Improvement of the acid stability of Bacillus licheniformis alpha amylase by site-directed mutagenesis

The objective of this work was to improve the acid stability of alpha amylase from Bacillus licheniformis (BLA) under acidic conditions by site-directed mutagenesis. Based on the analysis of three dimensional structure of BLA, five histidine residues at positions 281, 289, 293, 316, and 327 in BLA were substituted by arginine residues and aspartic acid residues, respectively. Ten mutants H281R/D, H289R/D, H293R/D, H316R/D, and H327R/D were obtained and H293R, H316R, and H327R were active at pH 4.5 and 6.5. Triple mutations of BLA was modified for the construction of H293R/H316R/H327R. Compared with wild type, which lost the activity, H293R, H316R, H327R, and H293R/H316R/H327R could maintain 8, 10, 20, 31% of the initial activity when incubated at pH 4.5 and 70 °C for 40 min, respectively. The results combined with three-dimensional structure analysis demonstrated that H293R, H316R, H327R, and H293R/H316R/H327R showed an improved acid stability under low pH condition as a result of the interactions of electrostatic fields, hydrogen bonding, and hydrophilcity. This work provides the theoretical basis and background data on the improvement of acid stability in BLA for satisfying the industrial requirements by protein engineering, which is beneficial to molecular modification of other industrial enzymes for acid-tolerance ability.