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

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.