Novel xylanolytic triple domain enzyme targeted at feruloylated arabinoxylan degradation

A three catalytic domain multi-enzyme; a CE1 ferulic acid esterase, a GH62 alpha-L-arabinofuranosidase and a GH10 beta-D-1,4-xylanase was identified in a metagenome obtained from wastewater treatment sludge. The capability of the CE1-GH62-GH10 multi-enzyme to degrade arabinoxylan was investigated to examine the hypothesis that CE1-GH62-GH10 would degrade arabinoxylan more efficiently than the corresponding equimolar mix of the individual enzymes. CE1-GH62-GH10 efficiently catalyzed the production of xylopyranose, xylobiose, xylotriose, arabinofuranose and ferulic acid (FA) when incubated with insoluble wheat arabinoxylan (WAX-I) (k(cat) = 20.8 +/- 2.6 s(-1)). Surprisingly, in an equimolar mix of the individual enzymes a similar k(cat) towards WAX-I was observed (k(cat) = 17.3 +/- 3.8 s(-1)). Similarly, when assayed on complex plant biomass the activity was comparable between CE1-GH62-GH10 and an equimolar mix of the individual enzymes. This suggests that from a hydrolytic point of view a CE1-GH62-GH10 multi-enzyme is not an advantage. Determination of the melting temperatures for CE1-GH62-GH10 (71.0 +/- 0.05 degrees C) and CE1 (69.9 +/- 0.02), GH62 (65.7 +/- 0.06) and GH10 (71 +/- 0.05 degrees C) indicates that CE1 and GH62 are less stable as single domain enzymes. This conclusion was corroborated by the findings that CE1 lost (similar to)50% activity within 2 h, while GH62 retained (similar to)50% activity after 24 h, whereas CE1-GH62-GH10 and GH10 retained (similar to)50% activity for 72 h. GH62-GH10, when appended to each other, displayed a higher specificity constant (k(cat)/K-m = 0.3 s(-1) mg(-1) ml) than the individual GH10 (k(cat)/K-m = 0.12 s(-1) +/- 0.02 mg(-1) ml) indicating a synergistic action between the two. Surprisingly, CE1-GH62, displayed a 2-fold lower k(cat) towards WAX-I than GH62, which might be due to the presence of a putative carbohydrate binding module appended to CE1 at the N-terminal. Both CE1 and CE1-GH62 released insignificant amounts of FA from WAX-I, but FA was released from WAX-I when both CE1 and GH10 were present, which might be due to GH10 releasing soluble oligosaccharides that CE1 can utilize as substrate. CE1 also displayed activity towards solubilized 5-O-trans-feruloyl-alpha-L-Araf (k(cat) = 36.35 s(-1)). This suggests that CE1 preferably acts on soluble oligosaccharides.