A Novel Dimeric Exoglucanase (GH5_38): Biochemical and Structural Characterisation towards its Application in Alkyl Cellobioside Synthesis.
Mpho S MafaHeinrich W DirrSamkelo MalgasRui Werner Maçedo KrauseKonanani RashamuseMpho Stephen MafaPublished in: Molecules (Basel, Switzerland) (2020)
An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked β-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards β-glucan, with a Km of 7.9 mg/mL and a kcat of 117.2 s-1, compared to lichenin which had a Km of 21.5 mg/mL and a kcat of 70.0 s-1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% β-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit.
Keyphrases
- mass spectrometry
- liquid chromatography
- ionic liquid
- tandem mass spectrometry
- high resolution mass spectrometry
- capillary electrophoresis
- high performance liquid chromatography
- gas chromatography
- simultaneous determination
- protein protein
- amino acid
- growth hormone
- binding protein
- single molecule
- case control
- cell wall
- solar cells