Cloning, characterization and functional expression of an alkalitolerant type C feruloyl esterase from Fusarium oxysporum.
A hypothetical protein FoFaeC-12213 of Fusarium oxysporum was found to have high amino acid sequence identity with known type C feruloyl esterases (FAEs) containing a 13-amino acid conserved region flanking the characteristic G-X-S-X-G motif of a serine esterase. The putative FAE from the genomic DNA was successfully cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in Pichia pastoris X-33 to confirm that the enzyme exhibits FAE activity. The molecular weight (62 kDa) and pI (6.8) were in agreement with the theoretical calculated values indicating the correct processing of the secretion signal in P. pastoris. The recombinant FAE was purified to its homogeneity and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme is a type C FAE showing broad hydrolytic activity against the four methyl esters of hydroxycinnamic acids and strong preference for the hydrolysis of n-propyl ferulate. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from Trichoderma longibrachiatum (a maximum of 67% total FA released after 1-h incubation). The esterase showed broad pH stability making it an important candidate for alkaline applications such as pulp treatment in the paper industry.
Moukouli
Maria
M
BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, 15700 Athens, Greece.
Topakas
Evangelos
E
Christakopoulos
Paul
P
D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003373:Coumaric Acids / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D003373:Coumaric Acids; D011994:Recombinant Proteins; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase