• acid stability;
  • Bacillus licheniformis alpha amylase;
  • characterization;
  • error-prone PCR;
  • protein structure



The purpose of this research was to obtain the mutant of Bacillus licheniformis alpha amylase (BLA) with an improved acid stability and elucidate the difference in catalytic mechanism under acidic conditions between wild-type and mutant BLAs.

Methods and Results

The stability of BLA under acid condition was enhanced through direct evolution using error-prone polymerase chain reaction. Two mutation sites, T353I and H400R, were obtained in BLA. To identify the mutation of amino acids in Thr353Ile/His400Arg related to its acid stability, single mutants Thr353Ile and His400Arg were obtained via site-directed mutagenesis. Among the resulting mutant enzymes, the kcat/Km values of the mutants Thr353Ile, His400Arg and Thr353Ile/His400Arg under pH 4·5 were 3·5-, 6·0- and 11·3-fold higher, respectively, than that of the wild-type. Thr353Ile/His400Arg exhibited stronger tolerance towards a lower pH without obvious difference in thermostability when compared with wild-type.


The results combined with three-dimensional structure analysis of mutant BLAs demonstrated that Thr353Ile/His400Arg showed an improved acid stability under low pH condition as a result of the interactions of hydrogen bonding, hydrophobicity, helix propensity and electrostatic fields.

Significance and Impact of Study

It provides theoretical basis and background data for the improvement of acid stability in BLA by protein engineering.