Stability and identification of active-site residues of carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea

Abstract

Determination of the apparent pKa's of purified carboxymethylcellulases from Aspergillus niger and Cellulomonas biazotea at different temperatures and in the presence of dioxane indicated two side chain carboxyl groups which controlled the limiting rate in both organisms. The thermostability of both enzymes slightly decreased with increasing pH from 5 to 75 but was unaffected in the presence of 0.5 mmol/L Mn2+. The CMCase from C. biazotea had an activation energy of 35 kJ/mol and a half-life of 89 min in the presence of 8 mol/L urea at 40 degrees C. The half-life of CMCase from A. niger in 8 mol/L urea and at 37 degrees C was 125 min as determined by a 0-9 mol/L transverse urea gradient PAGE. The CMCases from A. niger and C. biazotea had the same thermostabilities in the absence of CMC although the enzyme from the former was more thermostable in the presence of the substrate. The CMCase from A. niger was also more efficient in hydrolyzing CMC than the enzyme from C. biazotea.

Thermostability and thermodynamic characterization of sprouted pearl millet ?-amylases for its biotechnological applications

“…The enthalpy of activation of thermal denaturation ΔH (inact) , which is the amount of energy required to bring the enzyme to the activated state for the subsequent denaturation at a given temperature, Gibbs free energy of activation of the thermal denaturation (ΔG (inact) ) and the entropy of activation of the denaturation (ΔS (inact) ) were calculated through the following equations (Siddiqui et al, 1997;Riaz et al, 2007).…”

“…Thermal denaturation of enzymes requires an input of a minimum amount of energy known as activation energy of denaturation (Ea (D) ) which must be supplied to the native enzyme in order to initiate the denaturation process. The denaturation process proceeds through an unstable intermediate transition (or activated) state, U (Siddiqui et al, 1997). As long as the initial input of energy is less than the Ea (D) the unstable intermediate (U) can fold back into the native state (N) upon cooling (Saqib et al, 2010).…”

Kinetics of enhanced ethanol productivity using raw starch hydrolyzing glucoamylase from Aspergillus niger mutant produced in solid state fermentation

“…The enzymes from both cultures were purified by a combination of ammonium sulphate precipitation and gel-filtration chromatography with a 25-and 35-fold increase in the specific activity, respectively, as described earlier (Siddiqui et al 1997).…”

“…*These parameters were determined after Siddiqui et al (1997). Free energy of activation for irreversible inactivation of glucoamylase (DG*) ¼ )RT ln(k d h/K B T); enthalpy of irreversible inactivation of glucoamylase (DH*) ¼ E a ) RT; and entropy of irreversible inactivation of glucoamylase (DS*) ¼ DH* ) DG*/T were determined after Rashid and Siddiqui (1998).…”

Cobalt has Enhancing Effect on Extracellular Lipases Isolated from Pseudomonas aeroginosa JCM5962(T)

“…The metal salts used in the study wereFeSO 4, CuSO 4, ZnSO 4, CaCl 2, CoCl 2, HgCl 2, BaCl 2, NaCl, Nh 4 Cl, MgCl 2, KCL,LiCl, whereas the inhibitors were EDTA (Ethylenediaminetetracetic acid) and SDS (sodium dodecyl sulphate). The effect of metals and inhibitors was studied by incubation ofenzyme in the presence of different concentrations of metals or the inhibitor according tomodified method of Siddiquiet al 10 . Seven different concentrations (0.1 mM, 0.2mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 20mM) were prepared by dissolving appropriate amount in double distilled water.…”