Jiayi Song

DNA directed immobilization enzyme on polyamidoamine tethered magnetic composites with high reusability and stability

The development of new strategies for stabilizing or improving the activities of enzymes has attracted considerable interest because of the wide range of potential applications and high cost of the native enzymes. In this study, a novel trypsin immobilization procedure in which the enzyme was immobilized using polyamidoamine (PAMAM) dendrimer modified magnetic nanoparticles as carriers through DNA-directed immobilization was developed for the first time. The optimal DNA base pairs and the optimal generation of PAMAM on the enzymatic activity were 24 bases and G3.0PAMAM, respectively. The trypsin binding capacity of the immobilized trypsin was 74.6 mg g-1, and the Km and Vmax values were 1.23 mM and 468.35 μmol min-1 mg protein-1, respectively. The immobilized trypsin reactor exhibited excellent reusability and stability properties without significant loss in enzymatic activity. Notably, the high level of enzymatic activity remained at more than 63% after 82 cycles, with only a slight decrease (above 88%) after 14 weeks of continuous use at one-week intervals. The high reversibility and reproducibility of this trypsin dynamic immobilization strategy were also investigated. The significantly improved digestion performance of the immobilized trypsin composites was further demonstrated by digesting cytochrome C, myoglobin and glycated hemoglobin, with sequence coverages of 78%, 99% and 88%, respectively, which were higher than those obtained for the free enzyme. This system therefore shows great potential in high throughput enzymatic assays and proteome analysis.

A self-directed and reconstructible immobilization strategy: DNA directed immobilization of alkaline phosphatase for enzyme inhibition assays

This study focused on the development of a common method for the reversible and self-directed immobilization of enzymes based on a DNA-directed immobilization (DDI) technique. The successful anchoring of alkaline phosphatases to the surfaces of magnetic nanoparticles was confirmed using confocal laser scanning microscopy, thermogravimetric analysis and a vibrating sample magnetometer. The length of the DNA linker was optimized, with a base number of 24 providing maximum efficiency for the enzymolysis. Notably, the surface of alkaline phosphatase-functionalized magnetic nanoparticles could be regenerated using a mild dehybridization process of DNA. Furthermore, the resulting single-stranded probe DNA-modified magnetic nanoparticles could be reused to immobilize the alkaline phosphatase, which suggested that the DNA-functionalized surface of magnetic nanoparticles exhibited good reversibility. The biological activity of anchored alkaline phosphatases is evaluated in an enzyme inhibition assay. The results revealed that theophylline exhibited greater inhibitory activity than L-phenylalanine. The proposed protocol demonstrates a simple, mild and economic pathway for fabricating enzyme modified nanomaterial and can be applied in the high-throughput screening of inhibitors.

High Activity and Convenient Ratio Control: DNA-Directed Coimmobilization of Multiple Enzymes on Multifunctionalized Magnetic Nanoparticles

The development of new methods for fabricating artificial multienzyme systems has attracted much interest because of the potential applications and the urgent need for multienzyme catalysts. Controlling the enzyme ratio is critical for improving the cooperative enzymatic activity in multienzyme systems. Herein, we introduce a versatile strategy for fabricating a multienzyme system by coimmobilizing horseradish peroxidase (HRP) and glucose oxidase (GOx) on magnetic nanoparticles multifunctionalized with dopamine derivatives through DNA-directed immobilization. This multienzyme system exhibited precise enzyme ratio control, high catalytic efficiency, magnetic retrievability, and enhanced stability. The enzyme ratio was conveniently adjusted, as required, by regulating the quantity of functional groups on the multifunctionalized nanoparticles. The optimal mole ratio of GOx/HRP was 2:1. The Michaelis constant Km and specificity constant (kcat/Km, where kcat is the catalytic rate constant) of the multienzyme system were 1.41 mM and 5.02 s-1 mM-1, respectively, which were approximately twice the corresponding values of free GOx&HRP. The increased bioactivity of the multienzyme system was ascribed to the colocalization of the involved enzymes and the promotion of DNA-directed immobilization. Given the wide variety of possible enzyme associations and the high efficiency of this strategy, we believe that this work provides a new route for the fabrication of artificial multienzyme systems and can be extended for a wide range of applications in diagnosis, biomedical devices, and biotechnology.

Attachment of enzymes to hydrophilic magnetic nanoparticles through DNA-directed immobilization with enhanced stability and catalytic activity

The major barriers to the use of enzymes in practical applications are their insufficient stability and easy inactivation under processing conditions. In this work, we developed a promising platform that allows for the simple and effective immobilization of enzymes on hydrophilic polydopamine (PDA) modified magnetic nanoparticles through DNA directed immobilization. Taking α-chymotrypsin (ChT) as an example, the well-designed immobilized enzyme in this study exhibited excellent kinetic performance, and the apparent Vmax and catalytic efficiency (kcat/Km) values of the DNA immobilized enzyme were 15.77 mM min−1 mg protein−1 and 4.05 s−1 mM−1, which were more than 6.0- and 5.9-fold enhanced compared to that of the free enzyme, respectively. The DNA immobilized enzyme exhibited promising thermal stability, which could preserve promising enzymatic activities of about 83% after 60 min incubation at 60 °C. The DNA immobilized enzyme also exhibited excellent long-term storage and incubation stability, which could preserve more than 89% of the initial activity at 4 °C for 35 days and more than 77% after 47 h of incubation, respectively. In addition, the DNA immobilized ChT exhibited excellent reusability, which showed a high degree of activity (more than 84%) after 10 cycles. Notably, the enzyme immobilization procedure exhibited high reversibility and reproducibility, and the magnetic nanoparticle surfaces were successfully regenerated and cycled through DNA strand displacement reactions for subsequent enzyme immobilization, which could retrieve more than 98% of the enzymatic activity. A significantly improved protein digestion efficiency was achieved with this immobilized ChT within 10 min, and the obtained sequence coverages were more than 1.3-fold higher when compared to that obtained by conventional in-solution digestion for 12 h. Therefore, this work exhibits a promising alternative platform for the efficient immobilization of industrially important enzymes and their broad applications.

Efficient immobilization of enzymes onto magnetic nanoparticles by DNA strand displacement: a stable and high-performance biocatalyst

Enzymes usually have poor thermal and operational stability as well as limited reuse cycles, which greatly limit their practical applications. This study reports a novel strategy for enzyme immobilization based on toehold-mediated DNA strand displacement on modified magnetic nanoparticles using alkaline phosphatase (ALP) and horseradish peroxidase (HRP) as different model enzymes. The immobilized enzyme procedure by DNA strand displacement exhibited high reversibility and reproducibility, and the mild, convenient approach achieved successive enzyme immobilization through triggering a toehold-mediated DNA strand displacement reaction. The immobilized HRP exhibited promising thermal stability after 50 min incubation at 50 °C and 60 °C, which were about 6.5- and 9.7-fold greater than the free enzyme, respectively. Notably, the immobilized enzyme exhibited excellent long-term storage stability, and the enzymatic activities were about 34.4- and 24.9-fold greater than free HRP after storage at 4 °C for 68 days and at room temperature for 9 days, respectively. The immobilized enzyme also preserved high performance towards long-term incubation stability, compared to the free enzyme, and excellent reusability, which showed a high degree of activity (more than 73%) after 5 cycles. Thus, the developed strategy exhibited a promising alternative platform with high magnetic responsiveness and significantly enhanced properties for the immobilization of important enzymes and their broad applications.

Multifunctional magnetic particles for effective suppression of non-specific adsorption and coimmobilization of multiple enzymes by DNA directed immobilization

The overall stability and activity of immobilized enzyme systems have suffered from non-specific adsorption immobilization of enzymes at the surface of carriers through electrostatic and hydrophobic interactions. Elimination of these non-specific adsorptions of enzymes on the surface of carriers is critical for enzyme reactions. Herein, for the first time, we have prepared zwitterion-functionalized magnetic particles with amino, phosphonate, and thiol functional groups to coimmobilize glucose oxidase and horseradish peroxidase by DNA directed immobilization. The zwitterionic surface of the multifunctional magnetic particles could efficiently suppress non-specific adsorption of different kinds of enzymes, and the immobilized multienzyme catalyst without non-specifically adsorbed enzymes exhibited excellent enzymatic activity, stability, and reusability compared with those of free and non-specifically adsorbed enzymes. The immobilized multienzymes maintained 87% of their enzymatic activity after two weeks of storage at 4 °C and 58% of their enzymatic activity after 90 min of thermal incubation at 60 °C. Furthermore, the immobilized multienzymes exhibited more than 87% enzymatic activity after reuse for 10 cycles. The apparent Km and catalytic efficiency (kcat/Km) values of the immobilized multienzymes were 12.6 mM and 4.03 s-1 mM-1, respectively, which were 0.2- and 11.8-fold better than those of the free enzymes, indicating effective cascade efficiency and substrate affinity. The optimal ratio of GOx and HRP was 1 : 5, and the prepared immobilized enzymes could detect low concentrations of glucose (0.5 μM) with excellent selectivity. Therefore, we believe that the strategy developed in this study can be widely applied in biotechnology, industrial catalysis, and biomedical engineering.

DNA-directed trypsin immobilization on a polyamidoamine dendrimer-modified capillary to form a renewable immobilized enzyme microreactor

A novel type of trypsin capillary microreactor was developed based on a DNA-directed immobilization (DDI) technique applied to a fused-silica capillary modified with polyamidoamine (PAMAM) dendrimers. Trypsin binding to the inner wall of the capillary was confirmed by confocal laser scanning microscopy. The properties of the trypsin-DNA conjugated, PAMAM-modified capillary microreactor were investigated by monitoring hydrolysis of Nα-benzoyl-L-arginine ethyl ester. Through the hybridization and dehybridization of the DNA, the inner wall of the capillary functionalized with trypsin can be regenerated, thus indicating the renewability of this enzyme microreactor. In addition, these results demonstrated that introduction of PAMAM enabled higher amounts of trypsin to be immobilized, markedly improving the enzymolysis efficiency, compared with traditional modified capillaries. The digestion performance of the trypsin capillary microreactor was further evaluated by digesting cytochrome C, and a peptide numbers of 8, and a sequence coverage of 59% were obtained. This renewable and efficient immobilized trypsin capillary microreactor combines advantages of both DDI technology and PAMAM, and is potentially adaptable to high-throughput enzyme assays in biochemical and clinical research.