Cross‐linked enzyme aggregates of recombinant Candida antarctica lipase B for the efficient synthesis of olvanil, a nonpungent capsaicin analogue

Abstract

Despite the proven therapeutic role of capsaicin in human health, its usage is still hampered by its high pungency. In this sense, nonpungent capsaicin analogues as olvanil are a feasible alternative to the unpleasant sensations produced by capsaicin while maintaining a similar pharmacological profile. Olvanil can be obtained by a lipase‐catalyzed chemoenzymatic process. In the present work, recombinant Candida antarctica lipase B (CALB) was expressed in Pichia pastoris and subsequently immobilized by cross‐linked enzyme aggregate (CLEA) methodology for the synthesis of olvanil. The CALB‐CLEAs were obtained directly from the fermentation broth of P. pastoris without any purification step in order to assess the role of the contaminant proteins of the crude extract as co‐feeders. The CALB‐CLEAs were also bioimprinted to enhance the catalytic performance in olvanil synthesis. When CALB was precipitated with isopropanol, the obtained CALB‐CLEAs exhibited the highest activity in the synthesis of olvanil, regardless of the glutaraldehyde concentration. The maximum product synthesis was found at 72 hr obtaining 6.8 g L−1 of olvanil with a reaction yield of 16%. When CALB was bioimprinted with olvanil, the synthesis was enhanced 1.3 times, reaching 10.7 g L−1 of olvanil at 72 hr of reaction with a reaction yield of 25%. Scanning electron microscopy images indicated different morphologies of the CLEAs depending on the precipitating agent and the template used for bioimprinting. Recombinant CALB‐CLEAs obtained directly from the fermentation broth are a suitable alternative to commercial enzymatic preparations for the synthesis of olvanil in organic medium.

Advances in Recombinant Lipases: Production, Engineering, Immobilization and Application in the Pharmaceutical Industry

“…The immobilized preparation was used in the kinetic resolution of racemic 1-phenyethanol and five racemic amines using shaken flasks and continuous-flow packed-bed microreactors with good yields and high enantiomeric excess (ee > 99%, for all) [144]. Furthermore, the recombinant CALB expressed in K. phaffii was immobilized by cross-linked enzyme aggregate (CLEA) technology and tested for the synthesis of olvanil [145].…”

Microbial lipases and their industrial applications: a comprehensive review

“…So, the CLEAs appeared as a substitute to the procedures defined in the literature already. The precipitating agents like salts, acids, organic solvents addition by the precipitated enzymes using the protein precipitation procedure [ 179 , 186 ], and in the mixture holding the enzyme without distressing its active three-dimensional structure. As a result, the lacking need for it to have the topmost clarity and obtaining the desired enzyme, and reducing the cost of immobilization and time [ 187 ].…”

Multicatalytic Hybrid Materials for Biocatalytic and Chemoenzymatic Cascades—Strategies for Multicatalyst (Enzyme) Co-Immobilization

“…Another interesting methodology to enhance CLEAs activity is called bioimprinting, in which a substrate, substrate-analog, cofactor, or cofactor-analog molecule is added to the initial enzyme solution inducing the bound-enzyme conformation. The presence of ligands stabilizes active enzyme conformation over the crosslinking formation and can be removed by washing [139,153]. Diaz-Vidal et al used olvanil as a bioimprint molecule on the production of Candida antarctica lipase B (CAL-B) CLEAs.…”