Search

Abstract: A new soil isolate, Fusarium proliferatum ECU2042, was found capable of producing a ginsenoside Rg3 hydrolase (a special β-glucosidase) which is useful for production of a very potent antitumor agent Rh2. By investigating its fermentation progress, the optimal time for β-glucosidase biosynthesis was determined as 72h. The crude β-glucosidase was partially purified by 49.4-fold from the cell-free extract with an activity yield of 13%. The catalytic performance of the partially purified β-glucosidase was examined, giving optima of pH and temperature at pH 5.0–6.0 and 50°C. It was found that the F. proliferatum β-glucosidase can also hydrolyze cellobiose, but not active towards a frequently used artificial substrate p-nitrophenyl β-d-glucopyranoside (pNPG). In a lab-scale preparation, the bio-hydrolytic reaction was carried out at 50°C for 24h in 50ml NaAc/HAc buffer (100mM, pH 5.0) containing 50mg Rg3 and 12mg lyophilized crude enzyme extract, giving the ginsenoside Rh2 in 60.3% conversion. [Copyright &y& Elsevier]

Abstract: A novel β-glucosidase from Fusarium proliferatum ECU2042 (FPG) was successfully purified to homogeneity with a 506-fold increase in specific activity. The molecular mass of the native purified enzyme (FPG) was estimated to be approximately 78.7kDa, with two homogeneous subunits of 39.1kDa, and the pI of this enzyme was 4.4, as measured by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50°C, respectively. The enzyme was stable at pH 4.0–6.5 and temperatures below 60°C, and the deactivation energy (E d) for FPG was 88.6kJmo1−1. Moreover, it was interesting to find that although the purified enzyme exhibited a very low activity towards p-nitrophenyl β-d-glucoside (pNPG), and almost no activity towards cellobiose, a relatively high activity was observed on ginsenoside Rg3. The enzyme hydrolyzed the 3-C, β-(1→2)-glucoside of ginsenoside Rg3 to produce ginsenoside Rh2, but did not sequentially hydrolyze the β-d-glucosidic bond of Rh2. The K m and V max values of FPG for ginsenoside Rg3 were 2.37mM and 0.568μmol (hmg protein)−1, respectively. In addition, this enzyme also exhibited significant activities towards various alkyl glucosides, aryl glucosides and several natural glycosides. [Copyright &y& Elsevier]