Characteristics of Crosslinking Polymers Play Major Roles in Improving the Stability and Catalytic Properties of Immobilized Thermomyces lanuginosus Lipase

Abstract

This study aimed to improve the stability and catalytic properties of Thermomyces lanuginosus lipase (TLL) adsorbed on a hydrophobic support. At the optimized conditions (pH 5 and 25 °C without any additions), the Sips isotherm model effectively fitted the equilibrium adsorption data, indicating a monolayer and the homogenous distribution of immobilized lipase molecules. To preserve the high specific activity of adsorbed lipase, the immobilized lipase (IL) with a moderate loading amount (approximately 40% surface coverage) was selected. Polyethylenimine (PEI) and chitosan (CS) were successfully applied as bridging units to in situ crosslink the immobilized lipase molecules in IL. At the low polymer concentration (0.5%, w/w) and with 1 h incubation, insignificant changes in average pore size were detected. Short-chain PEI and CS (MW ≤ 2 kDa) efficiently improved the lipase stability, i.e., the lipase loss decreased from 40% to <2%. Notably, CS performed much better than PEI in maintaining lipase activity. IL crosslinked with CS-2 kDa showed a two- to three-fold higher rate when hydrolyzing p-nitrophenyl butyrate and a two-fold increase in the catalytic efficiency in the esterification of hexanoic acid with butanol. These in situ crosslinking strategies offer good potential for modulating the catalytic properties of TLL for a specific reaction.

Microbial Lipases and Their Potential in the Production of Pharmaceutical Building Blocks

“…( II ) Representation of lipases immobilized in a hydrophobic surface crosslinked with an ionic polymer. In the presence of surfactant binding points are also lost, however here it is shown that in contrast to I there is no enzymatic desorption [ 161 , 162 ].…”

“…However, the possible gain in stability obtained in the derivative through these modifications is not only inevitably accompanied by an increase in the complexity of the immobilization protocol and the reuse of the support, it also involves changes in the activity or selectivity of the derivative, so the modification process should be optimized under those variables in a particular reaction [ 161 ]. This was evidenced in a recent study, in which the authors improved the stability and catalytic properties of Thermomyces lanuginosus lipase (TLL) adsorbed on a hydrophobic support by using additives that function as cross-linking units, allowing the loss of lipase to be reduced from 40% to <2% of the adsorbed enzyme, with only 0.5% w / w of the corresponding additive, further achieving in the case of CS the increase in catalytic efficiency by two-fold [ 162 ]. On the other hand, as an example of the use of these derivatives in API production, the PEI modification of an octyl-agarose derivative of the lipase Eversa ® in the hydrolysis of methyl rac-2-hydroxy-2-phenylacetic acid (also a precursor of cephalosporin, antineoplastics, etc.…”