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.

Development of a rapid assay for ß-etherase activity using a novel chromogenic substrate.

Biocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as ß-etherases, capable of breaking ß-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying ß-etherase activity was developed based on the ß-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate ß-(ρ-nitrophenoxy)-α-acetovanillone (PNPAV), was chemically synthesized. Kintetic monitoring of ρ-nitrophenolate release at 410 nm over 10 min, using recombinant LigF from Sphingobium sp SYK-6, LigF-AB and LigE-AB from Althererytrobacter sp B11, yielded enzimatic activities of 404. 3 mU/mg, 72 mU/mg, and 50 mU/mg, respectively. This method is applicable in a pH range of 7.0-9.0, with a sensitivity of up to 50 ng of enzyme, exhibiting no interference with lipolytic, glycolytic, proteolytic, and oxidoreductase enzymes.

Cloning, expression, and biochemical characterization of ß-etherase LigF from Altererythrobacter sp. B11.

Lignin, a complex heteropolymer present in plant cell walls, is now recognized as a valuable renewable resource with potential applications in various industries. The lignin biorefinery concept, which aims to convert lignin into value-added products, has gained significant attention in recent years. ß-etherases, enzymes that selectively cleave ß-O-4 aryl ether bonds in lignin, have shown promise in lignin depolymerization. In this study, the ß-etherase LigF from Altererythrobacter sp. B11 was cloned, expressed, purified, and biochemically characterized. The LigF-AB11 enzyme exhibited optimal activity at 32 °C and pH 8.5 when catalyzing the substrate PNP-AV. The enzyme displayed mesophilic behavior and demonstrated higher activity at moderate temperatures. Stability analysis revealed that LigF-AB11 was not thermostable, with a complete loss of activity at 60 °C within an hour. Moreover, LigF-AB11 exhibited excellent pH stability, retaining over 50 % of its activity after 1 h under pH conditions ranging from 3.0 to 11.0. Metal ions and surface impregnation agents were found to affect the enzyme's activity, highlighting the importance of considering these factors in enzymatic processes for lignin depolymerization. This study provides valuable insights into the biochemical properties of LigF-AB11 and contributes to the development of efficient enzymatic processes for lignin biorefineries. Further optimization and understanding of ß-etherases will facilitate their practical application in the valorization of lignin.

A sensitive pH indicator-based spectrophotometric assay for PHB depolymerase activity on microtiter plates.

A continuous spectrophotometric assay for the screening of PHB depolymerase activity in microtiter plates was developed. We evaluated crystalline PHB in the suspension and coated it with the addition of a pH indicator to detect the breakage of the ester bond by proton titration. The reaction rate and the concentration of the recombinant PhaZ1 from Paucimonas lemoignei PHB depolymerase presented a linear correlation. A comparison of the proposed method with the turbidimetric method adapted to the microtiter plates revealed that the use of indicators increases the response signal by at least 5-fold, resulting in increased sensitivity and better signal-to-noise ratio. Furthermore, the proposed method offers a wide range of pH from 5.0 to 9.2 by using different buffer-indicator pairs and was employed for the screening of PHB-depolymerase activity on 140 bacterial strains isolated from Lake Chapala. Eleven strains were positive for PHB-depolymerase activity, which were ACSLRF-27, ACPLRF-6, and ACPLRF-5 (16S rRNA sequence alignment revealed 99-100% similarity with Actinomadura geliboluensis strain A8036, Streptomyces cavourensis strain NRRL 2740, and Streptomyces coelicolor strain DSM 40233, respectively); these that showed the highest activities. In conclusion, the method was successfully applied for finding new strains and for quantifying the PHB depolymerases activity with crystalline PHB.

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.