A three catalytic domain multi-enzyme; a CE1 ferulic acid esterase, a GH62 α-l-arabinofuranosidase and a GH10 β-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) (kcat = 20.8 ± 2.6 s-1). Surprisingly, in an equimolar mix of the individual en... More
A three catalytic domain multi-enzyme; a CE1 ferulic acid esterase, a GH62 α-l-arabinofuranosidase and a GH10 β-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) (kcat = 20.8 ± 2.6 s-1). Surprisingly, in an equimolar mix of the individual enzymes a similar kcat towards WAX-I was observed (kcat = 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 °C) and CE1 (69.9 ± 0.02), GH62 (65.7 ± 0.06) and GH10 (71 ± 0.05 °C) indicates that CE1 and GH62 are less stable as single domain enzymes. This conclusion was corroborated by the findings that CE1 lost ˜50% activity within 2 h, while GH62 retained ˜50% activity after 24 h, whereas CE1-GH62-GH10 and GH10 retained ˜50% activity for 72 h. GH62-GH10, when appended to each other, displayed a higher specificity constant (kcat/Km = 0.3 s-1 mg-1 ml) than the individual GH10 (kcat/Km = 0.12 s-1 ± 0.02 mg-1 ml) indicating a synergistic action between the two. Surprisingly, CE1-GH62, displayed a 2-fold lower kcat 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-α-l-Araf (kcat = 36.35 s-1). This suggests that CE1 preferably acts on soluble oligosaccharides.,Copyright © 2019 Elsevier Inc. All rights reserved.
