Towards cost-effective drug discovery: Reusable immobilized enzymes for neurological disease research.

Enzyme handling and utilization bears many challenges such as their limited stability, intolerance of organic solvents, high cost, or inability to reuse. Most of these limitations can be overcome by enzyme immobilization on the surface of solid support. In this work, the recombinant form of human cholinesterases and monoamine oxidases as important drug targets for neurological diseases were immobilized on the surface of magnetic non-porous microparticles by a non-covalent bond utilizing the interaction between a His-tag terminus on the recombinant enzymes and cobalt (Co2+) ions immobilized on the magnetic microparticles. This type of binding led to targeted enzyme orientation, which completely preserved the catalytic activity and allowed high reproducibility of immobilization. In comparison with free enzymes, the immobilized enzymes showed exceptional stability in time and the possibility of repeated use. Relevant Km, Vmax, and IC50 values using known inhibitors were obtained using particular immobilized enzymes. Such immobilized enzymes on magnetic particles could serve as an excellent tool for a sustainable approach in the early stage of drug discovery.

Towards Greener and More Cost-efficient Biosynthesis of Pharmaceuticals and Fragrance Molecules.

Enzymes are natural catalysts which are gaining momentum in chemical synthesis due to their exquisiteselectivity and their biodegradability. However, the cost-efficiency and the sustainability of the overall biocatalytic process must be enhanced to unlock completely the potential of enzymes for industrial applications. To reach this goal, enzyme immobilization and the integration into continuous flow reactors have been the cornerstone of our research. We showed key examples of the advantages of those tools for the biosynthesis of antivirals, anticancer drugs, and valuable fragrance molecules. By combining new strategies to immobilize biocatalysts, innovative bioengineering approaches, and process development, the performance of the reactions could be boosted up to 100-fold.

Assessment of the efficiency and stability of enzymatic membrane reaction utilizing lipase covalently immobilized on a functionalized hybrid membrane.

Enzyme immobilization in membrane bioreactors has been considered as a practical approach to enhance the stability, reusability, and efficiency of enzymes. In this particular study, a new type of hybrid membrane reactor was created through the phase inversion method, utilizing hybrid of graphene oxide nanosheets (GON) and polyether sulfone (PES) in order to covalently immobilize the Candida rugosa lipase (CRL). The surface of hybrid membrane was initially modified by (3-Aminopropyl) triethoxysilane (APTES), before the use of glutaraldehyde (GLU), as a linker, through the imine bonds. The resulted enzymatic hybrid membrane reactors (EHMRs) were then thoroughly analyzed by using field-emission scanning electron microscopy (FE-SEM), contact angle goniometry, surface free energy analysis, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, attenuated total reflection (ATR), and energy-dispersive X-ray (EDX) spectroscopy. The study also looked into the impact of factors such as initial CRL concentration, storage conditions, and immobilization time on the EHMR's performance and activity, which were subsequently optimized. The results demonstrated that the CRLs covalently immobilized on the EHMRs displayed enhanced pH and thermal stability compared to those physically immobilized or free. These covalently immobilized CRLs could maintain over 60% of their activity even after 6 reaction cycles spanning 50 days. EHMRs are valuable biocatalysts in developing various industrial, environmental, and analytical processes.

Synthesis of hydrophilic glyceryl monocaffeate with economical catalyst cation-exchange resin Amberlyst-35.

BACKGROUND: Caffeic acid (CA) has anti-oxidation and anti-inflammatory. However, the poor hydrophilicity of CA limits its biological activities. In this work, hydrophilic glyceryl monocaffeate (GMC) was synthesized by esterification using different caffeoyl donors (deep eutectic solvent and solid CA). Cation-exchange resins were used as the catalysts. The effects of reaction conditions were also investigated. RESULTS: The mass transfer limitation of esterification was eliminated using deep eutectic solvent. Compared with the previous catalysts (immobilized lipase Novozym 435), an economic cation-exchange resin, Amberlyst-35 (A-35), showed good catalytic performance for GMC preparation. The activation energies of GMC synthesis and CA conversion were 43.71 kJ mol-1 and 43.07 kJ mol-1 , respectively. The optimal reaction conditions were a temperature reaction of 90 °C, catalyst load of 7%, glycerol/CA molar ratio of 5:1 (mol mol-1 ), and reaction time of 24 h, which resulted in a maximum GMC yield and CA conversion of 69.75 ± 1.03% and 82.23 ± 2.02%, respectively. CONCLUSION: The results of the work showed a promising alternative for the synthesis of GMC. © 2023 Society of Chemical Industry.

Enzymatic cofactor regeneration systems: A new perspective on efficiency assessment.

Nowadays, the specificity of enzymatic processes makes them more and more important every year, and their usage on an industrial scale seems to be necessary. Enzymatic cofactors, however, play a crucial part in the prospective applications of enzymes, because they are indispensable for conducting highly effective biocatalytic activities. Due to the relatively high cost of these compounds and their consumption during the processes carried out, it has become crucial to develop systems for cofactor regeneration. Therefore, in this review, an attempt was made to summarize current knowledge on enzymatic regeneration methods, which are characterized by high specificity, non-toxicity and reported to be highly efficient. The regeneration of cofactors, such as nicotinamide dinucleotides, coenzyme A, adenosine 5'-triphosphate and flavin nucleotides, which are necessary for the proper functioning of a large number of enzymes, is discussed, as well as potential directions for further development of these systems are highlighted. This review discusses a range of highly effective cofactor regeneration systems along with the productive synthesis of many useful chemicals, including the simultaneous renewal of several cofactors at the same time. Additionally, the impact of the enzyme immobilization process on improving the stability and the potential for multiple uses of the developed cofactor regeneration systems was also presented. Moreover, an attempt was made to emphasize the importance of the presented research, as well as the identification of research gaps, which mainly result from the lack of available literature on this topic.

Simple Enzyme Immobilization for Flow Chemistry? An Assessment of Available Strategies for an Acetaldehyde-Dependent Aldolase.

Enzyme immobilization is a technology that enables (bio-)catalysts to be applied in continuous-flow systems. However, there is a plethora of immobilization methods available with individual advantages and disadvantages. Here, we assessed the influence of simple and readily available methods with respect to the performance of 2-deoxy-d-ribose-5-phosphate aldolase (DERA) in continuous-flow conditions. The investigated immobilization strategies cover the unspecific attachment to carriers via epoxides, affinity-based attachment via metal ion affinity, StrepTag™-StrepTactin™ interaction as well as the covalent affinity attachment of an enzyme to a matrix tethered by the HaloTag®. The metal-ion-affinity-based approach outperformed the other methods in terms of immobilized activity and stability under applied conditions. As most enzymes examined today already have a HisTag for purification purposes, effective immobilization may be applied, as simple as a standard purification, if needed.

Comparative performance and reusability studies of lipases on syntheses of octyl esters with an economic approach.

A suitable immobilized lipase for esters syntheses should be selected considering not only its cost. We evaluated five biocatalysts in syntheses of octyl caprylate, octyl caprate, and octyl laurate, in which conversions higher than 90% were achieved. Novozym®ï»¿ 435 and non-commercial preparations (including a dry fermented solid) were selected for short-term octyl laurate syntheses using different biocatalysts loadings. By increasing the biocatalyst's loading the lipase's reusability also raised, but without strict proportionality, which resulted in a convergence between the lowest biocatalyst loading and the lowest cost per batch. The use of a dry fermented solid was cost-effective, even using loadings as high as 20.0% wt/wt due to its low obtaining cost, although exhibiting low productiveness. The combination of biocatalyst's cost, esterification activity, stability, and reusability represents proper criteria for the choice. This kind of assessment may help to establish quantitative goals to improve or to develop new biocatalysts.

Optimization and comparison of the production of galactooligosaccharides using free or immobilized Aspergillus oryzae ß-galactosidase, followed by purification using silica gel.

The aim of this study was to optimize and compare the production of galactooligosaccharides (GOSs) by free and cotton cloth-immobilized Aspergillus oryzae ß-galactosidase, and perform economical evaluation of production of GOSs (100%) between them. Using the response surface method, the optimal reaction time (3.9 h), initial lactose concentration (57.13%), and enzyme to lactose ratio (44.81 U/g) were obtained for the free enzyme, which provided a GOSs yield of 32.62%. For the immobilized enzyme, the optimal yield of GOSs (32.48%) was obtained under reaction time (3.09 h), initial lactose concentration (52.74%), and temperature (50.0 ℃). And it showed desirable reusability during five successive enzymatic reactions. The recovery rate of GOSs (100%) is 65% using silica gel filtration chromatography. The economical evaluation showed almost no difference in the manufacturing cost for the GOSs (100%) between these two systems, and that the recovery rate had a great impact on the cost.

New frontiers in enzyme immobilisation: robust biocatalysts for a circular bio-based economy.

This tutorial review focuses on recent advances in technologies for enzyme immobilisation, enabling their cost-effective use in the bio-based economy and continuous processing in general. The application of enzymes, particularly in aqueous media, is generally on a single use, throw-away basis which is neither cost-effective nor compatible with a circular economy concept. This shortcoming can be overcome by immobilising the enzyme as an insoluble recyclable solid, that is as a heterogeneous catalyst.

Rapid Assessment of Pepsin Column Activity for Reliable HDX-MS Studies.

Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) is a widely used technique to probe protein structural dynamics, track conformational changes, and map protein-protein interactions. Most HDX-MS studies employ a bottom-up approach utilizing the acid active protease pepsin to digest the protein of interest, often utilizing immobilized protease in a column format. The extent of proteolytic cleavage will greatly influence data quality and presents a major source of variation in HDX-MS studies. Here, we present a simple cocktail of commonly available peptides that are substrates of pepsin and can serve as a rapid check of pepsin column activity. The peptide-based assay requires no system modifications and provides an immediate readout to check and benchmark pepsin activity across different HDX-MS platforms.

qDNase assay: A quantitative method for real-time assessment of DNase activity on coated surfaces.

DNase coatings show great potential to prevent biofilm formation in various applications of the medical implant, food and marine industry. However, straightforward and quantitative methods to characterize the enzymatic activity of these coatings are currently not available. We here introduce the qDNase assay, a quantitative, real-time method to characterize the activity of DNase coatings. The assay combines (1) the use of an oligonucleotide probe, which fluoresces upon cleavage by coated DNases, and (2) the continuous read-out of the fluorescent signal within a microplate fluorometer format. The combination of these two properties results in a real-time fluorescent signal that is used to directly quantify the activity of DNase coatings. As a proof of concept, bovine DNase I coatings were immobilized on titanium by means of chemical grafting and their activity was estimated at 3.87 × 10-4 U. To our knowledge, the qDNase assay provides the first approach to report the activity of a DNase coating in absolute DNase activity units. This assay will not only serve to compare existing DNase coating methods more accurately, but will also enable the rational design of new DNase coating methods in the future.

Fusion and secretory expression of an exo-inulinase and a d-allulose 3-epimerase to produce d-allulose syrup from inulin.

BACKGROUND: This study developed a feasible catalytic method for d-allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers. RESULTS: d-Allulose 3-epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo-inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing 12 residues (GSAGSAAGSGEF). At 55 °C and pH 8.0, and as with the addition of 1 mmol L-1 Mn2+ , the CSCA-linkerE-DAE fusion enzyme obtained through high cell-density cultivation displayed a maximal exo-inulinase activity of 21.8 U mg-1 and resulted in a yield of 6.3 g L-1 d-allulose and 39.2 g L-1 d-fructose using 60 g L-1 inulin as the raw material. Catechol-modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA-immobilized CSCA-linkerE-DAE (8 U g-1 ) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L-1 d-allulose and 32.7 g L-1 D-fructose from 60 g L-1 inulin. CONCLUSION: This study shed light on a feasible and cost-effective approach for the production of syrup containing d-allulose and D-fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. © 2020 Society of Chemical Industry.

Bio-based and cost effective method for phenolic compounds removal using cross-linked enzyme aggregates.

This work aimed at presenting a green method using a new source of peroxidase isolated from Raphanus sativus var. niger (RSVNP) in immobilized form, for the treatment of wastewater. To ensure stability and enzymatic activity in the biodegradation process, RSVNP was immobilized as a cross-linked enzyme aggregate (CLEAs). With more than 29% of recovered activity and 85% aggregation yield, acetone was selected as the best precipitating agent. The formed protein aggregates required 2% (v/v) of glutaraldehyde (GA) concentration and a ratio of 9:1 (v/v) enzyme (E) amount to cross-linker (E/GA). Compared to the free enzyme, RSVNP-CLEAs were found more chemically and thermally stable and exhibited good storage stability for more than 8 weeks. In addition, RSVNP-CLEAs were evaluated for their ability to remove phenol and p-cresol from aqueous solution by varying several operating conditions. A maximal yield (98%) of p-cresol conversion was recorded after 40 min; while 92% of phenol was degraded after 1 h duration time. The reusability of RSVNP-CLEAs was tested, displaying 71% degradation of phenol in the third batch carried out and more than 54% was achieved for p-cresol after four successive reuses in the presence of hydrogen peroxide at 2 mM concentration.

Simple and Cost-effective Enzymatic Detection of Cholesterol Using Flow Injection Analysis.

A flow-injection analytical (FIA) system was developed for the determination of cholesterol concentrations based on enzymatic reactions that occurred in a cholesterol oxidase (CHOx)-immobilized, fused-silica capillary followed by electrochemical detection. The production of hydrogen peroxide from cholesterol in an enzymatic reaction catalyzed by CHOx was subsequently oxidized electrochemically at an electrode. Our FlA system demonstrated its cost-effectiveness and utility at an applied potential of 0.6 V (vs. Ag/AgCl), a flow rate of 100 µL/min and, under optimal conditions, the resulting signal demonstrated a linear dynamic range from 50 µM to 1.0 mM with a limit of detection (LOD) of 12.4 µM, limit of quantification (LOQ) of 44.9 µM, and the coefficient of variation of 5.17%. In addition, validation of our proposed system using a reference HDL-cholesterol kit used for clinical diagnosis suggested our FIA system was comparable to commercial kits for the determination of the cholesterol incorporation amount in various aqueous liposomal suspensions. These good analytical features achieved by FIA could make the implementation of this methodology possible for on-line monitoring of cholesterol in various types of samples.

Immobilization of mannanase on sodium alginate-grafted-ß-cyclodextrin: An easy and cost effective approach for the improvement of enzyme properties.

Partially purified ß-mannanase was immobilized on the modified matrix of sodium alginate-grafted-ß-cyclodextrin. The Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction characterization proved that ß-cyclodextrin (ß-CD) was successfully grafted with sodium alginate. After successful immobilization, yield of enzyme was found 91.5%, pH and temperature optima were increased, 6.0 to 7.0 and 50 °C to 55 °C respectively. Immobilized mannanase was able to reuse 15 times and retained its 70% activity, meanwhile the immobilized enzyme showed 60% activity after 30 days of storage at 4 °C. Immobilization also increased the thermostability and half-life of the enzyme when compared to the free mannanase. During the comparison of adsorption isotherm and kinetic models, Langmuir isotherm and pseudo-first order kinetics were observed to be the best fit model for the confirmation of immobilization.

Bioconjugation as a smart immobilization approach for α-amylase enzyme using stimuli-responsive Eudragit-L100 polymer: a robust biocatalyst for applications in pharmaceutical industry.

Enzymes are powerful versatile biocatalysts, however, industrial application of enzymes is usually hampered by their susceptibility. Bio-inspired Eudragit-α-amylase conjugate (E-AC) was proposed as a biocatalyst for various pharmaceutical and industrial applications. In this study, α -Amylase (E.C. 3.2.1.1) was immobilized by covalent conjugation to Eudragit L-100 under mild conditions. The effect of polymer, carbodiimide and enzyme concentrations on optimization of (E-AC) was investigated. In addition, characterization of the free α -Amylase and E-AC with regard to pH, temperature, kinetic parameters, reusability and operational and storage conditions was carried out. Results showed a shift of the optimum pH of E-AC towards the alkaline side whereas, E-AC exhibited higher thermal stability at all tested temperatures. The kinetic parameters, Km values were 2.87 mg/ml and 3.15 mg/ml and Vmax values were 8.35 mg/ml/min and 8.98 mg/ml/min for free and E-AC, respectively. E-AC retained 85% of the initial activity after five consecutive amylolytic cycles, thus emphasizing its powerful potentials. Operational storage and thermal stability were highly improved as well for E-AC conjugate with an 11.6 stabilization factor in comparison to the free α-amylase. In this study, Eudragit L-100 polymer was successfully used as smart immobilization support to create a reversibly soluble-insoluble enzyme biocatalyst to enforce and extend biotechnological applications of α-amylase in the pharmaceutical industry.

Directly covalent immobilization of Candida antarctica lipase B on oxidized aspen powder by introducing poly­lysines: An economical approach to improve enzyme performance.

In our previous study, we could achieve high soluble expression of Candida antarctica lipase B (CalB) in E. coli by fusion poly­amino acid tags on CalB (pCalB). Herein, we are surprised to find that pCalB can be easily and directly covalent binding on a simply oxidized aspen powder (OAP) by the aid of poly­lysine tags. Under the optimal conditions, 72.9 ±â€¯3.6% of the total protein could be immobilized, and the activity recovery of immobilized pCalB (pCalB-OAP) was 98.9 ±â€¯3.8%. The analysis of scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) indicated that OAP was a suitable carrier for enzyme immobilization. The immobilized pCalB-OAP could exhibit excellent thermal stabilities, and it retained a residual activity of 58.4 ±â€¯2.8% at 55 °C, whereas only 21.2 ±â€¯2.2% of its initial activity for free pCalB was observed. And it could also display a nice tolerance for the changes of pH environment, compared with that of free pCalB. The results that pCalB-OAP could retained 73.6 ±â€¯2.9% of their initial activity in (R, S)-NEMPAME hydrolysis after the tenth cycles, suggested that pCalB-OAP could be effectively recycled. The immobilization strategies established here were simple and inexpensive.

Cost analysis of oil cake-to-biodiesel production in packed-bed micro-flow reactors with immobilized lipases.

Biodiesel production depends to a great extent on the use of cheap raw materials, since biodiesel itself is a mass product, not a high-value product. New processing methods, such as micro-flow continuous processing combined with enzymatic catalysis, open doors to the latter. As reported here, the window of opportunity in enzyme-catalyzed biodiesel production is the conversion of waste cooking oil. The main technological challenge for this is to obtain efficient immobilization of the lipase catalyst on beads. The beads can be filled into tubular reactors where designed packed-bed provide porous channels, forming micro-flow. It turns out, that in this way, the immobilization costs become the decisive economic factor. This paper reports a solution to that issue. The use of oil cake enables economic viability, which is not given by any of the commercial polymeric substrates used so far for enzyme immobilization. The costs of immobilization are mirrored in the earnings and cash flow of the new biotechnological process.

Low-cost mussel inspired poly(Catechol/Polyamine) modified magnetic nanoparticles as a versatile platform for enhanced activity of immobilized enzyme.

Owing to dopamine's excellent adhesion ability and easy modification, it has been widely applied for enzyme immobilization, while the high cost of dopamine and low activity recovery of immobilized enzyme highly impede large-scale application of immobilized enzyme. We herein developed a low-cost and ideal activity recovery enzyme immobilization strategy based on magnetic nanoparticles by replacing dopamine with cheap Catechol/tetraethylene pentamine (CPA) binary system and introducing spacer-arms. In brief, CPA was first polymerized and deposited on the surface of magnetic nanoparticles with a modified mussel-inspired method, and the generated poly(CPA) layer was further functionalized with ethylene glycol diglycidyl ether (EGDE) molecules as spacer-arms for enzyme immobilization. Subsequently, lipases as model enzymes were firmly immobilized on the surface of such amino-epoxy functionalized magnetic materials through ion exchange and covalent attachment with 180.6 mg/g support of loading capacity and 69.2% of activity recovery under the optimized conditions. Furthermore, the immobilized lipase exhibited the improved tolerance rang of pH, temperature and storage stability as well as excellent reusability. Most strikingly, the theoretical simulation and secondary structure analysis of immobilized lipase revealed that the biocompatible microenvironment and flexible tethering at interface could effectively improve performance of the immobilized enzyme and stability. Thus, this novel immobilized enzyme strategy will open up a new perspective for the development of enzyme immobilization and lower the cost of immobilized enzyme in large-scale industrial application.

Biodegradation of Cibacron Blue 3GA by insolubilized laccase and identification of enzymatic byproduct using MALDI-ToF-MS: Toxicity assessment studies by Daphnia magna and Chlorella vulgaris.

The presented paper describes a detailed study on the use of immobilized laccase for effective degradation of Cibacron Blue 3GA dye. The amount of laccase loading on the cyclic carbonate groups containing poly(hydroxyethyl methacrylate-co-vinylene carbonate), p(HEMA-co-VC), microbeads was 27.8 mg g-1, and the retained immobilized enzyme activity was 73% compared to free enzyme. The toxicity of the dye and its byproducts were studied using Daphnia magna as test organism. The micro-algal growth inhibition was also studied using a green micro algae "Chlorella vulgaris". MALDI-ToF-MS was used to verify dye degradation byproducts. After 60 min of incubation period, Cibacron Blue 3GA (CB3GA) and its byproducts disappeared from the medium. After 60-min enzymatic treatment, the non-toxic nature of medium was confirmed by toxicity studies. On the other hand, the initial byproducts of the dye seemed to be more toxic than the later formed dye products. It should be noted that the information obtained from this study can be beneficial for understanding the initial degradation byproducts toxicities of the enzymatically treated dyes to provide information about environmental protection.