RESUMEN
High amounts of toxic textile dyes are released into the environment due to coloring and wastewaters treatment processes' inefficiency. To remove dyes from the environment and wastewaters, researchers focused on applying immobilized enzymes due to mild reaction conditions and enzyme nontoxicity. Laccases are oxidases with wide substrate specificity, capable of degradation of many different dye types. Laccase from Streptomyces cyaneus was expressed on the surface of Saccharomyces cerevisiae EBY100 cells. The specific activity of surface-displayed laccase was increased by toluene-induced lysis to 3.1 U/g of cell walls. For cell wall laccase immobilization within hydrogel beads, alginate was modified by dopamine using periodate oxidation and reductive amination and characterized by UV-Vis, FTIR, and NMR spectroscopy. Cell wall laccase was immobilized within alginate and dopamine-alginate beads additionally cross-linked by oxygen and laccase. The immobilized enzyme's specific activity was two times higher using dopamine-alginate compared to native alginate beads, and immobilization yield increased 16 times. Cell wall laccase immobilized within dopamine-alginate beads decolorized Amido Black 10B, Reactive Black 5, Evans Blue, and Remazol Brilliant Blue with 100% efficiency and after ten rounds of multiple-use retained decolorization efficiency of 90% with Evans Blue and 61% with Amido Black.
Asunto(s)
Alginatos/química , Colorantes/aislamiento & purificación , Dopamina/química , Lacasa/química , Pared Celular/química , Streptomyces/enzimologíaRESUMEN
Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of ß-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.