Laccases are blue copper oxidases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. They are currently seen as highly interesting industrial enzymes because of their broad substrate specificity. A positive strain was isolated and characterized as nonspore forming Basidiomycetes Pleurotus sp. Laccase activity was determined using ABTS as substrate. Laccase was purified by ionexchange and gel filtration chromatography. The purified laccase was a monomer showed a molecular mass of 40 ± 1 kDa as estimated by SDS-PAGE and a 72-fold purification with a 22% yield. The optimal pH and temperature were 4.5 and 65°C, respectively. The K
m and V
max values are 250 (mM) and 0.33 (μmol/min), respectively, for ABTS as substrate. Metal ions like CuSO4, BaCl2, MgCl2, FeCl2, ZnCl2 have no effect on purified laccase whereas HgCl2 and MnCl2 moderately decrease enzyme activity. SDS and sodium azide inhibited enzyme activity, whereas Urea, PCMB, DTT, and mercaptoethanol have no effect on enzyme activity. The isolated laccase can be used in development of biosensor for detecting the phenolic compounds from the effluents of paper industries.
Ridge gourd () is consumed as a vegetable after peeling off the skin which is a domestic waste. peel (LAP) was observed to be a good source of fiber (20.6 %) and minerals (7.7 %). Amino acid analysis revealed presence of the highest content of Carnosine followed by aspartic acid and aminoadipic acid. Antioxidant activity of different extracts showed that ethyl acetate extract was more potent when compared to other solvent extractions. It exhibited a significant amount of phenolic acids like p-coumaric acid (68.64 mg/100 g of dry weight) followed by gallic acid (34.98 mg/100 g of dry weight), protocatechuic acid (30.52 mg/100 g of dry weight) in free form and ferulic acid (13.04 mg/100 g of dry weight) in bound form.
Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff's base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 o C to 70 o C. The immobilized lipase showed 20-25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ~60% when incubated with 30% butanol. The kinetic studies revealed increase in K M value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the V max of immobilized enzyme (142.8 µmol min −1 mg −1 ), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 o C in TrisHCl buffer pH 7.0 without any significant loss in enzyme activity.
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