Production of o-diphenols by immobilized mushroom tyrosinase

Journal of Biotechnology 139 (2009) 163–168

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Production of o-diphenols by immobilized mushroom tyrosinase María Elisa Marín-Zamora a , Francisco Rojas-Melgarejo a , Francisco García-Cánovas b , Pedro Antonio García-Ruiz a,∗ a Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA). Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain b Grupo de Investigación de Enzimología (GENZ). Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain

a r t i c l e

i n f o

Article history: Received 14 March 2008 Received in revised form 10 September 2008 Accepted 30 October 2008 Keywords: Tyrosinase Agaricus bisporus Immobilization Cinnamic carbohydrate esters Tyrosinase extraction o-Diphenols production

a b s t r a c t The o-diphenols 4-tert-butyl-catechol, 4-methyl-catechol, 4-methoxy-catechol, 3,4-dihydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acid were produced from the corresponding monophenols (4-tert-butyl-phenol, 4-methyl-phenol, 4-methoxy-phenol, p-hydroxyphenylpropionic acid and p-hydroxyphenylacetic acid) using immobilized mushroom tyrosinase from Agaricus bisporus. In all cases the yield was Rdiphenol ≥ 88–96%, which, according to the literature, is the highest yield so far, obtained using tyrosinase. The reaction was carried out in 0.5 M borate buffer pH 9.0 which was used to minimize the diphenolase activity of tyrosinase by complexing the o-diphenols generated. Hydroxylamine and ascorbic acid were also present in the reaction medium, the former being used to reduce mettyrosinase to deoxytyrosinase, closing the catalytic cycle, and the latter to reduce the o-quinone produced to o-diphenol. Inactivation of the tyrosinase by ascorbic acid was also minimized due to the formation of an ascorbic acid–borate complex. Concentrations of the o-diphenolic compounds obtained at several reaction times were determined by gas chromatography–mass spectrometry (GC–MS) and UV–vis spectroscopy. The experimental results are discussed. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Mushroom tyrosinase is a copper enzyme which catalyses the hydroxylation of monophenols to o-diphenols and the oxidation of the latter to o-quinones, using oxygen (Gómez-Fenoll et al., 2001a,b). Tyrosinase has been used previously to synthesise a variety of molecules of a diphenolic nature that are considered beneficial for human health, including l-dopa, an important and expensive medicament for use with Parkinson’s disease (Ates et al., 2007; Carvalho et al., 2000; Pialis and Saville, 1998; Seetharam and Saville, 2002; Ullrich and Hofrichter, 2007), although yields have never exceeded 30% (Octavio de Faria et al., 2007). The active site of tyrosinase from Agaricus bisporus exists in three intermediate states (depending on the copper ion valence and its bond with molecular oxygen): deoxy-tyrosinase (Cu(I)2 ) (Ed ), oxy-tyrosinase (Cu(II)2 O2 −2 ) (Eox ) and met-tyrosinase (Cu(II)2 ) (Em ). The met-tyrosinase form, Em , of the enzyme is predominant in vivo. This form, Em , can bind o-diphenols and monophenols, in the first case oxidizing the o-diphenol (D) to release the o-

∗ Corresponding author. Tel.: +34 968364814; fax: +34 968364147. E-mail address: [email protected] (P.A. García-Ruiz). 0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2008.10.014

quinone, and being converted into the deoxy-tyrosinase form, Ed . However when Em binds to a monophenol (M), an inactive Em M complex is formed. The deoxy-tyrosinase form, Ed , is able to bind reversibly with molecular oxygen, producing the oxy-form, Eox , which can act on both monophenols and o-diphenols, in the first case to form o-diphenols and o-quinones and in the second case o-quinones (Gómez-Fenoll et al., 2004a; Octavio de Faria et al., 2007; Rodríguez-López et al., 2001). Therefore, for monophenolase activity, it is essential that the Em form pass to Eox (Eox is the only form that can catalyze the hydroxylation of monophenols) (GómezFenoll et al., 2004b; Kahn et al., 1999; Kahn and Andrawis, 1986; Rodríguez-López et al., 2001; Yamazaki and Itoh, 2003). This occurs when a small quantity of o-diphenol is accumulated in the medium, which permits the above mentioned transformations, while the time taken for this concentration to be accumulated corresponds to the initial lag period observed in the monophenolase activity of tyrosinase before the steady state is reached (Rodríguez-López et al., 2001). When tyrosinase acts on a monophenol, the o-diphenol is obtained. This may be released to the medium (Rodríguez-López et al., 2001) or the enzymatic oxidation may continue to form o-quinone, which may also produce o-diphenol in the medium through non-enzymatic reactions, although in the steady state, only

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a very small part of the o-diphenol is accumulated in the medium (D/M »0.06 for several D/M pairs) (Gómez-Fenoll et al., 2001a,b; Rodríguez-López et al., 2001; Ros et al., 1994). The use of ascorbic acid (AH2 ) permits all the o-quinone formed to be reduced, regenerating the corresponding o-diphenol (Ates et al., 2007; Carvalho et al., 2000; Pialis and Saville, 1998; Seetharam et al., 2002). This makes it possible to obtain o-diphenols as long as AH2 is not totally used up, since, if this were the case, the enzyme would oxidise the o-diphenol to o-quinone. Very high quantities of AH2 would be needed if an acceptable yield of o-diphenol is to be attained, since the system would enter in a continuous regime of futile cycles (the o-diphenol obtained by reduction of the o-quinone competes with the monophenol to be re-oxidised by the enzyme, which means some of it would be oxidized (again) to o-quinone and would have to be reduced (again), before new monophenol molecules are hydroxylated increasing the final concentration of o-diphenol). However, high ascorbic acid concentrations would inactivate the enzyme (Andrawis and Varda, 1990; Octavio de Faria et al., 2007; Pialis and Saville, 1998), and the AH2 may even be oxidised by tyrosinase (ascorbate oxidase activity; Ho et al., 2003; Ros et al., 1995). This, together with the fact that a competitive inhibition of the enzyme occurs when high o-diphenol concentrations are reached in the medium (Pialis and Saville, 1998), explains why high yields of o-diphenol have never been obtained (Ates et al., 2007; Carvalho et al., 2000; Octavio de Faria et al., 2007; Pialis and Saville, 1998; Seetharam et al., 2002). It has been reported that the borate anion in the presence of o-diphenol gives rise to a stable borate–diphenol complex (Mochizuki et al., 2002; Yamazaki and Itoh, 2003; Yasunobu and Norris, 1957; Yoshino et al., 1979; Waite, 1984). The presence of small borate concentrations in the reaction medium when tyrosinase acted on monophenols caused a prolongation in the lag period (Yasunobu and Norris, 1957), this inhibition being attributed to the formation of a borate–diphenol complex: the borate complexes the o-diphenol and diminishes the concentration of free o-diphenol in the medium, and so Em cannot pass to Ed . To avoid the blocking of the reaction through a deficiency of o-diphenol, external reductants have been used, such as hydroxylamine (NH2 OH) (Kahn and Andrawis, 1986; Yamazaki and Itoh, 2003), which reduces Em to Ed , and, in the presence of oxygen, Eox is formed. This work describes how high yields of several o-diphenols can be obtained from their corresponding monophenols, using mushroom tyrosinase immobilized on the crosslinked d-sorbitol hexacinnamate (Marín-Zamora et al., 2006). The reaction medium used was borate buffer 0.5 M (pH 9), 1 mM monophenol, 6.7 mM hydroxylamine (Yamazaki and Itoh, 2003), and ascorbic acid in excess. The ascorbic acid reduces the o-quinone generated. The borate complexes the o-diphenol and the ascorbic acid, preventing both from competing with the monophenol as substrates, thereby minimizing the appearance of futile cycles and enzyme inhibition. The hydroxylamine reduces the Em form of the enzyme to Ed , closing the catalytic cycle. Using immobilized tyrosinase permitted us to study different reaction times, since the reaction could be stopped simply by removing the reaction medium from the reactor, leaving the reactor ready for use in subsequent cycles, which represents a considerable economic saving. This method, demonstrates the practicality of using tyrosinase for the synthesis of o-diphenols, increasing the yields previously obtained with similar methods from 30% to 85%. The procedure could also be used to obtain other similar compounds of a o-diphenolic nature, such as l-dopa or dopamine, both of which are expensive to produce and of great interest for human health.

2. Materials and methods Fruit bodies of the mushroom A. bisporus were supplied by Mercadona (Spain) and used to obtain fresh tyrosinase. 4-tert-Butylphenol (TBP), 4-methyl-phenol (MP), 4-methoxyphenol (MOP), p-hydroxyphenylpropionic acid (HPPA), p-hydroxyphenylacetic acid (HPAA), 4-tert-butyl-catechol (TBC), 4-methyl-catechol (MC), 3,4-dihydroxyphenylpropionic acid (DHPPA), 3,4-dihydroxyphenylacetic acid (DHPAA), ascorbic acid (AH2 ), hydroxylamine (NH2 OH) and p-nitrophenol (PNP) were purchased from Sigma (Spain). All other chemicals were of analytical grade and supplied by Fluka (Spain), Panreac (Spain), J.T. Baker (Holland), Sigma (Spain) and Lab-Scan (Ireland). Ultrapure water from a Milli-Q system (Millipore Corp.) was used throughout this research. 2.1. Photoreactive prepolymers preparation The preparation of totally cinnamoylated derivative of d-sorbitol followed a modified version of the method proposed by Van Cleve (Van Cleve, 1963; Rojas-Melgarejo et al., 2004a), in which 0.02 mol of d-sorbitol was dissolved in 100 ml of pyridine. The mixture was heated at 60 ◦ C for 1 h to ensure complete dissolution. After cooling to room temperature, 0.15 mol of cinnamic acid chloride was added. The reaction was allowed to proceed at room temperature for 4 h, after which the resulting mixture was poured into vigorously stirred water. The precipitate obtained, after decanting and filtering this mixture, was dissolved in chloroform and purified by adding, one drop at a time, to vigorously shaken hexane. The solid obtained was redissolved and reprecipitated before being dried on P2 O5 at reduced pressure. The hexacinnamate of d-sorbitol was characterized in previous works (Marín-Zamora et al., 2005; Rojas-Melgarejo et al., 2004a) from various experimental analyses: 1 H NMR and 13 C NMR, distortionless enhancement by polarization transfer (DEPT) spectra, different two-dimensional experiments (COSY and C/H ratio) and infrared spectra of the prepared compound. In the present work total esterification was confirmed by 1 H NMR and 13 C NMR. 2.2. Tyrosinase extraction Mushroom tyrosinase was extracted as previously described (Marín-Zamora et al., 2006). Briefly: before use, the fruit bodies of the mushroom A. bisporus were lyophilized, ground mechanically and stored at −18 ◦ C. To extract the fresh tyrosinase enzyme, 600 mg of lyophilized-ground mushroom was added to 16 ml of a 30 mM aqueous solution of PNP (pH 7.0), magnetically stirred for 30 min at 4 ◦ C and finally centrifuged at 4000 rpm for 5 min. The supernatant (9 ml) which contained the tyrosinase activity was collected and equilibrated to pH 5.5 by adding 1 ml of a 0.9 M aqueous solution of NaH2 PO4 and 0.1 M of H3 PO4 . The solids were totally eliminated by means of a second centrifugation. 2.3. Tyrosinase immobilization Microperl Industrial (Type A, 0.6–1.0 mm diameter) glass beads manufactured by Sovitec Iberica S.A. (Barcelona, Spain) and supplied by Jaque (Murcia, Spain), were used as inert matrix for tyrosinase immobilization. Before use, the glass beads were washed and degreased. A chloroform solution of totally cinnamoylated derivative of d-sorbitol (the immobilization support) at 5 g l−1 was prepared, in which the glass beads were immersed. A prepolymer film was formed on the beads when the solvent was eliminated by evaporation (Marín-Zamora et al., 2006; Rojas-Melgarejo et al., 2004b). After drying, the prepolymer film was polymerized by irra-

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diation in the ultraviolet zone for 15 min using an Osram HOL-125W mercury vapour lamp providing a power of 1.6 mW cm−2 as determined by a Nover-Laser power/energy monitor (OPHIR Optronics Ltd.). To immobilize fresh tyrosinase, 10 ml of tyrosinase extract (pH 5.5) was added to a syringe containing 22 g of glass beads covered with the immobilization support. The immobilization was allowed to proceed for 1 h at 4 ◦ C. After immobilization the enzyme solution was withdrawn and the immobilized enzyme was thoroughly rinsed in distilled water. The quantity of immobilized fresh tyrosinase was determined as in previous works (Marín-Zamora et al., 2007a,b), obtaining a value of 1.18 ␮g mgsupport −1 . 2.4. Enzymatic o-diphenol production Syringes containing 22 g of glass beads, covered with the cross-linked cinnamoylated derivative of d-sorbitol and adsorbed tyrosinase, were used as small packed bed continuous reactors, with recirculation and descending flow (11.5 ml), for o-diphenol production. The enzymatic reaction was carried out in 0.5 M borate buffer pH 9.0 medium, and the monophenol and hydroxylamine (NH2 OH) concentrations were 1 mM and 6.7 mM, respectively. The concentration of ascorbic acid (AH2 ) was always enough to reduce all the quinones produced, using an initial concentration of 5.5 mM, except for the substrate MOC when an initial concentration of 15 mM was needed. In each case, an excess of AH2 was confirmed by the absence of colour formation due to non-reduced o-quinone. In order to avoid the effect of second substrate oxygen on the enzyme activity and catechol production, the concentration of oxygen was kept constant in the assay medium (0.26 mM). The reaction was allowed to proceed until the desired reaction time and was stopped by removing the medium from the reactor. The pH value of the medium was the adjusted to 6.5 for TBC, MC and MOC, 3.0 for DHPPA and 0.5 for DHPAA, by adding the necessary amount of an aqueous solution of HCl, in order to facilitate the extraction of diphenolic compounds. For that, 10 ml of diethyl ether was added (twice). The phases were separated and the organic phase collected, dried with anhydrous Na2 SO4 and filtered through Watman no. 1 filter paper. The diethyl ether was evaporated at room temperature in a rotavapor system. The trimethylsilyl derivatives obtained were analysed by GC–MS, and the o-quinone generated by oxidation with excess periodate was measured immediately by UV–vis spectrophotometry, as indicated in the following sections. 2.5. o-Diphenol determination 2.5.1. UV–vis spectrophotometry Spectrophotometric measurements were made with a PerkinElmer Lambda 35 UV/Vis Spectrophotometer controlled by a PC running with the software Lambda 35. The o-diphenol quantity produced in each enzymatic reaction was determined by oxidation of the sample with excess periodate, obtaining the corresponding o-quinone, and measuring its absorbance immediately at the characteristic wavelength: 400 nm in the case of o-quinones generated from TBC, MC and DHPPA, 420 nm for MOC and 395 nm for DHPAA. For this, after diethyl ether evaporation, 4 ml of sodium phosphate buffer 50 mM, pH 6.8, were added to the dry flask and the sample was gently stirred. Finally, a 2 ml aliquot was deposited in a quartz cuvette as blank; then, 0.5 ml of 0.2 M periodate (in excess) was added and the absorbance of the o-quinone generated was measured. The o-diphenol concentration produced was calculated by extrapolating the corresponding calibration curve, which was obtained from known concentrations of the corresponding o-diphenol (0.1–1.2 mM), prepared in the same conditions (11.5 ml in 0.5 M borate buffer pH 9, 5.5 mM AH2 and 6.7 mM NH2 OH)

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Table 1 Gas chromatogram retention time (tr ) and mass spectra M+ and M-15 peaks for the TMS derivatives of the monophenols and o-diphenols studied. Compound

tr (min)

M+ (m/z)

TBP MP MOP HPPA HPAA TBC MC MOC DHPPA DHPAA Phenanthrene

8.3 ± 0.1 5.0 ± 0.1 7.3 ± 0.1 16.0 ± 0.2 13.8 ± 0.2 12.0 ± 0.1 9.2 ± 0.1 11.7 ± 0.1 19.2 ± 0.2 17.3 ± 0.2 16.8 ± 0.1

222.2 180.1 196.1 310.2 296.2 310.2 268.2 284.2 398.3 384.2 178.1

± ± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

M-15 (m/z) 207.2 165.1 181.1 295.2 281.2 295.2 253.2 269.2 383.3 369.2

± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

and submitted to the same extraction (at an appropriate pH) and oxidation processes as described above. The calibration curve equations were A400 = 1.011 × [TBC] ± 0.013, A400 = 1.134 × [MC] ± 0.016, A420 = 0.791 × [MOC] ± 0.003, A400 = 1.002 × [DHPPA] ± 0.010 and A395 = 0.670 × [DHPAA] ± 0.008 for the o-diphenols TBC, MC, MOC, DHPPA and DHPAA respectively. Triplicate assays were realized in all cases, and the mean value was considered. 2.5.2. Gas chromatography–mass spectrometry (GC–MS) analyses Mass spectra were obtained with a Finnigan Trace GC Ultra gas chromatograph coupled to a Finnigan Polaris Q mass spectrometer and an automatic Termo Finnigan AS 2000 sampler. GC was performed with a 30 m long and 0.25 mm diameter RTX-5MS column. Helium was used as the carrier gas, at a flow rate of 0.3 ml min−1 . The temperature of the oven was programmed from 100 ◦ C to 220 ◦ C at 6 ◦ C/min and then remained isothermal (tTotal = 40 min). The injection volume was 2 ␮l. In this case, identification and quantification of the o-diphenols studied were carried out preparing the corresponding TMS volatile derivatives and analyzing them on the GC–MS instrument. TMS volatile derivatives were prepared by adding to the dry flask (after diethyl ether evaporation): 600 ␮l of pyridine as solvent (containing phenanthrene as internal standard), 800 ␮l of hexamethyldisilazane and 200 ␮l of trimethylchlorosilane, following Makita’s method (Rodríguez-López et al., 2001; Sweely et al., 1963). The samples were centrifuged and filtered before analysis. Identification of TBC, MC, MOC, DHPPA and DHPA in the samples was confirmed by their mass spectra (MS) and by their GC–MS chromatograms and comparing them with those of authentic samples. For o-diphenol (TBC) quantification, a calibration curve was made preparing samples of known concentration (0.1–1.2 mM), in the same conditions (11.5 ml in 0.5 M borate buffer pH 9, 5.5 mM AH2 and 6.7 mM NH2 OH) and submitting them to the same process of extraction and preparation of the TMS derivatives as mentioned above. The relation between TBC peak area and phenanthrene peak area was determined (ATBC /APhenanthrene ), and the value was extrapolated in the corresponding calibration straight line: ATBC :APhenanthrene = 4.027 × [TBC] ± 0.114. Triplicate assays were realized in all cases, the mean value being considered in all cases. 2.6. o-Diphenol identification and quantification The o-diphenols produced and also their corresponding initial monophenols (when conversion was not complete) were identified by gas chromatography–mass spectrometry (GC–MS), comparing the retention time and mass spectra of the peaks in the chromatograms with those of authentic samples (Table 1). The characteristic MS fragments were the molecular ion M+, and the M-15 peak corresponding to the loss of the methyl group.

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Fig. 1. A: 4-Methoxy-catechol ( ), dihydroxyphenylpropionic acid ( ) and 4-tertbutyl-catechol ( ) produced by immobilized mushroom tyrosinase versus reaction time. Insert B: 4-Methyl-catechol ( ) and dihydroxyphenylacetic acid ( ) produced by immobilized mushroom tyrosinase versus reaction time. Data expressed as percentage of maximum theoretical concentration of o-diphenol that can be obtained (1 mM).

After identification of the compounds, the gas chromatogram (GC) can be used to follow the reaction (see Fig. 1AM of “Additional Material”). For example, in TBC production, a large peak (TBP) can be observed at the beginning of the reaction, along with a smaller one (TBC), while at longer reaction times the TBP peak diminishes and the TBC peak increases. At the end of the reaction, the TBP peak has almost disappeared and the TBC peak is very big. GC–MS and UV–vis spectrophotometry was used to quantify odiphenols as described above. The results pointed to a high degree of agreement between the two methods. 3. Results and discussion 3.1. o-Diphenol production The o-diphenols, TBC, MC, MOC, DHPPA and DHPAA, were produced by immobilized tyrosinase acting on the monophenols TBP, MP, MOP, HPPA and HPAA, respectively. Several reaction times were studied in each case, and the corresponding enzymatic reaction yield of o-diphenol was determined for each reaction time (Fig. 1). The maximum reaction yields (Rmax ) were very high and almost the same for all the o-diphenols produced: Rmax  95% for MC, DHPPA and DHPAA, and Rmax  88% in the case of TBC and MOC. 3.1.1. Reaction time The time necessary to reach maximum conversion, t (Rmax ), was different for each o-diphenol, increasing from 0.75 to 3, 3, 5 and 7 h for MOC, MC, DHPPA, DHPAA and TBC, respectively. This order coincided with the increasing ı4 values of monophenols in the 13 C NMR experiments, which were 149.5, 152.7, 153.5, 154.3, and 152.7 ppm for MOP, MP, HPPA, HPAA and TBP, respectively. That is, the reaction time was shorter as the nucleophilic power of the oxygen from the aromatic hydroxy group on C-4 of the substrate increased. This enabled the oxygen to attack the copper atoms of the enzyme active site more readily, the nucleophilic power being related with the higher electron donor capacity of the side chain of the substrate

Scheme 1. Kinetic reaction mechanism proposed for the transformation of monophenol (M) into o-diphenol (D): A. Enzymatic steps. B. Non enzymatic steps. Em , mettyrosinase; Ed , deoxytyrosinase; Eox , oxytirosinase; M, monophenol; D, odiphenol; Q, o-quinone; N, hydroxylamine; AH2 , ascorbic acid; B, borate; Em M, complex of Em with M; Em D, complex of Em with D; Em N, complex of Em with N; Eox M, complex of Eox with M; Eox D, complex of Eox with D; Eox –M, complex of Eox covalent bound to M; DB, complex of borate with D; AH2 B, complex of borate with AH2 .

(Espín et al., 2000, 1998a,b). In the case of TBP, the steric effect was the most important factor and the reaction time was almost the longest. In all cases, o-diphenol was produced rapidly at the beginning of the reaction, until about 70–80% conversion was reached, after which it took a very long time to improve on this value and reach maximum conversion. For industrial purposes, it would probably not be considered cost effective to wait so long to obtain a 10–15% increase in the yield, in which case optimal reaction times could be considered as 0.75, 2, 2, 2 and 5 h for MOC, MC, DHPPA, DHPAA and TBC, respectively (although obviously longer if the yield was to be increased further). 3.1.2. Reactions scheme A scheme of the enzymatic reactions that take place in the odiphenol production process is presented in Scheme 1A. In the box of Scheme 1A the thick lines represent the desired reaction pathway to produce o-diphenols (D), while the dotted lines outside the box, represent the reactions to minimize (diphenolase activity and dead-end complex formation) to optimize the process. The reactions that have no influence in the kinetic of the o-diphenol production are represented in Scheme 1B as non-enzymatic reactions.

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The action of the Eox form of tyrosinase on the monophenol (M) (step 1) generates the complex Eox M, which is converted to Em D (step 2). Part of the o-diphenol is released from this intermediate (step 3) and is (mostly) removed from the medium by complexation with borate (step 11), while another part is oxidised to o-quinone (step 4) and then reduced by the AH2 (step 10) to o-diphenol, which is also protected from the action of the enzyme by the borate (step 12). The Em formed in step 3 has to be converted again to the Eox form, which is achieved by reacting Em with hydroxylamine (step 5), producing Ed , which reacts with O2 (step 7), forming Eox , thus initiating the following o-hydroxylation cycle. 3.1.2.1. Hydroxylamine effect. In the scheme represented in Scheme 1A, the transformation of Em into Ed depends on a reduction step. To avoid using the o-diphenol generated as reductant (k2 in step 3 and k3 in step 4 corresponding to diphenolase activity) an excess of hydroxylamine is used (step 5 and step 6), which facilitates the monophenolase activity of the enzyme (Kahn and Andrawis, 1986; Yamazaki and Itoh, 2003). As is shown in this scheme, the o-diphenol and monophenol compete for the Em form of the enzyme (steps 3 and 9, respectively), although the high concentration of hydroxylamine (step 5) enables the Em form of the enzyme to be transformed into Ed , minimizing the reactions of Em with the o-diphenol and monophenol. Thus, the hydroxylamine prevents the diphenolase activity and the dead end complex Em M (step 9). The transformation of Ed into Eox occurs through binding to the oxygen of the solution (step 7). 3.1.2.2. Ascorbic acid effect. The function of AH2 is to reduce the o-quinone produced to o-diphenol without intervening in the enzymatic reaction or the kinetic of the process. To accomplish this, the concentration of AH2 in the medium must be the minimum necessary to ensure that all the o-quinone formed is reduced to o-diphenol. In experiments in which the concentration of AH2 was not sufficient (not shown) the yield of o-diphenol fell due to the accumulation of o-quinone (which was not reduced because the AH2 available in the medium had been used up) or of o-quinone derivates from non-enzymatic reactions such as that with hydroxylamine (Kahn and Andrawis, 1986; Kahn et al., 1999a,b). For example, when 10 mM of AH2 was used to obtain MOC, a yield of 81% was reached after 0.5 h reaction time, while an AH2 concentration of 3.5 mM at the same reaction time only produced a yield of 52% (30% lower). 3.1.2.3. Borate effects. 3.1.2.3.1. Concerning the o-diphenol. The sequestering of the odiphenol by borate to form the corresponding complex (step 11) (Mochizuki et al., 2002; Yamazaki and Itoh, 2003; Yasunobu and Norris, 1957; Yoshino et al., 1979; Waite, 1984) reduces the number of futile cycles, since the concentration of the non-complexed odiphenol in equilibrium with the diphenol–borate complex is very low, and so only this small part may be oxidised by the enzyme. This stabilisation of the o-diphenol by complexation with borate means that the Eox form of the enzyme has practically no o-diphenol available for the step 8, which favours the monophenol (step 1) in its competition with o-diphenol and permits high yields of o-diphenol. 3.1.2.3.2. Concerning ascorbic acid. AH2 in the presence of borate forms a complex (step 12) (Obi et al., 1998; Roomi and Tsao, 1998; Tsao and Young, 1996) and only a small free fraction remains in the medium to act as reductant. Therefore, borate permits a reserve of AH2 throughout all the process, while its free concentration is insufficient for it to act as substrate of the enzyme. The step-wise addition of AH2 , therefore, is unnecessary to achieve the desired result.

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If all the AH2 necessary for the process were added at the beginning in the absence of borate, it concentration would lead to its acting as substrate and it would inactivate the enzyme (Andrawis and Varda, 1990; Octavio de Faria et al., 2007; Pialis and Saville, 1998). No case of inactivation was observed in the presence of borate, high yields always being obtained. 3.1.3. Kinetic analysis The reaction rate of this system in the steady state is described by the following equations: (a) Reaction rate related with the o-quinone formation, vQ : 0

vQ0 =

Q Vmax [M]0 Km + [M]0

(1)

(b) Reaction rate related with the o-diphenol formation, vD : 0

vD0 =

D Vmax [M]0 Km + [M]0

(2)

v0 is the initial reaction rate; Vmax the maximum reaction rate; Km the Michaelis constant; [M]0 is the initial monophenol concentration. The deduction of Eqs. (1) and (2) can be consulted in “Additional Material”. Since the reduction of the o-quinone (Q) by the ascorbic acid is instantaneous, the final reaction rate in the o-diphenol production is vQ + vD . 0 0 The reaction progress with the time achieving the transformation of monophenol, M, into diphenol, D, according to the integrated Michaelis–Menten equation: [Q] = −Km t [D] = −Km t

 2.3 t

 2.3 t

log

[M]0 [M]0 − [Q]

log

[M]0 [M]0 − [D]

 

Q + Vmax

(3)

D + Vmax

(4)

4. Conclusion A new method has been described to obtain high yields (88–96%) of o-diphenols, which are compounds of great commercial interest. The method consists of using tyrosinase immobilized on the crosslinked hexacinnamate of d-sorbitol, in a reaction medium consisting of borate buffer, hydroxylamine, ascorbic acid and the corresponding monophenol. Among the advantages of the method are (i) the Em form of the enzyme can be converted to the Ed form without the intervention of o-diphenol; (ii) presumably the reduced consumption of ascorbic acid as a result of minimizing the futile cycles caused by diphenolase activity through the formation of an o-diphenol–borate complex (which diminishes competition with the monophenol for the Eox form of the enzyme); (iii) inactivation of enzyme by ascorbic acid is avoided since the ascorbic acid is complexed with borate, and most importantly; (iv) high yields of o-diphenol are obtained. Acknowledgements This work was supported by a grant from the Ministerio de Educación y Cultura (Spain) Projects MAT2004-01893 and BIO2006-15363, Consejería de Ciencia, Tecnología, Industria y Comercio (Murcia, Spain) Projects 2103 SIU0043 and BIO-BMC 06/01-0004. MEMZ gratefully acknowledges a grant from Fundación Caja Murcia.

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