Recombinant DNA-methyltransferase M1.Bst19I from <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> 19: Purification,properties, and amino acid sequence analysis

The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5′-GCATC-3′) in Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW that carries a tandem of thermo inducible promoters P _R /P _L from phage λ. Highly purified enzyme has been isolated by chromatography on various resins from Escherichia coli cells where it is accumulated in a soluble form. The study of M1.Bst19I properties has revealed that the enzyme has a temperature optimum at 50°C and demonstrates maximal activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5′-GCATC-3′. Kinetic parameters of M1.Bst19I DNA methylation reaction have been determined as follows: Km for λ DNA is 0.68 ± 0.07 μM, Km for S-adenosyl-L-methionine is 2.02 ± 0.31 μM. Catalytical constant (k _cat) is 1.8 ± 0.05 min⁻¹. Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and other α-N6-DNA-methyltransferases has allowed us to suggest the presence of two types of the enzymes containing ATG or ATC triplets in the recognition sequence.     

Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> S7

Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40°C. K _m values for NO₃ ⁻ (110 μM) and for ClO₃ ⁻ (138 μM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.     

Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI

DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases. Gene M1.BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers. It has been shown that M1.BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min⁻¹. K _{mphage is λ DNA}—0.053 ± 0.007 μM, and K _mSAM is 5.1 ± 0.3 μM.     

Recombinant DNA-methyltransferase M1.BspACI from <Emphasis Type="Italic">Bacillus psychrodurans</Emphasis> AC: Purification and properties

A restriction-modification system from Bacillus psychrodurans AC (recognition sequence 5′-CCGC-3′) comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. The bspACIM1 gene was cloned in the pJW2 vector and expressed in Escherichia coli cells. High-purity M1.BspACI preparation has been obtained by chromatography on different carriers. M1.BspACI has a temperature optimum of 30°C and demonstrates maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the recognition sequence 5′-CCGC-3′. The kinetic parameters of M1.BspACI DNA methylation are as follows: K _m for phage λ DNA is 0.053 μM and K _m for S-adenosyl-L-methionine is 5.1 μM. The catalytic constant (k _cat) is 0.095 min⁻¹.     

Endo-β-Glucanase from Acetobacter xylinum: Purification and Characterization

A cellulose-producing acetic acid bacterium, Acetobacter xylinum KU-1, abundantly produces an extracellular endo-β-glucanase (EC 3.2.1.4) in the culture broth. The enzyme was purified to homogeneity by DEAE- and CM- Toyopearl 650M ion-exchange chromatography, Butyl-Toyopearl 650M hydrophobic chromatography, and Toyopearl HW-50 gel filtration. The purified enzyme showed the maximum activity at pH 5 and 50°C: it was stable up to 50°C at pH 5, activated by Co²⁺, and competitively inhibited by Hg²⁺; the apparent K _i was 7 μM. The molecular weight of the enzyme was determined to be about 39,000 by sodium dodesyl sulfate/polyacrylamide gel electrophoresis, and about 41,000 by Toyopearl HW-50 gel filtration; the enzyme is monomeric. The enzyme hydrolyzed carboxymethylcellulose with an apparent K _m of 30 mg/ml and V _max of 1.2 μM/min. It hydrolyzed cellohexaose to cellobiose, cellotriose and cellotetraose, and also cellopentaose to cellobiose and cellotriose, but did not act on cellobiose, cellotriose, or cellotetraose. Received: 3 October 1996 / Accepted: 5 November 1996     

Purification, characterization, and primary structure of a monofunctional catalase from Methanosarcina barkeri

Methanosarcina barkeri is a strictly anaerobic, cytochrome-containing, methane-forming archaeon. We report here that the microorganism contains a catalase, which was purified and characterized. The enzyme with an apparent molecular mass of 190 kDa was shown to be composed of four identical subunits of apparent molecular mass of 54 kDa. The heme-containing enzyme did not exhibit peroxidase activity, which indicates that it is a monofunctional catalase. This is substantiated by the primary structure, which is related to that of other monofunctional catalases rather than to that of bifunctional catalase-peroxidases. The enzyme showed an [S]_0.5V for H₂O₂ of 25 mM and an apparent V _max of 200,000 U/mg; it was inhibited by azide ([I]_0.5V = 1 μM) and cyanide ([I]_0.5V = 5 μM) and inactivated by 1,2,4-aminotriazole. The activity was almost independent of the pH (between pH 4 and 10) and the temperature (between 15 °C and 55 °C). Comparison of the primary structure of monofunctional catalases revealed that the enzyme from M. barkeri is most closely related to the monofunctional catalase of Dictyostelium discoideum. Received: 29 December 1998 / Accepted: 1 March 1999     

Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 α-amylase

The starch-binding domain of Bacillus sp. strain TS-23 α-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel–chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70°C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K _m value. Starch-binding assays showed that the K _d and B _max values for the fusion enzyme were 2.3 μM and 0.35 μmol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris–HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption–elution cycle with a purification of 11.4-fold.     

Effects of gibberellic acid and abscisic acid on α-amylase activity and ultrastructure of aleurone cells ofAvena sativa L.

The effects of GA₃ and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied. α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA₃ solution or 100 μM GA₃+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA₃+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA₃+10 μM ABA.     

Purification and characterization of an intracellular β-glucosidase from the protoplast fusant of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4,000 with 0.01 mM CaCl₂. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular (β-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 5.4 and temperature of 65°C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0–6.6. Na⁺, K⁺, Ca²⁺, Mg²⁺ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe³⁺. The enzyme activity was strongly inhibited by glucose, the end product of glucoside hydrolysis. The K _m and V _max values against salicin as substrate were 0.035 mM and 1.7215 μmol min⁻¹, respectively.     

Biochemical controls of citrate synthase in chickpea bacteroids

Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear. The K _m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by NADH was linear competitive with respect to acetyl-CoA (K _is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K _is = 56 ± 3.8 μM and K _ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD⁺ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely to be relieved by NAD⁺ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle. Received: 14 July 1999 / Accepted: 20 September 1999     

A novel membrane-bound flavocytochrome c sulfide dehydrogenase from the colourless sulfur bacterium Thiobacillus sp. W5

A novel membrane-bound sulfide-oxidizing enzyme was purified 102-fold from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5 by means of a six-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 40 and 11 kDa. The smaller subunit contains covalently bound haem c, as was shown by haem staining. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide-oxidizing heterodimer contains one molecule of haem c and one molecule of flavin. It appeared that the sulfide-oxidizing enzyme is a member of a small class of redox proteins, the flavocytochromes c, and is structurally most related to the flavocytochrome c sulfide dehydrogenase of the green sulfur bacterium Chlorobium limicola. The pH optimum of the enzyme is 8.6. At pH 9, the V _max was 2.1 ± 0.1 μmol cytochrome c (mg protein)^–1 min^–1, and the K _m values for sulfide and cytochrome c were 1.7 ± 0.4 μM and 3.8 ± 0.8 μM, respectively. Cyanide inhibited the enzyme by the formation of an N-5 adduct with the flavin moiety of the protein. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product. Received: 15 October 1996 / Accepted: 7 January 1997     

Chromate reduction by Rhodobacter sphaeroides

Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration. The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K _m of 15±1.3 μM CrO₄ ²⁻ and a V _max of 420±50 μmol min⁻¹ mg protein⁻¹ was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO₄ ²⁻ and 100±9.6 μmol CrO₄ ²⁻ min⁻¹ mg protein⁻¹ for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203. Received 05 January 2000/ Accepted in revised form 27 May 2000     

Preparation and some properties of cholesterol oxidase from Rhodococcus sp. R14-2

Rhodococcus sp. R_14-2, isolated from Chinese Jin-hua ham, produces a novel extracellular cholesterol oxidase (COX). The enzyme was extracted from fermentation broth and purified 53.1-fold based on specific activity. The purified enzyme shows a single polypeptide band on SDS-PAGE with an estimated molecular weight of about 60 kDa, and has a pI of 8.5. The first 10 amino acid residues of the NH₂-terminal sequence of the enzyme are A-P-P-V-A-S-C-R-Y-C, which differs from other known COXs. The enzyme is stable over a rather wide pH range of 4.0–10.0. The optimum pH and temperature of the COX are pH 7.0 and 50°C, respectively. The COX rapidly oxidizes 3β-hydroxysteroids such as cholesterol and phytosterols, but is inert toward 3α-hydroxysteroids. Thus, the presence of a 3β-hydroxyl group appears to be essential for substrate activity. The Michaelis constant (Km) for cholesterol is estimated at 55 μM; the COX activity was markedly inhibited by metal ions such as Hg²⁺ and Fe³⁺ and inhibitors such as p-chloromercuric benzoate, mercaptoethanol and fenpropimorph. Inhibition caused by p-chloromercuric benzoate, mercuric chloride, or silver nitrate was almost completely prevented by the addition of glutathione. These suggests that -SH groups may be involved in the catalytic activity of the present COX.     

Inorganic carbon uptake by an Antarctic sea-ice diatom,Nitzschia frigida

There have been no studies to date on the mechanisms of inorganic carbon acquisition by Antarctic microalgae. Consequently, we have examined inorganic carbon (DIC) use inNitzschia frigida, a diatom typical of the Antarctic bottom-ice community. The K_0.5(CO₂) of photosynthesis in this organism was estimated to be 1.09 μM at pH 7.5. The internal concentration of DIC was approximately 4050 μM at an external [DIC] of 45 μM. At air-equilibration levels of inorganic carbon this would be sufficient for a ten-fold accumulation ratio of CO₂. Cells ofN. frigida are capable of carbon-dependent photosynthesis at rates that exceed that expected from uncatalysed CO₂ supply to the cell. About 25% of the total carbonic anhydrase activity appears to be associated with the cell surface inN. frigida. These results support the hypothesis thatN. frigida, like many microalgae from temperate waters, has an active carbon-concentrating mechanism, associated with the ability to utilize external HCO ₃ ⁻ for photosynthesis.     

Heterologous expression,purification, and characterization of xylose reductase from <Emphasis Type="Italic">Candida shehatae</Emphasis>

The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His₆-tagged fusion protein. The recombinant XR had K_m values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was specific for d-xylese.     

The two 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition

Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 μM for phosphoenolpyruvate and 500 μM for erythrose 4-phosphate. The rate constant was calculated as 6 s^–1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (K _i of 0.9 μM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate. Received: 29 December 1997 / Accepted: 3 March 1998     

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.     

Characteristics of protection by MgADP and MgATP of α3β3γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis

MgADP and MgATP binding to catalytic sites of βY341W-α₃β₃Γ subcomplex of F₁-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α₃β₃Γ sub-complex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 μM and 12 mM, and 46 μM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K _d of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α₃β₃Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F₁-ATPases.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Purification and characterization of the α-glucosidase produced by thermophilic fungus <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> CBMAI 756

An α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0–9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na⁺, Ba²⁺, Co²⁺, Ni²⁺, Mg²⁺, Mn²⁺, Al³⁺, Zn²⁺, Ca²⁺ this enzyme maintained 90–105% of its maximum activity and was inhibited by Cr³⁺, Ag⁺, and Hg²⁺. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and α-PNPG. The K_m measured for the α-glucosidase was 0.07 μM, with a V_max of 318.0 μmol/min/mg.