Khawar Sohail Siddiqui

Partial and complete alteration of surface charges of carboxymethylcellulase by chemical modification: thermostabilization in water-miscible organic solvent

Abstract Carboxymethylcellulase was purified from Aspergillus niger to homogeneity level. The native and subunit molecular weights were found to be 42 and 45 kDa, respectively. The purified CMCase was modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of dimethylamine hydrochloride (DIMAM) and ethylenediamine dihydrochloride (EDAM) as nucleophiles. The amino groups of DIMAM were further modified by acetic anhydride for the complete elimination of surface charges (double modification, DM). The specificity constants (V max /K m ) of EDAM (1.1), DIMAM (1.2) and DM-A (1.0) were increased as compared with native enzyme (0.16). Partial neutralization of surface negative charges (DIMAM), reversal of surface negative charges (EDAM) and complete neutralization of negative plus positive charges (DM-A), of CMCase had a drastic effect on the thermostability determined in the aqueous buffer, pH 5.2. Gibbs activation free energies of denaturation ( ΔG* ) of native, EDAM, DIMAM and DM-A at 80°C were 110, 107, 102 and 103 kJ mol −1 , respectively, whereas enthalpy of denaturation ( ΔH* ) of native, EDAM, DIMAM and DM-A at 80°C were 143, 144, 213 and 197 kJ mol −1 , respectively. The entropies of denaturation (ΔS*) of native, EDAM, DIMAM and DM-A at 80°C were 91, 105, 316 and 265 Jmol −1 K -1 , respectively, indicating highly disordered conformations of all the transition states of modified CMCases. On the other hand, the thermostability of doubly modified CMCase in 50% (v/v) aqueous dioxan (DM-D) was dramatically increased with concomitant activation with increasing temperatures up to 95°C as compared with the native CMCase under similar conditions. The analysis of thermodynamic parameters revealed that ΔS* of denaturation became negative implying a highly ordered transition state of DM-D in the presence of a solvent of higher hydrophobicity. We wish to propose that at least monomeric enzymes could be made significantly more thermostable in water-miscible organic solvents by totally converting the charged surface groups to non-polar ones by double modification .

Carboxyl group modification significantly altered the kinetic properties of purified carboxymethylcellulase from Aspergillus niger

Carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 was purified by a combination of ammonium sulphate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography on FPLC with 9-folds increase in specific activity. Native and subunit molecular weights were found to be 36 kDa each. The purified CMCase was modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of glycinamide for 15 min (GAM15) and glycinamide plus cellobiose for 75 min (GAM75). Similarly, the enzyme was modified by EDC in the presence of ethylenediamine dihydrochloride plus cellobiose for 75 min (EDAM75). The neutralization (GAM15 and GAM75) and reversal (EDAM75) of negative charges of carboxyl groups of CMCase had profound effect on the specificity constant (k(cat)/K(m)), pH optima, pK(a)s of the active-site residues and thermodynamic parameters of activation. The specificity constants of native, GAM15, GAM75, and EDAM75 were 143, 340, 804, and 48, respectively. The enthalpy of activation (DeltaH(#)) of Carboxymethylcellulose (CMC) hydrolysis of native (50 and 15 kJ mol(-1)) and GAM15 (41 and 16 kJ mol(-1)) were biphasic whereas those of GAM75 (43 kJ mol(-1)) and EDAM75 (41 k J mol(-1)) were monophasic. Similarly, the entropy of activation (DeltaS(#)) of CMC hydrolysis of native (-61 and -173 J mol(-1) K(-1)) and GAM15 (-91 and -171 J mol(-1) K(-1)) were biphasic whereas those of GAM75 (-82 J mol(-1) K(-1)) and EDAM75 (-106 J mol(-1) K(-1)) were monophasic. The pH optima/pK(a)s of both acidic and basic limbs of charge neutralized CMCases increased compared with those of native enzyme. The CMCase modification in the presence of glycinamide and absence of cellobiose at different pHs periodically activated and inhibited the enzyme activity indicating conformational changes. We believe that the alteration of the surface charges resulted in gross movement of loops that surround the catalytic pocket, thereby inducing changes in the vicinity of active site residues with concomitant alteration in kinetic and thermodynamic properties of the modified CMCases.

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheKm andkcat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.

Activity and thermostability of carboxymethylcellulase from Aspergillus niger is strongly influenced by noncovalently attached polysaccharides

Removal of non-covalently attached polysaccharides from carboxymethylcellulase (CMCase) of Aspergillus niger improved its activity but decreased its thermostability and protease resistance. The activation energy profile of the hydrolysis of carboxymethylcellulose (CMC) was triphasic with increasing values of 17,-55 and-562 kJ/mol for polysaccharide-free and 19, -21 and -207 kJ/mol for polysaccharide-complexed CMCase. The specificity constant (Vmax/Km) of polysaccharide-free CMCase was 1.41 compared to polysaccharide-complexed CMCase which was only 0.68. The polysaccharide free CMCase had lower thermostability (‘melting point’ = 82°C) and higher protease susceptibility compared to polysaccharide-complexed CMCase (‘melting point’>100°C).

Kinetic analysis of the active site of an intracellular β-glucosidase fromCellulomonas biazotea

Purified β-glucosidase fromCellulomonas biazotea had an apparentKm andV for 2-nitrophenyl β-d-glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of β-glucosidase was 65 kJ/mol. The inhibition by Mn2+vs. oNPG of parental β-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 µmol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pKa’s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of β-glucosidase decreased in the presence of 10 % ethanol. The half-life of β-glucosidase in 1.75 mol/L urea at 35 °C was 145 min, as determined by 0–9 mol/L transverse urea gradient-PAGE.

Carboxy‐group modification: high‐temperature activation of charge‐neutralized and charge‐reversed β‐glucosidases from Aspergillus niger

Purified β‐glucosidase from Aspergillus niger NIAB280 was chemically modified by 1‐ethyl‐3‐(3‐dimethyl‐aminopropyl)‐carbodi‐imide (EDC) in the presence of glycinamide (GAM) as nucleophile under various conditions to study the role of carboxy groups in the catalytic mechanism of this enzyme. β‐Glucosidase was inactivated by the binding of one mol of EDC per mol of the enzyme with a second‐order rate constant of 4.77 × 10−2 mM min−1. Glucose, as competitive inhibitor, partly protected the active‐site carboxy group against chemical modification, with a Kd of 3.64 mM. The pH dependence of chemical modification by EDC showed that first‐order rate constants decreased with increasing pH, indicating that the proton donating group is a carboxy group. The pKa values of the acidic and basic limbs of the native enzyme were 2.9 and 6.5 respectively. β‐Glucosidase was modified by EDC in the presence of GAM and ethylenediamine dihydrochloride (EDAM) as nucleophiles for 60 min. The effects of neutralization (GAM) and reversal (EDAM) of the negative charges of surface carboxy groups on the kinetic properties of the enzyme were also studied. Native β‐glucosidase, GAM and EDAM had Vmax/Km values of 0.73, 1.22 and 0.60 respectively at 40°C. Interestingly, the activation energy profiles of native β‐glucosidase (103 and 79 kJ/mol) were biphasic, whereas those of GAM (137, 101 and 30 kJ/mol) and EDAM (285, 100 and 29 kJ/mol) were triphasic, indicating significant activation of modified β‐glucosidases at temperatures higher than 50°C. The pKa values of both the active‐site carboxy groups as well as the pH optima of GAM and EDAM were also significantly decreased compared with those of the native β‐glucosidase.

Purification and the effect of manganese ions on the activity of carboxymethylcellulases from Aspergillus niger and Cellulomonas biazotea.

Carboxymethylcellulases (CMCases) fromAspergillus niger andCellulomonas biazotea were purified by a combination of ammonium sulfate precipitation, anion-exchange and gel-filtration chromatography with a 12- and 9-fold increase in the purification factor. The native and subunit molar mass of CMCase fromA. niger were 40 and 25–57 kDa, respectively, while those fromC. biazotea were 23 and 20–30 kDa, respectively. Low concentrations of Mn2+ activated the enzymes from both organisms (mixed activation) with apparent activation constants of 0.80 and 0.45 mmol/L of CMCases fromA. niger andC. biazotea, respectively, while at higher CMC concentrations Mn2+ inhibited the enzymes (mixed and partial uncompetitive inhibition). The reason for this complex behavior is that more than one Mn2+ bind to the same enzyme form with the apparent average inhibition constants of 2.7 and 1.3 mmol/L for CMCases fromA. niger andC. biazotea, respectively.

The stability of extracellular β-glucosidase fromAspergillus niger is significantly enhanced by non-covalently attached polysaccharides

The removal of noncovalently bound polysaccharide coating from the extracellular enzymes ofAspergillus niger, by the technique of compartmental electrophoresis, had a very dramatic effect on the stability of β-glucosidase. The polysaccharide-β-glucosidase complex was extremely resistant to proteinases and far more stable against urea and temperature as compared with polysaccharide-free β-glucosidase. The β-glucosidase-polysaccharide complex was 18-, 36-, 40-, and 82-fold more stable against chymotrypsin, 3 mol/L urea, total thermal denaturation and irreversible thermal denaturation, respectively, as compared with polysaccharide-free β-glucosidase. The activation energy of polysaccharide-complexed β-glucosidase (55 kJ/mol) was lower than polysaccharide-free enzyme (61 kJ/mol), indicating a slight activation of the enzyme by the polysaccharide. No significant difference could be detected in the specificity constant (V/Km) for 4-nitrophenyl β-d-glucopyranoside between polysaccharide-free and polysaccharide-complexed β-glucosidase. We suggest that the function of these polysaccharides secreted by fungi includingA. niger might be to protect the extracellular enzymes from proteolytic degradation, hence increasing their life span.

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.

Short Communication: Purification and characterization of an intracellular β-glucosidase from Cellulomonas biazotea

β-Glucosidase (EC 3.2.1.21) from Cellulomonas biazotea NIAB442 was purified by a combination of ammonium sulphate precipitation and ion-exchange chromatography with a 17-fold increase in specific activity. The native and subunit molecular weights of β-glucosidase were 355 kDa and 92 kDa respectively. The apparent K m and V max values for p-nitrophenyl-β-D-glucopyranoside (pNPG) were 4.25 mM and 1.526 U/mg protein respectively. The optimum temperature of β-glucosidase activity was 38 °C and the pH optimum was 6.6. The thermostability of β-glucosidase decreased with an increase in pH from 5 to 7. MnCl 2 and NaCl inhibited β-glucosidase activity.