title: High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification.
abstract: The mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high-cell-density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross-flow-filtration (50 kDa cut-off membrane). It was further purified in one-step anion-exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9-fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS-PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany. sven.richter@itb.uni-stuttgart.de
authors.forename: Sven
authors.initials: S
authors.lastname: Richter
authors.forename: Jens
authors.initials: J
authors.lastname: Nieveler
authors.forename: Holger
authors.initials: H
authors.lastname: Schulze
authors.forename: Till T
authors.initials: TT
authors.lastname: Bachmann
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D018533:Biomass; D020134:Catalytic Domain; D004390:Chlorpyrifos / Q000031:analogs & derivatives / Q000494:pharmacology; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation / Q000187:drug effects; D000432:Methanol / Q000378:metabolism / Q000494:pharmacology; D009154:Mutation / Q000235:genetics*; D009559:Nippostrongylus / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism; D010843:Pichia / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors; D014162:Transfection
chemical_list: D002800:Cholinesterase Inhibitors; D011994:Recombinant Proteins; C009618:O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphate; D000110:Acetylcholinesterase; D004390:Chlorpyrifos; D010100:Oxygen; D000432:Methanol