Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars.
Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).
Jose
Joachim
J
Pharmazeutische und Medizinische Chemie, Naturwissenschaftlich-Technische Fakultät III, Universität des Saarlandes, P. O. Box 151150, 66041 Saarbrücken, Germany. j.jose@mx.uni-saarland.de
von Schwichow
Steffen
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002241:Carbohydrates / Q000096:biosynthesis* / Q000737:chemistry; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007064:L-Iditol 2-Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D002241:Carbohydrates; D011993:Recombinant Fusion Proteins; D007064:L-Iditol 2-Dehydrogenase