title: A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.
abstract: An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.
authors.affiliation: Institut für Lebensmittelchemie, Leibniz Universität Hannover, Hannover, Germany annabel.nieter@lci.uni-hannover.de.
authors.forename: Annabel
authors.initials: A
authors.lastname: Nieter
authors.affiliation: Institut für Lebensmittelchemie, Leibniz Universität Hannover, Hannover, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Haase-Aschoff
authors.affiliation: Institut für Lebensmittelchemie, Leibniz Universität Hannover, Hannover, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kelle
authors.affiliation: Institut für Lebensmittelchemie, Leibniz Universität Hannover, Hannover, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Linke
authors.affiliation: Institut für Lebensmittelchemie, Leibniz Universität Hannover, Hannover, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Krings
authors.affiliation: SternEnzym GmbH & Co. KG, Ahrensburg, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Popper
authors.affiliation: Institut für Lebensmittelchemie, Leibniz Universität Hannover, Hannover, Germany.
authors.forename: Ralf G
authors.initials: RG
authors.lastname: Berger
mesh_terms: D002109:Caffeic Acids / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002726:Chlorogenic Acid / Q000378:metabolism; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003373:Coumaric Acids / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014588:Ustilago / Q000201:enzymology*
chemical_list: D002109:Caffeic Acids; D003373:Coumaric Acids; D004271:DNA, Fungal; D002726:Chlorogenic Acid; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C025374:chlorogenic acid esterase