Purification and Characterization of an Endo-(1,3)-β- d -Glucanase from Trichoderma longibrachiatum

Abstract

A laminarinase [endo-(1,3)-p-D-glucanasel has been purified from Trichoderma longibrachiatum cultivated with D-glucose as the growth substrate. The enzyme was found to hydrolyze laminarin to oligosaccharides varying in size from glucose to pentaose and to lesser amounts of larger oligosaccharides. The enzyme was unable to cleave laminaribiose but hydrolyzed triose to laminaribiose and glucose. The enzyme cleaved laminaritetraose, yielding laminaritriose, laminaribiose, and glucose, and similarly cleaved laminaripentaose, yielding laminaritetraose, laminaritriose, laminaribiose, and glucose. The enzyme cleaved only glucans containing 0-1,3 linkages. The pH and temperature optima were 4.8 and 55°C, respectively. Stability in the absence of a substrate was observed at temperatures up to 50°C and at pH values between 4.9 and 9.3. The molecular mass was determined to be 70 kilodaltons by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis, and the pl was 7.2. Enzyme activity was significantly inhibited in the presence of HgCl2, MnCl2, KMnO4, and N-bromosuccinimide. The Km of the enzyme on laminarin was 0.0016%, and the Vmax on laminarin was 3,170 ,umol of glucose equivalents per mg of the pure enzyme per min.

Characterization of a 29-kDa β-1,3-glucanase fromTrichoderma harzianum

“…The denaturing agents SDS and Hg 2+ strongly inhibited the enzyme activity. The complete inhibition by mercuric ions may indicate the importance of sulfhydryl groups in enzyme functions, as reported for purified β‐1,3‐glucanase from T. harzianum (TC) and T. longibrachiatum [6,17]. These results suggest that each β‐1,3‐glucanase produced by T. harzianum (TC) is different and probably encoded by different genes.…”

“…The effect of laminarin concentration on the 29‐kDa β‐1,3‐glucanase activity was examined from a Michaelis‐Menten plot using a non‐linear regression data analysis program [11]. The apparent K M (1.72 mg ml −1 ) was lower than the K M values of the 70‐kDa β‐1,3‐glucanases from T. harzianum (CECT 2413) (3.3 mg ml −1 ), and was higher than those found for 36‐kDa β‐1,3‐glucanases from T. harzianum (TC) (1.18 mg ml −1 ) and T. longibrachiatum (0.016 mg ml −1 ) [5,6,17].…”

Characterization of a 29-kDa β-1,3-glucanase from Trichoderma harzianum

“…The denaturing agents SDS and Hg 2 strongly inhibited the enzyme activity. The complete inhibition by mercuric ions may indicate the importance of sulfhydryl groups in enzyme functions, as reported for puri¢ed L-1,3-glucanase from T. harzianum (TC) and T. longibrachiatum [6,17].…”

“…L-1,3-Glucanases of low molecular mass have also been reported previously in di¡erent fungi The e¡ect of laminarin concentration on the 29-kDa L-1,3-glucanase activity was examined from a Michaelis-Menten plot using a non-linear regression data analysis program [11]. The apparent K M (1.72 mg ml 31 ) was lower than the K M values of the 70-kDa L-1,3-glucanases from T. harzianum (CECT 2413) (3.3 mg ml 31 ), and was higher than those found for 36-kDa L-1,3-glucanases from T. harzianum (TC) (1.18 mg ml 31 ) and T. longibrachiatum (0.016 mg ml 31 ) [5,6,17].…”

Purification and Characterization of an Endo‐1,3‐β‐Glucanase from Aspergillus fumigatus

“…min-' . mg protein -') (Reichelt and Fleet, 1981;Sanchez et al, 1982;Notario, 1982;Usui et al, 1985;Tangarone et al, 1989;de la Cruz et al, 1995). The increase of V,,,, with the size of the substrate, indicates that the number of 1,3-/?-glucosidic bonds/molecule of substrate influence directly the activity of the 74-kDa endo-p-glucanase, suggesting a putative repetitive attack where the enzyme cleaves several times the glucan chain before dissociation (Robyt and French, 1970;Thoma, 1976).…”