The GH51 α-l-arabinofuranosidase from Paenibacillus sp. THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans.

Background: Conceptually, multi functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi functional enzymes are quite rare and are generally multi domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both α l arabinofuranosidase and β d xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality. Results: THSAbf is a GH51 α l arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °C and active over a broad pH range (4–7). THSAbf preferentially releases para nitrophenyl from the l arabino furanoside ( k cat / K M = 1050 s − 1 mM − 1 ) and to some extent from d galactofuranoside and d xyloside. THSAbf is active on 4 O methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg − 1 , respectively) and on sugar beet branched and linear arabinans (1.1 ± 0.24 and 1.8 ± 0.1 U mg − 1 ). Further investigation revealed that like the Alicyclo - bacillus sp. A4 α l arabinofuranosidase, THSAbf also displays endo xylanase activity, cleaving β 1,4 bonds in heteroxy lans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the α l arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi functionality

Valorisation of wheat bran to produce natural pigments using selected microorganisms

International audiencePigments are compounds with highly diverse structures and wide uses, which production is increasing worldwide. An eco-friendly method of bioproduction is to use the ability of some microorganisms to ferment on renewable carbon sources. Wheat bran (WB) is a cheap and abundant lignocellulosic co-product of low recalcitrance to biological conversion. Microbial candidates with theoretical ability to degrade WB were first preselected using specific databases. The microorganisms were Ashbya gossypii (producing riboflavin), Chitinophaga pinensis (producing flexirubin), Chromobacterium vaccinii (violacein) and Gordonia alkanivorans (carotenoids). Growth was shown for each on minimal salt medium supplemented with WB at 5 g.L−1. Activities of the main enzymes consuming WB were measured, showing leucine amino-peptidase (up to 8.45 IU. mL−1) and β-glucosidase activities (none to 6.44 IU. mL−1). This was coupled to a FTIR (Fourier Transform Infra-Red) study of the WB residues that showed main degradation of the WB protein fraction for C. pinensis, C. vaccinii and G. alkanivorans. Production of the pigments on WB was assessed for all the strains except Ashbya, with values of production reaching up to 1.47 mg.L−1. The polyphasic approach used in this study led to a proof of concept of pigment production from WB as a cheap carbon source

Investigation of the specificity of an alpha-L-arabinofuranosidase using C-2 and C-5 modified alpha-L-arabinofuranosides

International audienceThe synthesis of three novel glycosyl donors presenting the same scaffold as alpha-L-arabinofuranose but modified at the C-2 or C-5 positions has been achieved. Furthermore, chemoenzymatic syntheses using the alpha-L-arabinofuranosidase AbfD3 and these unnatural furanosides were investigated. The use of the novel p-nitrophenyl furanoside donors revealed that AbfD3 can perform transglycosylation with the C-5 deoxygenated donor but not with the C-2 modified one. These results emphasize the vital role for OH-2 in AbfD3 substrate recognition

Effect of lignin content on a GH11 endoxylanase acting on glucuronoarabinoxylan-lignin nanocomposites

International audienceThe effects of lignin content on the activity and action pattern of GH11 endoxylanase from Thermobacillus xylanilyticus were investigated using in vitro reconstituted non-covalent glucuronoarabinoxylan-model lignin (GAX-DHP) nanocomposites. Four types of nanocomposites were prepared, each displaying different lignin contents. Variations in the DHP (model lignin) polymerization process were induced by increasing the coniferyl alcohol concentration. Examination of the morphology of the nanocomposites revealed globular particles enrobed in a matrix. The size of these particles increased in line with the lignin concentration. Physicochemical characterization of the in vitro reconstituted GAX-DHPs strongly suggested that increased particle size is directly related to the solubility and reactivity of coniferyl alcohol, as reflected by changes in the amount of ˇ-O-4 linkages. Evaluation of the impact of the GH11 endoxylanase on the GAX-DHP nanocomposites revealed a negative correlation between the proportion and organization patterns of DHP in the nanocomposites and enzyme activity

The hemicellulolytic enzyme arsenal of Thermobacillus xylanilyticus depends on the composition of biomass used for growth

Background: Thermobacillus xylanilyticus is a thermophilic and highly xylanolytic bacterium. It produces robust and stable enzymes, including glycoside hydrolases and esterases, which are of special interest for the development of integrated biorefineries. To investigate the strategies used by T. xylanilyticus to fractionate plant cell walls, two agricultural by-products, wheat bran and straw (which differ in their chemical composition and tissue organization), were used in this study and compared with glucose and xylans. The ability of T. xylanilyticus to grow on these substrates was studied. When the bacteria used lignocellulosic biomass, the production of enzymes was evaluated and correlated with the initial composition of the biomass, as well as with the evolution of any residues during growth. Results: Our results showed that T. xylanilyticus is not only able to use glucose and xylans as primary carbon sources but can also use wheat bran and straw. The chemical compositions of both lignocellulosic substrates were modified by T. xylanilyticus after growth. The bacteria were able to consume 49% and 20% of the total carbohydrates in bran and straw, respectively, after 24 h of growth. The phenolic and acetyl ester contents of these substrates were also altered. Bacterial growth on both lignocellulosic biomasses induced hemicellulolytic enzyme production, and xylanase was the primary enzyme secreted. Debranching activities were differentially produced, as esterase activities were more important to bacterial cultures grown on wheat straw; arabinofuranosidase production was significantly higher in bacterial cultures grown on wheat bran. Conclusion: This study provides insight into the ability of T. xylanilyticus to grow on abundant agricultural by-products, which are inexpensive carbon sources for enzyme production. The composition of the biomass upon which the bacteria grew influenced their growth, and differences in the biomass provided resulted in dissimilar enzyme production profiles. These results indicate the importance of using different biomass sources to encourage the production of specific enzymes

Probing a family GH11 endo-beta-1,4-xylanase inhibition mechanism by phenolic compounds: Role of functional phenolic groups

International audiencePhenolic compounds generated from lignin degradation during the pre-treatment step in the process of producing bioethanol from lignocellulosic biomass are known to be inhibitory to enzymatic hydrolysis and fermentation. The inactivation mechanism of a GH11 endoxylanase (Tx-Xyl) by several phenolic compounds varying in their hydroxyl and methoxyl radical content was investigated. Apparent kinetic inactivation parameters were measured as an approximate index of the inhibitory effects. All the tested aromatic compounds had strong negative impact on enzyme activity and kinetic analysis revealed non competitive multi-site inhibition mechanism. The interactions between Tx-Xyl and the phenolic compounds were further studied by steady-state (tryptophan) fluorescence spectroscopy. Changes in lambda(max) of emission and quenching of fluorescence intensity indicated changes in the microenvironment of tryptophan residues. In agreement with the kinetic parameters, the fluorescence derived binding constants evidenced higher enzyme-phenolics interaction affinity with increasing phenolic hydroxyl radical content, suggesting clear correlations of such radicals with the inhibitory effects. Results indicated that the inhibitory effects of phenolic compounds on Tx-Xyl activity are most likely brought about by conformational alterations of the enzyme protein inducing steric inactivation

Action of a GH51 alpha-L-arabinofuranosidase on wheat derived arabinoxylans and arabino-xylooligosaccharides.

International audienceThe substrate specificity of an arabinofuranosidase (AbfD3) from family 51 of glycoside hydrolase classification was investigated in order to precisely evaluate its catalytic abilities. AbfD3 activity on destarched wheat bran was poor and less than 1% of total arabinose was released. AbfD3 was also tested on arabinoxylans derived from destarched wheat bran that present different degrees of polymerization, A/X ratios, ferulic acid content and solubility. Results indicated that AbfD3 can hydrolyze polymeric arabinoxylans, even if this action was moderate when compared to the efficient hydrolysis of oligosaccharides. The limited action of AbfD3 on polymeric arabinoxylans is discussed with regard to the heterogeneous distribution of the arabinose residues along the xylan main chain, the insolubility of arabinoxylans and to the presence of disubstituted xylose or feruloylated arabinose