Study of Glucoamylase from Corticium rolfsii.

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Corticium rolfsii AHU 9627 secretes strong raw-starch-saccharifying enzyme (RSSE) that is superior to other enzymes. The RSSE consisted of five forms of glucoamylase (G1-G5) and a small amount of α-amylase. These glucoamylases, showed nearly identical characteristics, except that G4 and G5 were unable to hydrolyze raw starch. A cDNA coding for C. rolfsii glucoamylase G2 was cloned. This clone (CG 15) contains an entire coding region for a polypeptide of 579 residues, which has catalytic domain and starch-binding domain like other glucoamylases from filamentous fungi. Some differences were observed in the starch-binding domain and in the linker region between catalytic domain and the starch-binding domain. The obtained cDNA was introduced into Saccharomyces cerevisiae AH 22. The transformants acquired starch-saccharifying ability. The amount of secreted glucoamylase, however, was very small (0.001 U/mL). Therefore the cDNA was introduced into Aspergillus pryzae for better production. Through optimization of the culture conditions, the amount of recombinant glucoamylase obtained in the culture supernatant reached 100 mg/L (3.5 U/mL). The glucoamylase G2 secreted from A. oryzae (G2A0) had almost the same specific activity as native G2 from C. rolfsii. Thermal and pH stabilities of G2A0, however, were significantly lower than those of native G2. To clarify domain relationships and to compare their properties with those of other glucoamylase, two chimeric gluco-amylases whose domains were interchanged with that of Aspergillus awamori var. kawachi gluco-amylase (GAI) were made, and their enzymatic characteristics were investigated. The chimeric glucoamylases showed the pH and thermal stabilities similar to those of glucoamylases from which their catalytic domain derived. Native G2, GAI secreted from A. oryzae, and two chimeric gluco-amylases showed a similarly good rate of hydrolysis of raw potato starch, although G2A0 showed a significantly lower rate of hydrolysis.
日本応用糖質科学会の論文

Study of Glucoamylase from Corticium rolfsii.

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