Biotransformation of Aromatic Methyl Groups to Aldehyde Groups Using Laccase of Gloephyllum Stratum MTCC-1117

Laccases has been produced by white rot fungi are involved in lignin containing natural substrates wheat-straw, bagasse, saw-dust, corn cob and coir dust particle on the production of laccase enzyme in the aqueous cultivation medium of Gloephyllum stratum MTCC1117. The approach involved concentration of aqueous ltrate by ultraltration and anion exchange chromatography on DEAE (diethyl aminoethyl cellulose). From SDS-PAGE analysis the molecular mass of the puried enzyme is 57 kDa. The K m and k cat values of the laccase are found to be 18 μM and 0.34 s -1 using 2,6-dimethoxyphenol as the substrate, giving a k cat /K m value is 1.70 x 10 3 M -1 s -1 . The pH and temperature optimum were 4.5 and 40 °C respectively. The puried enzyme has yellow colour and does not show absorption band around 610 nm found in blue laccases. Moreover the conversion of methylbenzene to benzaldehyde in the lack of mediator molecules, property exhibited by yellow laccases.

The fungal strain was procured from the Microbial Type Culture Collection Center and Gene Bank, Institute of Microbial Technology, Chandigarh and was maintained on agar slant as reported in MTCC Catalogue of strains-2000 [19]. The growth medium for the fungal strain G. stratum MTCC-1117 consisted of malt extract 20.0 g and agar 20.0 g in 1.0 L Milli-Q water and pH of the medium was 6.5.
The liquid culture medium reported by Coll et al [28] was used for screening the fungal strain for the production of extracellular laccase in the liquid culture medium. This medium consisted of yeast extract 0.5 g, asparagine 1.0 g, glucose 10.0 g, MgSO 4 .7H 2 O and FeSO 4 .7H 2 O 0.01 g in 1.0 L of Milli-Q water. The Ortho and paradiphenols, aminophenol, polyphenols, polyamines, lignin and arylamines and some of the inorganic ions are the substrates for laccases.it is the most promising enzyme of oxidoreductases group for industrial applications [8][9]. The biotechnological importance of laccases have increased after the discovery that oxidizable reaction substrate range could be further extended in the presence of small readily oxidizable molecules called mediators [10][11]. During the last two decades, laccases have turned out to be the most promising enzymes for industrial uses [8][9] having applications in food, pulp and paper, textile, cosmetics industries and in synthetic organic chemistry [12][13][14][15]. Laccases puri ed from different sources exhibit different properties and are suitable for different applications. Keeping these points in view, the authors have initiated studies on puri cation, characterization and biotechnological applications of laccases from different sources [16][17][18]. G. stratum MTCC-1117 is a white rot fungus isolated from the twigs of Ficus bengalensis [19]. Being a white rot fungus, it is expected to secrete lignolytic enzymes. The special status of the laccase among lignolytic enzymes has prompted the authors to screen this fungus for the secretion of the laccase. In addition, there are reports [20][21] that blue laccase-secreting fungal strains secrete yellow laccases in the presence of culture media containing natural lignin-containing substrates but studies on yellow laccases are rare [22][23][24][25]. Moreover, blue laccases transform methylbenzene to benzaldehyde in the presence of mediator molecules [26][27].
Conversion of methylbenzene to benzaldehyde in the absence of mediator molecules has not been reported so far. The objective of this communication is to purify and characterize a laccase from the liquid culture growth medium of Gloephyllum stratum MTCC-1117 containing natural lignin-containing substrate wheat straw particles and to demonstrate the conversion of methylbenzene to benzaldehyde by the laccase in the absence of mediator molecules. liquid culture medium containing natural lignin substrate like wheat straw, saw dust, coir dust, corn cob, and bagasse particle were separately prepared by adding 0.5 g of one of the natural lignin substrates to 25 ml of the above mentioned growth medium in 100 ml culture asks which were sterilized. The sterilized growth medium was inoculated with small piece of mycelium (0.5 cm ´ 0.5 cm) under aseptic condition and the fungal culture was grown under stationary culture condition at 30 0 C in a BOD incubator. In order to monitor the production of laccase in the liquid culture medium 0.5 mL aliquots of the growth medium were withdrawn at a regular intervals of 24 hrs and ltered through sterilized Millipore lter 0.22 mm. The ltered extract was analyzed for the activity of the laccase using DMP as the substrate by the method [28] given below in the assay section. Secretion of the laccase in the liquid culture medium by G. stratum MTCC-1117 was determined by plotting the enzyme unit per milliliter of the growth medium against the number of days after inoculation of the fungal mycelia. Each point on the curve is an average of the three measurements. The growth medium for the control experiment has the same composition except that no natural lignolytic substrate has been added. In order to optimize the condition for maximum production of the laccase by G. stratum MTCC-1117 in the liquid culture medium, the amount of best inducer wheat straw particle was varied from 100-1000 mg in 25 mL of the growth medium. The amount of the inducer in the growth medium which gives the maximum height of the enzyme activity peak was taken as the optimal amount of the inducer.

Enzyme assay
The assay solution 1.0 mL for ABTS as the substrate [28] contained 0.5 mM ABTS in 0.1 M sodium acetate buffer pH 5.0 at 25 ºC for DMP as the substrate [29]1.0 mL solution contained 1.0 mM DMP in 50 mM sodium malonate buffer pH 4.5 at 37 ºC and for Syringaldazine (3,5-dimethoxy-4hydroxybenaldehyde azine) as the substrate [30] contained 0.1 mM syringaldazine in 50 mM sodium phosphate buffer pH 6.0 at 50 ºC. In case of ABTS, the reaction was monitored by measuring the absorbance change at λ=420 nm and using the molar extinction coe cient [28] value of 36.0 mM −1 cm −1 . In case of DMP, the reaction was monitored by measuring the absorbance change at λ=468 nm and using the molar extinction coe cient [29] value of 49.6 mM −1 cm −1 . In case of syringaldazine, the reaction was monitored by measuring the absorbance change at λ=530 nm and using molar extinction coe cient [30] value 64.0 mM −1 cm −1 . The UV/Vis spectrophotometer Hitachi (Japan) model U-2000 tted with electronic temperature control unit was used for absorbance measurement. The least count of absorbance measurement was 0.001 absorbance unit. One enzyme unit produced 1 μmol of the product per min under the speci ed assay conditions.

Puri cation of laccase enzyme
For the puri cation of the laccase, G. stratum MTCC-1117 was grown in ten 100 mL culture asks each containing 25 mL sterilized growth medium containing optimal amount 800 mg of the inducer wheat straw particles under stationary culture condition in a BOD incubator at 30 °C. The maximum activity of the laccase appeared on seventh day of the inoculation of the fungal mycelia. On the seventh day, all the cultures in the ten asks were pooled, mycelia were removed by ltration through four layers of cheese cloth. The culture ltrate was concentrated by using Amicon concentration cell model-8200 with PM-10 ultra ltration membrane, molecular weight cut-off value 10 kDa. The concentrated enzyme was dialysed against 10 mM Na 2 HPO 4 /NaH 2 PO 4 buffer (pH 6.5) in a volume ratio 1:1000 with three changes at the intervals of 8 h. 10-millilitre enzyme sample containing 1.87 mg/mL protein was loaded on to the DEAE column (size 1.0×38.0 cm) which was pre-equilibrated with 10 mM Na 2 HPO 4 /NaH 2 PO 4 buffer (pH 6.5), and the ow rate was 18 mL/h. The column was washed with 60 mL of the same buffer. The enzyme was eluted by applying linear gradient of 0.0 to1.0 M NaCl in the same buffer (50 mL buffer + 50 mL buffer with 1.0 M NaCl). The fractions of 5.0 mL size were collected and analyzed for the laccase activity [28]. The protein estimation was done by the lowery method [31]. All laccase-active fractions were combined and concentrated by putting it in a dialysis bag and covering the dialysis bag with powdered sucrose at 4 °C.

UV/Vis Spectrum of the Puri ed Enzyme
The UV/Vis spectrum of the puri ed laccase 0.56 mg/mL was recorded in 50 mM sodium phosphate buffer pH 6.5 at 30 °C, using UV/Vis spectrophotometer Hitachi (Japan) model U-2000 as mentioned in the assay section.

Steady-State Enzyme Kinetics
The steady-state enzyme kinetics of the puri ed laccase was studied using DMP as the substrate following the method as mentioned in the assay section [29]. K m and k cat values for the enzyme were determined from the linear regression of double reciprocal plot. The pH and temperature optima of the enzyme were determined by measuring the steady-state velocities of the enzyme catalyzed reaction in the solutions of varying pH/temperature keeping the other parameters xed and drawing graphs of steadystate velocities versus the variable parameter.

Native-PAGE and Zymogram Analysis
The native polyacrylamide gel electrophoresis of puri ed enzyme was done using the reported method [35]. The composition of resolving and stacking gel was similar to that used in SDS-PAGE, except SDS was absent. The molecular weight marker was the bovine serum albumin 66 kDa. Two sets of native gel were done. One set was strained with Coomassie Brilliant Blue R-250 and the other set was used for zymogram [36] preparation. For the preparation of zymogram, 100-mM DMP solution was made in 10 mM sodium tartrate buffer pH 5.0. The native gel was dipped in zymogram solution for 5 min, and a brown band appeared. The zymogram was removed from the DMP solution and was washed thrice with 10 mM sodium tartrate buffer pH 5.0 at the interval of 5 min.

Oxidation of Veratryl Alcohol to Veratraldehyde
This study was made by recording the UV/Vis spectra at zero time and after 30 min of 1.0 mL of the solution containing 0.10 mM veratryl alcohol, and 10.6 μg of the laccase in 100 mM sodium malonate buffer pH 4.5 at 30 °C. The time course of the conversion of veratryl alcohol to verataldehyde was monitored by observing absorbance increase at 310 nm due to verataldehyde [36] formation in the solution of the above composition.
Selective oxidation of the aromatic methyl group to aldehyde in the absence of mediators The bioconversion of methylbenzene to benzaldehyde [26][27] was done in 2 mL of 10 mM sodium phosphate buffer pH 6.5 containing 100 μL methylbenzene and 11.6 μg of puri ed laccase in the absence of any mediator kept in a 25-mL conical ask which was stirred vigorously for 3 min. The reaction solution was extracted thrice with 2 mL of n-hexane 20-microlitres of the n-hexane extracted was injected in Waters HPLC Model 600E using spherisorb C 18 5 UV, 4.5×250 mm column. The eluant phase was methanol/ water in the ratio 1:1 (v/v) at the ow rate of 1.0 mL/min. The detection was made using Waters UV detector model 2487 at λ=254 nm.

Results And Discussion
The results of the secretion of laccase by G. stratum MTCC-1117 in the liquid culture growth medium natural lignin containing substrate wheat straw, bagasse, corn cob, saw dust and coir dust and a control in which no natural lignin containing substrate has been added are shown in Fig. 1. Though the laccase was secreted in the liquid culture medium in the absence of natural lignin-containing substrates, the level of enzyme units secreted was low in comparison to the levels of enzyme units secreted in the presence of the natural lignin-containing substrates. The maximum level of enzyme units secreted in the liquid culture medium was in the presence of wheat straw particles. In order to optimize the secretion of the laccase in the presence of wheat straw particles, secretion of the laccase in the presence of different amounts of wheat straw particles were studied. The results are shown in Fig. 2. The maximum level of the laccase was secreted in the liquid culture medium containing 800 mg of the wheat straw particles per 25 mL of the culture medium.
The results of the puri cation of the laccase from the culture ltrate of the fungal strain are summarized in Table 1. It involved concentration by ultra ltration and an ion exchange chromatography on DEAE cellulose. The elution pro le of the enzyme from DEAE cellulose column is shown in Fig. 3. The enzyme bound to DEAE cellulose in 10 mM sodium phosphate buffer pH 6.0 and was eluted by the linear gradient of NaCl in the range 300 to 500 mM. The fraction numbers 6 to 10 were combined and concentrated to 1 mL by keeping the combined portion in a dialysis bag and covering the bag with powdered sucrose at 4 °C. The concentrated enzyme sample was analysed by SDS-PAGE, zymogram and native PAGE preparation. The results of SDS-PAGE, zymogram and native PAGE preparation are shown in Fig. 4A-C, respectively. In Fig. 4A, lane 1 contains molecular weight markers and lane 2 contains the puri ed enzyme. The appearance of single protein band in SDS-PAGE indicates that the enzyme sample is pure. In Fig. 4B lane 1 a single protein band in the puri ed enzyme in the zymogram analysis. Figure 3C of native PAGE coincides with the position of the puri ed laccase in the native PAGE. The relative molecular mass of the puri ed laccase determined from the analysis of SDS-PAGE was 57 kDa. The relative molecular asses of laccases puri ed from the culture ltrate of Ganoderma lucidium, Chaetomium thermophilium, Neurospora crassa, and Gaeumannomyces graminis var. tritici are 68, 77, 64.8 and 60 kDa [28,[38][39][40] respectively. Thus the relative molecular mass of G. stratum MTCC-1117 laccase is in the same range as reported for other fungal laccases [41].
The Michaelis-Menten and double reciprocal plots for the puri ed enzyme using DMP as the variable substrate [28] are shown in Fig. 5A, B respectively. The K m and k cat values determined from the double reciprocal plot using DMP as the substrate of the enzyme were 18 µM and 0.34 s − 1 , respectively, giving k cat /K m 1.70 × 10 3 M − 1 s − 1 . The range of K m values reported [41] for fungal laccases using DMP as the substrate are 8-14,720 µM. However the K m value for this laccase compares well with the K m of laccase puri ed from Armillaria mellea Lac-1 [42]. The value of k cat determined for the puri ed laccase has been found to be low in comparison to the values of k cat reported for other fungal laccases [42]. It is worth mentioning over here that the catalytic performances of laccases span several orders of magnitude for different substrates and are characteristic for speci c proteins [42]. The results of the variation of the activity of the puri ed laccase with the variation of pH of the reaction solution are shown in Fig. 6. The determined pH optimum of the enzyme was 4.0. The pH optima reported in the literature [42] for laccases using DMP as the substrate are in the range 3.0-8.0 pH units. The pH optimum of the puri ed laccase compares well with pH optima of laccases of Ceriporiopsis subvermispora L1, C. subvermispora L2, Daedalea quercina and Lentinula edodes LCC 1 [42].
The results of variation of the activity of the puri ed laccase as a result of variation of the temperature of the reaction solution of enzyme catalysed reaction are shown in Fig. 7. The calculated temperature optimum was 35 °C. The range of temperature optima reported for other laccases are 25-80 °C. The laccases of Pleurotus ostreatus POXA 3b and P. ostreatus POXC also have temperature optima at 35 °C [42] . The puri ed laccase does not possess a blue colour as visually observed in cases of blue laccases. Instead, it has a yellow colour. It has already been reported in the literature that some fungal strains, which secrete blue laccase in submerged liquid cultures, secrete blue laccases when grown in solid-state fermentation containing natural lignin substrate, wheat straw [20]. The yellow laccases lack absorption band around 610 nm observed in cases of blue laccases. The UV/Vis spectrum of the puri ed laccase was recorded and is shown in Fig. 8. The authors could not detect the blue absorption band around 610 nm in the puri ed laccase spectrum, indicating that it is a yellow laccase. It has also been reported that yellow laccases which oxidize non-phenolic substrates without the presence of electron transfer mediators [20][21] which are essential for the oxidation of non-phenolic substrates by blue laccases. The puri ed laccase was tested for its property of the oxidation of veratryl alcohol, a non-phenolic substrate which is not oxidized by blue laccases in the absence of the mediator molecules. The results are shown in Fig. 8. Figure 8 (a) is the spectrum of a solution containing veratryl alcohol and the puri ed enzyme just after addition of the enzyme. Figure 8 (b) is the spectrum of the same solution after 30 min of the addition of the puri ed enzyme. Veratryl alcohol has very low absorption at λ = 310 nm whereas the oxidation product of veratryl alcohol, veratraldehyde, has absorption maximum at λ = 310 nm. The comparison of the spectra a and b given in Fig. 8 clearly shows that the enzyme has oxidized veratryl alcohol to veratraldehyde in the absence of any mediator molecule. The time course of the oxidation of veratryl alcohol to veratraldehyde monitored by increase in absorbance at λ = 310 nm due to the formation of veratraldehyde is shown in Fig. 8 (c) as the insert. Thus the puri ed laccase exhibits the property exhibited by yellow laccases and can be termed as a yellow laccase.
One of the applications of the laccases in organic synthesis is in the selective oxidation of the aromatic methyl group to the corresponding aldehyde. The chemical routes of this conversion are inconvenient because the reaction has to be stopped at the aldehyde stage so that carboxylic acid could not be formed. Moreover, they require drastic reaction conditions which pollute the environment. The conversion done with laccase occurs under milder conditions, yield is > 90% and the process is ecofriendly. Laccases are the preferred enzymes because they cycle on oxygen and generate no side products except water. The use of blue laccases for this purpose has been studied [26][27] but they require mediator molecules like ABTS [27] and 1-hydroxy-1Hbenzotrizole (HOBT) [28]. As has been discussed above, the puri ed laccase oxidizes veratryl alcohol, a non-phenolic substrate to veratraldehyde without the presence of the mediator molecules. The potential of the puri ed laccase as a biocatalyst for the conversion of aromatic methyl group to the corresponding aldehyde in the absence of the mediator molecule was tested using methylbenzene as the substrate. The results are shown in Fig. 8. Figure 8a is the HPLC chromatogram of the n-hexane extract of the product formed by the reaction of the enzyme with methylbenzene in the absence of any mediator molecule. Figure 8b, c is the chromatograms of the standard samples of benzaldehyde and methylbenzene, respectively. The retention time of the standard sample of methylbenzene was 4.05 min, and the retention time of the standard sample of benzaldehyde was 2.96 min. Thus the retention time of the product of the enzyme-catalysed reaction 3.09 coincides with the retention time of benzaldehyde 2.96 min, showing that the product of enzyme-catalysed reaction is benzaldehyde. Figure 8 clearly demonstrates that the puri ed enzyme catalyses the oxidation of methylbenzene to benzaldehyde in the absence of mediator molecules like ABTS and HOBT as reported in cases of blue laccases [27][28] for the same conversion. The puri ed enzyme is a better biocatalyst than blue laccases for the above conversion because it does not require the mediator molecules for the above conversion.

Conclusions
Secretion of laccase was studied in presence of different lignin containing substrate (wheat straw, saw dust, coir dust, corn cob, and bagasse) in aqueous culture medium. The maximum level of laccases secreted in the liquid culture medium (25 mL) was in the presence of 800 mg wheat straw particles. Thus in this communication a yellow laccase from a new fungal strain G. stratum MTCC-1117 has been puri ed and characterized. Puri ed yellow laccases catalyse the oxidation of methylbenzene to benzaldehyde in the absence of mediator molecules like ABTS and HOBT [27][28]. Thus puri ed yellow laccase is a better biocatalyst than blue laccases.

Declarations
Availability of data and materials Author RS have procured and interpreted the data himself from experiment performed in the Chemistry lab Maharana Pratap P.G. College, Jungle Dhusan Gorakhpur. The materials required for the experiment were available in the lab.
Ethics approval and consent to participate:

Consent for publication
Author is agree to publish the manuscript in the "Journal of Biological Research-Thessaloniki".

Competing interests
The authors declare that they have no competing interests.

Author's Contribution's
The author RS himself performs all the experiments and the result reported in the manuscript.