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.
It is well known that the central nervous system (CNS) has a limited regenerative capacity and that many therapeutic molecules cannot cross the blood brain barrier (BBB). The use of biomaterials has ...emerged as an alternative to overcome these limitations. For many years, biomedical applications of chitosan have been studied due to its remarkable biological properties, biocompatibility, and high versatility. Moreover, the interest in this biomaterial for CNS biomedical implementation has increased because of its ability to cross the BBB, mucoadhesiveness, and hydrogel formation capacity. Several chitosan-based biomaterials have been applied with promising results as drug, cell and gene delivery vehicles. Moreover, their capacity to form porous scaffolds and to bear cells and biomolecules has offered a way to achieve neural regeneration. Therefore, this review aims to bring together recent works that highlight the potential of chitosan and its derivatives as adequate biomaterials for applications directed toward the CNS. First, an overview of chitosan and its derivatives is provided with an emphasis on the properties that favor different applications. Second, a compilation of works that employ chitosan-based biomaterials for drug delivery, gene therapy, tissue engineering, and regenerative medicine in the CNS is presented. Finally, the most interesting trends and future perspectives of chitosan and its derivatives applications in the CNS are shown.
Carrier-free immobilization of Candida rugosa lipase (CRL) and polymers containing primary amino groups were cross-linked using carbodiimide. To accomplish this, the free carboxyl groups of the ...enzyme were activated with carbodiimide-succinimide in organic medium, and then the activated proteins were cross-linked with different polyethylenimines (PEIs). The effect of the cross-linker chain length, the amount of added bovine serum albumin (BSA), and carbodiimide concentration on the catalytic properties of resulting cross-linked enzyme aggregates (CLEAs) was investigated. The CLEAs’ size, shape, specific activity, activity recovery, thermostability and enantioselectivity significantly varied according to the preparation procedure. The highest thermostable CRL-CLEA preparation was obtained with 1.3 kDa polyethyleneimine as cross-linker, 10 mg of BSA and 28 mM of carbodiimide. This preparation is 1.3-fold more active and thermostable than CLEAs prepared by the traditional method of amino cross-linking with glutaraldehyde, and retains 60% of residual activity after 22 h at 50 °C. Additionally, the CRL-CLEA preparation showed an enantioselectivity of 91% enantiomeric excess (ee). This immobilization procedure provides an alternative strategy for CLEA production, particularly for enzymes where the traditional method of cross-linking via lysine residues leads to enzyme inactivation.
The presenilin genes (PSEN1 and PSEN2) are mainly responsible for causing early-onset familial Alzheimer's disease, harboring ~300 causative mutations, and representing ~90% of all mutations ...associated with a very aggressive disease form. Presenilin 1 is the catalytic core of the γ-secretase complex that conducts the intramembranous proteolytic excision of multiple transmembrane proteins like the amyloid precursor protein, Notch-1, N- and E-cadherin, LRP, Syndecan, Delta, Jagged, CD44, ErbB4, and Nectin1a. Presenilin 1 plays an essential role in neural progenitor maintenance, neurogenesis, neurite outgrowth, synaptic function, neuronal function, myelination, and plasticity. Therefore, an imbalance caused by mutations in presenilin 1/γ-secretase might cause aberrant signaling, synaptic dysfunction, memory impairment, and increased Aβ42/Aβ40 ratio, contributing to neurodegeneration during the initial stages of Alzheimer's disease pathogenesis. This review focuses on the neuronal differentiation dysregulation mediated by PSEN1 mutations in Alzheimer's disease. Furthermore, we emphasize the importance of Alzheimer's disease-induced pluripotent stem cells models in analyzing PSEN1 mutations implication over the early stages of the Alzheimer's disease pathogenesis throughout neuronal differentiation impairment.
Xanthophyllomyces dendrorhous is one of the most attractive natural sources of astaxanthin. The yeast produces the compound intracellularly, therefore, it is necessary to evaluate cell rupture ...methods using clean and efficient technologies for its extraction and it subsequent use in industries. At present work, two non‐conventional high‐pressure extraction methods were evaluated for astaxanthin recovery: Supercritical fluid extraction and microfluidisation. Results: Effect of pressure (15, 30, and 45 Megapascals; MPa), Temperature (313 and 343 °K) and usage of co‐solvent were studied in supercritical extraction process, meanwhile the effect of microfluidisation process (five stages and 160 MPa) for cell rupture and its combination with conventional technics (lithic enzymes, glass beads and ultrasonication) for the recovery of astaxanthin were evaluated. Supercritical fluid extraction presented a higher astaxanthin recovery, 54% yield extraction, at 32.5 MPa and 313 °K, and using ethanol as co‐solvent, compared to a 31% yield extraction by mean microfluidisation process at 160 MPa combined with 15 min of sonication (amplitude of 80%).
Non‐conventional high‐pressure extraction methods for astaxanthin recovery from Xanthophyllomyces dendrorhous.
The effects of different concentrations of Hofmeister salts on the hydrolytic activity on triglycerides and the secondary structure of lipase B from Candida antarctica (CALB) were investigated. ...Structural changes after short- and long-time incubation at high salt concentrations were determined using circular dichroism (CD), fluorescence, and RMSD-RMSF simulations. At 5.2 M NaCl, the hydrolytic activity of CALB on tributyrin (TC4) and trioctanoin (TC8) was enhanced by 1.5 (from 817 ± 3.9 to 1228 ± 4.3 U/mg)- and 8.7 (from 25 ± 0.3 to 218 ± 2.3 U/mg)-folds compared with 0.15 M NaCl, respectively at pH 7.0 and 40 °C. An activity activation was seen with other salts tested; however, long-time incubation (24 h) did not result in retention of the activation effect for any of the salts tested. Secondary structure CD and fluorescence spectra showed that long-time incubation with NaCl, KCl, and CsCl provokes a compact structure without loss of native conformation, whereas chaotropic LiCl and CaCl2 induced an increase in the α-helical content, and kosmotropic Na2SO4 provoked a molten globule state with rich β-sheet content. The RMSD-RMSF simulation agreed with the CD analysis, highlighting a principal salt-induced effect at the α-helix 5 region, promoting two different conformational states (open and closed) depending on the type and concentration of salt. Lastly, an increase in the interfacial tension occurred when high salt concentrations were added to the reaction media, affecting the catalytic properties. The results indicate that high-salt environments, such as 2–5.2 M NaCl, can be used to increase the lipolytic activity of CALB on TC4 and TC8.
Display omitted
•The high concentration of NaCl increased the hydrolytic activity of CALB on triglycerides of short and medium chain length.•CALB can be catalogued as a halotolerant lipase maintaining its activity and native structure at high salt concentrations.•CALB is a robust lipase able to restore its activity after 24h incubation at high salt concentrations.•The major effect of NaCl concentration was at the α-helix 5 of CALB promoting open and closed states (RMSD-RMSF simulation).
Capsaicin analogues are typically synthetized by condensation of the amide group of vanillylamine with a fatty acid derivative. The enzyme of choice to perform this reaction is
Candida antarctica
...lipase B; however, this enzyme is unable to react with the phenolic substiruents of the vanillyl ring. So far, this can only be achieved chemically, resulting in capsaicin esters with higher lipophilicity and less irritation than the parent compound. In need of searching for new capsaicin derivatives, we investigated the transesterification of the phenolic OH group of capsaicin and several capsaicin analogues by
Candida antarctica
lipase A (CALA) with vinyl esters. Capsaicin esters were successfully synthetized with a reaction yield of 80.6% and 57.5% with vinyl butyrate and vinyl laurate, respectively. When the reactions were performed with capsaicin analogues of different acyl chain lengths, CALA exhibited a noticeable transesterification preference for medium-length capsaicin analogues. In an attempt to explore the catalytic limits of CALA, we performed the transesterification of capsaicin from
Capsicum
oleoresin. The results showed similar transesterification yields to those obtained with semi-pure capsaicin. Within our knowledge, this is the first report to achieve the synthesis of phenolic OH esters of capsaicin and capsaicin analogues by enzymatic means.
Display omitted
•Bulk compound chocolate products were developed as potential functional food.•Saturated fatty acids were less bioavailable using structured lipids.•The in vitro lipid digestion was ...reduced as well as its caloric impact.•Serum lipids were reduced in healthy C57BL/6J mice.
The bioavailability impact of serum lipids in compound chocolate products based on structured lipids was studied. Compound chocolate products containing fat with and without structured lipids were digested in vitro under simulated gastrointestinal lipolysis conditions and were studied in vivo in healthy C57BL/6J mice. The in vitro digestion results show that products containing structured lipids, milk compound chocolate filling and white compound coating, significantly reduced the release rate of Free Fatty Acids (FFA) and improved the caloric reduction between 12.49% and 13.71% compared to products without structured lipids, suggesting that FFA were not absorbed. Animal feeding studies revealed no adverse effects on the compound products intake; in fact, these products reduced total cholesterol, LDL-c, VLDL-c and triacylglycerols. The present work shows the relevance of developing functional compound chocolate as providing a potential healthy initiative through the biological effect of the bioactive ingredients incorporated.
Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, ...polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon
Natronococcus
sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6 %). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea:
Natronococcus
sp. TC6,
Halobacterium
sp. NRC-1 and
Haloarcula marismortui
were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.
Biologically active peptides produced from fish wastes are gaining attention because their health benefits. Proteases produced by halophilic microorganisms are considered as a source of active ...enzymes in high salt systems like fish residues. Hence, the aim of this study was the bioprospection of halophilic microorganisms for the production of proteases to prove their application for peptide production.
Halophilic microorganisms were isolated from saline soils of Mexico and Bolivia. An enzymatic screening was carried out for the detection of lipases, esterases, pHB depolymerases, chitinases, and proteases. Most of the strains were able to produce lipases, esterases, and proteases, and larger hydrolysis halos were detected for protease activity. Halobacillus andaensis was selected to be studied for proteolytic activity production; the microorganism was able to grow on gelatin, yeast extract, skim milk, casein, peptone, fish muscle (Cyprinus carpio), and soy flour as protein sources, and among these sources, fish muscle protein was the best inducer of proteolytic activity, achieving a protease production of 571 U/mL. The extracellular protease was active at 50°C, pH 8, and 1.4 M NaCl and was inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of H. andaensis was used to hydrolyze fish muscle protein for peptide production. The peptides obtained showed a MW of 5.3 kDa and a radical scavenging ability of 10 to 30% on 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and a ferric reducing ability of plasma.
The use of noncommercial extracellular protease produced by H. andaensis for biologically active peptide production using fish muscle as the protein source presents a great opportunity for high-value peptide production.
How to cite: Delgado-García M, Flores-Gallegos AC, Kirchmayr M, et al. Bioprospection of proteases from Halobacillus andaensis for bioactive peptide production from fish muscle protein. Electron J Biotechnol 2019;39. https://doi.org/10.1016/j.ejbt.2019.03.001.
PDF
