Hyperthermostable mutants of Bacillus licheniformis α-amylase:

thermodynamic studies and structural interpretation N.Declerck1,5, M.Machius2,4, R.Chambert3, G.Wiegand2, We have previously described hyperthermostable mutants of the highly thermostable α-amylase from Bacillus licheniformisR.Huber2 and C.Gaillardin1 (BLA); an enzyme of considerable industrial interest used for1Laboratoire de Ge´ne´tique Mole´culaire et Cellulaire, INRA-CNRS U537, starch hydrolysis. Two substitutions in the amino acid sequence,78850 Thiverval-Grignon, France, 2Max-Planck-Institut fu¨r Biochemie, D-85152 Planegg-Martinsried, Germany and 3Laboratoire de Ge´ne´tique et His133Tyr and Ala209Val (named therein H133Y and A209V, Membranes, Institut Jacques Monod, Universite ´ Paris-VII, 75005 Paris, respectively) have been identified that can together increase France the half-life of the enzyme at 90°C up to 10-fold (Declerck4Present address: University of Texas, Southernwestern Medical Center, et al., 1990; Joyet et al., 1992). In a previous paper, weHoward Hughes Medical Institute, Dallas TX 75235, USA described the introduction of all possible amino acids at5To whom correspondence should be addressed position 133 and 209 through site-directed mutagenesis, and This paper provides further understanding of the thermo- compared their resistance to thermal inactivation at high dynamic and structural features determining the stability temperatures. It was found that, at both positions, the stabilizing of Bacillus licheniformis α-amylase (BLA) at two crucial or destabilizing effect of the substitutions depends on the positions, His133 and Ala209. Results of protein modelling nature of the inserted amino acids, the most stabilizing having and saturated site-directed mutagenesis at position 133 and hydrophobic side-chains. Combining these results with protei