The copurification of beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58.
Two enzyme complexes, each with beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21), beta-xylosidase (beta-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-beta-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75,400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the beta-xylosidase, beta-glucosidase, and 1,3-beta-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl beta-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the beta-glucosidase and 1,3-beta-glucanase of the major complex had inhibition constants which differed by more than 80,000 times. In addition, the inhibition on the 1,3-beta-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.
D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D043326:Glucan 1,3-beta-Glucosidase; D005959:Glucosidases / Q000302:isolation & purification*; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology*; D014995:Xylosidases / Q000302:isolation & purification* / Q000378:metabolism; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism
D005959:Glucosidases; D006026:Glycoside Hydrolases; D014995:Xylosidases; D001617:beta-Glucosidase; C026579:exo-1,4-beta-D-xylosidase; D043326:Glucan 1,3-beta-Glucosidase