Purification of an extracellular beta-glucosidase from a strain of Trichoderma reesei containing multiple bgl1 genes

Cellulose, the most abundant renewable resource is a potential source of fuel and food. Its utilization may require a better understanding of the cellulases, especially β-glucosidase, which forms glucose from cellooligosaccharides. Many cellulolytic organisms either produce little extracellular β-glucosidase or retain a significant fraction of it in a cell-bound state. Recently a strain of Trichoderma reesei with an increased gene dosage for β-glucosidase has been reported. This strain produces a level of extracellular β-glucosidase some 50-fold greater than the parent strain. The most effective method of converting cellulose to fermentable sugars has been enzymatic saccharification, hence a clear picture of the role of the β-glucosidase and its properties are essential. Elucidating properties of the enzyme may minimize the problems that affect cellulose utilization.; Successive anion and cation exchange chromatographies were used to purify the enzyme. Evidence for homogeneity was provided by the increase in specific activity and by SDS gel electrophoresis. Kinetic properties, optimum pH and stability were determined for the purified enzyme. White egg-laying chickens were injected biweekly into the pectoral muscle at different sites with a solution of β-glucosidase (0.3 mg/mL) mixed with an equal volume of Freund's complete adjuvant biweekly. Eggs laid by the immunized hen were collected and anti-β-glucosidase antibody was isolated and purified from egg yolk by a series of precipitations with polyethyleneglycol. Female Muscovy ducks were immunized as described above. One week after each injection blood was collected and pooled. The method described by Higgins et al. (1995) was used to purify the duck antibody. Purified β-glucosidase and duck whole serum were sent to Cocalico Biologicals, Inc. Reamstown, PA, for the production of rabbit anti-β-glucosidase and rabbit anti-duck serumglobulin antibodies, respectively. Samples were emulsified in Freund's complete adjuvant and inoculated subcutaneously into New Zealand White × Lop Ear rabbits for three consecutive weeks. The rabbits were bled 2 weeks after the last injection and sera were stored at –20°C. Protein-G affinity columns were used to purify the antibodies.; The values of Km and Vmax of β-glucosidase were determined to be 0.14 mM and 54 units/mg using p-nitrophenyl β-D-glucopyranoside as substrate. The enzyme is optimally active from pH 4.5 to 5.0 and loses its activity at lower pHs. The isoelectric point was found to be pH 8.4. A molecular mass of 76 kDa was determined by SDS gel electrophoresis. Inhibition constants for glucose and 1-deoxynojirimycin were 638 μM and 0.62 μM respectively. ELISA and Western blotting confirmed specificity and sensitivity of the different antibodies against β-glucosidase. Affinity purification of β-glucosidase by hydro succinimidyl-agarose chromatography has shown for every 20 mg of antibody, 0.4 mg of β-glucosidase was bound. Elimination of contaminants and reduction of traditional purification steps were possible, achieving the same specific activity value of 43.5 units/mg of protein for the purified β-glucosidase.