Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome.
BACKGROUND
The importance of the accessory enzymes such as α-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails.


RESULTS
Three fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60 °C while AFase-D3 had unique properties as it showed optimal activity at 25 °C as well as the ability to maintain substantial activity at temperatures as high as 90 °C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70 °C. The maximum activity was observed at a pH profile between pH 4.0-6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH 5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31 mM and 0.43 mM, Kcat range between 131 s- 1 and 219 s- 1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175 U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-β-L-arabinopyranoside and pNP-β-L-mannopyranoside respectively, and both hydrolysed pNP-β-D-glucopyranoside.


CONCLUSION
All three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study.
['Fortune|Brent|B|', 'Mhlongo|Sizwe|S|', 'van Zyl|Leonardo Joaquim|LJ|0000-0001-9364-8671', 'Huddy|Robert|R|', 'Smart|Mariette|M|', 'Trindade|Marla|M|']
D000076282:Composting D004795:Enzyme Stability D006026:Glycoside Hydrolases D006358:Hot Temperature D006863:Hydrogen-Ion Concentration D006868:Hydrolysis D007527:Isoenzymes D007700:Kinetics D054892:Metagenome D010802:Phylogeny D010957:Plasmids D013379:Substrate Specificity
Compost Metagenomics Thermostability Arabinofuranosidase