Split Enzyme-Based Biosensors for Structural Characterization of Soluble and Insoluble β-Glucans

Int J Mol Sci. 2021 Feb 4;22(4):1576. doi: 10.3390/ijms22041576.

Abstract

β-Glucan is widely distributed in various plants and microorganisms and is composed of β-1,3-linked d-glucose units. It may have a branched short or long side chain of glucose units with β-1,6- or β-1,4-linkage. Numerous studies have investigated different β-glucans and revealed their bioactivities. To understand the structure-function relationship of β-glucan, we constructed a split-luciferase complementation assay for the structural analysis of long-chain β-1,6-branched β-1,3-glucan. The N- and C-terminal fragments of luciferase from deep-sea shrimp were fused to insect-derived β-1,3-glucan recognition protein and fungal endo-β-1,6-glucanase (Neg1)-derived β-1,6-glucan recognition protein, respectively. In this approach, two β-glucan recognition proteins bound to β-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 β-glucan. To test the applicability of this assay, β-glucan and two β-glucan recognition proteins were mixed, resulting in an increase in the luminescence intensity in a β-1,3-glucan with a long polymer of β-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 β-1,3-glucan and long-chain β-1,6-branched β-1,3-glucan in various soluble and insoluble β-glucans.

Keywords: BGRP; endo-β-1,6-glucanase; luciferase; β-1,3-glucan; β-1,6-glucan; β-glucan recognition protein.

MeSH terms

  • Animals
  • Biosensing Techniques / methods*
  • Cell Wall / metabolism*
  • Glycoside Hydrolases / metabolism*
  • Luciferases / metabolism
  • Substrate Specificity
  • beta-Glucans / chemistry*
  • beta-Glucans / metabolism*

Substances

  • beta-Glucans
  • Luciferases
  • Glycoside Hydrolases