Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity, enantioselectivity, and conformation

Abstract

Lipase from Burkholderia cepacia (lipase BC) and lipase B from Candida antarctica (CALB) show an increase of the transesterification activity in toluene (up to 2.4- and 1.7-fold, respectively), when lyophilized with 18-crown-6. Nevertheless, the increase was observed only for low (less than 100) 18-crown-6/lipase molar ratio, while at higher ratios, the activity decreased for both enzymes to values lower than those obtained in the absence of the additive. In 1,4-dioxane, the activation is lower for lipase BC (1.7-fold) and for CALB (1.5-fold). Concerning enantioselectivity, tested in the kinetic resolution of 6-methyl-5-hepten-2-ol, only in the case of CALB, an effect of the additive (the E value varied from about 120 to 280) was observed. In water, 4% (w/w) of 18-crown-6 caused a loss of activity in the hydrolysis of p-nitrophenyl laurate of about 88 and 99.75%, compared to that observed in the absence of the crown ether for CALB and lipase BC, respectively. These data and the conformational analysis of both lipases, carried out by FT/IR spectroscopy indicate that the enzyme inactivation in water and in organic solvents at 18-crown-6/lipase molar ratios, higher than 100 might be due to conformational changes caused by the additive. Instead, at molar ratios lower than 100, 18-crown-6 might increase the activity - particularly, in toluene - thanks to the fact that in its presence, the enzyme has an hydrogen bonds pattern, more similar to that in water. This suggests that the additive would be able to provide the enzyme with more water.

Development of effective nanobiocatalytic systems through the immobilization of hydrolases on functionalized carbon-based nanomaterials

“…The analysis of the Amide I band at approximately 1700-1600 cm À1 (mainly due to the C=O stretching vibration) makes it possible to obtain information on the effect of the immobilization on the secondary structure of the protein (Secundo et al, 2007). The Amide I band consists of several overlapping components that are assigned to different secondary structure elements.…”

Immobilization of Candida antarctica Lipase B on fumed silica

“…Much effort has been focused on strategies to overcome this issue, including enzyme immobilization on porous and non-porous solid supports 12,13 , chemical modification of the enzymes' surfaces to improve compatibility with solvents 14 , protein engineering 2 , and enzyme co-lyophilization with different types of excipients, such as cyclodextrins 10,15 , crown ethers [15][16][17] , and inorganic salts [18][19][20] . Specifically, the co-lyophilization of enzymes with inorganic salts from aqueous solution, termed salt activation, has been remarkably successful.…”

Electrostatic‐induced interfacial assembly of enzymes with nanosheets: Controlled orientation and optimized activity

“…Table 2 provides a comparison of the secondary structure features of pristine and bound PPL by analyzing their ATR- À1 are respectively assigned to the components of b-sheets, random coil, a-helix, and b-turns according to previous report. 40 It can be seen from Table 2 that the contents of these basic components in PPLLDHNSs-0.5 and PPL-LDHNSs-13 are almost consistent with pristine PPL, indicative of the complete preservation of PPL active conformation in the electrostatic-induced interfacial assembly with LDHNSs. It is confirmed that, in the interfacial adsorption, what has changed is only the PPL orientation rather than protein conformation.…”