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Characterization, subsite mapping and partial amino acid sequence of glucoamylase from the filamentous fungus Trichoderma reesei
Author(s) -
Fagerstrom R.,
Kalkkinen N.
Publication year - 1995
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1995.tb00333.x
Subject(s) - trichoderma reesei , chemistry , hydrolysis , substrate (aquarium) , pullulan , enzyme kinetics , starch , stereochemistry , molecular mass , affinities , enzyme , chromatography , biochemistry , cellulase , active site , polysaccharide , biology , ecology
The pH optimum, temperature‐dependence, thermal stability, substrate specificity and subsite affinities of the 66 kDa, pI 4.0 glucoamylase of the filamentous fungus Trichoderma reesei were determined. It had a pH optimum of 5.5 and a temperature optimum (5 min reaction time) of 70 degrees C with soluble starch as substrate. Thermal‐inactivation studies revealed that the glucoamylase is relatively thermostable up to 60 degrees C. Metal ions and EDTA tested at 5 mM concentrations had no significant effect, and beta‐cyclodextrin only slightly inhibitory effects, on the digestion of soluble starch. Estimated Km and kcat. values for soluble starch where 0.11 mg.ml‐1 and 28.5 s‐1 respectively. Hydrolysis of pullulan (Km 14 mg.ml‐1 and kcat. = 6.6 s‐1) indicated substantial activity towards 1,6‐O‐glucosidic bonds. From ratios of kinetic parameters of malto‐ and isomalto‐oligosaccharides, it was apparent that the glucoamylase showed approx. 3‐fold higher selectivity towards isomalto‐oligosaccharides than most other reported fungal glucoamylases. Substrate binding affinities were calculated from kinetic data for the linear series of malto‐ and isomalto‐oligosaccharides. The results were in good agreement with other reported glucoamylases. The main difference was that subsite 1 showed a slightly negative free energy of binding with malto‐oligosaccharides, whereas most other glucoamylases show a positive free energy at this subsite. A set of peptides obtained from purified glucoamylase by tryptic digestion where sequenced. They covered approx. 17% of the total amino acid sequence as estimated from molecular mass on SDS/PAGE. Some of the sequences were tentatively aligned to known glucoamylase sequences. They showed about 60% identity with the extensively studied Aspergillus glucoamylase.