Split Enzyme-Based Biosensors for Structural Characterization of Soluble and Insoluble ß-Glucans.
Int J Mol Sci
; 22(4)2021 Feb 04.
Article
em En
| MEDLINE
| ID: mdl-33557290
ABSTRACT
ß-Glucan is widely distributed in various plants and microorganisms and is composed of ß-1,3-linked d-glucose units. It may have a branched short or long side chain of glucose units with ß-1,6- or ß-1,4-linkage. Numerous studies have investigated different ß-glucans and revealed their bioactivities. To understand the structure-function relationship of ß-glucan, we constructed a split-luciferase complementation assay for the structural analysis of long-chain ß-1,6-branched ß-1,3-glucan. The N- and C-terminal fragments of luciferase from deep-sea shrimp were fused to insect-derived ß-1,3-glucan recognition protein and fungal endo-ß-1,6-glucanase (Neg1)-derived ß-1,6-glucan recognition protein, respectively. In this approach, two ß-glucan recognition proteins bound to ß-glucan molecules come into close proximity, resulting in the assembly of the full-length reporter enzyme and induction of transient luciferase activity, indicative of the structure of ß-glucan. To test the applicability of this assay, ß-glucan and two ß-glucan recognition proteins were mixed, resulting in an increase in the luminescence intensity in a ß-1,3-glucan with a long polymer of ß-1,6-glucan in a dose-dependent manner. This simple test also allows the monitoring of real-time changes in the side chain structure and serves as a convenient method to distinguish between ß-1,3-glucan and long-chain ß-1,6-branched ß-1,3-glucan in various soluble and insoluble ß-glucans.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
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Parede Celular
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Beta-Glucanas
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Glicosídeo Hidrolases
Limite:
Animals
Idioma:
En
Revista:
Int J Mol Sci
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Japão