All chemicals were analytical grade. Curcumin (>90%, natural) was purchased by Roth (Karlsruhe, Germany). 3,4-dimethoxybenzaldehyde and geranyl acetate from Sigma-Aldrich (Taufkirchen, Germany). 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and p-nitrophenyl butanoate were obtained from ICN Biochemicals (Muenchen, Germany). Diethyl ether, ethyl acetate, toluene and n-pentane were from Karl Roth (Karlsruhe, Germany), and solvents (all MS grade) used for HPLC-MS were from Carlo Erba Reactifs (Peypin, France).
Immobilized lipase (triacylglycerol hydrolase, EC 18.104.22.168 (Novozyme_435, 5000 U·g−1)) from Candida antarctica and laccase from Agaricus bisporus (6.8 U·mg−1) were from Sigma-Aldrich (Taufkirchen, Germany), and esterase from porcine liver (lyophilisate, 15 U·mg−1) was from Sigma Aldrich (Taufkirchen, Germany). Recombinant feruloyl esterase from Pleurotus eryngii (PeFaeA) and chlorogenic acid esterase from Ustilago maydis (UmChlE) were selected from our own stocks. Two further laccases were isolated from fungal culture supernatants, as described below. The strains were purchased from the Centraalbureau voor Schimmelcultures (Meripilus giganteus CBS 561.86) and from the German Collection of Microorganisms and Cell Cultures (Funalia trogii, DSMZ), respectively.
3.3. Cultivation of Fungi
The culture supernatant of M. giganteus
was provided according to the paper of Schmidt et al
]. Submerged pre-culture of F. trogii
was inoculated with the same structure of M. giganteus
,except that for the main cultures, the expression of laccases was induced either by the addition of three grams per 100 mL−1
wheat bran and CuSO4
(300 µM final concentration) to the culture medium of F. trogii
or 300 µM CuSO4
solely in the case of M. giganteus
. At the time of maximum laccase activity (ABTS activity, pH 3.0), cultivation was stopped and the culture supernatant harvested and stored at −20 °C, unless used immediately for laccase isolation and purification.
3.4. Laccase Isolation and Purification
The laccase from M. giganteus
was isolated according to the protocol of Schmidt et al.
]. In brief, the supernatant was frozen at −20 °C, thawed and centrifuged at 25,000× g
. After filtration using a 0.45-µM polyester filter (CHROMAFIL PET-45/25, Macherey-Nagel, Dueren, Germany) and concentration using an ultra-filtration module (30-kDa cut-off, PES, Sartorius, Goettingen, Germany), the laccase was purified using fast protein liquid chromatography (Biologic Duoflow TM, Bio-Rad, Hercules, CA, USA) at 4 °C. First, a weak anion exchange column was applied (HiPrep 16/10 DEAE, 16 × 100 mm fast flow, GE Healthcare, Munich, Germany). Concentrated laccase fractions were submitted to a second purification using size exclusion chromatography (Superdex 75 10/300 GL column, GE Healthcare, Munich, Germany). Active fractions were pooled and adjusted to the activity required.
Laccase from F. trogii was purified as follows: The culture supernatant was frozen at −20 °C, thawed and centrifuged at 5000× g at 4 °C for 15 min. The supernatant was filtered (0.45 µM, Chromafil Pet-45/25, Dueren, Germany), concentrated using an ultrafiltration module (30-kDa cut-off, PES, Sartorius, Goettingen, Germany) and subjected to fast protein liquid chromatography (Biologic Duoflow TM, Bio-Rad, Hercules, United States) at 4 °C. Twenty five milliliters of concentrated solution were purified on a HiPrep 16/10 DEAE, 16 × 100 mm fast flow column with a flow rate of 3 mL·min−1 (GE Healthcare, Munich, Germany) with 20 mL running Buffer A (50 mM, potassium phosphate, pH 6.5) and eluted with 5% Buffer B (50 mM potassium phosphate, pH 6.5 + 1 M NaCl). Purification was controlled using SDS-PAGE electrophoresis.
SDS-PAGE was performed using 12% (w/v) polyacrylamide gels. Samples were diluted in native loading buffer (0.05 M Tris/HCl pH 6.8, 0.1% bromophenol blue, 10% glycerol, 2% SDS) and applied to electrophoresis. Proteins were stained with ready-to-use Instant Blue solution (0.1%, Expedeon, Cambridge, UK). Laccase activity staining was performed directly on the gel using ABTS (5 mM in 100 mM sodium phosphate buffer pH 4.5).
3.5. Analysis of Amino Acid Sequence
The identity of the purified laccases, as well as the sequence of lccAbi were deduced from the amino acid sequence of tryptic peptides of cut out protein bands from SDS gel electrophoresis. De-staining and tryptic digestions of the respective protein bands were carried out as described elsewhere [19
]. Tryptic peptides were analyzed by means of nano-LC EASY-nLC II (Bruker Daltronik, Bremen, Germany) equipped with a 20-mm pre-column (C18-A1 3PCS; ThermoFisher Scientific, Dreieich, Germany) and a capillary column (0.1 mm × 150 mm) packed with Magic C18 AQ (3-mm particle size, 200-Å pore size; Michrom Bioresources, Inc., Auburn, CA, USA) eluted by a linear gradient (300 nL·min−1
) of water and acetonitrile, each with 0:1% formic acid v/v from 95% water to 95% acetonitrile within 25 min and held for 15 min. The amino acid sequences elucidated were subjected to protein database (NCBI, Mascot search algorithm). Sequences were aligned using the ClustalW2 multiple sequence alignment database.
3.7. Curcumin Transformation
3.7.1. Chemical Acetylation of Curcumin
Acetylated curcumins were synthesized chemically as reference compounds using acetic anhydride. One mmol of curcumin was dissolved in 150 mL ethyl acetate and mixed for 20 min. After dissolving was completed, four mmol acetic anhydride were carefully added. After six hours, the reaction was stopped by adding one drop H2O2. Reaction yield (consumption of curcumin) and product identification were carried out by LC-MS.
3.7.2. Lipase-Catalyzed Acetylation of Curcumin
Before each experiment, ethyl acetate and toluene as the solvent and the acyl donor (vinyl or geranyl acetate) were stored over Na2SO4. The reaction was carried out in 2 mL ethyl acetate and toluene (10:90) in sealed 30-mL glass vials at 40 °C with continuous stirring using a glass magnetic stir bar (150 rpm). The powdered CAL was added to a final concentration of five mg·mL−1. Sodium acetate buffer 50 mM, pH 6, was added at 4% to the reaction solution. Over the incubation, time samples were taken, filtered using 0.45-µm filter (Chromafil PET-45/25, Macherey-Nagel) and then analyzed directly by LC-MS.
3.7.3. Monoacetyl Curcumin Degradation
The two-phase reaction system was made up of 2.0 mL of 0.5 mM monoacetyl curcumin (concentration calculated according to a curcumin standard) together with a minor impurity of diacetyl curcumin (concentration not affected by the laccase present) in 2.0 mL diethyl ether and 2.0 mL respective enzyme solution (1.19 U·mL−1, ABTS-assay, 30 °C, pH 3) in 50 mM sodium phosphate buffer, pH 5.5, under continuous vortexing at 1300 rpm (Heidolph, Germany) for 20 h at room temperature. A control sample with the buffer, but without enzyme, was treated under the same conditions. After separation of the diethyl ether phase, the aqueous buffer was re-extracted three times with 2.0 mL diethyl ether, and the combined organic phases were dried over night with Na2SO4. The degradation rate of acetyl curcumin was determined by LC-MS, and volatile degradation products, such as vanillin acetate, were analyzed by GC-MS.
3.7.4. Esterase-Catalyzed Deacetylation of Vanillin Acetate
Three esterases were used for the hydrolysis of vanillin acetate. The hydrolysis was carried out in a glass vial containing 2 mL of 1 mM vanillin acetate dissolved in diethyl ether/hexane and 2 mL sodium phosphate buffer 50 mM, pH 5 and 6.5. The reactions were initiated by adding enzyme solution with an activity of one U·mL−1 to the reaction mixture and placed in a vortex shaker at 37 °C for five hours. The samples were extracted three times with diethyl ether, and the combined fractions were dried over sodium sulfate and analyzed by GC-MS using the external standard, 3,4-dimethoxybenzaldehyde (final concentration 125 mg·L−1). The reproducibility of three repeated transformations showed a relative standard deviation of typically 3%.
3.9. Liquid Chromatography/Mass Spectrometry
For the identification of vanillin, curcumin and acetylated curcumins, as well as for the determination of the molar mass of the expected oxidation/polymerization products thereof, high performance liquid chromatography coupled to a triple quadrupole mass analyzer was used (Varian 212 LC pump, Pro Star 325 UV-Vis detector, 320 TQ-MS mass spectrometer). The MS was conducted simultaneously in the ESI positive and negative mode with a scan range of m/z 110–500 or m/z 300–1200, respectively.
The MS parameters for ESI(+)/ESI(−) were: capillary voltage +30 V/−40 V, needle voltage 5000 V/−4500 V, nebulizer gas (N2) 379 kPa, drying gas 207 kPa at 350 °C. For HPLC, water and acetonitrile (MS-grade), both containing 0.1% formic acid, were used as the mobile phase, and the following linear gradient was used: 10% acetonitrile for three minutes, up to 90% acetonitrile within 20 min, hold for five minutes and back to start conditions. The separation was performed on an RP-18 HD column (Eurosphere 100-C18-5-HD, 250 × 4 mm, 5 µm, Macherey-Nagel) at a flow rate of 0.3 mL per minute. Additionally, UV absorption was monitored at 280 and 425 nm.