Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo-and holo-TK activity and structure to a range of commonly used polar organic co-solvents: acetonitrile (MeCN), nbutanol (nBuOH), ethyl acetate (EToAc), isopropanol (iPrOH), and tetrahydrofuran (THF).The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not correlated to global conformational stability. The cosolvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts.Aggregation was not found to be the driving mechanism of enzyme inactivation, but was in some cases an additional impact of solvent-induced local or global unfolding.TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) MeCN, or 6% (v/v) nBuOH over 3 hours. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops.