Cell Wall Anchoring of a Bacterial Chitosanase in Using a Food-Grade Expression System and Two Versions of an LP TG Anchor.

Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in WCFS1. The expression and cell-surface display efficiency driven by the food-grade -based system were compared with those obtained from the based pSIP system in terms of enzyme activities and their localisation on cells. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest enzymatic activity of CsnA-displaying cells was obtained from the strain carrying the -based expression plasmid with short cell wall anchor S. However, the attachment of chitosanase on cells via the long anchor L was shown to be more stable compared with the short anchor after several repeated reaction cycles. CsnA displayed on cells is catalytically active and can convert chitosan into chito-oligosaccharides, of which chitobiose and chitotriose are the main products.