Engineering the hydrophobic residues of a GH11 xylanase impacts its adsorption onto lignin and its thermostability.

This study aimed to characterise the parameters governing the non-specific adsorption of a xylanase from Thermobacillus xylanilyticus (Tx-Xyn11) onto lignin isolated from maize stems. Such adsorption may be due to hydrophobic interactions between Tx-Xyn11 and lignin. Our strategy was to mutate hydrophobic residues present on the surface of Tx- Xyn11 into non-hydrophobic residues. Three mutants (P1, P2, and P3) with altered hydrophobic regions were produced and characterised. The thermostability of the P1 mutant was largely decreased compared with the thermostable Tx-Xyn11. The rate of adsorbed enzyme onto lignin was reduced to a similar extent for the P1 and P2 mutants, whereas the adsorption of the P3 mutant was less affected compared with that of Tx-Xyn11. When considered separately, the hydrophobic residues did not affect xylanase adsorption onto lignin. The addition of Tween 20 also led to the decreased adsorption of Tx-Xyn11 onto lignin. These results suggest that hydrophobic interactions are a key parameter in the interaction of Tx-Xyn11 with isolated lignin.

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.

A thermostable feruloyl-esterase from the hemicellulolytic bacterium Thermobacillus xylanilyticus releases phenolic acids from non-pretreated plant cell walls.

A gene (Tx-est1) encoding a thermostable feruloyl-esterase was isolated from the genome of the gram-positive hemicellulolytic thermophilic bacterium Thermobacillus xylanilyticus. This gene contains an open reading frame of 1,020 bp encoding a protein with molecular mass of 37.4 kDa, similar to feruloyl-esterases from cellulolytic bacteria and fungi. The recombinant enzyme Tx-Est1 was expressed and produced in Escherichia coli. Tx-Est1 contains the conserved putative lipase residues Ser 202, Asp 287, and His 322 which act as catalytic triad in its C-terminus part. Purified Tx-Est1 was active against phenolic acid derivatives and stable at high temperatures. Optimal activity was observed at 65 °C and the optimal pH was around 8.5. The kinetic parameters of the esterase were determined on various substrates. The enzyme displayed activity against methyl esters of hydrocinnamic acids and feruloylated arabino-xylotetraose, exhibiting high specificity and affinity for the latter. Our results showed that Tx-Est1 is a thermostable feruloyl-esterase which could be useful to hydrolyze arabinoxylans from graminaceous plant cell walls as the enzyme is able to release phenolic acids from a lignocellulose biomass.