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 ac tivity towards xylan, The cloning of a DNA fragment of this microorgan ism in several Streptomyces strains permitted us to overproduce both p roteins, N-terminal sequence analyses, immunoblot assays, and time cou rse overproduction experiments allowed us to ensure that both xylanase s were encoded by the same gene and that the smallest form (35 kDa) or iginated from the large one (45 kDa) by proteolytic cleavage on the C terminus. The production of both forms was studied in different strain s carrying the gene in a multicopy plasmid. The best production was ob tained 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 an d characterized biochemically. The optimal pH and temperature, for bot h, were 6.3 and 60 degrees C, respectively, in 7.5-min assays, Both pr oteins were highly stable in a wide range of pHs (4 to 10) and tempera tures (4 to 50 degrees C); nevertheless, after 1-h incubations, both e nzymes lost most of their activity at temperatures over 55 to 60 degre es C. Endoxylanolytic activity was demonstrated in both enzymes, but n o beta-xylosidase activity was detected.