RESUMO
The genome of Kitasatospora setae KM-6054, a soil actinomycete, has three genes encoding chitosanases belonging to GH46 family. The genes (csn1-3) were cloned in Streptomyces lividans and the corresponding enzymes were purified from the recombinant cultures. The csn2 clone yielded two proteins (Csn2BH and Csn2H) differing by the presence of a carbohydrate-binding domain. Sequence analysis showed that Csn1 and Csn2H were canonical GH46 chitosanases, while Csn3 resembled chitosanases from bacilli. The activity of the four chitosanases was tested in a variety of conditions and on diverse chitosan forms, including highly N-deacetylated chitosan or chitosan complexed with humic or polyphosphoric acid. Kinetic parameters were also determined. These tests unveiled the biochemical diversity among these chitosanases and the peculiarity of Csn3 compared with the other three enzymes. The observed biochemical diversity is discussed based on structural 3D models and sequence alignment. This is a first study of all the GH46 chitosanases produced by a single microbial strain.
Assuntos
Variação Genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Streptomycetaceae/enzimologia , Quitosana/metabolismo , Clonagem Molecular , Glicosídeo Hidrolases/classificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Streptomyces lividans/genética , Streptomyces lividans/isolamento & purificação , Streptomyces lividans/metabolismoRESUMO
Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.
Assuntos
Quitosana/química , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/química , Animais , Cristalografia , Humanos , Hidrólise , Peso Molecular , Mutagênese Sítio-Dirigida , FenótipoRESUMO
The csnR gene, localized at the beginning of an operon, csnR-K, which organization is conserved through many actinomycete genomes, was previously shown to repress the transcription of the chitosanase gene csnA in Streptomyces lividans. However, knowledge on the function of the whole csnR-K operon in the metabolism of chitosan (an N-deacetylated derivative of chitin) remained limited. Mutants of S. coelicolor A3(2) harboring partial or total deletions of the csnR-K operon were analyzed for their capacity to uptake glucosamine oligosaccharides (GlcN)n. The csnR-K operon was autoregulated by CsnR repressor and its transcription was inducible by GlcN oligosaccharides. The operon controlled the uptake of GlcN oligosaccharides in S. coelicolor A3(2), with a minor contribution to the consumption of monomeric GlcN but not chitin-related N-acetylated derivatives. The deletion of the whole operon abolished the uptake of GlcN oligosaccharides. The CsnEFG transporter encoded by this operon is the front door for the assimilation of chitosan-derived hydrolysis products in S. coelicolor A3(2). The ATP-binding component MsiK was essential for CsnEFG transport function. Also, deletion of msiK abolished the induction of csnA transcription by GlcN oligosaccharides.
Assuntos
Quitosana/metabolismo , Oligossacarídeos/metabolismo , Óperon , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Transporte Biológico/genética , Quitina/metabolismo , Deleção de Genes , Glucosamina/metabolismo , Glicosídeo Hidrolases/genética , Hidrólise , MutaçãoRESUMO
The agricultural sector is responsible for an important part of Canadian greenhouse gas (GHG) emissions, 8 % of the 747 Mt eq. CO(2) emitted each year. The pork industry, a key sector of the agrifood industry, has had a rapid growth in Canada since the middle 1980s. For this industry, slurry storage accounts for the major part of methane (CH(4)) emissions, a GHG 25 times higher than carbon dioxide (CO(2)) on a 100-year time horizon. Intending to reduce these emissions, biofiltration, a process effective to treat CH(4) from landfills and coal mines, could be effective to treat CH(4) from the pig industry. Biofiltration is a complex process that requires the understanding of the biological process of CH(4) oxidation and a control of the engineering parameters (filter bed, temperature, etc.). Some biofiltration studies show that this technology could be used to treat CH(4) at a relatively low cost and with a relatively high purification performance.
