Review of using diacetic functionalized polymers in solubilizing enzymes in organic solvents

Enzymes have evolved to function in aqueous systems. However, a dichotomy exists in industrial biocatalysis where most of the substrates and products are more soluble in various organic solvents. Thus, it would be desirable to develop enzymes capable of dissolving and catalysing reactions in organic solvents. One approach to this end involves the directed evolution of enzymes to function in non-aqueous environments. But, this approach requires the generation of large mutant libraries difficult for small laboratories to effectively screen. Other approaches involve the modification of enzymes with hydrophobic groups such as hydrophobic polymers to aid in their solubilization in organic solvents. Working in this direction, Hijazi and coworkers reports in Biotechnology and Bioengineering the potential of using noncovalent interactions between poly(2-oxazoline) (POx) terminated with 2,2’-imino diacetic acid (IDA) to aid in the solubilization and conferment of higher enzymatic activity to a variety of enzymes in industrially relevant organic solvents: chloroform and toluene. Specifically, dynamic light scattering measurements revealed that IDA groups were important for aiding formation of soluble noncovalent polymer-enzyme conjugates (PEC) of molecular size, ~ 10 nm. In comparison, POx terminated with -OH group resulted in the formation of non-soluble aggregates of size ~ 140 nm. The approach was shown to be effective in solubilizing lysozyme, horseradish peroxidase (HRP), laccase, α-chymotrypsin, catalase and alcohol dehydrogenase to a concentration of 2 mg/mL, yielding clear solutions in chloroform and toluene. Furthermore, the stoichiometry of polymer binding to enzyme for solubilizing it was obtained and showed a general trend of higher polymer binding for higher molecular weight enzymes. Efforts in understanding whether polymer modification of enzyme or solubilization of PEC in organic solvents led to reduced enzymatic activity in water revealed that noncovalent conjugation of polymer to enzyme was generally well-tolerated but the same is not true for solubilization of PEC in organic solvent. Except for HRP, both laccase and α-chymotrypsin exhibited reduced enzymatic activity in water after extraction of PEC from organic solvent. Nevertheless, PEC of HRP, laccase and α-chymotrypsin exhibited enhanced activity in organic solvents compared to the native enzyme in the same solvents. Hence, the work demonstrated that noncovalent modification of enzymes by IDA terminated polymer is a feasible approach for aiding the solubilization of enzymes in organic solvents. Furthermore, PEC formed exhibited enhanced enzymatic activity compared to native enzymes in organic solvent; thereby, opening up the approach's potential use in non-aqueous enzymology. However, the reported work could be further augmented by more detailed exploration of toluene as an organic solvent for PEC as well as testing the feasibility of the approach is solubilizing more classes of enzymes in different organic solvents.