IDENTIFICATION OF SECRETED CELLULASES AND HEMICELLULASES FROM A NATIVE ISOLATE OF Cellulomonas sp. AND RECOMBINANT EXPRESSION OF A GH10 ENDOXYLANASE.

PICCINNI, FLORENCIA; ONTAÑON ORNELLA M.; GHIO, SILVINA; TALIA, PAOLA; CAMPOS, ELEONORA

San Miguel de Tucumán

Congreso; XII Congreso Argentino de Microbiologia general; 2017

We have isolated a novel bacterial strain from the genus Cellulomonas, named B6, from aforest soil consortium. Cellulomonas sp. B6 has the ability to degrade both cellulose and xylan and togrow in a minimal saline medium supplemented with either carboxymethylcellulose (CMC), orlignocellulosic biomass, such as sugar cane residue (SCR), extruded barley straw (BSE) and extrudedwheat straw (WSE).In order to identify the secreted enzymes responsible for the activity, we undertook a shotgunproteomics approach. Extracellular proteins from a culture grown on WSE were precipitated and boththe whole secretome as well as 1D-SDS-PAGE fractions were analyzed by mass spectrometry(CEQUIBIEM, FCEN, UBA). Identified proteins were compared to the previously obtained annotatedgenome of Cellulomonas sp. B6 and analyzed manually using the dbCAN algorithm from the CAZydatabase. Potential enzymatic activity was assigned using the BLAST algorithm from NCBI.We identified two potential exoglucanases of GH6 and GH48 families, eight GH10 and oneGH11 xylanases, four potential GH9 and one GH6 endoglucanases and a GH74 xyloglucanase. Onlysome of these enzymes had been previously identified under other growth conditions, such as CMC orSCR, suggesting that growth on different biomasses can result in differential expression and secretionof hydrolytic enzymes.In order to evaluate the activity of some of these enzymes, a GH10 with a carbohydratebinding module CBM2 was selected. We amplified and cloned the coding gene and the protein washeterologously expressed without its signal peptide as a His-tag N-terminal fusion and purified byaffinity chromatography. The activity of the purified enzyme, named rGH10XynC, was evaluated onxylan and lignocellulosic biomass. We confirmed the EC 3.2.1.8 xylanase activity by hydrolysis ofxylan to xylobiose (X2) and xylose (X1). Optimal activity was observed at 50°C in a pH ranging from 5to 7,5. Activity on extruded barley straw (BSE) also resulted in conversion to xylo-oligosacharides, X2and X1, demonstrating that rGH10XynC is active on the xylan contained in biomass.These results suggest that Cellulomonas sp. B6 is a good source of enzymes with potentialindustrial applications- such as the production of paper pulp, animal feed and second generationbioethanol.