Improvement of Cellulomonas fimi endoglucanase CenA by multienzymatic display on a decameric structural scaffold.

The development of multifunctional particles using polymeric scaffolds is an emerging technology for many nanobiotechnological applications. Here we present a system for the production of multifunctional complexes, based on the high affinity non-covalent interaction of cohesin and dockerin modules complementary fused to decameric Brucella abortus lumazine synthase (BLS) subunits, and selected target proteins, respectively. The cohesin-BLS scaffold was solubly expressed in high yield in Escherichia coli, and revealed a high thermostability. The production of multienzymatic particles using this system was evaluated using the catalytic domain of Cellulomonas fimi endoglucanase CenA recombinantly fused to a dockerin module. Coupling of the enzyme to the scaffold was highly efficient and occurred with the expected stoichiometry. The decavalent enzymatic complexes obtained showed higher cellulolytic activity and association to the substrate compared to equivalent amounts of the free enzyme. This phenomenon was dependent on the multiplicity and proximity of the enzymes coupled to the scaffold, and was attributed to an avidity effect in the polyvalent enzyme interaction with the substrate. Our results highlight the usefulness of the scaffold presented in this work for the development of multifunctional particles, and the improvement of lignocellulose degradation among other applications. KEY POINTS: • New system for multifunctional particle production using the BLS scaffold • Higher cellulolytic activity of polyvalent endoglucanase compared to the free enzyme • Amount of enzyme associated to cellulose is higher for the polyvalent endoglucanase.

Insights into the xylan degradation system of Cellulomonas sp. B6: biochemical characterization of rCsXyn10A and rCsAbf62A.

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.

Secretome profile of Cellulomonas sp. B6 growing on lignocellulosic substrates.

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.

Cloning and expression of a novel, moderately thermostable xylanase-encoding gene (Cflxyn11A) from Cellulomonas flavigena.

The Cfl xyn11A gene, encoding the endo-1,4-beta-xylanase Cfl Xyn11A from Cellulomonas flavigena, was isolated from a genomic DNA library. The open reading frame of the Cfl xyn11A gene was 999 base pairs long and encoded a polypeptide (Cfl Xyn11A) of 332 amino acids with a calculated molecular mass of 35,110Da. The Cfl xyn11A gene was expressed in Escherichia coli and the recombinant enzyme, with an estimated molecular weight of 31kDa was purified and xylanase activity was measured. Cfl Xyn11A showed optimal activity at pH 6.5 and 55 degrees C. The enzyme demonstrated moderate thermal stability as Cfl Xyn11A maintained 50% of its activity when incubated at 55 degrees C for 1h or at 45 degrees C for 6h. This is the first report describing the cloning, expression and functional characterization of an endo-1,4-beta-xylanase-encoding gene from C. flavigena. Cfl Xyn11A may be suitable for industrial applications in the food and feed industries, or in the pre-treatment of lignocellulosic biomass required to improve the yields of fermentable sugars for bioethanol production.

Purification and characterization of two sugarcane bagasse-absorbable thermophilic xylanases from the mesophilic Cellulomonas flavigena.

We report the purification and characterization of two thermophilic xylanases from the mesophilic bacteria Cellulomonas flavigena grown on sugarcane bagasse (SCB) as the only carbon source. Extracellular xylanase activity produced by C. flavigena was found both free in the culture supernatant and associated with residual SCB. To identify some of the molecules responsible for the xylanase activity in the substrate-bound fraction, residual SCB was treated with 3 M guanidine hydrochloride and then with 6 M urea. Further analysis of the eluted material led to the identification of two xylanases Xyl36 (36 kDa) and Xyl53 (53 kDa). The pI for Xyl36 was 5.0, while the pI for Xyl53 was 4.5. Xyl36 had a Km value of 1.95 mg/ml, while Xyl53 had a Km value of 0.78 mg/ml. In addition to SCB, Xyl36 and Xyl53 were also able to bind to insoluble oat spelt xylan and Avicel, as shown by substrate-binding assays. Xyl36 and Xyl53 showed optimal activity at pH 6.5, and at optimal temperature 65 and 55 degrees C, respectively. Xyl36 and Xyl53 retained 24 and 35%, respectively, of their original activity after 8 h of incubation at their optimal temperature. As far as we know, this is the first study on the thermostability properties of purified xylanases from microorganisms belonging to the genus Cellulomonas.

Influence of various fermentation variables on exo-glucanase production in Cellulomonas flavigena

The influence of carbon and nitrogen sources on the production of exo-glucanase was investigated. The enzyme production was variable according to the carbon or nitrogen source used. Levels of beta-cellobiohydrolase (CBH) were minimal in the presence of even low concentrations of glucose. Enzyme production was stimulated by other carbohydrates and thus is subject to carbon source control by easily metabolizable sugars. In Dubos medium, on cellobiose, the cellobiohydrolase titres were 2-to 110-fold higher with cells growing on monomeric sugars and 2.7 times higher than cells growing on other disaccharides. alpha-Cellulose was the most effective inducer of beta-cellobiohydrlase and filter paperase (FPase) activities, followed by kallar grass straw. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of Cellulomonas flavigena. Nitrates were the best nitrogen sources and supported greater cell mass, cellobiohydrolase and FPase production. During growth on alpha-cellulose containing 8-fold sodium nitrate concentration, maximum volumetric productivities (Qp) of beta-cellobiohydrolase and FPase were 87.5 and 79.5 IU/l./h respectively and are significantly higher than the values reported for some other potent fungi and bacteria.

Influence of high salinities on the degradation of diesel fuel by bacterial consortia.

Microbial communities from three Argentinean saline soils were extracted and tested for their ability to degrade diesel fuel in liquid culture at salinities between 0% and 25%. In each case, the degradation process was continuously monitored by measuring oxygen consumption. Two communities (CR1 and CR2) showed nearly equal degrees of degradation across a salinity range of 0%-10% (the former degrading about 63% of the diesel fuel and the latter about 70% after 53 and 80 d, respectively). Furthermore, the degree of degradation was not significantly lower in the presence of 17.5% salt (58% and 65% degraded, respectively). A third community (El Zorro) showed a maximum turnover at 5% salt (79% diesel fuel degraded) and significant degradation (66%) at a salinity of 10%. However, the degree of degradation by this community clearly dropped at 0% and 15% salt. None of the communities were able to degrade diesel fuel in the presence of 25% salt, but the living cell counts showed that components of the microbial population survived the long-term exposure. The surviving portion is obviously sufficient to allow substantial restoration of the original community, as verified by the BIOLOG method. Isolates of the CR1 community were identified as members of the genera Cellulomonas, Bacillus, Dietzia, and Halomonas. In light of our investigations, the bioremediation of contaminated saline soils should be quite possible if the salinity of the soil water is lower than 15% or if it is reduced below this limit by the addition of water.