GH36 α-galactosidase from Lactobacillus plantarum WCFS1 synthesize Gal-α-1,6 linked prebiotic α-galactooligosaccharide by transglycosylation.

Deepesh, Panwar and Shubhashini, A. and Sachin, R. Chaudhari and Harish Prashanth, K. V. and Mukesh, Kapoor (2020) GH36 α-galactosidase from Lactobacillus plantarum WCFS1 synthesize Gal-α-1,6 linked prebiotic α-galactooligosaccharide by transglycosylation. International Journal of Biological Macromolecules, 144. pp. 334-342. ISSN 0141-8130

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Abstract

α-Galactosidases are potent industrial glycoside hydrolases which are relatively less explored for their transglycosylation potential, especially from Lactobacillus genera. A GH36 α-galactosidase from Lactobacillus plantarum WCFS1 was cloned and over expressed in Hi-control Escherichia coli BL21(DE3). Ni-NTA affinity gel chromatography resulted in purified α-galactosidase (LpαG; specific activity 3077.35 U mg−1) having a monomeric weight of ~80 kDa with 29.3% yield. Size exclusion chromatography of LpαG showed native molecular mass of ~240.5 kDa. LpαG displayed optimum activity at pH 6 and 37 °C. The Km, Vmax and kcat/Km of LpαG towards pNPαGal were found to be 0.93 mM and 714.3 μmol ml−1 min−1 and 12,075 s−1 mM−1, respectively. LpαG displayed maximum transglycosylation activity towards melibiose substrate (as both donor and acceptor) and synthesized majorly a trisaccharide with 0.26 mg ml−1 yield. Nuclear magnetic resonance (NMR) characterization revealed that trisaccharide consist of only single species of α-linked galactooligosaccharide (manninotriose; α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp) with α-(1 → 6) regioselectivity. Manninotriose displayed prebiotic property by supporting the growth of probiotic L. plantarum WCFS1 and Bifidobacteria adolescentis DSM 20083.

Item Type:	Article
Uncontrolled Keywords:	GH36 α-galactosidase Characterization Transglycosylation α-Galactooligosaccharides Prebiotic
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 07 Enzyme Biochemistry
Divisions:	Dept. of Biochemistry Protein Chemistry and Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	15 Jun 2020 07:07
Last Modified:	15 Jun 2020 07:07
URI:	http://ir.cftri.res.in/id/eprint/14393

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