Hybrid Bacillus amyloliquefaciens X Bacillus licheniformisα‐Amylases

A series of 33 single and mosaic hybrid alpha-amylases was constructed from the genes amyBA or amyLI, encoding the alpha-amylases from Bacillus amyloliquefaciens (AmyBA) and Bacillus licheniformis (AmyLI). The hybrid proteins, consisting of the entire alpha-amylase sequence with a variable portion of AmyBA or AmyLI origin, were characterized in order to find enzymes with new properties (thermostability, temperature and pH optima, and substrate specificity), and to localize the amino acid sequence regions responsible for the changes. The thermostability of the AmyBA/AmyLI (AL-type) hybrid proteins correlated with the position and the length of the hybrid sequence. The hybrid enzymes fell into six groups retaining, in comparison to AmyBA, a certain value of the extra-thermostability of AmyLI or becoming more thermolabile than AmyBA. Four regions are proposed to contain thermostability determinants (TSDs). They map between amino acid residues 34-76, 112-142, 174-179 and 263-276 of the respective hybrid enzymes, indicating the dominance of the N-terminal half of AmyLI for these hybrid enzymes' resistance against irreversible inactivation. Two (TSD3 and TSD4) coincide with regions I and II that had already been suggested to stabilise AmyLI [Suzuki, Y., Ito, N., Yuuki, T., Yamagata, H. & Udake, S. (1989) J. Biol. Chem. 264, 18,933-18,938]. The temperature dependence of activity of the AL-type hybrid alpha-amylases was compared at pH 6.4 and pH 7.6 and the hybrid enzymes of one thermostability group were found to have similar temperature responses. A hybrid region between residues 34-76 is demonstrated to correlate with the alpha-amylases' substrate specificity, i.e. either hydrolysis or accumulation of maltohexaose. This region was therefore named the G6G5 region. The exchange of internal sequences between residues 17-201 of AmyBA by the AmyLI counterpart in ALA-type mosaic hybrid alpha-amylases, with one exception (ALA99-429), unexpectedly destabilized the respective ALA-type hybrids. Two of these hybrid alpha-amylases (ALA17-151 and ALA76-151) were less thermostable than AmyBA, while others (ALA112-151, ALA112-201) were enzymically inactive. These data support specific roles of the predicted A1-B domain portion between residues 17-201 of those Bacillus alpha-amylases probably for correct folding and enzymic activity.