Secretory production in Escherichia coli of a GH46 chitosanase from Chromobacterium violaceum, suitable to generate antifungal chitooligosaccharides
| Autor: | Thalles B. Grangeiro, Christiana de Fátima Bruce da Silva, Mayara Itala Geronimo de Azevedo, Celli Rodrigues Muniz, Ana C.S. Gadelha, Simone T. Oliveira, Matheus S. Girão, Cleverson D.T. Freitas, Rômulo Farias Carneiro, José E. Monteiro-Júnior, Davi Coe Torres, Celso Shiniti Nagano |
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| Rok vydání: | 2020 |
| Předmět: |
Antifungal Agents
Glycoside Hydrolases Oligosaccharides Chitin 02 engineering and technology medicine.disease_cause Biochemistry Chitooligosaccharides Chitosan 03 medical and health sciences chemistry.chemical_compound Structural Biology Escherichia coli medicine Amino Acid Sequence Chitosanase Molecular Biology Polyacrylamide gel electrophoresis Mycelium 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology Molecular mass Chemistry Chromobacterium Hydrolysis General Medicine Hydrogen-Ion Concentration 021001 nanoscience & nanotechnology biology.organism_classification Chromobacterium violaceum Molecular Weight Enzyme 0210 nano-technology |
| Zdroj: | Repositório Institucional da Universidade Federal do Ceará (UFC) Universidade Federal do Ceará (UFC) instacron:UFC |
| ISSN: | 0141-8130 |
| DOI: | 10.1016/j.ijbiomac.2020.09.221 |
| Popis: | A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and β-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases. |
| Databáze: | OpenAIRE |
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