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To clone and express the γ-polyglutamic acid (γ-PGA) synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications. [ABSTRACT FROM AUTHOR]

Ruta graveolens (the common rue) has been used for various therapeutic purposes, including relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on main drug-metabolizing enzymes , effects of an aqueous extract of the aerial part of rue and its ingredients on cytochrome P450 (P450/CYP), uridine diphosphate (UDP)- glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H):quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg) induced hepatic Cyp1a and Cyp2b activities and protein levels in males and females. This treatment increased hepatic UDP-glucuronosyltransferase activity only in males. However, NAD(P)H:quinone oxidore- ductase activity remained unchanged. Based on the contents of rutin and furanocoumarins of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of furanocoumarins (Fmix) increased Cyp2b activities in males. The mixture of rutin and F mix increased Cyp1a and Cyp2b activities. These results revealed that rutin and Fmix contributed at least in part to the P450 induction by rue [ABSTRACT FROM AUTHOR]

Plant endophytes are among the most important resources of biologically active metabolites. Twenty-three endophyte strains residing in Trachelospermum jasminoides were cultivated in vitro with the cultures assayed for the fibrinolytic substance production. As a result, the culture of Verticillium sp. Tj33 was shown to be the most active. A fibrinolytic enzyme designated as verticase was subsequently purified from the supernatant of Verticillium sp. culture broth by a combination of DEAE-52, Sephadex G-75 and hydrophobic column chromatographies. Verticase, with its molecular mass of 31 kDa and pI of 8.5, was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Verticase is an enzyme that hydrolyzes fibrin directly without activation of plaminogen. It was stable in a broad pH range from 4 through to 11 with the optimal reaction pH value and temperature shown to be around 9–10 and 50–60 °C, respectively. The fibrinolytic activity of verticase was severely inhibited by phenylmethylsulfony fluoride, indicating that verticase was a serine protease. [ABSTRACT FROM AUTHOR]

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37°C, respectively). The metal ions Cu2+ and Fe3+ (at a concentration of 5 mmol liter-1) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-L-arabinofuranose (FAA) as a substrate, the enzyme exhibited a Km of 0.0953 mmol liter-1 and a Vmax of 86.27 mmol liter-1 min-1 mg-1 of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-α-L-arabinofuranosyl)-(1→3)-O-βD-xylopyranosyl-(1→4)-D-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase. [ABSTRACT FROM AUTHOR]

We present the in vitro expression and purification of N-terminal fragments of human perlecan in insect cells. Three tailored fragments of human perlecan cDNA were introduced into the polyhedrin locus of baculovirus expression vectors (BEVs) encoding amino acids 1–196 (domain I), 1–404 (domain I+11a) and 1–506 (domain I+IIab). The integrity of the BEVs was checked by DNA sequencing, polymerase chain reaction, restriction enzyme analysis and Southern blotting. Northern hybridization and metabolic labeling with [35S]methionine showed that expression of the perlecan-(I–404) and the -(1–506)- peptide was successful, but in the case of tile perlecan-(1–196)-peptide no recombinant protein was produced. lmmunoblotting sbowed that both the (l–404)-peptide and (1–506)-peptide are recognized by 95J 10, a monoclonal antibody that was previously raised against perlecan-(24–404)-peptide expressed in Escherichia coli Gel permeation and anion-exchange chromatograhy were applied to purify the recombinant proteins. Glycosaminoglycans were demonstrated to be present. Deglycosylation with chondroitinase ABC showed that the perlecan-(1–404) peptide was glycosylated with chondroitin sulfate residues. Consistent with these results, glycosaminoglycans isolated from the perlecan-(1–404)-peptide were identified as chondroitin sulfate by agarose gel electrophoresis. Furthermore the perlecan (1–404)- peptide showed affinity to immobilized basic fibroblast growth factor. The availability of baculovirus- derived recombinant perlecan fragments will facilitate domain-specific investigation of the structural and functional properties of perlecan in the future. Kewords: perlecan; structure; baculovirus expression; chondroitin sulfate; basic fibroblast-growth factor [ABSTRACT FROM AUTHOR]

1. H+-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity. 2. The enzyme from Rb. capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53000 and 48000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants. 3. Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R. R. & Earle, S. R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York] that the soluble component associated with H+-transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb. capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli. 4. The dependence of the rate of H- transfer by purified H+-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5'-AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex. 5. In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R. R. & Earle, S. R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H+-transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive. [ABSTRACT FROM AUTHOR]

A pregastric lipase was purified from calf pharyngeal tissues. The purification procedure was based on chromatographies on octyl-Sepharose and lentil-lectin-Sepharose followed by gel filtration. The final preparation, with an overall recovery of 26% of activity, gave a single protein band on dodecyl sulfate/polyacrylamide gel electrophoresis with a Mr of 55 000. The Mr on gel filtration was 44–48 000. The discrepancy may be due to the fact that pregastric lipase is a glycoprotein containing &assymp;10% (w/w) of carbohydrate. The pI was around 7.0 and the enzyme protein is characterized by a high content of branched, aliphatic amino acid residues. The NH2-terminal amino acid sequence is: H2N-Phe-Leu/(Ile)-Gly-. Rabbit antibodies to the purified preparation detected only one component in the crude starting material in immuno-blotting experiments. Preincubation with antiserum resulted in loss of enzyme activity, showing that the antibodies were directed against the lipase. [ABSTRACT FROM AUTHOR]

DNA-dependent RNA polymerase III has been purified to homogeneity from the filamentous fungus Podospota comata. The enzyme was extracted at low ionic strength, separated from the polymerases I and II by DEAE-Sephadex chromatography and purified by heparin-Sepharose and phosphocellulose chromatography; 0.1–0.2 mg highly purified homogenous enzyme with a specific activity of 220 units/mg could be obtained from 2 kg wet mycelium. The subunit composition of the enzyme was determined after sodium dodecyl sulphate/ polyacrylamide gel electrophoresis: thirteen putative subunits of molecular weight 174000 (a), 129000 b). 87000 (c), 50000 (d), 39000 (e), 23500 (f), 21000 (g), 19000 (h), 17000 (i), 16500 (j), 13500 (k), 11000 (l) and 10000 (m) were identified. All of the polypeptide components of the enzyme are present in about integral stoichiometric amounts as judged by dye binding. The presence of subunit Mr = 87000 in a molar ratio 1:1 is necessary to obtain very active enzyme. Thirteen homologous subunits were observed in a preparation of RNA polymerase III from Podospora anserine, which is a related species. Only subunit i is different in the two species. [ABSTRACT FROM AUTHOR]

Rat liver microsomal glutathione 5-transferase was activated with N-ethylmaleimide, solubilized with Triton X-100, and purified by chromatography on hydroxyapatite and CM-Sepharose. A 36-fold purification resulted in a 36% yield, indicating that the giutathione S-transferase accounts for 2.5 – 3% of the original microsomal protein. The purified protein moved as a band with an apparent molecular weight of 14000 on sodum dodecyl sulphate gel electrophoresis and appeared to be nearly homogeneous. The complex formed between the purified microsomal glutathione S-transferase and Triton X-100 has a sedimentation coefficient of 3.2 S, a partial specific volume of 0.844 cm3/g, and a Stokes radius of 5.5 nm. The complex has a molecular weight of 127000 and contains three or four polypeptide chains and 112–134 detergent molecules. Antibodies directed against soluble glutathione S-transferases A, B and C do not react with the purified microsomal enzyme. This finding, together with differences in molecular weight and substrate specificity, demonstrate that the microsomal glutathione 5-transferase is an enzyme distinct from the cytosolic glutathione 5-transferases. [ABSTRACT FROM AUTHOR]

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 µM, 18 µM, and 50 µM respectively. [ABSTRACT FROM AUTHOR]

A method for the isolation of T4-amber-N82-induced DNA ligase is described which results in a nearly homogeneous enzyme preparation after two column chromatographic steps. The enzyme is detected during the purification by its ability to form a stable acid-precipitable enzyme-adenylate complex. Some properties of the assay, such as the effect of salt, temperature and incubation time, are presented. The isolated enzyme and its adenylate complex are characterized by acrylamide gel electrophoresis under native and denaturing conditions, as well as by isoelectric focusing. The purified enzyme exhibits a molecular weight of approximately 60000. Isoelectric focusing yields at least 5 protein components, which are able to form an enzyme-adenylate complex. The main activity possesses a pI of 6. The enzyme preparation is capable of repairing T5+ DNA known to contain about 4 or 5 single-strand breaks, to circularize A DNA and to join HindIII and EcoRI fragments. [ABSTRACT FROM AUTHOR]

Arginyl-tRNA synthetase from baker's yeast (Saccharomyces cerevisiae, strain 836) was obtained pure by a large-scale preparative method, which involves four chromatographic columns and one preparative polyacrylamide gel electrophoretic step. The enzyme has a high specific activity (9000 U/ mg) and consists of a single polypeptide chain of molecular weight approximately 73000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Amino acid analysis of the enzyme permitted calculation of the absorption coefficient of arginyl-tRNA synthetase At 280nm1mg/ml = 1.26). Concerning kinetic parameters of the enzyme we found the following Km values: 0.28 μM, 300 μM, 1.5 μM for tRNAIIIArg,ATP and arginine in the aminoacylation reaction, and 1400 μM, 2.5 μM, and 50 &0mu;M for ATP, arginine and PPi in the ATP-PPi exchange reaction. Arginyl-tRNA synthetase requires tRNAIIIArg to catalyse the ATP-PPi exchange reaction. [ABSTRACT FROM AUTHOR]

A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

Uronic acid dehydrogenase was purified to homogeneity. After a 338-fold purification a yield of 16% was achieved with a specific activity of 81 μmol NADH formed min-1 mg protein-1. The purity of the enzyme was controlled by disc electrophoresis, sodium dodecylsulfate electrophoresis and ultracentrifugation. A molecular weight of 60000 was determined by gel chromatography and by ultracentrifugation. The native enzyme is composed of two subunits, their molecular weight being 30000 as estimated by sodium dodecylsulfate electrophoresis. The subunits as such are inactive. The absorption spectrum with a maximum at 278 nm shows no evidence for a prosthetic group. For catalytic activity no SH groups and no metals seem to be necessary. The Michaelis constants determined with the pure enzyme are for glucuronic acid Km=0.37 mM, galacturonic acid Km=54μM and NAD+ (with glucuronic acid) Km=80μM. A weak reverse reaction could be observed with glucaric acid lactones at acidic pH. NADH is competitive with NAD+. The inhibitor constant is Ki=60 μM. The NAD+ binding site seems to be of lower specificity than the uronic acid binding site. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase was isolated from fresh human donor kidneys which had been thoroughly perfused free from blood. The isolation procedure involved biospecific affinity chromatography on a sulfanilamide—agarose column and yielded one soluble form of the enzyme, which was homogeneous with respect to sedimentation in the ultracentrifuge, electrophoresis, isoelectric focusing and immunodiffusion. The renal enzyme had an amino acid composition and behaved chromatographically, electrophoretically, and immunochemically like the erythrocyte form human carbonic anhydrase C, isolated by the same technique. The kinetic behaviour of the renal enzyme was similar to that of human carbonic anhydrase C when compared by the stopped-flow pH-indicator technique. The results therefore suggest that the cytoplasmic carbonic anhydrase of the human kidney is very similar, if not identical, to the high-activity erythrocyte form of human carbonic anhydrase C. [ABSTRACT FROM AUTHOR]

NAD kinase was purified from pigeon liver by an improved procedure which included chromatography on phosphocellulose. The resultant preparation was homogeneous as judged by gel electrophoresis, but electrofocusing gave indications of heterogeneity. The enzyme appeared to be of molecular weight 270000, and to consist of subunits of molecular weight 34000; it may therefore be an octomer. Kinetic studies over a wide range of substrate concentrations revealed departures from Michaelis­Menten behaviour with the substrate NAD+; these were interpreted tentatively in terms of negative homotropic interactions between identical binding sites, since thermal and chemical inactivation studies revealed no evidence for more than one type of catalytic site. The significance of the kinetics and of the type of inhibition produced by NADPH is discussed in terms of the regulation of NAD kinase activity in vivo. [ABSTRACT FROM AUTHOR]

Candida Albicans was able to produce a keratinolytic proteinase (KPase) when cultivated in a medium containing human stratum corneum as a nitrogen source. The KPase was purified to 108.5-fold by ion-exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration through Sephacryl S-200, while the isoelectric point was determined to be at pH 4.5. The enzyme had an optimum pH of 4.0 and was "inactive" below pH 2.5 and above pH 6.0. The activity of KPase after preincubation at various temperatures was stable up to 50°C. The keratinolytic activity was not affected by the addition of nonionic detergents and divalent cations. The enzyme was a glycoprotein and contained a high content of aspartic acid residues (172/1000) Pepstatin and chymostatin inhibited the activity in a dose-dependent manner; however, neither the other group specific inhibitors tested nor the pepsin specific inhibitors, DAN or EPNP, showed any effect on the enzyme. From these inhibitory profiles, this enzyme was determined to be a carboxyl proteinase such as cathepsin D. Among the various substrates for proteolytic enzymes, KPase digested human stratum corneum as much as albumin and hemoglobin. In the three fractions (water soluble, keratin filamentous, and membranous) prepared from human stratum corneum, the keratin filamentous fraction was more susceptible to degradation by KPase than the other two fractions were. KPase also digested much less human fingernail (13%) than human stratum corneum, but did not show any signs of there being any digestion of human scalp hair. These studies suggest that KPase from C. albicans may play an important role in superficial infection by affecting the human stratum corneum of the skin and nail. [ABSTRACT FROM AUTHOR]

A kinin-forming-enzyme in human skin extract was further purified by successive column chromatography on DEAE-cellulose, Hydroxylapatite-cellulose and Sepharose-4B. By these procedures, 2.7 mg of purified enzyme was obtained from 10 gin of original skin. The purified material was homogeneous as ascertained by cellulose acetate membrane electrophoresis, sodium dodecyl sulfate polyacrylamide gel disc electrophoresis and ultra-centrifugation. It had an S20,w, value of 4.3 and an apparent molecular weight of 104,000 as measured by gel filtration on Sephadex G-200. The purified enzyme was comparatively heat-stable, but was unstable below pH values of 5 and above pH 9. It possessed arginine or lysine esterolytic activity, but not tyrosine or tryptophane esterolytic activity and denatured proteolytic activity. This enzyme was not affected by metal ion, cystein, glutathion or ρ-chloromercuribenzoate, but was strongly inhibited by α-N-ρ-tosyl-L-lysine chioromethyl ketone or soybean-trypsin inhibitor. It was also inhibited by &alph;1-antitrypsin, but not by α2-macroglobulin. This enzyme was confirmed to be immunologically distinct from human plasma, urinary or pancreas kallikrein. [ABSTRACT FROM AUTHOR]

Radioactive leucine was incorporated by Neurospora crassa mitochondria in vivo in the presence of cycloheximide. When the membrane protein of these mitochondria was chromatographically separated on oleyl polymethacrylic acid resin, a number of fractions were obtained which differ with respect to their contents of radioactivity and cytochromes. The highest specific radioactivity was found in the fraction containing cytochrome aa3. This fraction proved to be a pure and enzymatically active cytochrome oxidase. Its ratio of absorbance at, 280 nm (ox)/ 443 nm (red.) was 2.1. By means of sodium dodecylsuffate gel-electrophoresis, this enzyme was separated into five polypeptides with molecular weights of 30000, 20000, 13000, 10000, and 8000. Only the polypeptide with the molecular weight 20000 displayed a high specific radioactivity. [ABSTRACT FROM AUTHOR]

Extracts of mycelium of Mucor rouxii have been shown to contain two forms of pyruvate kinase named Type I and II. They can be separated by DEAE-cellulose chromatography and by electrophoresis in polyacrylamide gel. Extracts from ungerminated sporangiospores or yeast-like cells contained only the Type I form. Pyruvate kinase Type II appears during aerobic germination of spores or during conversion of yeast-like cells to mycelium. Both pyruvate kinases are similar in theft molecular weight, pH range of activity, Km with respect to substrates and their response to activators (Mn++ and fructose 1,6-diphosphate). [ABSTRACT FROM AUTHOR]

1. β-Methylcrotonyl-CoA-carboxylase was purified 120-fold from an Achromobacter species. When the ceils were grown on isovaleric acid, the enzyme was formed mainly during exponential growth. The enzyme can be labeled by addition of radioactive biotin to growing cells. A former procedure of purification was improved by using adsorption to calcium phosphate gel and chromatography on Sephadex G-200. Enzyme homogeneous by both sedimentation and Tiselius electrophoresis was thus obtained in a yield of 40-70 % and was crystallized in a form resembling crystalline propionyl-CoA-carboxylase. 2. The molecular weight of the enzyme as determined by sedimentation velocity is 760000 daltons. The biotin content of 1,27 μmoles/g indicates 4 moles of biotin are bound to 1 mole of protein as is also the case for propionyl-CoA-carboxylase and pyruvate carboxylase. 3. Glutamic and aspartic acid comprise 23% of the amino acids in the enzyme. Extrapolation of electrophoretic mobility as a function of pH indicates the isoelcctric point of the enzyme is in the vicinity of pH 3.5. Such a value would mean that most of the carboxyl groups of the acidic residues are free. 4. The usual methods of preparations for electron microscopy, even with preceding fixation steps, cause degradation of the enzyme since only particles of about 1/4 of the expected size could be seen. However, ultracentrifugation did not indicate any dissociation of the enzyme into subunits. [ABSTRACT FROM AUTHOR]

The ethanolic extract of 8-week-old parsley plants was chromatographed on Dowex-1×2 with a concave gradient using the buffer system of Hurlbert. After further purification on carboncelite columns, paper chromatography and high-voltage electrophoresis, 120 μmoles of a homogeneous UDP­sugar fraction were obtained. The sugars present in this fraction were identified after acid or enzymatic hydrolysis (Crotalus adamanteus venom, acid phosphatase) by various chemical and enzymatic tests. A modified Dische test which is specific for apiose and a quantitative deterruination of D­xylose with D­glucose—oxidase were developed. According to the analytical investigations the UDP­sugar fraction had the following composition (μmol): UDP-D-glucose (84), UDP-D-galactose (27), UDP-D-xylose (4), UDP-L-arabinose (2), UDP­fructose (0.42), UDP-L-rhamnose (2.4), and UDP­apiose (0.18). The presence of UDP­apiose in parsley strongly corroborates the proposed biosynthetic pathway for the formation of D-apiose via DDP-D-glucuronic acid. [ABSTRACT FROM AUTHOR]

The purification of a phosphodiesterase from Bothrops atrox venom by chromatography on phosphocellulose and affinity chromatography with O-(4-nitrophenyl)-O'-phenyl-thiophosphate ester coupled to activated Sepharose is described. Phosphatases are removed by chromatography on hydroxyapatite. The enzyme is a single-chain protein with a molecular weight of approx 130000 as judged by dodecylsulfate-gel electrophoresis and ultracentrifugation It has a specific activity of 3.3 μmol bis(4-nitrophenyl)phosphate hydrolyzed min-1 mg-1 at 24 °C. [ABSTRACT FROM AUTHOR]

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn(2+) and the reducing agents ß -mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg(2+), Zn(2+), and Cu(2+) as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost. [ABSTRACT FROM AUTHOR]