Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg²⁺, Co²⁺, and Mn²⁺ were required for maximum activity of the enzyme. The K_m values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while K_cat values were 2.3 × 10² s^-1 and 0.5 × 10² s^-1, respectively.     

A bifunctional beta-xylosidase-xylose isomerase from Streptomyces sp. EC 10

beta-Xylosidase (1,4-beta-D-xylan xylohydrolase EC 3.2.1.37) and xylose isomerase (D-xylose ketol-isomerase EC 5.3.1.5) produced by Streptomyces sp. strain EC 10, were cell-bound enzymes induced by xylan, straw, and xylose. Enzyme production was subjected to a form of carbon catabolite repression by glycerol. beta-Xylosidase and xylose isomerase copurified strictly, and the preparation was found homogeneous by gel electrophoresis after successive chromatography on DEAE-Sephacel and gel filtration on Biogel A. Streptomyces sp. produced apparently a bifunctional beta-xylosidase-xylose isomerase enzyme. The molecular weight of the enzyme was measured to be 163,000 by gel filtration and 42,000 by SDS-PAGE, indicating that the enzyme behaved as a tetramer of identical subunits. The Streptomyces sp. beta-xylosidase was a typical glycosidase acting as an exoenzyme on xylooligosaccharides, and working optimally at pH 7.5 and 45 degrees C. The xylose isomerase optimal temperature was 70 degrees C and maximal activity was observed in a broad range pH (5-8). Enhanced saccharification of arabinoxylan caused by the addition of the enzyme to endoxylanase suggested a cooperative enzyme action. The first 35 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of xylose isomerase produced by other microorganisms but not with other published N-terminal sequences of beta-xylosidases.     

Localization and Characterization of alpha-Glucosidase Activity in Brettanomyces lambicus

Brettanomyces lambicus was isolated and identified from a typical overattenuating Belgian lambic beer and exhibited extracellular and intracellular alpha-glucosidase activities. Production of the intracellular enzyme was higher than production of the extracellular enzyme, and localization studies showed that the intracellular alpha-glucosidase is mostly soluble and partially cell wall bound. Both intracellular and extracellular enzymes were purified by ammonium sulfate precipitation, gel filtration (Sephadex G-150, Sephadex G-200, Ultrogel AcA-44), and ion-exchange chromatography (sulfopropyl-Sephadex C-50, (carboxymethyl-Sephadex C-50). The intracellular alpha-glucosidase exhibited optimum activity at 39 degrees C and pH 6.2. The extracellular enzyme exhibited optimum catalytic activity at 40 degrees C and pH 6.0. The molecular masses of purified intracellular and extracellular alpha-glucosidases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 72,500 and 77,250, respectively. For both enzymes there was a decrease in the rate of hydrolysis with an increase in the degree of polymerization, and both enzymes hydrolyzed dextrins isolated from lambic wort (degrees of polymerization, 3 to 9 and more than 9). The K(m) values for p-nitrophenyl-alpha-d-glucopyranoside, maltose, and maltotriose for the intracellular enzyme were 0.9, 3.4, and 3.7 mM, respectively. The K(i) values for both enzymes were between 28.5 and 57 muM for acarbose and between 7.45 and 15.7 mM for Tris. These enzymes are probably involved in the overattenuation of spontaneously fermented lambic beer.     

Production, purification, and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus

Enhancement of the productivity of xylanase and beta-xy-losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N-methyl-N-nitro-N-nitro-soguanidine (NTG). The hyperxylanolytic mutant (NG-13), which showed good clearing on the surface of the xylan-agar plate, secretes xylanase and beta-xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG-13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45 degrees C though its activity was maximal at 50 degrees C. The molecular weight of the enzyme was estimated to be 4.3 x 10(4) by SDS-polyacrylamide gel electrophoresis and 5 x 10(4) by gel filtration on Sephadex G-75. The kinetic data showed that the K(m) and V(max) values for xylan were 1 x 10(-3)M and 19.6 mumol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K(+)and was inactivated by thiol reagents such as Hg(2+), p-hydroxymercuribenzoate (PHMB), 3', 5'-dithiobis (2'-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM).     

Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489.

An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60 degrees C) or pH 6.8 (80 degrees C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn2+, Co2+, or Mg2+ for thermal stability (stable for 1 h at 82 degrees C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80 degrees C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85 degrees C, respectively). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed significantly from the T. saccharolyticum B6A-RI xylose isomerase suggested that one or more of the observed amino acid changes was responsible for this observed difference.     

Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site.

Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.     

Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-0

A Bacillus spp. strain SPS-0, isolated from a hot spring in Portugal, produced an extracellular xylanase upon growth on wheat bran arabinoxylan. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange, gel filtration, and affinity chromatography. The optimum temperature and pH for activity was 75 degrees C and 6.0. Xylanase was stable up to 70 degrees C for 4 h at pH 6.0 in the presence of xylane. Xylanase was completely inhibited by the Hg(2+) ions. beta-Mercaptoethanol, dithiothreitol, and Mn(2+) stimulated the xylanase activity. The products of birchwood xylan hydrolysis were xylose, xylobiose, xylotriose, and xylotetraose. Kinetic experiments at 60 degrees C and pH 6.0 gave V(max) and K(m)values of 2420 nkat/mg and 0.7 mg/ml.     

Aminoacyl-tRNA synthetases from Bacillus stearothermophilus. Asymmetry of substrate binding to tyrosyl-tRNA synthetase.

The interaction of L-tyrosine, L-tyrosyladenylate and tRNA-Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by equilibrium dialysis, gel filtration and fluorescence spectroscopy. The enzyme, which consists of two identical subunits (mol. wt 2 x 44000), binds only a single molecule of L-tyrosine per dimer with a K-d of 2 x 10-5 M at pH 7.8 and 23 degrees C. The tyrosyl-tRNA synthetase--tyrosyladenylate complex which was isolated by gel filtration also has one adenylate bound per dimeric enzyme molecule. In contrast, two tRNA-Tyr molecules bind per enzyme dimer, but the two binding sites are not equivalent having K-d values of 2 x 10-7 M and 1.3 x 10-6 M respectively at pH 6.5 and 25 degrees C. Since crystallographic analysis of the free enzyme [2] shows that the monomer is the asymmetric unit, the data indicate that substrate binding induces asymmetry in the enzyme.     

Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp at pH 9 and 65 degrees C. The enzyme was purified and concentrated in a single adsorption step onto a cation exchanger and is made of a single polypeptide with an apparent M(r) of 43,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Xylanase T-6 is an endoxylanase that completely degrades xylan to xylose and xylobiose. The pIs of the purified protein were 9 and 7 under native and denaturing conditions, respectively. The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10. At 65 degrees C and pH 7, the enzyme was stable for more than 10 h; at 65 degrees C and pH 9, the half-life of the enzyme was approximately 6 h. Kinetic experiments at 55 degrees C gave Vmax and Km values of 288 U/mg and 1.63 mg/ml, respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by Zn2+, Cd2+, and Hg2+. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region of xylanase A from the alkalophilic Bacillus sp. strain C-125.     

A novel thermostable alpha-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: purification and characterization

High levels of an extracellular alpha-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH(4))(2)SO(4) fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric alpha-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5-5.5 and 65 degrees C, respectively. The purified enzyme retains more than 90% of its activity at 45 degrees C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-alpha-galactopyranoside, raffinose and stachyose and very similar K(m) values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn(++) ions activates enzyme activity, whereas inhibitory effects can be observed with Ca(++), Zn(++) and Hg(++). Five min incubation at 65 degrees with 10 mM Ag(+) results in complete inactivation of the purified alpha-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the alpha-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.     

Purification and characterization of a new xylanase (APX-II) from the fungus Aureobasidium pullulans Y-2311-1.

Aureobasidium pullulans Y-2311-1 produced four major xylanases (EC 3.2.1.8) with pI values of 4.0, 7.3, 7.9, and 9.4 as revealed by isoelectric focusing and zymogram analysis when grown for 4 days on 1.0% oat spelt xylan. The enzyme with a pI of 9.4 was purified by ammonium sulfate precipitation, chromatography on a DEAE-Sephadex A-50 column, and gel filtration with a Sephadex G-75 column. The enzyme had a mass of about 25 kDa as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The purified enzyme had a Km of 7.6 mg . ml(-1) and Vmax of 2,650 micromol . min(-1) . mg(-1) for birchwood xylan at 28 degrees C and pH 4.5. It lacked activity towards carboxymethylcellulose, cellobiose, starch, mannan, p-nitrophenyl (pNP)-beta-D-xylopyranoside, pNP-beta-D-glucopyranoside, pNP-alpha-D-glucopyranoside, pNP-beta-D-cellobioside, pNP-beta-D-fucopyranoside, or pNP-alpha-D-galactopyranoside. The predominant end products of birchwood xylan or xylohexaose hydrolysis were xylobiose and xylose. The enzyme had the highest activity of pH 4.8 and 54 degrees C. Sixty percent of the activity remained after the enzyme had been incubated at 55 degrees C and pH 4.5 for 30 min. The sequence of the first 68 amino acid residues at the amino terminus showed homology to those of several other xylonases. Immunoblot analysis with antiserum raised against the purified xylanase revealed that two immunologically related polypeptides of 25 and 22 kDa were produced in A. pullulans cultures containing oat spelt xylan or xylose as carbon sources but not in cultures containing glycerol or glucose.     

Isolation and properties of an extracellular beta-glucosidase from the polycentric rumen fungus Orpinomyces sp. strain PC-2.

An extracellular beta-glucosidase (EC 3.2.1.21) was purified from culture filtrate of the anaerobic rumen fungus Orpinomyces sp. strain PC-2 grown on 0.3% (wt vol-1) Avicel by using Q Sepharose anion-exchange chromatography, ammonium sulfate precipitation, chromatofocusing ion-exchange chromatography, and Superose 12 gel filtration. The enzyme is monomeric with a M(r) of 85,400, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has a pI of 3.95, and contains about 8.5% (wt vol-1) carbohydrate. The N terminus appears to be blocked. The enzyme catalyzes the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (PNPG). The Km and Vmax values with cellobiose as the substrate at pH 6.0 and 40 degrees C are 0.25 mM and 27.1 mumol.min-1 x mg-1, respectively; with PNPG as the substrate, the corresponding values are of 0.35 mM and 27.7 mumol.min-1 x mg-1. Glucose (Ki = 8.75 mM, with PNPG as the substrate) and gluconolactone (Ki = 1.68 x 10(-2) and 2.57 mM, with PNPG and cellobiose as the substrates, respectively) are competitive inhibitors. Optimal activity with PNPG and cellobiose as the substrates is at pH 6.2 and 50 degrees C. The enzyme has high activity against sophorose (beta-1,2-glucobiose) and laminaribiose (beta-1,3-glucobiose) but has no activity against gentiobiose (beta-1,6-glucobiose). The activity of the beta-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.     

Isolation and Some Properties of a beta-d-Xylosidase from Clostridium acetobutylicum ATCC 824

A beta-d-xylosidase from C. acetobutylicum ATCC 824 was purified by column chromatography on CM-Sepharose, hydroxylapatite, Phenyl Sepharose, and Sephadex G-200. The enzyme had an apparent molecular weight of 224,000 as estimated by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme consisted of two subunits of 85,000 and one subunit of 63,000 daltons. It exhibited optimal activity at pH 6.0 to 6.5 and 45 degrees C. the enzyme had an isoelectric point of 5.85. It hydrolyzed p-nitrophenylxyloside readily with a K(m) of 3.7 mM. The enzyme hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 6 units by cleaving a single xylose from the chain end. It showed little or no activity against xylan, carboxymethyl cellulose, and other p-nitrophenylglycosides.     

Purification and some kinetic properties of catalase from parsley (Petroselinum hortense Hoffm., Apiaceae) leaves

In this study, catalase (CAT: EC 1.11.1.6) was purified from parsley (Petroselinum hortense) leaves; analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps, including preparation of homogenate, ammonium sulfate fractionation, and fractionation by DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 9.5% and had a specific activity of 1126 U (mg proteins)(-1). The overall purification was about 5.83-fold. A temperature of 4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured at 240 nm. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acryl amide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for the enzyme. The molecular weight was found to be 183.29 kDa by Sephadex G-200 gel filtration chromatography. The stable pH, optimum pH, and ionic strength were determined for phosphate and Tris-HCl buffer systems. In addition, K(M) and V(max) values for H(2)O(2), at optimum pH and 25 degrees C, were determined by means of Lineweaver-Burk plots.     

Partial Purification and Some Properties of Tyrosine Phenol-Lyase from Aeromonas phenologenes ATCC 29063

Tyrosine phenol-lyase was purified 32-fold from Aeromonas phenologenes ATCC 29063, the organism that produces phenol in refrigerated haddock. The purification procedure included ammonium sulfate fractionation, protamine sulfate treatment, and column chromatography with Sephadex G-200, diethyl-aminoethyl-cellulose, and hydroxyapatite. The enzyme was found to be thermally inactivated at temperatures above 40 degrees C. The optimum pH of the enzyme was found to be pH 8.5. The Michaelis constants for l-tyrosine and pyridoxal phosphate were 2.3 x 10 M and 3.2 x 10 M, respectively. The molecular weight of tyrosine phenol-lyase was found by gel filtration and electrophoresis to be approximately 380,000.     

xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana.

The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892. The native enzyme was a homotetramer with a molecular weight of 200,000. This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km. The T. neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases. Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures.     

An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Purification and characterization of alpha-L-arabinofuranosidase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produced an alpha-L-arabinofuranosidase when grown in the presence of L-arabinose, sugar beet arabinan, or oat spelt xylan. At the end of a fermentation, about 40% of the activity was extracellular, and enzyme activity in the cell-free supernatant could reach 25 U/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 20000, and the enzyme was purified eightfold by anion-exchange and hydrophobic interaction chromatographies. The molecular weight of T-6 alpha-L-arabinofuranosidase was 256,000, and it consisted of four identical subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The native enzyme had a pI of 6.5 and was most active at 70 degrees C and at pH 5.5 to 6.0. Its thermostability at pH 7.0 was characterized by half-lives of 53, 15, and 1 h at 60, 65, and 70 degrees C, respectively. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave a Vmax, a Km, and an activation energy of 749 U/mg, 0.42 mM, and 16.6 kcal/mol, (ca. 69.5 kJ/mol), respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by 1 mM Hg2+. T-6 alpha-L-arabinofuranosidase released L-arabinose from arabinan and had low activity on oat spelt xylan. The enzyme acted cooperatively with T-6 xylanase in hydrolyzing oat spelt xylan, and L-arabinose, xylose, and xylobiose were detected as the end reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties and function of malate enzyme from Pseudomonas putida

Malate enzyme (L-malate: NADP+ oxidoreductase (oxalacetate-decarboxylating, EC 1.1.1.40)) has been purified from Pseudomonas putida to 99 per cent homogeneity by heat, ammonium suphate fractionation, gel filtration and anion exchange chromatography. Sodium dodecylsulphate-(SDS)-polyacrylamide disc gel electrophoresis analysis showed an approximate tetrameric subunit with a molecular weight of 52,000. The purified enzyme showed a pH optimum between 8.0 and 8.5 (for Tris-HCl buffer) and required bivalent cations for catalysis; monovalent ions like K+ and NH4+ acted as very effective activators. The temperature-activity relationship for the malate enzyme from 35-80 degrees C showed broken Arrhenius plots with an inflexion at 65 degrees C. The enzyme halflife was 30s at 85 degrees C. The enzyme showed hyperbolic kinetics for both substrates with apparent Km values of 4.0 X 10(-4) M and 2.3 X 10(-5) M for L-malate and NADP+ respectively. From the study of the effects of some compounds on the enzyme, the physiological significance of those produced by fumarate, succinate and oxalacetate can be emphasized.     

Purification and characterisation of lignin peroxidase from Pycnoporus sanguineus MTCC-137

Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass 40 kDa. The K(m)(, kcat) and k(cat)/K(m) values of the enzyme using veratryl alcohol and H2O2 as the substrate were 61 microM, 2.13 s(-1), 3.5 x 10(4) M(-1) s(-1) and 71 microM, 2.13 s(-1), 3.0 x 10(4) M(-1) s(-1) respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25 degrees C.