Chemical modification of biocatalyst for function in supercritical CO2: In silico redesign of stable lipase

Abstract

Graphical Abstract HighlightsPTMs are introduced as a protein stabilization strategy in supercritical condition Lysine acetylation prevents enzyme denaturation at high temperatures in water Acetylation is not so useful for stabilization of enzyme in supercritical CO 2 Methylation and carboxylation efficiently stabilize enzyme in supercritical CO 2 Modification of arginine residues is not so satisfactory in enzyme stabilization Abstract:In spite of excellent properties of supercritical CO 2 in enzyme catalyzed reactions, destabilizing effects of CO 2 molecules on the enzyme structure limits the industrial applications of this green solvent in the field of biocatalysis. Here, based on the substantial role of charged surface residues such as lysines in enzyme inactivation, we introduced for the first time, Post Translational Modifications (PTMs), a famous concept in molecular biology, as a protein stabilization strategy in supercritical condition. Lysine groups were modified using PTM templates to find out more about the exact mechanism of enzyme inactivation in supercritical CO 2 and to explore a new way for protein stabilization in this solvent. We used MD simulation as common tool for in situ examining enzyme structure in supercritical fluids, for the investigation of structural consequence of modifications. Different modifications including acetylation, methylation, phosphorylation, and carboxylation have been applied on the model enzyme. For comparison to CALB structure in supercritical CO 2 , the acetylated enzyme was also simulated in aqueous solvent at 300 and 353 K. Interestingly, acetylation of lysine residues prevents enzyme from denaturation at high temperatures in water, which is in agreement with experimental observation in aqueous solution. However, acetylation is not so useful for stabilization of enzyme in supercritical CO 2 . In contrast, methylation and carboxylation efficiently stabilize enzyme in supercritical CO 2 . Phosphate groups in phosphorylated lysines destabilize enzyme by formation of excess hydrogen bonds by inappropriate groups and perturb enzyme conformation. Moreover, it was found that modification of surface arginine residues was not so satisfactory in stabilization of the enzyme. This finding supports the mechanism of lipase inactivation through direct interaction of CO 2 molecules on lysine residues and formation of carbamates. We think this new exploration not only can open new window for developing enzyme catalyzed reaction in supercritical fluids, but it can shed some light on the mechanism of enzyme inactivation in scCO 2 .

Ionic Liquids as Tool to Improve Enzymatic Organic Synthesis

“…Although they failed to improve stability of the enzyme in scCO 2 , thermal stability of the enzyme in water was successfully improved by this modification. 367 Hydrophobic ionic liquids generally act as good reaction media for lipase-catalyzed reaction, which, on the contrary, hydrophilic ILs give poor or no reaction. As described in this chapter, introduction of alkyl ether moiety in the cationic part of ILs with combined hydrophobic anion such as Tf 2 N anion seems to be a sure way to design ionic liquids suited for lipase-catalyzed reaction.…”

“…On the other hand, this was prevented by the IL, hence the desired reaction proceeded smoothly. It has now been firmly established that addition of IL as a cosolvent is essential to achieve the lipase-catalyzed transesterification in the scCO 2 solvent system. − …”

“…Monhemi and Reza Housaindokht recently reported an interesting attempt to prevent denaturation of enzyme in scCO 2 : the authors made an attempt to modify the lipase protein which would be tolerant to a scCO 2 solvent system by the acetylation of lysin residue side chains. Although they failed to improve stability of the enzyme in scCO 2 , thermal stability of the enzyme in water was successfully improved by this modification . Hydrophobic ionic liquids generally act as good reaction media for lipase-catalyzed reaction, which, on the contrary, hydrophilic ILs give poor or no reaction.…”

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

“…177 The succinylated cellulase showed a 1.6-fold increase in Avicel cellulose conversion in 15% (v/v) aqueous solution of [BMIM][Cl] after 170 h of saccharification and reduced lignin inhibition with 1 wt % lignin, whereas the acetylated cellulase showed a small decrease in cellulose conversion compared to the unmodified enzyme. In addition, Monhemi and Housaindokht 178 used molecular dynamics simulation for the investigation of the structural consequence of modifications on enzyme structure in supercritical fluids. Different modifications including acetylation, methylation, phosphorylation, and carboxylation were applied on the model enzyme, CALB.…”