title: Cultivation and purification of two stereoselective imine reductases from Streptosporangium roseum and Paenibacillus elgii.
abstract: The reductive amination is one of the most important reactions in the synthesis of chiral amines. Imine reductases (IREDs) are novel enzymes that catalyze the asymmetric reduction of imines and reductive aminations using NADPH as hydride donor. In this study, we have developed a simple method to produce two enantiocomplementary IREDs from Streptosporangium roseum DSM 43021 (R-IRED-Sr) and Paenibacillus elgii (S-IRED-Pe). The proteins were expressed efficiently in Escherichia coli (E. coli) JW5510 at the 4-L-cultivation scale and were purified to 95% homogeneity in two steps by immobilized metal ion affinity and anion-exchange chromatography. The total protein yield was about 9 g per liter of E. coli culture and resulted in 150-220 mg purified IRED per liter of E. coli culture. The bioactivity of both IREDs was measured by the depletion of the NADPH cofactor in the reduction of model substrates 2-methylpyrroline (R-IRED-Sr) and 3,4-dihydroisoquinoline (S-IRED-Pe). High level reducing activity was found demonstrating the production of correctly folded and active IRED proteins. Specific activities of about 2.58 U/mg and 0.24 U/mg for the R- and S-selective IREDs were obtained, being in agreement with activities reported in the literature.
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Maike
authors.initials: M
authors.lastname: Lenz
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Philipp N
authors.initials: PN
authors.lastname: Scheller
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Sven M
authors.initials: SM
authors.lastname: Richter
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hauer
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569, Stuttgart, Germany. Electronic address: bettina.nestl@itb.uni-stuttgart.de.
authors.forename: Bettina M
authors.initials: BM
authors.lastname: Nestl
mesh_terms: D039903:Actinobacteria / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D056507:Paenibacillus / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases
keywords: Affinity chromatography; Cultivation; Enzyme purification; High level overexpression; Imine reductase; Ion-exchange chromatography