In the past decades, the production of biopharmaceuticals has gained high interest due to its great sensitivity, specificity, and lower risk of negative effects to patients. Biopharmaceuticals are ...mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-asparaginase (L-asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from
Escherichia coli
and
Erwinia chrysanthemi
. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production.
Key points
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Microbial enzyme applications as biopharmaceutical and in food industry
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Biosynthesis process: from the microorganism to bioreactor technology
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Enzyme activity and kinetic properties: crucial for the final application
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The biocatalytic performance of immobilized enzyme systems depends mostly on the intrinsic properties of both biomolecule and support, immobilization technique and immobilization ...conditions. Multi-walled carbon nanotubes (MWCNTs) possess unique features for enzyme immobilization by adsorption. Enhanced catalytic activity and stability can be achieved by optimization of the immobilization conditions and by investigating the effect of operational parameters.
Laccase was immobilized over MWCNTs by adsorption. The hybrid material was characterized by Fourier transformed infrared (FTIR) spectroscopy, scanning and transmission electron microscopy (SEM and TEM, respectively). The effect of different operational conditions (contact time, enzyme concentration and pH) on laccase immobilization was investigated. Optimized conditions were used for thermal stability, kinetic, and storage and operational stability studies.
The optimal immobilization conditions for a laccase concentration of 3.75μL/mL were a pH of 9.0 and a contact time of 30min (522 Ulac/gcarrier). A decrease in the thermal stability of laccase was observed after immobilization. Changes in ΔS and ΔH of deactivation were found for the immobilized enzyme. The Michaelis–Menten kinetic constant was higher for laccase/MWCNT system than for free laccase. Immobilized laccase maintained (or even increased) its catalytic performance up to nine cycles of utilization and revealed long-term storage stability.
Laccase Activation in Deep Eutectic Solvents Toledo, Mariah L; Pereira, Matheus M; Freire, Mara G ...
ACS sustainable chemistry & engineering,
07/2019, Volume:
7, Issue:
13
Journal Article
Peer reviewed
Open access
The research on alternative solvents and cosolvents is relevant when envisioning the improvement of biocatalytic reactions. Among these solvents and cosolvents, deep eutectic solvents (DES) may be ...considered as customizable new reaction media for biocatalysis. Accordingly, in this work, 16 DES aqueous solutions, as well as the individual DES components at the same conditions, have been investigated in laccase-catalyzed reactions. Cholinium- and betaine-based DES formed with polyols at different molar ratios and concentrations were evaluated. The results reported show that in the presence of most DES the laccase activity is preserved and, with a particular DES, enhanced up to 200%. Molecular docking studies demonstrated that while most DES components establish hydrogen bonds with the enzyme amino acids, those that establish stronger interactions with the enzyme (expressed by absolute values of docking affinity energies) lead to an enhanced laccase activity. Finally, the laccase stability was evaluated in additional tests under extreme storage temperatures (60 °C and −80 °C). Although no significant protection to high temperatures was afforded by DES, an enhanced laccase activity when stored at low temperatures was found, at least up to 20 days. Combining experimental results and molecular docking, this work shows that DES can be designed as cosolvents to improve biocatalytic reactions.
The increased resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious health concerns. This has stimulated interest in the development of bio-based ...therapeutics with limited resistance, namely, essential oils (EOs) or antimicrobial peptides (AMPs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control compounds for comparison purposes. The biomolecules were initially screened for their antibacterial efficacy using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000-1250 µg/mL) to induce the same effects as the AMPs (500-7.8 µg/mL) or EOs (365.2-19.7 µg/mL). Pexiganan and CLO were the most effective biomolecules, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5-7.8 µg/mL and 39.3-19.7 µg/mL, respectively), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were observed to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these new biomolecules as replacements for the conventional antibiotics and allowed us to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.
•Eriochrome black T as monomer to produce imprinted materials.•On-site electropolymerization of Eriochrome black T.•Reducing the electroactivity of chloramphenicol in acetonitrile/water ...medium.•Poly(eriochrome black T) formed for chloramphenicol detection.•Application to chloramphenicol determination in water from fish activities.
A novel device for monitoring chloramphenicol (CAP) on-site is described, making use of commercial carbon screen-printed electrodes (C-SPEs) modified with a suitable sensing element. This element consisted of a molecularly imprinted polymer (MIP), produced in-situ, by electro-polymerization. The monomers used herein were Eriochrome black T (EBT), and polymerization was conducted in acetonitrile. Raman spectroscopy followed the chemical changes occurring at each stage of the carbon surface modification.
The device performance was assessed by evaluating the changes in electron transfer properties of a standard redox probe Fe(CN)63−/Fe(CN)64− by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV). SWV and EIS electrochemical techniques were used to calibrate the system, having standard solutions prepared under different background media (electrolyte or water coming from a home fish tank). A wide linear range was observed, with linear responses of current/resistance against log (CAP concentration) down to 10nM.
Overall, the results obtained revealed that all modifications carried out on the sensing element were effective. The final sensor provided reproducible and accurate readings and was all assembled in-situ, in a very simple and straightforward approach, most likely suitable for scaling-up, directing towards its subsequent commercial use.
Novel liquid supports for enzyme immobilization and reuse based on aqueous biphasic systems (ABS) constituted by cholinium‐based ionic liquids (ILs) and polymers for the degradation of dyes are here ...proposed. The biocatalytic reaction for dye decolorization using laccase occured in the biphasic medium, with the enzyme being “supported” in the IL‐rich phase and the dye and degradation products being enriched in the polymer‐rich phase. An initial screening of the laccase activity in aqueous solutions of ABS constituents, namely cholinium dihydrogen citrate (ChDHC), cholinium dihydrogen phosphate (ChDHP), cholinium acetate (ChAcet), polypropylene glycol 400 (PPG 400), polyethylene glycol 400 (PEG 400) and K2HPO4 was carried out. Compared to the buffered control, a relative laccase activity of up to 170%, 257%, and 530% was observed with PEG 400, ChDHP, and ChDHC, respectively. These ABS constituents were then investigated for the in situ enzymatic biodegradation of the Remazol Brilliant Blue R (RBBR) dye. At the optimized conditions, the ABS constituted by PPG 400 at 46 wt% and ChDHC at 16 wt% leads to the complete degradation of the RBBR dye, further maintaining the enzyme activity. This ABS also allows an easy immobilization, recovery, and reuse of the biocatalyst for six consecutive reaction cycles, achieving a degradation yield of the dye of 96% in the last cycle. In summary, if properly designed, high enzymatic activities and reaction yields are obtained with ABS as liquid supports, while simultaneously overcoming the safety and environmental concerns of conventional organic solvents used in liquid–liquid heterogeneous reactions, thus representing more sustainable biocatalytic processes.
Magnetically actuated microscopic artificial cilia were implemented in millimeter‐scale culture wells for Scenedesmus subspicatus, and they were able to increase the growth rate of the microalgae by nearly ten times. Hydrophobicity of the cilia surface was also found to significantly influence the enhancement of growth. The technique of creating and controlling the artficial cilia can be easily mastered and adopted in labs without microfabrication infrastructure.
The appearance and quick spread of the new severe acute respiratory syndrome coronavirus disease, COVID-19, brought major societal challenges. Importantly, suitable medical diagnosis procedures and ...smooth clinical management of the disease are an emergent need, which must be anchored on novel diagnostic methods and devices. Novel molecular diagnostic tools relying on nucleic acid amplification testing have emerged globally and are the current gold standard in COVID-19 diagnosis. However, the need for widespread testing methodologies for fast, effective testing in multiple epidemiological scenarios remains a crucial step in the fight against the COVID-19 pandemic. Biosensors have previously shown the potential for cost-effective and accessible diagnostics, finding applications in settings where conventional, laboratorial techniques may not be readily employed. Paper- and cellulose-based biosensors can be particularly relevant in pandemic times, for the renewability, possibility of mass production with sustainable methodologies, and safe environmental disposal. In this review, paper-based devices and platforms targeting SARS-CoV-2 are showcased and discussed, as a means to achieve quick and low-cost PoC diagnosis, including detection methodologies for viral genomic material, viral antigen detection, and serological antibody testing. Devices targeting inflammatory markers relevant for COVID-19 are also discussed, as fast, reliable bedside diagnostic tools for patient treatment and follow-up.
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