The catalytic and kinetic characterization of Bacillus subtilis MK775302 milk clotting enzyme: comparison with calf rennet as a coagulant in white soft cheese manufacture.

BACKGROUND: Calf rennet is considered the traditional source of milk clotting enzyme (MCE). However, increasing cheese consumption with decreasing the calf rennet supply had encouraged the quest for new rennet alternatives. The purpose of this study is to acquire more information about the catalytic and kinetic properties of partially purified Bacillus subtilis MK775302 MCE and to assess the role of enzyme in cheese manufacture. RESULTS: B. subtilis MK775302 MCE was partially purified by 50% acetone precipitation with 5.6-fold purification. The optimum temperature and pH of the partially purified MCE were 70 °C and 5.0, respectively. The activation energy was calculated as 47.7 kJ/mol. The calculated Km and Vmax values were 36 mg/ml and 833 U/ml, respectively. The enzyme retained full activity at NaCl concentration of 2%. Compared to the commercial calf rennet, the ultra-filtrated white soft cheese produced from the partially purified B. subtilis MK775302 MCE exhibited higher total acidity, higher volatile fatty acids, and improved sensorial properties. CONCLUSIONS: The partially purified MCE obtained in this study is a promising milk coagulant that can replace calf rennet at a commercial scale to produce better-quality cheese with improved texture and flavor.

Molecular characterization, catalytic, kinetic and thermodynamic properties of protease produced by a mutant of Bacillus cereus-S6-3.

The proteolytic strain Bacillus cereus-S6-3 was subjected to mutagenic treatments viz. UV irradiations and methyl methane sulfonate (MMS). The obtained mutant strain, B. cereus-S6-3/UM90 showed 1.34 fold over the parent strain. Molecular characterization of proteases from the parent (PP/S6-3) and mutant (PM/UM90) strains indicated that they were consisted of two domains and binds a zinc ion and 4 calcium ions in the active site. Amino acid sequence alignment of PM/UM90 protease showed 19 amino acid residues were substituted compared to that of the wild-type enzyme. However, both proteases contained equal number of aromatic and hydrophobic amino acids. Protease from PM/UM90 showed an effective improvement in thermal properties in terms of reaction temperature, t1/2, the values of kd, activation energy (Ea), and decimal reduction time (D) within the temperature range from 60 to 80 °C. In addition, the kinetic and thermodynamic parameters for substrate hydrolysis (i.e., Km, Vmax, ΔH*, ΔG*, ΔS*, kcat, Vmax/Km, kcat/Km, ΔG*E-T and ΔG*E-S) showed a significant improvement of the catalytic efficiency for PM/UM90 protease. Furthermore, the correlation between thermodynamic properties and the patterns of amino acid substitution of wild-type enzyme to has been discussed.

Structural characterization, catalytic, kinetic and thermodynamic properties of Keratinase from Bacillus pumilus FH9.

Bacillus pumilus FH9 keratinase was purified to homogeneity with a 59.9% yield through a series of three steps. The purified enzyme was a monomeric protein with a molecular mass around 50kDa and containing 7.3% carbohydrates. The pure B. pumilus FH9 keratinase was optimally active at pH 9.0 and 60°C. The calculated activation energy for keratin hydrolysis was 24.52kJmol-1 and its temperature quotient (Q10) was 1.19. The calculated values of thermodynamic parameters for keratin hydrolysis were as follows: ΔH*=21.75kJmol-1, ΔG*=65.86kJmol-1 ΔS*=-132.46Jmol-1K-1, (ΔG*E-S)=4.74kJmol-1 and ΔG*E-T=-11.254kJmol-1. The pure keratinase exhibited Km, Vmax, kcat and kcat/Km of 5.55mg/ml keratin, 5882Umgprotein-1 323.54s-1 and 58.28 (s-1/mgml-1). The calculated half-life time at 50, 60, 70 and 80°C was 90.69, 59.1, 16.62 and 9.48min, respectively. Similarly, the thermodynamic parameters for irreversible thermal inactivation at temperature ranging from 50 to 80°C were determined. The pure enzyme was stimulated by Ca2+ and Mg2+. However, Zn2+, EDTA, Co2+ and Hg2+ significantly inhibited the enzyme activity. The purified enzyme was able to hydrolyze different substrates showing its higher proteolytic activity on casein, bovine serum albumin, and collagen, followed by feather, horn and wool.

Catalytic, kinetic and thermodynamic properties of stabilized Bacillus stearothermophilus alkaline protease.

Bacillus stearothermophilus alkaline protease was conjugated to several oxidized polysaccharides of different chemical structure. The conjugates were evaluated for the kinetic and thermodynamic stability. The conjugated enzyme with oxidized pectin had the highest retained activity (79.5%) and the highest half-life (T1/2) at 50°C and pH 9.0. Compared to the native protease, the conjugated preparation exhibited lower activation energy (Ea), lower deactivation constant rate (kd), higher T1/2, and higher D values (decimal reduction time) within the temperature range of 50-60°C. The thermodynamic parameters for irreversible inactivation of native and conjugated protease indicated that conjugation significantly decreased entropy (ΔS*) and enthalpy (ΔH*) of deactivation. The calculated value of activation energy for thermal denaturation (Ead) for the conjugated enzyme was 20.4KJmole-1 higher over the native one. The results of thermodynamic analysis for substrate hydrolysis indicated that the enthalpy of activation (ΔH*) and free energy of activation (free energy of substrate binding) ΔG*E-S and (ΔG*), (free energy of transition state) ΔG*E-T values were lower for the modified protease. Similarly, there was significant improvement of kcat, kcat/Km values. The enzyme proved to be metalloprotease and significantly stimulated by Ca2+ and Mg2+ whereas Hg2+, Fe3+ Cu2+ and Zn2+ inhibited the enzyme activity. There was no pronounced effect on substrate specificity after conjugation.

Structural characterization, catalytic, kinetic and thermodynamic properties of Aspergillus oryzae tannase.

Tannase (EC.3.1.1.20) from Aspergillus oryzae was purified using ammonium sulphate precipitation (75%), gel filtration chromatography through Sephadex G-100, and G-200. The purified enzyme was monomeric protein with a molecular mass of 106kDa. The activation energy for tannic acid hydrolysis was 32.6kJmol-1 and its temperature quotient (Q10) was 1.0. The pKa1 and pKa2 values of acidic and basic limbs of the active site residues were 4.6 and 6.4. The calculated values of thermodynamic parameters for tannic acid hydrolysis, were as follows: ΔH*=30.02kJmol-1, ΔG*=59.75kJmol-1 ΔS*=-95.90Jmol-1K-1, (ΔG*E-S)=3.66kJmol-1 and ΔG*E-T -12.61kJmol-1. The pure enzyme exhibited Km, Vmax and kcat of 4.13mM, 3507Umgprotein-1 and 551.4s-1. The calculated half-life time at 40, 45, 50, 55, 60, and 70°C was 955.15, 142.0, 30.28, 17.88, 8.23 and 2.95min, respectively. The thermodynamic parameters for irreversible thermal inactivation at different temperatures (40-70°C) were determined. The enzyme was activated by Ca2+, and Mg2+ while Hg2+, Fe2+, and Cu2+ strongly inhibited it. Hydrolysis of tannic acid by the pure enzyme indicated that gallic acid was the end-product.

Catalytic, kinetic and thermodynamic properties of Bacillus pumilus FH9 keratinase conjugated with activated pectin.

Bacillus pumilus FH9 keratinase was covalently coupled to several oxidized polysaccharides. The conjugates were evaluated for the retained activity, kinetic and thermodynamic stability. Among all preparations, the conjugated enzyme with oxidized pectin had the highest recovered activity (71.75%) and the highest thermal stability at 60°C (t1/2=333 min). Compared to the native enzyme, the conjugated preparation exhibited higher optimum temperature, lower activation energy (Ea), lower deactivation constant rate (kd), higher t1/2, and higher decimal reduction time values (D) within the temperature range of 50-80°C. The thermodynamic parameters (ΔH*, ΔG*, ΔS*) of irreversible thermal denaturation for the native and conjugated keratinase were also evaluated. The values of enthalpy of activation (ΔH*), free energy of activation (ΔG*), and free energy of transition state binding (ΔG*E-T) for keratin hydrolysis were lower for the conjugated enzyme. Moreover, there was highly significant impact on improving the values of Vmax/Km, kcat, kcat/Km, and ΔG*E-S for the modified enzyme. Both native and conjugated enzymes were slightly activated by CaCl2 and MgCl2. However, the inhibitory effects of EDTA, HgCl2 and ZnSO4 were more pronounced with the native enzyme.

Catalytic and thermodynamic properties of glycosylated Bacillus cereus cyclodextrin glycosyltransferase.

Cyclodextrin glycosyltransferase (CGTase) was covalently coupled to five oxidized polysaccharides differing in structure and chemical nature. The conjugates were evaluated for the retained activity, kinetic and thermodynamic stability. The conjugated CGTase with oxidized dextran (MW 47000) had the highest retained specific activity (70.05%) and the highest half-life (T1/2) at 80°C. Compared to the native enzyme, the conjugated preparation exhibited higher optimum temperature, lower activation energy (Ea), lower deactivation constant rate (kd), higher T1/2, and higher D values (decimal reduction time) within the temperature range of 60-80°C. The values of thermodynamic parameters for irreversible inactivation of native and conjugated CGTase indicated that conjugation significantly decreased entropy (ΔS*) and enthalpy of deactivation (ΔH*). The results of thermodynamic analysis for cyclodextrin production from starch indicated that The enthalpy of activation (ΔH*) and free energy of activation (ΔG*), (free energy of transition state) ΔG*E-T and (free energy of substrate binding) ΔG*E-S values were lower for the conjugated CGTase. Similarly, there was significant impact on improvement of kcat, kcat/Km values. Both native and conjugated enzyme produce α-cyclodextrin from starch.

Catalytic properties of the immobilized Aspergillus tamarii xylanase.

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher Km (Michaelis constant), lower Vmax (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60 degrees C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60 degrees C for the immobilized enzyme is about 6.0 x 10(-3), which is lower than that of the free enzyme (7.77 x 10(-3) min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.