Optimisation of soy flour fermentation parameters to produce ß-glucosidase for bioconversion into aglycones.

The solid state fermentation (SSF) parameters of defatted soybean flour (DSF) with Aspergillus oryzae IOC 3999/1998 or Monascus purpureus NRRL 1992 was evaluated using a rotational central composite experimental design to optimise the production of ß-glucosidase and convert glycosidic isoflavones in aglycones. Variables investigated were initial pH of DSF, volume of water added to 10 g of DSF and incubation temperature. ß-Glucosidase activity was measured using the synthetic substrate, p-nitrophenyl-ß-D-glucoside. The content of isoflavones was determinate by ultra performance liquid chromatography. The highest production of ß-glucosidase for both strains occurred when adding 10 mL of water to the DSF, incubating at 30 °C and using 6.0 as the initial DSF pH. A. oryzae IOC 3999/1998 expressed ß-glucosidase activity at 10.7 times higher than M. purpureus NRRL 1992. The DSF fermentation was more efficient in converting isoflavones with M. purpureus NRRL 1992.

Purification and characterization of an extracellular beta-glucosidase from Monascus purpureus.

An extracellular beta-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A 22 central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at 50 degrees and pH 5.5. The beta-glucosidase showed moderate thermostability, was inhibited by HgCl2, K2CrO4, and K2Cr2O7, whereas other reagents including beta- mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-beta-D-glucopyranoside, and maltose indicates that the beta-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-beta-Dcellobiose. beta-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.