucosaminide, the pH optimum was 3.7, the optimum temperature was 45°C, and the Km was 0.24 mM. For p-nitrophenyl β-N-acetyl-D-galactosaminide, these were pH 3.4, 45°C, and 0.15 mM, respectively. The enzyme liberated non-reducing terminal β-Iinked N-acetyl-D-glucosamine or N-acetyl-D-galactosamine from various 2-aminopyridyl derivatives of oligosaccharides of N-glycan or glycolipid type except of GM2-tetrasaccharide. As the enzyme was stable around pH 3.5–5.5, it may be useful for long time reactions around the optimum pH. 相似文献
20.
《Bioscience, biotechnology, and biochemistry》2013,77(3):560-565
Partial acid hydrolysis of Saccharomyces cerevisiae mannan gave 2-O-α-d-Manp-d-Man (1), 3-O-α-d-Manp-d-Man (2), 6-O-α-d-Manp-d-Man (3), O-α-d Manp-(1→2)O-α-d-Manp-(1→2)-d-Man (4), O-α-d-Manp-(1→2)-O-α-d-Manp-(1→6)-d-Man (5), O-α-d Manp-(1→6)-6-O-α-d-Manp-(1→6)-d-Man (6), O-α-d Manp-(1→2)-O-α-d-Manp-(1→2)-6-O-α-d-Manp-(1→6)-d-Man (7), O-α-d-Manp-(1→2)-O-α-d-Manp-(1→6)-O-α-d-Manp-(1→6)-d-Man (8), and O-α-d-Manp-(1→6)-O-[α-d-Manp-(1→2)]-O-α-d-Manp-(1→6)-d-Man (9). 相似文献