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.
Daroit
Daniel J
DJ
Laboratorio de Bioquimica e Microbiologia Aplicada, Departamento de Ciencia de Alimentos (ICTA), Universidade Federal do Rio Grande do Sul, 91501-970, Porto Alegre, RS, Brazil.
Simonetti
Aline
A
Hertz
Plinho F
PF
Brandelli
Adriano
A
D004795:Enzyme Stability; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D032741:Monascus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D005656:Fungal Proteins; D001617:beta-Glucosidase