|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1995 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1994 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Calorimetric and steady-state kinetic studies were done to investigate the structure and function of fungal glucoamylases.
(1) Thermal unfolding of two forms of Aspergillus niger glucoamylase was observed by adiabatic differential scanning calorimetry (DSC) at pH 7. The DSC traces of the larger form of the enzyme, G1, and the shorter form which lacks the C-terminal starch-binding domain of G1, G2, could be resolved by assuming two-state unfolding of five and four independent components, respectively. The thermal unfolding of the starch-binding domain was found to be reversible, but that of the catalytic domain was irreversible. The DSC observation of G1 and G2 in the presence of beta-cyclodextrin or 1-deoxynojirimycin showed that there was no domain interaction between the starch-binding domain and catalytic domain.
(2) Steady-state inhibitory kinetic studies of Aspergillus glucoamylase showed that gluconolactone and 1-deoxynojirimycin were non-competitive type and competitive type inihi
bitor, respectively. These results indicate that gluconolactone binds to a site other than the active site of the enzyme forming nonproductive ternary complex of the enzyme, substrate, and gluconolactone, but 1-deoxynojirimycin binds to the active site (most probably subsite 2) of the enzyme without forming the ternary complex.
(3) The subsite structure of Rhizopus glucoamylase was identified for the hydrolytic reaction of isomaltooligosaccharides at 25ﾟC and at pH4.5. The arrangement of the subsite affinities A_i's (i is the subsite number) was evaluated as follows : A_1=-6.0, A_2=13.8, A_3=4.6, A_4=1.5, A_5=0.6, A_6=-0.5, and A_7=0.2 (in kj mol^<-1>) unit). The intrinsic rate constant for the hydrolytic reaction in the productive binding mode of substrates, k_<int>, was 10.2 s^<-1>. Larger values of K_m for isomaltooligosaccharides compared with those for maltooligosaccharides were attributed to smaller values A_1 and A_2 compared with those for maltooligosaccharides, and smaller values of k_0 were attributed t smaller values of A_1 and k_<int>.
(4) Hydrolytic reactions of maltooligosaccharides (degree of polymerization n=3-7) and isomaltooligosaccharides (n=3-7) by Rhizopus glucoamylase were observed by HPLC at pH 4.5,25ﾟC.It was confirmed that initial rates of the decrease of n-mer substrate and the increase of the products, glucose and (n-1)-mer oligosaccharide, are the same for all the substrates, indicating that the reaction mode of the enzyme is "random attack, " and no "multiple attack" or "single chain attack" is operating. Less