Strategies for discovery and improvement of enzyme function: state of the art and opportunities.
Bottom Line: Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non-conventional media, using new substrates for old enzymes, active-site imprinting, altering temperature, etc.Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem.The present review attempts to highlight some of these achievements and potential opportunities.
Affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi - 110 016, India.Show MeSH
Mentions: Use of enzymes in organic solvents has now become a major thrust area on biocatalysis since it allows solubilization of insoluble substrates and modification of enzyme properties (Klibanov, 2001). However, the choice of an organic solvent requires considerations of enzyme behaviour, partitioning of substrate and product from the enzyme, reaction equilibrium, hydrolytic side reactions, etc. (Martineka et al., 1981; Halling, 1994; Heinemann et al., 2003). At present there exist no precise rules for making choice of an appropriate solvent; however, certain studies have correlated enzyme behaviour to various solvent properties (Kaul and Banerjee, 2008), particularly logP (Laane et al., 1987). The scope and breadth of enzyme‐catalysed reactions in organic media has been extensively reviewed (Zaks and Russel, 1988; Gupta, 1992; Carrea and Riva, 2008; Doukyua and Ogino, 2010) and the techniques have been applied particularly to lipases. Interestingly, lipases catalyse esterification in organic medium (Fig. 7A) and hydrolysis in aqueous medium (Fig. 7B), both reactions yielding opposite enantiomers of the product under different conditions (Breitgoff et al., 1986; Terao et al., 1988; Wang and Wong, 1988). Lipase‐catalysed transesterification reactions have largely been executed in hydrophobic solvents; however, a study describing asymmetric transesterification of glycerol with acyl donors (such as vinyl benzoate) utilized hydrophilic solvents (Kato et al., 1999b; 2000c) (Fig. 8). About 40 commercially available lipase sources were screened for the asymmetric transesterification reaction and finally CHIRAZYME L‐2 (Candida antarctica) provided access to (R)‐α‐monobenzoyl glycerol in 1,4‐dioxane. The study therefore highlighted a novel yet simple single step route to optically active chiral building block from a prochiral substrate. Since one of the major impediments to use non‐aqueous enzymology is maintaining high catalytic activity of enzymes, one may use approaches such as enzyme modification, inclusion of additives and molecular imprinting to enhance enzyme properties. Dissolving the enzyme in a solution of ligand (usually a substrate or its analogue) and subsequent lyophilization results in an enzyme preparation with embossed active site capable of retaining memory upon placement in anhydrous solvent (due to more rigid active‐site conformation). The memory is however lost if the enzyme is placed in aqueous medium since the proteins become more flexible (due to high dielectric constant of water) and tend to retain their original active‐site conformation. This provides a means to fine tune not only enzyme activity and specificity (Rich and Dordick, 1997) but also its enantioselectivity (Okahata et al., 1995). For successful implementation of this approach, the ligand must be completely soluble in aqueous medium (from which the enzyme is lyophilized). However, most of the compounds of commercial interest are practically insoluble in water which significantly limits the applicability of molecular imprinting. Overcoming this limitation requires one to modify the ligand or the aqueous medium in order to enhance its solubility to achieve improved enzyme performance in organic solvents (Rich et al., 2002). Ionic liquids have been used as a replacement of organic solvents and offer the advantage of allowing solubilization of both hydrophobic and hydrophilic molecules (Kragl et al., 2002; Park and Kazlauskas, 2003; van Rantwijk et al., 2003; Song, 2004). These liquids are generally known to be mild and significantly enzyme‐friendly and have allowed enzymes to exhibit improved yield and selectivity, e.g. lipase‐B from C. antarctica (CAL‐B) when used in ionic liquids resulted in significantly higher yield and regioselectivity for acylation of glucose (99% yield and 93% selectivity) compared with the reaction performed in organic solvents (73% yield and 76% selectivity) (Park and Kazlauskas, 2001).
Affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi - 110 016, India.