首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
A Monascus pilosus strain was selected for production of intracellular alpha-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Galactose was one of the best enzyme inducers. The enzyme was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography and was demonstrated to be homogeneous by slab gel electrophoresis. The molecular weight of this enzyme, estimated by gel filtration, was about 150,000. The optimum conditions for the enzyme reaction was pH 4.5 to 5.0 at 55 degrees C. The purified enzyme was stable at 55 degrees C or below and in buffer at pH 3 to 8. The activity was inhibited by mercury, silver, and copper ions. The kinetics of this enzyme, with p-nitrophenyl-alpha-d-galactoside as substrate, was determined: K(m) was about 0.8 mM, and V(max) was 39 mumol/min per mg of protein. Enzymatic hydrolysis of melibiose, raffinose, and stachyose was analyzed by thin-layer chromatography.  相似文献   

2.
A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50 degrees C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu(2+). The Michaelis constant (K(m)) and V(max) for dimethoate were 1.25 mM and 292 micromol min(-1) mg of protein(-1), respectively.  相似文献   

3.
An endogenous inhibitor of thiol protease from adult Paragonimus miyazakii was found to occur during the gel filtration on Sephacryl S-300. The partially purified inhibitor was specific for thiol proteases such as ficin and papain. The inhibitor also suppressed tosyl-L-lysine alpha-naphthylester hydrolytic activity of Paragonimus thiol protease. The molecular weight of the inhibitor was found to be 430,000 by the gel filtration. This inhibitor was thermostable and resistant to trypsin and glycosidase digestions.  相似文献   

4.
From Bacillus sphaericus ATCC 12123 an aryl acylamidase (EC 3.5.1.13) was purified to homogeneity by ion exchange chromatography, gel filtration, and polyacrylamide gel electrophoresis. The enzyme is inducible by various phenylamides of the acylanilide, phenylcarbamate, and methoxysubstituted phenylurea type. It has a molecular weight of 75,000. Enzyme activity was inhibited by sulfhydryl reagents, several metal ions, and 3,4-dichloroaniline (a product of linuron degradation). A requirement for divalent metal ions in enzyme activity could not be demonstrated. In the presence of 6 M urea an irreversible inactivation of the enzyme occurred. The hydrolysis of L-alanine-4-nitroanilide was competitively inhibited by puromycin.  相似文献   

5.
No change was observed in the viscosity of samples of mucilage from Dicerocaryum zanguebarium when treated with 5 mM ascorbic acid. Mucilage was treated with ascorbic acid in the presence of copper and iron, and a mixture of the two ions. The effect of treatment was followed by gel filtration of products on a Sepharose 6B column and by measuring the amount of reducing sugars generated by the dinitrosalicylic acid (DNSA) method. The molecular weight of treated samples was lower than that of untreated controls. The amount of reducing groups was found to increase. When the mucilage was treated in a system containing hydrogen peroxide and copper ions, the polymer was also degraded, yielding fragments of lower molecular weight as reflected by the broadened gel filtration peak.  相似文献   

6.
Extracellular xylanase (EC 3.2.1.8) from Streptomyces sp. K37 was purified 33.53 by ultrafiltration and cation exchange chromatography followed by gel filtration chromatography. The optimum pH and temperature for purified xylanase were found to be pH 6.0 and 60 degrees C. The Km and V(max) values of the purified xylanase were 15.4 mg ml(-1) and 0.67 micromole reducing sugar min(-1) ml(-1). High performance liquid chromatography (HPLC) gel filtration of the purified xylanase eluted xylanase activity as a peak corresponding to the molecular weight of about 24.3 kDa while the molecular weight determined by SDS-PAGE was found to be 26.4 kDa. The purified xylanase of Streptomyces sp. K37 was found to be endoxylanase and non arabinose liberating enzyme and was highly glycosylated (73.97%).  相似文献   

7.
Pyruvate decarboxylase (PDC) was prepared from four-day-old pea seedlings by a procedure which involves extraction of plant material with a phosphate buffer, fractionation of the extract with ammonium sulphate, desalting by dialysis or gel filtration on Sephadex G-25 column, chromatography on DEAE cellulose and filtration on Sepharose 4B. The PDC preparation activity 10 000 U g-1 protein was about 600 fold higher than that of the sodium phosphate buffer extract. According to the enzyme behaviour during the gel filtration on different carriers the molecular mass of pea PDS was estimated at about 106. Magnesium ions and thiamine pyrophosphate were found to be coenzymes of PDC. Cofactors can be removed by 48 h dialysis followed by chromatography on DEAE cellulose. Apoenzym is activated optimally with the concentration of cofactors of 0.002 M. Magnesium ions can be replaced in their activation function by ions of Fe2+, Ni2+, Co2+, Zn2+ and Mn2+. Another ions,i.e. Ba2+, Ca2+, Cd2+, Hg2+ and Cu2+ are inactive. Assumption about the relation between the ion diameter and degree of activation is formulated.  相似文献   

8.
The existence of two active siter per molecule of L-phenylalanine:tRNA ligase from Escherichia coli K(-10) has been demonstrated by isolation of the E-aminoacyl adenylate and tel filtration and the nitrocellulose filter assay at pH 5.0 revealed the same stoichiometry for the E-tRNAPhe comples as protection against degradation by snake venom phosphodiesterase and equilibrium gel filtration at pH 7.5. Using a fluorescence titration technique, it was found that the dissociation constant for ligase-tRNAPhe complex is decreased 20-fold when the hydrogen ion concentration is changed from pH 6.0 to pH 5.0. The existence of two active sites binding the aminoacyl adenylate intermediate was demonstrated by gel filtration and retention on DEAE-cellulose filters. "Burst" experiments indicated that two sites were involved in a rapid ATP consumption at conditions of catalytic amino acid activation. Furthermore, it was observed that the activated amino acid could be transferred from both sites to cognate tRNA.  相似文献   

9.
A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.  相似文献   

10.
We searched for a new aggregation factor, and found one we named 3315-AF in the culture filtrate of Streptomyces sp. strain No. A-3315. 3315-AF was purified by active carbon treatment, ethanol precipitation, gel filtration on Sepharose 2B, ether extraction, silica gel chromatography and gel filtration on Sephadex LH-20. 3315-AF was found to be a triglyceride which consists of myristic acid, pentadecanoic acid, and palmitic acid. The aggregation activity of 3315-AF was maximum around pH 8.0 at 30°C and the activity increased by addition of metallic ions such as calcium and cobalt. Hyaluronic acid, ovalbumin, BSA, and casein inhibited the aggregation activity. 3315-AF aggregated Proteus vulgaris and HeLa cells as well as Serratia marcescens and weakly aggregated Saccharomyces cerevisiae, Candida albicans, C. neoformans, and Leukemia P388, but it was inert to human erythrocytes and Sarcoma 180.  相似文献   

11.
Water-soluble polysaccharide fractions VO1-VO4 were isolated from the squeezed berries of snowball tree (Viburnum opulus) by successive extraction with water at various temperatures and pH and with aqueous solutions of ammonium oxalate. These fractions were purified by ion-exchange chromatography on DEAE cellulose, and the homogeneity of the purified polysaccharides was determined by gel filtration on Sephacryl S-500. Acidic polysaccharides close to pectins in their sugar composition were found in all the extracts (fractions VO1-1, VO2-1, VO3-2, and VO4-2). Residues of galacturonic acid, galactose, arabinose, and (to a lesser extent) rhamnose are their main constituents. Neutral polysaccharides composed mainly of galactose and mannose residues were additionally found in fractions extracted with acidified water (pH 4.0) and with aqueous ammonium oxalate solutions. Partial acidic hydrolysis and digestion with pectinase of acidic polysaccharides indicated that their carbohydrate backbone consists of alpha-1,4-linked residues of D-galacturonic acid. NMR spectra of acidic polysaccharides (fractions VO3-2 and VO3-3) confirmed this and demonstrated that their side oligosaccharide chains are composed of beta-1,4-linked galactopyranose residues and of terminal and 2,5- and 3,5-substituted residues of alpha-arabinofuranose at a Gal: Ara ratio of 3:1. Some polysaccharides from V. opulus were found to possess an immunostimulating activity: they enhance phagocytosis, in particular, the phagocytic index and the secretion of lysosomal enzymes with peritoneal macrophages. Calcium ions were found to be necessary for the appearance of the stimulating effect of acidic polysaccharides from V. opulus.  相似文献   

12.
The tetrahydropteroylglutamate methyltransferase from green beans (Phaseolus vulgaris) has been purified 80-fold by ion exchange chromatography and gel filtration. Optimal methyl transfer is found at pH 6.5 and 39 degrees C. Even at 0 degrees C, however, a considerable catalytic rate is observed. The Michaelis-Menten constants for homocysteine and 5-methyltetrahydropteroylglutamate are 0.43mM and 2.4 mM, respectively. Magnesium ions enhance the activity. Even purified preparations appear to contain traces of magnesium ions firmly bound, since a residual activity is found without addition of magnesium salts. Though the reaction requires anaerobiosis, an excess of reducing agents is inhibitory. The molecular weight of the transferase, determined by gel filtration, is 40 000 +/- 6%.  相似文献   

13.
Angiotensin converting enzyme [EC 3.4.15.1] was shown to exist in two distinct forms in bovine seminal plasma. The higher molecular weight form of the enzyme (angiotensin convering enzyme I) was purified to homogeneity by Sephadex G-200 gel filtration, and DEAE-Sepharose, blue Sepharose, and concanavalin A-Sepharose column chromatography. Final recovery of the enzyme was 9.0. The molecular weight of the enzyme was estimated to be 8 x 10(5) by the gel filtration method. A value of 4.6 x 10(5) was obtained for the reduced and denatured enzyme by dodecylsulfate polyacrylamide gel electrophoresis. The Stokes' radius, diffusion coefficient, and intrinsic viscosity of the purified enzyme were determined to be 95 A, 2.3 x 10(-7) cm2/s, and 6.76 ml/g. The enzyme had a specific activity of 105.12 mumol/min/mg protein for hippurylhistidylleucine. The Km value for hippurylhistidylleucine was found to be 20 mM. Studies with EDTA suggest that metal ions which are tightly bound are required for its activity. The enzyme was inhibited by some heavy metal ions but did not required sulfhydryl groups for its activity. Trypsin treatment of the urea-denatured enzyme produced a catalytically active fragment with an Mr of 30,000. Chemical hydrolysis of the native enzyme did not produce any active fragment.  相似文献   

14.
Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.  相似文献   

15.
Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine brain has been purified 68,000-fold to an electrophoretically homogeneous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33,900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revealed a biphasic saturation curve being described by higher-degree rational polynomials.  相似文献   

16.
An active β-amylase was purified from germinated rice seeds by precipitation with ammonium sulfate, acid treatment, chromatographies on DEAE-cellulose and DEAE-Sephadex A-50, and gel filiations on Sephadex G-75. The purified enzyme was homogeneous in disc electrophoretic analysis.

The molecular weight was estimated to be approximately 53,000 by thin-layer gel filtration and polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 5.0 by disc electrofocusing.

The optimum pH was found to be in the pH range of 5.5 to 6.5. The Km value for soluble starch was 3 mg/ml. The enzyme was inhibited by sulfhydryl reagents or heavy metal ions.

The active β-amylase was oxidatively dimerized by treatment with 0.3 m ferricyanide in 3 m urea. The dimerized enzyme was thought to be one of inert β-amylases in ungerminated rice seeds.  相似文献   

17.
The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106kDa (gel filtration) and 55kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, AβNA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3, Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species.  相似文献   

18.
1. An acid aspartic proteinase in the regressing tadpole tail was purified about 800-fold with a 36% recovery. 2. The mol. wt of the enzyme was found to be 42,000 on gel filtration and 38,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively. 3. The purified enzyme had a maximum activity at pH 3.5 and an apparent Km of 0.084% with acid-denatured hemoglobin as substrate. 4. The enzyme activity was strongly inhibited by pepstatin. In addition, diazoacetylnorleucine methyl ester inactivated the enzyme in the presence of cupric ions. 5. The enzyme was identified as a cathepsin D (EC. 3.4.23.5)-like proteinase.  相似文献   

19.
A xylanase, which produces exclusively xylobiose from oat spelt and birch xylans, was isolated from the culture medium of Aeromonas caviae ME-1. The enzyme (xylanase V) was purified by ammonium sulfate fractionation, hydrophobic interaction, and ion-exchange and gel filtration chromatographies. The homogeneity of the final preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrofocusing. The molecular mass and isoelectric point of the xylanase were 46 kDa and 5.4, respectively. Xylanase V had a maximum activity at a pH of 6.8 and at a temperature between 30 and 37 degrees C. It was relatively stable at a pH between 5.0 and 8.6 and a temperature between 25 and 37 degrees C. When soluble birch xylan was used as the substrate, the enzyme had a K(m) and V(max) of 2 mg/ml and 182 mumol of xylose equivalent liberated . min . mg of protein, respectively. By the action of xylanase V on xylans (from oat spelt and birch), only one product corresponding to xylobiose was observed by thin-layer chromatography. The xylanase V putative product was confirmed to be xylobiose by acid and enzymatic hydrolyses. The xylanase had neither beta-xylosidase, alpha-l-arabinofuranosidase, cellulase, nor beta-1,3-xylanase activities. Xylotriose was the shortest substrate which the enzyme could attack. These findings suggest that xylanase V is a novel enzyme that cleaves a xylobiose unit from one of the ends of xylans, probably by an exomechanism.  相似文献   

20.
Oxytocinase (cystyl-aminopeptidase) [EC 3.4.11.3] was isolated from monkey placenta in a purified form by a six-step prodedure comprising extraction from monkey placenta homogenate, ammonium sulfate fractionation, repeated chromatography on hydroxylapatite, chromatography on a column of DEAE-cellulose and gel filtration on a column of Sephadex G-200. The purified enzyme showed a single band on polyacrylamide disc electrophoresis. Oxytocin was inactivated by this enzyme preparation. The enzyme hydrolyzed several aminoacyl-beta-naphthylamides. A terminal amino group was required for enzyme activity. The molecular weight of the purified enzyme was estimated to be 87,000 by gel filtration and 83,000 by sodium dodecyl sulfate gel electrophoresis. Other properties of the enzyme, the effects of metal ions and various chemical reagents on the enzyme activity, the pH optimum, and Km values for a number of aminoacyl-beta-naphthylamides were also examined.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号