Abstract
Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl2. Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics.
Key points
• Thermophilic exo-arabinanase from family GH93
• Molecular basis of arabinan depolymerization
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Funding
This work was financially supported by the São Paulo Research Foundation (FAPESP), grants #2014/18714-2, #2014/06923-6, and #2017/00525-0 to FS; #2017/16089-1 to TAG, #2019/01165-0 to JV; #2015/50590-4 to FMS and #2019/06663-8 to BO; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grants #442352/2014-0 to LCO; 305748/2017-3 and 428527/2018-3 to FMS and #443916/2014-4, and #302627/2018-9 to FS. This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Finance Code 001. The molecular dynamics simulations and molecular docking were performed at the CENAPAD-SP (Centro Nacional de Processamento de Alto Desempenho em São Paulo), project UNICAMP/FINEP-MCT.
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JV, BO, ALG, ASL, EVM, GF, AM, and BAF performed the experiments and data interpretation and drafted the manuscript. TAG and FMS performed experiments and data interpretation related to CZE studies. MASK, IP, and LCO performed experiments and data interpretation related to molecular docking and SAXS. ALG, JV, and FS performed the phylogenetic analyses and data interpretation. JV, LCO, and IP participated in the design of the study and data interpretation and reviewed the manuscript. FS conceived the study, participated in data interpretation, and reviewed the manuscript. All authors read and approved the final manuscript.
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Velasco, J., Oliva, B., Gonçalves, A.L. et al. Functional characterization of a novel thermophilic exo-arabinanase from Thermothielavioides terrestris. Appl Microbiol Biotechnol 104, 8309–8326 (2020). https://doi.org/10.1007/s00253-020-10806-6
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DOI: https://doi.org/10.1007/s00253-020-10806-6