Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

Physiochemical properties and kinetics of glucoamylase produced from deoxy-d-glucose resistant mutant of Aspergillus niger for soluble starch hydrolysis

Glucoamylases (GAs) from a wild and a deoxy-d-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS–PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: k_cat = 343 and 727 s⁻¹, K_m = 0.25 and 0.16 mg mL⁻¹, k_cat/K_m (specificity constant) = 1374 and 4510 mg mL⁻¹ s⁻¹, respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.     

Substrate specificity of a recombinant d-lyxose isomerase from Providencia stuartii for monosaccharides

The specific activity and catalytic efficiency (k_cat/K_m) of the recombinant putative protein from Providencia stuartii was the highest for d-lyxose among the aldose substrates, indicating that it is a d-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for d-lyxose isomerization was observed at pH 7.5 and 45 °C in the presence of 1 mM Mn²⁺. The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as d-lyxose, d-mannose, l-ribose, d-talose, and l-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for d-xylulose among all pentoses and hexoses. Thus, d-lyxose was produced at 288 g/l from 500 g/l d-xylulose by d-lyxose isomerase at pH 7.5 and 45 °C for 2 h, with a conversion yield of 58 % and a volumetric productivity of 144 g l^− 1 h^− 1. The observed k_cat/K_m (920 mM^− 1 s^− 1) of P. stuartiid-lyxose isomerase for d-xylulose is higher than any of the k_cat/K_m values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of d-lyxose.     

Purification and some properties of α-amylase from post-harvest Pachyrhizus erosus L. tuber

α-Amylase, a starch splitting enzyme, was purified to homogeneity from post-harvest Pachyrhizus erosus L. tuber by successive chromatography on DEAE- and CM-cellulose columns. Purification achieved was 110 fold from the crude extract with a yield of 22.8%. SDS-PAGE showed a molecular weight of 40 kDa for the enzyme. The enzyme is of α-type as it lost total activity in the presence of EDTA, a chelating agent. It is a glycoprotein that contains 2.6% sugar as estimated by the phenol-sulfuric acid method. The enzyme displayed optimum activity at pH 7.3 and 37 °C with an apparent K_m value of 0.29% for starch as substrate. The enzyme was strongly inhibited by Cu²⁺, Fe²⁺ and Zn²⁺, moderately by Li²⁺, Hg⁺ and Cd²⁺ and slightly by Ag⁺, Mg²⁺ and K⁺. Calcium ion almost doubled the activity whereas Fe³⁺, Mn²⁺ and Na⁺ enhanced it appreciably.     

Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-β-GlcNAc) hydrolysis was five; the optimum temperature was 50 °C; the K_m was 0.18 mM, V_max was 37.6 μmol min⁻¹ mg⁻¹ and activation energy (E_a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30–45 °C for 40 min, but its activity decreased significantly when the temperature exceeded 50 °C. Heavy metal ions, Ag⁺ and Hg²⁺, at a concentration of 0.25 mM and Zn²⁺ and Cu²⁺, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The β-NAHA had only one active site for binding both pNP-β-GlcNAc and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-β-GalNAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon–Webb plots.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi)

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21 kDa and 21.5 kDa, respectively, as estimated by SDS–PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35 °C and 40 °C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45 °C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca²⁺ and Mg²⁺ and inactivated by Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Al³⁺, Ba²⁺ and Co²⁺ to different degrees. Apparent K_m values of trypsin A and B were 2.18 μM and 1.88 μM, and K_cat values were 81.6 S⁻¹ and 111.3 S⁻¹ for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.     

Artocarpus integer leaf protease: Purification and characterisation

The presence of a protease in Artocarpus integer leaves, which are traditionally used as a meat tenderiser, was verified by the presence of a band at 69 kDa, using caseinolytic zymography. Purification by temperature phase partitioning with Triton X-114, ammonium sulphate precipitation and gel filtration chromatography yielded a preparation with a 12-fold increase in enzyme purity and a final specific activity of 76.67 U/mg. The cysteinic nature of this enzyme was confirmed through inhibition of enzyme activity by E-64 and iodoacetamide and enhancement of activity by cysteine and 2-mercaptoethanol. The protease retained 70% of its activity over a broad pH range (pH 6–12), with optimal activity recorded at pH 10 and 40 °C. The enzyme was stable at temperatures up to 70 °C, with 80% of its activity intact. Addition of 5 mM Ca²⁺ stimulated enzyme activity and a kinetic study of the enzyme yielded K_m and V_max values of 0.304 mg/mL and 0.735 mg/mL/min, respectively.     

An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl⁻ form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h at 37 °C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a V_max and K_m of 0.64 mM/mg/min and 29 mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45 °C and pH 8.0, respectively. The enzyme had half-lives (T_1/2) of 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca²⁺ (18%) and Mg²⁺ (12%) at 30 mM. The lipase was inhibited by Co²⁺, Cu²⁺, Zn²⁺, Fe² even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) completely inactivated the original lipase. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45 min, as compared to 91.5% when incubated with Ca²⁺ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.     

Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus

β-Galactosidase, commonly named lactase, is one of the most important enzymes used in dairy processing; it catalyzes the hydrolysis of lactose to its constituent monosaccharides glucose and galactose. Here, a thermostable β-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. sub-tilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified β-galactosidase appeared as a single protein band in sodium dodecyl sulfate-PAGE gel with a molecular mass of approximately 70 kDa. Its isoelectric point, determined by polyacryl-amide gel isoelectric focusing, was close to 5.1. The optimum temperature and pH for this β-galactosidase activity were 70°C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70°C were 50 and 9 h, respectively, and the K_m and V_max values were 2.96 mM and 6.62 μmol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe²⁺, Zn²⁺, Cu²⁺, Pb²⁺, and Sn²⁺, inhibited its activity. Thiol reagents had no effect on the enzyme activity, and sulfhydryl group blocking reagents inactivated the enzyme. This enzyme possessed a high level of transgalactosylation activity in hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galactooligosaccharides in milk processing.     

A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification,biochemical properties and industrial applications

A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg⁻¹, with approximately 1840 fold purification. Analysis of the protein by SDS–PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC–MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a K_m value of 1.73 mM at 37 °C. The energy of activation (E_a) calculated in the range of 35 to 60 °C, using Arrhenius equation, was determined to be 11.32 kcal mol⁻¹. The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40 °C. K_m and E_a for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol⁻¹, respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60 °C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology.     

Isolation and properties of 5′-nucleotidase isolated from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

The enzyme 5′-nucleotidase of jumbo squid (Dosidicus gigas) mantle was purified and its SDS–PAGE showed a single band of 33 kDa, whereas a protein with a molecular mass of 107 kDa was detected by gel filtration suggesting a homotrimeric nature of this enzyme. Subunits of the named enzyme were not linked by covalent bonds. Isoelectric focusing of this enzyme showed a pI of 3.6–3.8 and presented a hyperbolic kinetics with V_max of 1.16 μM/min/mg of protein, K_m of 1.49 mM, K_cat of 3.48 μM of P_ι s⁻¹ and K_cat/K_m relation of 356.52 ((mol/L)⁻¹ s⁻¹). Purified enzyme preferred AMP as substrate (by 6.7-folds) than IMP, showing a K_m of 6.34 mM, V_max of 0.19 μM/min/mg of protein a K_cat of 0.3388 mol of P_ι s⁻¹ and K_cat/K_m relation of 53.44 ((mol/L)⁻¹ s⁻¹). The low K_m in relation to purified AMP deaminase of the same organism suggested a high contribution of 5′-nucleotidase in AMP degradation in jumbo squid mantle.     

A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K_m and k_cat of the purified proteinase were 27 μM and 12 s⁻¹, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca²⁺, Mg²⁺ and Sr²⁺ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.     

Purification and characterisation of exo- and endo-inulinase from Aspergillus ficuum JNSP5-06

Three exoinulinases (Exo-I, Exo-II, and Exo-III) and two endoinulinases (Endo-I and Endo-II) were purified from the culture broth of Aspergillus ficuum JNSP5-06 by ammonium sulphate precipitation, DEAE-cellulose column chromatography, Sepharose CL-6B column chromatography and preparative electrophoresis. The molecular weights of Exo-I, Exo-II, Exo-III, Endo-I, and Endo-II were determined to be 70 kDa, 40 kDa, 46 kDa, 34 kDa, and 31 kDa, respectively. Using inulin as the substrate, their K_m values were 43.1 mg/ml, 31.5 mg/ml, 25.3 mg/ml, 14.8 mg/ml, and 25.6 mg/ml, respectively. These five inulinases were stable below 50 °C with optimum activity at 45 °C, and were stable at a pH range of 4–8 with an optimum pH at 4.5 for exoinulinase and at 5.0 for endoinulinase. The inulinase activity was completely inhibited by Ag⁺ and strongly inhibited by Fe²⁺ and Al³⁺, whereas K⁺, Ca²⁺, Li²⁺, EDTA and urea had no significant influence on the inulinase activity.     

Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5′-AMP sepharose. Specific activity of 2.5 U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS–PAGE showed a single band with 87 kDa molecular mass, native PAGE proved a band of 178 kDa, whereas gel filtration detected a 180 kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with V_max of 1.16 μM/min/mg, K_m of 13 mM, K_cat of 3.48 μM.s⁻¹ and a K_cat/K_m of 267 (mol/L)⁻¹.s⁻¹. The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Purification and characterization of a leucine aminopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

An aminopeptidase was purified from the skeletal muscle of common carp (Cyprinus carpio) to homogeneity, with 1160-fold purification and a yield of 4.3%. The purification procedure consisted of ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, Phenyl Sepharose and Macro-Prep High Q columns. The molecular mass of the enzyme was estimated to be 105 kDa and 100 kDa by SDS-PAGE under reducing conditions and gel filtration chromatography, respectively, suggesting it to be a monomer. The enzymatic activity was optimal at 35 °C and pH 7.0. The metal-chelating agents EDTA, EGTA and 1,10-phenanthroline specifically inhibited the enzyme activity while inhibitors of other proteinases did not show much effect, indicating that it was a metalloproteinase. Furthermore, bestatin, a specific aminopeptidase inhibitor strongly inhibited its activity. Divalent cations Mn²⁺, Mg²⁺ and Ba²⁺ slightly enhanced the enzymatic activity, while Co²⁺, Cu²⁺, Zn²⁺, Ca²⁺ and Fe²⁺ inhibited the activity to different extents. In addition, a sulfhydryl reagent was necessary to maintain its activity. Substrate specificity study revealed that the purified enzyme preferentially hydrolyzed Leu-MCA, followed by Arg-MCA, Ala-MCA and Tyr-MCA and it was thus regarded as a leucine aminopeptidase (LAP, EC 3.4.11.1). The apparent K_m and V_max values of the enzyme were 4.6 μM and 9.6 μmol min⁻¹ mg⁻¹, respectively for substrate Leu-MCA. This is the first report concerning purification and characterization of LAP from freshwater fish.     

Characterisation of trypsin purified from the viscera of Tunisian barbel (Barbus callensis) and its application for recovery of carotenoproteins from shrimp wastes

Trypsin was purified from the viscera of barbel by precipitation using ammonium sulphate (0-80%), Sephadex G-100, and Mono Q-Sepharose ion exchange chromatography. The trypsin was purified 27-fold, with 79 U/mg specific activity and 31% recovery. The enzyme had a molecular weight of 24 kDa; purified trypsin appeared as a single band on native-PAGE. The optimum pH and temperature for enzyme activity were pH 10.0 and 55 °C with BAPNA used as a substrate. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPYSQ. The Michaelis-Menten constant (K_m) and catalytic constant (k_cat) values of the enzyme were 0.018 mM and 1.21 s⁻¹, respectively. The study also investigated the effects of purified trypsin on the recovery of carotenoproteins from shrimp (Parapenaeus longirostris) shells through hydrolysis using 1.0 U barbel trypsin/g shrimp shells for 1 h at 30 °C. The freeze-dried carotenoproteins recovered contained 71.09% protein, 16.47% lipid, 7.78% ash, and 1.79% chitin.     

Purification and characterization of cell wall-bound peroxidase from vanilla bean

A cold-active ionically bound cell wall peroxidase was purified from a polyvinylpolypyrrolidone extract of mature vanilla beans by ultra filtration of 10 kDa and gel filtration chromatography on Sephacryl S-200. The M_r was 46.5 kDa determined by electrophoresis on SDS-PAGE, while native gel filtration confirmed tetramer enzyme form of approximately 186 kDa. The optimum pH and temperature were 3.8 and 16 °C, respectively, as determined with guaiacol as the substrate (K_m 3.8 mmol/L). The pI was approximately 7.7. The POD was inhibited by 1,4-dithiothreitol, β-mercaptoethanol and sodium azide. The POD showed decreasing activity in the presence of ascorbic acid, NaEDTA and sodium dodecyl sulfate at 1 mmol/L. The enzyme lost 80% of its activity in the presence of 20% ethanol. These results will permit better understanding of POD role in vanilla curing process.