Characterization, subsite mapping and partial amino acid sequence of glucoamylase from the filamentous fungus Trichoderma reesei.
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.
Fagerström
R
R
Research Laboratories, Alko Ltd, Helsinki, Finland.
Kalkkinen
N
N
D000595:Amino Acid Sequence; D001665:Binding Sites; D002845:Chromatography; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000493:pharmacokinetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D055585:Physical Phenomena; D010825:Physics; D012995:Solubility; D013213:Starch / Q000737:chemistry; D014242:Trichoderma / Q000201:enzymology*; D014357:Trypsin / Q000737:chemistry
D007477:Ions; D008670:Metals; D009844:Oligosaccharides; D010455:Peptides; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase; D014357:Trypsin