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

摘要

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 °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_m value was 4.0 mgml~(-1)(carboxymethylcellulose)and 5.1 mgml~(-1)(hydroxyethylcellulose)with V_(max)of 0.3 and 0.19 mu molmin~(-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 mgl~(-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).p-Glucosidase was purified 5-fold after acetone precipitation,affinity chromatography with Whatman cellulose CC31 and gel exclusion on Sepharose 4B.The K_m value was 84.2 mu M(methyl-umbelliferyl-beta-D-glucopyranoside)and the V_(max)4.4mu molmin~(-1)ml~(-1).All of the transition metals inhibited beta-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.