Protein solubility enhancement by directed evolution: Characterization of wild-type and mutant S-hydroxynitrile lyase from Manihot esculenta expressed in Escherichia coli and comparative expression in several hosts

Hydroxynitrile lyases are mainly plant enzymes that catalyze the addition of cyanide to aldehyde or ketones resulting in chiral cyanohydrins which are valuable intermediates in industry. MeHNL was expressed in E. coli as a low in vivo soluble enzyme therefore; protein engineering strategies were employed to improve its solubility in this versatile host. Mutagenesis reactions resulted in mutations in some hot spots on the surface and inside the protein and changed the solubility of this originally low-soluble enzyme drastically in E. coli so that the expression profiles of the mutants in soluble and insoluble fractions were completely different. Saturation mutagenesis of the buried residue His provided more soluble mutants with hydrophobic substitutions, while causing only minor structural changes, proved by biophysical evaluations. The specific activity of highly soluble mutants was elevated several times in the cell-free extract, while the catalytic efficiency of the mutants was affected only slightly in their purified form. The stability of the mutants differed from that of the wild-type at high temperatures and at pH>8. The findings challenge the rationale of producing recombinant proteins in E. coli at 37°C.
As a part of attempts to clarify the mechanism of this phenomenon, we have studied the possibility of expression of a highly active and soluble mutant as well as wild-type enzyme in several expression systems ranging Pichia pastoris, Leishmania tarentolae and two cell-free translations, including an E. coli lysate and wheat germ translation system. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes in eukaryotic hosts, while those of E. coli exhibited totally different levels. The different activity levels in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system.