Purification and characterization of novel raw-starch-digesting and cold-adapted alpha-amylases from Eisenia foetida

Novel raw-starch-digesting and cold-adapted alpha-amylases (Amy I and Amy II) from the earthworm Eisenia foetida were purified to electrophoretically homogeneous states. The molecular weights of both purified enzymes were estimated to be 60,000 by SDS-PAGE. The enzymes were most active at pH 5.5 and 50 degrees C and stable at pH 7.0-9.0 and 50-60 degrees C. Both Amy I and II exhibited activities at 10 degrees C. The enzymes were inhibited by metal ions Cu(2+), Fe(2+), and Hg(2+), and hydrolyzed raw starch into glucose, maltose and maltotriose as end products.     

Disaccharidase activities in camel small intestine: biochemical investigations of maltase-glucoamylase activity

Disaccharidases (maltase, cellobiase, lactase, and sucrase), alpha-amylase, and glucoamylase in the camel small intestine were investigated to integrate the enzymatic digestion profile in camel. High activities were detected for maltase and glucoamylase, followed by moderate levels of sucrase and alpha-amylase. Very low activity levels were detected for lactase and cellobiase. Camel intestinal maltase-glucoamylase (MG) was purified by DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of camel small intestinal MG4 and MG6 were estimated to be 140,000 and 180,000 using Sephacryl S-200. These values were confirmed by SDS-PAGE, where the two enzymes migrated as single subunits. This study encompassed characterization of MGs from camel intestine. The Km values of MG4 and MG6 were estimated to be 13.3 mM and 20 mM maltose, respectively. Substrate specificity for MG4 and MG6 indicated that the two enzymes are maltase-glucoamylases because they catalysed the hydrolysis of maltose and starch with alpha-1,4 and alpha-1,6 glycosidic bonds, but not sucrose with alpha-1,2 glycosidic bond which was hydrolyzed by sucrase-isomaltase. Camel intestinal MG4 and MG6 had the same optimum pH at 7.0 and temperature optimum at 50 degrees C and 40 degrees C, respectively. The two enzymes were stable up to 50 degrees C and 40 degrees C, followed by strong decrease in activity at 60 degrees C and 50 degrees C, respectively. The effect of divalent cations on the activity of camel intestinal MG4 and MG6 was studied. All the examined divalent cations Ca(2+), Mn(2+), Mg(2+), Co(2+) and Fe(3+) had slight effects on the two enzymes except Hg(2+) which had a strong inhibitory effect. The effect of different inhibitors on MG4 and MG6 indicated that the two enzymes had a cysteine residue.     

Molecular cloning and biochemical characterization of the first archaeal maltogenic amylase from the hyperthermophilic archaeon Thermoplasma volcanium GSS1

Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes belonging to glycoside hydrolase family 13, have been studied extensively, but their physiological roles in microbes and evolutionary relationships with other amylolytic enzymes remain unclear. Here, we report the biochemical properties of a thermostable archaeal MAase from Thermoplasma volcanium GSS1 (TpMA) for the first time. The primary structure and catalytic properties of TpMA were similar to those of MAases, such as possession of an extra domain at its N-terminal and preference for CD over starch. TpMA showed high thermostability and optimal activity at 75 degrees C and 80 degrees C for beta-CD and soluble starch, respectively. The recombinant TpMA exists as a high oligomer in a solution and the oligomeric TpMA was dissociated into dimer and monomer mixture by a high concentration of NaCl. The substrate preference and thermostability of TpMA were significantly dependent on the oligomeric state of the enzyme. However, TpMA exhibited distinguishable characteristics from those of bacterial MAases. The transglycosylation pattern of TpMA was opposite to that of bacterial MAases. TpMA formed more alpha-1,4-glycosidic linked transfer product than alpha-1,6-linked products. Like as alpha-amylases, notably, TpMA has a longer subsite structure than those of other CD-degrading enzymes. Our findings in this study suggest that TpMA, the archaeal MAase, shares characteristics of both bacterial MAases and alpha-amylases, and locates in the middle of the evolutionary process between alpha-amylases and bacterial MAases.     

Formation of covalent beta-linked carbohydrate-enzyme intermediates during the reactions catalyzed by alpha-amylases

Porcine pancreatic and Bacillus amyloliquefaciens alpha-amylases were examined for the formation of covalent carbohydrate intermediates during reaction. The enzymes were precipitated and denatured by adding 10 volumes of acetone. When these denatured enzymes were mixed with methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and chromatographed on BioGel P-2, no carbohydrate was found in the protein void volume peak. When the enzymes were added to the methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and allowed to react for 15s at 1 degrees C and then precipitated and denatured with 10 volumes of acetone, (3)H-labeled carbohydrates were found in the BioGel P-2 protein void volume peak, indicating the formation of enzyme-carbohydrate covalent intermediates. (1)H NMR analysis of the denatured enzyme from the reaction with methyl alpha-maltooligosaccharide glycosides confirmed that carbohydrate was attached to the denatured enzyme. (1)H NMR saturation-transfer analysis further showed that the carbohydrate was attached to the denatured enzyme by a beta-configuration. This configuration is what would be expected for an enzyme that catalyzes the hydrolysis of alpha-(1-->4) glycosidic linkages by a two-step, S(N)2 double-displacement reaction to give retention of the alpha-configuration of the substrates at the reducing-end of the products.     

Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme

Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-) ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0) values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower (0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of another affinity site specific to the polysaccharides.     

Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.     

Purification and characterization of two alkaline, thermotolerant alpha-amylases from Bacillus halodurans 38C-2-1 and expression of the cloned gene in Escherichia coli

A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 10-11, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding alpha-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.     

Various physico-chemical properties of the amylolytic complex from Bacillus mesentericus]

Some physico-chemical properties of the Bacillus mesentericus amylolytic complex were studied, and optimal conditions of starch hydrolysis (pH 7.5-8.0; 45 degrees C) were found. The half-life of amylases at 50 degrees was 75 min. The heat stability of the enzymes increased in the presence of Ca2+ ions. Amylase was stable at pH 7-9 and readily inactivated at pH below 6.0. By physical and chemical characteristics the complex was close to analogous preparations from Bacillus alkalophilic strains. Isoelectrofocusing revealed that the complex consisted at least of two amylolytic enzymes.     

Isolation,characterization and inhibition by acarbose of the alpha-amylase from Lactobacillus fermentum: comparison with Lb. manihotivorans and Lb. plantarum amylases

Extracellular alpha-amylase from Lactobacillus fermentum (FERMENTA) was purified by glycogen precipitation and ion exchange chromatography. The purification was approximately 28-fold with a 27% yield. The FERMENTA molecular mass (106,000 Da) is in the same range as the ones determined for L. amylovorus (AMYLOA), L. plantarum (PLANTAA) and L. manihotivorans (MANIHOA) alpha-amylases. The amino acid composition of FERMENTA differs from the other lactobacilli considered here, but however, indicates that the peptidic sequence contains two equal parts: the N-terminal catalytic part; and the C-terminal repeats. The isoelectric point of FERMENTA, PLANTAA, MANIHOA are approximately the same (3.6). The FERMENTA optimum pH (5.0) is slightly more acidic and the optimum temperature is lower (40 degrees C). Raw starch hydrolysis catalyzed by all three amylases liberates maltotriose and maltotretaose. Maltose is also produced by FERMENTA and MANIHOA. Maltohexaose FERMENTA catalyzed hydrolysis produces maltose and maltotriose. Finally, kinetics of FERMENTA, PLANTAA and MANIHOA using amylose as a substrate and acarbose as an inhibitor, were carried out. Statistical analysis of kinetic data, expressed using a general velocity equation and assuming rapid equilibrium, showed that: (1) in the absence of inhibitor k(cat)/Km are, respectively, 1x10(9), 12.6x10(9) and 3.2x10(9) s(-1) M(-1); and (2) the inhibition of FERMENTA is of the mixed non-competitive type (K(1i)=5.27 microM; L(1i)=1.73 microM) while the inhibition of PLANTAA and MANIHOA is of the uncompetitive type (L(1i)=1.93 microM and 1.52 microM, respectively). Whatever the inhibition type, acarbose is a strong inhibitor of these Lactobacillus amylases. These results indicate that, as found in porcine and barley amylases, Lactobacillus amylases contain in addition to the active site, a soluble carbohydrate (substrate or product) binding site.     

Presence of rhodanese in the cytosolic fraction of the fruit bat (Eidolon helvum) liver

Rhodanese was isolated and purified from the cytosolic fraction of liver tissue homogenate of the fruit bat, Eidolon helvum, by using ammonium sulphate precipitation and CM-Sephadex C-50 ion exchange chromatography. The specific activity was increased 130-fold with a 53% recovery. The K(m) values for KCN and Na(2)S(2)O(3) as substrates were 13.5 +/- 2.2mM and 19.5 +/- 0.7 mM, respectively. The apparent molecular weight was estimated by gel filtration on a Sephadex G-100 column to be 36,000 Da. The optimal activity was found at a high pH (pH 9.0) and the temperature optimum was 35 degrees C. An Arrhenius plot of the heat stability data consisted of two linear segments with a break occurring at 35 degrees C. The apparent activation energy values from these slopes were 11.5 kcal/mol and 76.6 kcal/mol. Inhibition studies on the enzyme with a number of cations showed that Mg(2+), Mn(2+), Ca(2+), and Co(2+) did not affect the activity of the enzyme, but Hg(2+) and Ba(2+) inhibited the enzyme.     

Purification and Characterization of a Maltotetraose-Forming Alkaline (alpha)-Amylase from an Alkalophilic Bacillus Strain, GM8901

An alkalophilic bacterium, Bacillus sp. strain GM8901, grown at pH 10.5 and 50(deg)C, produced five alkaline amylases in culture broth. At an early stage of the bacterial growth, amylase I (Amyl I) was produced initially and then, as cultivation progressed, four alkaline amylases, Amyl II, Amyl III, Amyl IV, and Amyl V, were produced from proteolytic degradation of Amyl I. A serine protease present in the culture medium was believed to be involved in Amyl I degradation. We purified Amyl I from the culture supernatant by ammonium sulfate precipitation, heparin-Sepharose CL-6B column chromatography, phenyl-Toyopearl column chromatography, and Mono Q HR5/5 high-performance liquid chromatography. The molecular weight of Amyl I was estimated to be about 97,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amyl I had an extremely high optimal pH of 11.0 to 12.0 and was stable in a broad pH range of 6.0 to 13.0. Amyl I had an optimal temperature of 60(deg)C and was stable up to 50(deg)C. Thermostability was increased in the presence of Ca(sup2+) and soluble starch. The enzyme required metal ions such as Ca(sup2+), Mg(sup2+), Cu(sup2+), Co(sup2+), Ag(sup+), Zn(sup2+), and Fe(sup2+) for its enzyme activity and was inhibited by 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. According to the mode of action of Amyl I on starch, Amyl I was classified as an (alpha)- and exo-amylase. Amyl I produced maltotetraose predominantly from starch via intermediates such as maltohexaose and maltopentaose.     

Alginate beads as an affinity material for alpha amylases

Calcium-alginate beads were found to bind a variety of enzymes in a nonspecific fashion. However, alpha amylases from porcine pancreas, Bacillus subtilis (BAN 240L) and wheat germ bound at a significant level and B. subtilis and wheat germ amylases could be eluted with 1M maltose. The wheat germ alpha amylase could be purified 45 fold with 70% recovery. The SDS - PAGE pattern showed significant purification by this single step strategy.     

Characterization and large-scale production of recombinant <Emphasis Type="Italic">Streptoverticillium platensis</Emphasis> transglutaminase

Recombinant Streptomyces platensis transglutaminase (MtgA) produced by the Streptomyces lividans transformant 25-2 was purified by ammonium sulfate fractionation, followed by CM-Sepharose CL-6B fast flow, and blue-Sepharose fast flow chromatography. The purification factor was ~33.2-fold, and the yield was 65%. The molecular weight of the purified recombinant MtgA was 40.0 KDa as estimated by SDS-PAGE. The optimal pH and the temperature for the enzyme activity were 6.0 and 55 degrees C, respectively, and the enzyme was stable at pH 5.0-6.0 and at temperature 45-55 degrees C. Enzyme activity was not affected by Ca(2+), Li(+), Mn(2+), Na(+), Fe(3+), K(+), Mg(2+), Al(3+), Ba(2+), Co(2+), EDTA, or IAA but was inhibited by Fe(2+), Pb(2+), Zn(2+), Cu(2+), Hg(2+), PCMB, NEM, and PMSF. Optimization of the fermentation medium resulted in a twofold increase of recombinant MtgA activity in both flasks (5.78 U/ml) and 5-l fermenters (5.39 U/ml). Large-scale productions of the recombinant MtgA in a 30-l air-lift fermenter and a 250-l stirred-tank fermenter were fulfilled with maximal activities of 5.36 and 2.54 U/ml, respectively.     

Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.     

Purification and partial characterization of fructosyltransferase and invertase from Aspergillus niger AS0023

Fructosyltransferase (EC.2.4.1.9) and invertase (EC.3.2.1.26) have been purified from the crude extract of Aspergillus niger AS0023 by successive chromatographies on DEAE-sephadex A-25, sepharose 6B, sephacryl S-200, and concanavalin A-Sepharose 4B columns. On acrylamide electrophoresis the two enzymes, in native and denatured forms, gave diffused glycoprotein bands with different electrophoretic mobility. On native-PAGE and SDS-PAGE, both enzymes migrated as polydisperse aggregates yielding broad and diffused bands. This result is typical of heterogeneous glycoproteins and the two enzymes have proved their glycoprotein nature by their adsorption on concanavalin A lectin. Fructosyltransferase (FTS) on native PAGE migrated as two enzymatically active bands with different electrophoretic mobility, one around 600 kDa and the other from 193 to 425 kDa. On SDS-PAGE, these two fractions yielded one band corresponding to a molecular weight range from 81 to 168 kDa. FTS seems to undergo association-dissociation of its glycoprotein subunits to form oligomers with different degrees of polymerization. Invertase (INV) showed higher mobility corresponding to a molecular range from 82 to 251 kDa, on native PAGE, and from 71 to 111 kDa on SDS-PAGE. The two enzymes exhibited distinctly different pH and temperature profiles. The optimum pH and temperature for FTS were found to be 5.8 and 50 degrees C, respectively, while INV showed optimum activity at pH 4.4 and 55 degrees C. Metal ions and other inhibitors had different effects on the two enzyme activities. FTS was completely abolished with 1 mM Hg(2+) and Ag(2+), while INV maintained 72 and 66% of its original activity, respectively. Furthermore, the two enzymes exhibited distinctly different kinetic constants confirming their different nature. The K(m) and V(m) values for each enzyme were calculated to be 44.38 mM and 1030 micromol ml(-1)min(-1) for FTS and 35.67 mM and 398 micromol ml(-1) min(-1) for INV, respectively. FTS and INV catalytic activity was dependent on sucrose concentration. FTS activity increased with increasing sucrose concentrations, while INV activity decreased markedly with increasing sucrose concentration. Furthermore, INV exhibited only hydrolytic activity producing exclusively fructose and glucose from sucrose, while FTS catalyzed exclusively fructosyltransfer reaction producing glucose, 1-kestose, nystose and fructofuranosyl nystose. In addition, at 50% sucrose concentration FTS produced fructooligosaccharides at the yield of 62% against 54% with the crude extract.     

Significant differences in the activities of alpha-amylases in the absence and presence of polyethylene glycol assayed on eight starches solubilized by two methods

Starch is a reserve chemical source of the energy of the sun found in plants as a water-insoluble granule that differs in their chemical and physical properties, depending on the source. The granules can be solubilized by heating in water or by treatment with various reagents, such as 1M NaOH. alpha-Amylases are widely distributed enzymes that initiate the hydrolysis of starch into low molecular weight maltodextrins. We recently found that the activities of a single alpha-amylase on two different starches were significantly different. We then determined the activities of Bacillus amyloliquefaciens and porcine pancreas alpha-amylases, using eight different starches, solubilized by two methods: autoclaving at 121 degrees C and 1M NaOH at 20 degrees C. There were significant differences in the activities of both of the amylases on all eight of the starches. Previously, it had been found that polyethylene glycol (PEG) stabilized and activated the activities of both enzymes, using a soluble amylose as the substrate. Addition of PEG to the enzymes greatly increased the activities on the eight starches, but the activities still differed significantly. The different activities with the starches were hypothesized as differences in the amounts of secondary and tertiary structures that are partially retained when the different starches are solubilized; the activities on addition of PEG is hypothesized as the formation of highly active species from a series of less active forms.     

Production and immobilization of D-aminoacylase of Alcaligenes faecalis DA1 for optical resolution of N-acyl-DL-amino acids.

The production of D-aminoacylase by Alcaligenes faecalis DA1 was induced 5- to 50-fold by N-acetyl-D-amino acids. This strain produced about 443 units of D-aminoacylase and 52 units of L-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-DL-leucine as the carbon source. The D-aminoacylase was partially purified by Fractogel DEAE 650 column chromatography and then immobilized on another Fractogel DEAE 650 column. The catalytic activity of the immobilized D-aminoacylase was 2,650 units per milliliter of gel. The Km values for the free and the immobilized enzymes were found to be 1.00 and 0.22 mM, respectively, using N-acetyl-D-methionine as a substrate. The optimal reaction pH and temperature for both soluble and immobilized enzyme were around 8.0 and 45 degrees C, respectively. The free enzyme was stable in the pH range from 5.0 to 11.0, whereas the immobilized enzyme tended to detach from the gel at pH values higher than 9.0. Both forms of enzyme were stable up to 40 degrees C. When used for the optical resolution of N-acetyl-DL-methionine, the immobilized enzyme maintained 90% initial activity after 17 days of continuous operation at 45 degrees C. The process of purification and immobilization of D-aminoacylase described in this report is very effective and easy to scale up.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.