Regulation and characterization of xylanolytic enzymes of Thermoanaerobacterium saccharolyticum B6A-RI

During growth on xylan and xylose Thermoanaerobacterium saccharolyticum B6A-RI produced endoxylanase, beta-xylosidase, arabinofuranosidase, and acetyl esterase, and the first three activities appeared to be produced coordinately. During nonlimiting growth on xylan, these enzyme activities were predo...

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Veröffentlicht in:Applied and Environmental Microbiology 1993-03, Vol.59 (3), p.763-771
Hauptverfasser: Lee, Y.E, Lowe, S.E, Zeikus, J.G
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Sprache:eng
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Zusammenfassung:During growth on xylan and xylose Thermoanaerobacterium saccharolyticum B6A-RI produced endoxylanase, beta-xylosidase, arabinofuranosidase, and acetyl esterase, and the first three activities appeared to be produced coordinately. During nonlimiting growth on xylan, these enzyme activities were predominantly cell associated; however, during growth on limiting concentrations of xylan, the majority of endoxylanase activity was extracellular rather than cell associated. Endoxylanase, beta-xylosidase, and arabinofuranosidase activities were induced by xylan, xylose, and arabinose, respectively. Acetyl esterase activity was constitutive, and endoxylanase activity was catabolite repressed by glucose. Extracellular endoxylanase existed as a high-molecular-weight complex (molecular weight, more than 10(6). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms, the crude endoxylanase complex was composed of at least six activity bands. Endoxylanase was purified by gel filtration with Sephacryl S-300 and affinity chromatography with xylan coupled to Sepharose CL-4B preequilibrated to 45 degrees C with 50 mM sodium acetate buffer (pH 4.0) and eluted with 0.1% soluble xylan. A single area of endoxylanase activity was identified on the zymogram; when this activity was analyzed by SDS-PAGE, it was composed of a major protein with a molecular weight of approximately 160,000 and a minor protein with a molecular weight of approximately 130,000. The endoxylanase activity stained with Schiff's reagent, indicative of glycoproteins, displayed a specific activity of 41 U/mg of protein on xylan, and had pH and temperature optima of 6.0 and 70 degrees C, respectively.
ISSN:0099-2240
1098-5336
DOI:10.1128/aem.59.3.763-771.1993