high specific activity
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2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.prot103127
Author(s):  
Edward A. Greenfield

In an antigen capture assay for hybridoma screening, the detection method identifies the presence of the antigen. Often this is achieved by labeling the antigen directly. In this assay, the polyvinyl chloride (PVC) wells of a high-binding-capacity ELISA plate are first coated with an affinity-purified rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are “captured” on the coated PVC surface and detected by screening with biotin- or histidine (His)–tagged antigen. The antigen can be labeled to a high specific activity and thus very little antigen is required for this procedure.


Author(s):  
Li-Jian Zhou ◽  
Li-Bin Guo ◽  
Wei Wei ◽  
Zhi-Xiang Lv ◽  
Ye-Wang Zhang

Chondroitin AC lyase (ChSaseAC) is one of the essential polysaccharides lyases in low molecular chondroitin sulfate production. In this work, a novel PrChSaseAC from Pedobacter rhizosphaerae was successfully cloned, expressed in Escherichia coli. After optimizing the induction, the recombinant PrChSaseAC could be expressed efficiently at 0.1 mM IPTG, 25°C, and 12 h induction. Then, it was purified with Ni-NTA affinity chromatography. The characterization of the purified PrChSaseAC showed that it had high specific activity and good storage stability, which would favor the production of low molecular weight chondroitin sulfate. It also displayed activity toward chondroitin sulfate C and hyaluronic acid. PrChSaseAC had the highest activity at pH 7.5, 37°C, 10 mM Ca2+, and 5 mg/ml of chondroitin sulfate A. Molecular docking of substrate and enzyme showed the interactions between the enzyme and substrate; it revealed that the enzyme showed high activity to CS-A and hyaluronic acid, but lower activity to CS-C attributed to the structure of the binding pocket. The high stability and specific activity of the enzyme will benefit the industrial production or clinical treatment.


2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ling Zheng ◽  
Zilong Guo ◽  
Shengsheng Cao ◽  
Benwei Zhu

AbstractThe cold-active pectate lyases have drawn increasing attention in food and biotechnological applications due to their ability to retain high catalytic efficiency under lower temperatures, which could be helpful for energy saving, cost reduction and flavor preservation. Herein, a new cold-tolerant pectate lyase (ErPelPL1) gene from Echinicola rosea was cloned and heterologously expressed in Escherichia coli. Interestingly, ErPelPL1 retained high catalytic activity even at a low temperature (4 °C). ErPelPL1 exhibited optimal activity at 35 ℃, pH 8.0 with 1 mM of Ca2+. It showed high specific activity towards polygalacturonic acid (34.7 U/mg) and sodium polygalacturonate (59.3 U/mg). The combined thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC) and electrospray ionization mass spectrometry (ESI-MS) results indicated that ErPelPL1 endolytically degraded pectic substances into the oligosaccharides with degrees of depolymerization (Dps) of 1–6. In conclusion, this study mainly conducted biochemical characterization and product analysis of a cold-tolerant pectate lyase. Therefore, it provides a promising enzyme candidate for food and biotechnological applications. Graphical Abstract


2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Liya Wang ◽  
Lucia Unger ◽  
Hanan Sharif ◽  
Staffan Eriksson ◽  
Vinzenz Gerber ◽  
...  

Abstract Background Thymidine kinase 1 (TK1) plays a key role in the synthesis of deoxythymidine triphosphate (dTTP) and is thus important for DNA replication and cell proliferation. The expression of TK1 is highest during S-phase, and it is rapidly degraded after mitosis. In cancer cells, TK1 is upregulated, resulting in leakage of excess TK1 into the blood. Consequently, serum TK1 has been used as a diagnostic and prognostic cancer biomarker, mainly in human medicine. The aims of this work were to characterize equine TK1 and to evaluate its suitability as a serum biomarker for equine lymphoma. Results Equine TK1 was cloned, expressed in E. coli and affinity purified. The purified recombinant horse TK1 showed broad substrate specificity, phosphorylating pyrimidine deoxyribo- and ribonucleosides and, to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor. Serum TK1 activity was measured in samples collected from horses with confirmed or suspected lymphoma and control horses with and without concurrent diseases. Serum TK1 activity levels were significantly higher in horses with lymphoma (p <  0.0005) and suspected lymphoma (p <  0.02) and in tumour-free groups with diverse diseases (p <  0.03) than in controls without concurrent diseases. There was a significant difference between the lymphoma group and the tumour-free group with diverse diseases (p <  0.0006). Furthermore, receiver operating characteristic analysis revealed a sensitivity of 0.86, a specificity of 0.95 and an AUC (area under the curve) of 0.92 compared to the controls without concurrent diseases, with a sensitivity of 0.97, a specificity of 0.71 and an AUC of 0.88 when compared with the tumour-free group with diverse diseases. Conclusion Equine TK1 showed high specific activity and broader substrate specificity than human TK1. Anticancer and antiviral thymidine analogues were efficiently phosphorylated by horse TK1, suggesting that these analogues might be good candidates for chemotherapy in horses. Serum TK1 activity was significantly higher in horses with lymphoma than in controls. ROC analysis indicated that serum TK1 could serve as a promising cancer biomarker in horses.


Catalysts ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1410
Author(s):  
Anastasia Sedova ◽  
Lenka Rucká ◽  
Pavla Bojarová ◽  
Michaela Glozlová ◽  
Petr Novotný ◽  
...  

Industries such as mining, cokemaking, (petro)chemical and electroplating produce effluents that contain free cyanide (fCN = HCN + CN−). Currently, fCN is mainly removed by (physico)chemical methods or by biotreatment with activated sludge. Cyanide hydratases (CynHs) (EC 4.2.1.66), which convert fCN to the much less toxic formamide, have been considered for a mild approach to wastewater decyanation. However, few data are available to evaluate the application potential of CynHs. In this study, we used a new CynH from Exidia glandulosa (protein KZV92691.1 designated NitEg by us), which was overproduced in Escherichia coli. The purified NitEg was highly active for fCN with 784 U/mg protein, kcat 927/s and kcat/KM 42/s/mM. It exhibited optimal activities at pH approximately 6–9 and 40–45 °C. It was quite stable in this pH range, and retained approximately 40% activity at 37 °C after 1 day. Silver and copper ions (1 mM) decreased its activity by 30–40%. The removal of 98–100% fCN was achieved for 0.6–100 mM fCN. Moreover, thiocyanate, sulfide, ammonia or phenol added in amounts typical of industrial effluents did not significantly reduce the fCN conversion, while electroplating effluents may need to be diluted due to high fCN and metal content. The ease of preparation of NitEg, its high specific activity, robustness and long shelf life make it a promising biocatalyst for the detoxification of fCN.


Author(s):  
Daniela Ordaz-Pérez ◽  
José Antonio Fuentes-Garibay ◽  
Martha Guerrero-Olazarán ◽  
José María Viader-Salvadó

2021 ◽  
Vol 8 ◽  
Author(s):  
Reinhard Heinke ◽  
Eric Chevallay ◽  
Katerina Chrysalidis ◽  
Thomas E. Cocolios ◽  
Charlotte Duchemin ◽  
...  

Thulium-167 is a promising radionuclide for nuclear medicine applications with potential use for both diagnosis and therapy (“theragnostics”) in disseminated tumor cells and small metastases, due to suitable gamma-line as well as conversion/Auger electron energies. However, adequate delivery methods are yet to be developed and accompanying radiobiological effects to be investigated, demanding the availability of 167Tm in appropriate activities and quality. We report herein on the production of radionuclidically pure 167Tm from proton-irradiated natural erbium oxide targets at a cyclotron and subsequent ion beam mass separation at the CERN-MEDICIS facility, with a particular focus on the process efficiency. Development of the mass separation process with studies on stable 169Tm yielded 65 and 60% for pure and erbium-excess samples. An enhancement factor of thulium ion beam over that of erbium of up to several 104 was shown by utilizing laser resonance ionization and exploiting differences in their vapor pressures. Three 167Tm samples produced at the IP2 irradiation station, receiving 22.8 MeV protons from Injector II at Paul Scherrer Institute (PSI), were mass separated with collected radionuclide efficiencies between 11 and 20%. Ion beam sputtering from the collection foils was identified as a limiting factor. In-situ gamma-measurements showed that up to 45% separation efficiency could be fully collected if these limits are overcome. Comparative analyses show possible neighboring mass suppression factors of more than 1,000, and overall 167Tm/Er purity increase in the same range. Both the actual achieved collection and separation efficiencies present the highest values for the mass separation of external radionuclide sources at MEDICIS to date.


2021 ◽  
Author(s):  
Peter Wied ◽  
Francesco Carraro ◽  
Juan Bolivar ◽  
Christian Doonan ◽  
Paolo Falcaro ◽  
...  

Hydrogen bonded organic frameworks (HOFs) with enzymes incorporated during their bottom-up synthesis represent functional biocomposites with promising applications in catalysis and sensing. High enzyme loading while preserving high specific activity is fundamental for development, but to combine these biospecific features with a porous carrier is an unmet challenge. Here, we explored synthetic incorporation of D-amino acid oxidase (DAAO) with metal-free tetraamidine/tetracarboxylate-based BioHOF-1. Comparison of different DAAO forms in BioHOF-1 incorporation revealed that N-terminal enzyme fusion with the positively charged module Zbasic2 (Z-DAAO) promotes the loading (2.5-fold; ~500 mg g-1) and strongly boosts the activity (6.5-fold). To benchmark the HOF composite with metal-organic framework (MOF) composites, Z-DAAO was immobilized into the zeolitic imidazolate framework-8 (ZIF-8), the relatively more hydrophilic analogue metal azolate framework-7 (MAF-7). While sensitivity to the framework environment limited the activity of DAAO@MAF-7 (3.2 U mg-1) and DAAO@ZIF-8 (≤ 0.5 U mg-1), the activity of DAAO@BioHOF-1 was comparable (~45%) to that of soluble DAAO (50.1 U mg-1) and independent of the enzyme loading (100 – 500 mg g-1). The DAAO@BioHOF-1 composites showed superior activity with respect to every reported carrier for the same enzyme and excellent stability during solid catalyst recycling. Collectively, our results show that the fusion of the enzyme with a positively charged protein module enables the synthesis of highly active HOF biocomposites suggesting the use of genetic engineering for the preparation of biohybrid systems with unprecedented properties.


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