Overproduction, purification, and biochemical characterization of a xylanase (Xys1) from Streptomyces halstedii JM8
| Autor: | Alberto Ruiz-Arribas, Ramón I. Santamaría, Pilar Sánchez, Ana Lila Garda, José M. Fernández-Abalos |
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| Rok vydání: | 1995 |
| Předmět: |
DNA
Complementary Molecular Sequence Data Molecular cloning Applied Microbiology and Biotechnology Streptomyces Plasmid Enzyme Stability Amino Acid Sequence Overproduction chemistry.chemical_classification Ecology biology Streptomycetaceae biology.organism_classification Xylan Endo-1 3-beta-Xylosidase Enzyme Xylosidases chemistry Biochemistry Xylanase Actinomycetales Sequence Alignment Food Science Biotechnology Plasmids Research Article |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 0099-2240 |
| Popis: | Streptomyces halstedii JM8, isolated from straw, produces and secretes into the culture supernatant at least two proteins with hydrolytic activity towards xylan. The cloning of a DNA fragment of this microorganism in several Streptomyces strains permitted us to overproduce both proteins. N-terminal sequence analyses, immunoblot assays, and time course overproduction experiments allowed us to ensure that both xylanases were encoded by the same gene and that the smallest form (35 kDa) originated from the large one (45 kDa) by proteolytic cleavage on the C terminus. The production of both forms was studied in different strains carrying the gene in a multicopy plasmid. The best production was obtained with Streptomyces parvulus transformed with the plasmid pJM9, a pIJ702 derivative, which yielded 144 U/ml. Both forms of the xylanase were purified with a fast-performance liquid chromatography system and characterized biochemically. The optimal pH and temperature, for both, were 6.3 and 60°C, respectively, in 7.5-min assays. Both proteins were highly stable in a wide range of pHs (4 to 10) and temperatures 14 to 50°C); nevertheless, after 1-h incubations, both enzymes lost most of their activity at temperatures over 55 to 60°C. Endoxylanolytic activity was demonstrated in both enzymes, but no β-xylosidase activity was detected. This research was supported by a grant from the Comisión Interministerial de Ciencia y Tecnología (BIO92-0173). A.R.-A. and J.M.F.-A. had fellowships from the Ministerio de Educación y Ciencia (Spain); A.G. had a fellowship from the Instituto de Cooperación Iberoamericana. |
| Databáze: | OpenAIRE |
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