Isolation, purification and characterisation of an endoglucanase and β-glucosidase from an anaerobic sulphidogenic bioreactor

Two endoglucanases and a beta-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 degrees 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 K value was 4.0 mg ml(-1) (carboxymethylcellulose) and 5.1 mg ml(-1) (hydroxyethylcellulose) with V-max of 0.3 and 0.19 mu mol 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 I-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). beta-Glucosidase was purified 5-fold after acetone precipitation, affinity chromatography with Whatman cellulose CC31 and gel exclusion on Sepharose 4B. The K value was 84.2 mu M (methyl-umbelliferyl-beta-D-glucopyranoside) and the V-max 4.4 mu mol min(-1) ml(-1). All of the transition metals inhibited P-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. (c) 2006 Elsevier Inc. All rights reserved.