Immobilization of Lactobacillus acidophilus β-galactosidase on chitosan obtained from the shells of the African giant snail, Achatina achatina
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Abstract
High enzyme activity and reusability are the major factors that limit enzyme application in the industry. This study explored the properties of Lactobacillus acidophilus β-galactosidase immobilized on Achatina chitosan, for improved enzyme reusability in industry. β-Galactosidase was produced from environmentally well-adapted Lactobacillus acidophilus. The enzyme was purified by ion exchange chromatography using DEAE-cellulose and had a molecular weight of 43kDa. Mg2+ was a major positive effector of the β-galactosidase activity. Chitin was extracted from Achatina shells by demineralization and deproteination, and deacetylated to chitosan. The chitin and chitosan yields were 74.64% and 58.60%. However, a hypochlorite-decolorized chitin, deacetylated to chitosan, gave a yield of 71%. FTIR spectra of chitin showed major bands at 711 cm-1, 855 cm-1, 1082 cm-1, and 1438 cm-1 for chitin and for chitosan at 6778 cm-1, 711 cm-1, 851 cm-1, 1082 cm-1 and 1436 cm-1. The β-galactosidase was immobilized on chitosan beads by adsorption and covalent linkage using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), and glutaraldehyde, separately. The enzyme had optimal temperature and pH of 70°C and 5.5. The Michaelis-Menten constant (KM) and maximal velocity (Vmax) of the free and immobilized β-galactosidase were 0.262/0.251mM, and 270.27/290 μmol/min using p-NPG as substrate, and 0.53/10.02 mM and 250/275 μmol/min for lactose as substrate, respectively. Covalent immobilization by glutaraldehyde improved the β-galactosidase activity more than adsorption, in comparison to EDC/NHS. The results show that extracellular β-galactosidase from Lactobacillus acidophilus, isolated from dairy wastewater, can be immobilized on chitosan support produced using cheaply available Achatina shell chitosan for greater reusability.
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