A novel thermophilic recombinant obligate xylobiohydrolase (AcGH30A) from Acetivibrio clariflavus orchestrates the deconstruction of xylan polysaccharides.

GH30 xylobiohydrolases, an expanding enzyme category, need deeper insights for optimal use. The primary aim of this study was to characterize a new xylobiohydrolase, AcGH30A of GH30 family from Acetivibrio clariflavus. The gene encoding AcGH30A was cloned using pET28a(+) vector and expressed in E. coli BL21(DE3) cells. AcGH30A was purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of AcGH30A showed molecular mass of ~58 kDa. AcGH30A showed optimum temperature 80 °C and optimum pH 7.0. AcGH30A was stable (maintaining >80 % of control activity) in pH range, 4-7 and temperature range, 30 °C -70 °C when incubated for 90 min. AcGH30A displayed melting temperature, 72 °C and half-life, 21 days at 4 °C. The enzyme activity of AcGH30A was enhanced by 10 mM Ca and Mg ions by 25 % and 21 %, respectively, whereas 10 mM Co, Zn, Fe, and Cu ions significantly reduced it. AcGH30A showed activity against various xylan polysaccharides displaying highest V, 139 U.mg and K, 0.71 mg.ml against 4-O-methyl glucuronoxylan under optimum conditions. TLC, HPLC and LC-MS analyses of AcGH30A hydrolyzed products from xylan substrates revealed the release of sole product, xylobiose, confirming it as an obligate xylobiohydrolase. AcGH30A being a highly thermostable enzyme can be potentially utlilized in various biotechnological applications.