Affinity Chromatographic Purification of β-Glucosidase of Candida Guilliermondii

Abstract

A 8-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethylcneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacryl-amide disc electrophoresis of the purified enzyme gave a single band. A K_m of 1.25 × 10^?4M was obtained using p_-nitrophenyl-β-D_-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at a temperature of 37°C. Enzyme activity was completely inhibited by Hg⁺⁺, Pb⁺⁺, and Zn⁺⁺ ions. The molecular weight of the enzyme is 48, 000 as estimated by sucrose density gradient centri-fugation.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Studies of monoamine oxidases: properties of the enzyme in bovine and rabbit brain mitochondria

Brain mitochondria were prepared from rabbit and bovine cerebral cortex and the purity and intactness of the preparation assessed through the use of enzyme markers and electron microscopy. Enzymatic properties of monoamine oxidase were studied in the purified mitochondrial preparations which were essentially devoid of major contamination by other organelles, especially microsomes. Five substrates were used for characterization of the enzyme: dopamine, kynuramine, serotonin, tryptamine and tyramine. It was found that there was considerable substrate variation in the properties, but in general, the two species showed similar characteristics. The more pertinent findings were: (1) apparent K_m values ranged from 1.1 ± 10^?5m for tryptamine to 2.5 ± 10^?4m for dopamine; (2) substrate specificity from V_max values in decreasing order was tyramine > dopamine > kynuramine > serotonin > tryptamine for the bovine enzyme and tyramine > kynuramine > dopamine > serotonin > tryptamine for rabbit; (3) there appeared to be three distinct pH optima according to substrate: pH 7.5 for phenylethylamines, pH 8.2–8.5 for the indolylamines and pH 9.1 for kynuramine; and (4) the activity with tyramine was highly sensitive to increased oxygen tension while kynuramine showed no sensitivity. It is proposed that the properties of monoamine oxidase, a membrane-bound enzyme, might be influenced by the microenvironment and results are also discussed in terms of multiple forms or multiple activity sites on a single form.     

Isolation and Purification of Ascorbate Oxidase from Acremonium sp. HI-25

A screening test was undertaken to isolate microorganisms that produced ascorbate oxidase. The enzyme activity was found in a culture filtrate of a fungal strain (HI-25), newly isolated from a soil sample. Based on the morphological characteristics, this isolate was identified as Acremonium sp. From the examinations of cultural conditions, optimum conditions for enzyme production were found; strain HI-25 was aerobically cultured by a jar fermenter at 25°C in a medium containing 5% glycerol, 2% defatted soybeans, 0.1% monosodium L-glutamate, 0.1% KH₂PO₄, 0.02% MgSO₄ ·7H₂O, and 0.01% KCl, pH 6.0. After cultivation, an ascorbate oxidase was purified from the culture filtrate by an ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and Butyl-Toyopearl, and gel filtration twice on Sephadex G-100. The purification was 850-fold with an activity yield of 8.8%. The purified enzyme gave a single band on SDS polyacrylamide gel electrophoresis, and had a molecular weight of 80,000 by SDS polyacrylamide gel electrophoresis and 76,000 by native gel filtration. This enzyme was most active at pH 4.0, 45°C, and was most stable between pH 6.0–10.0 and at temperatures below 60°C.     

PURIFICATION AND CHARACTERIZATION OF MUTANASE PRODUCED BY Paenibacillus curdlanolyticus MP-1

Mutanases are enzymes that catalyze hydrolysis of α-1,3-glucosidic bonds in various α-glucans. One of such glucans, mutan, which is synthesized by cariogenic streptococci, is a major virulence factor for induction of dental caries. This means that mutan-degrading enzymes have potential in caries prophylaxis. In this study, we report the purification, characterization, and partial amino acid sequence of extracellular mutanase produced by the MP-1 strain of Paenibacillus curdlanolyticus, bacterium isolated from soil. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular mass 134 kD, while native gel filtration chromatography confirmed that the enzyme was a monomer of 142 kD. Mutanase showed a pH optimum in the range from pH 5.5 to 6.5 and a temperature optimum around 40–45°C. It was thermostable up to 45°C, and retained 50% activity after 1 hr at 50°C. The enzyme was fully stable at a pH range of 4 to 10. The enzyme activity was stimulated by the addition of Tween 20, Tween 80, and Ca²⁺, but it was significantly inhibited by Hg²⁺, Ag⁺, and Fe²⁺, and also by p-chloromercuribenzoate, iodoacetamide, and ethylenediamine tetraacetic acid (EDTA). Mutanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-1,3-glucans. It also showed binding activity to insoluble α-1,3-glucans. The N-terminal amino acid sequence was NH₂-Ala-Gly-Gly-Thr-Asn-Leu-Ala-Leu-Gly-Lys-Asn-Val-Thr-Ala-Ser-Gly-Gln. This sequence indicated an analogy of the enzyme to α-1,3-glucanases from other Paenibacillus and Bacillus species.     

Alkaline phosphatase from the excretory system of the grasshopper,Poekilocerus bufonius

Alkaline phosphatase from the excretory system of the grasshopper, Poekilocerus bufonius was purified with ammonium sulphate fractionation and chromatography on Bio-Gel A-0.5 m. The specific activity of the enzyme is 152 units/mg of protein. The enzyme is a tetramer and the M_r value of the subunit is 72,000 ± 2500 as shown by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 9.6 and an apparent K_m value of 0.28 × 10⁻³ M. The activity of the enzyme reached a maximum at 75°C and the enzyme showed stability at 65°C. The enzyme was inhibited by Ca²⁺, Na⁺ and Fe³⁺ and was stimulated by Zn²⁺, Mn²⁺ and Mg²⁺.     

Purification and properties of dipeptidyl peptidase IV from Streptococcus mitis ATCC 9811

Dipeptidyl peptidase IV (EC 3.4.14.—) from Streptococcus mitis ATCC 9811 was purified to a specific activity of 56.2 units/mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 120,000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 53,000 was calculated. The purified enzyme has an optimum pH between 6.0 and 8.7 and an isoelectric point of 4.0. The K_m value toward glycylprolyl-p-nitroanilide is about 6.0 × 10^?5m. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-proline from X-Pro-p-nitroanilides. Inhibition of this enzyme was achieved with Hg²⁺, Pb²⁺, Zn²⁺, EDTA, and diisopropyl phosphorofluoridate, but not with N-ethyl-maleimide and sulfhydryl inhibitors.     

Thermostable alkaline protease from newly isolated <Emphasis Type="Italic">Vibrio</Emphasis> sp.: extraction,purification and characterisation

An extracellular alkaline protease-producing Vibrio sp. was isolated from mangrove sediments of Vellar estuary. A 9.36-fold purification was achieved by a three-step purification procedure and the molecular weight of the enzyme was determined as 33 kDa by SDS-PAGE. The enzyme was active in a broad range of pH (6.0–11.0) and temperature (30–70°C), the optimum being at pH 9.0 and temperature 55°C. The enzyme was stable at alkaline pH range of 9–11 and up to a temperature of 60°C, after incubation for 1 h. Metals like Co²⁺, Hg²⁺, Ni²⁺ and Cu²⁺ inhibited the enzyme activity, whereas Fe²⁺, Ca²⁺ and Mn²⁺ were found to enhance the activity. The protease was found to be highly stable in the presence of oxidizing agents like H₂O₂, detergents such as SDS and Triton-X-100 and also some of the commonly used commercial detergents. The organic solvents like xylene, isopropanol, hexane and benzene were found to enhance as well as stabilize the enzyme activity. The extracellular production of the enzyme, the pH and thermal stability, and the stability in presence of oxidants, surfactants, commercial detergents and organic solvents, altogether suggest that it can be used as a laundry additive.     

Purification and kinetic characterization of polygalacturonase - 3 from palmyrah palm (Borassus flabellifer L)

Polygalacturonase-3 was isolated and purified to homogeneity from palmyrah palm (Borassus flabellifer L.) fruit using Con A-Sepharose affinity column. The purified enzyme migrated as a single band on native and SDS–polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme was estimated to be 66 kDa by size elution chromatography. Optimum polygalacturonase activity as a function of pH and temperature was determined using polygalacturonic acid as substrate. Optimum pH and temperature values ranged between the pH?4.0–5.0 and temperature 30–40 °C. At the optimum pH and temperature, the K_m and V_max values were determined by Lineweaver–Burk method. The value K_m (0.33 mM) reveals that polygalacturonase has significant reactivity towards polygalacturonic acid. The enzyme showed varied responses towards divalent and monovalent metal ions. Ca²⁺ activated the polygalacturonase-3 enzyme protein. Both teepol and cetyltrimethylammonium bromide inhibited polygalacturonase-3 activity by 44 %, while 2-mercaptoethanol stimulated the enzyme marginally.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.     

Purification and partial characterization of NAD aminohydrolase from Aspergillus oryzae NRRL447

Aspergillus oryzae aminohydrolase free acid phosphodiesterase catalyzes nicotinamide adenine dinucleotide to deamino-NAD and ammonia. The enzyme was purified to homogeneity by a combination of acetone precipitation, anion exchange chromatography and gel filtration chromatography. The enzyme was purified 230.5 fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of MW 94 kDa. The enzyme displayed maximum activity at pH 5 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable up to 40 °C. The enzyme activity was enhanced slightly by addition of Na⁺ and K⁺, whereas inhibited strongly by addition of Ag⁺, Mn²⁺, Hg²⁺ and Cu²⁺ to the reaction mixtures. The enzyme hydrolyzes several substrates, suggesting a probable non-specific nature. The enzyme catalyzes the hydrolytic cleavage of amino group of NAD, adenosine, AMP, CMP, GMP, adenosine, cytidine and cytosine to the corresponding nucleotides, nucleosides or bases and ammonia. The substrate concentration–activity relationship is the hyperbolic type and the apparent K_m and K_cat for the tested substrates were calculated.     

The partial purification and characterization of a gibberellin C-20 hydroxylase from immature Pisum sativum L. seeds

A gibberellin (GA) C-20 hydroxylase that catalyses the conversion of GA₅₃ to GA₄₄ was purified from developing pea embryos by ammonium-sulfate precipitation, gel filtration and anion-exchange column chromatography. The purification was about 270-fold and 15% of the enzymic activity was recovered. The relative molecular mass was 44000 by Sephadex G-200 gel filtration. The apparent Michaelis constant was 0.7 M and the isoelectric point was 5.6–5.9. The enzymic activity was optimal at pH 7.0 2-Oxoglutarate and ascorbate were required for activity. Low concentrations of Fe²⁺ stimulated the reaction, but externally added Fe²⁺ was not essential, even in the most purified preparation. Catalase and bovine serum albumin also stimulated. Dithiothreitol preserved the activity during purification but was not needed during incubation. In fact, the simultaneous presence of dithiothreitol and Fe²⁺ in the incubation mixture was inhibitory to the purified enzyme. The cofactor requirements are typical for those of 2-oxoglutarate-dependent dioxygenases.When the incubation time was long enough, GA₅₃ was converted to both GA₄₄ and GA₁₉. The proportions of these two products remained constant throughout the purification, but this does not necessarily mean that their formations is catalysed by a single enzyme. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the final preparation contained several proteins. Although the most prominent protein band was located within the range expected for the enzyme on the grounds of its molecular weight, this band did not represent the enzyme, since it separated from the GA C-20 hydroxylase activity on ultrathin-layer isoeletric focusing.Abbreviation BSA bovine serum albumin - DEAE diethylaminoethyl - DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - GA_n gibberellin A_n - HPLC high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Prolindehydrogenase aus keimenden farnsporen

A soluble enzyme which converts proline to glutamic acid using NAD as coenzyme was isolated from young prothallia and spores of the fern Anemia phyllitidis. The purification was about 36-fold. The pH optimum is between 10·2 and 10·7; the K_m for proline is 4·6 × 10⁻⁴ M and for NAD 3·4 × 10⁻⁴ M. There are no multiple forms of this enzyme, as proved by gel electrophoresis.     

Isolation of 3-methylcrotonyl-coenzyme A carboxylase from bovine kidney

3-Methylcrotonyl-CoA carboxylase (MCase), an enzyme of the leucine oxidation pathway, was highly purified from bovine kidney. The native enzyme has an approximate molecular weight of 835,000 as measured from exclusion limits by polyacrylamide gel electrophoresis at pH 7.3. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated two subunits, identified as a biotin-free subunit (A subunit; M_r = 61,000) and a biotin-containing subunit (B subunit; M_r = 73,500). The biotin content of the enzyme was 1 mol/ 157,000 g protein, consistent with an AB protomeric structure for the enzyme. The isoelectric point of the enzyme was found to be 5.4. Maximal MCase activity was found at pH 8 and 38 °C in the presence of Mg²⁺ and an activating monovalent cation such as K⁺. Kinetic constants (K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 75 μm; ATP, 82 μm; HCO₃^?, 1.8 mm. Certain acyl-CoA derivatives, including crotonyl-CoA, (2Z)-3-ethylcrotonyl-CoA, and acetoacetyl-CoA, were also substrates for the enzyme. Some data on inhibition of the enzyme by acyl-CoA derivatives, and sulfhydryl- and arginyl-reagents, are presented.     

Trehalase from the cellular slime mold Dictyostelium discoideum: Purification and characterization of the homogeneous enzyme from myxamoebae

Trehalase (α-α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) was solubilized from myxamoebae of the cellular slime mold Dictyostelium discoideum by a freeze-thaw cycle and was subsequently purified to homogeneity using the techniques of ethanol fractionation, molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide disc gel electrophoresis. The 1000-fold purified enzyme had a specific activity of about 104 units/mg, which was accompanied by a net recovery of 5 to 7% of the original activity. The purified enzyme was maximally active at pH 5.5, showed high specificity for trehalose, and exhibited a typical hyperbolic response as a function of trehalose concentration with a K_m of 1.2 mm. The enzyme was maximally active at 50 °C and had an energy of activation of 12–13 kcal/mol. Thermal stability studies demonstrated that full enzymatic activity was recovered following a 5-min incubation of trehalase at temperatures up to 45–50 °C. Analysis of various compounds for inhibitory effects indicated that Tris and urea were slightly effective, reducing enzymatic activity by 28 and 6% at concentrations of 100 and 10 mm, respectively. Of five heavy metals tested, HgCl₂ was the most inhibitory, reducing activity by 58% when present at a final concentration of 1.0 mm. Enzymatic activity was not affected by any adenine derivative examined (e.g., ATP, ADP, AMP, cAMP, adenosine, and adenine). The molecular weight of the native enzyme was determined by molecular sieve chromatography, pore gradient electrophoresis, and electrophoresis as a function of acrylamide concentration. All three methods yielded a value of about 10⁵ ± 5 × 10³. Estimation of the subunit or monomer molecular weight by sodium dodecyl sulfate-gel electrophoresis indicated a value of 95–100 × 10³. The isoelectric point as determined in 7.5% polyacrylamide gels with pH 3–10 ampholytes was 7.2–7.3. The purified enzyme adsorbed to concanavalin A-Sepharose in the presence of KCl (0.1 m) and was eluted with α-methylmannoside, thereby suggesting an association between trehalase and carbohydrate. In agreement with this conclusion was the observation that trehalase could be specifically stained for carbohydrate with the Alcian blue and periodic acid-Schiff's reagents following polyacrylamide disc gel electrophoresis.     

Identity of Phosphodiesterase and Phosphomonoesterase Activities with Nuclease P1 (a Nuclease from Penicillium citrinum)

Enzymatic properties of a purified Penicillium nuclease (designated as nuclease P₁) were investigated. The enzyme activities for RNA, heat-denatured DNA, native DNA, 3′-AMP and 2′-AMP showed a great degree of similarity with respect to the following properties: a) Range of stable pH (5~8), b) temperature optima (at around 70°C), c) thermostability (about 50% inactivation at 67°C, pH 6.0 for 15 min, d) effect of metal ions and SH inhibitors, e) requirement of Zn²⁺, f) protection from the heat-inactivation by albumin and Zn²⁺, g) inactivation on standing in the cold and reactivation on heating, h) sensitivity to protease, and i) competitive relationship between substrates in the enzyme reaction. Moreover, the ratio of enzyme activities in several mutants of Penicillium citrinum was constant. From these results, together with constant ratio of the specific activities throughout purification, it is concluded that a single enzyme might be responsible for both phosphodiesterase and phosphomonoesterase functions.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

A simple purification procedure of D-amino-acid oxidase from <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

D-amino-acid oxidase (EC 1.4.3.3) was purified about 1480-fold from the yeast Candida guilliermondii using chromatofocusing method. The purification procedure gave an enzyme preparation which is greater than 90% homogenous on SDS-polyacrylamide gels with a specific activity of 11.54 U/mg at 30°C with D-proline as substrate with the yield of total activity 9.3%. The molecular weights of subunit and native enzyme were determined to be 38.4 and 78.6 kDa by SDS-polyacrylamide gel electrophoresis and gel-filtration, respectively, suggesting that the native enzyme exists as a homodimer. A single molecular form with an isoelectric point of 6.85 was detected in analytical isoelectrofocusing. The optimum pH and temperature were 8.0 and 33°C. An enzyme shows stability in the pH range from 7.4 to 9.0 and at the temperature no higher than 38°C. Activation energy for D-amino-acid oxidase reaction was calculated to be 60 kJ/mol at 30°C. The strict D-isomer specificity of the enzyme is confirmed, since no reaction could be detected with L-amino acids, and a large number of D-amino acids could be substrates for this enzyme. K _m and V _max values were determined for D-proline and D-alanine, which, among 22 tested, were the best substrates of the enzyme. D-amino-acid oxidase from the yeast C. guilliermondii is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalently, bound FAD. The enzyme is completely inhibited by sodium benzoate, SH-oxidizing agents, but not by sodium azide, toluene or chloroform.     

Purification and characterization of an acid phosphatase from Trichoderma harzianum

An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55°C. The enzyme retained 60% of its activity after incubation at 55°C for 60 min. The K _m and V _max values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min^?1, respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.