Direct Immobilization Research Articles

Direct purification and immobilization of his-tagged enzymes using unmodified nickel ferrite NiFe2O4 magnetic nanoparticles

Purification of valuable engineered proteins and enzymes can be laborious, costly, and generating large amount of chemical waste. Whilst enzyme immobilization can enhance recycling and reuse of enzymes, conventional methods for immobilizing engineered enzymes from purified samples are also inefficient with multiple-step protocols, regarding both the carrier preparation and enzyme binding. Nickel ferrite magnetic nanoparticles (NiFe2O4 MNPs) offer distinct advantages in both purification and immobilization of enzymes. In this work, we demonstrate the preparation of NiFe2O4 MNPs via a one-step solvothermal synthesis and their use in direct enzyme binding from cell lysates. These NiFe2O4 MNPs have showed an average diameter of 8.9 ± 1.7 nm from TEM analysis and a magnetization at saturation (Ms) value of 53.0 emu g–1 from SQUID measurement. The nickel binding sites of the MNP surface allow direct binding of three his-tagged enzymes, d-phenylglycine aminotransferase (d-PhgAT), Halomonas elongata ω-transaminase (HeωT), and glucose dehydrogenase from Bacillus subtilis (BsGDH). It was found that the enzymatic activities of all immobilized samples directly prepared from cell lysates were comparable to those prepared from the conventional immobilization method using purified enzymes. Remarkably, d-PhgAT supported on NiFe2O4 MNPs also showed similar activity to the purified free enzyme. By comparing on both carrier preparation and enzyme immobilization protocols, use of NiFe2O4 MNPs for direct enzyme immobilization from cell lysate can significantly reduce the number of steps, time, and use of chemicals. Therefore, NiFe2O4 MNPs can offer considerable advantages for use in both enzyme immobilization and protein purification in pharmaceutical and other chemical industries.

Synergistic co-catalytic nanozyme system for highly efficient one-pot colorimetric sensing at neutral pH: Combining molybdenum trioxide and Fe(III)-Modified covalent triazine framework

This study investigates the co-catalytic capabilities of MoO3 nanosheets in enhancing the enzyme-like catalytic activity of a two-dimensional ultrathin Fe(III)-modified covalent triazine framework (Fe-CTF) under neutral pH conditions. The unique physicochemical surface properties and two-dimensional structures of Fe-CTF enable the direct immobilization of native enzymes (glucose oxidase (GOD) and xanthine oxidase (XOD)) through adsorption, eliminating the need for chemical processes. Efficient immobilization of the native enzymes within the Fe-CTF/GOD(XOD) hybrid is achieved through multipoint attachment involving various interactions. The Fe-CTF/MoO3 co-catalytic system exhibits enzyme-mimicking activity at neutral pH and, when combined with the high catalytic activity of the immobilized native enzymes, enables the development of a colorimetric method for glucose detection. This method demonstrates excellent facilitation, rapidity, sensitivity, and selectivity, with a linear detection range of 50–1000 μM and a limit of detection of 8.8 μM for glucose. Furthermore, a straightforward one-pot colorimetric method is established for screening XOD inhibitors. The inhibitory potential of a crude extract derived from Chinese water chestnut peel on XOD activity is evaluated using this method. The findings of this study pave the way for the utilization of nanozyme/native enzyme hybrids in pH-neutral conditions for one-pot colorimetric sensing. This work contributes to the advancement of enzyme-based sensing technologies and holds promise for various applications in biosensing and biomedical research.

Design of a novel multi-layer enzyme membrane reactor for low-fouling, tailored production of oligodextran

Enzymatic conversion processes face challenges in controlling oligosaccharide molecular weight (Mw). Enzymatic membrane reactors (EMRs) with immobilized enzymes address this, but direct enzyme immobilization on the membrane surface can lead to deactivation and reduced hydrolysis efficiency. This study proposes a novel EMR configuration: a three-layer structure. An electrospun porous fibrous layer, modified with PDA, TA, and APTES, serves as a mechanical support layer. A commercial separation membrane is positioned below. This configuration enhances enzyme activity and selectivity. Using a “fouling-induced” technique, immobilized activity of the enzyme (i.e. dextranase) significantly increased to 11.5 µmol-isomaltose/min, surpassing incubation-immobilized dextranase (0.075 µmol-isomaltose/min). The additional layer preserves catalytic patterns, reducing fouling and ensuring high selectivity. The EMR configuration excels in producing low Mw oligosaccharides. The catalytic layer achieves 11.3 µmol-isomaltose/min, while the membrane exhibits exceptional selectivity and stability. The hydrophilic RC10 membrane with small pores performs best. This study highlights the potential of the EMR system for efficient production of stable low Mw oligosaccharides. Insights into optimizing enzyme immobilization strategies and membrane selection benefit enzymatic conversion processes.

Graphene oxide nanosheets immobilised on honeycomb pore structure of pomelo peel for enhanced removal of methylene blue.

Herein, for the first time, a simple, green, direct immersion immobilisation method is reported for graphene oxide (GO) nanosheets using pomelo peel (PL) as a substrate. A GO/PL porous sponge was successfully prepared and used to remove methylene blue (MB) from dye wastewater. Considering the porosity, hydrophilicity and elasticity of the PL, the PL was placed in a GO aqueous suspension through direct immersion immobilisation. The carboxyl and hydroxyl groups in the peel could effectively capture and immobilise the GO nanosheets. GO was adsorbed into the PL pores and dispersed throughout the PL. Finally, the prepared super-hydrophilic and elastic GO/PL exhibited excellent adsorption performance towards MB in dye wastewater. The adsorption results revealed that the adsorption behaviour was consistent with the Langmuir isotherm and quasi-secondary kinetic models. The maximum equilibrium adsorption capacity of GO/PL towards MB was 124.2mg/g, exceeding the values obtained for most of the previously reported adsorbents. Moreover, after five consecutive adsorption-desorption cycles, GO/PL retained 75% of its initial adsorption capacity. Mechanistic analysis revealed that pore filling, electrostatic attraction, ion exchange and hydrogen bonding interactions are the primary driving forces facilitating MB adsorption.

Optimization of the immunorecognition layer towards Brucella sp. on gold surface for SPR platform

A successful immunosensor is characterized by a proper antibody immobilization and orientation in order to enhance the antigen recognition. In this work, a thorough characterization of the antibody functionalized gold surface is performed to set up the best conditions to implement in an optical platform for the detection of Brucella sp. Two different strategies are evaluated, based on a random immobilization and on an oriented one: a direct antibody immobilization on carboxylic mixed polyethylene (PEG) self-assembled monolayer (SAM) or only carboxylic PEG SAM interface is compared to an oriented immobilization on a layer of protein G on the same PEG SAM interfaces. X-ray Photoelectron Spectroscopy (XPS), Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and contact angle (CA) are used to chemically characterize the gold functionalized surface and ToF-SIMS is also used to confirm the right antibody orientation. Optical characterization is applied to monitor the functionalization steps and fluorescence measurements are used to set up the proper experimental conditions and also to detect Brucella bacteria on the surface. Best results are obtained with a 10 ng/μl incubation solution of antibody immobilized, in an oriented way, on a mixed PEG SAM interface.

Electrochemical properties of hydroxyapatite immobilization material for potential cytosensor fabrication

Aim: The biorecognition unit of an electrochemical biosensor requires molecules that are immobilised to serve as a bridge between the recognition unit and the transducing surface. Unique materials that enhance immobilisation of biorecognition molecules and improve electrochemical signal transduction are important in overcoming challenges based on the sensitivity of biosensors. In this regard, the electrochemical properties (EPs) of hydroxyapatite (HAp) material for the direct immobilisation of cells was investigated. Methods: Snail shell HAp (SHAp) material was synthesised from Achatina achatina snail shells and phosphate-containing solutions. The SHAp material was characterised using X-ray diffractometry (XRD), Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy to determine the structural configuration, after which it was blended with a conductive polymer [poly(3,4-ethylenedioxythiophene): poly-4-styrene sulfonate (PEDOT: PSS)] to improve the electrochemical responses. The SHAp/PEDOT: PSS blend was used to modify a screen-printed carbon electrode (SPCE) by drop-casting, followed by seeding of pheochromocytoma (PC 12) and human embryonic kidney (HEK)-293T cells on the modified SPCE to record the EP using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Red blood cells (RBCs) were used as a control. Results: The CV analysis showed lower peak currents for HEK 293T (50 µA) and PC 12 (120 µA) compared to the RBC (230 µA). Also, the EIS showed impedance values of 0.70 for HEK 293T, 0.62 for PC 12, and 0.52 mΩ for RBC. The findings indicate that SHAp/PEDOT: PSS enables the differentiation of cell proliferation signals through voltammetric and impedimetric measurements. Conclusions: The unique current and impedance differences among the cells could serve as potential markers for rapid cell detection.

Toward the early diagnosis of tuberculosis: A gold particle-decorated graphene-modified paper-based electrochemical biosensor for Hsp16.3 detection

Tuberculosis (TB) currently remains a major life-threatening disease as it can be fatal if not treated properly or in a timely manner. Herein, we first describe a disposable and cost-effective paper-based electrochemical biosensor based on a gold particle-decorated carboxyl graphene (AuPs/GCOOH)-modified electrode for detecting heat shock protein (Hsp16.3), which is a specific biomarker indicating the onset of TB infection. The device pattern was first engineered to facilitate detection procedures and printed on low-cost filter paper to create hydrophobic and hydrophilic regions using a wax printing technique. Immunoassays proceeded in a half-sandwich format because it is a reagent-less approach and requires no labeling step. The fabrication of the immunosensor began with GCOOH drop casting, the electrochemical deposition of AuPs, and the establishment of a biorecognition layer against Hsp16.3 utilizing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-sulfo standard chemistry. The appearance of Hsp16.3 resulted in a substantial decrease in the electrochemical signal response of the redox probe employed [Fe (CN)6]3−/4− due to the created immunocomplexes that possess insulation properties. GCOOH enables direct antibody immobilization, and AuPs enhance the electrochemical properties of the sensor. This proposed immunosensor, while requiring only a miniscule sample volume (5 μL), achieved superior performance in terms of the limit of detection, measuring at 0.01 ng/mL. Our platform was confirmed to be highly specific to Hsp16.3 and can rapidly detect TB-infected sera without necessitating any pre-enrichment (20 min), making it an alternative and particularly suitable for the early diagnosis of TB in resource-scarce countries.

Direct covalent immobilizaion of chiral phosphines to hollow mesoporous polystyrene nanospheres (HMPNs) for efficient heterogeneous enantioselective [2+3] cycloaddition

Low-cost and convenient strategy for the direct covalent immobilizaion of chiral phosphines into structurally superior solid supports is desirable for efficient heterogeneous enantioselective catalysis. In this paper, a universal and convenient method for the direct covalent immobilization of chiral phosphines into HMPNs is developed via Friedel-Craft alkylation to fabricate supported chiral phosphines at a low cost. Using (S)-2,2´-bis(diphenylphosphino)-1,1´-binaphthyl [(S)-BINAP] and (R,R)-diphenylbis-[(o-anisyl)-methylenephosphine] [(R,R)-DIPAMP] as examples, the as-fabricated HMPNs-supported (S)-BINAP and (R,R)-DIPAMP, namely BINAP@HMPNs and DIPAMP@HMPNs, possess a well-shaped spherical morphology with hollow interior and thin mesoporous shell, enabling reactants to easily access to catalytic sites. In enantioselective [2+3] cycloaddition, DIPAMP@HMPNs exhibits good yields (70–86 %) with excellent enantioselectivities (94–98 %ee). Furthermore, DIPAMP@HMPNs shows a good mechanical stability of spherical morphology and mesoporous structure in its reuses. Moreover, the oxidation of DIPAMP leads to the gradual deactivation of DIPAMP@HMPNs including yield and enantioselectivity, which can be restored by the reduction of tetramethyldisiloxane (TMDS)/Ti(OiPr)4.

Direct immobilization of Se(IV) from acidic Se(IV)-rich wastewater via ferric selenite Co-precipitation

The attenuation of acidic Se(IV)-rich wastewater, including those associated with acid mine drainage (AMD) and nonferrous metallurgical wastewater (NMW), presents a serious environmental challenge. This study investigates the effects of diverse factors from pH values to Se(IV)/Fe(III) molar ratios, initial Se(IV) concentrations, and alkali neutralization agents on the direct co-precipitation of ferric selenites in AMD and NMW systems involving different orders of Fe(III) and alkali addition. Our results show that amorphous sulfate-substituted ferric (hydrogen) selenite and Se(IV)-bearing ferrihydrite-schwertmannite are the major Se(IV)-attenuation solids except that gypsum is an additional phase in the NMW system with Ca(OH)2 neutralization. Produced ferric selenites achieve 98–99.8% of Se(IV) immobilization under optimal conditions of pH 4.5, Se(IV)/Fe(III) molar ratios of 0.0625–0.5, and initial Se(IV) concentrations of 0.15–1.3 mmol·L-1. Moreover, completing FeSO4+ and FeHSeO32+/FeSeO3+ complexes as well as different ferric selenite co-precipitates are shown to collectively control aqueous Se(IV) remaining. Specifically, three distinct trends of aqueous Se(IV) concentrations separately correspond to changes in the four factors. The co-precipitation in the NMW system via pH adjustment followed by Fe(III) addition is more efficient for Se(IV) fixation than that in the AMD system because of minimal complexation, concurrent Fe(III) hydrolysis, and enhanced ferric selenite co-precipitation in the former.

Directional immobilization of phage on the palladium-based nanozyme for colorimetric detection of Cronobacter sakazakii in powdered infant formula

Cronobacter sakazakii in powdered infant formula (PIF) can cause infections in newborns that are life-threatening. Establishing a High-performance evaluation method for C. sakazakii in the PIF is urgently needed. In this study, a chromogenic system based on phage-directed immobilized nanozyme for colorimetric measurement of C. sakazakii in PIF was constructed. Initially, our acquired phage EspYZU15 was electrostatically immobilized on the surface of a palladium-based nanozyme to produce EspYZU15 directionally immobilized Pd (EspYZU15@Pd). EspYZU15@Pd possesses outstanding peroxidase-like properties, catalyzing the chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. The ability of EspYZU15@Pd to recognize and trap host bacteria allows C. sakazakii to specifically inhibit the TMB chromogenic process catalyzed by EspYZU15@Pd. Based on this detection strategy, a visual quantification method for C. sakazakii ranging from 3.2 × 102 to 3.2 × 107 CFU mL−1 was developed, yielding a low limit of detection (LOD) of 3.6 × 101 CFU mL−1. This method was able to successfully detect C. sakazakii in PIF, with outstanding recoveries ranging from 95.8% to 104.9%. These characteristics indicate that the EspYZU15@Pd-based chromogenic system has considerable promise for the colorimetric determination of C. sakazakii in PIF.

Electrochemical Fine-Tuning of the Chemoresponsiveness of Langmuir-Blodgett Graphene Oxide Films.

Graphene oxide has been widely deployed in electrical sensors for monitoring physical, chemical, and biological processes. The presence of abundant oxygen functional groups makes it an ideal substrate for integrating biological functional units to assemblies. However, the introduction of this type of defects on the surface of graphene has a deleterious effect on its electrical properties. Therefore, adjusting the surface chemistry of graphene oxide is of utmost relevance for addressing the immobilization of biomolecules, while preserving its electrochemical integrity. Herein, we describe the direct immobilization of glucose oxidase onto graphene oxide-based electrodes prepared by Langmuir-Blodgett assembly. Electrochemical reduction of graphene oxide allowed to control its surface chemistry and, by this, regulate the nature and density of binding sites for the enzyme and the overall responsiveness of the Langmuir-Blodgett biofilm. X-ray photoelectron spectroscopy, surface plasmon resonance, and electrochemical measurements were used to characterize the compositional and functional features of these biointerfaces. Covalent binding between amine groups on glucose oxidase and epoxy and carbonyl groups on the surface of graphene oxide was successfully used to build up stable and active enzymatic assemblies. This approach constitutes a simple, quick, and efficient route to locally address functional proteins at interfaces without the need for additives or complex modifiers to direct the adsorption process.

Enhancing the purification performance of layered mica for thallium-containing wastewater by mechanochemical activation – An ingenious insight into the pathway of lattice state immobilization

The deep treatment of monovalent thallium (Tl+) and the study of Tl+ incorporation mechanism are of great value for water safety. However, current research objectives have focused on how to oxidize or precipitate Tl+, while neglecting to investigate the underlying mechanisms of the direct immobilization and stable storage state of Tl+. Therefore, the present study examines in detail how Tl+ is directly incorporated and stably stored by silicate minerals. In this study, an idea is proposed to enhance the purification of Tl-containing wastewater by using mechanically activated mica to stabilize Tl+ in the silicate structure in the form of lattice residue state. Elucidation of the removal mechanism was first performed with focus on Tl+ exchange with interlayer K+ and H(H2O)+ inside the silicate mineral based on the quantitative data and a series of characterizations such as XRD, XPS, FTIR, SEM-EDS and TG. As adsorbents for Tl-containing wastewater, both activated phlogopite and biotite for the purification of target wastewater demonstrated excellent performances at a dose of 2 g/L and a reaction time of 240 min, reaching quantitatively down to 23.88 μg/L and nearly 0 μg/L respectively, from an initial Tl+ concentration of 1203 μg/L, so that the effluent concentrations would meet the requirements of US EPA and Chinese discharge standards for industrial wastewater and drinking water. In addition, density functional theory (DFT) calculation was used to quantify the adsorption energy of Tl+ being solidified in the interlayer structure to further give insight into the mechanism of Tl+ immobilization by silicate mineral.

Chromoionophoric probe imbued porous polymer monolith as a Three-in-One Solid-state Naked-eye sensor for the selective sensing and recovery of Ultra-trace Lead, Mercury, and cadmium ions from Industrial/Environmental samples

This article presents a simple, low-cost, eco-benign, and portable naked-eye solid-state sensor for the selective and sensitive colorimetric detection and recovery of ultra-trace concentrations of Pb(II), Hg(II) and Cd(II) from environmental/industrial sources. For solid-state sensing, an indigenously synthesized polymer monolith, i.e., poly(VAn-EGDMA), in-built with high surface area and interlinked meso-/macro-pore network serves as a template for the voluminous and uniform decoration of chromoionophoric probe molecules, i.e., 2-((1H-benzo[d]imidazol-2-yl)diazenyl)-4-methoxyphenol (IDMP) onto the monolith perforations via. direct immobilization technique. The structural and surface topological properties of poly(VAn-EGDMA) monolith and probe-loaded polymer, i.e., IDMP@poly(VAn-EGDMA) sensor, are characterized through FE-SEM, HR-TEM, EDAX, XPS, p-XRD, SAED, FT-IR, BET and BJH analysis. The IDMP@poly(VAn-EGDMA) sensor forms a selective/stable metal complex through visually imposing enhanced charge transfer color transitions from its original canary yellow to dark denim blue, brownish green and lapis blue for varying concentrations of Pb(II), Hg(II) and Cd(II), respectively. The sensor reusability has been tested by decomplexing the IDMP-Mn+ complex with 0.1 M HCl and repeating the sensor performance consistently for eight consecutive cycles. The IDMP@poly(VAn-EGDMA) sensor exhibits pH-dependent ion-sensing, demonstrating a wide linear response range for Pb(II) (0–200 ppb), Hg(II) (0–150 ppb), and Cd(II) (0–150 ppb), with a detection limit of 0.44, 0.56, and 0.52 ppb, respectively. The three-in-one sensor exhibits pH-dependent sequential selectivity, chemical/thermal stability, and data reliability/reproducibility, thus proving effective for on-spot sensing of various sources of contaminated samples.

Protein engineering for natural product biosynthesis: expanding diversity for therapeutic applications

In this dispensation of the fourth industrial revolution, protein engineering has become a popular approach for increasing enzymatic activity, stability, and titer in the biosynthesis of natural products. This is attributed to its numerous advantages (over direct isolation from plants or via chemical synthesis), including decreasing or eliminating reaction byproducts, high precision, moderate handling of intricate and chemically unstable chemicals, overall reusability, and cost efficiency. Recently, protein engineering tools have advanced to redesign and enhance natural product biosynthesis. These methods include direct evolution, substrate engineering, medium engineering, enzyme engineering and immobilization, structure-assisted protein engineering, and advanced computational. Recent successes in implementing these emerging protein engineering technologies were critically discussed in this article. Also, the advantages, limitations, and applications in industrial and medical biotechnology were discussed. Last, future research directions and potential were also highlighted.

Improving toxic dye removal and remediation using novel nanocomposite fibrous adsorbent

Materials with regulated nanoscale morphology provide capabilities to remove dangerous pollutants from wastewater, by adsorption. In this study, the chitosan-based composite fibrous adsorbent was fabricated and used as a novel and facile eliminator in the process of removing cationic methyl orange (MO) from the water medium. Different methods were applied to check the properties of the desired composite fibrous adsorbent. The removal value showed that the chitosan-embedded composite fibrous adsorbent has super characteristics and can be removed the organic dye from the water. The influence of pH, time, temperature, eliminator quantity, and MO content was explored. With increasing time and eliminator quantity, the adsorption efficiency increased. The composite fibrous adsorbent with the advantage of its high functionality and combined micro-nanomorphology features, has emerged as a very promising candidate for obtaining versatile and robust adsorbents. This work presents how the tunned synthesis of composite fibrous adsorbent tailored their nanoarchitecture giving rise to adsorption capacities toward cleaning aqueous samples polluted with MO dye. The composite fibrous adsorbent was prepared by the direct immobilization method, with normal aging temperature, in order to study the effect of synthesis conditions on the adsorption properties of MO dye. The solution acidity was exhibited as the key factor, and a suitable pH of 5.50 was selected based on the efficiency. The competing ions were not adversely affected in the dye adsorption as defined by the stable bonding mechanism. The adsorption data were highly fitted with the Langmuir adsorption model with monolayer coverage. The determined maximum adsorption was 175.45 mg/g, which was comparable with the other forms of materials. The adsorbed MO dye elution was evaluated using ethanol and then the composite fibrous adsorbent was ready to use for MO dye adsorption after washing with water without significant loss in its functionality. Therefore, we explored the tunned morphology of composite fibrous adsorbent for obtaining performant adsorbents for the elimination of refractory pollutants in wastewater.

Influence of Immobilization Strategies on the Antibacterial Properties of Antimicrobial Peptide-Chitosan Coatings.

It is key to fight bacterial adhesion to prevent biofilm establishment on biomaterials. Surface immobilization of antimicrobial peptides (AMP) is a promising strategy to avoid bacterial colonization. This work aimed to investigate whether the direct surface immobilization of Dhvar5, an AMP with head-to-tail amphipathicity, would improve the antimicrobial activity of chitosan ultrathin coatings. The peptide was grafted by copper-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry by either its C- or N- terminus to assess the influence of peptide orientation on surface properties and antimicrobial activity. These features were compared with those of coatings fabricated using previously described Dhvar5-chitosan conjugates (immobilized in bulk). The peptide was chemoselectively immobilized onto the coating by both termini. Moreover, the covalent immobilization of Dhvar5 by either terminus enhanced the antimicrobial effect of the chitosan coating by decreasing colonization by both Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Relevantly, the antimicrobial performance of the surface on Gram-positive bacteria depended on how Dhvar5-chitosan coatings were produced. An antiadhesive effect was observed when the peptide was grafted onto prefabricated chitosan coatings (film), and a bactericidal effect was exhibited when coatings were prepared from Dhvar5-chitosan conjugates (bulk). This antiadhesive effect was not due to changes in surface wettability or protein adsorption but rather depended on variations in peptide concentration, exposure, and surface roughness. Results reported in this study show that the antibacterial potency and effect of immobilized AMP vary greatly with the immobilization procedure. Overall, independently of the fabrication protocol and mechanism of action, Dhvar5-chitosan coatings are a promising strategy for the development of antimicrobial medical devices, either as an antiadhesive or contact-killing surface.

Rational immobilization of l-threonine aldolase from Bacillus nealsonii for efficiently synthesis of l-syn-3-[4-(methylsulfonyl)] phenylserine

l-syn-3-[4-(methylsulfonyl)] phenylserine is a key industrial intermediate for the synthesis of florfenicol, thiamphenicol, and other products. l-Threonine aldolase can synthesize l-syn-3-[4-(methylsulfonyl)] phenylserine from glycine and 4-methylsulphonyl benzaldehyde directly. However, the selectivity of free l-threonine aldolase was difficult to meet the requirement of industrialization. Here, we selected the suitable site to insert His tag as immobilized site to improve the selectivity of l-threonine aldolase based on the molecular dynamics simulation and prior protein engineering. The phenanthroline was coupled to the surface of magnetic nanoparticles coated with silicon and thus enabled the selective and oriented immobilization of His-tagged l-threonine aldolase. Due to magnetic nanoparticles coated with silicon as supports, it was easy to separate the residual reactant and impurities from the immobilized enzyme by magnetic attraction. Moreover, the immobilization was combined with the purification of free enzymes through directional immobilization, which avoided heterozyme linking supports and improved the utilization efficiency of supports. Our study demonstrates an effective approach: how to analyze and select enzyme-targeted ligation sites in conjunction with previous modifications of engineered bacteria, and the immobilized enzyme obtained also provided a good candidate for the industrial production of l-syn-3-[4-(methylsulfonyl)] phenylserine.

Partial purification of crude lipase extract from Yarrowia lipolytica: Precipitation, aqueous two-phase systems (ATPS), and immobilization methods

Efforts have been concentrated on developing alternative methods of enzyme purification that are less costly and highly efficient. In this work, we evaluated three different methods for lipase purification from Yarrowia lipolytica, such as precipitation using ammonium sulfate, ethanol, or acetone; aqueous two-phase systems (ATPS) based on polyethylene glycol (PEG) and potassium phosphate; and direct immobilization. It was impossible to obtain stable precipitates of the enzyme due to the low concentration of total protein and the presence of biosurfactant produced by the microorganism. Different mixture compositions were selected for the partitioning study. Three ATPS showed selective partitioning of the target enzymes, i.e., lipase and protease migrated to opposite phases. In the ATPS composed of 13 wt% PEG-4000 and 10 wt% salts, it was possible to achieve a purification factor for lipase of 4.2. Purification by immobilization performed by lipase-lipase interactions showed three lipases of distinct sizes in the crude extract. In the immobilization method by hydrophobic supports, phenyl-agarose and butyl‑agarose were more selective in immobilizing than octyl-agarose. In the ion exchange immobilization method, only the lipases identified at 66 kDa and 41 kDa have an attraction for DEAE-agarose (anionic) and sulfopropyl-agarose (cationic) matrices.

Electrodeposited Carbonyl Functional Polymers as Suitable Supports for Preparation of the First-Generation Biosensors.

The aim of this electrochemical study was to ascertain which type of electrochemically deposited carbonyl functionalized polymer represents the most suitable electrode substrate for direct covalent immobilization of biological catalysts (enzymes). For this purpose, a triad of amperometric biosensors differing in the type of conductive polymers (poly-vanillin, poly-trans-cinnamaldehyde, and poly-4-hydroxybenzaldehyde) and in the functioning of selected enzymes (tyrosinase and alkaline phosphatase) has been compared for the biosensing of neurotransmitters (dopamine, epinephrine, norepinephrine, and serotonin) and phenyl phosphates (p-aminophenyl phosphate and hydroquinone diphosphate). The individual layers of the polymers were electrochemically deposited onto commercially available screen-printed carbon electrodes (type C110) using repetitive potential cycling in the linear voltammetric mode. Their characterization was subsequently performed by SEM imaging and attenuated total reflectance FTIR spectroscopy. Molecules of enzymes were covalently bonded to the free carbonyl groups in polymers via the Schiff base formation, in some cases even with the use of special cross-linkers. The as-prepared biosensors have been examined using cyclic voltammetry and amperometric detection. In this way, the role of the carbonyl groups embedded in the polymeric structure was defined with respect to the efficiency of binding enzymes, and consequently, via the final (electro)analytical performance.

Formation of a Conducting Polymer by Different Electrochemical Techniques and Their Effect on Obtaining an Immunosensor for Immunoglobulin G

In this work, a conducting polymer (CP) was obtained through three electrochemical procedures to study its effect on the development of an electrochemical immunosensor for the detection of immunoglobulin G (IgG-Ag) by square wave voltammetry (SWV). The glassy carbon electrode modified with poly indol-6-carboxylic acid (6-PICA) applied the cyclic voltammetry technique presented a more homogeneous size distribution of nanowires with greater adherence allowing the direct immobilization of the antibodies (IgG-Ab) to detect the biomarker IgG-Ag. Additionally, 6-PICA presents the most stable and reproducible electrochemical response used as an analytical signal for developing a label-free electrochemical immunosensor. The different steps in obtaining the electrochemical immunosensor were characterized by FESEM, FTIR, cyclic voltammetry, electrochemical impedance spectroscopy, and SWV. Optimal conditions to improve performance, stability, and reproducibility in the immunosensing platform were achieved. The prepared immunosensor has a linear detection range of 2.0-16.0 ng·mL-1 with a low detection limit of 0.8 ng·mL-1. The immunosensing platform performance depends on the orientation of the IgG-Ab, favoring the formation of the immuno-complex with an affinity constant (Ka) of 4.32 × 109 M-1, which has great potential to be used as point of care testing (POCT) device for the rapid detection of biomarkers.