Cloning and expression of a GH10 xylanase from Cellulomonas sp. B6 for application in lignocellulosic biomass deconstruction

DIANA CAICEDO; ORNELLA ONTAÑON; JULIANA TOPALIAN; ELEONORA CAMPOS; LAURA E. NAVAS

Mendoza

Congreso; SAIB 2022; 2022

Lignocellulosic biomass contains mainly cellulose, hemicellulose and lignin. Xylanases (EC 3.2.1.8) are a group of depolymerizing enzymes that catalyze the hydrolysis of the β-1,4 glycosidic linkage in the backbone of xylan (XYN) (the main component of hemicellulose in terrestrial plants). These enzymes are classified in CAZy database as glycoside hydrolases (GH) in several families, mainly GH10 and GH11. The main objective of this work was to characterize the activity of CsXyn10C, a GH10 xylanase from Cellulomonas sp. B6 and to study the contribution of its appended carbohydrate binding module from family 13 (CBM13) to its activity. CsXyn10C was previously identified in the supernatant of Cellulomonas sp. B6 cultures, using lignocellulosic biomass as sole carbon source. We amplified the coding sequence for the mature protein from total genomic DNA, with and without the sequence coding for the CBM13: CsXyn10CCBM and CsXyn10Ccat, respectively, and cloned them as amino-terminal His-tagged fusions. Both enzymes were expressed in E coli Arctic Express and purified by Niquel- affinity chromatography in a soluble form. Both forms of the enzyme were active towards beechwood xylan although CsXyn10Ccat showed higher specific activity (6.75 and 12.46 IU/nmol for CsXyn10CCBM and CsXyn10Ccat, respectively). Both showed the highest activity at 50 °C (while active in a range from 40 ºC to 55 ºC) and at close to neutral pH, displaying more than 50% activity in a wide range of pH (4 to 9). Regarding thermal stability, at 50 °C, CsXyn10CCBM was more stable than CsXyn10Ccat, suggesting that the CBM might stabilize the protein. Nevertheless, at 45 °C no differences were observed as the two forms of the enzyme retained above 60% activity after 4 hours of preincubation.Overall, CsXyn10C is an attractive candidate to be assayed in xylan-valorization bioprocesses that require mild temperature conditions in a wide range of pH.