Long chain capsaicin analogues synthetized by CALB-CLEAs show cytotoxicity on glioblastoma cell lines.

Glioblastoma is one of the most lethal tumors, displaying striking cellular heterogeneity and drug resistance. The prognosis of patients suffering from glioblastoma after 5 years is only 5%. In the present work, capsaicin analogues bearing modifications on the acyl chain with long-chain fatty acids showed promising anti-tumoral activity by its cytotoxicity on U-87 and U-138 glioblastoma multiforme cells. The capsaicin analogues were enzymatically synthetized with cross-linked enzyme aggregates of lipase B from Candida antarctica (CALB). The catalytic performance of recombinant CALB-CLEAs was compared to their immobilized form on a hydrophobic support. After 72 h of reaction, the synthesis of capsaicin analogues from linoleic acid, docosahexaenoic acid, and punicic acid achieved a maximum conversion of 69.7, 8.3 and 30.3% with CALB-CLEAs, respectively. Similar values were obtained with commercial CALB, with conversion yields of 58.3, 24.2 and 22% for capsaicin analogues from linoleic acid, DHA and punicic acid, respectively. Olvanil and dohevanil had a significant cytotoxic effect on both U-87 and U-138 glioblastoma cells. Irrespective of the immobilization form, CALB is an efficient biocatalyst for the synthesis of anti-tumoral capsaicin derivatives. KEY POINTS: • This is the first report concerning the enzymatic synthesis of capsaicin analogues from docosahexaenoic acid and punicic acid with CALB-CLEAs. • The viability U-87 and U-138 glioblastoma cells was significantly affected after incubation with olvanil and dohevanil. • Capsaicin analogues from fatty acids obtained by CALB-CLEAs are promising candidates for therapeutic use as cytotoxic agents in glioblastoma cancer cells.

Design for preparation of more active cross-linked enzyme aggregates of Burkholderia cepacia lipase using palm fiber residue.

A new design of cross-linked enzyme aggregates (CLEAs) of Burkholderia cepacia lipase (BCL) based mainly on the use of lignocellulosic residue of palm fiber as an additive was proposed. Different parameters for the preparation of active CLEAs in the hydrolysis of olive oil, such as precipitation agents, crosslinking agent concentration, additives, and coating agents were investigated. The highest activity yield (121.1 ± 0.1%) and volumetric activity (1578.1 ± 2.5 U/mL) were achieved for CLEAs prepared using the combination of a coating step with Triton® X-100 and polyethyleneimine plus the use of palm fiber as an additive. The variations of the secondary structures of BCL-CLEAs were analyzed by second-derivative infrared spectra, mainly indicating a reduction of the α-helix structure, which was responsible for the lipase activation in the supramolecular structure of the CLEAs. Thus, these results provided evidence of an innovative design of BCL-CLEAs as a sustainable and biocompatible opportunity for biotechnology applications.

Carrier-bound and carrier-free immobilization of type A feruloyl esterase from Aspergillus niger: Searching for an operationally stable heterogeneous biocatalyst for the synthesis of butyl hydroxycinnamates.

Feruloyl esterases synthesize butyl hydroxycinnamates, molecules possessing interesting biological properties, nonetheless, they exhibit a low stability under synthesis conditions in organic solvents, restricting its use. To enhance its operational stability in synthesis, we immobilized type A feruloyl esterase from Aspergillus niger (AnFAEA) using several carrier-bound and carrier-free strategies. The most active biocatalysts were: 1) AnFAEA immobilized on epoxy-activated carriers (protein load of 0.6 mgenzyme x mg-1carrier) that recovered 91 % of the initial hydrolytic activity, and 2) AnFAEA aggregated and cross-linked in the presence of 5 mg of BSA and 15 mM of glutaraldehyde (AnFAEA-amino-CLEAs), which exhibited 385 % of its initial hydrolytic activity; both using 4-nitrophenyl butyrate as substrate. The AnFAEA-amino-CLEAs were 12.7 times more thermostable at 60 °C than the AnFAEA immobilized on epoxy-activated carrier, thus AnFAEA-amino-CLEAs were selected for further characterization. Interestingly, during methyl sinapate hydrolysis (pH 7.2 and 30 °C), AnFAEA-amino-CLEAs KM was 15 % higher, while during butyl sinapate synthesis the KM was reduced in 63 %, both compared with the soluble enzyme. The direct esterification of butyl sinapate at solvent free conditions using sinapic acid 50 mM, reached 95 % conversion after 24 h employing AnFAEA-amino-CLEAs, which could be used for 10 cycles without significant activity losses, demonstrating their outstanding operational stability.

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.