A GH 8 ENDOGLUCANASE FROM Paenibacillus sp. A59 FOR APPLICATION IN BIOPROCESSES

BRADANINI M; GHIO S; ONTAÑON ORNELLA M.; GARRIDO M; CAMPOS E

Congreso; 54° Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2018

In order to deal with the energy problems affecting the modern world, sustainable alternatives to fossil fuels are being under study. Secondgeneration bioethanol production still requires to overcome cost issues and technical limitations because the process requires highly effectivecellulases and hemicellulases and efficient fermentative microorganisms. One of the most studied approaches is the prospecting of bacteriacapable of degrading lignocellulolytic biomass to obtain enzymes to use during the saccharification process. In this work, an endoglucanase fromthe hemicellulolytic isolate Paenibacillus sp. A59 was expressed as recombinant protein, purified in a soluble form and biochemicallycharacterized. The protein, which we named Cel8Pa, has 409 amino acids including a conserved domain from glycosyl hydrolases family 8. Byhomology with previously characterized proteins, we identified the aminoacids Glu95 and Asp156 as the catalytic residues. The tertiary structurecorresponded to a regular (alpha/alpha) 6 barrel formed by six inner and six outer alpha helices, determined by molecular modeling. The matureprotein fused to a 6 His N-terminal tag was expressed in E. coli, and purified in a native form by IMAC, with an apparent molecular weight of46 kDa. To determine the substrate specificity of Cel8Pa we evaluated its activity on several substrates. It had activity on barley beta-glucan(BG) (45 UI/mg), phosphoric acid swollen cellulose (PASC) (14 UI/mg), chitosan (5 UI/mg) and carboxymethyl cellulose (CMC) (4 UI/mg),while it did not have beta-glucosidase activity. The resulting main products obtained from BG hydrolysis were cello-oligosaccharides (COS)with a degree of polymerization (DP) ≥ β, while PASC, CMC, C4, C5 and C6 were completely hydrolyzed to cellotriose and cellobiose,identified by TLC assays. Cel8Pa did not hydrolyze insoluble cellulosic substrates such as bacterial cellulose, Avicel or filter paper, inaccordance to its endoglucananase activity profile. Noteworthy, long-term assays (17 h) on xylan resulted in xylose, xylobiose and xylooligosaccharides (XOS) of DP≥γ, although no beta-xylosidase activity was observed at any time, indicating Cel8Pa has also low levels ofendoxylanase activity (0.4 UI/mg). The optimal reaction conditions on BG were 40°C and pH 4.5, maintaining 50% of activity after 30 hoursunder these conditions. Kinetic studies indicated a Vmax of 197 ± 70 IU/μM, a KM of 0.99 ± 0.47 μM and the catalytic constant Kcat of 215.18sec-1. We also assayed Cel8a activity on barley straw (pre-treated by extrusion), which resulted in COS/XOS of different DP after 17 h ofhydrolysis. In conclusion, Cel8Pa is an endoglucanase/endoxylanase enzyme, active at moderate temperatures and acid pH, which may be usedas part of an enzymatic cocktail in simultaneous saccharification and co-fermentation processes (SSCF) for lignocellulosic biomassdeconstruction