Overproduction, purification, and biochemical characterization of a xylanase (Xys1) from Streptomyces halstedii JM8.
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 degrees C, respectively, in 7.5-min assays. Both proteins were highly stable in a wide range of pHs (4 to 10) and temperatures (4 to 50 degrees C); nevertheless, after 1-h incubations, both enzymes lost most of their activity at temperatures over 55 to 60 degrees C. Endoxylanolytic activity was demonstrated in both enzymes, but no beta-xylosidase activity was detected.
Ruiz-Arribas
A
A
Departamento de Micrbiología y Genética, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Spain.
Fernández-Abalos
J M
JM
Sánchez
P
P
Garda
A L
AL
Santamariá
R I
RI
D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D010957:Plasmids; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase