Shashi Kant

Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323.

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS-PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0-5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3h. The addition of Mn(2+), K(+), Zn(2+), Ca(2+) and Al(3+) inhibited the enzyme activity; it increased in the presence of Mg(2+) and Cu(2+) ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K(m) and V(max) values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T(650) from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.

Purification of pectin methylesterase from Lycopersicon esculentum and its application.

Pectin methylesterase (PME) (3.1.1.11) is the pectin degrading enzyme which catalyses the hydrolysis of pectin methylester group, resulting in de-esterification. PME is widely distributed in plants, fungi, yeast and bacteria. In the present study, PME was extracted from tomato by using 8.8% NaCl (4°C). The crude enzyme precipitated with 60% ammonium sulphate resulted in 1.02 fold purification of the enzyme. The purification was done by ion exchange chromatography using DEAE-Cellulose column. This resulted in 1.82 fold purification of the enzyme. The molecular weight of purified enzyme was determined by SDS-PAGE which was found to be 34.0 kDa. During characterization of the purified enzyme, the maximum activity was found at temperature 50°C, pH 6.5, reaction time 45 min. Citrus pectin was the best substrate for maximum enzyme activity. The enzyme did not require any metal ion to express its activity, enzyme was found to be very stable at 4°C and at 50°C the enzyme was stable upto 2 h as it retained 70% of its activity. The K(m) and V(max) values of the enzyme were found to be 0.115 mg/ml and 1.03 μmol/ml/min. PME enhanced the pectin degradation process in apple juice clarification in combination with polygalacturonase and increased %T(650) from 1.7% to 5.6%.

Designing a cost-effective and dual-functional muslin-based anion exchanger for defluoridation

AbstractIn the present study, muslin was modified by graft copolymerization with poly(4-vinyl pyridine) using γ-ray initiation method. The graft copolymers, thus synthesized, were further functionalized by reaction with 2-chloroethanol. The resultant materials, having pyridinium ring and exchangeable Cl−, were evaluated for the removal of fluoride ions from the simulated water samples. The materials exhibited high fluoride uptake and the maximum uptake was observed at pH 4.0, 20°C and 10 ppm of the fluoride ions. The maximum retention capacity of 7.7 mg/g was observed when fluoride uptake was studied up to 10 cycles. The data generated fit the pseudo-second-order kinetics and Langmuir isotherm. The efficacy of the functionalized muslin was evaluated as an antimicrobial agent against a bacterium (Bacillus aureus) and a fungus (Aspergillus niger). It was observed to be effective to inhibit the growth of both the microbes.