Characterization of the xylanolytic core of Cellulomonas sp. B6 by recombinant expression of five extracellular xylanases

ORNELLA ONTAÑON; MERCEDES GARRIDO ; JULIANA TOPALIAN; DIANA MARIA CAICEDO LAURIDO; LAURA NAVAS; ELEONORA CAMPOS

Simposio; Lignobiotech 2022; 2022

Cellulomonas sp. B6 is a cellulolytic soil strain with high potential for lignocellulosedeconstruction and biomass valorization. By growth on wheat bran, sugar cane straw orwheat straw, it is able to secrete its complete repertoire of extracellular xylanases(seven GH10 and one GH11) as well as xylan-debranching enzymes. The genomiccharacterization of the strain revealed that the genes encoding these xylanases are notlocated in common regions and are rarely part of carbohydrate utilization loci.The main objective of this work was to characterize the individual activity ofCellulomonas sp. B6 xylanases as a way to understand their role in the polysaccharidesdeconstruction core of the genus.Thus, four GH10 (CeXyn10A, CeXyn10B, CeXyn10C, CeXyn10D) and one GH11(CeXyn11A) were expressed as recombinant proteins in E coli Rossetta-pet28 system,purified by affinity chromatography and characterized according to their xylanolyticactivity. None of the enzymes hydrolyzed cellulosic substrates, demonstrating highspecificity towards xylan. All the xylanases showed optimal activity at neutral pH range(4.5 to 8) and moderate temperature range (30-45°C), with the exception of CeXyn10Band CeXyn10A, which performed better at 55 and 50°C, respectively, and were themost thermostable. Interestingly, they were the only two modular GH10 expressed withtheir carbohydrate-binding modules (cbm22 and cbm2, respectively). Under optimalconditions, xylanase activities ranged from 80 to 450 U/mg for GH10 and ~ 60 U/mg forGH11, determined as xylose equivalents (reducing sugars) released from beechwoodxylan by DNS assay. The preferred substrates were arabinoxylan for GH10 and122glucuronoxylan for GH11, as shown by the release of reducing sugars. The xylanolyticactivity dropped when the number of substitutions in glucuronoxylan increased andwhen arabinoxylan was linked to pectin residues. The hydrolysis products of CeXyn11Awere xylooligosaccharides (XOS) of variable length (DP > 2) while GH10 xylanasesreleased a similar profile of shorter XOS, xylobiose and xylose (X1).CeXyn10A was the most active and thermostable xylanase. It showed a classicalMichaelis-Menten kinetics on xylan, with a Vmax above 1000 µmol/min/mg. It alsohydrolyzed the xylan fraction of barley straw into XOS and X1.Based on the obtained results, the xylanases from Cellulomonas sp. B6 have differentperformance and, therefore, could play different roles in the bacterial xylanolytic system,possibly related to adaptation to the environment. Additionally, they showed interestingfeatures as biocatalysts for producing emerging prebiotics (short soluble XOS) and forbiorefining purposes.