Characterization and cold-adaptation mechanism of a cold-active α-amylase from Thermoascus crustaceus JCM12803

Abstract:

[Objective] To mine the genetic resource of novel cold-active α-amylases and reveal their cold-adaptation mechanism are of importance to deepen our understanding of cold-active enzyme and provide key information for the molecular improvement of α-amylase.[Methods] Based on the genome sequence of Thermoascus crustaceus JCM12803, we cloned an α-amylase-encoding gene (Tcamy), and inserted it into the expression vector pPIC9. The gene product was heterologously expressed in Pichia pastoris GS115 and characterized. By using amino acid sequence analysis and homologous modeling, we studied the cold-adaptation mechanism of TcAmy in viewpoint of sequence-structure-function relationship.[Results] TcAmy is a typical cold-active α-amylase, showing optimal activity at 35℃ and remaining 27% maximal activity even at 0℃. Sequence and structure analysis indicated that in comparison to thermostable counterparts, TcAmy has low N-glycosylation degree, decreased Pro and Arg contents and increased Gly content, and less disulfide bridges and ionic bonds.[Conclusion] We obtained a novel cold-active α-amylase, with low N-glycosylation degree, specific amino acid composition and intermolecular interactions, all contributing to its cold-adapted property.