Short‐chain alcohols inactivate an immobilized industrial lipase through two different mechanisms

Abstract

Broadly used in biocatalysis as acyl acceptors or (co)‐solvents, short‐chain alcohols often cause irreversible loss of enzyme activity. Understanding the mechanisms of inactivation is a necessary step toward the optimization of biocatalytic reactions and the design of enzyme‐based sustainable processes. The functional and structural responses of an immobilized enzyme, Novozym 435 (N‐435), exposed to methanol, ethanol, and tert‐butanol, are explored in this work. N‐435 consists of Candida antarctica lipase B (CALB) adsorbed on polymethacrylate beads and finds application in a variety of processes involving the presence of short‐chain alcohols. The nature of the N‐435 material required the development of an ad hoc method of structural analysis, based on Fourier transform infrared microspectroscopy, which was complemented by catalytic activity assays and by morphological observation by transmission electron microscopy. The inactivation of N‐435 was found to be highly dependent on alcohol concentration and occurs through two different mechanisms. Short‐chain alcohols induce conformational changes leading to CALB aggregation, which is only partially prevented by immobilization. Moreover, alcohol modifies the texture of the solid support promoting the enzyme release. Overall, knowledge of the molecular mechanisms underlying N‐435 inactivation induced by short‐chain alcohols promises to overcome the limitations that usually occur during industrial processes.

Enzymatic Transesterification of Atlantic Salmon (Salmo salar) Oil with Isoamyl Alcohol

“…In this case, the alcohol had a positive effect on the reaction kinetics, facilitating the formation of a homogeneous suspension of reactants and biocatalysts [ 42 ]. Similar results were obtained by other researchers [ 18 , 36 , 38 ] when, during two or three stepwise additions of alcohol to the transesterification medium, the yield of biodiesel was increased up to 10% when methanol and ethanol were used as acyl acceptors. When using higher alcohols (propanol, isobutanol, 1-butanol), Deng et al [ 40 ] increased the yield of biodiesel over four times when alcohol was gradually introduced into the reaction media.…”

“…With an increase in the molar ratio of alcohol to oil from 3:1 to 6:1, the transesterification degree increases. This ratio also depends on the type of raw material used [ 18 , 35 , 36 ]. However, with an increase in the molar ratio higher than 6:1, the transesterification degree slightly decreased.…”

Synthesis of Biorenewable Terpene Monomers Using Enzymatic Epoxidation under Heterogeneous Batch and Continuous Flow Conditions

“…A slight decrease in enzyme reactivity was observed (see Section S17) being ascribed to the presence of oxidative inhibition along with the formation of short-chain alcohols (EtOH) that proved to be harmful to the enzyme (see Scheme ). ,− To avoid the generation of EtOH, the possibility of directly using acetic acid and conducting the catalysis in 2-methyl-tetrahydrofuran (Me-THF) was evaluated. A long-run experiment for the epoxidation of (+)-3-carene by Novozym 435/H 2 O 2 was conducted (CH 3 COOH: 5.0 equiv, H 2 O 2 : 1.2 equiv; see Figures S4 and S5).…”

Current Trends in Acetins Production: Green versus Non-Green Synthesis

“…Another issue is the inhibition of lipase-catalyzed reactions by short-chain alcohols [ 119 ]. Since lipase has a considerably higher affinity toward short-chain alcohols than water, the alcohols molecules generated as a by-product in the acetins production could gradually replace water molecules on the lipase surface [ 119 , 120 ]. This could disrupt intra-protein hydrophobic interactions, resulting in the collapse of the enzyme structure following the irreversible deactivation of lipase.…”

“…Thus, an improvement of lipase against short-chain alcohols inhibition is required by mining alcohol stable lipases, mutagenesis of existed enzymes, or process engineering. In addition to the kinetic and molecular inhibition, short-chain alcohol could also deactivate lipase by releasing it from solid support [ 119 ].…”