Expression and purification of the 5'-nucleotidase YitU from Bacillus species: its enzymatic properties and possible applications in biotechnology
| Autor: | Natalia Pavlovna Zakataeva, Victoria S. Skripnikova, Yuliya R. Yusupova, Alexandr D. Kivero |
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| Rok vydání: | 2019 |
| Předmět: |
Bacillus amyloliquefaciens
Riboflavin Flavin mononucleotide Gene Expression Bacillus Bacillus subtilis Applied Microbiology and Biotechnology 5'-nucleotidase Substrate Specificity 03 medical and health sciences Nucleotidases chemistry.chemical_compound Bacterial Proteins Nucleotidase Escherichia coli 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) producers Nucleotide Cloning Molecular Biotechnologically Relevant Enzymes and Proteins 5'-Nucleotidase Purine Nucleotides Riboflavin producers 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology 030306 microbiology Chemistry General Medicine biology.organism_classification Ribonucleoside Aminoimidazole Carboxamide Recombinant Proteins 5′-Nucleotidases (EC 3.1.3.5) Kinetics Biochemistry Ribonucleosides Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology |
| ISSN: | 1432-0614 |
| Popis: | 5’-Nucleotidases (EC 3.1.3.5) are enzymes that catalyze the hydrolytic dephosphorylation of 5′-ribonucleotides and 5′-deoxyribonucleotides to their corresponding nucleosides plus phosphate. In the present study, to search for new genes encoding 5′-nucleotidases specific for purine nucleotides in industrially important Bacillus species, “shotgun” cloning and the direct selection of recombinant clones grown in purine nucleosides at inhibitory concentrations were performed in the Escherichia coli GS72 strain, which is sensitive to these compounds. As a result, orthologous yitU genes from Bacillus subtilis and Bacillus amyloliquefaciens, whose products belong to the ubiquitous haloacid dehalogenase superfamily (HADSF), were selected and found to have a high sequence similarity of 87%. B. subtilis YitU was produced in E. coli as an N-terminal hexahistidine-tagged protein, purified and biochemically characterized as a soluble 5′-nucleotidase with broad substrate specificity with respect to various deoxyribo- and ribonucleoside monophosphates: dAMP, GMP, dGMP, CMP, AMP, XMP, IMP and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate (AICAR-P). However, the preferred substrate for recombinant YitU was shown to be flavin mononucleotide (FMN). B. subtilis and B. amyloliquefaciens yitU overexpression increased riboflavin (RF) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) accumulation and can be applied to breed highly performing RF- and AICAR-producing strains. |
| Databáze: | OpenAIRE |
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