The feruloyl esterase system of Talaromyces stipitatus: determining the hydrolytic and synthetic specificity of TsFaeC.
The active site of the recombinant Talaromyces stipitatus type-C feruloyl esterase (TsFaeC) was probed using a series of C1-C4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of k(cat) and k(cat)/K(m) values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl group transfer to l-arabinose, using as a reaction system a ternary water-organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.
Biotechnology Laboratory, Chemical Engineering Department, National Technical University of Athens, 5 Iroon Polytechniou Str, Zografou Campus, 15700 Athens, Greece.
Christina
C
Vafiadi
Evangelos
E
Topakas
Paul
P
Christakopoulos
Craig B
CB
Faulds
D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D032901:Talaromyces / Q000201:enzymology*; D013696:Temperature
D003373:Coumaric Acids; D011994:Recombinant Proteins; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase