Part:BBa_K5089009

ABO2449(H243R) is an α/β hydrolase mutant with a H243R mutation to improve enzyme stability at acidic pH. Our team designed these mutants for application in acidic compost environments but can be used by other iGEM teams that require an esterase.

We designed these mutants inspired by Chen et al. (2019) who explored amino acid substation to arginine residues within the active site of Bacillus acidopullulyticus pullulanase to shift its pH optimum to acidic pH (from pH 5.0 – 4.0). By analyzing the hydrogen bonding network involving the catalytic residues within the active site, they identified a set of residues that formed hydrogen bonds with residues that directly interact with the catalytic residue (1).

The rationale for this approach is that arginine’s side chain can stabilize the deprotonated catalytic serine which in turn lowers the overall pKa of the enzyme towards acidic pH (1). Using this approach, we identified potential amino acid targets, including the S146R mutation in ABO2449.

References: 1. Chen, A., Xu, T., Ge, Y., Wang, L., Tang, W., & Li, S. 2019. Hydrogen-bond-based protein engineering for the acidic adaptation of Bacillus acidopullulyticus pullulanase. Enzyme and Microbial Technology, 124, 79–83. https://doi.org/10.1016/j.enzmictec.2019.01.010