The maltodextrin transport system and metabolism inLactobacillus acidophilusNCFM and production of novel α-glucosides through reverse phosphorolysis by maltose phosphorylase


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Abstract

A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichiacoli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing β-glucose 1-phosphate (β-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and β-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mMβ-Glc 1-P and 50 mM glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or L-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be α-Glcp-(1→4)-Glcp (maltose), α-Glcp-(1→4)-GlcNp (maltosamine), α-Glcp-(1→4)-GlcNAcp (N-acetyl maltosamine), α-Glcp-(1→4)-Manp, α-Glcp-(1→4)-Xylp and α-Glcp-(1→4)- L-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

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