CHARACTERIZATION, SUBSITE MAPPING AND PARTIAL AMINO-ACID-SEQUENCE OF GLUCOAMYLASE FROM THE FILLAMENTOUS FUNGUS TRICHODERMA-REESEI

The pH optimum, temperature-dependence, thermal stability, substrate s pecificity and subsite affinities of the 66 kDa, pI 4.0 glucoamylase o f the filamentous fungus Trichoderma reesei were determined, It had a pH optimum of 5.5 and a temperature optimum (5 min reaction time) of 7 0 degrees C with soluble starch as substrate. Thermal-inactivation stu dies revealed that the glucoamylase is relatively thermostable up to 6 0 degrees C. Metal ions and EDTA tested at 5 mM concentrations had no significant effect, and beta-cyclodextrin only slightly inhibitory eff ects, on the digestion of soluble starch. Estimated K-m and k(cat.) va lues for soluble starch where 0.11 mg . ml(-1) and 28.5 s(-1) respecti vely. Hydrolysis of pullulan (K-m 14 mg . ml(-1) and k(cat.) = 6.6 s(- 1)) indicated substantial activity towards 1,6-O-glucosidic bonds. Fro m ratios of kinetic parameters of malto- and isomalto-oligosaccharides , it was apparent that the glucoamylase showed approx. 3-fold higher s electivity towards isomalto-oligosaccharides than most other reported fungal glucoamylases. Substrate binding affinities were calculated fro m kinetic data for the linear series of malto- and isomalto-oligosacch arides. The results were in good agreement with other reported glucoam ylases. The main difference was that subsite 1 showed a slightly negat ive free energy of binding with malto-oligosaccharides, whereas most o ther glucoamylases show a positive free energy at this subsite. A set of peptides obtained from purified glucoamylase by tryptic digestion w here sequenced. They covered approx. 17% of the total amino acid seque nce as estimated from molecular mass on SDS/PAGE. Some of the sequence s were tentatively aligned to known glucoamylase sequences. They showe d about 60% identity with the extensively studied Aspergillus glucoamy lase.