Abstract
Background Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum.ResultsManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. ConclusionThis study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially ‘prebiotic’ oligosaccharides. This approach, with the enzyme of interest being displayed on the cell surface of a food-grade organism, may also be applied in production processes relevant for food industry.
Highlights
IntroductionLactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recog‐ nized as safe) status and long history of use in food applications
Lactobacillus plantarum is considered as a potential cell factory because of its GRAS status and long history of use in food applications
One of the most attractive features of cell-surface display is that enzyme molecules are simultaneously synthesized and self-immobilized on the bacterial cell surface, and the living whole-cell biocatalyst can be obtained from the cultivation [2,3,4]
Summary
Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recog‐ nized as safe) status and long history of use in food applications. Anchoring of a secreted enzyme to the bacterial cell wall enables the direct use of microbial cells as immobilized biocatalyst, simultaneously with or immediately after the fermentation step. There are two different ways of anchoring a secreted protein to the bacterial surface: via covalent attachment to the cell membrane or the cell wall, or noncovalently via a protein domain that interacts strongly with components of the cell wall or the membrane. Both systems have been used in lactic acid bacteria (LAB), primarily in Lactococcus lactis and different lactobacilli [6, 7]. Two major differences exist between these two methods of covalent attachment: (1) The protein is attached either to the membrane or the cell wall, and sortase-mediated anchoring results in a more peripherally displayed protein. (2) The protein is attached to the cell surface via its N terminus with the lipobox approach, while the sortase-mediated cell wall anchor attaches the protein via its C terminus
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