In vitro effects of some drugs on catalase purified from human skin

Catalase enzyme (H202: oxidoreductase; E.C. 1.11.1.6) was purified from human skin homogenate using ammonium sulfate precipitation and DEAE-Sephadex A50 ion exchange chromatography at 4 degrees C and some characteristics of the enzyme were investigated. The human skin enzyme, having a specific activity of 1354.5 EU/mg proteins was purified with a yield of 43.13% and 1110-fold. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band for the enzyme. Inhibition by piroxicam, ketoprofen, diclofenac sodium, sulfamethoxazole and nidazole occurred with I50 values of 0.414, 1.29, 1.8, 3.83, and 8.64 mM, respectively.     

An Improved Assay Method for Antifouling Substances Using the Blue Mussel,Mytilus edulis

The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37°C and pH 8.5 in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10 mm Tris-HCl, 7 mm 2-mercaptoethanol, 7 mm MnCl₂ and 50 mm NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5′-GACGTC-3′, cuts between T and C and produces a 3′-tetranucleotide extension in the presence of MnCl₂, as it does in the presence of MgCl₂.     

Milk-clotting Enzyme from Microorganisms: V. Purification and Crystallization of Mucor Rennin from Mucor pusillus var. Lindt.1

A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg²⁺, Ag⁺, Zn²⁺, and KMnO₄.     

Milk-clotting Enzyme from Microorganisms: V. Purification and Crystallization of Mucor Rennin from Mucor pusillus var. Lindt

A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg²⁺, Ag⁺, Zn²⁺, and KMnO₄.     

Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg²⁺, while Mn²⁺ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K _m and V _max values of 2.5 and 26 μmol/mg per minute, respectively.     

Purification,Crystallization and Some Properties of Extracellular Pullulanase from Aerobacter aerogenes

Extracellular pullulanase was purified and crystallized from the culture fluid of Aerobacter aerogenes. Pullulanase was purified by means of ammonium sulfate fraction, DEAE-cellulose column chromatography and Sephadex column chromatography. Crystalline pullulanase was formed when saturated ammonium sulfate solution was added to the purified enzyme solution. The crystalline enzyme appeared as colorless fine rods. On ultracentrifugation analysis, the enzyme showed a single sharp and symmetrical Schlieren peak. The sedimentation coefficient, s_20,w was 4.39S. Polyacrylamide gel electrophoresis at pH 8.4 gave a main band with two sub-bands and the molecular weight of the main enzyme was estimated to be 66,000 from Polyacrylamide gel electrophoresis and to be 58,000 from sedimentation equilibrium. The optimum pH and temperature for the enzyme action were pH 6.5 and 50°C, respectively.     

Purification and characterization of a lysosomal aspartic protease with cathepsin D activity from the mosquito

A lysosomal aspartic protease with cathepsin D activity, from the mosquito, Aedes aegypti, was purified and characterized. Its isolation involved ammonium sulfate (30–50%) and acid (pH 2.5) precipitations of protein extracts from whole previtellogenic mosquitoes followed by cation exchange chromatography. Purity of the enzyme was monitored by SDS-PAGE and silver staining of the gels. The native molecular weight of the purified enzyme as determined by polyacrylamide gel electrophoresis under nondenaturing conditions was 80,000. SDS-PAGE resolved the enzyme into a single polypeptide with M_r = 40,000 suggesting that it exists as a homodimer in its non-denatured state. The pI of the purified enzyme was 5.4 as determined by isoelectric focusing gel electrophoresis. The purified enzyme exhibits properties characteristic of cathepsin D. It utilizes hemoglobin as a substrate and its activity is completely inhibited by pepstatin-A and 6M urea but not by 10 mM KCN. Optimal activity of the purified mosquito aspartic protease was obtained at pH 3.0 and 45°C. With hemoglobin as a substrate the enzyme had an apparent K_m of 4.2 μ M. Polyclonal antibodies to the purified enzyme were raised in rabbits. The specificity of the antibodies to the enzyme was verified by immunoblot analysis of crude mosquito extracts and the enzyme separated by both non-denaturing and SDS-PAGE. Density gradient centrifugation of organelles followed by enzymatic and immunoblot analyses demonstrated the lysosomal nature of the purified enzyme. The N-terminal amino acid sequence of the purified mosquito lysosomal protease (19 amino acids) has 74% identity with N-terminal amino acid sequence of porcine and human cathepsins D.     

Purification and Characterization of a Cathepsin L-Like Enzyme from the Body Wall of the Sea Cucumber Stichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS–PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine

7-amido-4-methylcoumarin with K _m (69.92 μM) and k _cat (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 °C. It showed thermal stability below 40 °C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.     

Purification and properties of aldose reductase form Pachysolen tannophilus

Aldose reductase (EC 1.1.1.21) from Pachysolen tannophilus IFO 1007 was purified 15 fold from the crude enzyme in a yield of 0.9% by pH 5 treatment, protamine sulfate precipitate, ammonium sulfate fractionation, and G-100 gel chromatography. The purified enzyme was entirely homogeneous on disc gel electrophoresis. The optimum pH and temperature were 5–6 and 50°C, and it was stable at pH 6–8 and up to 35°C. Its activity was enhanced slightly by Na₂SO₄, glycylglycine, glutathione, and cysteine, and inhibited remarkably by SH inhibitors such as AgNO₃, HgCl₂, lead acetate and iodo-acetate. Its K_m values were determined ad follows: 0.97 mM for d-glyceraldehyde, 1.7 mM for dl-glyceraldehyde, 3.5 mM for d-erythrose, 12 mM for d-xylose, 18mM for l-arabinose, 25 mM for galactose, 33 mM for valeraldehyde, 33 mM for 2-deoxy-d-glucose, 50 mM for propionaldehyde, 67 mM for d-ribose, 200 mM for d-mannose, and 280 mM for acetaldehyde. The enzyme also reduced glucose, l-sorbose, butylaldehyde, and benzaldehyde. Its molecular weight was estimated to be 40,650 by sedimentation equilibrium, 40,000 by SDS polyacrylamide gel electrophoresis and 43,000 by Sephadex G-200 column chromatography.     

Purification and properties of methylmalonyl coenzyme A mutase from human liver

Methylmalonyl coenzyme A (CoA) mutase has been purified to apparent homogeneity from human liver by a procedure involving column chromatography on DEAE-cellulose, Matrex-Gel Blue A, hydroxylapatite, and Sephadex G-150. The overall purification achieved is 500- to 600-fold, yield 3–5%. Electrophoresis of the native purified protein on nondenaturing polyacrylamide gels shows a single diffuse band coincident with the enzyme activity; dodecyl sulfate/polyacrylamide gels show a single protein band with an apparent molecular weight of 77,500. The native protein has a molecular weight of approximately 150,000 by Sephadex G-150 chromatography, suggesting that it is composed of two identical subunits. The activity of the purified enzyme is stimulated only slightly (10–20%) by the addition of its cofactor, adenosylcobalamin, indicating that the purified enzyme is largely saturated with coenzyme. The spectrum of the enzyme is consistent with the presence of about 1 mole of adenosylcobalamin per mole of subunit. The enzyme displays complex kinetics with respect to dl-methylmalonyl CoA; substrate inhibition by l-methylmalonyl CoA appears to occur. The enzyme activity is stimulated by polyvalent anions (PO₄^3? > SO₄^2? > Cl^?); monovalent cations are without effect, but high concentrations of divalent cations are inhibitory. The enzyme activity is insensitive to N-ethylmaleimide, is rapidly destroyed at temperatures > 50 °C, and shows a broad pH optimum around pH 7.5.     

Purification and Characterization of Fatty Acid Cyclooxygenase from Human Platelets

Abstract

The fatty acid cyclooxygenase (EC 1.14.99.1) that produces the prostaglandin, thromboxane, and prostacyclin precursor (PGHp), was solubilized from human platelet microsomes in 20 sucrose and 1.0% Triton X-100. The enzyme was purified 300-fold by electrofocusing, Sephadex G-200 gel filtration, and hydrophobic chromatography on ethyl agarose. The cyclooxygenase catalyzed the conversion of arachidonic acid to prostaglandin endoperioxide, PGH₂, that was trapped at ?25°C and separated on TLC at ?20°C. PGH₂ was hydrolyzed to HHT in acidic pH, or was chemically converted to PGE₂ in slightly alkaline pH in the absence of cofactors. The enzyme showed a broad pH optimum in the range of 7–9. Hemin containing substances such as methemoglobin were absolutely required as cofactors, while tryptophan, epinephrine, phenol, and hydro-quinone stimulated the PGH₂ formation. Metal ions, such as Zn²⁺ and Cd²⁺ inhibited the enzyme reaction at 0.1 to 1 mM.

The molecular weight of the purified enzyme was estimated at 79,432 by sodium dodecyl sulfate disc gel electrophoresis at pH 8.0. The properties of the human platelet enzyme was generally similar to the sheep vesicular enzyme in the method of solubilization, pH optimum, and molecular weight.     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

Purification and characterization of fibrinolytic alkaline protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. BLB

Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH₄)₂SO₄ and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was observed at pH 9.5 and 50°C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50°C. The enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus.     

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     

A Review of: “HPLC of Biological Macromolecules (Methods and Applications) K.M. Goodring F.E. Regnier,Eds Marcel Dekker,New York, 1990; hardbound, 676 pages, $150”

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0–9.0) and temperature (30–90°C). From the thermal inactivation studies in the range 60–75°C, the half-life (t_1/2) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol^?1. It showed higher specificity with catechol (K_m = 8 mM) as compared to 4-methylcatechol (K_m = 10 mM). Among metal ions and reagents tested, Cu²⁺, Fe²⁺, Hg²⁺, Mn²⁺, Ni²⁺, protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K⁺, Na⁺, Co²⁺, kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

Alkaline Serine Protease AprE Plays an Essential Role in Poly-γ-glutamate Production during Natto Fermentation

A thermostable chitosanase, purified 156-fold to homogeneity in an overall yield of 12.4%, has a molecular weight of about 29,000±2,000, and is composed of monomer. The enzyme degraded soluble chitosan, colloidal chitosan, and glycol chitosan, but did not degrade chitin or other β-linked polymers. The enzyme activity was increased about 2.5-fold by the addition of 10 mM Co²⁺ and 1.4-fold by Mn²⁺. However, Cu²⁺ ion strongly inhibited the enzyme. Optimum temperature and pH were 60°C and 6.5, respectively. The enzyme was stable after heat treatment at 80°C for 30 min or 70°C for 60 min and fairly stable in protein denaturants as well. Chitosan was hydrolyzed to (GlcN)₄ as a major product, by incubation with the purified enzyme. The effects of ammonium sulfate and organic solvents on the action pattern of the thermostable chitosanase were investigated. The amounts of (GlcN)₃-(GlcN)₆ were increased about 30% (w/w) in DAC 99 soluble chitosan containing 10% ammonium sulfate, and (GlcN)₁ was not produced. The monophasic reaction system consisted of DAC 72 soluble chitosan in 10% EtOH also showed no formation of (GlcN)₁, however, the yield of (GlcN)₃ ～ (GlcN)₆ was lower than DAC 99 soluble chitosan-10% ammonium sulfate. The optimal concentration of ammonium sulfate to be added was 20%. At this concentration, the amount of hexamer was increased by over 12% compared to the water-salt free system.     

Purification and properties of the glucose-6-phosphate-dependent form of human placental glycogen synthase.

Glycogen synthase in the glucose-6-phosphate (glucose-6-P)-dependent form was purified over 10,000-fold from an extract of term human placenta. The purified enzyme shows a single protein band on polyacry1amide-gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme activity in the presence of glucose-6-P is increased by the single addition of Mg²⁺, Ca²⁺, or Mn²⁺ and is reduced by the addition of either sulfate or phosphate. Addition of either Mg²⁺, Ca²⁺, or Mn²⁺ relieves the inhibition by sulfate or phosphate. The enzyme activity in the absence of glucose 6-P is greatly increased by the addition of MnSO₄, CoSO₄, and NiSO₄ and is increased to a lesser extent by MgSO₄, CaSO₄, and FeSO₄. The activation of the glucose-6-P-dependent form of the enzyme by these metal sulfates in the absence of glucose-6-P has never been reported. MnSO₄, which shows homotropic cooperativity, is the best activator among the various metal sulfates tested. The human placental glucose-6-P-dependent form of glycogen synthase (D form) can be converted to the glucose-6-P-independent form (I form) of the enzyme by incubating the partially purified glycogen synthase, which is copurified with synthase phosphatase, with Mn²⁺. This conversion can be reversed by the addition of cyclic AMP-dependent protein kinase. The synthase D to synthase I converting system from human placenta is unique in its stringent requirement for Mn²⁺.     

Purification and Properties of 2-Ketogluconate Reductase from Gluconobacter liquefaciens.

2-Ketogluconate reductase (2KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was purified about 1000-fold by a procedure involving ammonium sulfate fractionation and column chromatographies using DEAE-cellulose, hydroxylapatite, and Sephadex gel The purified enzyme gave a single band on polyacrymamide gel electrophoresis. NADP was specifically required for the oxidation reaction of gluconic acid. Using gel filtration a molecular weight of about 110,000 was estimated for the enzyme. The pH optimum for the oxidation of gluconic acid (GA) to 2-ketogluconic acid (2KGA) by the enzyme was 10.5 and for the reduction of 2KGA was 6.5. The optimum temperature of the enzyme was 50 C for both reactions of oxidation and reduction. The enzyme was stable at pH between 5.0 and 11.0 and at temperature under 50°C, The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ions, but remarkably stimulated by manganese ions (1×10^?3 m). Km value of the enzyme for GA was 1.3×10^?2 m and for 2KGA was 6.6×10^?3 _m. Km values for NADP and NADPH₂ were 1.25×10^?5 and 1.52×10^?5 m respectively.     

Partial Purification and Characterization of A Novel Histidine Decarboxylase from Enterobacter aerogenes DL-1

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca²⁺ and Mn²⁺ showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver–Burk plot showed that K _m and V _max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.     

Characterization,Gene Cloning,and Sequencing of a Fungal Phytase,PhyA, From Penicillium oxalicum PJ3

A phytase from Penicillium oxalicum PJ3, PhyA, was purified near to homogeneity with 427-fold increase in specific phytase activity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis of the purified enzyme indicated an estimated molecular mass of 65 kD. The optimal pH and temperature of the purified enzyme were pH 4.5 and 55°C, respectively. The enzyme activity was strongly inhibited by Ca²⁺, Cu²⁺, Zn²⁺, and phenylmethylsulfonyl fluoride (PMSF). The K_m value for sodium phytate was 0.545 mM with a V_max of 600 U/mg of protein. The phyA gene was cloned, and it contains an open reading frame of 1,383 with a single intron (118 bp), and encodes a protein of 461 amino acids.