Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors.
The gene for phytase from Escherichia coli was sequenced and compared with the appA gene. It was found to be a mutant derivative of the appA gene. After fusion with a C-terminal His-tag, phytase was purified by affinity chromatography and the enzymatic properties were analyzed. To develop a system for overexpression and extracellular production of phytase in E. coli, factors affecting the expression and secretion such as promoter type, host strain and selection pressure were analyzed. Using a secretion system based on the controlled expression of the kil gene, the expression of phytase was improved and the enzyme was released into the culture medium at a high level. An effective fermentation strategy based on fed-batch operation was developed.
Miksch
G
G
Department of Fermentation Engineering, Faculty of Technology, University of Bielefeld, 33501 Bielefeld, Germany. gmi@fermtech.techfak.uni-bielefeld.de
Kleist
S
S
Friehs
K
K
Flaschel
E
E
D010832:6-Phytase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D019149:Bioreactors; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005285:Fermentation; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000135:Acid Phosphatase; D010832:6-Phytase; C406788:appA protein, E coli