Abstract
Using solid-state fermentation, production of an industrially important pectin lyase from a fungal strain Fusarium oxysporum MTCC 1755 was attempted, which was further subjected to purification and characterization. The enzyme was purified by three steps, namely ammonium sulfate fractionation, cation-exchange chromatography on CM cellulose followed by gel filtration chromatography using Sephadex G-100 column. A 16-fold purification with 31.2% yield and 3.2 U/mg specific activity was achieved. The optimum pH of the purified enzyme was 9.0 and stability ranged from pH 5.0–7.0 for 24 h. Optimum temperature of purified enzyme was found to be 40 °C while temperature stability ranged from 10 to 50 °C for 30 min. The Km and kcat of the enzyme was 1.75 mg/ml and 83.3 s−1, respectively. The purified enzyme was found to be highly stimulated by Ca2+ ions while sugars like mannitol and sorbitol, and salts like NaCl and CaCl2 enhanced the thermostability. The purified pectin lyase was found suitable for retting of Crotolaria juncea fiber.
Highlights
Pectic substances are high molecular weight, negatively charged, acidic, complex glycosidic macromolecules that are present in the plant kingdom
Using solid-state fermentation, production of an industrially important pectin lyase from a fungal strain Fusarium oxysporum MTCC 1755 was attempted, which was further subjected to purification and characterization
The enzyme was purified by three steps, namely ammonium sulfate fractionation, cation-exchange chromatography on CM cellulose followed by gel filtration chromatography using Sephadex G-100 column
Summary
Pectic substances are high molecular weight, negatively charged, acidic, complex glycosidic macromolecules (polysaccharides) that are present in the plant kingdom. A group of enzymes popularly known as pectinases are associated with degradation of pectin and includes exo- and endo-polygalacturonases (PGs, E.C 3.2.115), pectin methyl esterases (PME.E.C 3.2.1.11), pectate lyases (PL,E.C 4.2.2.9) and pectin lyases (PNL 4.2.2.10) These enzymes are widely distributed in higher plants and microorganisms (Whitaker 1990) and are of prime importance for plants as they help in cell wall extension and softening of some plant tissues during maturation and storage (Aguilar and Huirton 1990; Sakai 1992). SSF has advantage over SmF as it aims to achieve the highest substrate concentration for fermentation by bringing a cultivated fungus or bacterium into close contact with the insoluble substrate and is economically more feasible It involves use of abundant and cheap substrates, which are mostly the waste products, such as wheat bran, sugarcane bagasse and orange peel (Meenakshisundaram 2012; Tivkaa et al 2013; Kashyap et al 2003; Martin et al 2004). Purification and enzymatic characterization of an alkaline pectin lyase from Fusarium oxysporum MTCC 1755 with its possible application in retting have been discussed
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