BEHAVIOR AND SUPPORTIVE EVIDENCE OF A LARGE XYLANASE-CONTAINING MULTIENZYME COMPLEX OF TEPIDIMICROBIUM XYLANILYTICUM BT14

Paripok Phitsuwan, Ely Morag, Chakrit Tachaapaikoon, Patthra Pason, Akihiko Kosugi, Khanok Ratanakhanokchai

Abstract


Cellular behaviors and a xylanolytic-cellulolytic enzyme system of Tepidimicrobium xylanilyticum BT14 towards xylan-rich plant biomass degradation were characterized. During the exponential growth phase, the bacterial cells were bound tightly to the growth substrate where the degradation zones appeared mostly around the cells, indicating that the xylanolytic-cellulolytic enzyme system was linked to the cell surfaces. Interestingly, several cells appeared to secrete extracellular matrix to connect to their neighbors, and the matrix disappeared when cells passed to the stationary growth phase. Cationized-ferritin staining resulted in a dense assembly of bulbs, protuberance-like structures on the growing bacterial cell surfaces. The cell-associated proteins derived by sonication contained predominated xylanase with relatively low carboxymethyl-cellulase (CMCase) activities, suggesting that the xylanolytic-cellulolytic enzyme system occurred as a cell-associated enzyme. By means of gel-filtration chromatography, a high molecular mass protein with the estimated size of 2000 kDa was retrieved from the cell-associated enzymes, and it appeared as a single protein band on non-denaturing gel. However, more bands were obtained after the protein was boiled with sodium dodecyl sulfate and β-mercaptoethanol – which contained 4 xylanases and one CMCase – suggesting that these proteins were organized as a multienzyme complex (MEC) in natural form. Additionally, the predominated xylanolytic MEC preferred binding to cellulose rather than xylan.

Keywords


Cellulase; Multienzyme complex; Plant biomass; Polysaccharide binding; Tepidimicrobium xylanilyticum; Xylanase

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