Hybrid Bacillus amyloliquefaciens X Bacillus licheniformisα‐Amylases

Abstract

A series of 33 single and mosaic hybrid a-amylases was constructed from the genes amyBA or amyLI, encoding the a-amylases from Bacillus amyloliquefaciens (AmyBA) and Bacillus lichenifomis (AmyLI). The hybrid proteins, consisting of the entire a-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 ldcalize the amino acid sequence regions responsible for the changes.The thermostability of the AmyBAlAmyLI (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 I1 that had already been suggested to stabilise AmyLI [Suzuki, Y., Ito, N., Yuuki, T., Yamagata, H. & Udake, S. (1989) J. Biol. Chern. 264, 18 933 -18 9381. The temperature dependence of activity of the AL-type hybrid a-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 a-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 a-amylases, with one exception (ALA99-429), unexpectedly destabilized the respective ALA-type hybrids. Two of these hybrid a-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 a-amylases probably for correct folding and enzymic activity.

Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 α-amylase

“…Therefore, we selected those asparagines that are exposed to solvent and/or have relatively high B-factors (Table 2). It had been shown that the structural determinants contributing to the overall thermostability of ␣-amylases concentrate in domain B and at its interface with the central A domain [25][26][27][28]. Three out of four selected Asn residues are located in domain B (Asn129, Asn112, and Asn119).…”

Kinetic Stabilization of Bacillus licheniformis α-Amylase through Introduction of Hydrophobic Residues at the Surface

“…Nevertheless, a wealth of information has been acquired in various laboratories providing insights into the origin of BLA stability (11-16). Several hundred BLA variants have been constructed and characterized and have led to the identification of protein regions and residues that are important for thermostability (13,(17)(18)(19)(20). BLA is also one of the few proteins for which detailed biochemical studies on the mechanism of irreversible thermal inactivation have been carried out.…”

“…The derivation of comprehensive and accurate thermodynamic parameters for the folding/unfolding process of BLA is therefore not straightforward. Nevertheless, a wealth of information has been acquired in various laboratories providing insights into the origin of BLA stability (11)(12)(13)(14)(15)(16). Several hundred BLA variants have been constructed and characterized and have led to the identification of protein regions and residues that are important for thermostability (13,(17)(18)(19)(20).…”

An analysis of temperature adaptation in cold active, mesophilic and thermophilic Bacillus α-amylases

“…Conrad et al [39] identified another thermostabilitydetermining segment comprising residues 34-76. Part of this region contributes to the interface between domains A and B.…”