Characterization of three tannases from Clostridium butyricum active on oak bark extract

Other conference contribution, 2022

Tannins are secondary metabolites that are especially enriched in bark, which serves as the outer defense layer of trees. The tannin molecules impede microbial attack as they bind to and precipitate proteins. Tannins are grouped into condensed and hydrolysable tannins, where the latter are composed of a glucose unit ester linked to ellagic or galloyl moieties. As the name implies, hydrolysable tannins can be cleaved and enzyme called tannases (EC 3.1.1.20) exist that are able to selectively target galloyl units. Tannases could be used to facilitate further use of the bark by alleviating the inhibitory effect of tannins, for example in bioethanol production. However, only a few tannases have been studied to date.

In this work, we explored the multiplicity of tannases found in the gut bacterium Clostridium butyricum. This bacterium encodes three tannases, which were biochemically characterized using model substrates and oak bark extract, and furthermore structurally. We showed that the three tannases differed in substrate preference on model substrates and also displayed activity upon oak bark extract where they released gallic acid and glucose. The structure of one of the tannases was solved as 2.2 Å resolution and showed a similar catalytic site as the structures of the few previously solved tannases, and the other two enzymes were modelled using AlphaFold2. The main differences between the enzymes and previously studied tannases were the “cap” covering the active site as well as several large insert regions of unknown function.

In conclusion, we showed that the multiplicity of tannases in the genome of C. butyricum appears to reflect different biological roles, based on the enzymes displaying functional differences on the substrates as well as distinct structural features.