Immobilized Dipeptidase in Manganese Ion-Loaded Polyethylenimine-Induced Calcium Phosphate Nanocrystals for Carnosine Synthesis.

Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.

Alloyed Trimetallic Nanocomposite as an Efficient and Recyclable Solid Matrix for Ideonella sakaiensis Lipase Immobilization.

In this work, a trimetallic (Ni/Co/Zn) organic framework (tMOF), synthesized by a solvothermal method, was calcinated at 400 and 600 °C and the final products were used as a support for lipase immobilization. The material annealed at 400 °C (Ni-Co-Zn@400) had an improved surface area (66.01 m2/g) and pore volume (0.194 cm3/g), which showed the highest enzyme loading capacity (301 mg/g) with a specific activity of 0.196 U/mg, and could protect the enzyme against thermal denaturation at 65 °C. The optimal pH and temperature for the lipase were 8.0 and 45 °C but could tolerate pH levels 7.0-8.0 and temperatures 40-60 °C. Moreover, the immobilized enzyme (Ni-Co-Zn@Lipase, Ni-Co-Zn@400@Lipase, or Ni-Co-Zn@600@Lipase) could be recovered and reused for over seven cycles maintaining 80, 90, and 11% of its original activity and maintained a residual activity >90% after 40 storage days. The remarkable thermostability and storage stability of the immobilized lipase suggest that the rigid structure of the support acted as a protective shield against denaturation, while the improved pH tolerance toward the alkaline range indicates a shift in the ionization state attributed to unequal partitioning of hydroxyl and hydrogen ions within the microenvironment of the active site, suggesting that acidic residues may have been involved in forming an enzyme-support bond. The high enzyme loading capacity, specific activity, encouraging stability, and high recoverability of the tMOF@Lipase indicate that a multimetallic MOF could be a better platform for efficient enzyme immobilization.

Electrostatically Mediated In Situ Polymerization for Enzyme Immobilization and Activation.

Enzyme immobilization in nanoparticles is of interest for boosting their catalytic applications, yet rational approaches to designs achieving both high enzyme loading and activation remain a challenge. Herein, we report an electrostatically mediated in situ polymerization strategy that simultaneously realizes enzyme immobilization and activation. This was achieved by copolymerizing cationic monomers with a cross-linker in the presence of the enzyme lipase (anionic) as the template, which produces enzyme-loaded nanogels. The effects of different control factors such as pH, lipase dosage, and cross-linker fraction on nanogel formation are investigated systematically, and optimal conditions for enzyme loading and activation have been determined. A central finding is that the cationic polymer network of the nanogel creates a favorable environment that not only protects the enzyme but also boosts enzymatic activity nearly 2-fold as compared to free lipase. The nanogels improve the stability of the lipase to tolerate a broader working range of pH (5.5-8.5) and temperature (25-70 °C) and allow recycling such that after six cycles of reaction, 70% of the initial activity is conserved. The established fabrication strategy can be applied generally to different cationic monomers, and most of these nanogels exhibit adequate immobilization and activation of lipase. Our study confirms that in situ polymerization based on electrostatic interaction provides a facile and robust strategy for enzyme immobilization and activation. The wide variety of ionic monomers, therefore, features great potential for developing functional platforms toward satisfying enzyme immobilization and demanding applications.

Efficient expression of γ-glutamyl transpeptidase in Bacillus subtilis via CRISPR/Cas9n and its immobilization.

In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. KEY POINTS: • Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide. • Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system. • The process of immobilizing BlGGT with epoxy resin was optimized.

Paddy straw saccharification using immobilized laccase on magnetized multiwall carbon nanotubes.

The effective recovery of the immobilized enzymes using magnetic carriers has led to growing interest in this technology. The objective of this research was to evaluate the efficiency of immobilized laccase on magnetized multiwall carbon nanotubes (m-MWCNTs) in terms of stability and reusability. Laccases were efficiently adsorbed onto magnetized multiwall carbon nanotubes (m-MWCNTs) synthesized using water. The concentration of 7 mg laccase/mL was found to be ideal for immobilization. The optimal activity of both free and immobilized laccases was observed at pH 5, while for the latter, the optimal temperature was shifted from 40 to 50 °C. Compared to the free laccase, the immobilized laccase exhibited a greater range of stability at more extreme temperatures. At the fourth cycle of reactions, the immobilized laccase exhibited more than 60% relative activity in terms of reusability. Based on the fourier-transform infrared spectroscopy (FTIR) peak at 2921 cm-1, saccharification of paddy straw using immobilized laccase verified lignin degradation. The easy recovery of the immobilized laccase on m-MWCNTs lends credence to its potential use in biomass hydrolysis.

Mechanically stirred enzymatic membrane reactor containing HRP immobilized on Tau-SiO2@Fe3O4-GO nanocomposite for removal of tetracycline in synthetically concocted wastewater.

The process of mechanically stirred membrane reactor containing the suspension of horseradish peroxidase (HRP) immobilized on synthesized nanocomposite (Tau-SiO2@Fe3O4-GO) was designed for continuous degradation of tetracycline. The immobilized HRP was characterized in terms of kinetic parameters and catalytic activities as these parameters were improved highly through immobilization. The stability indices including pH and temperature were investigated in parallel. The immobilized HRP was more tolerable to pH changes as compared to free HRP and the optimum temperature obtained at 40 °C. The reusability of HRP was promoted by immobilization as far as 70% of initial activity after ten cycles. The enzymatic degradation of optimum concentration of tetracycline was carried out in batch condition and 100% of tetracycline removed after 30 min. The results also showed that higher concentration of H2O2 exhibited more oxidation of tetracycline. The optimal ratio of HRP/H2O2 was also obtained at 0.005. The simultaneous process including separation and the biocatalytic degradation established in the membrane stirrer reactor concluded that no amount of tetracycline was observed in the permeate stream coming from the membrane after 30 min of operation.

Lactose hydrolysis in packed-and fluidized-bed reactors using a recombinant ß-galactosidase immobilized on magnetic core-shell capsules.

The objective of this study was to develop a bioprocess for lactose hydrolysis in diverse dairy matrices, specifically skim milk and cheese whey, utilizing column reactors employing a core-shell enzymatic system featuring ß-galactosidase fused to a Cellulose Binding Domain (CBD) tag (ß-galactosidase-CBD). The effectiveness of reactor configurations, including ball columns and toothed columns operating in packed and fluidized-bed modes, was evaluated for catalyzing lactose hydrolysis in both skim milk and cheese whey. In a closed system, these reactors achieved lactose hydrolysis rates of approximately 50% within 5 h under all evaluated conditions. Considering the scale of the bioprocess, the developed enzymatic system was capable of continuously hydrolyzing 9.6 L of skim milk while maintaining relative hydrolysis levels of approximately 50%. The biocatalyst, created by immobilizing ß-galactosidase-CBD on magnetic core-shell capsules, exhibited exceptional operational stability, and the proposed bioprocess employing these column reactors showcases the potential for scalability.

A catalytic membrane approach as a way to obtain sweet and unsweet lactose-free milk.

The growing need in the current market for innovative solutions to obtain lactose-free (L-F) milk is caused by the annual increase in the prevalence of lactose intolerance inside as well as the newborn, children, and adults. Various configurations of enzymes can yield two distinct L-F products: sweet (ß-galactosidase) and unsweet (ß-galactosidase and glucose oxidase) L-F milk. In addition, the reduction of sweetness through glucose decomposition should be performed in a one-pot mode with catalase to eliminate product inhibition caused by H2O2. Both L-F products enjoy popularity among a rapidly expanding group of consumers. Although enzyme immobilization techniques are well known in industrial processes, new carriers and economic strategies are still being searched. Polymeric carriers, due to the variety of functional groups and non-toxicity, are attractive propositions for individual and co-immobilization of food enzymes. In the presented work, two strategies (with free and immobilized enzymes; ß-galactosidase NOLA, glucose oxidase from Aspergillus niger, and catalase from Serratia sp.) for obtaining sweet and unsweet L-F milk under low-temperature conditions were proposed. For free enzymes, achieving the critical assumption, lactose hydrolysis and glucose decomposition occurred after 1 and 4.3 h, respectively. The tested catalytic membranes were created on regenerated cellulose and polyamide. In both cases, the time required for lactose and glucose bioconversion was extended compared to free enzymes. However, these preparations could be reused for up to five (ß-galactosidase) and ten cycles (glucose oxidase with catalase).

Enhancing the sustainable immobilization of laccase by amino-functionalized PMMA-reinforced graphene nanomaterial.

Human health and the environment are negatively affected by endocrine-disrupting chemicals (EDCs), such as bisphenol A. Therefore, developing appropriate remediation methods is essential for efficiently removing phenolic compounds from aqueous solutions. Enzymatic biodegradation is a potential biotechnological approach for responsibly addressing water pollution. With its high catalytic efficiency and few by-products, laccase is an eco-friendly biocatalyst with significant promise for biodegradation. Herein, two novel supporting materials (NH2-PMMA and NH2-PMMA-Gr) were fabricated via the functionalization of poly(methylmethacrylate) (PMMA) polymer using ethylenediamine and reinforced with graphene followed by glutaraldehyde activation. NH2-PMMA and NH2-PMMA-Gr were utilized for laccase immobilization with an immobilization yield (IY%) of 78.3% and 82.5% and an activity yield (AY%) of 81.2% and 85.9%, respectively. Scanning electron microscope (SEM) and Fourier-transform infrared (FTIR) were used to study the characteristics of fabricated material supports. NH2-PMMA-Gr@laccase exhibited an optimal pH profile from 4.5 to 5.0, while NH2-PMMA@laccase exhibited optimum pH at 5.0 compared to a value of 4.0 for free form. A wider temperature ranges of 40-50 °C was noted for both immobilized laccases compared to a value of 40 °C for the free form. Additionally, it was reported that immobilized laccase outperformed free laccase in terms of substrate affinity and storage stability. NH2-PMMA@laccase and NH2-PMMA-Gr@laccase improved stability by up to 3.9 and 4.6-fold when stored for 30 days at 4 °C and preserved up to 80.5% and 86.7% of relative activity after ten cycles of reuse. Finally, the degradation of BPA was achieved using NH2-PMMA@laccase and NH2-PMMA-Gr@laccase. After five cycles, NH2-PMMA@laccase and NH2-PMMA-Gr@laccase showed that the residual degradation of BPA was 77% and 84.5% using 50 µm of BPA. This study introduces a novel, high-performance material for organic pollution remediation in wastewater that would inspire further progress.

UCST-Type Soluble Immobilized Cellulase: A New Strategy for the Efficient Degradation and Improved Recycling Performance of Wastepaper Cellulose.

This paper reports an innovative study that aims to address key issues in the efficient recycling of wastepaper cellulose. The research team utilized the temperature-responsive upper critical solution temperature (UCST) polymer P(NAGA-b-DMA) in combination with the LytA label's affinity for choline analogs. This innovative approach enabled them to successfully develop a novel soluble immobilized enzyme, P(NAGA-b-DMA)-cellulase. This new enzyme has proven highly effective, significantly enhancing the degradation of wastepaper cellulose while demonstrating exceptional stability. Compared with the traditional insoluble immobilized cellulase, the enzyme showed a significant improvement in the pH, temperature stability, recycling ability, and storage stability. A kinetic parameter calculation showed that the enzymatic effectiveness of the soluble immobilized enzyme was much better than that of the traditional insoluble immobilized cellulase. After the immobilization reaction, the Michaelis constant of the immobilized enzyme was only increased by 11.5%. In the actual wastepaper degradation experiment, the immobilized enzyme was effectively used, and it was found that the degradation efficiency of wastepaper cellulose reached 80% of that observed in laboratory conditions. This novel, thermosensitive soluble immobilized cellulase can efficiently catalyze the conversion of wastepaper cellulose into glucose under suitable conditions, so as to further ferment into environmentally friendly biofuel ethanol, which provides a solution to solve the shortage of raw materials and environmental protection problems in the paper products industry.

Novel Approach for the Isolation and Immobilization of a Recombinant Transaminase: Applying an Advanced Nanocomposite System.

The increasing application of recombinant enzymes demands not only effective and sustainable fermentation, but also highly efficient downstream processing and further stabilization of the enzymes by immobilization. In this study, a novel approach for the isolation and immobilization of His-tagged transaminase from Chromobacterium violaceum (CvTA) has been developed. A recombinant of CvTA was simultaneously isolated and immobilized by binding on silica nanoparticles (SNPs) with metal affinity linkers and additionally within poly(lactic acid) (PLA) nanofibers. The linker length and the nature of the metal ion significantly affected the enzyme binding efficiency and biocatalytic activity of CvTA-SNPs. The formation of PLA nanofibers by electrospinning enabled rapid embedding of CvTA-SNPs biocatalysts and ensured enhanced stability and activity. The developed advanced immobilization method reduces the time required for enzyme isolation, purification and immobilization by more than fourfold compared to a classical stepwise technique.

Recent trends using natural polymeric nanofibers as supports for enzyme immobilization and catalysis.

All the disciplines of science, especially biotechnology, have given continuous attention to the area of enzyme immobilization. However, the structural support made by material science intervention determines the performance of immobilized enzymes. Studies have proven that nanostructured supports can maintain better catalytic performance and improve immobilization efficiency. The recent trends in the application of nanofibers using natural polymers for enzyme immobilization have been addressed in this review article. A comprehensive survey about the immobilization strategies and their characteristics are highlighted. The natural polymers, e.g., chitin, chitosan, silk fibroin, gelatin, cellulose, and their blends with other synthetic polymers capable of immobilizing enzymes in their 1D nanofibrous form, are discussed. The multiple applications of enzymes immobilized on nanofibers in biocatalysis, biosensors, biofuels, antifouling, regenerative medicine, biomolecule degradation, etc.; some of these are discussed in this review article.

Nanotechnology and enzyme immobilization: a review.

The synergy between enzymes and nanotechnology (nano-biocatalysts) has created some of the most promising biomaterials fabricated by synergistically incorporating advanced nano-biotechnology. The incorporation of enzymes into nanotechnology is of great significance for making nanomaterials that are rarely harmful to the environment. However, the unique/specific physicochemical characteristics and supramolecular nature ascribed to functional nanostructures (nanomaterials), have made them novel, interesting, and exceptional matrices for the creation of nano-biocatalysts. These have a lot of potential for improving the enzyme stability, function, efficiency, kinetic characteristics, vulnerability to diffusional constraints, and engineering performance in bioprocessing. Hence, the nano-biocatalysts developed contain exceptional properties with many potential applications in diverse fields. This review covers a wide range of the nanotechnology and enzyme technology involved in producing nano-biocatalysts, including different mechanisms, strategies in nanomaterial enzyme immobilization, and various nanocarriers, as well as recent developments in controlling enzyme activity. The vast range of potential applications of nano-biocatalysts in various fields, including food, pharmaceuticals, biofuels, and bioremediation, has been discussed.

Metal Azolate Coordination Polymer-Enabled High Payload and Non-Destructive Enzyme Immobilization for Biocatalysis and Biosensing.

The biomineralized metal-organic frameworks (MOFs) as protective layers help enhance the robustness of enzymes for biocatalysis. Despite great efforts, it is still challenging to develop a recyclable system with high payload and tolerance to harsh conditions. Here, we report a facile surface charge-independent strategy based on Zn-based coordination polymer (ZnCP) for nondestructive immobilization of enzyme. The ZnCP outcompetes most of the previously reported MOFs, in terms of high-payload enzyme packaging. Moreover, benefiting from the hydrophilicity of ZnCP, the entrapped enzymes (e.g., positive cytochrome C and negative glucose oxidase) maintained high catalytic activity, resembling their native counterparts. Notably, compared with ZIF-8, such enzyme-incorporated ZnCP (enzyme@ZnCP) is more tolerant to acidic pH, which imparts the enzyme with good recyclability, even in acid species-generated catalytic reactions, thus broadening its application in biocatalysis. The feasibility of enzyme@ZnCP for protein packaging, enzyme cascade catalysis, and biosensing was also validated. Altogether, enzyme@ZnCP demonstrates high enzyme payload, operational stability, and preservation of enzymatic activity, affording a versatile platform to accommodate bioactive enzyme for biocatalysis and biosensing.

Overview on immobilization of enzymes on synthetic polymeric nanofibers fabricated by electrospinning.

The arrangement and type of support has a significant impact on the efficiency of immobilized enzymes. 1-dimensional fibrous materials can be one of the most desirable supports for enzyme immobilization. This is due to their high surface area to volume ratio, internal porosity, ease of handling, and high mechanical stability, all of which allow a higher enzyme loading, release and finally lead to better catalytic efficiency. Fortunately, the enzymes can reside inside individual nanofibers to remain encapsulated and retain their three-dimensional structure. These properties can protect the enzyme's tolerance against harsh conditions such as pH variations and high temperature, and this can probably enhance the enzyme's stability. This review article will discuss the immobilization of enzymes on synthetic polymers, which are fabricated into nanofibers by electrospinning. This technique is rapidly gaining popularity as one of the most practical ways to fibricate polymer, metal oxide, and composite micro or nanofibers. As a result, there is interest in using nanofibers to immobilize enzymes. Furthermore, present research on electrospun nanofibers for enzyme immobilization is primarily limited to the lab scale and industrial scale is still challanging. The primary future research objectives of this paper is to investigate the use of electrospun nanofibers for enzyme immobilization, which includes increasing yield to transfer biological products into commercial applications.

Mechanism and Kinetics of Lipase-Catalyzed Polycondensation of Glycerol and Sebacic Acid: Influence of Solvent and Temperature.

The mechanism for theCandida antacticalipase B (CALB)-catalyzed polycondensation of glycerol and sebacic acid in polar solvents was proposed based on the profile of formation and consumption of the glyceridic species in the reaction media and on the occurrence of the acyl migration reaction. The acyl migration is mainly responsible for the esterification of the secondary hydroxyl of glycerol and in an opposite way to the regioselective CALB-catalyzed esterification of primary hydroxyls. The enzymatic esterification of glycerol primary hydroxyls occurs preferentially up to carboxylic acid conversions of approximately 0.60-0.75 with rate constants in the range of 0.07-1.44 L mol-1 h-1, depending on the solvent. Above carboxylic acid conversions of 0.60-0.75, acyl migration occurs in parallel to enzymatic esterification with rate constants of approximately 0.04-0.12 h-1 and is the rate-limiting step of the polymerization. The hydrogen bonding accepting ability of the solvents is the main parameter that dictates the enzymatic catalysis rate. However, the magnitude of the polymer-solvent interaction governs the polymer chain growth. Acetonitrile has a lower hydrogen bonding accepting ability and a less favorable polymer-solvent interaction compared with the other polymer-solvent pairs, and polycondensation achieves the highest enzymatic rate constant of approximately 0.84-1.44 L mol-1 h-1; however, low molar mass polymers with Mn = 1.4 kDa were formed. On the other hand, acetone has intermediate hydrogen bonding accepting ability and optimal intermediate polymer-solvent interactions and, therefore, an intermediate enzymatic rate constant of approximately 0.41-0.52 L mol-1 h-1, and the highest molar mass polymers with Mn = 4.9-9.4 kDa were obtained.

Precipitation-based microscale enzyme reactors coupled with porous and adhesive elastomer for effective bacterial decontamination and membrane antifouling on-demand.

Bacterial contamination of water environments can cause various troubles in various areas. As one of potential solutions, we develop enzyme-immobilized elastomer, and demonstrate the uses of enzyme reactions on-demand for effective microbial decontamination and antifouling. Asymmetrically-structured elastomer is prepared by combining two polydimethylsiloxane (PDMS) layers with different degrees of crosslinking: highly-crosslinked and lightly-crosslinked PDMS layers. At the surface of highly-crosslinked PDMS layer, porous structure with average diameter of 842 nm is formed by dissolving pre-packed and entrapped latex beads. Lightly-crosslinked PDMS on the other side, due to its adhesive nature, enables iterative attachments on various materials under either dry or wet condition. Glucose oxidase (GOx) is immobilized by using the pores at the surface of highly-crosslinked PDMS matrix via a ship-in-a-bottle protocol of precipitation-based microscale enzyme reactor (p-MER), which consists of GOx adsorption, precipitation and chemical crosslinking (EAPC). As a result, crosslinked enzyme aggregates (CLEAs) of GOx not only are well entrapped within many pores of highly-crosslinked PDMS layer (ship-in-bottle) but also cover the external surface of matrix, both of which are well connected together. Highly-interconnected network of CLEAs themselves effectively prevents enzyme leaching, which shows the 25% residual activity of GOx under shaking at 200 rpm for 156 days after 48% initial drop of loosely-bound p-MER after 4 days. In presence of glucose, the underwater attachment of biocatalytic elastomer demonstrates the generation of hydrogen peroxide via p-MER-catalyzed glucose oxidation, exhibiting effective biocidal activities against both gram-positive S. aureus and gram-negative E. coli. Adhesion-induced GOx-catalyzed reaction also alleviates the biofouling of membrane, suggesting its extendibility to various engineering systems being suffered by biofouling. This study of biocatalytic elastomer has demonstrated its new opportunities for the facile and on-demand enzyme-catalyzed reactions in various environmental applications, such as bactericidal treatment, water treatment/purification, and pollutant degradation.

A Platinized Carbon Fiber Microelectrode-Based Oxidase Biosensor for Amperometric Monitoring of Lactate in Brain Slices.

BACKGROUND: Direct and real-time monitoring of lactate in the extracellular space can help elucidate the metabolic and modulatory role of lactate in the brain. Compared to in vivo studies, brain slices allow the investigation of the neural contribution separately from the effects of cerebrovascular response and permit easy control of recording conditions. METHODS: We have used a platinized carbon fiber microelectrode platform to design an oxidase-based microbiosensor for monitoring lactate in brain slices with high spatial and temporal resolution operating at 32 °C. Lactate oxidase (Aerococcus viridans) was immobilized by crosslinking with glutaraldehyde and a layer of polyurethane was added to extend the linear range. Selectivity was improved by electropolymerization of m-phenylenediamine and concurrent use of a null sensor. RESULTS: The lactate microbiosensor exhibited high sensitivity, selectivity, and optimal analytical performance at a pH and temperature compatible with recording in hippocampal slices. Evaluation of operational stability under conditions of repeated use supports the suitability of this design for up to three repeated assays. CONCLUSIONS: The microbiosensor displayed good analytical performance to monitor rapid changes in lactate concentration in the hippocampal tissue in response to potassium-evoked depolarization.

Tuning Immobilized Enzyme Features by Combining Solid-Phase Physicochemical Modification and Mineralization.

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.

Design and Evaluation of a Lactate Microbiosensor: Toward Multianalyte Monitoring of Neurometabolic Markers In Vivo in the Brain.

Direct in vivo measurements of neurometabolic markers in the brain with high spatio-temporal resolution, sensitivity, and selectivity is highly important to understand neurometabolism. Electrochemical biosensors based on microelectrodes are very attractive analytical tools for continuous monitoring of neurometabolic markers, such as lactate and glucose in the brain extracellular space at resting and following neuronal activation. Here, we assess the merits of a platinized carbon fiber microelectrode (CFM/Pt) as a sensing platform for developing enzyme oxidase-based microbiosensors to measure extracellular lactate in the brain. Lactate oxidase was immobilized on the CFM/Pt surface by crosslinking with glutaraldehyde. The CFM/Pt-based lactate microbiosensor exhibited high sensitivity and selectivity, good operational stability, and low dependence on oxygen, temperature, and pH. An array consisting of a glucose and lactate microbiosensors, including a null sensor, was used for concurrent measurement of both neurometabolic substrates in vivo in the anesthetized rat brain. Rapid changes of lactate and glucose were observed in the cortex and hippocampus in response to local glucose and lactate application and upon insulin-induced fluctuations of systemic glucose. Overall, these results indicate that microbiosensors are a valuable tool to investigate neurometabolism and to better understand the role of major neurometabolic markers, such as lactate and glucose.