Development and Application of a High-Throughput Functional Metagenomic Screen for Glycoside Phosphorylases.
Cell Chem Biol
; 26(7): 1001-1012.e5, 2019 07 18.
Article
em En
| MEDLINE
| ID: mdl-31080075
ABSTRACT
Glycoside phosphorylases (GPs) catalyze the reversible phosphorolysis of glycosidic bonds, releasing sugar 1-phosphates. To identify a greater range of these under-appreciated enzymes, we have developed a high-throughput functional screening method based on molybdenum blue formation. In a proof-of-principle screen focused on cellulose-degrading GPs we interrogated â¼23,000 large insert (fosmid) clones sourced from microbial communities inhabiting two separate environments and identified seven novel GPs from carbohydrate active enzyme family GH94 and one from GH149. Characterization identified cellobiose phosphorylases, cellodextrin phosphorylases, laminaribiose phosphorylases, and a ß-1,3-glucan phosphorylase. To demonstrate the versatility of the screening method, varying substrate combinations were used to identify GP activity from families GH13, GH65, GH112, and GH130 in addition to GH94 and GH149. These pilot screen and substrate versatility results provide a screening paradigm platform for recovering diverse GPs from uncultivated microbial communities acting on different substrates with considerable potential to unravel previously unknown degradative pathways within microbiomes.
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Base de dados:
MEDLINE
Assunto principal:
Fosforilases
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Glicosídeos
Idioma:
En
Revista:
Cell Chem Biol
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Canadá