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Title: Isolation, purification and characterisation of an endoglucanase and β-glucosidase from an anaerobic sulphidogenic bioreactor
Authors: Oyekola, Oluwaseun 
Ngesi, N 
Whiteley, CG 
Keywords: Anaerobic sludge digestion;Endoglucanase;β-Glucosidase;Purification
Issue Date: 2007
Publisher: Elsevier
Source: Enzyme and Microbial Technology, Volume 40, Issue 4, 5 March 2007, Pages 637–644
Abstract: Two endoglucanases and a β-glucosidase have been isolated, purified and characterised from an anaerobic sulphidogenic bioreactor. The enzymes, associated predominantly with the organic particulate matter, exhibited a pH optima of 6 and 6.5, respectively and temperature optima of 50 °C. Under such conditions the endoglucanases remained stable and exhibited no decrease in activity after 60 min while only 30% glucosidase remained after the same period. The endoglucanases were purified 13- and 25-fold after sonication, PEG concentration and DEAE chomatography. They were inhibited slightly by increasing concentrations of sulphate but stimulated some 4–6.5-fold by sulphide levels above 400 mg l−1. The Km value was 4.0 mg ml−1 (carboxymethylcellulose) and 5.1 mg ml−1 (hydroxyethylcellulose) with Vmax of 0.3 and 0.19 μmol min−1 ml−1, respectively. Divalent ions like Cu, Ni and Zn proved to be inhibitory while Fe, Mg and Ca stimulated the enzyme at concentrations above 400 mg l−1. Volatile fatty acids such as acetic, propionic and butyric acid proved slightly inhibitory to endoglucanases with 20–40% inhibition occurring at concentrations of 800 mg l−1. β-Glucosidase was purified 5-fold after acetone precipitation, affinity chromatography with Whatman cellulose CC31 and gel exclusion on Sepharose 4B. The Km value was 84.2 μM (methyl-umbelliferyl-β-d-glucopyranoside) and the Vmax 4.4 μmol min−1 ml−1. All of the transition metals inhibited β-glucosidase above 200 mg l−1 while the volatile fatty acids afforded similar effects to those of the endoglucanases. Acetic acid enhanced activity at lower concentrations.
Appears in Collections:Eng - Journal articles (DHET subsidised)

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