Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl-saccharide esters.
The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Hannover, Germany.
Sebastian
S
Kelle
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Hannover, Germany.
Annabel
A
Nieter
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Hannover, Germany.
Ulrich
U
Krings
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Hannover, Germany.
Katerina
K
Zelena
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Hannover, Germany.
Diana
D
Linke
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Hannover, Germany.
Ralf G
RG
Berger
D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003373:Coumaric Acids / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000737:chemistry*; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000096:biosynthesis* / Q000737:chemistry*; D010843:Pichia / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014908:Triticum / Q000737:chemistry
D003373:Coumaric Acids; D004952:Esters; D062385:Hydroxybenzoates; D011994:Recombinant Proteins; D008320:Maltose; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; C017616:phenolic acid
Pleurotus sapidus; basidiomycete; esterification; feruloyl esterase; functional expression, Pichia pastoris