Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome

Abstract

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 K M range between 0.31 mM and 0.43 mM, K cat 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. Electronic supplementary material The online version of this article (10.1186/s12896-019-0510-1) contains supplementary material, which is available to authorized users.

Xylanolytic Extremozymes Retrieved From Environmental Metagenomes: Characteristics, Genetic Engineering, and Applications

“…It finds applications in biomass conversion, food and feed industries, and environmental waste management (Wilkens et al, 2017;Verma et al, 2019). Metagenomic approaches have brought out very few α-L-arabinofuranosidases from different environmental sources (Fortune et al, 2019). Recently, three α-L-arabinofuranosidases (AFaseH4, AFaseE3, and AFaseD3) have been reported from the high-temperature compost metagenome (Matsuzawa et al, 2015;Fortune et al, 2019).…”

“…Metagenomic approaches have brought out very few α-L-arabinofuranosidases from different environmental sources (Fortune et al, 2019). Recently, three α-L-arabinofuranosidases (AFaseH4, AFaseE3, and AFaseD3) have been reported from the high-temperature compost metagenome (Matsuzawa et al, 2015;Fortune et al, 2019). On characterization, all three display optimum activity at pH in the acidic range of pH 4.0-5.0 and at 40 • C. However, sequence analysis reveals that AFaseH4and AFaseE3-encoding genes share 100 and 99% identity with the existing sequences, respectively, while the ORF-encoding AFaseD3 shares 77% identity with the AFAse of a Paenibacillus taihuensis ORF (Kolinko et al, 2018).…”

“…Metagenomic approaches have brought out very few α-L-arabinofuranosidases from different environmental sources ( Fortune et al, 2019 ). Recently, three α-L-arabinofuranosidases (AFaseH4, AFaseE3, and AFaseD3) have been reported from the high-temperature compost metagenome ( Matsuzawa et al, 2015 ; Fortune et al, 2019 ). On characterization, all three display optimum activity at pH in the acidic range of pH 4.0–5.0 and at 40°C.…”