Role of enzyme immobilization in the formulation of enzymes for single use

Immobilized enzymes find application in diverse areas of biosciences as biofuels, biosensors and biocatalysis. Development of a immobilization for an enzyme implies a careful selection and optimization of different parameters that have a critical influence on the final properties, e.g. activity, stability [1,2]. Enzyme stabilization via immobilization is a common target and it is achievable when properly performed [2]. There is the general idea that the use of enzyme in free soluble form is associated to single use due to the simplicity and significant lower cost, whereas the use of immobilized preparations is generally associated to facilitate retention, recycling or continuous use. Nevertheless, there are some constraints where immobilization is a fundamental tool to enable the single use of the enzyme under conditions otherwise not-applicable. Among others, these conditions involve the preservation of enzyme activity during extraction, purification, storage and transport, stabilization under condition of use and easy separation from the medium where it is applied. This communication presents some examples of immobilization strategies and benefits for the application of oxidative enzymes. Glucose oxidase and D-amino acid oxidase were immobilized into porous materials made from natural organic polymers or synthetic organic polymers [3,4]. Under the conditions of use, that involves diluted application and existence of gas-liquid interfaces, the free enzymes have half-lifetime lower than 5 min whereas proper immobilized preparations within porous materials did not show decrease of activity during a time span of observation of 40 h. The storage of the enzyme formulation was also facilitated, immobilized enzymes were stable under simple storage conditions whereas free enzymes required prevention microbiological contamination and tuning of buffer conditions to avoid protein precipitation. Among different strategies, the use of reversible immobilization was evaluated to enable the reuse of support and facilitate an immobilization on/off character. The development reversible one step purification-immobilization simplified the production and application of the immobilizate format compared with the soluble counterpart [3]. Finally, the immobilization was used as a compartmentalization strategy, two enzymes (oxidase-catalase, oxidase-peroxidase) or a pair of oxidase - oxygen probe can be used when they are confined into solid materials compared to incompatible conditions of use in soluble format [5]. Co-joined design of polymeric materials and immobilization strategies is recognized as important for the efficient cost-effective formulation of enzyme immobilizates. [1] Bolivar, J.M., Eisl, I., Nidetzky, B.(2016) Catalysis Today, 259, pp. 66-80. [2] Rodrigues, R.C., Ortiz, C., Berenguer-Murcia, A., Torres, R., Fernández-Lafuente, R. (2013) Chemical Society Reviews, 42 (15), pp. 6290-6307. [3] Wiesbauer, J., Bolivar, J.M., Mueller, M., Schiller, M., Nidetzky, B. (2011) ChemCatChem, 3 (8), pp. 1299-1303. [5] Bolivar, J.M., Consolati, T., Mayr, T., Nidetzky, B. (2013) Biotechnology and Bioengineering, 110 (8), pp. 2086-2095. [4] Bolivar, J.M., Consolati, T., Mayr, T., Nidetzky, B. (2013) Trends in Biotechnology, 31 (3), pp. 194-20

Dual-lifetime referencing (DLR): a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts

<p>Abstract</p> <p>Background</p> <p>Industrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier). Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR) for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation.</p> <p>Results</p> <p>The DLR method employs fluorescein as pH-sensitive luminophore and Ru(II) tris(4,7-diphenyl-1,10-phenantroline), abbreviated Ru(dpp), as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance). The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under conditions of continuous measurement. During hydrolysis of cephalosporin C by the immobilizate in a stirred reactor with bulk pH maintained at 8.0, the intraparticle pH dropped initially by about 1 pH unit and gradually returned to the bulk pH, reflecting the depletion of substrate from solution. These results support measurement of intraparticle pH as a potential analytical processing tool for proton-forming/consuming biotransformations catalyzed by carrier-bound immobilized enzymes.</p> <p>Conclusions</p> <p>Fluorescein and Ru(dpp) constitute a useful pair of luminophores in by DLR-based intraparticle pH monitoring. The pH range accessible by the chosen DLR system overlaps favorably with the pH ranges at which enzymes are optimally active and stable. DLR removes the restriction of working with static immobilized enzyme particles, enabling suspensions of particles to be characterized also. The pH gradient developed between particle and bulk liquid during reaction steady state is an important carrier selection parameter for enzyme immobilization and optimization of biocatalytic conversion processes. Determination of this parameter was rendered possible by the presented DLR method.</p

New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme

<p>Abstract</p> <p>Background</p> <p>The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD⁺pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD⁺. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.</p> <p>Results</p> <p>The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.</p> <p>Conclusion</p> <p>We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of <it>rac</it>-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.</p

A review on the environmental impact of phosphogypsum and potential health impacts through the release of nanoparticles

Many industrial by-products have been disposed along coastlines, generating profound marine changes. Phosphogypsum (PG) is a solid by-product generated in the production of phosphoric acid (PA) using conventional synthesis methods. The raw material, about 50 times more radioactive as compared to unperturbed soils, is dissolved in diluted sulfuric acid (70%) forming PG and PA. The majority of both, reactive hazardous elements and natural radionuclides, remain bound to the PG. A nonnegligible fraction of PG occurs as nanoparticles (<0.1 μm). When PG are used for e.g., agriculture or construction purposes, nanoparticles (NPs) can be re-suspended by Aeolian and fluvial processes. Here we provide an overview and evaluation of the geochemical and radiological hazardous risks associated with the different uses of PG. In this review, we show that NPs are important residues in both raw and waste materials originating from the uses of phosphate rock. Different industrial processes in the phosphate fertilizer industries are discussed in the context of the chemical and mineralogical composition as well as size and reactivity of the released NP. We also review how incidental NPs of PG impact the global environment, especially with respect to the distribution of rare earth elements (REEs), toxic elements such as As, Se, and Pb, and natural radionuclides. We also propose the application of advanced techniques and methods to better understand formation and transport of NPs containing elements of high scientific, economic, and environmental importance

Usefulness of sputum gram stain for etiologic diagnosis in community-acquired pneumonia: a systematic review and meta-analysis

Background: implementation of sputum Gram stain in the initial assessment of community-acquired pneumonia (CAP) patients is still controversial. We performed a systematic review and meta-analysis to investigate the usefulness of sputum Gram stain for defining the etiologic diagnosis of CAP in adult patients. Methods: we systematically searched the Medline, Embase, Science Direct, Scopus and LILACS databases for full-text articles. Relevant studies were reviewed by at least three investigators who extracted the data, pooled them using a random effects model, and carried out quality assessment. For each bacterium (Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Gram-negative bacilli), pooled sensitivity, specificity, positive and negative likelihood ratios were reported. Results: after a review of 3539 abstracts, 20 articles were included in the present meta-analysis. The studies included yielded 5619 patients with CAP. Pooled sensitivity and pooled specificity of sputum Gram stain were 0.59 (95% CI, 0.56-0.62) and 0.87 (95% CI, 0.86-0.89) respectively for S. pneumoniae, 0.78 (95% CI, 0.72-0.84) and 0.96 (95% CI, 0.94-0.97) for H. influenzae, 0.72 (95% CI, 0.53-0.87) and 0.97 (95% CI, 0.95-0.99) for S. aureus, and 0.64 (95% CI, 0.49-0.77) and 0.99 (95% CI, 0.97-0.99) for Gram-negative bacilli. Conclusion: Sputum Gram stain test is sensitive and highly specific for identifying the main causative pathogens in adult patients with CAP

Conjugating his-tagged proteins to magnetic nanoparticles: tips and challenges

Resumen del póster presentado al 5th Multistep Enzyme Catalyzed Processes Congress (MECPC), celebrado online del 13 al 16 de septiembre de 2021.The histidine tag (His-tag) is one of the most used affinity-tag for protein purification due to its small size and versatility. Agarose and sepharose beads containing nitriloacetic acid (NTA) transition metal derivatives are widely used for the purification of His-tagged proteins, thanks to their high affinity to the His-tag genetically fused to the protein of interest [1]. The same chemistry can be used to conjugate enzymes to magnetic nanoparticles (MNPs) with the aim of tuning their activity by magnetic heating [2, 3]. Within the frame of the FET-OPEN project HOTZYMES (https://www.hotzymes.eu), different MNPs have been synthetized and coated with polyacrylic acid and dimercaptosuccinic acid, and then were further functionalized with NTA-Cu2+ as His-tag chelating agent. Different proteins were expressed as His-tag variants and immobilized on the MNPs, including monomeric (superfolded GFP), dimeric (C. violaceum transaminase, CvTA; C. uda cellobiose phosphorylase, CuCbP), and tetrameric (B. stearothermophilus alcohol dehydrogenase, ADH) variants. While for the monomeric protein selected as model no difficulties in the bioconjugation processes were observed, when using dimeric or tetrameric enzymes the aggregation of the MNPs occurs very easily due to crosslinking between the nanoparticles. This colloidal destabilization of the MNPs is favored due to its high surface area and the presence of several tags per enzyme molecule. To avoid this situation, different strategies have been developed: saturation of the binding site of the nanoparticles, presence of a small percentage of imidazole in reaction, changes in the incubation conditions (pH, ionic strength, …). First positive results confirm that by playing with different factors it is possible to conjugate different His-tagged enzymes to very different MNPs in terms of size, shape, surface area, and colloidal stability. Actually, it is possible to avoid protein and MNPs aggregation while obtaining good activity yields for the conjugated enzymes and maintaining the magnetic heating capacity of the MNPs.The research for this work has received funding from the European Union (EU) project HOTZYMES (grant agreement n° 829162) under EU’s Horizon 2020 Programme Research and Innovation actions H2020-FETOPEN-2018-2019-2020-01. Authors also thank Spanish MINECO project BIO2017-84246-C2-1-R, DGA and Fondos Feder (Bionanosurf E15_17R).Peer reviewe

Multiple Binding Sites for Fatty Acids on the Potassium Channel KcsA

Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein-protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ~0.1-1 ?M

The Microenvironment in Immobilized Enzymes: Methods of Characterization and Its Role in Determining Enzyme Performance

The liquid milieu in which enzymes operate when they are immobilized in solid materials can be quite different from the milieu in bulk solution. Important differences are in the substrate and product concentration but also in pH and ionic strength. The internal milieu for immobilized enzymes is affected by the chemical properties of the solid material and by the interplay of reaction and diffusion. Enzyme performance is influenced by the internal milieu in terms of catalytic rate (&ldquo;activity&rdquo;) and stability. Elucidation, through direct measurement of differences in the internal as compared to the bulk milieu is, therefore, fundamentally important in the mechanistic characterization of immobilized enzymes. The deepened understanding thus acquired is critical for the rational development of immobilized enzyme preparations with optimized properties. Herein we review approaches by opto-chemical sensing to determine the internal milieu of enzymes immobilized in porous particles. We describe analytical principles applied to immobilized enzymes and focus on the determination of pH and the O2 concentration. We show measurements of pH and [O2] with spatiotemporal resolution, using in operando analysis for immobilized preparations of industrially important enzymes. The effect of concentration gradients between solid particle and liquid bulk on enzyme performance is made evident and quantified. Besides its use in enzyme characterization, the method can be applied to the development of process control strategies