Purification and some properties of a xylanase from Aspergillus sydowii MG49

Aspergillus sydowii MG49 produces a 30-kDa exosplitting xylobiohydrolase during growth on xylan. A specific chemical modification and substrate protection analysis of purified xylanase provided evidence that tryptophan and carboxy and amino groups are present at the catalytic site of this enzyme. Thermal inactivation of the xylanase occurs because of irreversible polymolecular aggregation, which is slower in the presence of glycerol.     

Purification and characterization of an endo-1,3-beta-glucanase from Aspergillus fumigatus

An endo-1,3-beta-glucanase was purified from a cell wall autolysate of Aspergillus fumigatus. This beta-glucanase activity was associated with a glycosylated 74-kDa protein. Using a sensitive colorimetric assay and a high-performance anion-exchange chromatography with a pulsed electrochemical detector for product analysis, it was shown that the endoglucanase hydrolysed exclusively linear 1,3-beta-glucan chains, had an optimum pH of 7.0 and an optimum temperature of 60 degrees C. A substrate kinetic study gave a Km value of 0.3 mg/ml for soluble (laminarin and laminari-oligosaccharides) and 1.18 mg/ml for insoluble (curdlan) 1,3-beta-glucan. Laminari-oligosaccharide degradation, analysed by HPLC, showed that the endoglucanase bind to the subtrate at several positions and suggested that the active site of the enzyme recognized five glucose units linked by a 1,3-beta bond. The association of the present endo-1,3-beta-glucanase with the cell wall of A. fumigatus suggests a putative role for this enzyme during cell-wall morphogenesis.     

Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae

Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.     

Purification and characterization of virginiamycin M1 reductase from Streptomyces virginiae

Virginiamycin M1 (VM1), produced by Streptomyces virginiae, is a polyunsaturated macrocyclic lactone antibiotic belonging to the virginiamycin A group. S. virginiae possesses an activity which stereospecifically reduces a 16-carbonyl group of VM1, resulting in antibiotically inactive 16R-dihydroVM1. The corresponding VM1 reductase was purified to homogeneity from crude extracts of S. virginiae in five steps, with 5,650-fold purification and 23% overall yield. The N-terminal amino acid sequence was determined to be MAIKLVIA. The purified enzyme showed an apparent Mr of 73,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 280,000 by native molecular sieve high-performance liquid chromatography, indicating the tetrameric nature of the native enzyme. NADPH served as a coenzyme for the reduction, with a Km value of 0.13 mM, but NADH did not support the reaction, even at a concentration of 5 mM, indicating the NADPH-specific nature of the enzyme. The Km for VM1 was determined to be 1.5 mM in the presence of 2 mM NADPH. In the reverse reaction, only 16R-dihydroVM1, not the 16S-epimer, served as a substrate, with a less than 0.1% overall reaction rate compared to that of the forward reaction, confirming that the VM1 reductase participates solely in VM1 inactivation in vivo.     

Purification and characterization of endothelin-1 degradation activity from porcine kidney

In order to identify the membrane-bound peptidase that is responsible for the degradation of endothelin (ET), an endothelin-1 (ET-1) degradation enzyme was solubilized from membrane fractions of porcine kidney with 1% Triton X-100, and subsequently purified by column chromatographies, i.e., diethylamino-Sepharose ion exchange, gel permeation, Con A Sepharose and hydroxyapatite chromatography. On DEAE-Toyopearl ion exchange column chromatography, the ET degradation enzyme and aminopeptidase were separated, but ET degradation enkephalinase activities were not separable. In order to separate ET degradation enzyme and enkephalinase, the active fractions were loaded on each of the column chromatographies: sephacryl S-200, Con A Sepharose or hydroxyapatite. The ET degradation activities were co-migrated with enkephalinase activities on all of the three chromatographies. In addition, the ET degradation activities were inhibited by thiorphan, phosphoramidon and EDTA, which are known to inhibit enkephalinase. These results suggest that ET degradation activity in the membrane fractions of the kidney is related to enkephalinase and may be involved in the degradation of ET-1 in vivo.     

Purification and characterization of the glucoamylase produced by a strain of Aspergillus flavus

A strain of Aspergillus flavus isolated from an agricultural soil in Egypt produced a gluycoamylase which when purified had a molecular weight of 51,300 +/- 800 Daltons. The optimum pH for activity was 4 and the optimum temperature was 60 degrees C. The enzyme was stable at 70 degrees C for 15 min but denatured at 90 degrees C over 30 min. The Km value with soluble starch was 2.85 mg ml-1, and 10 mM HgCl2 inhibited the enzyme. It was possible to store the enzyme for at least 1 year at -20 degrees C without significant loss in activity.     

Purification and characterization of glucoamylase produced by Aspergillus niger in solid state fermentation

Electrocardiographic changes, left ventricular wall motion abnormalities, myocardial ischemia, myocytolysis and arrhythmias have been well documented in patients with cerebral bleed. These complications may be related to stimulation of autonomic nervous system and central nervous system. We report a case of a 38-year-old back woman without previous heart disease, taken to emergency unit with headache and subarachnoid Haemorrhage. One day after, she complained of retroesternal pain. An electrocardiographic tracing showed significant and diffuse ST-T wave abnormalities. The patient remained stable with no neurologic or cardiac deficits. She was treated with bed rest, nimodipine, isossorbide propranolol and is symptomless six months of follow-up.     

Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus

The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40-55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5-5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatography. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.     

Purification and characterization of EDTA monooxygenase from the EDTA-degrading bacterium BNC1

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O2 were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH2 and O2. The FMN reductase provided EDTA monooxygenase with FMNH2 by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35 degreesC. Kms were 34.1 microM for uncomplexed EDTA and 8.5 microM for MgEDTA2-; this difference in Km indicates that the enzyme has greater affinity for MgEDTA2-. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH2-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.     

Purification and characterization of beta-N-acetylgalactosaminidase from Bacillus sp. AT173-1

Beta-N-Acetylgalactosaminidase [EC 3.2.1.53] was purified to homogeneity from the culture media of Bacillus sp. AT173-1. The enzyme has a molecular weight of 48,000 as estimated by SDS-PAGE under reducing conditions and an isoelectric point of 4.3. The enzyme requires dithiothreitol as an activator and is most active at pH 6.0. Analysis of its substrate specificity using 2-aminopyridine-labeled oligosaccharides as substrates revealed the enzyme specifically hydrolyzes beta-N-acetylgalactosaminyl linkages of GalNAcbeta1-4Galbeta1-4Glc, GalNAcbeta1-3Gal alpha1-4Galbeta1-4Glc, and N-glycans terminating with beta-N-acetylgalactosamine residues but not those with beta-N-acetylglucosamine residues. The enzyme is thus a novel beta-N-acetylgalactosaminidase with practically no beta-N-acetylglucosaminidase activity.     

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.     

Purification and characterization of MAR1. A mitochondrial associated ribonuclease from Leishmania tarentolae

A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mitochondrial extract of Leishmania tarentolae and the gene cloned. The purified nuclease has a Km of 100-145 +/- 33 nM and a Vmax of 1.8-2.9 +/- 2 nmol/min, depending on the RNA substrate, and yields a 3'-OH and a 5'-phosphate. Cleavage was limited to several specific sites in the substrate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was affinity-purified. The cleavage specificity of the recombinant enzyme from L. tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820-kilobase pair chromosome and contains an open reading frame of 579 nucleotides. The 18-amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Data base searching revealed two homologues of MAR1 corresponding to unidentified open reading frames in Caenorhabditis elegans (GenBankTM accession number Z69637) and Archaeoglobus fulgidus (GenBankTM accession number AE000943). The function of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be determined.     

Purification and substrate specificities of two alpha-L-arabinofuranosidases from Aspergillus awamori IFO 4033

alpha-L-Arabinofuranosidases I and II were purified from the culture filtrate of Aspergillus awamori IFO 4033 and had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum temperature of 60 degreesC and exhibited stability at pH values from 3 to 7 and at temperatures up to 60 degrees C. The enzymes released arabinose from p-nitrophenyl-alpha-L-arabinofuranoside, O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-x ylopyranose, and arabinose-containing polysaccharides but not from O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L-arabinofuranosyl-(1-->3)-O-b eta-D-xylopyranosyl-(1-->4)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyra nose. alpha-L-Arabinofuranosidase I also released arabinose from O-beta-D-xylopy-ranosyl-(1-->4)-[O-alpha-L-arabinofuranosyl- (1-->3)]- O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose. However, alpha-L-arabinofuranosidase II did not readily catalyze this hydrolysis reaction. alpha-L-Arabinofuranosidase I hydrolyzed all linkages that can occur between two alpha-L-arabinofuranosyl residues in the following order: (1-->5) linkage > (1-->3) linkage > (1-->2) linkage. alpha-L-Arabinofuranosidase II hydrolyzed the linkages in the following order: (1-->5) linkage > (1-->2) linkage > (1-->3) linkage. alpha-L-Arabinofuranosidase I preferentially hydrolyzed the (1-->5) linkage of branched arabinotrisaccharide. On the other hand, alpha-L-arabinofuranosidase II preferentially hydrolyzed the (1-->3) linkage in the same substrate. alpha-L-Arabinofuranosidase I released arabinose from the nonreducing terminus of arabinan, whereas alpha-L-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan.     

Primary structure and characterization of an exopolygalacturonase from Aspergillus tubingensis

From the culture fluid of the hyphal fungus Aspergillus tubingensis, an exopolygalacturonase with a molecular mass of 78 kDa, an isoelectric point in the pH-range 3.7-4.4 and a pH optimum of 4.2 was purified. The enzyme has been characterized as an exopolygalacturonase [poly(1,4-alpha-D-galacturonide)galacturonohydrolase] that cleaves monomer units from the non-reducing end of the substrate molecule. K(m) and Vmax for polygalacturonic acid hydrolysis were 3.2 mg ml-1 and 3.1 mg ml-1 and 255 U mg-1 and 262 U mg-1 for the wild-type and recombinant enzymes, respectively. The kinetic data of exopolygalacturonase on oligogalacturonates of different degree of polymerization (2-7) were interpreted in terms of a subsite model to obtain more insight into catalysis and substrate binding. On oligogalacturonates of different degrees of polymerization (2-7), the Michaelis constant (K(m)) decreased with increasing chain length (n). The Vmax value increased with chain length up to n = 4, then reached a plateau value. The enzyme was competitively inhibited by galacturonic acid (Ki = 0.3 mM) as well as by reduced digalacturonate (Ki = 0.4 mM). The exopolygalacturonase gene (pgaX) was cloned by reverse genetics and shows only 13% overall amino acid sequence identity with A. niger endopolygalacturonases. The exopolygalacturonase is most related to plant polygalacturonases. Only four small stretches of amino acids are conserved between all known endogalacturonases and exopolygalacturonases. Expression of the pgaX gene is inducible with galacturonic acid and is subject to catabolite repression. A fusion between the promoter of the A. niger glycolytic gene encoding pyruvate kinase and the pgaX-coding region was used to achieve high level production of exopolygalacturonase under conditions where no endopolygalacturonases were produced.     

Purification and characterization of phosphatidylglycerolphosphate synthase from Schizosaccharomyces pombe

The enzyme CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase (phosphatidylglycerolphosphate synthase; PGPS4; EC 2.7.8.5) is located in the mitochondrial inner membrane and catalyzes the committed step in the cardiolipin branch of phospholipid synthesis. Previous studies revealed that PGPS is the most highly regulated enzyme in cardiolipin biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. In this work, we report the purification to homogeneity of PGPS from S. pombe. The enzyme was solubilized from the mitochondrial membrane of S. pombe with Triton X-100. The solubilized enzyme, together with the associated detergent and intrinsic lipids, had a molecular mass of 120 kDa, as determined by gel filtration. The enzyme was further purified using salt-induced phase separation, gel filtration, and ionic exchange, hydroxylapatite, and affinity chromatographies. The procedure yielded a homogeneous protein preparation, evidenced by both SDS-polyacrylamide gel electrophoresis (PAGE) and agarose isoelectric focusing under nondenaturing conditions. The purified enzyme had an apparent molecular mass of 60 kDa as determined by SDS-PAGE. The enzyme showed a strong dependence on lipid cofactors for activity in vitro. While both phosphatidic acid and CDP-diacylglycerol appeared to be activators, the most significant activation was observed with cardiolipin. The possible physiological significance of the lipid cofactor effect is discussed. This is the first purification of a eucaryotic PGPS enzyme to date, and the first purification of a phospholipid biosynthetic enzyme from S. pombe.     

Purification and characterization of a cytotoxin from Enterobacter cloacae

OBJECTIVE: To document our evolving surgical management of colonoscopic perforation and examine factors crucial to the improvement of patient care. DESIGN: We conducted a computer-based retrospective analysis of medical records (1980 through 1995). MATERIAL AND METHODS: Among 57,028 colonoscopic procedures performed, 43 patients (0.075%, or 1 perforation in 1,333 procedures) had a colonic perforation. Two additional patients were treated after colonoscopy performed elsewhere. The outcomes analyzed included surgical morbidity and mortality. RESULTS: Twenty-six women and 19 men who ranged in age from 28 to 85 years (median, 69) were treated for colonic perforation. More than 80% of perforations occurred during the latter half of the study period because of the increased volume of colonoscopic procedures (8 perforations among 12,581 examinations from 1980 through 1987 versus 35 perforations among 44,447 colonoscopies from 1988 through 1995). Emergency laparotomy was performed in 42 patients (93%). Perforations occurred throughout the colon: right side = 10; transverse = 9; and left side = 23. Three patients without evidence of peritoneal irritation fared well with nonoperative management. Most patients underwent primary repair or limited resection in conjunction with end-to-end anastomosis. In 14 patients (33%), an ostomy was created. One patient underwent laparotomy without further treatment. Intra-abdominal contamination ranged from none (31%) to local soiling (48%) to diffusely feculent (21%). Postoperative complications occurred in 12 patients and were associated with older age (P = 0.01), large perforations (P = 0.03), and prior hospitalization (P = 0.04). No postoperative deaths occurred. CONCLUSION: Despite a consistently low risk of colonic perforation, the increasing use of colonoscopy in our practice has resulted in an increased number of iatrogenic colonic perforations. In order to minimize morbidity and mortality, prompt operative intervention is the best strategy in most patients. Non-operative management is warranted in carefully selected patients without peritoneal irritation.     

Purification and characterization of the D-hydantoinase from Bacillus circulans

A D-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from Bacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0-10.0 and a temperature of 75 degrees C. The enzyme is the most stable in a pH range of 8.5-9.5 and up to 60 degrees C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.     

Purification and characterization of trehalose phosphorylase from Catellatospora ferruginea

Trehalose phosphorylase was purified from the cell extracts of Catellatospora ferruginea. The enzyme had an apparent molecular weight of 400,000 by gel filtration and 98,000 by SDS-PAGE, suggesting that the enzyme was a tetramer. The enzyme was specific for trehalose in phosphorolysis and specific for beta-D-glucose 1-phosphate in synthesis. In addition to D-glucose, D-xylose and D-fucose were also possible sugar acceptors during synthesis. Phosphate ions were a key to the activity and stability of the enzyme, controlling the equilibrium of the reversible reaction and the heat stability of the enzyme. The enzyme was strongly inhibited by p-chloromercuribenzoate and pyridoxal phosphate. The enzyme was inactivated by heat or by storage frozen with ammonium chloride and lithium chloride.     

Purification and characterization of a prolidase from Aureobacterium esteraromaticum

An EDTA-insensitive prolidase (proline dipeptidase, EC 3.4.13.9) was isolated from a cell-free extract of Aureobacterium esteraromaticum IFO 3752. The enzyme was purified almost to homogeneity using acetone precipitation, hydrophobic chromatography, ion-exchange chromatography, and gel-permeation chromatography. The enzyme has a molecular weight of about 440,000 by gel permeation chromatography, and about 40,000 by SDS polyacrylamide gel electrophoresis. The isoelectric point was 4.6. The enzyme hydrolyzed aminoacylprolines such as Ser-Pro. Thr-Pro, Gly-Pro, Ala-Pro, Ile-Pro, Leu-Pro, and Pro-Pro. It also hydrolyzed Gly-Hyp and Pro-Hyp. The rate of hydrolysis for Pro-Hyp was the highest among the substrates tested. Optimum pH for hydrolyzing Pro-Hyp was 9.0 and the enzyme was stable in the pH range from 5 to 10. The optimum temperature was estimated to be 45 degrees C using 10 min of reaction. At least 90% of the initial activity remained after 30 min of incubation at 60 degrees C. p-Chloromercuribenzoic acid and o-phenanthrolin inhibited the enzyme's activity while EDTA did not. Addition of Mn2+ ion did not stimulate activity. These results suggest either that the metal ion in the enzyme may be tightly bound to the polypeptide chain, or that the enzyme is not a metallo-enzyme but a thiol-enzyme.