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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(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
3.
Yu Gao Wataru Saburi Yodai Taguchi 《Bioscience, biotechnology, and biochemistry》2013,77(11):2097-2109
ABSTRACTMaltose 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 kcat(app)/Km(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. 相似文献
4.
Seiya Watanabe Yuki Utsumi Shigeki Sawayama Yasuo Watanabe 《Bioscience, biotechnology, and biochemistry》2016,80(11):2151-2158
d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The kcat/Km value with d-arabinose (1.27 min?1 mM?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae. 相似文献
5.
《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
6.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2520-2524
The mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of Fugu pardalis was investigated by HPLC. The values for the half inhibitory concentration of tetrodotoxin (1.6 μM) binding to this protein (1.2 μM) for saxitoxin, and of saxitoxin (0.47 μM) binding to that (0.30 μM) for tetrodotoxin were 0.35±0.057 μM and 81±16 μM (n=2), respectively. 相似文献
7.
Keisuke Kitamura Kazuyoshi Okubo Kazuo Shibasaki 《Bioscience, biotechnology, and biochemistry》2013,77(5):1083-1085
Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (ko) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (KM), and plots of l/ko against the initial concentration of a nucleophile (no) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor. 相似文献
8.
Oliver Spadiut Christian Leitner Tien-Chye Tan Roland Ludwig Christina Divne 《Biocatalysis and Biotransformation》2013,31(1-2):120-127
Site-directed mutagenesis was used to enhance the catalytic activity of pyranose 2-oxidase (P2Ox) from Trametes multicolor with different substrates. To this end, threonine at position 169 was replaced by glycine, alanine and serine, respectively. Using oxygen as electron acceptor the mutant T169G was equally active with d-glucose and d-galactose, whereas wild-type recombinant P2Ox only showed 5.2% relative activity with the latter substrate. When d-galactose was used as electron donor in saturating concentrations, T169G showed a 4.5-fold increase in its catalytic efficiency kcat/KM for the alternative electron acceptor 1,4-benzoquinone and a nine-fold increased kcat/KM value with the ferricenium ion compared with wt recP2Ox. Variant T169S showed an increase in its catalytic efficiency both with 1,4-benzoquinone (3.7 times) as well as with the ferricenium ion (1.4 times) when d-glucose was the substrate. 相似文献
9.
《Bioscience, biotechnology, and biochemistry》2013,77(2):188-193
The β-d-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only β- d-glucosidase activity but also β- d-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47,000–48,000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45°C, respectively. The enzyme was stable up to 40°C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the Km values for p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-fucoside were 1.3mm and 0.7 mm, respectively. This enzyme had also transferase activity for the β-d-fucosyl group but not for the β-d-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of β-d-glucosidase from other bacteria, actinomycetes, and plants. 相似文献
10.
Nanao Hayashi Masanori Masuoka Kazuyuki Maeshima Takashi Sakao Mandai Tabata Hisashi Komae 《Bioscience, biotechnology, and biochemistry》2013,77(6):1397-1399
A new intracellular peptidase, which we call “d-peptidase S,” was purified from Nocardia orientalis IFO 12806 (ISP 5040). The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight and the isoelectric point were estimated to be 52,000 and 4.9, respectively. The optimum pH for the hydrolysis of d-leucyl-d-leucine was 8.0 to 8.1, and the optimum temperature was 36°C. The purified enzyme usually hydrolyzed the peptide bonds preceding the hydrophobic D-amino acids of dipeptides. Tri- and tetra-peptides extending to the amino terminus of such peptides were also hydrolyzed. Therefore, the enzyme is a carboxylpeptidase-like peptidase specific to d-amino acid peptides. The Km values for d-leucyl-d-leucine and l-leucyl-d-leucine were 0.21 × 10-3 and 0.44 × 10-3 m respectively. The activity was inhibited by several sulfhydryl reagents and two chelators, 8-hydroxyquinoline and o-phenanthroline. 相似文献
11.
Andrea Angeli Fatmah A. S. Alasmary Sonia Del Prete Sameh M. Osman Zeid AlOthman William A. Donald 《Journal of enzyme inhibition and medicinal chemistry》2018,33(1):680-685
The activation of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii (TweCAδ) was investigated using a panel of natural and non-natural amino acids and amines. The most effective activator of TweCAδ was d-Tyr (KA of 51?nM), whereas several other amino acids and amines, such as L-His, L-Trp, d-Trp, dopamine and serotonin were submicromolar activators (KAs from 0.51 to 0.93?µM). The most ineffective activator of TweCAδ was 4-amino-l-Phe (18.9?µM), whereas d-His, l-/d-Phe, l-/d-DOPA, l-Tyr, histamine, some pyridyl-alkylamines, l-adrenaline and aminoethyl-piperazine/morpholine were moderately potent activators (KAs from 1.34 to 8.16?µM). For any δ-CA, there are no data on the crystal structure, homology modelling and the amino acid residues that are responsible for proton transfer to the active site are currently unknown making it challenging to provide a detailed rational for these findings. However, these data provide further evidence that this class of underexplored CA deserves more attention. 相似文献
12.
《Bioscience, biotechnology, and biochemistry》2013,77(7):1520-1526
Synthetic D- and L-amino acid type cationic 9-mer peptides (all sequences were synthesized as D- or L-amino acids) derived from the active sites of insect defensins were tested for their ability to modify the growth of blood-stream form African trypanosomes in vitro. One of them, the D-type peptide A (RLYLRIGRR-NH2), irreversibly suppressed proliferation of the Trypanosoma brucei brucei GUTat3.1 parasite. The presence of negatively charged phosphatidylserine on the surface of the parasites was demonstrated, suggesting electrostatic interaction between the peptide and the phospholipids. Furthermore, this peptide was found to alter trypanosome membrane-potentials significantly, an effect apparently due to the removal of the parasite’s plasma membrane. The potential toxic effects of D-peptide A on mammalian cells was assessed using human brain microvascular endothelial cells. Only minor effects were found when the endothelial cells were exposed for 16 h to peptide concentrations of less than 200 μM. These findings suggest that insect defensin-based peptides represent a potentially new class of membrane-disrupting trypanocidal drugs. 相似文献
13.
Keiji Yano Naoki Higashi Satoshi Nakamura Kei Arima 《Bioscience, biotechnology, and biochemistry》2013,77(9):1363-1365
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. 相似文献
14.
The electrophoretically homogeneous glucomannan isolated from konjac flour was composed of d-glucose and d-mannose residues in the approximate ratio of 1: 1.6. Controlled acid hydrolysis gave 4-O-β-d-mannopyranosyl-d-mannose, 4-O-β-d-mannopyranosyl-d-glucoseT 4-O-β-d-glucopyranosyl-d-glucose(cellobiose), 4-O-β-d-glucopyranosyl-d-mannose(epicellobiose), O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-glucopyranosyl- (1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-mannopyranosyl-(1→4)-O-β-d-glucopy- ranosyl-(1→4)-d-mannose and O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-mannose. 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.
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
17.
Ichizo Shinoda Yasuharu Nosho Ken Otagiri Hideo Okai Sakuzo Fukui 《Bioscience, biotechnology, and biochemistry》2013,77(7):1785-1890
In order to investigate the steric requirement of the C-terminal hydrophobic amino acid of the bitter peptide, Arg-Arg-Pro-Pro-Phe-Phe, for the production of bitterness, we prepared analogs of it containing d-phenylalanine. in place of l-phenylalanine. The analogs with l-phenylalanine at the C-terminal exhibited stronger bitterness than those with d-phenylalanine at the C-terminal. We confirmed that the configuration of the C-terminal hydrophobic amino acid is important for the increase in bitterness. 相似文献
18.
19.
Jie Cheng Jingchuan Zhu Bo Liu Zhouxiong Liao Zhonghong Lai 《Molecular simulation》2013,39(8):625-630
Diameter and wall thickness of self-assembled peptide nanotube of cyclo[(-d-Ala-l-Ala)4-] were characterised by molecular simulation. In order to verify the existence of peptide nanotube of cyclo[(-d-Ala-l-Ala)4-], cyclo[(-d-Ala-l-Ala)4-] was firstly synthesised through Fmoc solid-phase synthesis method and then self-assembled in trifluoroacetic acid. Based on the results of experiment, the single nanotube structure was further characterised by molecular dynamics (MD) employing the COMPASS force field. The results indicate that cyclo[(-d-Ala-l-Ala)4-] is self-assembled into nanotube bundles of about 0.5 μm in diameter and 10 μm in length; the inner and outer diameter of the single nanotube is 8.5 and 15.9 Å, respectively, and the nanotube wall thickness is 3.3 Å. 相似文献
20.
The mechanism of asymmetric production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 was examined by investigating the properties of the enzymes involved in the hydrolysis of dl-5-substituted hydantoins. The enzymatic production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 involved the following two successive reactions; the d-isomer specific hydrolysis, i.e., the ring opening of d-5-substituted hydantoins to d-form N-carbamyl amino acids by an enzyme, d-hydantoin hydrolase (d-HYD hydrolase), followed by the d-isomer specific hydrolysis, i.e., the cleavage of N-carbamyl-d-amino acids to d-amino acids by an enzyme, N-carbamyl-d-amino acid hydrolase (d-NCA hydrolase).l-5-Substituted hydantoins not hydrolyzed by d-HYD hydrolase were converted to d-form 5- substituted hydantoins through spontaneous racemization under the enzymatic reaction conditions.It was proposed that almost all of the dl-5-substituted hydantoins were stoichiometrically and directly converted to the corresponding d-amino acids through the successive reactions of d-HYD hydrolase and d-NCA hydrolase in parrallel with the spontaneous racemization of l-5-substituted hydantoins to those of dl-form. 相似文献