Characterization of an exo-beta-D-fructosidase from Streptococcus mutans Ingbritt.

An extracellular enzyme beta-D-fructosidase was purified from the culture supernatant of Streptococcus mutans Ingbritt and characterized. The molecular weight of the enzyme was 127,000 as determined by SDS-polyacrylamide gel electrophoresis. The enzyme was specific for levan which mainly consists of beta-(2,6)-linked D-fructose and was also able to hydrolyze inulin, sucrose and raffinose at the activities of 13, 9 and 5% of that hydrolyzing levan, respectively. The pH optima for levan, inulin and sucrose were approximately 5.5, 6.0 and 5.0, respectively. The enzyme was optimally reactive at 55 C for levan. The enzyme was inhibited by Fe3+, Hg2+ and Zn2+ and not by either anionic or non-ionic detergents. Paper chromatographic analysis revealed that the enzyme attacked levan by an exo-type mechanism.     

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7.

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 degrees C, respectively. Chi-A was stable in the range of pH 5-10 up to 40 degrees C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulans WL-12.     

Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by nonionic detergents, poly(ethylene glycol) and high-molecular-weight dextran

Dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5) (3 IU/ml culture supernatant) was obtained by a modification of the method of Robyt and Walseth (Robyt, J.F. and Walseth, T.F. (1979) Carbohydr. Res. 68, 95-111) from a nitrosoguanidine mutant of Leuconostoc mesenteroides NRRL B-512F selected for high dextransucrase production. Dialyzed, concentrated culture supernatant (crude enzyme) was treated with immobilized dextranase (EC 3.2.1.11) and chromatographed on a column of Bio-Gel A-5m. The resulting, purified enzyme lost activity rapidly at 25 degrees C or on manipulation, as did the crude enzyme when diluted below 1 U/ml. Both enzyme preparations could be stabilized by low levels of high-molecular-weight dextran (2 micrograms/ml), poly(ethylene glycol) (e.g., 10 micrograms/ml PEG 20 000), or nonionic detergents (e.g., 10 micrograms/ml Tween 80). The stabilizing capacity of poly(ethylene glycol) and of dextran increased with molecular weight. Calcium had no stabilizing action in the absence of other additions, but reduced the inactivation that occurred in the presence of 0.5% bovine serum albumin or high concentrations (greater than 0.1%) of Triton X-100. In summary, dextransucrase could be stabilized against activity losses caused by heating or by dilution through the addition of low concentrations of nonionic polymers (dextran, PEG 20000, methyl cellulose) or of nonionic detergents at or slightly below their critical micelle concentrations.     

InhA-like protease secreted by Bacillus sp. S17110 inhabited in turban shell

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around 50 degrees . Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of Ca2+, Zn2+, Mg2+, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.     

Identification, purification, and characterization of a secretory serine protease in an Indian strain of Leishmania donovani

An aprotinin sensitive serine protease was identified in the culture supernatant of the Indian strain of Leishmania donovani (MHOM/IN/1983/AG83). The protease was subsequently purified and characterized. The apparent molecular mass of the enzyme was 115 kDa in SDS-PAGE under non-reducing condition, while on reduction it showed a 56 kDa protein band indicating that the protease is a dimeric protein. The purified enzyme was optimally active at the pH and temperature of 7.5 and 28 degrees C, respectively. Assays of thermal stability indicated that the enzyme preserved 59% of activity even after pretreatment at 42 degrees C for 1 h. The purified protease was not glycosylated and its isoelectric pI was 5.0. N-alpha-p-tosyl-L-arginine methylester (TAME) appeared to be relatively better substrate among the commonly used synthetic substrates. The enzyme was inhibited by Ca(2+) and Mn(2+), but activated by Zn(2+). The protease could play important role(s) in the pathogenesis of visceral leishmaniasis or kala-azar.     

Purification and some properties of dextransucrase from Streptococcus bovis 148

The dextransucrase produced by Streptococcus bovis 148 was purified about 20.8-fold to electrophoretic homogeneity (specific activity 246.3 units/mg) from the culture supernatant. Molecular weight of the native enzyme was estimated to be 600 kDa, whereas the molecular weight per subunit was 150 kDa. The dextran synthesized consisted chiefly of α-1,6-glucosidic linkage, containing small numbers of branches that had the α-1,3-glucosidic linkage.     

Purification and characterization of beta-1,3-xylanase from a marine bacterium, Vibrio sp. XY-214

beta-1,3-Xylanase was purified to gel electrophoretic homogeneity and 83-fold from a cell-free culture fluid of Vibrio sp. XY-214 by ammonium sulfate precipitation and successive chromatographies. The enzyme had a pl of 3.6 and a molecular mass of 52 kDa. The enzyme had the highest level of activity at pH 7.0 and 37 degrees C. The enzyme activity was completely inhibited by Cu2+, Hg2+, and N-bromosuccinimide. The enzyme hydrolyzed beta-1,3-xylan to produce mainly xylotriose and xylobiose but did not act on xylobiose, p-nitrophenyl-beta-D-xyloside, beta-1,4-xylan, beta-1,3-glucan, or carboxymethyl cellulose.     

Glycoprotein biosynthesis in Saccharomyces cerevisiae. Purification of the alpha-mannosidase which removes one specific mannose residue from Man9GlcNAc

A soluble form of the specific alpha-mannosidase from Saccharomyces cerevisiae, which catalyzes the following reaction, was purified at least 100,000-fold by conventional chromatography procedures: (Formula: see text). The purified enzyme migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of about 60 kDa in the absence of reducing agent, and as two bands of about 44.5 kDa and 22.5 kDa in the presence of reducing agent. The apparent molecular weight of the soluble enzyme is about 75,000 by gel filtration on Sephacryl S-200. The specific alpha-mannosidase does not require the addition of divalent cation for activity, but it is inhibited by Tris, EDTA, Mn2+, Co2+, Zn2+, and Mg2+. The inhibition caused by EDTA can be reversed completely by Ca2+ and partially by Mg2+, but not by other divalent cations. The soluble alpha-mannosidase arises from a larger hydrophobic form of the enzyme which is found in the detergent phase during partition in Triton X-114. The formation of the soluble enzyme, which is recovered in the aqueous phase during partition in Triton X-114, is time- and temperature-dependent and is prevented by pepstatin, but not by other protease inhibitors. These results indicate that the purified soluble alpha-mannosidase represents the catalytically active domain of the enzyme which has been proteolytically released from its membrane-bound form.     

米曲霉LY-128广谱有机磷农药水解酶的纯化和鉴定*

米曲霉LY-128的培养物经硫酸铵分级沉淀,Sephadex G-100 凝胶过滤, DEAE-Sephrose CL-6B 和Sephadex G-100层析手段,获得了电泳纯的广谱有机磷农药水解酶。通过SDS-PAGE 和IEF电泳测得其分子量为62 kDa, 等电点为pH 5.2。该酶的最适反应温度为45℃,最适 pH 6.8, 在50℃以下及pH6.0～9.5 范围内活性稳定。Hg2+、Fe3+、对氯高汞苯甲酸、碘乙酸和N-乙基马来酰亚胺对该酶有强烈的抑制作用,而Cu2+、 巯基乙醇、二硫苏糖醇、二硫赤藓糖醇、谷光甘肽和去污剂对酶有不同程度的激活作用。底物的专一性实验表明,该酶不仅可以作用于含P-O键的有机磷农药;而且也能水解含P-S键的有机磷农药。以甲基对硫磷和内吸磷为底物的Km值分别为52祄ol、236 祄ol; Vmax分别为317祄ol min-1 mg-1、179 祄ol min-1 mg-1;Kcat分别为1152 s-1、650 s-1。     

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40 degrees C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu2, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.     

Purification and characterization of a thermostable glucoamylase from Chaetomium thermophilum

Thermostable amylolytic enzymes are currently being investigated to improve industrial processes of starch degradation. A thermostable extracellular glucoamylase (exo-1, 4-alpha-D-glucanohydrolase, E.C.3.2.1.3) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by using ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography, and Phenyl-Sepharose Fast Flow chromatography. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 64 kDa. The glucoamylase exhibited optimum catalytic activity at pH 4.0 and 65 degrees C. It was thermostable at 50 degrees C and 60 degrees C, and retained 50% activity after 60 min at 65 degrees C. The half-life of the enzyme at 70 degrees C was 20 min. N-terminal amino acid sequencing (15 residues) was AVDSYIERETPIAWN. Different metal ions showed different effects on the glucoamylase activity. Ca2+, Mg2+, Na+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, and Hg2+ cause obvious inhibition. These properties make it applicable to other biotechnological purposes.     

Purification and characterization of methylenetetrahydrofolate reductase from human cadaver liver

Methylenetetrahydrofolate reductase from human cadaver liver was purified to homogeneity. The purified enzyme had a molecular mass of 150 kDa. On SDS-polyacrylamide gel electrophoresis it was dissociated into a single fragment with a molecular mass of 39 kDa. In contrast, fresh lymphocyte enzyme extract showed a major band with a molecular mass of 75 kDa and a minor band of 39 kDa. Fresh liver enzyme was inhibited by S-adenosylmethionine while the purified enzyme from human cadaver liver was not inhibited. These observations suggest that human methylenetetrahydrofolate reductase is composed of two identical subunits of 75 kDa each but is cleaved into a major single band due to autolysis in cadaver liver. The purified cadaver enzyme was a FAD-specific protein. The pH optimum was 6.6 for methylenetetrahydrofolate-NADPH oxidoreductase, 6.5 for methyltetrahydrofolate-menadione oxidoreductase, and 7.2 for NADP-menadione oxidoreductase. The Km values of human liver methylenetetrahydrofolate reductase were 17 microns for NADPH and 38 microns for methyltetrahydrofolate in the reduction of menadione, and 12 microns for NADPH in the reduction of methylenetetrahydrofolate.     

Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum

A thermostable superoxide dismutase (SOD) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum strain CT2 was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-sepharose, phenyl-sepharose hydrophobic interaction chromatography. The pure SOD had a specific activity of 115.77 U/mg of protein and was purified 7.49-fold, with a yield of 14.4%. The molecular mass of a single band of the enzyme was estimated to be 23.5 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 94.4 kDa, indicating that this enzyme was composed of four identical subunits of 23.5 kDa each. The SOD was found to be inhibited by NaN3, but not by KCN and H2O2. Atomic absorption spectrophotometric analysis showed that the content of Mn was 2.05 microg/mg of protein and Fe was not detected in the purified enzyme. These results suggested that the SOD in C. thermophilum was the manganese superoxide dismutase type. N-terminal amino acid sequencing (10 residues) was KX (X is uncertain) TLPDLKYD. The N-terminal amino acid sequencing homologies to other MnSod also indicated that it was a manganese-containing superoxide dismutase. The SOD exhibited maximal activity at pH 7.5 and optimum temperature at 60 C. It was thermostable at 50 and 60 C and retained 60% activity after 60 min at 70 C. The half-life of the SOD at 80 C was approximately 25 min and even retained 20% activity after 30 min at 90 C.     

A phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5: purification and characterization of conditions for optimal activity with an artificial substrate

Phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5 was purified from buffered yeast extract culture supernate by ion-exchange chromatography followed by fractionation by manganous chloride and ammonium sulphate precipitation steps. Enzyme activity was assayed by hydrolysis of p-nitrophenylphosphorylcholine and confirmed by release of radioactivity from tritiated L-alpha-dipalmitoylphosphatidylcholine labelled in the methyl groups of choline. After SDS-PAGE, the purified preparation yielded a single band upon Coomassie-blue staining. This protein migrated with an apparent Mr of 50,000-54,000. Phospholipase C activity was maximal at pH greater than or equal to 8.4 and was enhanced in the presence of sorbitol and of several nonionic detergents but was eliminated by SDS. EDTA, Cu2+, Fe2+ and Zn2+ inhibited enzyme activity, whereas Ba2+, Ca2+, Co2+, Mg2+ and Mn2+ restored activity to EDTA-treated material. No haemolytic activity was demonstrated with the purified enzyme.     

Purification and characterization of a detergent-requiring membrane-bound metalloendopeptidase from porcine brain.

A detergent-requiring metalloendopeptidase cleaving a progastrin-C-terminal peptide (progastrin-(88-101)) mainly at the Arg95-Gly96 bond was solubilized from porcine cerebral vesicular membranes and purified to homogeneity as examined by PAGE. The purified enzyme had a molecular mass of approximately 76 kDa as estimated by both SDS/PAGE and Sephacryl S-300 gel filtration. It hydrolyzed progastrin-(88-101) peptide, BAM-12P, and bradykinin fairly specifically, and more efficiently than various other neuropeptides and related oligopeptides examined as substrates. It was inactive in the absence of detergents, and required certain detergents such as Triton X-100 or Lubrol PX for activity. Its optimum pH was about 6.5 and was strongly inhibited by metal-chelating agents such as EDTA, EGTA, and o-phenanthroline. It was extremely sensitive to EDTA and was completely inhibited even by 0.3 microM EDTA; the activity was fully restored by addition of a 10-fold higher concentration of Zn2+, CO2+, or Mn2+ ions over EDTA. On the other hand, dynorphin A-(1-13) peptide, a strong inhibitor of neurolysin, failed to inhibit the enzyme. The various characteristics indicated that the present enzyme is a unique membrane-bound metalloendopeptidase.     

Purification and properties of mouse liver coproporphyrinogen oxidase

Coproporphyrinogen oxidase was purified to homogeneity from mouse liver. The specific activity of the pure enzyme was 3500 nmol.h-1.mg-1; its apparent molecular mass (35 kDa) was confirmed by immunological characterization of the enzyme in a trichloroacetic-acid-precipitated total-liver-protein extract. The native enzyme appeared to be a dimer of 70 kDa as determined by gel filtration under nondenaturating conditions. The Km value for coproporphyrinogen III was 0.3 microM. The purified enzyme was activated by neutral detergents and phospholipids (affecting both Vmax and Km) but inhibited by ionic detergents. Reactivity toward sulfhydryl agents suggested the possible involvement of (an) SH group(s) for the activity. When compared to the previously purified coproporphyrinogen oxidases (from bovine liver and yeast), the mouse liver coproporphyrinogen oxidase appears to share many common catalytic properties with both enzymes. However, its apparent molecular mass is very different from that of the bovine liver enzyme (71.6 kDa) but identical to that found for the yeast (Saccharomyces cerevisiae) enzyme.     

Purification and characterization of histidinol dehydrogenase from cabbage

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.     

Purification and characterization of an invertase produced by Aspergillus ochraceus TS.

Purification and characterization of an extracellular invertase produced by Aspergillus ochraceus TS are reported. The enzyme was purified (42-fold) from culture filtrate by salt precipitation, ion-exchange and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single band of molecular mass 66 kDa. The molecular mass of the native enzyme was found to be 130 kDa by gel filtration. The purity of the protein was also checked against its antiserum raised in rabbits by two-dimensional immunodiffusion in agarose gel and Western blot that showed a single band. It is a glycoprotein with mannose as its carbohydrate residue. The enzyme showed high affinity for sucrose with a Km of 3.5 mM. The amino acid analysis revealed a high proportion of acidic residues but it had a low content of cysteine, histidine and arginine comparable to other fungal invertases.     

The effect of neutral polymers on glutamate dehydrogenase activity of mouse liver mitochondria upon administration of endotoxin

It is shown that neutral polymers administered intraperitoneally to intact animals considerably affect glutamate dehydrogenase activity in the liver cell mitochondria as well as in the supernatant. Of the tested polymers, only polyvinyl methylacetamide and dextran inhibit a decrease in the level of mitochondrial enzyme activity which develops with administration of endotoxin. Polyvinyl pyrrolidone with molecular weight of 100 kDa, polyvinyl methylacetamide, dextran and polyvinyl caprolactam prevent an increase of glutamate dehydrogenase activity in the supernatant in case of endotoxin administration to animals. It is possibly a result of the effect of the mitochondrial structure stabilization by the above polymers. A physiological effect of polyvinyl pyrrolidone revealed as an effect on the activity level of mitochondrial glutamate dehydrogenase and in the supernatant after endotoxin administration to animals, depends on the molecular weight of the polymer.     

A developmentally regulated hyaluronidase of Haemonchus contortus

The trichostrongylid nematode Haemonchus contortus released a hyaluronic acid-degrading enzyme during in vitro development from the third (L3) to fourth (L4) larval stage. The enzyme did not degrade chondroitin sulfate A. Enzyme activity was optimal between pH 4.0 and 6.0, and the enzyme was inhibited by high concentrations of NaCl; the divalent cations Cu2+, Zn2+, Ca2+, and Mn2+ were not inhibitory. The hyaluronidase had a molecular mass estimated at 57 kDa by sucrose density gradient centrifugation and at 111 kDa by substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (reducing and nonreducing conditions), suggesting the formation of a dimer during the electrophoretic separation conditions. The level of hyaluronidase released during in vitro development peaked between 24 and 48 hr in culture and then gradually decreased, with little or no activity present in the 168-hr culture fluid. The enzyme was not detected in culture fluid from 24-hr incubations of either the mid-L4 stage (obtained from sheep 7 days postinfection) or the adult stage (obtained from sheep 30-35 days postinfection). The temporal expression of the hyaluronidase suggested a role for this enzyme in the early stages of the L3-L4 developmental process.