Site-saturation mutagenesis at amino acid 329 of Klebsiella pneumoniae halophilic α-amylase affects enzymatic properties.

Halophilic α-amylases possess optimal activity in high salt concentrations. Therefore, they can be used in many extreme conditions in industrialised production. In the present work, a halophilic α-amylase (KP) from Klebsiella pneumoniae was characterised, and it exhibited a high specific activity of 3512 U/mg under optimal conditions of 2 M NaCl at 50°C and pH 6.5, but only 97 U/mg in the absence of salt. Furthermore, threonine at position 329 (Thr-329) was found to be related to the non-halophilic properties of KP according to PCR-based site-saturation mutagenesis. The activity of a mutant KP in which this threonine was replaced by aspartic acid was improved 14.6-fold compared with the native enzyme under salt-free conditions, and was increased by 14.8% in the absence of salt. Additionally, the optimal enzymatic properties of KP, including pH and temperature, were altered very little by the amino acid replacement. A further three halophilic α-amylases displayed similar mutational results. The findings provide a reference for bidirectional transformation of KP and similar halophilic enzymes.