Strategies Used for Enhanced Enzyme Production

Desirable Attributes of CeUulase for Hydrolysis of Cellulose Strategies Used for Enhanced Enzyme Production... 

The stability and activity of lipases can be improved by the pretreatment of an enzyme prior to its application. The pretreatment strategy involves exposure of an enzyme to substrate and its analogs, organic solvents, and salts. These pretreatments enhance catalytic performance by keeping the active sites in open conformation. Methanol inactivation and high price are major drawbacks for lipases in their successful use for biodiesel production. Pretreatments improve the catalytic performance, methanol tolerance, as well as stability of lipases (Guldhe et al., 2015). 

In this chapter we have seen that enzymatic catalysis is initiated by the reversible interactions of a substrate molecule with the active site of the enzyme to form a non-covalent binary complex. The chemical transformation of the substrate to the product molecule occurs within the context of the enzyme active site subsequent to initial complex formation. We saw that the enormous rate enhancements for enzyme-catalyzed reactions are the result of specific mechanisms that enzymes use to achieve large reductions in the energy of activation associated with attainment of the reaction transition state structure. Stabilization of the reaction transition state in the context of the enzymatic reaction is the key contributor to both enzymatic rate enhancement and substrate specificity. We described several chemical strategies by which enzymes achieve this transition state stabilization. We also saw in this chapter that enzyme reactions are most commonly studied by following the kinetics of these reactions under steady state conditions. We defined three kinetic constants—kai KM, and kcJKM—that can be used to define the efficiency of enzymatic catalysis, and each reports on different portions of the enzymatic reaction pathway. Perturbations... 

The biosynthesis of geosmin is of interest because an enhanced understanding of the pathways and enzymes involved may support the development of effective off-flavor control strategies. Increased biochemical information could also enhance the use of biochemical systems to produce large amounts of geosmin for the flavor and fragrance industries, which may be interested in this compound to provide a desirable earthy note to certain products (88). Currently, difficulties associated with the synthesis of the three chiral carbons renders the chemical synthesis of geosmin extremely difficult (88). 

Another example of a possible strategy for genetic enhancement of cell function in a broad sense is the research presently in progress by Valkanos, Stephanopolous, and Sinskey at MIT to reduce lactic acid production in mammalian cells by eliminating specifically the enzyme needed to produce lactic acid. This strategy may lead to cells that use carbon more efficiently and produce fewer inhibitory products in the medium. Such a cell line would be of interest in many different contexts. 

An additional strategy that enhances metabolic adaptability is evolution of alternative enzymes that synthesize the same product using different substrates (see Table 1). Alternative enzymes allow bacteria to take advantage of reactants that may be in sufficient supply only occasionally in a fluctuating environment. For example, E. coli contains two enzymes that catalyze oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX in the heme biosynthesis pathway. FlemF uses O2 HemN is a radical 5-adenosylmethionine enzyme that catalyzes a complex 02-independent reaction. HemN contains an 02-sensitive Fe-S cluster. HemN is active only under anoxic conditions, and HemF is active only when O2 is available, but E. coli is able to synthesize heme in either case. 

---