Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase.

Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)). These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain. This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions. In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity. Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases. Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues.