Gaofei Liu,Tiantong Cheng,Jianlin Chu,Sui Li,Bingfang He
Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) fromAneurinibacillus migulanusAM007 was cloned and heterologously expressed inEscherichia coliBL21(DE3). TheAmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0–9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived fromBrevibacillus borstelensisLK01 andAmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio ofBbPyNP:AmPNP (2:20 μg/mL). Thus, the new trimericAmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.Keywords:trimeric nucleoside phosphorylase;thermostability;purine nucleoside analogs;enzyme ratio
Ye Yang,Ruiqi Zhang,Bingnan Zhou,Jiayi Song,Ping Su,Yi Yang
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.
Glutathione-capped water-soluble CuInS/ZnS quantum dots (QDs) were prepared by a microwave-assisted method. Their fluorescence, with excitation/emission peaks at 380/570 nm, is found to be quenched by hydrogen peroxide (H 2 O 2 ) that is produced by the uricase catalyzed oxidation of uric acid (UA) and oxygen. The findings are used in a quenchometric method for the determination of UA. The effects of different ligands on the QDs, of pH value, buffers, enzyme ratio and reaction time were optimized. The detection limit for UA is 50 nM which is lower than other QD-based method, and the detection ranges extends from 0.25–4.0 μM. The assay is simple and sensitive, and no further modification of the QDs is required.
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