Journal article
Novel xylanolytic triple domain enzyme targeted at feruloylated arabinoxylan degradation
Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark1
Department of Biotechnology and Biomedicine, Technical University of Denmark2
Enzyme Technology, Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark3
Novozymes A/S4
LLa-Bioeconomy5
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.
Language: | English |
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Year: | 2019 |
Pages: | 109353 |
ISSN: | 18790909 and 01410229 |
Types: | Journal article |
DOI: | 10.1016/j.enzmictec.2019.05.010 |
ORCIDs: | 0000-0002-0425-9875 , Holck, Jesper , Pilgaard, Bo , Meyer, Anne S. and Wilkens, Casper |