In Vitro outside Living Cells

Lipases are enzymes that catalyze the in vivo hydrolysis of lipids such as triacylglycerols. Lipases are not used in biological systems for ester synthesis, presumably because the large amounts of water present preclude ester formation due to the law of mass action which favors hydrolysis. A different pathway (using the coenzyme A thioester of a carboxylic acid and the enzyme synthase [Blei and Odian, 2000]) is present in biological systems for ester formation. However, lipases do catalyze the in vitro esterification reaction and have been used to synthesize polyesters. The reaction between alcohols and carboxylic acids occurs in organic solvents where the absence of water favors esterification. However, water is a by-product and must be removed efficiently to maximize conversions and molecular weights. 

The reaction mechanism for the polymerization of a hydroxyalkanoic acid (Eqs. 2-243 through 2-246) is a chain polymerization, often called an activated monomer polymerization. The active site of lipase is its serine a-amino acid unit, which contains a hydroxyl group. The acyl carbon of the hydroxyalkanoic acid undergoes nucleophilic attack by the hydroxyl group of serine to form lipase-activated monomer (Eq. 2-243). Initiation consists of reaction 

Among other in vitro enzymatic polymerizations that have been studied are the oxidative polymerizations of 2,6-disubstituted phenols to poly(p-phenylene oxide)s (Sec. 2-14b) catalyzed by horseradish peroxidase [Higashimura et al., 2000b] and the polymerization of P-cellobiosyl fluoride to cellulose catalyzed by cellulase [Kobayashi, 1999 Kobayashi et al., 2001], 

In vitro enzymatic polymerizations have the potential for processes that are more regio-selective and stereoselective, proceed under more moderate conditions, and are more benign toward the environment than the traditional chemical processes. However, little of this potential has been realized. A major problem is that the reaction rates are slow compared to non-enzymatic processes. Enzymatic polymerizations are limited to moderate temperatures (often no higher than 50-75°C) because enzymes are denaturated and deactivated at higher temperatures. Also, the effective concentrations of enzymes in many systems are low because the enzymes are not soluble. Research efforts to address these factors include enzyme immobilization to increase enzyme stability and activity, solubilization of enzymes by association with a surfactant or covalent bonding with an appropriate compound, and genetic engineering of enzymes to tailor their catalytic activity to specific applications. 

A supercritical fluid (SCF) is a substance above its critical temperature and critical pressure. The critical temperature is the highest temperature at which a substance can exist as a gas. The critical pressure is the pressure needed at the critical temperature to liquify a gas. Above the critical temperature and critical pressure, a substance has a density characteristic of a liquid but the flow properties of a gas, and this combination offers advantages as a reaction solvent. The liquidlike density allows the supercritical fluid to dissolve substances, while the gaslike flow properties offer the potential for fast reaction rates. Supercritical carbon dioxide (SCCO2) has a critical temperature of 31°C and critical pressure of 73 atm. 

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