EFFECT ON THERMOSTABILITY AND CATALYTIC ACTIVITY OF INTRODUCING DISULFIDE BONDS INTO ASPERGILLUS-AWAMORI GLUCOAMYLASE

Two additional disulfide bonds and three combined thermo-stabilizing m utations were introduced into Aspergillus awamori glucoamylase to test their effects on enzyme thermostability and catalytic properties. The single cysteine mutations N20C, A27C, T72C and A471C were made and co mbined to produce the double cysteine mutations N20C/A27C and T72C/A47 1C. The double cysteine mutants were expressed efficiently in Saccharo myces cerevisiae, and disulfide bonds formed spontaneously after ferme ntation. At 50 degrees C, the single mutants N20C and A27C had decreas ed specific activity, whereas the specific activity of the double muta nts N20C/A27C and T72C/A471C were similar to wildtype glucoamylase. Th e N20C/A27C mutation increased thermostability, with an increased acti vation free energy of 1.5 kJ/mol at 65 degrees C, while the single mut ation A27C only slightly increased thermostability and N20C decreased it. The other disulfide bond-forming mutation T72C/A471C did not affec t thermostability at pH 4.5. The N20C/A27C mutation was separately com bined with two other thermo-stabilizing mutations, G137A and S436P The rmostabilities of all of the combined mutated glucoamylases were addit ive. N20C/A27C/G137A glucoamylase had higher specific activity than wi ld-type glucoamylase from 45 to 67.5 degrees C, The disulfide bond bet ween positions 20 and 27 connects the C-terminus of helix 1 and the fo llowing beta-turn, suggesting that this region is important for glucoa mylase thermostability.