Muhammad Hamid Rashid

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 .

Purification, kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.).

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k(m)=55 mg ml(-1), k(cat)=21s(-1), k(cat)/k(m)=0.38, while the thermodynamic parameters were: DeltaH*=52.6 kJ mol(-1), DeltaG*=71.2 kJ mol(-1), DeltaS*=-57 J mol(-1) K(-1), DeltaG*(E-S)=10.8 kJ mol(-1) and DeltaG*(E-T)=2.6 kJ mol(-1). The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53-63 degrees C were also determined. The half -life of SAI at 53 and 63 degrees C was 112 and 10 min, respectively. At 55 degrees C, surprisingly the half -life increased to twice that at 53 degrees C. DeltaG*, DeltaH* and DeltaS* of irreversible thermal stability of SAI at 55 degrees C were 107.7 kJ mol(-1), 276.04 kJ mol(-1) and 513 J mol(-1) K(-1), respectively.

Cost-efficient entrapment of β-glucosidase in nanoscale latex and silicone polymeric thin films for use as stable biocatalysts

β-Glucosidase is an ubiquitous enzyme which has enormous biotechnological applications. Its deficiency in natural enzyme preparations is often overcome by exogenous supplementation, which further increases the enzyme utilization cost. Enzyme immobilization offers a potential solution through enzyme recycling and easy recovery. In the present work Aspergillus niger β-glucosidase is immobilized within nanoscale polymeric materials (polyurethane, latex and silicone), through entrapment, and subsequently coated onto a fibrous support. Highest apparent activity (90 U g(-1) polymer) was observed with latex, while highest entrapment efficiency (93%) was observed for the silicone matrix. Immobilization resulted in the thermo-stabilization of the β-glucosidase with an increase in optimum temperature and activation energy for cellobiose hydrolysis. Supplementation to cellulases also resulted in an increased cellulose hydrolysis, while retaining more than 70% functional stability. Hence, the current study describes novel preparations of immobilized β-glucosidase as highly stable and active catalysts for industrial food- and bio-processing applications.

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.

Invertase from Hyper Producer Strain of Aspergillus niger: Physiochemical Properties, Thermodynamics and Active Site Residues Heat of Ionization

Here we report for the first time heat of ionization of invertase (E.C.3.2.1.26) active site residues from hyper-producer strain of Aspergillus niger (34.1 U ml(-1)), along with its physiochemical properties, kinetics and thermodynamics of stability-function. The Invertase showed great potential for industry as being highly efficient (k(cat) = 24167 s(-1) at 65 degrees C, pH 5.0) and stable (half life= 12 h at 56 degrees C).

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.

Improvement of Aspergillus oryzae for hyperproduction of endoglucanase: expression cloning of cmc-1 gene of Aspergillus aculeatus

FI-Carboxymethylcellulase (cmc1; family 12) is one of the endoglucanases of Aspergillus aculeatus and consists of single polypeptide chain of 221 amino acids. The cmc1 gene was expressed in Aspergillus oryzae niaD300 (niaD−) under promoter 8142. The plasmid pCMG14 carrying the cmc1 gene at PstI site was used as a source of the gene (920 bp) and Aspergillus oryzae was successfully transformed by the plasmid pNAN-cmc1 (harboring cmc1 gene). The plasmid was integrated in Aspergillus oryzae niaD300 genome at niaD locus and the transformed fungus constitutively produced very high amounts of endoglucanases when grown on glucose, maltose, soluble starch and wheat bran.

A simple and nondestructive method for the separation of polysaccharides from β-glucosidase produced extracellularly by Aspergillus niger

An apparatus based on electrophoresis has been devised that removes noncovalently bound polysaccharides from extracellular proteins of Aspergillus niger with concomitant partial beta-glucosidase purification and concentration. The apparatus consists of a series of three chambers separated by polyacrylamide gels. Dialyzed and concentrated crude extract of Aspergillus niger containing beta-glucosidase was poured into the middle chamber, while smaller anodic and cathodic chambers contained buffer. When electric current was applied, negatively charged protein-polysaccharide complexes moved toward the anode. Most of the negatively charged proteins, including beta-glucosidase, crossed the gel barrier into the anodic compartment, while neutral polysaccharides were either trapped in the gels or remained in the middle chamber. In this way, 125 ml of dialyzed and concentrated crude extract of Aspergillus niger was processed. Therefore, after 24 h of electrophoresis, 68% of the proteins and 90% of the beta-glucosidase activity, but only negligible amounts of polysaccharide, were transferred to the anodic chamber. The removal of high-molecular-weight polysaccharide from beta-glucosidase had a detrimental effect on the stability of the enzyme.

Kinetic and thermodynamic properties of an immobilized glucoamylase from a mesophilic fungus, Arachniotus citrinus.

Purified glucoamylase from Arachniotus citrinus was immobilized on polyacrylamide gel with 70% yield of immobilization. The immobilization improved the pH optima, temperature optima, values of K(m), V(max), and activation energy. Irreversible thermal denaturation studies of soluble and immobilized glucoamylase indicated that immobilization decreased the entropy and enthalpy of deactivation by magnitudes and made the immobilized glucoamylase thermodynamically more stable.

Shake Table Tests on Single-Story Dhajji Dewari Traditional Buildings

ABSTRACT This article presents shake table tests carried out on two reduced scaled models, including 1/3rd scaled single room model and 1/4th scaled two rooms house model Dhajji Dewari (timber-braced frame rubble stone masonry) traditional structures for seismic performance evaluation. Multiple excitations tests were performed on the model structures using natural accelerograms with increasing intensity to deform the structure from elastic to inelastic and extreme damage state. The structure observed damage states, acceleration, and displacement response and lateral force-deformation behavior are retrieved. The tested structure models showed an excellent performance under the earthquake ground motions; deforming to large lateral displacement without jeopardizing the global stability despite extreme level shaking and observed localized damages. The model under bi-directional loading, reproduced through diagonal excitation, was observed with relatively reduced lateral strength and lateral deformation, due to walls subjected to both in-plane and out-of-plane loading. The research highlights the importance of dynamic excitation on the seismic performance evaluation of Dhajji structures. Analytical models are used for the capacity evaluation of considered Dhajji buildings, which shows promising performance in calculating the building yield stiffness and strength. Displacement-based method is used for the intensity-based seismic performance assessment of individual building in the highest seismic zone (PGA = 0.40 g, Soil Type D), which shows that Dhajji building under orthogonal loading will not exceed even the Immediate Occupancy level under design level earthquake ground motions. However, the building can just exceed the Life Safety level in design level ground motions, under diagonal excitation.