Lipomyces kononenkoae LKA1 alpha-amylase Gln 443 is critical for enzyme activity.
Ricardo Cordero Otero (1), Sakkie Pretorius (2), Nivetha Ramachandran (1)
(1) Inst. for Wine Biotechnol., University of Stellenbosch, Victoria St., Stellenbosch, 7600, South Africa; (2) IWBT; The Australian Wine Research Institute, Adelaide, Australia
The alpha-amylase from Lipomyces kononenkoae LKA1 is active on alpha-1,4 and alpha-1,6 linkages in starch and related substrates. In addition to raw starch degradation, the enzyme exhibits considerable activity on pullulan. The catalytic domain of alpha-amylases consists of four conserved segments that constitute the active domain and are well conserved in LKA1. However, the invariant His 296 of the fourth conserved segment (TAKA amylase numbering) which has been highly conserved and well demonstrated in transition-state stabilisation in other alpha-amylases, is replaced by Gln 443 in LKA1. Site-specific mutagenesis of Gln 443 has confirmed the essential role of this amino acid in the substrate specificity of LKA1. The effect of mutations on the pH and temperature profile of the enzyme, and specificity towards substrates, will be discussed.