Engineering of Bacillus amyloliquefaciens -Amylase with Improved Calcium Independence and Catalytic Efficiency by Error-Prone PCR

Bacillus amyloliquefaciens -amylase (BAA) is one of the most important amylases and presents a wide range of applications in many processes involving starch-liquefaction. However, its activity and thermostability largely depend on the existence of calcium ions, which is a major obstacle for its wide-scale industrial applications. In the present study, to enhance the calcium independence of a mesophilic -amylase from B. amyloliquefaciens CICIM B2125, a novel calcium-independent mutant Q264S is evolved by directed evolution using error-prone polymerase chain reaction (PCR). Enzymatic properties and kinetic parameters of mutant Q264S were subsequently analyzed. Although the thermostability at 60 degrees C and optimum temperature of Q264S are slightly decreased, its calcium independence and catalytic efficiency are significantly improved, with no apparent changes of specific activity and optimum pH compared with those of the wild-type enzyme. Furthermore, three-dimensional structure analysis showed that the improved calcium independence and catalytic efficiency of Q264S are most likely to be a subtle balance among the hydrophobic interactions, electrostatic forces, and hydrogen bonds around calcium-binding sites and active-site regions. To the best of our knowledge, this is the first report on increasing calcium independence of BAA by error-prone PCR. These results could aid the understanding of mechanistic bases of calcium independence of BAA and thus facilitate the design and engineering of more efficient and robust calcium-independent BAA variants.