Engineering an industrial enzyme synthesizing pharmaceutical intermediates: A trade-off between protein solubility and stability

Protein engineering is being increasingly used for improving protein stability, enhancing the activity or overall performance of enzymes and organisms, altering enzyme substrate specificity, stereoselectivity and designing new activities. Numerous advantages of Escherichia coli as a versatile host render it as the first choice for recombinant gene expression but low or poor solubility of the expressed protein would be a bottleneck in many cases. Highly in vivo soluble mutants of an HNL from Manihot esculenta, a plant enzyme catalyzing reversible degradation of cyanohydrins as valuable building blocks, were created in E. coli. We employed structure-guided saturation mutagenesis and random mutagenesis to generate triple Lys-Pro (up to 8-fold increase) and His103Met (18.5-fold increase) mutants of this low soluble wild-type enzyme, respectively. We prepared and purified the mutant enzymes as well as the wild-type and studied their characteristics using biochemical and biophysical approaches in vitro. The mutations caused minor structural changes as monitored by Circular Dichroism, FT-IR and Fluorescence spectra and slightly affected catalytic efficiency (kcat/KM) (0.8-1.6-fold). The highly soluble single mutants showed high refolding yield in vitro, whilst the wild-type enzyme exhibited no refolding, as a proof of the merit of correct folding of these mutants in the complex environment within E. coli. On the other hand, this efficient solubilizing mutation conversely resulted in an important decrease in the stability of the enzyme at high temperatures and at pH values greater than 8 which might be described as a trade-off between solubility and stability. Likely these mutations assist the enzyme to overcome mis-folding by blocking the wrong interactions between the secondary structures of a monomer during the folding and subsequently an efficient folding into its native conformation.