Isolation and characterization of a cold-active,alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205?kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0–12.0) and retained 96% of its original activity at low temperatures (10–40°C) for 24?hr. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba²⁺, Ca²⁺, Na⁺, Zn²⁺, Mn²⁺, H₂O₂, and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.     

Biochemical characterization and structural insights into the high substrate affinity of a dimeric and Ca2+ independent Bacillus subtilis α-amylase

An extracellular amylase (AmyKS) produced by a newly isolated Bacillus subtilis strain US572 was purified and characterized. AmyKS showed maximal activity at pH 6 and 60°C with a half-life of 10 min at 70°C. It is a Ca²⁺ independent enzyme and able to hydrolyze soluble starch into oligosaccharides consisting mainly of maltose and maltotriose. When compared to the studied α-amylases, AmyKS presents a high affinity toward soluble starch with a K_m value of 0.252 mg ml⁻¹. Coupled with the size-exclusion chromatography data, MALDI–TOF/MS analysis indicated that the purified amylase is a dimer with a molecular mass of 136,938.18 Da. It is an unusual feature of a non-maltogenic α-amylase. A 3D model and a dimeric model of AmyKS were generated showing the presence of an additional domain suspected to be involved in the dimerization process. This dimer arrangement could explain the high substrate affinity and catalytic efficiency of this enzyme.     

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35?kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60?°C. It was determined that the enzyme had remained stable at the range of pH 7.0–10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20–80?°C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. K_M and V_max values were calculated as 0.197?mg/mL and 7.29?μmol.mL^?1.min^?1, respectively.     

PURIFICATION AND CHARACTERIZATION OF MUTANASE PRODUCED BY Paenibacillus curdlanolyticus MP-1

Mutanases are enzymes that catalyze hydrolysis of α-1,3-glucosidic bonds in various α-glucans. One of such glucans, mutan, which is synthesized by cariogenic streptococci, is a major virulence factor for induction of dental caries. This means that mutan-degrading enzymes have potential in caries prophylaxis. In this study, we report the purification, characterization, and partial amino acid sequence of extracellular mutanase produced by the MP-1 strain of Paenibacillus curdlanolyticus, bacterium isolated from soil. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular mass 134 kD, while native gel filtration chromatography confirmed that the enzyme was a monomer of 142 kD. Mutanase showed a pH optimum in the range from pH 5.5 to 6.5 and a temperature optimum around 40–45°C. It was thermostable up to 45°C, and retained 50% activity after 1 hr at 50°C. The enzyme was fully stable at a pH range of 4 to 10. The enzyme activity was stimulated by the addition of Tween 20, Tween 80, and Ca²⁺, but it was significantly inhibited by Hg²⁺, Ag⁺, and Fe²⁺, and also by p-chloromercuribenzoate, iodoacetamide, and ethylenediamine tetraacetic acid (EDTA). Mutanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-1,3-glucans. It also showed binding activity to insoluble α-1,3-glucans. The N-terminal amino acid sequence was NH₂-Ala-Gly-Gly-Thr-Asn-Leu-Ala-Leu-Gly-Lys-Asn-Val-Thr-Ala-Ser-Gly-Gln. This sequence indicated an analogy of the enzyme to α-1,3-glucanases from other Paenibacillus and Bacillus species.     

Purification and Some Properties of the Endo α-1,4 Polygalactosaminidase from Pseudomonas sp.

The endo α-1,4 polygalactosaminidase from Pseudomonas sp. 881 was purified from the culture nitrate by ethanol precipitation and sequential column chromatographies on CM-Sephadex C-25, Sephadex G-50 and Phenyl-Sepharose CL-4B. The purified enzyme was electrophoretically homogeneous and its molecular weight and isoelectric point were 31,000 and 6.7, respectively. The optimum pH and temperature for hydrolysis of polygalactosamine were 5.0 and 55°C, respectively. The enzyme was stable up to 45°C for 15min and from pH 4.0 to 7.6 at 37°C for 1 hr.

The Km value was 0.05% α-1,4 polygalactosamine and the V was 0.154μmol reducing sugar (galactosamine)/min/μg protein. This polygalactosaminidase was inhibited by Sn²⁺ , Fe²⁺ , Fe³⁺ , Hg²⁺, Cu²⁺ ions and SDS. The enzyme did not hydrolyze oligo galactosamines (n < tetramer) or N-acetyl-polygalactosamines. It acted only on oligo galactosamine (n > trimer) and polygalactosamine endogeneously so far tested.     

Purification and characterization of the amylase of B. subtilis NRRL B3411

The amylase of Bacillus subtilis NRRL B3411 has been purified and partially characterized. The specific activity can be increased from 300,000 units/g to 6,000,000 units/g with a 60% recovery of total units. The purified material consists of one major and one trace anodic component as determined by disc gel electrophoresis. The molecular weight was 48,000 as determined by bio-gel filtration; the molecular weight was 44,900 ± 2400 as determined by sedimentation equilibrium methods. This purified enzyme is stable at, 70°C in the presence of 0.01 M Ca⁺⁺ and 0.1 M NaCl over a broad pH range from 5.5–9.5. The pH activity profile indicates optimum activity at pH 6.0. This amylase exhibits maximum activity at 60°C. The enzyme is a liquefying α-amylase as determined by analysis of hydrolysis products and immunological studies.     

Production,Purification, and Characterization of α-Amylase from Solventogenic Clostridium sp. BOH3

A solventogenic strain of Clostridium sp. BOH3 produces extracellular α-amylase (7.15 U/mg protein) in reinforced clostridial medium supplemented with sugarcane bagasse hydrolysate (1 % w/v) and a small amount of starch (0.1 % w/v), which is essential for the expression of α-amylase. In the presence of α-amylase, BOH3 utilizes starch directly without any pretreatment and produces butanol almost equivalent (～90 %) to the production of butanol from glucose. α-Amylase can be purified from culture supernatant by using one-step weak anion exchange chromatography with a yield of 43 %. In peptide fingerprinting analysis, this enzyme shows homology with α-amylase produced by Clostridium acetobutylicum ATCC824. However, the molecular weight is 54 kDa, which is smaller than α-amylase of ATCC824 (84 kDa). This enzyme has optimum temperature at 45–50 °C and optimum pH at 4.5–5.5. Under this condition, the enzyme activity is 91.32 U/mg protein, and its K _m and V _max values are 1.71?±?0.02 mg/ml and 96.13?±?0.15 μmol/min/mg protein, respectively. Activity of this α-amylase can be enhanced (>1.5 times) by addition of Ca²⁺ and Co²⁺ and its activity can be maintained at an acidic pH (pH 3–5) for about 24 h. These unique characteristics suggest that this enzyme can be used for saccharification of starch for production of biofuel in one pot.     

Purification an α-galactosidase from Coriolus versicolor with acid-resistant and good degradation ability on raffinose family oligosaccharides

An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg^?1. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4–50 °C and at a pH range of 2–5. Among the metal ions tested, Cu²⁺, Cd²⁺ and Hg²⁺ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag⁺ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.     

Molecular cloning,expression, and biochemical characterization of a novel cold-active α-amylase from Bacillus sp. dsh19-1

A novel gene (ANK58566) encoding a cold-active α-amylase was cloned from marine bacterium Bacillus sp. dsh19-1 (CCTCC AB 2015426), and the protein was expressed in Escherichia coli. The gene had a length of 1302 bp and encoded an α-amylase of 433 amino acids with an estimated molecular mass of 50.1 kDa. The recombinant α-amylase (AmyD-1) showed maximum activity at 20 °C and pH 6.0, and retained about 35.7% of activity at 4 °C. The AmyD-1 activity was stimulated by Ca²⁺ and Na⁺. However, the chelating agent, EDTA, inactivated the enzyme. Moreover, AmyD-1 displayed extreme salt tolerance, with the highest activity in the presence of 2.0 M NaCl and 60.5% of activity in 5.0 M NaCl. The K_m, V_max and k_cat of AmyD-1 in 2.0 M NaCl were 2.8 mg ml⁻¹, 21.8 mg ml⁻¹ min⁻¹ and 933.5 s⁻¹, respectively, at 20 °C and pH 6.0 with soluble starch as substrate. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) revealed that the end products of starch hydrolysis by AmyD-1 were glucose, maltose, maltotriose, maltotetraose, and malt oligosaccharides. Thus, AmyD-1 is one of the very few α-amylases that can tolerate low temperatures and high salt concentrations, which makes it to be a potential candidate for research in basic and applied microbiology.

     

Purification and Properties of Mycodextranase from Streptomyces sp. J-13-3

An enzyme hydrolyzing nigeran (alternating α-l,3-and α-l,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by precipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-I00. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS–PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50°C. The enzyme activity was inhibited significantly by Hg⁺, Hg²⁺, and p-chloromercuribenzoic acid. The K_m (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the α-l,4-glucosidic linkages in nigeran.     

Biochemical properties of a serine protease from Aspergillus flavus and application in dehairing

The proteases are enzymes produced by several filamentous fungi with important biotechnological applications. In this work, a protease from Aspergillus flavus was characterized. The culture filtrate of A. flavus was purified to homogeneity by Sephacryl S-200 column chromatography followed by CM–cellulose. The molecular weight of the purified enzyme was estimated to be approximately 32?kDa by SDS–PAGE. The enzyme hydrolysed BTpNA (N-α-benzoyl-dl-tyrosyl-p-nitroanilide), azo-casein and casein as substrates. Optimal temperature and pH were 55?°C and 6.5, respectively. The enzyme was stimulated by Mg²⁺, Ca²⁺, Zn²⁺ and inhibited by Hg²⁺ and Ag²⁺ and Cu²⁺. The protease showed increased activity with detergents, such as Tween 80 and Triton X, and was stable to the reducing agents, such as β-mercaptoethanol. The protease activity was strongly inhibited in the presence of phenylmethylsulfonyl fluoride, indicating it is a serine protease. The enzyme entrapped in calcium alginate beads retained its activity for longer time and could be reused up to 10 times. The thermostability was increased after the immobilization and the enzyme retained 100% of activity at 45?°C after 60?min of incubation, and 90% of residual activity at 50?°C after 30?min. In contrast, the free enzyme only retained 10% of its residual activity after 60?min at 50?°C. The enzymatic preparation was demonstrated to be efficient in the capability of dehairing without destruction of the hide. The remarkable properties such as temperature, pH and immobilization stability found with this enzyme assure that it could be a potential candidate for industrial applications.     

Production,purification, characterization and usage of a detergent additive of endoglucanase from isolated halotolerant Amycolatopsis cihanbeyliensis mutated strain Mut43

The Amycolatopsis cihanbeyliensis Mut43, which is obtained by UV radiation, exhibited endoglucanase activity of 5.21?U/mL, which was ～2.3-fold higher than that of the wild strain (2.04?U/mL). The highest enzyme activity was obtained after 3 days of incubation at 32?°C, pH 7.0, 150?rpm, and 6% NaCl in a liquid medium containing 1.5% (w/v) wheat straw (0.25?mm of particle size) and 0.6% (w/v) yeast extract. Enzyme activity was eluted as a single peak (gel filtration chromatography), and Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the corresponding peak revealed a molar mass of 30?kDa. Zymogram analysis confirmed the presence of a single active endoglucanase component. The enzyme was purified to ～21-fold, and the mean overall yield was ～6%. The purified endoglucanase was active up to 80?°C and showed a half-life of 214?min at 60?°C in the absence of substrate at pH 8.0. The apparent K_m value for the purified endoglucanase was 0.70?mg/mL, while the V_max value was 6.20 Units/μg. Endoglucanase activity was reduced (25%) by treatment with 30?U of proteinase K/mg. The addition of Mg⁺² and Ca⁺² (5?mM) enhanced endoglucanase activity. Additionally, endoglucanase activity in the presence of 5?mM SDS or organic solvents was 75 and 50% of maximum activity, respectively. The high levels of enzyme production from A. cihanbeyliensis Mut43 achieved under batch conditions, coupled with the temperature stability, activity over a broad pH range, relatively high stability (70–80%) in the presence of industrial laundry detergents and storage half-lives of 45 days at +4?°C and 75 days at ?20?°C signify the suitability of this enzyme for industrial applications as detergent additive.     

Purification and Some Properties of a New Levanase from Bacillus sp. No. 71

A levanase from Bacillus sp. was purified to a homogeneous state. The enzyme had a molecular weight of 135,000 and an isoelectric point of pH 4.7. The enzyme was most active at pH 6.0 and 40°C, stable from pH 6.0 to 10.0 for 20 hr of incubation at 4°C and up to 30°C for 30 min of incubation at pH 6.0. The enzyme activity was inhibited by Ag ⁺, Hg^{2 +}, Cu^{2 +}, Fe^{3 +}, Pb²⁺, and p-chloromercuribenzoic acid. The enzyme hydrolyzed levan and phlein endowise to produce levanheptaose as a main product. The limit of hydrolysis of levan and phlein were 71% and 96%, respectively.     

Calorimetric measurement of enthalpy change in the isothermal helix--coil transition of poly-L-lysine in aqueous solution

The heat ΔH° for converting an uncharged lysine residue from a coil to an α-helical state in poly-L -lysine in 0.1N KCl has been determined calorimetrically to be ?1200 cal/mole at both 15°C and 25°C. Essentially the same value has been obtained for the conversion of an uncharged residue from a coil to a β-pleated sheet state. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH°, the observed Calorimetric heat was corrected for the heat of breaking the sample cell, the heal of dilution of HCl, the heat of neutralization of OH^? ion, and the heat of ionization of the ε-amino group in the random coil. The latter was obtained from similar Calorimetric measurements on poly-D ,L -lysine, which was shown to be a good model for the random coil form of poly-L -lysine. The measured transition heat was ～0.7 cal., which is only 7% of the total heat liberated when a 40 ml solution of 0.25% w/v poly-L -lysine is brought, from pH 11 to pH 7; nevertheless it could be determined with a precision of ±8%. The conformation of poly-L -lysine at pH 11 appears to be completely helical at 15°C, but a mixture of 90% α-helix, 5% β form, and 5% coil at 25°C. Since ΔH° ～ 0 for the α ? β conversion, the polymer behaves like one of 95% α-helix and 5% coil in the calorimeter at 25°C. At neutral pH, poly-L -lysine is an extended coil, like poly-D ,L -lysine.     

Asymmetric Reduction of Iminium Salt with Chiral Dihydropyridines

A gram positive bacterium (strain No. 109) isolated from soil as a producer of cyclodextrinase was identified as Bacillus coagulans. The cyclodextrinase from B. coagulans was purified to a homogeneous state by disc-electrophoresis after Streptomycin treatment, DEAE-Sephadex column chromatography, Ultrogel AcA44 gel filtration and hydroxyapatite column chromatography. The molecular weight of the enzyme was determined to be 6.2}10⁴ by sodium dodecyl-sulfate gel electrophoresis. The isoelectric point of the enzyme was pH 5.0. The enzyme was most active at pH 6.2 and 50°C, and stable up to 45°C at pH 7.0 and in the range of pH 6.0 ~ 7.3 at 40°C on 2 hr incubation. This enzyme hydrolyzed linear maltooligosaccharides (such as maltotetraose (G4), maltopentaose (G5) and maltohexaose (G6)) and α-, β- and α-cyclodextrins (CDs) faster than maltotriose (G3) and short chain amylose ( 18), but did not hydrolyze maltose. The rates of hydrolysis for polysaccharides (such as starch, amylose and amylopectin) were below 1 % as compared to that for β-CD. The Km values for G3, G4, G5, G6, short chain amylose ( 18) and α, β- and γ-CD were 4.5, 4.0,2.3,1.5,1.5,10,2.8 and 0.47 mM, respectively. The products with this enzyme had the α-configulation.     

Digestive amylase and pectinase activity in the larvae of alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil Hypera postica is a serious economic pest in most alfalfa grown in many countries worldwide. Digestive α-amylase and pectinase activities of larvae were investigated using general substrates. Midgut extracts from larvae showed an optimum activity for α-amylase against starch at acidic pH (pH 5.0). α-Amylase from larval midgut was more stable at mildly acidic pH (pH 5–6) than highly acidic and alkaline pH. The enzyme showed its maximum activity at 35°C. α-Amylase activity was significantly decreased in the presence of Ca²⁺, Mg²⁺ and sodium dodecylsulfate. On the contrary, K⁺ and Na⁺ did not significantly affect the enzyme activity. Zymogram analysis revealed the presence of one band of α-amylase activity in in-gel assays. Pectinase activity was assayed using agarose plate and colorimetric assays. Optimal pH for pectinase activity in the larval midgut was determined to be pH 5.0. Pectinase enzyme is more stable at pH 4.0–7.0 than highly acidic and alkaline pH. However, the enzyme was more stable at slightly acidic pH (pH 6.0) when incubation time increased. Maximum activity for the enzyme incubated at different temperatures was observed to be 40°C. Optimum pH activity for α-amylase and pectinase is not completely consistent with the pH prevailing in the larval midgut. This is the first report of the presence of pectinase activity in H. postica.     

A COMPARISON OF ALPHA-AMYLASES FROM THE LATEX OF THREE SELECTED SPECIES OF EUPHORBIA (EUPHORBIACEAE)

A technique for the partial purification of α-amylases from latex of Euphorbia heterophylla, E. marginata, and E. tirucalli is described. The enzymes were found to be similar to other higher plant amylases using the criteria of molecular weight, pH characteristics, kinetics, number of isozymes, and blue value-reducing value patterns. Carbohydrases other than α-amylases were not detectable in latex. The amylases were employed to examine their capacity to digest latex starch grains which are common components of the laticiferous cell in this genus. Laticifer starch grains are not susceptible to in vitro amylolysis. Removal of the starch grain membrane with Triton X-100, damaging the grain, or treating the grains with α-amylases from diverse biological sources had little effect upon hydrolysis. Grains incubated with pullulanase followed by α-amylase caused a slight but significant increase in hydrolysis of raw laticifer starch grains. These studies indicate that the nonarticulated laticifer in Euphorbia is a cul de sac for certain primary and secondary metabolic products and that the indigestible and morphologically complex starch grains in the latex have evolved to function in a secondary role within the laticifer.     

Enzymatic characterization of an extracellular glucoamylase from Tricholoma matsutake and its cloning and secretory expression in Pichia pastoris

ABSTRACT

A glucoamylase from the ectomycorrhizal fungus Tricholoma matsutake (TmGLA) was purified 33.2-fold to homogeneity as a single monomeric glycoprotein with a molecular mass of 63.9 kDa. Maximum activity was observed at 60°C and pH 5.0. The enzyme is active down to 50°C and in the pH range of 4.0–6.0, and its activity is strongly inhibited by Ag⁺. It degrades α-1,4- and α-1,6-glycosidic linkages in various polysaccharides. Its gene (TmGlu1) was cloned using information from the enzyme’s internal amino acid sequences and the whole genome sequence of T. matsutake NBRC 30605. The deduced amino acid sequence showed clear homology with those of GH family 15 proteins. Pichia pastoris transformed with TmGlu1 secreted the active enzyme in a glycosylated form, and its characteristics were the same as the native enzyme.     

Purification and characterization of a propanol-tolerant neutral protease from Bacillus sp. ZG20

Abstract

A propanol-tolerant neutral protease was purified and characterized from Bacillus sp. ZG20 in this study. This protease was purified to homogeneity with a specific activity of 26,655?U/mg. The recovery rate and purification fold of the protease were 13.7% and 31.5, respectively. The SDS-PAGE results showed that the molecular weight of the protease was about 29?kDa. The optimal temperature and pH of the protease were 45?°C and 7.0, respectively. The protease exhibited a good thermal- and pH stability, and was tolerant to 50% propanol. Mg²⁺, Zn²⁺, K⁺, Na⁺ and Tween-80 could improve its activity. The calculated K_m and V_max values of the protease towards α-casein were 12.74?mg/mL and 28.57?µg/(min mL), respectively. This study lays a good foundation for the future use of the neutral protease from Bacillus sp. ZG20.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.