title: Large-scale production and homogenous purification of long chain polysialic acids from E. coli K1.
abstract: The study of new biomaterials is the objective of many current research projects in biotechnological medicine. A promising scaffold material for the application in tissue engineering or other biomedical applications is polysialic acid (polySia), a homopolymer of alpha2,8-linked sialic acid residues, which represents a posttranslational modification of the neural cell adhesion molecule and occurs in all vertebrate species. Some neuroinvasive bacteria like, e.g. Escherichia coli K1 (E. coli K1) use polySia as capsular polysaccharide. In this latter case long polySia chains with a degree of polymerization of >200 are linked to lipid anchors. Since in vertebrates no polySia degrading enzymes exist, the molecule has a long half-life in the organism, but degradation can be induced by the use of endosialidases, bacteriophage-derived enzymes with pronounced specificity for polySia. In this work a biotechnological process for the production of bacterial polysialic acid is presented. The process includes the development of a multiple fed-batch cultivation of the E. coli K1 strain and a complete downstream strategy of polySia. A controlled feed of substrate at low concentrations resulted in an increase of the carbon yield (C(product)/C(substrate)) from 2.2 to 6.6%. The downstream process was optimized towards purification of long polySia chains. Using a series of adjusted precipitation steps an almost complete depletion of contaminating proteins was achieved. The whole process yielded 1-2g polySia from a 10-l bacterial culture with a purity of 95-99%. Further product analysis demonstrated maximum chain length of >130 for the final product.
authors.affiliation: Institut für Technische Chemie, Leibniz Universität Hannover, Callinstrasse 3, 30167 Hannover, Germany.
authors.forename: Bastian
authors.initials: B
authors.lastname: Rode
authors.forename: Christian
authors.initials: C
authors.lastname: Endres
authors.forename: Chen
authors.initials: C
authors.lastname: Ran
authors.forename: Frank
authors.initials: F
authors.lastname: Stahl
authors.forename: Sascha
authors.initials: S
authors.lastname: Beutel
authors.forename: Cornelia
authors.initials: C
authors.lastname: Kasper
authors.forename: Sebastian
authors.initials: S
authors.lastname: Galuska
authors.forename: Rudolf
authors.initials: R
authors.lastname: Geyer
authors.forename: Martina
authors.initials: M
authors.lastname: Mühlenhoff
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
authors.forename: Thomas
authors.initials: T
authors.lastname: Scheper
mesh_terms: D019149:Bioreactors; D004926:Escherichia coli / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008962:Models, Theoretical; D015394:Molecular Structure; D012794:Sialic Acids / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D012794:Sialic Acids; C021319:polysialic acid