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: Sulfation chemistry and sulfated scaffolds generated using these techniques find numerous applications in various industrial sectors ranging from analytical (Stalcup and Gahm, 1996) to pharmaceutical (Dorfman et al., 2006) and consumer products (Salka and Bator, 1990). However, majority of these scaffolds are prepared using chemical methods that suffer from numerous disadvantages. Although sulfation appears to be a single‐step reaction, its execution by chemical methods presents significant challenges (Al‐Horania and Desai, 2010). Lability of sulfate groups to harsh conditions (acidic pH and high temperature) coupled by lack of manoeuvrability following introduction of sulfate group complicates the process conditions. Introduction of a sulfate group allows only a few functional group transformations to be successfully executed forcing the design of the synthetic scheme to include sulfation as the final step. The above complications generally increase geometrically for poly‐sulfated scaffolds. Additionally, the chemical sulfation process is highly energy intensive which requires excessive cooling (exothermic reaction). A still larger segment of the consumer product industry uses sulfated detergents in most finished products such as shampoos, soaps, cleaners, etc. Most of these products (using sulfated esters and detergents) are contaminated by 1,4‐dioxane, a potential carcinogen that is introduced by a process of ethoxylation (Black et al., 2001). Federal policies governing consumer safety allow carcinogenic contaminants to be present in small amounts and do not compel the manufacturers to remove them completely, which has led to many companies being sued for negligence in the past (NewsInferno, 2001; PRLog Press Release, 2008). These problems can be circumvented by introducing an enzymatic step of sulfation, which would allow precise control over reaction selectivity and achieve sulfation of complex chemical scaffolds in a single step, without the need for employing protection and deprotection strategies, under mild reaction conditions. Sulfotransferases catalyse transfer of a sulfate group from a donor molecule to an acceptor (usually and alcohol or amine) (Negishi et al., 2001). Bacterial sources of the enzyme are known to utilize p‐nitropheynl sulfate (PNPS) as a donor which has relatively a low cost and favours process economics for industrial application and also allows execution of high‐throughput screening approach, since enzymatic sulfation can be easily monitored by measuring absorbance at 400 nm for formation of p‐nitrophenol (PNP) (Fig. 9). Screening of human faeces samples leads to identification of Eubacterium A‐44 as a producer of arylsulfotransferase (Kobashi et al., 1986) which has been used for sulfation to produce molecules of therapeutic interest, such as cholecystokinin (Hagiwara et al., 1990a), angiotensin‐II (Hagiwara et al., 1990b), tannins (Koizumi et al., 1991), among many others. Application of sulfotransferases to a broader range of molecules has been restricted mainly due to narrow substrate spectrum of these enzymes. This provides a favourable starting point for using discovery and engineering tools to this enzyme for executing a broad range of sulfation reactions of different templates with applications in chemical and pharmaceutical industry.
Affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi - 110 016, India.