Chitosan immobilized novel peroxidase from Azadirachta indica: Characterization and application.

In the present paper, a peroxidase was purified from the leaves of a medicinal tree, namely Azadirachta indica, to 45.2 folds with overall recovery of 61%. Based on the subunit size, the purified peroxidase was suggested to be a monomeric structure of size 50kDa and exhibited good thermostability as it was fully stable at 65°C for 1hr and also retained about 73% activity at 70°C till 30min. The substrate affinity was found to be in order of guaiacol>pyrogallol>o-dianisidine. The purified peroxidase was found to be insensitive towards high concentrations of Na+, Ca2+, Mg2+ and Mn2+. Heavy metals, namely Cs2+, Co2+ and Cd2+ activated the peroxidase while that of Hg2+ deactivated the peroxidase in concentration dependent manner. The purified peroxidase exhibited tolerance towards organic solvents in order of ethanol>butanol>isopropanol>acetone. Immobilization of purified peroxidase by entrapment into chitosan beads led to shift in its optimum pH from pH 5 to 7 and considerable enhancement in dye decolorization ability as compared to that of free enzyme. Thus, based on all the above properties, it may be suggested that the purified A. indica peroxidase is a promising candidate for industrial applications.

Immobilization of papaya laccase in chitosan led to improved multipronged stability and dye discoloration.

A purified papaya laccase was immobilized in chitosan beads using entrapment approach and its physico-chemical properties were investigated and compared with that of free enzyme. Increase in properties of the laccase such as optimum temperature (by 10 °C), thermostability (by 3-folds) and optimum pH (from 8.0 to 10.0) was observed after immobilization. Immobilization led to increased tolerance of enzyme to a number of metal ions (including heavy metals) and organic solvents namely, ethanol, isopropanol, methanol, benzene and DMF. The catalytic efficiency (Kcat/Km) of the immobilized enzyme was found to increase more than ten folds, in comparison to that of the free enzyme, with hydroquinone as substrate. Immobilization of laccase also led to improvement in dye decolorization such that the synthetic dye indigo carmine (50 µg/ml) was completely decolorized within 8h of incubation as compared to that of the free laccase which decolorized the same dye to only 56% under similar conditions. Thus, immobilization of laccase into chitosan beads led to tremendous improvement in various useful attributes of this enzyme thereby making it more versatile for its industrial exploitation.

Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation.

Laccase, widely distributed in bacteria, fungi, and plants, catalyzes the oxidation of wide range of compounds. With regards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesis while fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual function of laccases using in-silico molecular docking and dynamics simulation approaches. Modeling and superimposition analyses of one each representative of plant and fungal laccases, namely, Populus trichocarpa and Trametes versicolor, respectively, revealed low level of similarity in the folding of two laccases at 3D levels. Docking analyses revealed significantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as compared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites were found to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analyses for the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetrameric lignin model compound remains attached to the active site of fungal laccase throughout the simulation period, while it protrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basis of binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than that of plant. The overall data suggested a situation favorable for the degradation of lignin polymer by fungal laccase while its synthesis by plant laccase.

Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation.

An organic solvent and surfactant stable α-amylase from soybean seeds.

An organic solvent and surfactant stable α-amylase was obtained from soybean seeds. The direct and indirect effect of various organic solvents (non-polar, polar protic, and polar aprotic) and surfactants on the activity and stability of free enzyme was determined. The enzyme showed a very high catalytic efficiency and stabilization against most of the organic solvents and surfactants tested, except for few. Those organic solvents and surfactants (like chloroform, dimethyl formamide, n-butanol, and Tween 20), which caused an inhibition in enzyme activity, were used to study their effects on immobilized enzyme. The inhibitory effect was found to be decreased in immobilized enzyme as compared to free enzyme indicating that immobilization imparted stability to the enzyme. Moreover, the possibility of reuse of the enzyme in the presence of the organic solvents and surfactants was increased upon immobilization. The stability of soybean α-amylase towards organic solvents and surfactants shows that it is a potential candidate for use in organic-solvent biocatalysis as well as in detergent industries.

A highly efficient and thermostable α-amylase from soya bean seeds.

The α-amylase from soya bean seeds was purified by affinity precipitation, resulting in approx. 20-fold purification with approx. 84% recovery. The purified α-amylase had an optimum pH of 5.5, optimum temperature of 75 °C, Arrhenius energy of activation of 6.03 kcal/mol (1 kcal≈4.184 kJ) and a Km of 2.427 mg/ml (starch substrate). The enzyme had maximum substrate specificity for starch. Among the various metal ions tested, Co2+ and Mn2+ were found to be strong activators. The effect of thiol group modifying agents showed that the thiols of soya bean α-amylase are not directly involved in catalysis. The thermostability of the enzyme makes it suitable for starch liquefaction and the detergent industry respectively.

alpha-Amylase: an ideal representative of thermostable enzymes.

The conditions prevailing in the industrial applications in which enzymes are used are rather extreme, especially with respect to temperature and pH. Therefore, there is a continuing demand to improve the stability of enzymes and to meet the requirements set by specific applications. In this respect, thermostable enzymes have been proposed to be industrially relevant. In this review, alpha-amylase, a well-established representative of thermostable enzymes, providing an attractive model for the investigation of the structural basis of thermostability of proteins, has been discussed.