Abstract:
: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.
journal_name
Biotechnol Appl Biochemjournal_title
Biotechnology and applied biochemistryauthors
Fagerström R,Kalkkinen Ndoi
10.1111/j.1470-8744.1995.tb00333.xsubject
Has Abstractpub_date
1995-04-01 00:00:00pages
223-31issue
2eissn
0885-4513issn
1470-8744journal_volume
21pub_type
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