초록
<P><B>Abstract</B></P> <P>The aim of this study was to rationally engineer the acidic adaptation of <I>B. subtilis</I> Ca-independent alpha-amylase (Amy7C) by decreasing the pKa values of catalytic residues through mutations at active site. Within 4.5 Å of three catalytic residues of Amy7C, three mutations R172 K, A270 K and N271H were identified by computational homology modeling and pKa prediction analyses. Five single and double mutants consisting of these three mutations were constructed and characterized. Compared to the wild-type, all mutants shifted the pH optima and pH-activity profiles toward lower pH values without comprising the thermostablity. Double mutants showed simultaneous accumulation of advantageous mutations. The best mutant, A270 K/N271H showed 2 units decrease in optimum pH and about 3.94-fold increase of catalytic efficiency. Structural analysis suggested that the improved acidic adaptation could be attributed to the decreased pKa values of catalytic nucleophile and proton donor residues. Protein engineering of α-amylase for acidic adaptation here provides a successful example of the extent to which mutations near active site and computational models can be used for industrial enzyme improvements.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Three mutations decreasing pKa values of catalytic residues of Amy7C were designed. </LI> <LI> Three single and two double mutants were constructed and characterized. </LI> <LI> All mutants shifted pH optima and pH-activity profiles toward more acidic values. </LI> <LI> N271H decreased pH optimum by 2 units without comprising the catalytic efficiency. </LI> <LI> A270 K/N271H increased catalytic efficiency by 3.94-fold at optimum pH of 4.5. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>