Hydrolysis of Nonpolar n-Alkyl Ferulates by Feruloyl Esterases

Article pubs.acs.org/JAFC

Hydrolysis of Nonpolar n‑Alkyl Ferulates by Feruloyl Esterases Aline Schar̈ ,† Isabel Sprecher,† Evangelos Topakas,§ Craig B. Faulds,#,⊥ and Laura Nyström*,† †

Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zurich, Switzerland Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, 15700 Athens, Greece # Aix Marseille Université, INRA BBF UMR_A 1163 Biodiversité et Biotechnologie Fongiques, 13288 Marseille cedex 02, France ⊥ INRA BBF UMR_A1163, Polytech Marseille, 163 Avenue de Luminy, 13288 Marseille cedex 02, France §

ABSTRACT: Ferulic acid is one of the major phenolic acids in plants and can be found esterified to plant cell wall components, but also as long-chain n-alkyl and steryl esters. Microbial feruloyl esterases may play a role in the bioavailability of phenolic acids during human and animal digestion. It is therefore of interest if feruloyl esterases are capable of hydrolyzing nonpolar ferulic acid esters. A series of n-alkyl ferulates with increasing lipophilicity were enzymatically synthesized, and the kinetic constants of their hydrolysis by four feruloyl esterases and a lipase as control were determined. A decrease in Km and kcat could be observed with decreased substrate polarity for all of the feruloyl esterases. Only one feruloyl esterase and the control lipase showed hydrolytic activity toward octadecyl ferulate. These results led to the conclusion that lipophilic ferulates are poor substrates for known feruloyl esterases and more specific esterases/lipases need to be identified. KEYWORDS: feruloyl esterase, alkyl ferulates, A. niger feruloyl esterase, C. thermocellum feruloyl esterase, R. miehei lipase, ferulic acid

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intestinal bacterium Lactobacillus acidophilus12 and esterases with hydroxycinnamate-hydrolyzing activity characterized from intestinal Eschericia coli, Bifidobacterium lactis and Lactobacillus gasseri.13 The substrate specificity of feruloyl esterases from different origins and types is therefore of interest for a broad range of areas including the human digestion of plant materials containing phenolic acid esters. Feruloyl esterases can be classified into at least four groups, as suggested by Crepin and co-workers.14 Their activity on different hydroxycinnamic acid methyl esters, the capability to release 5,5′-diferulic acid from various substrates, and amino acid sequence similarities are key criteria for this grouping. The feruloyl esterase from Aspergillus niger (AnFaeA) is a typical representative of a type A feruloyl esterase, showing preference for methyl hydroxycinnamates with methoxy groups on the aromatic ring, such as ferulic and sinapic acid.15,16 Furthermore, AnFaeA shows structural similarities to lipases.17 However, AnFaeA did not show lipase activity on olive oil triglycerides and very little hydrolytic activity on diglycerides.18 Type B feruloyl esterases, such as the one from Myceliophthora thermophila,19 on the other hand, prefer methyl hydroxycinnamates with one or two hydroxyl groups such as p-coumaric acid or caffeic acid and show only very low to no activity against methyl sinapate.14 In addition, the type of sugar, the length of oligosaccharide chain, and the location of the ester link between the acid and the sugar have a strong impact on the specificity of feruloyl esterases.20 Thus, feruloyl esterases of different classes may show strongly varying activities toward a range of

INTRODUCTION In plant tissues, ferulic acid is one of the most abundant hydroxycinnamic acids.1 The phenolic acids in plants occur as soluble free acids, soluble conjugated phenolic acids, and insoluble bound phenolic acids.2 In wheat, for instance, the major group is the insoluble bound form, which is composed of phenolic acids bound to insoluble cell wall components,3 such as arabinoxylan or pectin.4 The soluble conjugated phenolates, such as the nonpolar alkyl ferulates, are covalently bound to low molecular weight components and can be analyzed through extraction and hydrolysis afterward.2 Prominent examples are steryl ferulates, where the phenolic acid is esterified to a plant sterol, which can be found, for example, in cereal grains, such as rice, wheat, and corn.5 Other nonpolar alkyl ferulates can be found in suberin waxes, a nonpolymeric extract of low polarity from suberized tissues.6 Ferulic acid esters of 1-alkanols in suberin waxes are long-chain (C16−C30) and mostly possess an even number of carbons in the alkyl chain.6,7 The occurrence of alkyl hydroxycinnamates in plants has been summarized recently and also includes, for example, alkyl ferulates with an alcohol chain length of C16−C30 in potato pulp. 8 Furthermore, these compounds are known for their antioxidant activity, which is dependent on the chain length and the type of hydroxycinnamic acid.9 Overall, phenolic acids can be found esterified to various compounds with very different properties influencing also their bioavailability. Feruloyl esterases have a significant impact on plant processing by not only improving the bioavailability of phytonutrients but also optimizing the saccharification of cereal derived raw materials for feed and bioethanol production.10 It has been shown that esterases extracted from human intestinal mucosa are capable of hydrolyzing esters of dietary hydroxycinnamic acids.11 Furthermore, a feruloyl esterase has been extracted and characterized also from a typical human © 2016 American Chemical Society

Received: Revised: Accepted: Published: 8549

June 15, 2016 September 5, 2016 September 7, 2016 September 7, 2016 DOI: 10.1021/acs.jafc.6b02694 J. Agric. Food Chem. 2016, 64, 8549−8554

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Journal of Agricultural and Food Chemistry substrates, and the examination of a representative selection of feruloyl esterases is required. Apart from methyl hydroxycinnamates, methyl esters of various phenylalkanoic and cinnamic acids have also been evaluated as substrates for feruloyl esterases.16,21,22 Although the influence of the acid moiety of the substrate on the feruloyl esterase activity has been studied several times, there are fewer studies available related to the effect of alcohol moiety on the enzyme activity. For two type C and one type B feruloyl esterases short-chain alkyl ester substrates up to butyl ferulate were evaluated,19,22−24 but for more lipophilic substrates the data are scarce. For example, the activity of type A feruloyl esterase from Aspergillus awamori against α-naphthyl esters was evaluated, and no activity was detected for acids longer than eight carbon atoms such as caprylic acid.25 However, the chain length of the fatty acid was varied, and the alcohol α-naphthol remained the same. Enzymatic activity of feruloyl esterases on lipophilic substrates is further influenced by cosolvents.26 For AnFaeA, the activity toward methyl ferulate decreased to around 60% if the buffer solution contained 5% DMSO (v/v). On the other hand, for the substrate p-nitrophenyl acetate, the activity increased to almost 180% by the addition of 5% DMSO. Therefore, for water-insoluble substrates a treatment with 10−30% DMSO was proposed beneficial to the activity of feruloyl esterases.26 Consequently, it is of interest if feruloyl esterases can also hydrolyze nonpolar n-alkyl ferulates, but this question has until now not been systematically evaluated for chain lengths longer than four. To approach this problem, a series of n-alkyl ferulates with increasing lipophilicity were synthesized and evaluated as substrates for four types of feruloyl esterases and one lipase as control.

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Figure 1. Structural formula of ferulic acid esters. For the enzymatic esterification n corresponds to 2, 5, 9, or 17, and for the hydrolysis by feruloyl esterases n equals 0, 1, 2, 5, 9 or 17. workers.29 In this procedure, n-hexane was evaporated, and 100 μL of the remaining alcohol including the ferulic acid and the n-alkyl ferulate were redissolved in 4 mL of methanol. After the addition of 666 μL of 0.6% KOH (0.6% (v/v) aqueous saturated KOH diluted in water), the methanol was washed 10 times with 3.2 mL of n-hexane to remove the unreacted alcohol. Finally, the methanol phase was acidified with 400 μL of 6 M aqueous hydrochloric acid, and the n-alkyl ferulates were extracted five times with 3.2 mL of n-hexane. For the octadecyl ferulate the following minor changes in the base−acid wash were made: 333 μL of 1.2% KOH, only five washings of the basic methanol, and the whole procedure was performed twice. Products were analyzed by NPHPLC (Luna HILIC column from Phenomenex, Torrance, CA, USA, isocratic flow of hexane and isopropanol (99:1) at 0.5 mL/min) equipped with a refractive index detector (RID) to control the removal of the free alcohol. Hydrolysis of n-Alkyl Ferulates by Feruloyl Esterases. An aliquot of a solution of n-alkyl ferulates in acetone was transferred into a glass tube, and the solvent was removed under a stream of nitrogen at 50 °C. The volume of substrate solution in acetone was calculated on the basis of the amount needed for the hydrolysis experiments in accordance with the concentration determined, as described below. First, the DMSO was added followed by the buffer to reach the total reaction volume; final concentrations were 5% DMSO, 1 mM MOPS, or 5 mM MES buffer and varying n-alkyl ferulate concentrations. The reactions with AnFaeA and MtFaeB were conducted at pH 6 with MES buffer and for the others (lipase, CtFae, ROFae) with MOPS buffer at pH 7. Concentrations of n-alkyl ferulates ranged from 3.5 μM to 6 mM, depending on the enzyme, and final protein concentrations were 1.5 nM, 0.6 nM, 35.2 nM, 0.9 nM, and 3.7 μM for AnFaeA, MtFaeB, CtFae, ROFae, and lipase, respectively. For each enzyme and substrate six or more different substrate concentrations were analyzed in triplicates. The sample was preheated in a water bath at 40 °C before the enzyme was added to start the hydrolytic reaction. After 15 min, the reaction was terminated again by the addition of acetonitrile in a ratio of 1:1 to the reaction volume and filtration for HPLC analysis. Quantification of Substrates and Ferulic Acid by RP-HPLC and Data Analysis. A standard substrate concentration was measured in the same way without incubation and enzyme addition to determine the substrate concentration in the acetone. The activity of the enzyme solution was periodically monitored with a standard assay based on methyl ferulate. If the activity decreased significantly, a new solution was prepared. Ferulic acid and n-alkyl ferulates were quantified by RP-HPLC as published earlier.28 Briefly, an xBridge Phenyl column from Waters was used with a gradient elution of 1% acetic acid in water and acetonitrile, water, butanol, acetic acid in a ratio of 88:6:4:2. Calibration was achieved for all ferulates by creating one calibration curve for ferulic acid, methyl ferulate, ethyl ferulate, and γ-oryzanol (0.006−2.6 nmol/injection). Kinetic constants were estimated by fitting them to Michaelis−Menten kinetics using SigmaPlot (version 12.5 Systat Software, Inc., San Jose, CA, USA), which includes an estimation of the standard error for the calculated parameters. The used molecular masses for the calculation of kcat were the following: 30 kDa for AnFaeA,27 39 kDa for MtFaeB,19 31.6 kDa for the lipase,30 and 29 kDa for CtFae and ROFae, according to the provided data sheets.

MATERIALS AND METHODS

Chemicals. Ferulic acid (≥99%), 3-(N-morpholino)propanesulfonic acid (MOPS; ≥99.5%), and 2-(N-morpholino)ethanesulfonic acid (MES; ≥99%) were obtained from Sigma-Aldrich, Buchs, Switzerland. Methyl ferulate (99%) and ethyl ferulate (98%) were purchased from Alfa Aesar, Karlsruhe, Germany. γ-Oryzanol was obtained from Wako Pure Chemical Industries, Osaka, Japan. All solvents used were of HPLC grade or of higher purity. Enzymes. Lipozyme RM IM was provided by Novozymes A/S, Bagsvaerd, Denmark. Feruloyl esterases from rumen microorganism, ROFae (600 U/mL where 1 U corresponds to 1 μmol of ferulic acid released from ethyl ferulate per minute at pH 6.5 and 40 °C), and from XynZ domain of Clostridium thermocellum, CtFae (10 U/mL where 1 U corresponds to 1 μmol of ferulic acid released from ethyl ferulate per minute at pH 6 and 50 °C), were obtained from Megazyme, Bray, Ireland. Recombinant feruloyl esterase type A from A. niger, AnFaeA, was produced according to the method of Juge and co-workers.27 The lyophilized enzyme was redissolved in buffer (MOPS, pH 6). The type B feruloyl esterase from Myceliophthora thermophila, MtFaeB, was prepared according to procedure of Topakas et al. without the chromatographic purification.19 Lipase from Rhizomucor miehei (≥20000 U/g) was purchased from Sigma-Aldrich. Protein contents of enzyme preparations were analyzed according to the Bradford assay using Bradford reagent from Sigma-Aldrich and bovine serum albumin as standard. Preparation of n-Alkyl Ferulates. Propyl, hexyl, decyl, and octadecyl ferulates (Figure 1) were enzymatically esterified using Lipozyme RM IM as published earlier.28 To remove the ferulic acid from the propyl ferulate, the reaction mixture in n-hexane was washed with water. After evaporation of the unreacted propanol and the solvent n-hexane at 50 °C, the propyl ferulate product was redissolved in acetone and ready for hydrolytic reactions. The other ferulates were purified by a base−acid wash adapted from that of Hakala and co-

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RESULTS AND DISCUSSION The kinetic constants using the Michaelis−Menten equation were determined for four feruloyl esterases and one control lipase using methyl, ethyl, propyl, hexyl, and decyl ferulate as 8550

DOI: 10.1021/acs.jafc.6b02694 J. Agric. Food Chem. 2016, 64, 8549−8554

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Journal of Agricultural and Food Chemistry

Table 1. Kinetic Constants of Feruloyl Esterases (Feruloyl Esterase Type A from A. niger (AnFaeA); Feruloyl Esterase Type B from M. thermophila (MtFaeB), Feruloyl Esterase from C. thermocellum (CtFae), and Feruloyl Esterase from Rumen Microorganism (ROFae)) and Lipase from R. miehei for Different n-Alkyl Ferulates methyl ferulate

ethyl ferulate

Km (μM) kcat (s−1) kcat/Km (mM−1 s−1) R2a nb

1123 (71)c 32.9 (1.2) 29 (2) 0.996 9

611 (60) 29.4 (1.2) 48 (5) 0.985 9

Km (μM) kcat (s−1) kcat/Km (mM−1 s−1) R2 n

51 (3.4) 8.8 (0.3) 173 (13) 0.988 9

48 (2.7) 11.2 (0.4) 236 (15) 0.991 9


2472 (170) 8.0 (0.3) 3.2 (0.3) 0.994 6

2578 (152) 5.7 (0.2) 2.2 (0.1) 0.996 6


134 (17) 33.5 (4.9) 250 (48) 0.962 8

149 (16) 30.7 (4.5) 206 (37) 0.973 8


413 (79) 0.002 (0.0002) 0.005 (0.001) 0.941 7

636 (168) 0.004 (0.0004) 0.006 (0.002) 0.939 7

propyl ferulate AnFaeA 245 (18) 44.6 (1.2) 182 (14) 0.989 11 MtFaeB 27 (1.7) 12.1 (0.4) 452 (32) 0.985 9 CtFae 1237 (358) 3.2 (0.5) 2.6 (0.8) 0.93 10 ROFae 81 (8) 31.7 (4.6) 391 (68) 0.976 9 Lipasee 1848g (401) 0.022 (0.0030) 0.012 (0.003) 0.979 10

hexyl ferulate

decyl ferulate

octadecyl ferulate

40 (3.9) 9.8 (0.3) 243 (25) 0.948 13

8 (2.0) 4.6 (0.3) 547 (136) 0.709 11e

ndd

10 (0.9) 8.9 (0.3) 906 (89) 0.918 13

>0f

nd

29 (5) 0.2 (0.006) 5.6 (0.9) 0.909 11

125 (27) 0.4 (0.02) 3.2 (0.7) 0.907 10

>0

27 (2.5) 6.1 (0.9) 225 (39) 0.937 13

3.3h (0.8) 2.6 (0.4) 780 (213) 0.636 11

nd

88 (25) 0.006 (0.0004) 0.07 (0.02) 0.811 9

146 (33) 0.010 (0.0009) 0.07 (0.02) 0.894 8

>0

a 2

R reflects the coefficient of determination between the experimental data and the calculated Michaelis−Menten kinetics. bn is the number of different substrate concentrations analyzed in triplicates. cNumbers in parentheses represent the estimated standard errors. dnd indicates the amount of ferulic acid released was below limit of detection. eAt one substrate concentration only duplicate results were available. f>0 indicates the amount of ferulic acid released was below limit of quantification. gKm was above tested substrate concentrations. hKm was below tested substrate concentrations.

and kcat/Km, respectively.31 This Km is slightly lower than the value determined in this study, which could be a result of the 5% DMSO in the reaction system, as shown for another feruloyl esterase.26 The turnover number measured here was quite low, which may result again from the DMSO addition, as was shown in an earlier study for AnFaeA, where the addition of 8% DMSO led to a decrease of 50% of the original activity.26 Moreover, the different molecular masses, which were determined earlier for AnFaeA, can lead to differences in kcat values depending on the method. The molar mass determined by mass spectroscopy was 29.7 kDa, whereas following SDSPAGE a molecular mass of 36 kDa was found.32 Furthermore, the kinetic constants of MtFaeB for methyl ferulate were determined earlier and were found to be 270 μM, 6.4 s−1, and 23.7 mM−1 s−1 for Km, kcat, and kcat/Km,, respectively.19 Compared to that study, the turnover number obtained matches quite well (8.8 s−1); however, Km found in this study is lower (51 μM). This difference may again result from the DMSO addition, as not all feruloyl esterases show the same effect of activity on the addition of this aprotic solvent.26 Overall, the determined kinetic constants for methyl ferulate as substrate are in the range that could be expected on the basis of previous results.

substrates (Table 1). For the substrate with the longest alkyl chain, the octadecyl ferulate, no hydrolysis could be measured for AnFaeA, MtFaeB, and ROFae, even if the incubation time was increased to 24 h. In contrast, CtFae and the control lipase liberated ferulic acid; however, the activity was too low to determine kinetic constants. Generally, Km and kcat values decreased with increasing chain length for the feruloyl esterases. Although with increasing lipophilicity of the substrate Km decreases more strongly compared to the kcat values, the catalytic efficiency kcat/Km is increasing mainly in the case of AnFaeA and MtFaeB. For CtFae and the control lipase the pattern was not as clear. Also, the coefficient of determination (R2) of the experimental data fitted to the Michaelis−Menten kinetics showed a decreasing trend with increasing chain length of the n-alkyl ferulate. The kinetic constants of the different feruloyl esterases for methyl ferulate differed quite strongly. MtFaeB and ROFae show very high affinity to methyl ferulate with Km values of 51 and 134 μM, respectively. On the other hand, AnFaeA and CtFae showed only low affinity toward methyl ferulate, even lower than R. miehei lipase. The kinetic constants for AnFaeA against methyl ferulate have been determined before and were found to be 780 μM, 70.74 s−1, and 91 mM−1 s−1 for Km, kcat, 8551


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The Michaelis constant decreased with an increasing lipophilicity of the substrate for all tested feruloyl esterases, which could have several reasons. First, as the solubility of the long-chain n-alkyl ferulates in the reaction system was very low, aggregation of substrate can be one source of error. The apparent Km in this case would represent the solubility of the substrate rather than the affinity of the enzyme to the substrate, because above the limit of solubility the substrate in solution would stay constant, even if the substrate amount would be increased. However, because the Michaelis constants determined in this study for decyl ferulate were quite different between the enzymes, ranging from 3.3 to 146 μM, this factor can be excluded. Second, a more pronounced decrease in Km with increasing lipophilicity compared to kcat indicates a reduced k−1 (rate constant for dissociation of the enzyme− substrate complex) or an increased k1 (rate constant for formation of the enzyme−substrate complex) for more hydrophobic substrates. This could lead to the hypothesis that a decreasing Km with increasing lipophilicity of the substrate not only is an indication for the specificity to the enzyme but also reflects the solubility of the substrate in the aqueous system. The substrate undergoes desolvation when binding to the enzyme, which is energetically more favored for less soluble substrates.34,35 Accordingly, the reverse process (k−1) is less favored. In this case, the declining Km may therefore be misleading with regard to the specificity of feruloyl esterases. On a mechanistic basis feruloyl esterases show similarities. All feruloyl esterases evaluated in this study, except ROFae, have been shown to have a catalytic triad in the active site,17,19,36 as well as the lipase.37 Therefore, a covalent enzyme−acyl intermediate is formed during the hydrolysis. Identical catalytic rate constants can result from a common acyl−enzyme intermediate and a rate-limiting deacylation.38 As the acyl group was always ferulic acid, the catalytic rate should always be similar if the deacylation is rate limiting. However, this was often the case only for short-chain ferulic acid esters. Examples are ROFae and AnFaeA, where similar kcat values for methyl, ethyl, and propyl ferulates were measured, whereas a decrease in rate constant was observed for longer chains. In this case, the rate-limiting step probably shifted partially or fully to the formation of the acyl−enzyme complex, which could be explained by a less suitable position of the long-chain ester for the nucleophilic attack of the catalytic serine. However, as the feruloyl esterases are structurally very different, one would have to study the interaction of the nonpolar substrate in more detail individually. Overall, this supports the hypothesis that longchain n-alkyl ferulates are poor substrates for feruloyl esterases. A systematic evaluation of the activity of feruloyl esterases from different classes on nonpolar n-alkyl ferulates was carried out to evaluate if microbial feruloyl esterases are capable of hydrolyzing naturally occurring n-alkyl ferulates. This led to the conclusion that for feruloyl esterases, nonpolar ferulic acid esters such as long-chain n-alkyl ferulates are very poor substrates. Only very little or no activity was determined for octadecyl ferulate. This conclusion is supported by earlier studies, which showed no activity of a feruloyl esterase against olive oil triglycerides or, in a second study, against long-chain (>C10) α-naphthyl esters. Further evaluations of more feruloyl esterases would support this conclusion. Finally, studies using biological samples containing long-chain n-alkyl ferulates would be of interest to evaluate the in vivo activity in a more complex environment. The change in n-alkyl ferulate concentration in

Several trends in the kinetic constants for the different feruloyl esterases could be observed for a varied lipophilicity of the ferulate substrate. There is a trend of a decreasing Michaelis constant (Km) with increasing lipophilicity of the substrate for all tested feruloyl esterases. Furthermore, the turnover number was also shown to decrease with increasing chain length of the alcohol. For CtFae the turnover number behaves in a similar way to the Michaelis constant, which results in a rather stable catalytic efficiency with varying lipophilicity of the substrate. If kcat decreases less than Km, the catalytic efficiency increases. This was the case for ROFae, where the catalytic efficiency is around 3 times higher for decyl ferulate than for methyl ferulate. For AnFaeA, the stronger decrease in Km than in kcat is most pronounced, leading to a much higher catalytic efficiency for decyl ferulate. The kinetic constants of MtFaeB for decyl ferulate could not be determined as hydrolysis was observed, but no clear change of initial reaction rate over the measured substrate concentrations could be observed. For MtFaeB, the kinetic constants have been determined earlier for also ethyl, propyl, and butyl ferulates.19 However, due to DMSO addition, comparisons are difficult between similar reaction systems, as discussed above for methyl ferulate. The lipase from R. miehei has been applied as positive control. For this lipase no clear trend within the kinetic constants concerning the lipophilicity of the substrate could be observed. The Michaelis constant and the turnover number of the lipase were at a maximum with propyl ferulate. Michaelis− Menten kinetics seemed also appropriate for the lipase, as low substrate concentrations and therefore monophasic conditions were applied. Although lipases and esterases often follow a similar mechanism, lipases differ from esterases due to their typical interfacial activation. This phenomenon is often explained through a repositioning of the lid, thus stabilizing the active site. Although a structure similar to a lid can be found in AnFaeA, the lid is stabilized in open conformation, which gives the esterase character to this enzyme.17 However, the R. miehei lipase seems to be a suitable control enzyme for the hydrolysis of n-alkyl ferulates, although its hydrolytic activity is low. For decyl ferulate, the Km was higher for CtFae and for the lipase compared to the other enzymes tested. Although this would indicate lower affinity, these were the two enzymes for which still some activity against octadecyl ferulate could be measured. Interestingly, the type A feruloyl esterase AnFaeA, which structurally resembles the R. miehei lipase,17,31 was not able to hydrolyze octadecyl ferulate. This might be explained by the structure of AnFaeA. Although the catalytic serine is exposed to the solvent in a large cavity, the surrounding region shows, similarly to carbohydrate-binding proteins, a highly negative electrostatic potential.17 Earlier it has also been shown that the catalytic efficiency of the same enzyme (AnFaeA earlier FAE-III) is generally higher for sugar esters than for methyl ferulate.20,33 Therefore, the findings of this study correspond well with the general idea of feruloyl esterases preferring polar ferulates. Furthermore, the coefficient of determination was very low for AnFaeA and ROFae with decyl ferulate, which is probably due to the fact that only a few samples below Km were measured. This also increases the relative error and therefore the uncertainty of the determined constants. A lower Km value for feruloyl esterases with decyl ferulate could therefore not directly be connected to a higher affinity for nonpolar substrates. 8552


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(16) Kroon, P. A.; Faulds, C. B.; Brezillon, C.; Williamson, G. Methyl phenylalkanoates as substrates to probe the active sites of esterases. Eur. J. Biochem. 1997, 248, 245−251. (17) Hermoso, J. A.; Sanz-Aparicio, J.; Molina, R.; Juge, N.; Gonzalez, R.; Faulds, C. B. The crystal structure of feruloyl esterase A from Aspergillus niger suggests evolutive functional convergence in feruloyl esterase family. J. Mol. Biol. 2004, 338, 495−506. (18) Aliwan, F. O.; Kroon, P. A.; Faulds, C. B.; Pickersgill, R.; Williamson, G. Ferulic acid esterase-III from Aspergillus niger does not exhibit lipase activity. J. Sci. Food Agric. 1999, 79, 457−459. (19) Topakas, E.; Moukouli, M.; Dimarogona, M.; Christakopoulos, P. Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila. Appl. Microbiol. Biotechnol. 2012, 94, 399−411. (20) Faulds, C. B.; Kroon, P. A.; Saulnier, L.; Thibault, J. F.; Williamson, G. Release of ferulic acid from maize bran and derived oligosaccharides by Aspergillus niger esterases. Carbohydr. Polym. 1995, 27, 187−190. (21) Topakas, E.; Christakopoulos, P.; Faulds, C. B. Comparison of mesophilic and thermophilic feruloyl esterases: characterization of their substrate specificity for methyl phenylalkanoates. J. Biotechnol. 2005, 115, 355−366. (22) Vafiadi, C.; Topakas, E.; Christakopoulos, P.; Faulds, C. B. The feruloyl esterase system of Talaromyces stipitatus: determining the hydrolytic and synthetic specificity of TsFaeC. J. Biotechnol. 2006, 125, 210−221. (23) Vafiadi, C.; Topakas, E.; Wong, K. K. Y.; Suckling, I. D.; Christakopoulos, P. Mapping the hydrolytic and synthetic selectivity of a type C feruloyl esterase (StFaeC) from Sporotrichum thermophile using alkyl ferulates. Tetrahedron: Asymmetry 2005, 16, 373−379. (24) Moukouli, M.; Topakas, E.; Christakopoulos, P. Cloning, characterization and functional expression of an alkali tolerant type C feruloyl esterase from Fusarium oxysporum. Appl. Microbiol. Biotechnol. 2008, 79, 245−254. (25) Koseki, T.; Takahashi, K.; Fushinobu, S.; Iefuji, H.; Iwano, K.; Hashizume, K.; Matsuzawa, H. Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence. Biochim. Biophys. Acta, Gen. Subj. 2005, 1722, 200−208. (26) Faulds, C. B.; Perez-Boada, M.; Martinez, A. T. Influence of organic co-solvents on the activity and substrate specificity of feruloyl esterases. Bioresour. Technol. 2011, 102, 4962−4967. (27) Juge, N.; Williamson, G.; Puigserver, A.; Cummings, N. J.; Connerton, I. F.; Faulds, C. B. High-level production of recombinant Aspergillus niger cinnamoyl esterase (FAEA) in the methylotrophic yeast Pichia pastoris. FEMS Yeast Res. 2001, 1, 127−132. (28) Schär, A.; Nyström, L. High yielding and direct enzymatic lipophilization of ferulic acid using lipase from Rhizomucor miehei. J. Mol. Catal. B: Enzym. 2015, 118, 29−35. (29) Hakala, P.; Lampi, A. M.; Ollilainen, V.; Werner, U.; Murkovic, M.; Wahala, K.; Karkola, S.; Piironen, V. Steryl phenolic acid esters in cereals and their milling fractions. J. Agric. Food Chem. 2002, 50, 5300−5307. (30) Wu, X. Y.; Jaaskelainen, S.; Linko, Y. Y. Purification and partial characterization of Rhizomucor miehei lipase for ester synthesis. Appl. Biochem. Biotechnol. 1996, 59, 145−158. (31) Faulds, C. B.; Molina, R.; Gonzalez, R.; Husband, F.; Juge, N.; Sanz-Aparicio, J.; Hermoso, J. A. Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger. FEBS J. 2005, 272, 4362−4371. (32) deVries, R. P.; Michelsen, B.; Poulsen, C. H.; Kroon, P. A.; vandenHeuvel, R. H. H.; Faulds, C. B.; Williamson, G.; vandenHombergh, J. P. T. W.; Visser, J. The faeA genes from Aspergillus niger and Aspergillus tubingensis encode ferulic acid esterases involved in degradation of complex cell wall polysaccharides. Appl. Environ. Microbiol. 1997, 63, 4638−4644. (33) Ralet, M. C.; Faulds, C. B.; Williamson, G.; Thibault, J. F. Degradation of feruloylated oligosaccharides from sugar-beet pulp and

comparison to the total liberated ferulic acid may be researched. Potentially, feruloyl esterases play a minor role in the natural decomposition and digestion of nonpolar n-alkyl ferulates compared to lipases.

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AUTHOR INFORMATION

Corresponding Author

*(L.N.) Phone: +41 44 632 91 65. Fax: +41 44 632 11 23. Email: [email protected]. Funding

This study was financially supported by the Swiss National Science Foundation, SNSF (Project 200021_141268), and ETH Zurich. Notes

The authors declare no competing financial interest.

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Hydrolysis of Nonpolar n-Alkyl Ferulates by Feruloyl Esterases

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