Purification and properties of an extracellular beta-xylosidase from a newly isolated Fusarium proliferatum

An extracellular beta-xylosidase from a newly isolated Fusarium proliferatum (NRRL 26517) capable of utilizing corn fiber xylan as growth substrate was purified to homogeneity from the culture supernatant by DEAE-Sepharose CL-6B batch adsorption chromatography, CM Bio-Gel A column chromatography, Bio-Gel A-0.5 m gel filtration and Bio-Gel HTP Hydroxyapatite column chromatography. The purified beta-xylosidase (specific activity, 53 U/mg protein) had a molecular weight of 91,200 as estimated by SDS-PAGE. The optimum temperature and pH for the action of the enzyme were 60 degrees C and 4.5, respectively. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It had a Km value of 0.77 mM (p-nitrophenol-beta-D-xyloside, pH 4.5, 50 degrees C) and was competitively inhibited by xylose with a Ki value of 5 mM. The enzyme did not require any metal ion for activity and stability. Comparative properties of this enzyme with other fungal beta-xylosidases are presented.     

Purification and characterization of a novel salt-tolerant protease from Aspergillus sp. FC-10, a soy sauce koji mold

A novel salt-tolerant protease produced by Aspergillus sp. FC-10 was purified to homogeneity through anion-exchange chromatography, preparative isoelectric-focusing electrophoresis, and gel filtration chromatography, with an overall recovery of 12.7%. This protease demonstrated an optimum pH range of 7.0-9.0 for activity, with a stable pH range of 5.0-9.0. The optimum process temperature at pH 7.0 was 65 degrees C. The enzyme has a molecular mass of 28 kDa and was deduced as a monomer with an isoelectric point of 3.75. Enzyme activity was strongly inhibited by 5 mM of HgCl(2) and FeCl(3), and significantly inhibited by 5 mM of CuSO(4), FeSO(4), and MnCl(2). The activity of this purified protease was inhibited by Na(2).EDTA; however, leupeptin, pepstatin A, PMSF, and E-64 did not affect the activity. Based on the N-terminal amino acid sequence and amino acid composition, this purified protease should be classified as a member of the deuterolysin family.     

Purification and characterization of a major secretory cellobiase, Cba2, from Cellulomonas biazotea

A novel cellobiase (Cba2) was purified from the culture supernatant of Cellulomonas biazotea and characterized. Cba2 appeared to be a major secretory cellobiase in C. biazotea as its enzymatic activity was estimated to represent over 40% of the total extracellular beta-glucosidase activity. The enzyme was purified over 260-fold subsequent to ammonium sulfate precipitation, gel-filtration chromatography, anion-exchange chromatography, and reversed-phase high-performance liquid chromatography. Cba2 was shown by SDS-PAGE to have a large molecular mass of 109 kDa, which makes it one of the largest secretory cellobiases characterized. Its homogeneity was confirmed by N-terminal amino acid sequencing. The K(m) and V(max) values were 0.025 mM and 0.0048 mM min(-1), respectively, for the Cba2 hydrolysis of p-nitrophenyl-beta-d-glucopyranoside, and 0.73 mM and 0.00033 mM min(-1), respectively, for the hydrolysis of cellobiose (at 37 degrees C and pH 7.0). The purified enzyme has a pH optimum of 4.8 and the optimum temperature for activity is 70 degrees C. In view of the secretory nature of Cba2 and the fact that it is a major component of secretory cellobiases of C. biazotea, it is potentially important in the enzymatic degradation of cellulose, and its availability as a recombinant protein may facilitate the studies of its biotechnological applications.     

A highly N-glycosylated chitin deacetylase derived from a novel strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2-4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1-10 mM Co(2+) and strongly inhibited by 10 mM Cu(2+). It required at least two GlcNAc residues for catalysis. When (GlcNAc)(6) was used as substrate, K(m) and V(max) were determined to be 1.1 mM and 54.6 μmol min(-1) respectively.     

Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

脆弱拟杆菌β—内酰胺酶的理化及酶学特性

Bacteroides fragilis 55 from clinical specimens was selected at random for beta-lactamase investigation of physico-chemical and enzymological properties. The enzyme was characterized as a cell-associated cephalosporinase with some penicillinase activity, the molecular weight of the enzyme being 43,000 and the pI 4.95. It could be inhibited by cefoxitin, PCMB, carbenicillin, sulbactam, clavulanic acid and cloxacillin. The optimum pH and temperature for enzyme reactions have been found to be 7.2 and 37 degrees C, respectively. The analysis of amino acid composition and parameters of enzyme kinetics has been described.     

Susceptibility of Rhodobacter sphaeroides to beta-lactam antibiotics: isolation and characterization of a periplasmic beta-lactamase (cephalosporinase).

Thirteen strains of the gram-negative, facultative phototrophic bacterium Rhodobacter sphaeroides were examined fro susceptibility to beta-lactam antibiotics. All strains were sensitive to the semisynthetic penicillins ampicillin, carbenicillin, oxacillin, cloxacillin, and methicillin, but 10 of the 13 strains were resistant to penicillin G, as well as a number of cephalosporins, such as cephalothin, cephapirin, and cephalosporin C. A beta-lactamase (EC 3.5.2.6) with strong cephalosporinase activity was detected in all of the resistant strains of R. sphaeroides. With strain Y-1 as a model, it was shown that the beta-lactamase was inducible by penicillin G, cephalosporin C, cephalothin, and to some minor extent, cephapirin. The beta-lactamase was located in the periplasmic space, from which it could be extracted by osmotic shock disruption. By using this fraction, the beta-lactamase was purified 34-fold to homogeneity by steps involving batch adsorption to and elution from DEAE-Sephadex A50, chromatography on Q-Sepharose, and preparative polyacrylamide gel electrophoresis. The molecular masses of the native and denatured enzymes were determined to be 38.5 kilodaltons by gel filtration and 40.5 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating a monomeric structure. The isoelectric point was estimated to be at pH 4.3. In Tris hydrochloride buffer, optimum enzyme activity was measured at pH 8.5. The beta-lactamase showed high activity in the presence of the substrates cephalothin, cephapirin, cephalosporin C, and penicillin G, for which the apparent Km values were 144, 100, 65, and 110 microM, respectively. Cephalexin, cepharidine, and cephaloridine were poor substrates. The beta-lactamase was strongly inhibited by cloxacillin and oxacillin but only slightly inhibited by phenylmethylsulfonyl fluoride or thiol reagents such as iodoacetate and p-chloromercuribenzoate.     

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillus phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5, a K(m) of 14 mM with alpha-D-Manp-(1-->6)-D-Manp as substrate. It was strongly inhibited by Mn(2+) and did not need Ca(2+) or any other metal cofactor of those tested. The enzyme cleaves specifically (1-->6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1-->3)-D-Manp 10 times faster than alpha-D-Manp-(1-->6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1-->2)-D-Manp. The activity towards (1-->3)-linked mannobiose is strongly activated by 1mM Ca(2+) and inhibited by 10mM EDTA, while (1-->6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities.     

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN₃, ascorbic acid, β-mercaptoethanol and dithreitol. The K_m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.     

Purification and characterization of a sea squirt beta-galactosidase

A beta-galactosidase was extracted from the internal organs of a sea squirt, Styela plicata, and purified 959-fold, with an 18% yield, by successive gel chromatography, anion-exchange chromatography, chromatofocusing, and affinity chromatography on a Con A-Sepharose column. The purified enzyme was fairly homogeneous, as judged on disc PAGE, SDS-PAGE, and gel chromatography on a Sephadex G-200 column. The molecular weight of the enzyme was estimated to be 77,000 and 75,000 by gel chromatography and SDS-PAGE, respectively, and its isoelectric point was determined to be 4.9 by the isoelectric focusing method. The enzyme was substantially stable in the pH range of 3.5 to 7.5, the optimum pH being 4.0. The enzyme was significantly inhibited by 9 mM HgCl2 and 9 mM DFP, while the inhibition by 0.9% PCMB was only 60% at 0 degrees C for 30 min. The purified beta-galactosidase apparently liberated galactose from a sea squirt antigen (H-antigen), two allergenically active glycopeptides (Gp-1 and Gp-2) derived from another sea squirt antigen (Gi-rep), asialo-ovomucoid glycopeptide, asialo-fetuin glycopeptide, GA1, CDH, and an ABEE-derivative (Gal beta 1----3ThrNAc-ABEE) of Gal beta 1----3GalNAc-ol isolated from bovine submaxillary gland mucin.     

Penicillium chrysogenum extracellular acid phosphatase: purification and biochemical characterization

An extracellular acid phosphatase (EC 3.1.3.2) from crude culture filtrate of Penicillium chrysogenum was purified to homogeneity using high-performance ion-exchange chromatography and size-exclusion chromatography. SDS-PAGE of the purified enzyme exhibited a single stained band at an Mr of approx. 57,000. The mobility of the native enzyme indicated the Mr to be 50,000, implying that the active form is a monomer. The isoelectric point of the enzyme was estimated to be 6.2 by isoelectric focusing. Like acid phosphatases from several yeasts and fungi the Penicillium enzyme was a glycoprotein. Removal of carbohydrate resulted in a protein band with an Mr of 50,000 as estimated by SDS-PAGE, suggesting that 12% of the mass of the enzyme was carbohydrate. The enzyme was catalytically active at temperatures ranging from 20 degrees C to 65 degrees C with a maximum activity at 60 degrees C and the pH optimum was at 5.5. The Michaelis constant of the enzyme for p-nitrophenyl phosphate was 0.11 mM and it was inhibited competitively by inorganic phosphate (ki = 0.42 mM).     

Purification and characterization of beta-N-acetylhexosaminidase from rice seeds

N-Acetyl-beta-D-hexosaminidase (beta-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sativa L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions (Fsub1;- F7sub7) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S- 300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNPGalNAc) as substrates, which are typical properties of beta-HexNAc'ase. The ratio of the pNP-GlcNAc'ase activity to the pNP-GalNAc'ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-beta-glucopyranoside, or pNP-beta-galactopyranoside. The enzyme showed K(M), V(max) and K(cat) for pNP-GlcNAc of 1.65mM, 79.49mM min(1), and 4.79 x 10(6) min(1), respectively. The comparison of kinetic values for pNPGlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNPGlcNAc of 5.0 and 50 degrees C, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and 20-40 degrees C. The enzyme activity was completely inhibited at a concentration of 0.1 mM HgCl(2) and AgNO(3), suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.     

Decolorization of the textile dyes using purified banana pulp polyphenol oxidase

Polyphenol oxidase (PPO) purified using DEAE-cellulose and Biogel P-100 column chromatography from banana pulp showed 12.72-fold activity and 2.49% yield. The optimum temperature and pH were found to be 30 degrees C and 7.0, respectively for its activity. Catechol was found to be a suitable substrate for banana pulp PPO that showed V(max), 0.041 mM min(-1) and K(m), 1.6 mM. The enzyme activity was inhibited by sodium metabisulfite, citric acid, cysteine, and beta-mercaptoethanol at 10 mM concentration. The purified enzyme could decolorize (90%) Direct Red 5B (160 microg mL(-1)) dye within 48 h and Direct Blue GLL (400 microg mL(-1)) dye up to 85% within 90 h. The GC-MS analysis indicated the presence of 4-hydroxy-benzenesulfonic acid and Naphthalene-1,2,3,6-tetraol in the degradation products of Direct Red 5B, and 5-(4-Diazenyl-naphthalene-1-ylazo)-8-hydroxy-naphthalene-2-sulfonic acid and 2-(4-Diazenyl-naphthalene-1-ylazo)-benzenesulfonic acid in the degradation products of Direct Blue GLL.     

Purification of a 51 kDa endo-β-N-acetylglucosaminidase from Staphylococcus aureus

A bacteriolytic enzyme obtained from the culture fluid of Staphylococcus aureus FDA 209P was purified to homogeneity utilizing dye-ligand affinity column chromatography, hydrophobic interaction high pressure liquid chromatography (HPLC) and hydroxyapatite HPLC. Subsequent characterizations indicated that the purified enzyme acted as endo-beta-N-acetylglucosaminidase. The molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was 51,000 and the isoelectric point was higher than 10. The optimum pH for the enzyme activity on whole cells of Micrococcus luteus as a substrate was 8.0. Some heavy metal cations (Cu2+ and Zn2+) inhibited the enzyme activity at a concentration of 0.1 mM and others (Ba2+, Mg2+ and Co2+) showed a stimulating effect at a concentration of 1 mM.     

Extracellular alpha galactosidase (E.C. 3.2.1.22) from Aspergillus ficuum NRRL 3135 purification and characterization

Extracellular alpha-galactosidase, a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system, was purified to homogeneity in five steps by ion exchange and hydrophobic interaction chromatography. The molecular mass of the enzyme was 70.8 Kd by SDS polyacrylamide gel electrophoresis and 74.1 Kd by gel permeation HPLC. On the basis of a molecular mass of 70.7 Kd, the molar extinction coefficient of the enzyme at 279 nm was estimated to be 6.1 X10(4) M-1 cm-1. The purified enzyme was remarkably stable at 0 degrees C. It had a broad temperature optimum and maximum catalytic activity was at 60 degrees C. It retained 33% of its activity after 10 min. at 65 degrees C. It had a pH optimum of 6.0. It retained 62% of its activity after 12 hours at pH 2.3. The Kms for p-nitrophenyl-alpha-D-galactopyranoside, o-nitrophenyl-alpha-D-galactopyranoside and m-nitrophenyl-alpha-D-galactopyranoside are: 1462, 839 and 718 microM. The enzyme was competitively inhibited by mercury (19.8 microM), silver (21.5 microM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM) and fructose (60.3 mM). It was inhibited non-competitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Purification and characterization of the extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius

The extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS-PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0-7.0. Under the conditions tested, the activity is maximal between 45 and 50 degrees C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.     

Purification and properties of midgut alpha-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae

Using soluble starch as a substrate five isoforms of alpha-amylase were identified in a crude extract of Morimus funereus larvae. The main alpha-amylase (termed AMF-3) was purified by gel filtration chromatography and anion exchange chromatography to obtain a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Its enzymatic purity was confirmed by an in-gel activity assay after SDS-PAGE. The purity of AMF-3 was increased 112-fold with a 15.4% yield. AMF-3 had apparent molecular masses of 33 and 31 kDa when analysed using SDS-PAGE and Superdex 75 FPLC gel filtration chromatography, respectively and a calculated isoelectric point of 3.2. Purified AMF-3 showed maximal activity at pH 5.2 and had an optimum activity temperature of 45 degrees C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 degrees C. AMF-3 exhibited a high affinity towards soluble starch with a K(m) value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl(2), while at higher concentrations its activity decreased. AMF-3 activity increased with increasing NaCl concentration. AMF-3 activity was significantly inhibited by alpha-amylase wheat inhibitor. Using a number of raw starch substrates maximum AMF-3 activity was achieved with horse-radish starch, in contrast to undetectable activity towards potato starch.