Purification and characterisation of thermo-alkaline pectinase enzyme from Hylocereus polyrhizus

The thermo-alkaline pectinase enzyme from Hylocereus polyrhizus was purified 232.3-fold with a 73.3 % recovery through ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. Ion exchange chromatography combined with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme was monomeric with a molecular weight of 34.2 kDa. The pectinase exhibited broad specificity towards polygalacturonic acid, arabinan, oat spelt xylan, and pNP-α-glucopyranoside. The optimum pH and temperature were 8.0 and 75 °C, respectively. This enzyme was stable over a wide pH range (3.0–11.0) and at relatively high temperature (85 °C for 1 h). The K_m and V_max values of pectinase towards polygalacturonic acid were 2.7 mg/ml and 34.30 U/mg proteins, respectively. In addition, the enzyme activity was inhibited by Ni²⁺, Al³⁺, and Fe²⁺ and was increased in the presence of Ca²⁺ and Mg²⁺ by 120 and 112 %, respectively. The purified pectinase demonstrated robust stability in response to surfactants and oxidising agents. EDTA, which is a powerful chelating agent, did not exert any significant effect on the enzyme stability. Thus, enzymes with these unique properties may be widely used in different types of industries and biotechnological applications.     

Induction,purification and characterization of malolactic enzyme from Oenococcus oeni SD-2a

The aim of this study was to purify a malolactic enzyme (MLE) from Oenococcus oeni (O. oeni) strain and determine its properties in detail. O. oeni SD-2a was cultivated in the ATB broth supplemented with 7 g/L l-malic acid for harvesting the cells. After harvest, the cells were washed and disrupted for purification of MLE. MLE was purified from the supernatant of the disrupted cells through protamine sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The purified MLE was identified using mass spectrometry. The MLE was purified by 43-fold with a yield of 0.42 % and possessed a specific activity of 419.2 U/mg. The purified enzyme with a nominal molecular mass of 59 kDa and a theoretical pI of 4.76 exhibited a maximum enzyme activity at 35 °C and pH 6.0, which retained over 50 % of its initial activity in the presence of 14 % (v/v) ethanol. Mn²⁺ was proven to be the most effective divalent cation to promote enzyme activity. Under the conditions of temperature 30 °C and pH 6.0, the K _m and V _max of MLE on l-malic acid were 12.5 × 10^?3 M and 43.86 μmol/(min × mg), respectively. Moreover, the purified enzyme exhibited a higher stability with 0.1 M NaCl in addition and had a half-life of 30 days at 4 °C.     

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.     

A Comparative Biochemical Characterization of Microbial Transglutaminases: Commercial vs. a Newly Isolated Enzyme from <Emphasis Type="Italic">Streptomyces</Emphasis> Sp.

A new microbial transglutaminase (MTGase or MTG, EC 2.3.2.13) from a Streptomyces sp. strain isolated from Brazilian soil samples was characterized in crude and purified forms. The aim of this work is to provide relevant information about a new transglutaminase and to compare its characteristics with the well-known commercial transglutaminase from Ajinomoto Co. Inc. (Activa® TG-BP). The enzyme from Streptomyces sp., in both crude and pure forms, exhibited optimal activity in the 6.0–6.5 pH range and at 35–40°C. The results for the commercial enzyme were the same. A second maximum of activity was observed at pH 10.0 with both the crude Streptomyces sp. enzyme and the commercial enzyme. This interesting fact has not been reported in the literature previously. The fact that this second maximum of activity does not appear on the purified form of the enzyme may suggest the presence of an isoenzyme on the crude extract. All of the enzymes tested were stable over the pH range from 4.5 to 8.0 and up to 45°C. The decline in activity of the commercial transglutaminase above 45°C and pH 8.0 was more gradual. The activities of all the MTG samples were independent of Ca⁺² concentration, but they were elevated in the presence of K⁺, Ba²⁺, and Co²⁺ and inhibited by Cu²⁺ and Hg²⁺, which suggests the presence of a thiol group in the MTG’s active site. The purified enzyme presented a K _m of 6.37 mM and a V _max of 1.7 U/mL, while the crude enzyme demonstrated a K _m of 6.52 mM and a V _max of 1.35 U/mL.     

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.     

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     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

Purification and characterization of an alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 and its application in mannooligosaccharides preparation having prebiotic potential

An alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 was purified 38.96-fold to homogeneity with specific activity of 2,288.90 ± 27.80 U mg^?1 protein and final recovery of 8.92 ± 0.09 %. The purified β-mannanase was an extracellular monomeric protein with a molecular mass of 50 kDa on SDS–PAGE. The first 20 N-terminal amino acid sequence of mannanase enzyme was MVVKKLSSFILILLLVTSAL. The optimal temperature and pH for enzyme were 65 °C and 8.8, respectively. It was completely stable at 60 °C for 3 h and retained >50 ± 1.0 % activity at 70 °C up to 3 h. The β-mannanase was highly stable between pH 5–10 and retained >85 % of the initial activity for 3 h. The metal ions Ni⁺², Co⁺², Zn⁺² and Mg⁺² enhanced the enzyme activity. The enzyme remained stable after 3 h of preincubation with most of the tested organic solvents. According to substrate specificity study, the purified mannanase had high specificity to locust bean gum which was degraded mainly to mannooligosaccharides (MOS) like mannotriose, mannotetraose and mannopentose. These MOS enhanced the growth of Lactobacillus casei but inhibited the growth of Salmonella enterica indicating potential prebiotic properties. The properties of the purified β-mannanase from B. nealsonii PN-11 make this enzyme attractive for biotechnological applications.     

Purification and characterization of hydrolytic and transgalactosyl α-galactosidase from Lactobacillus helveticus ATCC 10797

α-Galactosidase purified from Lactobacillus helveticus ATCC 10797 by fast performance liquid chromatography system using ion exchange and gel-filtration columns showed the K _m of 3.83 mM and V _max of 416.44 µmol/min/mg protein calculated from the substrate p-nitrophenyl-α-d-galactopyranoside. The molecular mass was 188 kDa by gel-filtration, but 90 kDa by SDS-PAGE, indicating a homodimer. The optimum temperature was 37 °C, and the optimum pH was at 6 with an acceptable stability between pH 4 and 8. This enzyme was activated by 10 mM monovalent ions such as K⁺, NH₄ ⁺, Li⁺, and CS⁺, while the activity was inhibited by divalent ions such as Cu²⁺, Zn²⁺, and Fe²⁺. Melibiose was hydrolyzed to glucose and galactose, raffinose to galactose and sucrose, while stachyose to galactose and sucrose. A novel source of α-galactosidase from L. helveticus possessing both hydrolytic activity to eliminate flatulence sugars and transgalactosylation activities to synthesize galacto-oligosaccharides is identified and characterized.     

Purification and characterisation of β‐mannanase from Lactobacillus plantarum (M24) and its applications in some fruit juices

β‐Mannanase was purified 2619.05‐fold from the Lactobacillus plantarum (M24) bacterium by ammonium sulphate precipitation and ion exchange chromatography (DEAE‐Sephadex). The purified enzyme gave two protein bands at a level of approximately 36.4 and 55.3 kDa in the SDS‐PAGE. The purified mannanase enzyme has shown its maximum activity at 50 °C and pH 8, and it has been also determined that the enzyme was stable at 5–11 pH range and over 50 °C. The V_max and K_m values have been identified as 82 mg mannan mL^?1 and 0.178 mm , respectively. The effects of some metal ions such as Fe²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ on the mannanase enzyme have been also investigated, and it has been determined that all metal ions had significant effects on the activation of the mannanase enzyme. In addition, the effectiveness of the purified mannanase enzyme on the clarification of some fruit juices such as orange, apricot, grape and apple has been investigated. During the clarification processes, the enzyme was more effective than crude extracts on the clarification of the peach juice with a ratio of 223.1% at most.     

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     

Lactobacillus plantarum 70810 from Chinese paocai as a potential source of β-galactosidase for prebiotic galactooligosaccharides synthesis

A novel food-grade strain Lactobacillus plantarum 70810 producing β-galactosidase with high transgalactosylation activity was isolated from Chinese paocai. The galactooligosaccharides (GOS) were synthesized by using this enzyme with a maximum yield of 44.3 % (w/w) from 400 g/L lactose at 45 °C for 10 h. The β-galactosidase from this strain was purified to homogeneity by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. It was a heterodimer arrangement of approximately 105 kDa composed of two subunits of 35 and 72 kDa. The optimal pH of the purified β-galactosidase was 8.0 for both o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose hydrolysis, and optimal temperature was 60 °C and 55 °C, respectively. Its K _m and V _max values for oNPG and lactose were 0.89 ± 0.05 mM, 194 ± 3.0 μmoL/min/mg protein, and 9.88 ± 0.16 mM, 15.88 ± 0.21 μmoL/min/mg protein, respectively. This enzyme was slightly inhibited by the hydrolysis products, that is, glucose and galactose. Since the β-galactosidase from L. plantarum 70810 exhibited higher transgalactosylation activity, strong affinity for lactose and low end-product inhibition, it was suggested to be a potential candidate for the synthesis of prebiotic GOS.     

Arginine aminopeptidase from white shrimp (Litopenaeus vannamei) muscle: purification and characterization

Aminopeptidases act on N-terminal of proteins and peptides produce free amino acids making an impact on the final flavor of foods. An arginine aminopeptidase (RAP) which preferred to hydrolyze basic amino acids from N-termini of peptides and proteins was purified to homogeneity from white shrimp (Litopenaeus vannamei) muscle. The molecular mass of RAP was estimated as 100 kDa on SDS-PAGE. Peptide mass fingerprinting analysis obtained 95 amino acid residues which was 100 and 77.9 % identical to puromycin-sensitive aminopeptidases from insect and zebrafish, respectively. Optimum pH and temperature of the RAP were 7.0 and 30 °C. RAP rapidly hydrolyzed fluorogenic substrates l-arginine 4-methylcoumaryl-7-amide (Arg-MCA) and Lys-MCA with K _m values of 2.7 and 4.9 μM, respectively. The enzyme can be strongly inhibited by puromycin, bestatin, and 1,10-phenanthroline and partially inhibited by ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). Moreover, the competitive inhibition of puromycin for RAP was confirmed, and K _i value was calculated as 0.07 nM. Metal ions of Zn²⁺ and Mn²⁺ significantly reactivated the inactive apoenzyme activity dialyzed by EDTA. All these results indicated that the purified enzyme is a metalloaminopeptidase which would possibly contribute to flavor development in shrimp muscle.     

A novel cyclodextrin glycosyltransferase from an alkalophilic Bacillus species: purification and characterization

Cyclodextrin glycosyltransferase (E.C. 2.4.1.19) of alkalophilic Bacillus sp. 7-12 was purified by ammonium sulfate precipitation, DEAE–cellulose column chromatography and Sepharose CL-6B column chromatography. The enzyme thus obtained consisted of a single band that did not dissociate into subunits by SDS–polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was determined to be 69,000 Da by SDS–PAGE. The enzyme was stable below 70 °C with an optimum activity at 60 °C, and was stable at a pH range of 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by MgCl₂, ZnCl₂, CuSO₄, Al₂(SO₄)₃, CoCl₂, AgNO₃, FeSO₄ and slightly inhibited by SnCl₂ and MnCl₂. CaCl₂, KCl, EDTA and DTT had no influence on the enzyme activity. For cyclodextrin production, up to 34% conversion to cyclodextrins was obtained from 10% starch. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.26:1:0.86.     

Alpha-Amylase from Persimmon Honey: Purification and Characterization

The α-amylase was extracted from pure persimmon honey and purified by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies. Molecular weight of purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively suggested that the purified enzyme was a monomer. Optimum pH of the enzyme was 6.0?7.0 and optimum temperature 40°C. The enzyme was extremely inactivated at pH was higher than 7.0 or lower than 5.0. Heat inactivation occurred at 40°C. This enzyme activated by Ca ²⁺ , Mn²⁺, PCMB, and DTNB, but inhibited by Ba²⁺, Fe³⁺, Hg²⁺, Mg²⁺, and iodoacetic acid. The purified enzyme was of α?-type by TLC analysis. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α?-1,4-linkages and with increasing percentage of α?-1,6-linkages in substrate similar to the results from commercially available honey.     

Purification and Characterization of a β-Amylase of Hendersonula toruloidea

β-Amylase produced by Hendersonula toruloidea was purified to homogeneity by salting out with ammonium sulphate, ion-exchange chromatography on DEAE-cellulose and gel-filtration on Sephadex G-75. The relative molecular mass of the enzyme was estimated to be 60,000 by gel filtration. The enzyme was optimally active at pH 6.0 and 60°C, stable between pH 6 and 8 (24 h) and retained 74% activity at 70°C (30 min). It was strongly activated by Na⁺ but inhibited by Hg²⁺, Zn²⁺ and Cu²⁺. The enzyme hydrolyzed amylopectin (Km 0.42 mg/ml) forming maltose, maltotetraose and unidentified maltooligosaccharide, and hydrolyzed soluble starch (Km 0.3 mg/ml) and glycogen (Km 0.5 mg/ml) forming maltose and unidentified maltooligosaccharide.     

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.     

An alkaline protease from Kocuria kristinae F7: properties and characterization of its hydrolysates from soy protein

An alkaline serine protease (SF1) from Kocuria kristinae F7 was purified. The molecular mass of the SF1 protease was estimated to be 57 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The N-terminal amino acid sequence of the first 14 amino acids of the SF1 protease showed low homology with bacterial proteases, suggesting that the enzyme had not been described previously. The SF1 protease exhibited maximal activity at pH 9.0 and 60 °C. The activity of the SF1 protease was enhanced by the presence of Ca²⁺ and Mg²⁺ ions. It showed high stability in the presence of NaCl and ethanol. Reverse-phase high-performance liquid chromatography (RP-HPLC) analyses indicated that soybean protein isolates treated with the SF1 protease generated four principal new hydrophilic peptides. Mass spectrometry analyses indicate that the distribution of molecular weight of these peptides was from 0.705 to 1.305 kDa. Hydrolysis of soybean protein isolates with the SF1 protease increased the level of total free amino acids, essential amino acids and flavor amino acids. The SF1 protease may decrease the bitterness of soy protein hydrolysates. The results showed that the SF1 protease of Kocuria kristinae F7 appears to be good candidate enzyme for potential application in acceleration of fermented soybean food ripening.     

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.