Characterization of a Thermostable Extracellular beta-Glucosidase with Activities of Exoglucanase and Transglycosylation from Paecilomyces thermophila

摘要

The purification and characterization of a novel extracellular beta-glucosidase from Paecilomyces thermophila J18 was studied.The beta-glucosidase was purified to 105-fold apparent homogeneity with a recovery yield of 21.7% by DEAE 52 and Sephacryl S-200 chromatographies.Its molecular masses were 116 and 197 kDa when detected by SDS-PAGE and gel filtration,respectively.It was a homodimeric glycoprotein with a carbohydrate content of 82.3%.The purified enzyme exhibited an optimal activity at 75 °C and pH 6.2.It was stable up to 65 °C and in the pH range of 5.0-8.5.The enzyme exhibited a broad substrate specificity and significantly hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (pNPG),cellobiose,gentiobiose,sophorose,amygdalin,salicin,daidzin,and genistin.Moreover,it displayed substantial activity on beta-glucans such as laminarin and lichenan,indicating that the enzyme has some exoglucanase activity.The rate of glucose released by the purified enzyme from cellooligosaccharides with a degree of polymerization (DP) ranging between 2 and 5 decreased with increasing chain length.Glucose and glucono-zeta-lactone inhibited the beta-glucosidase competitively with Ki values of 73 and 0.49 mM,respectively.The beta-glucosidase hydrolyzed pNPG,cellobiose,gentiobiose,sophorose,salicin,and amygdalin,exhibiting apparent K_m values of 0.26,0.65,0.77,1.06,1.39,and 1.45 mM,respectively.Besides,the enzyme showed transglycosylation activity,producing oligosaccharides with higher DP than the substrates when cellooligosaccharides were hydrolyzed.These properties make this beta-glucosidase useful for various biotechnological applications.