Regulation and Characterization of Xylanolytic Enzymes of Thermoanaerobacterium saccharolyticum B6A-RI

Abstract

During growth on xylan and xylose Thermoanaerobacterium saccharolyticum B6A-RI produced endoxylanase, 13-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, 13-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 highmolecular-weight complex (molecular weight, more than 106). When analyzed by sodium dodecyl sulfatepolyacrylamide 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°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 Schifis 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°C, respectively.

Location of xylanolytic enzymes in Chaetomium globosum

“…However, with increasing substrate concentration, the activity of both the enzymes was predominantly cell associated ( Table 3). Similar observations were also made in the case of Thermoanaerobacterium sacchurolyticum (LEE et al 1993).…”

Biology, ecology, and biotechnological applications of anaerobic bacteria adapted to environmental stresses in temperature, pH, salinity, or substrates.

“…Recently a new isolate, Thermoanaerobacterium saccharolyticum B6A-RI, was isolated from Frying Pan Springs in Yellowstone National Park (245). This organism has very active xylanases but is not cellulolytic, and it produces endoxylanase, I-xylosidase, arabinofuranosidase, acetyl esterase, and xylose isomerase, with the first three enzymes produced coordinately (247). Negatively charged cell surface structures were visualized in cells growing on xylan, and they coincided with the production of cell-bound endoxylanases and the ability of cells to bind tightly to xylan.…”

Purification and Properties of ArfI, an α- l -Arabinofuranosidase from Cytophaga xylanolytica

“…However, we regard this assessment of the cellular location of ENDOX components as a first approximation. Other workers have shown that the distribution of xylanase components can be affected by the amount and type of growth substrate, the culture conditions employed, and the growth phase of cells at the time of harvest (24,25,31,37).…”