Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile.
A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0+/-1.5 kDa, with a mass of 33+/-1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55-60 degrees C. The purified esterase was stable at the pH range 5.0-7.0. The enzyme retained 70% of activity after 7 h at 50 degrees C and lost 50% of its activity after 45 min at 55 degrees C and after 12 min at 60 degrees C. Determination of k(cat)/ K(m) revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5- O- trans-feruloyl-alpha- l-arabinofuranoside (NPh-5-Fe-Ara f) 2-fold more efficiently than NPh-2-Fe-Ara f. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.
Topakas
E
E
Biotechnology Laboratory, Chemical Engineering Department, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, 15700, Athens, Greece.
Stamatis
H
H
Biely
P
P
Christakopoulos
P
P
D020001:1-Butanol / Q000378:metabolism; D002109:Caffeic Acids / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003373:Coumaric Acids / Q000378:metabolism; D004043:Dietary Fiber / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability; D006586:Hexanes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D062385:Hydroxybenzoates / Q000378:metabolism; D007526:Isoelectric Point; D008970:Molecular Weight; D021122:Protein Subunits / Q000737:chemistry; D013175:Sporothrix / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D014995:Xylosidases / Q000378:metabolism
D002109:Caffeic Acids; D003373:Coumaric Acids; D004043:Dietary Fiber; D006586:Hexanes; D062385:Hydroxybenzoates; D021122:Protein Subunits; C026385:n-hexane; C042405:methyl caffeate; D020001:1-Butanol; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; D014995:Xylosidases; C017616:phenolic acid