In vivo selection for the enhancement of Thermotoga maritima exopolygalacturonase activity at neutral pH and low temperature.
The aim of this study was to develop an Escherichia coli-based metabolic selection system for the uncovering of new oligogalacturonate-active enzymes. Based on the expression of the specific permease TogMNAB, this system enabled the entry of oligogalacturonates into the cytoplasm of E. coli thus providing a modified strain usable for this purpose. This tool was used for the metabolic selection of Thermotoga maritima exopolygalacturonase (TmGalU) mutants enabling the uptake of sodium trigalacturonate as the sole carbon source by the bacterium. In only one round of error-prone PCR and selection, mutants of TmGalU with a 4-fold increased turnover at pH 7.0 and 2-fold more active at 37 degrees C than wild-type enzyme were isolated. These results show the versatility of this strain for the evolution of oligogalacturonate-active enzymes.
Drone
Jullien
J
Université de Nantes, Nantes Atlantique Universités, UMR CNRS 6204, Biotechnologie, Biocatalyse, Biorégulation, 2, rue de la Houssinière, BP 92208, F-44322 Nantes, France.
Dion
Michel
M
Tellier
Charles
C
Rabiller
Claude
C
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012641:Selection, Genetic; D016415:Sequence Alignment; D013400:Sugar Acids / Q000378:metabolism; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*; D014312:Trisaccharides / Q000378:metabolism
D013400:Sugar Acids; D014312:Trisaccharides; C065008:O-galactopyranosyluronic acid-(1-4)-O-galactopyranosyluronic acid-(1-4)-galactopyranosyluronic acid; D006026:Glycoside Hydrolases; C020136:exopolygalacturonase