Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

Biochemical Characterization of an Extracellular Heat‐Stable Protease from Serratia liquefaciens Isolated from Raw Milk

The protease Ser2 secreted by the psychrotrophic strain Serratia liquefaciens L53, a highly proteolytic strain isolated from Brazilian raw milk was purified and characterized. Using azocasein as substrate, Ser2 exhibited activity in a wide range of pH (5 to 10) and temperature (4 to 60 °C). The optimal activity was detected at pH 8.0 and at a temperature of 37 °C. This protease, still active at 4, 7, and 10 °C, was strongly inhibited by chelating agents and by dithiothreitol, a reducing agent. These results confirmed that Ser2 belongs to the peptidase family M10 and requires Ca²⁺, Zn²⁺, and disulfide bridges for stability. This protease is able to hydrolyze three kinds of casein in the preferential order of κ→ β→ α‐casein. Highly heat‐stable in skimmed, semi‐skimmed, and whole milk at 140°C with D‐values of 2.8, 3.9, and 4.5 min, respectively, Ser2 showed a residual activity between 87 and 100 percent after heat‐treatment of 65 °C for 30 min, 72 °C for 20 s, and 140 °C for 4 s that are commonly used in dairy industries. As the protease AprX that is mainly secreted by Pseudomonas genus, Ser2 could be one of the main causes of UHT milk destabilization during storage.     

Purification and characteristics of serine protease from the head of pacific white shrimp

Serine protease from the head of Pacific white shrimp was purified by the following techniques: ammonium sulfate fractionation, Q-Sepharose HP ion exchange chromatography, and Sephadex G-100 gel filtration. The molecular weight was estimated as 32.8 kDa using SDSPAGE. The optimum pH and temperature of the enzyme for the hydrolysis of casein were determined to be 10.0 and 40°C. It was stable at pH range from 8.0 to 11.0 and had good thermal stability. Pb²⁺, Ca²⁺, Mg²⁺, Cu²⁺, and Mn²⁺ could active the enzyme certainly when Zn²⁺ and Hg²⁺ strongly inhibited the activity. The enzyme was inhibited by the general serine protease inhibitor (PMSF) and the specific trypsin inhibitors (TLCK, SBTI). The modification of various amino acid modifiers for the purified enzyme determined that the enzyme active center included tryptophan, histidine, and serine, moreover, arginine had a certain relationship with the enzyme activity.     

Purification and characterization of cathepsin B from the gut of the sea cucumber (<Emphasis Type="Italic">Stichopus japonicas</Emphasis>)

Cathepsin B from the gut of sea cucumber (Stichopus japonicas) was purified 81-fold with a 3% recovery by ammonium sulfate fractionation and a series chromatography on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-Gel 3000 SWxl. The purified protein appeared as a single band on Native-PAGE but showed 2 bands of 23 and 26 kDa on SDS-PAGE. The optimum activity was found at pH 5.5 and 45°C. The enzyme was stable at pH 4.5–6.0 and the thermal stability was up to 50oC. The enzyme was strongly inhibited by E-64, iodoacetic acid, and antipain, demonstrating it is a cysteine protease containing sulfhydryl groups. Cu²⁺, Ni²⁺, and Zn²⁺ could strongly inhibit the enzyme activity. The amino acid sequences of the purified enzyme were acquired by mass spectrometer, which did not show any homology with previously described cathepsins, suggesting it may be a novel member.     

Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912

A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion‐exchange chromatography on Q‐ and S‐Sepharose (fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 8.4‐fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg^?1 protein. SDS—PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0–8.0 and retained over 60% activity at 70 °C after 30‐min incubation. It was highly significantly (P < 0.001) inhibited by Hg²⁺, appreciably (P < 0.01) inhibited by Ag⁺, Ba²⁺ and Pb²⁺ but highly significantly (P < 0.001) activated by Co²⁺, Mn²⁺ and Sr²⁺. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p‐chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: K_m = 0.33 mg ml^?1; V_max = 0.08 µmol ml^?1 min^?1. Copyright © 2004 Society of Chemical Industry     

Production optimization,purification, and characterization of a novel acid protease from a fusant by Aspergillus oryzae and Aspergillus niger

The production of a novel acid protease was enhanced by 44 % through statistical optimization. The cultural parameters, such as inoculum size, temperature, moisture content, and incubation time, were 8.59 × 10⁵ g^?1 dry koji, 31 °C, 57 %, and 86 h, respectively. This novel acid protease was purified by 17 folds with a recovery yield of 33.56 % and a specific activity of 4,105.49 U mg^?1. Far-UV circular dichroic spectra revealed that this purified protease contained 7.1 % α-helix, 64.1 % β-sheet, and 32 % aperiodic coil. This novel acid protease was active over the temperature range of 35–55 °C with optimum temperature of 40 °C and was stable in the pH range of 2.5–6.5 with optimum pH of 3.5. Mn²⁺ enhanced its activity while Co²⁺ showed inhibitory effect. With casein as substrate, the kinetic parameters of K _m, V _max, energy of activation (E _a), and attenuation index of inactivation velocity by heat inducing (λ) were 0.96 mg mL^?1, 135.14 μmol min^?1 mg^?1, 64.11 kJ mol^?1, and 0.59, respectively.     

Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.)

Background: γ‐Aminobutyric acid (GABA) is a non‐protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro. Results: The molecular mass of the enzyme estimated by Sephadex G‐100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg²⁺, Cu²⁺, Fe³⁺, aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA‐Na₂), L ‐cysteine and β‐mercaptoethanol. The K_m value of DAO was 0.23 mmol L^?1 for putrescine and 0.96 mmol L^?1 for spermidine. However, the enzyme did not degrade spermine. Conclusion: DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine. Copyright © 2011 Society of Chemical Industry     

Purification and characterization of Aspergillus oryzae LK-101 salt-tolerant acid protease isolated from soybean paste

A novel salt-tolerant acid protease was produced from Aspergillus oryzae LK-101 (AOLK-101). The AOLK-101 protease was purified to homogeneity by ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatographies in order. The specific activity and the purification ratio of the purified protease were 2,301 unit/mg and 11.6 fold, respectively, with 25 kDa of molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrpphoresis (SDS-PAGE). Its optimal pH and temperature were pH 6.5 and 50°C, respectively. This protease was relatively stable at pH 4.5–7.5, below 40°C, and up to 10% salt concentration. The protease was moderately inhibited by Ag²⁺ and Zn²⁺, and strongly by ethylenediamide tetraacetic acid (EDTA) and phenylmethysulfonyl fluoride (PMSF), but activated by Cu²⁺ and Mn²⁺. Therefore, the AOLK-101 protease was a serine protease based on the influence of metal ions and inhibitors. K _m , V _max , k _cat , and k _cat /K _m values of AOLK-101 protease for hammastein milk casein were 1.04 mg/mL, 124.84 unit/L, 163.5/sec, and 3.9×10⁶/m·sec, respectively.     

A preliminary study of the protease activities in germinating brown rice (Oryza sativa L.)

BACKGROUND: Proteases hydrolyse storage proteins to provide precursors for perpetuating species. The aim of this study was to investigate and characterise different proteases in germinating brown rice. RESULTS: The protease activity of brown rice increased sevenfold during 7 days of germination. It was highest on day 6 when determined at pH 3.5. With casein as substrate the proteases showed two catalytic groups: acidic proteases with an optimal pH of 3.5 and alkaline proteases with an optimal pH of 8.0. The acidic protease activity was inhibited by Ba²⁺ and Pb²⁺ but stimulated by Zn²⁺, while the alkaline protease activity was inhibited by Ca²⁺ and Pb²⁺ but stimulated by Mg²⁺ and Zn²⁺. SDS‐gelatin‐PAGE assay showed two protease activity bands at pH 3.5, while two different bands with higher molecular weights were observed at pH 8.0. Inhibition assay revealed that pepstatin A and E‐64 inhibited 67.63 and 38.26% respectively of the protease activity at pH 3.5, indicating the presence of aspartic and cysteine proteases. Metalloproteases played a major role under alkaline conditions (88.37% inhibition with EDTA). CONCLUSION: Germinated brown rice proteases fall into different classes with different properties. This study is helpful for their further purification. Copyright © 2010 Society of Chemical Industry     

Characterization of a β-Glucosidase from an Edible Mushroom,Lycoperdon Pyriforme

A β-glucosidase from Lycoperdon pyriforme, a wild edible mushroom, was characterized biochemically. The enzyme showed a maximum activity at pH 4.0 and 50°C when p-nitrophenyl-β-D-glucoside was used as a substrate. K_m and V_max values were calculated as 0.81 mM and 1.62 U/mg protein, respectively. The enzyme activity was conserved about 85% over a broad range of pH (3.0–9.0) at 4°C after 24 h incubation. The activity was fully retained after 60 min incubation at 20–40°C. Na⁺, Li⁺, Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺, and Cu²⁺ did not affect the enzyme activity and 0.25% sodium dodecylsulfate inhibited the enzyme activity approximately 76%. Ethylenediamine tetra-acetic acid, phenylmethanesulfonylfluoride, and dithiothreitol showed no or a little negative effect on the enzyme activity. The resistance of the enzyme to some metal ions, chemicals, and ethanol along with the pH stability, can make it attractive for future applications in industry.     

Helvellisin,a novel alkaline protease from the wild ascomycete mushroom Helvella lacunosa

A 33.5-kDa serine protease designated as helvellisin was isolated from dried fruiting bodies of the wild ascomycete mushroom Helvella lacunosa. It was purified by using a procedure which entailed ion exchange chromatography on DEAE-cellulose, CM-Sepharose, Q-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was characterized by unique N-terminal amino acid sequence, thermostability and pH stability. The protease exhibited a pH optimum of 11.0 and a temperature optimum of 65 °C, with about 40% activity remaining at 87 °C and pH 5 and 13. Helvellisin demonstrated a protease activity of 14 600 U/mg toward casein. The K_m of the purified protease for casein was 3.81 mg/ml at pH 11.0 and 37 °C. The V_max was 5.35 × 10^− 2 mg ml^− 1 min^− 1. It was adversely affected by phenylmethylsulfonyl fluoride, suggesting that it is serine protease. The activity of the protease was enhanced by Mg²⁺, Fe²⁺ and Mn²⁺, but was curtailed by Cu²⁺, Hg²⁺ and Fe³⁺. It was devoid of antifungal and ribonuclease activities.     

PURIFICATION OF A PROTEASE FROM AN ALKALOPHlLIC BACILLUS SUBTILIS CHZ1 ISOLATED FROM A ZIMBABWEAN HOT SPRING

A proteolytic enzyme produced by Bacillus subtilis CHZ1 was purified using ammonium sulfate precipitation, gel filtration and cationic exchange on S‐Sepharose fast flow column chromatography. Production of the protease was higher when the Bacillus strain was cultured in a synthetic medium, M162, supplemented with 0.3% (w/v) organic compared to inorganic nitrogen sources. Enzyme production was growth dependent and production was highest when tryptone was used as the nitrogen source. When run on SDS‐PAGE gel, the purified enzyme gave a 35 kDa band, suggesting that it consisted of one polypeptide chain. High enzyme activity was observed in the pH range of 6–10 with a maximum value at pH 8.0 when 0.5% (w/v) azocasein was used as the substrate. Optimum temperature for protease activity was found to be 60–80C, and the enzyme had considerable thermal stability for 5.5 h retaining about 90% activity after 5.5 h. At 2.5 mM concentration, PMSF, Ag⁺ and Hg⁺ inhibited activity of the protease. Metal cofactors like Mn²⁺, Mg²⁺ and Fe²⁺ increased the enzyme activity by about 20%. Zn²⁺, Cu²⁺ and Ca²⁺ did not affect the enzyme's activity. The pH and thermal stability as well as high specific activity of this enzyme can be exploited for industrial applications.     

ENZYMATIC PROPERTIES OF A PROTEASE FROM THE HEPATOPANCREAS OF SHRIMP, PENAEVS OMENTAIM

The protease purified from hepatopancreas of shrimp, Penaeus orientals, had high proteolytic activity in the pH range of 7.0 to 9.5. Temperature optimum for hydrolysis of casein was 70C. The protease was stable at neutral and alkaline pH and unstable at acidic pH. The apparent Michaelis‐Menten constant (K_m) and the turnover number (V_max) of the protease on hydrolysis of N‐CBZ‐L‐tyrosine p‐nitrophenyl ester (CBZ‐Tyr‐NE) and N‐CBZ‐L‐phenylalanine p‐nitrophenyl ester (CBZ‐Phe‐NE) ‐were similar, however, those for N‐CBZ‐L‐cysteine p‐nitropher.yl ester (CBZ‐Cys‐NE) were different. K_m and V_max for hydrolysis of casein by the protease were determined to be 0.31% and 5.21s^‐1, respectively. The N‐terminal sequence of the protease showed higher homology with the collagenase of crab and trypsins from Crustacea. Myosin heavy chain (MHC) was the primary substrate during proteolysis with the protease. Actin/tropomyosin were degraded progressively during 2 h incubation but to a lesser extent than MHC.     

Characterization of ginger proteases and their potential as a rennin replacement

BACKGROUND: Ginger rhizome (Zingiber officinale Roscoe) contains ginger proteases and has proteolytic activity. Ginger proteases have been used for tenderizing meat but rarely for milk clotting. The purpose of this study was to purify ginger proteases and to research their biochemical characteristics. RESULTS: The milk clotting activity (MCA) and proteolytic activity (PA) of the proteases was stable after storage at 4 °C for 24 h. The MCA and PA of fresh ginger juice with 0.2% L ‐ascorbic acid remained stable for 6 days at 4 °C. When under storage at ?80 °C for 2 months, the MCA and PA of the fresh ginger juice and acetone precipitate were still high. Two peaks with protease activity were purified from a DEAE FF ion‐exchange column; the specific activity (units mg^?1 protein) of the MCA (MCSA) and PA (PSA) for the first peak was significantly higher than the second peak (P < 0.05). The protease activity of the ginger proteases was significantly inhibited by E‐64, leupeptin, and iodoacetic acid. Zymography results showed that two protease fractions purified from ginger juice with 62 and 82 kDa had a higher PA against α‐ and β‐casein than against κ‐casein. CONCLUSION: The ascorbic acid addition significantly stabilized the MCA and PA of ginger proteases. The protease inhibition test suggested that ginger proteases belonged to the cysteine type. The biochemical characteristics of ginger protease described in this paper can provide useful information for making new milk curd products. Copyright © 2009 Society of Chemical Industry     

Purification and characterization of lysozyme from filipino venus, Ruditapes philippinarum

Lysozyme from Filipino venus (Ruditapes philippinarum) was purified by ion-exchange and gel filtration chromatography. The purification fold and yield were 3,402 and 32.4%, respectively. The molecular weight was determined to be 13.4 kDa by SDS-PAGE. The specific activity of lysozyme was 3.76×10⁵ units/mg protein with Micrococcus lysodeikticus as a substrate. The optimum temperature and pH of lysozyme were 75°C and 5.5, respectively. Lysozyme activity was decreased with about 45% after heat treatment for 30 min at 80°C, and completely inactivated at 100°C. It was activated by NaCl (10–70 mM), MgCl₂, and CaCl₂ (2–5 mM) whereas it was inhibited by ZnCl₂ (2–30 mM).     

Isolation,purification and properties of the peroxidase from the hull of Glycine max var HH2

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from soybean (Glycine max var HH2) hulls was purified to electrophoretic homogeneity by a combination of ammonium sulphate fractionation, DEAE‐Sephadex A‐50 chromatography, concanavalin A‐Sepharose 4B affinity chromatography and Bio‐Gel P‐60 gel filtration. The specific activity of purified peroxidase was about 57‐fold higher than that of crude extract. The yield was about 16.4%. The molecular weight of the enzyme was estimated to be 38 000 by SDS‐polyacrylamide gel electrophoresis. The peroxidase was a glycoprotein containing about 18.7% carbohydrate, approximately one‐quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.9. The enzyme was most active at pH 4.6 and 45°C, and was stable in the pH range 2.5–11.5. The enzyme could tolerate heating for 10 min at 75°C without being inactivated, and at 85°C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel thermostable enzyme. Fe ²⁺, Fe³⁺, Sn²⁺, CN ⁻ and N₃⁻ inhibited enzyme activity, while Hg²⁺, Ag⁺, Pb ²⁺, Cr³⁺, EDTA and SDS were not significantly inhibitory. © 1999 Society of Chemical Industry     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

PARTIAL PURIFICATION AND PROPERTIES OF PUMPKIN LIPOXYGENASE WITH CAROTENE-BLEACHING ACTIVITY

From locally grown pumpkins, an active lipoxygenase preparation with an active carotene-bleaching factor was partially purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. The enzyme had a pH optimum at 6.5 and was inactive at pH below 3 and above 10. The maximum activity occurred at 30°C. The apparent K_m determined in the presence of linoleate was 0.33 × 10^?3 M. The heavy metals Hg²⁺, Cu²⁺, Co²⁺, and Fe³⁺ were effective inhibitors of this enzyme. Cyanide, fluoride, and L-ascorbic acid also inhibited lipoxygenase activity. Carotene-bleaching activities operated strongly on the lipoxygenase fraction and slightly on the denatured lipoxygenase and hemoproteins treated with heat.     

Optimization of process conditions for the production of a prolylendopeptidase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 11414 in solid state fermentation

The effect of 8 factors [(with/without) daily mixing and moisture control, incubation time (t), temperature, ratio between dry substrate mass and bed’s cross section area (MA), inoculum size (spores/g), wheat germ content (WG), initial pH, and moisture content (M)] in the production of a prolyl endopeptidase (PEP) by Aspergillus niger ATCC 11414 in solid state fermentation (SSF) was tested. Contribution of all the factors was significant (p<0.05); main effects were those of MA, t, and M. The 4 interactions that presented high interaction severity indexes involved the WG. Under optimized conditions PEP and protease activity were 9.76±0.06 and 3.6×10⁶±1.5×10⁵ U/kg, respectively. The enzyme was partially purified (ammonium sulfate precipitation, dialysis, DEAE-Sepharose ionexchange); it has a molecular weight of 66 kDa (SDS-PAGE), and maximum activity was exhibited at pH 4 and 50°C. The enzyme is stable in a wide pH range (2.2–10) and at temperatures lower than 70°C.