共查询到20条相似文献,搜索用时 15 毫秒
1.
A low molecular weight 1,4-beta-D-glucan glucohydrolase from an extracellular culture filtrate of Thermomonospora sp. was purified to homogeneity. The molecular weight of the purified enzyme was 14.2 kDa by MALDI-TOF analysis and is in agreement with SDS-PAGE and gel filtration chromatography. The purified enzyme exhibited both endocarboxymethyl cellulase and endoxylanase activities. A kinetic method was employed to study the active site of the enzyme that hydrolyzes both carboxymethyl cellulose and xylan. The experimental data coincide well with the theoretical values calculated for the case of a single active site. Conformation and microenvironment at the active site was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde as the chemical initiator. Formation of isoindole derivative resulted in complete inactivation of the enzyme to hydrolyze both xylan and CMC as judged by fluorescence studies corroborating a single active site for the hydrolysis of xylan and CMC. 相似文献
2.
Conformation and microenvironment at the active site of 1,4-beta-D-glucan glucanohydrolase was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde. OPTA has been known to form a fluorescent isoindole derivative by cross-linking the proximal thiol and amino groups of cysteine and lysine. Modification of lysine of the enzyme by TNBS and of cysteine residue by PHMB abolished the ability of the enzyme to form an isoindole derivative with OPTA. Kinetic analysis of the TNBS and PHMB-modified enzyme suggested the presence of essential lysine and cysteine residues, respectively, at the active site of the enzyme. The substrate protection of the enzyme with carboxymethylcellulose (CMC) confirmed the involvement of lysine and cysteine residues in the active site of the enzyme. Multiple sequence alignment of peptides obtained by tryptic digestion of the enzyme showed cysteine is one of the conserved amino acids corroborating the chemical modification studies. 相似文献
3.
Satoshi Nakamura Yutaka Ishiguro Ryuichiro Nakai Kenji Wakabayashi Rikizo Aono Koki Horikoshi 《Journal of Molecular Catalysis .B, Enzymatic》1995,1(1)
Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a Km of 0.82 mg/ml and a Vmax of 280 μmol min−1 mg−1 for xylan at 50°C and pH 9.0. 相似文献
4.
M. Paterson-Beedle J. F. Kennedy F. A. D. Melo L. L. Lloyd V. Medeiros 《Carbohydrate polymers》2000,42(4)
An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated from an agro-industrial environment in the north-eastern region of Brazil. The extracellular polysaccharide produced from sugarcane molasses was hydrolysed with trifluoroacetic acid (mild and strong conditions) giving 88% of soluble material. The main monosaccharides present in the soluble fraction were glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0.1%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-methylhexitol (74.7%) and 2,3,-di-O-methylhexitol (17.7%). Enzyme hydrolysis of the polysaccharide with a cellulase confirmed the presence of (1→4)-β-
-glucopyranosyl residues. 相似文献
5.
6.
Labeo rohita 《Fish & shellfish immunology》2001,11(8):683
The immunostimulant β-1,3 glucan was fed at 0·1% in feed for 7 days to healthy and aflatoxin B1(AFB1)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial. The effects of AFB1, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied. A single intraperitoneal injection of AFB1at 1·25 mg kg−1body weight) caused a significant (P< 0·05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan. Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control. Feeding of glucan to AFB1-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A. hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB1exposed fish. Although feeding of glucan was able to increase specific immunity, al measured through haemagglutination titre against sheep red blood cells, and bacterial (E. tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen. 相似文献
7.
8.
Luiz C. Miletti Carla Marino Karina Mario Rosa M. de Lederkremer Walter Colli Maria Júlia M. Alves 《Carbohydrate research》1999,320(3-4)
An alternative and fast method for the purification of an exo-β-
-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β-
-galactofuranoside affinity chromatography system and specific elution with 10 mM
-galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM
-galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β-
-galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and Km and Vmax values of 0.311 mM and 17 μmol h−1 μg−1 respectively, when 4-nitrophenyl β-
-galactofuranoside was employed as the substrate. 相似文献
9.
Jorge Iulek Octvio Luiz Franco Mrcio Silva Christiane Trevisan Slivinski Carlos BlochJr Daniel John Rigden Maria Ftima Grossi de S 《The international journal of biochemistry & cell biology》2000,32(11-12)
Plant α-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the α-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13 756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional α-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus α-amylases was observed. The inhibitor is more effective against insect α-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional α-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised. 相似文献
10.
Yanan Cao Peilong Yang Pengjun Shi Yaru Wang Huiying Luo Kun Meng Zhifang Zhang Ningfeng Wu Bin Yao Yunliu Fan 《Enzyme and microbial technology》2007,41(6-7):835-841
A novel extracellular α-galactosidase, named Aga-F78, from Rhizopus sp. F78 ACCC 30795 was induced, purified and characterized in this study. This soybean-inducible α-galactosidase was purified to homogeneity by ammonium sulfate precipitation and fast protein liquid chromatography (FPLC), with a yield of 14.6% and a final specific activity of 74.6 U mg−1. Aga-F78 has an estimated relative molecular mass of 78 kDa from SDS-PAGE while native mass of 210 kDa and 480 kDa from non-denaturing gradient PAGE. This α-galactosidase had no N- or O-glycosylated. Amino acid sequences of three internal fragments were determined, and fragment 1, NQLVLDLTR, shared high homology with bacterial and fungal GH-36 α-galactosidases. The optimum pH and temperature on activity of Aga-F78 were 4.8 and 50 °C, respectively. The properties of pH and temperature stability, effect of ions and chemicals were also studied. Furthermore, the resistant to neutral and alkaline proteases and substrate specificity of natural substrates (melibiose, raffinose, stachyose and guar gum) were also studied to enlarged the application of Aga-F78 in more fields. Kinetic studies revealed a Km and Vmax of 2.9 mmol l−1 and 246.1 μmol (mg min)−1, respectively, using pNPG as substrate. To our knowledge, this is the first report of purification and characterization of α-galactosidase from Rhizopus with some special properties, which may aid its utilization in the food and feed industries. 相似文献
11.
Three bacterial strains isolated from oysters recovered at the Spanish Mediterranean coast have been phenotypically and genetically characterized. The results of the phylogenetic analysis based on almost complete 16S rDNA sequences clustered all three strains together with 99.9% average sequence similarity and situated them in the neighbourhood of the genera Stappia, Roseibium and Pannonibacter, Stappia aggregata being their closest neighbour with sequence similarities between 98.8% and 98.9%. DNA-DNA hybridization experiments using DNA of strains 5OM6T and S. aggregata CECT 4269T as reference DNAs confirmed the independent status at species level of the oyster isolates. Phenotypically, they can be distinguished from the closest relatives by the ionic requirements, growth temperatures and use of carbon compounds. We propose these oyster strains constitute a new species of Stappia, for which the name Stappia alba sp. nov. has been chosen, and strain 5OM6T (= CECT 5095T = CIP 108402T) as its type strain. 相似文献
12.
The extracellular β-agarase LSL-1 produced by an agar-liquefying, soil bacterium Acinetobacter sp., AG LSL-1 was purified to homogeneity by combination of ion-exchange and size exclusion chromatography with final yield of 44%. The enzyme has a specific activity of 397 U mg−1 protein and with a molecular mass of 100 kDa. The agarase was active in the pH range of 5.0–9.0, optimally at pH 6.0 and temperature between 25 °C and 55 °C and optimal at 40 °C. The enzyme retained 63% of native activity at 50 °C suggesting it is a thermostable. The activity of the agarase was completely inhibited by metal ions, Hg2+, Ag+ and Cu2+, whereas 25–40% of native activity was retained in the presence of Zn2+, Sn2+ and SDS. Neoagarobiose was the final product of hydrolysis of both agarose and neoagarohexaose by the purified agarase LSL-1. Based on the molecular mass and final products of agarose hydrolysis, the β-agarase LSL-1 may be further grouped under group III β-agarases and may be a member of GH-50 family. This is the first report on the purification and biochemical characterization of β-agarase from an agar-liquefying Acinetobacter species. 相似文献
13.
A new strain of Bacillus sp. I-3, isolated from natural soil samples, showed a high raw starch digesting activity towards potato starch. Upon optimization of various environmental and cultural conditions, the yield of α-amylase reached 642 U/mL. The kinetic characterization of partially purified enzyme exhibited the maximum activity at 70 °C, pH 7.0 and revealed a high thermostability in the presence of 10 mM CaCl2·2H2O where it could retain more than 90% residual activity at 70 °C after 3.5 h. At 80, 90 and 100 °C, the enzyme retained 80, 59 and 26% of its maximum activity after 2.5, 0.5 and 0.5 h, respectively. The enzyme preparation had a strong affinity towards raw potato starch granules and was almost completely adsorbed onto it. It also hydrolyzed raw potato starch at a concentration of 12.5% significantly in a short period of time of 12 h. 相似文献
14.
Jun Ogawa Junichi Mano Tairo Hagishita Sakayu Shimizu 《Journal of Molecular Catalysis .B, Enzymatic》2009,60(3-4):138-144
A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis. 相似文献
15.
The characterization of three bacterial strains isolated from cultured oysters and seawater at the Spanish Mediterranean coast has been performed. Strains were phenotypically and genetically characterized and the results led us to identify them as members of the genus Marinomonas. A phylogenetic analysis based on the almost complete 16S rDNA sequences clustered all three strains together (with sequence similarities around 99.8%) in the vicinity of M. communis and M. vaga sequences and distantly related to the other four species of the genus. The most closely related species was M. communis that shared 97.4-97.6% with the Mediterranean strains. DNA-DNA hybridizations were performed to clarify the taxonomic position of our isolates and the results confirmed their specific isolation, with interspecific binding ratios below 59%. We propose the bacteria to constitute a new Marinomonas species, i.e. M. aquamarina and strain 11SM4T (CECT 5080T, CIP 108405T, CCUG 49439T) as the type strain. 相似文献
16.
Toshihiko Ooi Hirokazu Sato Kenichiro Matsumoto Seiichi Taguchi 《Protein expression and purification》2009,67(2):126-131
To characterize an exo-β-1,3-glucanase (ExgP) of an isolated fungal strain with high laminarin degradation activity, identified as Penicillium sp. KH10, heterologous secretory expression of the ExgP was performed in Aspergillus oryzae. Deduced amino acid sequence of the exgP gene possibly consisted of 989 amino acids which showed high sequence similarity to those of fungal exo-β-1,3-glucanases belonging to the glycoside hydrolase (GH) family 55. Notably, the purified recombinant ExgP showed a single protein peak in the native state (by gel-permeation chromatographic analysis), but showed two protein bands in the denatured state (by SDS–polyacrylamide gel electrophoresis). These two polypeptides exhibited activity in a coexisting state even under reducing conditions, suggesting that non-covalent association of both polypeptides took place. Taken together with the nucleotide sequence information, the ExgP precursor (104 kDa) would be proteolytically processed (cleaved) to generate two protein fragments (42 and 47 kDa) and the processed products (polypeptide fragments) would be assembled each other by a non-covalent interaction. Moreover, one of the matured ExgP polypeptides was N-glycosylated by the post-translational modification. 相似文献
17.
A novel aerobic, Gram-negative, non-pigmented bacterium, GCM72(T), was isolated from the alkaline, low-saline ikaite columns in the Ikka Fjord, SW Greenland. Strain GCM72(T) is a motile, non-pigmented, amylase- and protease-producing, oxidase-positive, and catalase-negative bacterium, showing optimal growth at pH 9.2-10.0, at 15 degrees C, and at 3% (w/v) NaCl. Major fatty acids were C(12:0) 3-OH (12.2+/-0.1%), C(16:00) (18.0+/-0.1%), C(18:1)omega7c (10.7+/-0.5%), and summed feature 3 comprising C(16:1)omega7c and/or iso-C(15:0) 2-OH (36.3+/-0.7%). Phylogenetic analysis based on 16S rRNA gene sequences showed that isolate GCM72(T) was most closely related to Rheinheimera baltica and Alishewanella fetalis of the gamma-Proteobacteria with a 93% sequence similarity to both. The G+C content of DNA isolated from GCM72(T) was 49.9mol% and DNA-DNA hybridization between GCM72T and R. baltica was 9.5%. Fatty acid analysis and G+C content supports a relationship primarily to R. baltica, but several different features, such as a negative catalase-response and optimal growth at low temperature and high pH, together with the large phylogenetic distance and low DNA similarity to its closest relatives, lead us to propose a new genus, Arsukibacterium, gen. nov., with the new species Arsukibacterium ikkense sp. nov. (type strain is GCM72(T)). 相似文献
18.
Hiroyuki Nozaki Shinji Kuroda Kunihiko Watanabe Kenzo Yokozeki 《Journal of Molecular Catalysis .B, Enzymatic》2009,59(4):237-242
The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The Vmax value was 1.40 μmol min−1 mg−1, and the Km value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid. 相似文献
19.
A. Bajaj A. Pathak M.R. Mudiam S. Mayilraj N. Manickam 《Journal of applied microbiology》2010,109(6):2135-2143
Aim: To isolate bacteria capable of degrading endosulfan (ES) and the more toxic ES sulfate and to characterize their metabolites. Methods and Results: A Pseudomonas sp. strain IITR01 capable of degrading α‐ES and toxic ES sulfate was isolated using technical‐ES through enrichment culture techniques. No growth and no degradation were observed using β‐ES. Thin‐layer chromatography and gas chromatography‐mass spectrum analysis revealed the disappearance of both α‐ES and ES sulfate and the formation of hydroxylated products ES diol, ether and lactone. We show here for the first time the formation of aforementioned metabolites in contrast to ES hemisulfate yielded by an Arthrobacter sp. Metabolism of α‐ES and endosulfate was also observed using the crude cell extract of IITR01. The molecular mass of protein induced during the degradation of α‐ES and sulfate as substrate was found to be approximately 150 kDa as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Conclusion: We describe characterization of bacterium capable of degrading α‐ES and ES sulfate but not β‐ES. Genetic investigation suggests that a gene nonhomologous to the reported esd may be present in the strain IITR01. Significance and Impact of the Study: This study describes toxic ES degradation by a Pseudomonas species that may be utilized for the bioremediation of the industrial soils contaminated with ES residues. 相似文献
20.
Keiji Tsusaki Hikaru Watanabe Tomoyuki Nishimoto Takuo Yamamoto Michio Kubota Hiroto Chaen Shigeharu Fukuda 《Carbohydrate research》2009,344(16):2151-2156
The bacterial strain PP710, isolated from soil and identified as Paenibacillus species, produced a low-digestibility α-glucan containing a large amylase-resistant portion. This α-glucan was obtained in high yields from maltodextrin (dextrose equivalent 3) by using the condensed culture supernatant of the strain as the enzyme preparation. The water-soluble dietary fiber content of the low-digestibility α-glucan was 80.2%, and showed resistance to a rat intestinal enzyme preparation. The α-glucan was found to be a novel highly branched α-glucan by acid hydrolysis, NMR analysis, gel permeation chromatography, methylation analysis, and enzymatic digestion. 相似文献