Secretory production in Escherichia coli of a GH46 chitosanase from Chromobacterium violaceum, suitable to generate antifungal chitooligosaccharides

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 b...

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Bibliographic Details
Published in:International journal of biological macromolecules 2020-12, Vol.165 (Pt A), p.1482-1495
Main Authors: Azevedo, Mayara I.G., Oliveira, Simone T., Silva, Christiana F.B., Carneiro, Rômulo F., Nagano, Celso S., Gadelha, Ana C.S., Torres, Davi C., Monteiro-Júnior, José E., Girão, Matheus S., Muniz, Celli R., Freitas, Cleverson D.T., Grangeiro, Thalles B.
Format: Article
Language:English
Subjects:
Amino Acid Sequence - genetics
Antifungal Agents - chemistry
Chitin - analogs & derivatives
Chitin - biosynthesis
Chitin - chemistry
Chitin - genetics
Chitooligosaccharides
Chitosan - chemistry
Chitosanase
Chromobacterium - enzymology
Chromobacterium - genetics
Chromobacterium violaceum
Escherichia coli - genetics
Glycoside Hydrolases - biosynthesis
Glycoside Hydrolases - chemistry
Glycoside Hydrolases - genetics
Hydrogen-Ion Concentration
Hydrolysis
Molecular Weight
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Summary: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. •A GH46 chitosanase from Chromobacterium violaceum was produced in Escherichia coli.•The recombinant protein (CvCsn46) was purified and partially characterized.•CvCsn46 had an amphiprotic behavior, with highest activity at pH values 6.0 and 11.0.•The enzyme degraded chitosan through dual, endo- and exo-type hydrolytic activities.•Low molecular mass chitosan oligomers produced by CvCsn46 showed antifungal activity.
ISSN:0141-8130
1879-0003
DOI:10.1016/j.ijbiomac.2020.09.221