Characterization of acid and neutral alpha-mannosidases in bull semen and reproductive organs

Acid and neutral alpha-mannosidase activities were studied in the bull reproductive tissues, isolated spermatozoa, epididymal and seminal vesicle secretion and seminal plasma. The acid enzyme in the seminal plasma mainly derived from the epididymal secretion, while the neutral one was enriched in the sperm cells. The latter activity in the seminal plasma appears to be due to an enzyme released from the cytoplasmic droplets in the epididymis. The acid enzyme had a molecular weight of 220,000-320,000, pI 7.3-6.0 and an optimum at pH 4.0. It was sensitive to swainsonine but was stimulated by Zn2+. The neutral enzyme had a molecular weight of 360,000-460,000, pI 5.4-4.7 and showed double optima at pH 5.5 and 6.0-7.0. It was resistant to swainsonine but was markedly activated by Co2+ or Fe2+. The neutral enzyme was also more sensitive to thermal inactivation than the acid one.     

Beta-galactosidase of Penicillium chrysogenum: production, purification, and characterization of the enzyme

Intracellular beta-galactosidase from Penicillium chrysogenum NCAIM 00237 was purified by procedures including precipitation with ammonium sulfate, ion-exchange chromatography on DEAE-Sephadex, affinity chromatography, and chromatofocusing. These steps resulted a purification of 66-fold, a yield of about 8%, and a specific activity of 5.84 U mg(-1) protein. Some enzyme characteristics were determined using o-nitrophenyl-beta-d-galactopyranoside as substrate. The pH and temperature optimum of the activity were about 4.0 and 30 degrees C respectively. The K(m) and pI values were 1.81 mM and 4.6. beta-Galactosidase of P. chrysogenum is a multimeric enzyme of about 270 kDa composed of monomers with a molecular mass of 66 kDa.     

Purification and characterization of active recombinant human napsin A.

Recombinant human napsin A expressed in human embryonic kidney 293 cells was purified to homogeneity by a single-step procedure using part of napsin A propeptide as affinity ligand. N-Terminal amino-acid sequencing of the purified enzyme identified the mature form of napsin A. Treatment of purified napsin A with endoglycosidases F and H resulted in a decrease in its molecular mass from 39 kDa to approximately 37 kDa, confirming that napsin A is glycosylated. The kinetic properties were analyzed by using two fluorogenic synthetic substrates K(Dabsyl)-TSLLMAAPQ-Lucifer yellow (DS1) and K(Dabsyl)-TSVLMAAPQ-Lucifer yellow (DS3). The Km values obtained were 1.7 microM and 6.2 microM, respectively. A substrate-specificity study using a napsin A-targeted peptide library confirmed the preference of napsin A for hydrophobic residues at positions P1 and P1'. Adjacent positions, P2-P4 and P2'-P4', appeared less restricted in distribution of amino acids. A pH optimum between 4.0 and 5.5 at room temperature was determined. The purified enzyme was fully active for more than 10 h at pH 5.0 and 6.0, while a half-life of 4 h was determined at pH 7.0 and 37 degrees C.     

Purification and characterisation of a beta-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65 degrees C and was stable up to 60 degrees C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. Km and Vm values for these substrates were 0.5, 1.25 mM and 95, 91 mumol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucose and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with Ki of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a pI of 4.5 and appeared to be a glycoprotein.     

聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素

以苦瓜籽为材料,研究了聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素。等电点沉淀试验表明,柠檬酸、盐酸分别调节苦瓜种仁粗提液pH至6.0、4.0时,各有14.62%和32.49%的苦瓜种仁蛋白被沉淀。醋酸的等电点沉淀作用呈现阶段性特点,pH6.0和4.0时分别有26.17%和38.72%的苦瓜种仁蛋白被沉淀。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液(pH4.0),1%PAA选择性沉淀碱性蛋白(等电点pI为8.65~9.30)的最佳用量分别为100μL、120μL和100μL。醋酸调节苦瓜种仁粗提液pH分别至5.0、4.0和3.0,等电点沉淀后的上清液用PAA沉淀碱性蛋白,当PAA(1%)用量为160μL/mL提取液时,pH5.0和3.0样液分别有33.77%和43.56%蛋白质被沉淀;当PAA用量为120μL/mL提取液时,pH4.0样液中30.83%蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液(pH>9.0),当溶液NaCl浓度为3.0%时,溶液蛋白质浓度最高。PAA选择性沉淀的苦瓜种仁碱性蛋白经SephadexG-75柱层析分离,分别在175min和300min出现主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kD,pI值约为9.5,主峰Ⅱ的分子量约为10kD,pI值约为9.3。     

Purification and characterization of two endoglucanases from Melanocarpus sp. MTCC 3922

This study reports the purification and characterization of endoglucanases (EG I and EG II) from a newly isolated thermophilic fungus, Melanocarpus sp. MTCC 3922. The molecular weight of EG I and EG II as with SDS-PAGE and pI were approximately 40 and 50 kDa, and approximately 4.0 and 3.6, respectively. EG I and EG II were optimally active at 50 and 70 degrees C, and pH 6.0 and 5.0, respectively. EG I was active over a broad range of pH (5.0-7.0), whereas, loss of activity was observed as the temperature was increased from 50 to 80 degrees C. However, EG II was active over pH 4.0-6.0 and temperature 40-80 degrees C. The presence of mercaptoethanol and SDS inhibited the EG I activity but showed no negative effect on EG II. Both the endoglucanases showed higher activity against barley-beta-glucan as compared to CMC. Km values of EG I and EG II for barley-beta-glucan were lower than CMC. Turn over number (K(cat)) and catalytic efficiency (K(cat)/Km) values of both the endoglucanases were higher with barley-beta-glucan as substrate than CMC. EG I showed affinity for Avicel indicating the presence of cellulose binding domains (CBD) whereas, EG II was found to lack CBD.     

Properties and prenatal ontogeny of beta-D-mannosidase in selected goat tissues.

beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.     

L-asparaginase of Thermus thermophilus: Purification,properties and identificaation of essential amino acids for its catalytic activity

L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77°C. Chemical modification experiments implied the existence of histidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.     

Purification and characterization of beta-1,6-glucanase of Streptomyces rochei application in the study of yeast cell wall proteins

A beta-1,6-glucanase was purified to apparent homogeneity from a commercial yeast digestive enzyme prepared from Streptomyces rochei by a series of column chromatographies. The molecular mass of the purified enzyme was 60 kDa by SDS-PAGE. The purified enzyme had an optimum pH range from 4.0 to 6.0 and was stable in the same pH range. The enzyme was stable under 50 degrees C but lost almost all activity at 60 degrees C. The enzyme was specific to beta-1,6-glucan and had little activity towards beta-1,3-glucan and beta-1,4-glucan. When the beta-1,6-glucan was hydrolyzed with the purified enzyme for 5 h, the reaction products contained 20% glucose, 36% gentiobiose, and 44% other oligosaccharides, suggesting that the enzyme is an endo-type glucanase. When the purified enzyme was used for the digestion of the cell wall of Saccharomyces cerevisiae, cell-wall proteins covalently bound to the cell-wall glucan were recovered as soluble forms, suggesting that this enzyme is useful for analysis of yeast-cell wall proteins.     

Properties of acid ceramidase from human spleen

We have characterised ceramidase activity in extracts of human spleen from control subjects and from patients with Gaucher disease. In Triton X-100 extracts of control spleens, a broad pH optimum of pH 3.5-5.0 was found; no ceramidase activity was detectable at neutral or alkaline pH. About 45-60% of acid ceramidase could be extracted from spleen without detergents, but for complete extraction, Triton X-100 was required. For the radiolabelled substrate oleoylsphingosine, a Km of 0.22 +/- 0.09 mM and a Vmax of 57 +/- 11 nmol/h per mg protein was calculated in spleen from a control subject. Flat-bed isoelectric focussing in the presence of Triton X-100 revealed a pI of 6.0-7.0 for acid ceramidase; similar values were found for sphingomyelinase and glucerebrosidase. HPLC-gel filtration indicated that in the presence of Triton X-100, acid ceramidase has an Mr of about 100 kDa. In the absence of detergents, the enzyme forms high-molecular-weight aggregates. Similar aggregation behaviour was observed for sphingomyelinase, while the elution of beta-hexosaminidase was not affected by detergents. The elution profile of glucocerebrosidase was only slightly altered by Triton X-100. There was no difference in the properties of acid ceramidase present in spleen from control subjects and from patients with type I Gaucher disease.     

Purification and characterization of phosphotriesterases from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11

A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34-41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%-16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life=7.3 h) than that from SBL11 (half-life=6.4 h at 50 degrees C), while both showed the same temperature optimum of 37 degrees C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.     

Purification and properties of a chitinase from Penicillium oxalicum autolysates

A chitinase (EC. 3.2.1.14) from autolysed culture filtrate of Penicillium oxalicum was purified by precipitation with ammonium sulphate, gel filtration and ion exchange chromatographies. The purified enzyme showed a single protein band in SDS gel electrophoresis. The enzyme is an acidic protein with a pI of 4.5 and has a molecular weight of 54 900 as estimated from SDS gel electrophoresis and 21 500 from gel filtration. The optimum pH and temperature were 5.0 and 35°C, respectively. The enzyme was stable at temperatures up to 45°C and in a pH range between 4.0 and 6.0. The K_m was 2.5 mg ml^-1 for colloidal chitin, Hg²⁺ and Ag⁺ were effective inhibitors. The viscosimetric study carried out using carboxymethyl chitin as substrate revealed the endotype action of this enzyme.     

Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic archaeon, a Thermococcus sp

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 micrometers in diameter. The new isolate grew at temperatures between 60 and 95 degrees C (optimum, 85 degrees C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120 degrees C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100 degrees C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110 degrees C. The enzyme formed mainly alpha-cyclodextrin with small amounts of beta- and gamma-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.     

Purification and characterisation of a fructosyltransferase from Rhodotorula sp

The present work was devoted to investigations concerning the purification and characterisation of the fructooligosaccharide (FOS)-producing extracellular enzyme of Rhodotorula sp. LEB-V10. FOS are functional food ingredients showing prebiotic properties, meaning that it could stimulate selectively the growth and/or activity of probiotic bacteria in the gut. The purification of the enzyme was carried out according to the following sequential procedure: cell separation by centrifugation, recovering by ethanol precipitation and purification by anion exchange chromatography. The molecular weight was estimated to be 170 kDa by preparative gel filtration and 77 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, signifying that the native enzyme exists as a dimer. With sucrose as substrate, the data failed to fit the Michaelis-Menten behaviour, rather showing a sigmoid shape similar to that of the allosteric enzymes (cooperative behaviour), requiring high sucrose concentrations to obtain high reaction rates. The enzyme showed both fructofuranosidase (FA) and fructosyl-transferase (FTA) activities. The optimum pH and temperature for FA activity were found to be around 4.0 and 72-75 degrees C, respectively, while FTA showed optimum activity at pH 4.5 and 65-70 degrees C. Both activities were very stable at temperatures below 66 degrees C, while for FA, the enzyme was more stable at pH 4.0 and for FTA at pH 5.0.     

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense.

A cysteine protease (trypanopain-Tc) with cathepsin-L-like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31-32 kDa by SDS/PAGE and 66 kDa by gel chromatography. It has a pI 7.4 and a high affinity for concanavalin A. Trypanopain-Tc catalyses the limited proteolysis of a variety of protein substrates such as fibrinogen, serum albumin and trypanosome variant-surface glycoprotein. It has minimal or no activity against casein or elastin. A variety of peptidyl amidomethylcoumarins and peptidyl diazomethanes were used to test the specificity of trypanopain-Tc. The better substrates had Arg or Lys in P1 and hydrophobic amino acids in P2 and P3. The best substrate found for trypanopain-Tc was Z-Phe-Arg-NHMec (Z, benzyloxycarbonyl; NHMec, 7-amido-4-methylcoumarin). The kinetic constants for the hydrolysis of Z-Phe-Arg-NHMec were kcat = 17.4 s-1, Km = 4.4 microM, kcat/Km = 4.0 microM-1.s-1, which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z-Arg-Arg-NHMec) and cathepsin H (Arg-NHMec) were not hydrolysed by trypanopain-Tc under the conditions tested. The pH optimum of trypanopain-Tc against Z-Phe-Arg-NHMec was pH 6.0 but it showed a broad peak of activity extending well into the alkaline region. The enzyme was activated by low-molecular-mass thiol compounds and inhibited by cystatin, L-trans-epoxysuccinyl-4-guanidinobutane (E-64) and a variety of peptidyl diazomethanes. The most effective diazomethane inhibitors (Z-Leu-Leu-Met-CHN2, Z-Leu-Met-CHN2 and Z-Leu-Lys-CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vitro after 24-48 h incubation in greater than or equal to 20 microM [inhibitor]. However, it is not clear whether the trypanocidal activity of these inhibitors is a consequence of the inhibition of trypanopains or of some other essential proteolytic activities within the parasites.     

海枣曲霉β—葡萄糖苷酶的提纯与性质

A beta-glucosidase has been purified to electrophoretically homogeneity from the wheat bran culture of Aspergillus phoenicis by PEG 6000-phosphate biphasic separation, column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and SE-Sephadex C-50. The enzyme showed optimal activity at pH 5.0 and 60 degrees C. It was stable in the pH range of 4.0-7.5 and up to 55 degrees C. The enzyme activity was strongly inhibited by Ag+ and Hg2+. The molecular weight of the enzyme was 118000 as determined by SDS-PAGE and 195000 by gradient-PAGE. The isoelectric point was pI 3.95 as determined by PAGIF.     

Purification and characterization of beta-1,3-xylanase from a marine bacterium,Alcaligenes sp. XY-234

A beta-1,3-xylanase-producing bacterium, Alcaligenes sp. XY-234, was isolated from the marine environment. The organism produced endo-1,3-beta-xylanase at a high level in the culture fluid. The enzyme was purified 292-fold by ammonium sulfate precipitation and several column chromatographies. The final enzyme preparation appeared to be homogeneous on disc gel electrophoresis and SDS-PAGE with a molecular mass of 59 kDa, and the pI was 4.0. The enzyme hydrolyzed beta-1,3-xylan and larger xylooligosaccharides than xylobiose to give several xylooligosaccharides, but it could not hydrolyze xylobiose, p-nitrophenyl-beta-D-xyloside, and beta-1,4-xylan. The Km of the enzyme was 4.0 mg/ml. Optimal pH and temperature were 7.5 and 40 degrees C, respectively. It was stable from pH 6.0 to 10 and at a temperature of less than 40 degrees C. The enzyme was strongly inhibited by 1 mM HgCl(2)., AlCl(3), CuCl(2), FeCl(3), HgCl(2), Pb(CH(3)COO) (2), and N-bromosuccinimide.     

Purification and Characterization of an alpha-l-Arabinofuranosidase from Butyrivibrio fibrisolvens GS113

An alpha-l-arabinofuranosidase (EC 3.2.1.55) was purified from the cytoplasm of Butyrivibrio fibrisolvens GS113. The native enzyme had an apparent molecular mass of 240 kDa and was composed of eight polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.0, a pH optimum of 6.0 to 6.5, a pH stability of 4.0 to 8.0, and a temperature optimum of 45 degrees C and was stable to 55 degrees C. The K(m) and V(max) for p-nitrophenyl-alpha-l-arabinofuranoside were 0.7 mM and 109 mumol/min/mg of protein, respectively. The enzyme was specific for the furanoside configuration and also readily cleaved methylumbelliferyl-alpha-l-arabinofuranoside but had no activity on a variety of other nitrophenyl- or methylumbelliferyl glycosides. When the enzyme was incubated with cellulose, carboxymethyl cellulose, or arabinogalactan, no release of sugars was found. Arabinose was found as the hydrolysis product of oatspelt xylan, corn endosperm xylan, or beet arabinan. No activity was detected when either coumaric or ferulic acid ester linked to arabinoxylobiose was used as substrates, but arabinoxylobiose was degraded to arabinose and xylobiose. Since B. fibrisolvens GS113 possesses essentially no extracellular arabinofuranosidase activity, the major role of the purified enzyme is apparently in the assimilation of arabinose-containing xylooligosaccharides generated from xylosidase, phenolic esterase, xylanase, and other enzymatic activities on xylans.     

Isolation, Purification, and Characterization of the PR Oxidase from Penicillium roqueforti

The PR oxidase, an extracellular enzyme, involved in the conversion of PR toxin into PR acid, was purified from the culture broth of Penicillium roqueforti ATCC 48936. The enzyme has a pI of 4.5 and a molecular mass of approximately 88 kDa, and it is a monomer. The optimum pH for this enzyme is ca. 4.0, and the optimum temperature is 50°C.