Exploring the full natural diversity of single amino acid exchange reveals that 40–60% of BSLA positions improve organic solvents resistance

Abstract

Objectives: Protein engineering has been employed to successfully improve organic solvent resistance of enzymes. Exploration of nature’s full potential (how many beneficial positions/beneficial substitutions of the target enzyme) to improve organic solvent resistance of enzymes by a systematic study was performed. Results: We report the results of screening the previously generated BSLA (Bacillus subtilis lipase A)-SSM (site saturation mutagenesis) library (covering the full natural diversity of BSLA with one amino acid exchange) in presence of three cosolvents. The potential of single amino acid substitution that nature offers to improve the cosolvent resistance of BSLA was determined by analyzing the number of beneficial positions/substitutions, accessibility and chemical compositions. Conclusion: Lessons learned from analysis of BSLA-SSM library are: (1) 41–59% of BSLA positions with in total 4–10% of all possible substitutions improve the cosolvent resistance against TFE, DOx, and DMSO; (2) charged substitutions are preferred to improve DOx and TFE resistance whereas polar ones are preferred for DMSO; (3) charged substitutions on the surface predominantly improved resistance while polar ones were preferred in buried “regions”. (4) Interestingly, 58–93% of beneficial substitutions led to chemically different amino acids.[Figure not available: see fulltext.].