RESUMO
Valorization of the hemicellulose fraction of plant biomass is crucial for the sustainability of lignocellulosic biorefineries. The Cellulomonas genus comprises Gram-positive Actinobacteria that degrade cellulose and other polysaccharides by secreting a complex array of enzymes. In this work, we studied the specificity and synergy of two enzymes, CsXyn10A and CsAbf62A, which were identified as highly abundant in the extracellular proteome of Cellulomonas sp. B6 when grown on wheat bran. To explore their potential for bioprocessing, the recombinant enzymes were expressed and their activities were thoroughly characterized. rCsXyn10A is a GH10 endo-xylanase (EC 3.2.1.8), active across a broad pH range (5 to 9), at temperatures up to 55 °C. rCsAbf62A is an α-L-arabinofuranosidase (ABF) (EC 3.2.1.55) that specifically removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides (AXOS), xylan, and arabinan backbones, but it cannot act on double-substituted residues. It also has activity on pNPA. No differences were observed regarding activity when CsAbf62A was expressed with its appended CBM13 module or only the catalytic domain. The amount of xylobiose released from either wheat arabinoxylan or arabino-xylo-oligosaccharides increased significantly when rCsXyn10A was supplemented with rCsAbf62A, indicating that the removal of arabinosyl residues by rCsAbf62A improved rCsXyn10A accessibility to ß-1,4-xylose linkages, but no synergism was observed in the deconstruction of wheat bran. These results contribute to designing tailor-made, substrate-specific, enzymatic cocktails for xylan valorization. KEY POINTS: ⢠rCsAbf62A removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides, xylan, and arabinan backbones. ⢠The appended CBM13 of rCsAbf62A did not affect the specific activity of the enzyme. ⢠Supplementation of rCsXyn10A with rCsAbf62A improves the degradation of AXOS and xylan.
Assuntos
Cellulomonas , Xilanos , Cellulomonas/genética , Cellulomonas/metabolismo , Fibras na Dieta , Endo-1,4-beta-Xilanases/metabolismo , Glicosídeo Hidrolases/metabolismo , Hidrólise , Oligossacarídeos/metabolismo , Especificidade por Substrato , Xilanos/metabolismoRESUMO
One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides. KEY POINTS: ⢠Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised. ⢠Biomass-induced extracellular enzymes were identified by proteomic profiling. ⢠Combinations of extra and intracellular extracts were used for barley straw hydrolysis.
Assuntos
Cellulomonas , Biomassa , Endo-1,4-beta-Xilanases , Hidrólise , ProteômicaRESUMO
AIMS: Lignocellulosic biomass deconstruction is a bottleneck for obtaining biofuels and value-added products. Our main goal was to characterize the secretome of a novel isolate, Cellulomonas sp. B6, when grown on residual biomass for the formulation of cost-efficient enzymatic cocktails. METHODS AND RESULTS: We identified 205 potential CAZymes in the genome of Cellulomonas sp. B6, 91 of which were glycoside hydrolases (GH). By secretome analysis of supernatants from cultures in either extruded wheat straw (EWS), grinded sugar cane straw (SCR) or carboxymethylcellulose (CMC), we identified which proteins played a role in lignocellulose deconstruction. Growth on CMC resulted in the secretion of two exoglucanases (GH6 and GH48) and two GH10 xylanases, while growth on SCR or EWS resulted in the identification of a diversity of CAZymes. From the 32 GHs predicted to be secreted, 22 were identified in supernatants from EWS and/or SCR cultures, including endo- and exoglucanases, xylanases, a xyloglucanase, an arabinofuranosidase/ß-xylosidase, a ß-glucosidase and an AA10. Surprisingly, among the xylanases, seven were GH10. CONCLUSIONS: Growth of Cellulomonas sp. B6 on lignocellulosic biomass induced the secretion of a diverse repertoire of CAZymes. SIGNIFICANCE AND IMPACT OF THE STUDY: Cellulomonas sp. B6 could serve as a source of lignocellulose-degrading enzymes applicable to bioprocessing and biotechnological industries.
Assuntos
Proteínas de Bactérias/metabolismo , Cellulomonas , Lignina/metabolismo , Metaboloma/fisiologia , Biomassa , Cellulomonas/química , Cellulomonas/enzimologia , Cellulomonas/metabolismo , Cellulomonas/fisiologiaRESUMO
Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding ß-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active ß-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L-1 and 222 IU g-1, respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20 g L-1) as the sole carbon source for 48 h. Ethanol production was 5 g L-1 after 96 h of culture, which represented a yield of 0.41 g g-1 of substrate consumed (12 g L-1), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the ß-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.