Heterologous expression and characterization of a putative glycoside hydrolase family 43 arabinofuranosidase from Clostridium thermocellum B8
| Autor: | Eliane Ferreira Noronha, Brenda R. de Camargo, Servé W. M. Kengen, Nico J. Claassens, Betania Ferraz Quirino |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Subfamily Glycoside Hydrolases 030106 microbiology Clostridium thermocellum B8 Firmicutes Sequence Homology Bioengineering Dockerin Xylose Biology Applied Microbiology and Biotechnology Biochemistry Microbiology Substrate Specificity Clostridium thermocellum 03 medical and health sciences chemistry.chemical_compound Arabinofuranosidase Microbiologie Polysaccharides Arabinoxylan Glycoside hydrolase Amino Acid Sequence Peptide sequence VLAG Gene Expression Regulation Bacterial biology.organism_classification Kinetics 030104 developmental biology chemistry GH43 glycoside hydrolase Heterologous expression Biotechnology |
| Zdroj: | Enzyme and Microbial Technology, 109, 74-83 Enzyme and Microbial Technology 109 (2018) |
| ISSN: | 1879-0909 0141-0229 |
| Popis: | An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose deconstruction. Our research group has been working on mapping and characterization of glycoside hydrolases produced by C. thermocellum B8, that are critical for lignocellulosic biomass deconstruction. One of the identified genes expressed during growth on sugar cane bagasse and straw is axb8, which encodes a putative cellulosomal GH43_29 α-arabinofuranosidase (EC 3.2.1.55) that has not previously been characterized at the molecular or kinetic levels. The AxB8 predicted amino acid sequence presented GH43 and dockerin domains, as well as a family 6 carbohydrate-binding module (CBM6). Also, it is a close homologue of Firmicutes putatives α-arabinofuranosidases, including cellulosomal proteins. Multiple alignment analysis grouped AxB8 in a cluster with four uncharacterized putative GH43_29 subfamily enzymes, all containing dockerin type I domain and CBM6 modules. Purified heterologously expressed AxB8 showed activity against the synthetic substrates pNPX (p-nytrophenyl-β-D-xylopyranoside) and pNPA (p-nytrophenyl-α-L-arabinofuranoside), as well as against the natural substrate wheat arabinoxylan (WAX), with maximal activity at 50 °C and pH between 5.0 and 6.0. The WAX degradation profile by AxB8 is different from those typically seen for α-arabinofuranosidases, presenting mainly xylose as a hydrolysis product, instead of arabinose. In addition, unlike other GH43_29 enzymes already characterized, AxB8 did not present activity against arabinan. Kinetic parameters using pNPA as a substrate were Km of 23 ± 3 mM and kcat of 104 ± 7 s−1. Despite its activity against pNPX, we did not observe AxB8 saturation with this substrate. AxB8 is the first member in its clade to be characterized regarding kinetic parameters, and together with its closest homologues could represent a large group of glycoside hydrolases with particular properties within the GH43_29 subfamily. |
| Databáze: | OpenAIRE |
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