Lipase conformational flexibility

The low-temperature method has been applied to some primary and secondary alcohols (Fig. 1) For example, solketal, 2,2-dimethyl-1,3-dioxolane-4-methanol (3) had been known to show low enantioselectivity in the lipase-catalyzed resolution (lipase AK, Pseudomonas fluorescens, E = 16 at 23°C, 27 at 0oc) 2ia however, the E value was successfully raised up to 55 by lowering the temperature to —40°C (Table 1). Further lowering the temperature rather decreased the E value and the rate was markedly retarded. Interestingly, the loss of the enantioselectivity below —40°C is not caused by the irreversible structural damage of lipase because the lipase once cooled below —40°C could be reused by allowing it to warm higher than -40°C, showing that the lipase does not lose conformational flexibility at such low temperatures. 

It is worth noting that the enzyme can be withdrawn and recycled by using supercritical CO2. The success of the polymerizations carried out in organic solvents stems directly from the sustained activity of several lipases in organic solvents. In this respect, it must be noted that water has a manifold influence on the course of the polymerization. On the one hand, water can initiate the polymerization. On the other hand, a minimum amount of water has to be bound to the surface of the enzyme to maintain its conformational flexibility, which is essential for its catalytic activity [94]. Lipase-mediated polymerization cannot therefore be achieved in strictly anhydrous conditions. 

Peracetylated [3-CD has a beneficial action on the lipase-catalyzed enantioselec-tive transesterification of l-(2-furyl)ethanol in organic solvents. The use of CD as a regulator of lipase was assumed to be the result of increasing the conformational flexibility of the enzyme and undergoing host-guest complexation with the product, with an enhancement of the enantiomeric ratio E and the reaction rate [83]. 

Cygler, M., and J. D. Schrag. 1999. Structure and Conformational Flexibility of Candida Rugosa Lipase. Biochimica et Biophysica Acta (BBA)—Molecular and Cell Biology of Lipids 1441 (2-3) 205-214. 

Lipases are another important group of hydrolases. The most commonly used example is porcine pancreatic lipase (PPL). Lipases tend to function best at or above the solubility limit of the hydrophobic substrate. In the presence of water, the substrate forms an insoluble phase (micelles) the concentration at which this occurs is called the critical micellar concentration. The enzyme is activated by a conformational change that occurs in the presence of the micelles and results in the opening of the active site. Lipases work best in solvents that can accommodate this activation process. PPL is often used as a relatively crude preparation called pancreatin or steapsin. The active site in PPL has not been as precisely described as the one for PLE. There are currently two different models, but they sometimes make contradictory predictions. It has been suggested that the dominant factors in binding are the hydrophobic and polar pockets (sites B and C in Figure 2.29), but that the relative location of the catalytic site is somewhat flexible and can accommodate to the location of the hydrolyzable substituent. ... 

The pH of the reaction medium is another important factor for modulating both the activity and enantioselectivity of Lipase OF. The enzyme showed optimal hydrolysis activity at pH 4.0, while the enantioselectivity increased sharply with the decrease in medium pH from 4.0 to 2.2. Based on spectroscopic studies, the enhancement of the lipase activity and enantioselectivity at the lower pH could be attributed to the changes in the flexible and sensitive conformation of the lipase induced by tuning the biocatalyst microenvironment. Using a hybrid strategy by modulating pH and surfactant, enantiomer-enriched (5)-ketoprofen could be obtained with 95.5% ee and 39.1% yield from rac-ketoprofen chloroethyl ester (100 mM) at pH 2.5 in the presence of 0.5% (w/v) Tween-80 as a modulator. ... 

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