Production, purification and biochemical characterization of two laccase isoforms produced by Trametes versicolor grown on oak sawdust Fernando Martínez-Morales, Brandt Bertrand, Angélica A. Pasión Nava, Raunel Tinoco, Lourdes AcostaUrdapilleta & María R. Trejo-Hernández Biotechnology Letters ISSN 0141-5492 Biotechnol Lett DOI 10.1007/s10529-014-1679-y 1 23 Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media Dordrecht. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Biotechnol Lett DOI 10.1007/s10529-014-1679-y ORIGINAL RESEARCH PAPER Production, purification and biochemical characterization of two laccase isoforms produced by Trametes versicolor grown on oak sawdust Fernando Martı́nez-Morales • Brandt Bertrand • Angélica A. Pasión Nava • Raunel Tinoco • Lourdes Acosta-Urdapilleta • Marı́a R. Trejo-Hernández Received: 19 June 2014 / Accepted: 9 September 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Two laccase isoforms (lcc1 and lcc2) produced by Trametes versicolor, grown on oak sawdust under solid-state fermentation conditions, were purified and characterized. The two isoforms showed significant biochemical differences. Lcc1 and lcc2 had MWs of 60 and 100 kDa, respectively. Both isoforms had maximal activity at pH 3 with ABTS and 2,6-dimethyloxyphenol (DMP). Lcc1 was the most attractive isoform due to its greater affinity towards all the laccase substrates used. Lcc1 had Km values of 12, 10, 15 and 17 mM towards ABTS, DMP, guaiacol and syringaldazine, respectively. Lcc2 had equivalent values of 45, 47, 15 and 39 mM. The biochemical properties of lcc1 substantiate the potential of this enzyme for application in the treatment of F. Martı́nez-Morales
B. Bertrand
A. A. Pasión Nava
M. R. Trejo-Hernández (&) Laboratorio de Biotecnologı́a Ambiental, Centro de Investigación en Biotecnologı́a, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Chamilpa, CP 62209 Cuernavaca, Morelos, Mexico e-mail: mtrejo@uaem.mx R. Tinoco Instituto de Biotecnologı́a, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Chamilpa, CP 62210 Cuernavaca, Morelos, Mexico L. Acosta-Urdapilleta Centro de Investigaciones Biológicas, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Chamilpa, CP 62209 Cuernavaca, Morelos, Mexico contaminated water with low pH values and high phenolic content. Keywords Laccase characterization
Laccase isoforms
Laccase purification
Lignolytic enzymes
Solid-state fermentation Introduction Laccases (benzenediol:oxygen oxidoreductase, E.C. 1.10.3.2) are widely distributed multicopper enzymes and are mainly produced by white rot fungi such as Trametes versicolor (Poojary and Mugeraya 2012). They catalyze the oxidation of a wide range of aromatic compounds by the removal of electrons with the concomitant reduction of O2 to water and thus the application of these enzymes in many biotechnological processes is attracting great interest (Imran et al. 2012). The industrial applications of laccases include opportunities in the pulp and paper, environmental and food industries (Osma et al. 2010). Because laccases have potential in a number of industrial applications, numerous studies have been carried out to increase the production of laccases with high redox potential and high stability. Laccases have diverse biological functions (Baldrian 2006) one of which is an essential role in carbon recycling during lignin degradation (Theuerl et al. 2010). The tendency towards the effective use and 123 Author's personal copy Biotechnol Lett valuation of organic wastes, such as wastes from the agriculture and food industries, as substrates for solidstate fermentation (SSF) has increased. Moreover, the majority of these wastes contain lignin and/or cellulose or hemicellulose, that act as inducers of lignolytic activity (Bertrand et al. 2014). Most of these wastes have a high sugar content, making the production of these enzymes economical. The use of these types of wastes provides an alternative source of substrates and assists in solving environmental pollution problems (Risdianto et al. 2010). SSF is a promising method for culturing filamentous fungi to produce lignolytic enzymes because these fungi are grown under conditions that emulate their natural habitat, allowing them to produce certain enzymes in large quantities. Various agricultural substrates, such as barley bran and wheat bran, have been used in SSF for laccase production by white rot fungi (Neifar et al. 2011). The aim of this work was to investigate the production of T. versicolor laccases by SSF using oak sawdust as the substrate. Materials and methods All the chemicals in this investigation were purchased from Sigma-Aldrich. Synthetic culture media were obtained from Bioxon (Becton and Dickinson, Mexico). Organism and strain preservation The white-rot fungus T. versicolor HEMIM-9 was isolated from decayed oak (Quercus sp.) in Morelos, central Mexico, and kept in the culture collection of the Mycology Laboratory at the Centro de Investigaciones Biológicas, UAEM, México. An inoculum was obtained from a potato/dextrose/agar (PDA) plate with actively-growing mycelia. Solid medium as an inoculum for SSF Wheat grains were used as a colonization support, growth substrate, and energy source. Polypropylene bags were prepared with 350 g wheat grains, 2 % (w/ w) CaSO4, and 0.5 % (w/w) CaSO3. The bags were closed, sterilized for 1 h at 121 °C, cooled to room temperature and inoculated with 3 agar disks (15 mm diam.) containing the mycelium of T. versicolor. The 123 bags were incubated at 25 °C in the dark for 15 days or until complete colonization of the surface of the grains by the mycelium of T. versicolor HEMIM-9. Lignocellulosic substrate preparation for laccase production Oak sawdust (the lignocellulosic substrate) was chopped into 2.5–10 cm pieces and pasteurized by submerging in water maintained at 70 °C for 1 h. After draining, the sawdust contained approx. 65 % moisture. Two kg substrate was placed in polypropylene/ethylene bags with a micropore filter with dimensions of 4 9 4 cm and inoculated with 4 % (wet wt) of the colonized wheat grains. The inoculated sawdust was incubated at 22–24 °C for 9 days with 12 h periods of light and darkness. Enzyme extract After 9 days of incubation, the solid culture (2 kg) was submerged in 5 l 100 mM phosphate buffer (pH 6) and left to soak overnight. The supernatant was recovered by pressing the solid product and the obtained extract was filtered. Five ll 0.5 mM PMSF was added to the enzyme extract. The extract was then centrifuged for 10 min at 10,0009g to remove any solid particles from the supernatant. The extract was then frozen for 3 days, defrosted and then centrifuged once again to precipitate and remove excess polysaccharides. Afterwards, the supernatant was concentrated and dialyzed by ultrafiltration using a membrane with a 10 kDa cutoff. Analytical techniques Protein electrophoresis Proteins were electrophoresed using a 12 % SDSPAGE gel. Page Ruler pre-stained protein ladder plus (Fermentas) was used as the molecular weight marker standard. Zymograms were used for visualization of the laccase activity profiles. Samples were loaded into the wells and ran at 150 V for 1 h. Zymograms were washed with a 100 mM sodium acetate buffer solution (pH 5) to remove SDS before staining. Bands of laccase activity were detected by a reaction with 1 mM 2,6-dimethoxyphenol (DMP) dissolved in the same buffer. For denatured gels, laccase samples were Author's personal copy Biotechnol Lett mixed with 5 ll loading buffer (containing b-mercaptoethanol and SDS) and heated at 96 °C for 5 min in a dry bath. After electrophoresis, the gels were stained with Coomassie Blue R-250. Enzyme assays and laccase purification Laccase activity was determined at room temperature by oxidation of 1 mM ABTS in 100 mM sodium acetate buffer (pH 3.6) mixed with 50 ll enzyme in 1 ml. The oxidation of ABTS was determined at 436 nm (e = 29,300 M-1 cm-1). Enzyme activities were expressed in international units (U). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 lmol oxidized ABTS per min. For laccase purification, the supernatant was centrifuged at 10,0009g for 20 min to remove any solid particles. Extracellular laccase produced by T. versicolor HEMIM-9 was concentrated by ultra-filtration as previously described. To separate the laccase isoforms present in the supernatant, an anionic exchange column (25 9 2.5 cm) of DEAE-cellulose (Whatman DE-52) was used. The concentrated supernatant was loaded into the column and eluted with a linear gradient of NaCl from 0.1 to 1 M in 20 mM phosphate buffer (pH 6) at 3 ml min-1. Fractions of 6 ml were collected. The fractions with laccase activity were pooled, dialyzed and concentrated by ultrafiltration. Enzymatic characterization Effect of pH on enzymatic activity The effect of pH on enzyme activity was determined for laccase isoforms (lcc1 and lcc2) using a pH range of 3–8 (acetate buffer 3–6 and phosphate buffer 7–8). The activity was determined using ABTS, DMP, guaiacol (GUA) and syringaldazine (SYR). Kinetic parameters (apparent Km and Vmax)-effect of the substrate concentration Table 1 Purification steps of laccase isolated from T. versicolor HEMIM-9 grown in SSF on oak sawdust Purification step Laccase activity (U ml-1) Total activity (U) Yield (%) Crude extract 0.5 11,980 100 Ultrafiltration 4 5,240 44 DEAE-cellulose chromatography Lcc1 2 1,400 27 Lcc2 9 578 11 Bioinformatic analysis Laccase gene sequences were obtained from the genome of T. versicolor ATCC20869, http://genome. fungalgenomics.ca. The molecular weight of mature laccase proteins were predicted by removing the signal peptides and using the Compute pI/Mw tool from the ExPASy proteomics server (http://web.expasy.org/ compute_pi/). Results and discussion Industrial application of laccases requires the production of large amounts of enzyme at a low cost. Research in this area is focused on the search for efficient production systems. A suitable strategy to achieve this aim is the production of laccase by SSF using agro-industrial wastes as a support/substrate. The forestry industries produce very large volumes of wastes annually worldwide, and these wastes cause a serious disposal problem. In addition, great interest has been shown in the reutilization of renewable biomass because of energetic and environmental problems, and hence the industry is forced to find an alternative for the valorization of residual agricultural and forestry wastes. Forestry wastes are rich in soluble carbohydrates and also contain inducers of laccase synthesis, ensuring an efficient production of laccase (Bertrand et al. 2014). Laccase crude extract purification The kinetics constants from the Michaelis–Menten equation were determined using the linear regression methods of Lineweaver–Burk and Langmuir. The parameters were determined using ABTS (k = 436 nm), DMP (k = 468 nm), SYR (k = 530 nm) and GUA (k = 470 nm), at 0.0025 to 5 mM for each substrate. Laccase produced by SSF was obtained after successive extractions of the solid culture with a phosphate buffer solution of pH 6. The total activity of laccase present in the crude extract (12,100 U) was concentrated by ultrafiltration. The laccase yield obtained after the ultrafiltration process was 44 % and was 123 Author's personal copy Biotechnol Lett Fig. 1 T. versicolor laccase electrophoresis. a Gel zymogram of lcc1 and lcc2. Lane 1 crude laccase extract; lane 2 purified lcc2; and lane 3 purified lcc1. b Denatured gel. Lane 1 Molecular weight markers, lane 2 heterodimer lcc1 under denaturing gel conditions. Laccase bands are indicated by black stars (a) subsequently further purified. The ultra-filtered crude extract was purified using DEAE-cellulose (DE-52) (Table 1). Two laccase isoforms, lcc1 and lcc2, were detected in the crude laccase extract and were used for biochemical characterization. The apparent molecular weights for lcc1 and lcc2 were 60 and 100 kDa, respectively (Fig. 1a). The majority of laccases purified and characterized to date are monomeric. However, current knowledge of fungal laccases shows that several of these proteins are homodimeric or oligomeric. The typical molecular weights of laccases from the Trametes species range from 58 to 71 kDa (Baldrian 2006). Our results suggest that lcc2 exists as a heterodimer because, after denaturing the native band, SDS-PAGE revealed the appearance of two bands with molecular weights of 55.4 and 66.3 kDa, accompanied by the disappearance of the heavier band (100 kDa) (Fig. 1b). Similar results were also obtained with other fungal laccases (other basidiomycetes, mycorrhiza and ascomycetes). Thakker et al. (1992) reported a laccase from the ascomycete Monocillium indicum that had a single band of 100 kDa after gel filtration and this protein was resolved into three proteins on SDS-PAGE (24, 56 and 72 kDa). The white-rot fungus Phellinus ribis also produced a single form of 123 (b) laccase, and this protein was a dimer consisting of two 76 kDa subunits (Min et al. 2001). The complete genome of T. versicolor ATCC20869 is now available, and genome analysis reveals the presence of eight laccase genes (http://genome. fungalgenomics.ca) (Table 2). The estimated molecular weights of laccases bands detected in this study using SDS-PAGE were consistent with the theoretical molecular weights calculated for the laccases from the genome of T. versicolor ATCC20869. pH profile of lcc1 and lcc 2 Fungal laccases have activities over a wide pH range (2–10); this may be a useful characteristic for various industrial and biotechnological applications (Asgher et al. 2012). In this study, the optimum pH for enzymatic activity for both isoforms was at pH 3 (Fig. 2). Lcc1 and lcc2 activity with ABTS and 2,6dimethoxyphenol (DMP) substrates decreased with a corresponding increase in pH. Further analysis indicated that lcc1 had a higher specific activity towards ABTS and DMP compared to lcc2. The optimum pH for enzymatic activity with guaiacol (GUA) and syringaldazine (SYR) for both isoforms was at pH 5. Lcc1 also presented a higher specific activities compared to lcc2 towards GUA and SYR, respectively. Author's personal copy Biotechnol Lett Table 2 Gene and protein data of laccases from the genome of T. versicolor ATCC20869 Gene model Product Chromosomal location # of amino acids Signal peptide Theoretical mass (kDa) Trave2p4_000270 Laccase-1 Scaffold0001S:641872..643866 520 21 53.28 Trave2p4_000509 Laccase-2 Scaffold0001S:1265289..1267416 519 20 53.58 Trave2p4_004282 Trave2p4_004487 Laccase-1 Laccase-2 Scaffold0005S:1297140..1299827 Scaffold0006S:139680..141791 624 524 0 25 66.30 53.69 Trave2p4_011748 Laccase-3 Scaffold0028S:531347..534324 580 0 63.51 Trave2p4_011750 Laccase-3 Scaffold0028S:537693..539903 515 21 54.50 Trave2p4_013793 Laccase-5 Scaffold0043S:54282..56547 527 23 53.74 Trave2p4_013795 Laccase-4 Scaffold0043S:57936..60530 560 24 57.09 The molecular weight of mature laccase proteins were predicted by removing the signal peptides and using the Compute pI/Mw tool from the ExPASy proteomics server (http://web.expasy.org/compute_pi/) Table 3 Kinetic constants of purified lcc1 and lcc2 isoforms isolated from T. versicolor HEMIM-9 grown in SSF on oak sawdust Kinetic parameter/ substrate Fig. 2 Effect of pH on the specific activity of the two purified laccase isoforms (lcc1 and lcc2) produced by T. versicolor for ABTS, 2,6-dimethoxyphenol (DMP), syringaldazine (SYR) and guaiacol (GUA) each at 1 mM in 100 mM acetate buffer (3–6) and 100 mM phosphate buffer (7–8). Isoforms registered maximal laccase activity at pH 3 with ABTS and DMP. All experiments were repeated using three biological replicates Kinetic studies The values for Km and Vmax obtained for the two isoforms are shown in Table 3. Lcc1 from T. versicolor HEMIM-9 had a greater affinity than lcc2 Lcc1 Km (mM) Lcc2 Vmax (lmol min-1) Km (mM) Vmax (lmol min-1) 117 ABTS 12 18 45 DMP 10 21 47 47 GUA SYR 15 17 28 214 15 39 23 65 towards all four substrates. Lcc1 had a higher affinity for DMP compared to the results of More et al. (2011) with a Km of 38 mM and a Vmax of 20 lmol min-1 for the laccase in Pleurotus sp. Lcc1 also presented higher affinities than the major laccase from Trametes maxima produced under submerged culture conditions with Km values of 20 and 74 mM towards SYR and DMP, respectively, (Gutiérrez-Soto et al. 2011). The ability of this latter enzyme to decolorize nine commercially-significant textile dyes supported its implementation in wastewater remediation. Conclusions Current knowledge on the physicochemical properties of fungal laccases is based on data of purified proteins from wood-rotting white-rot basidiomycetes. Fungal laccases have been mainly described as monomeric proteins from these basidiomycetes. Some laccases, however, reveal a homodimeric structure with two identical subunits and a molecular weight typical for 123 Author's personal copy Biotechnol Lett monomeric laccases of different fungal species (Phellinus ribis, Pleurotus pulmonarius, Trametes villosa, Cantharellus cibarius, Rhizoctonia solani, Gaeumannomyces graminis, Monocillium indicum and Podospora anserine). The two laccase isoforms isolated in this study displayed different behaviors after pH profiling analysis. Lcc1 registered higher activities for the oxidation of the four mentioned substrates. Interestingly, the optimum pH for the oxidation of DMP and GUA was from 3 to 3.5. Normally, the optimum pH for the oxidation of these compounds is higher. In addition to the laccase isoform, lcc1, oxidizing DMP and GUA at acidic pH, this enzyme also had a high affinity for the substrates tested. Lcc1 also showed high affinity for SYR. The reactivity of the lcc2 isoform was less with a Km value of 39 lM. Our findings demonstrate that lcc1 has potential for application in the treatment of contaminated water with low pH values and high phenolic content; moreover, this isoform can be produced at low cost by SSF cultures. Acknowledgments BB and AAPN acknowledge the fellowship of the PROMEP project 103.5/07/2674. We are grateful to M.A. Francisco Abel Medrano-Vega from Laboratorio de Micologı́a at Centro de Investigaciones Biológicas, UAEM for the SSF cultures of T. versicolor used in this work. We are also grateful to M. B. Daniel Morales Guzmán for his excellent technical assistance. References Asgher M, Iqbal HMN, Asad MJ (2012) kinetic characterization of purified laccase produced from Trametes versicolor IBL-04 in solid state bio-processing of corncobs. Bio Res 9:5778–5781 Baldrian P (2006) Fungal laccases—occurrence and properties. FEMS Microbiol Rev 30:215–242 Bertrand B, Martı́nez-Morales F, Tinoco R, Rojas-Trejo S, Serrano-Carreón L, Trejo-Hernández M (2014) Induction 123 of laccases in Trametes versicolor by aqueous wood extracts. World J Microbiol Biotechnol 30:135–142 Gutiérrez-Soto JG, Salcedo-Martı́nez SM, Contreras-Cordero JF, Hernández-Luna CE (2011) Characterization of the major laccase from Trametes maxima CU1 and decolorization of nine commercially significant dyes by the enzyme. Water R&D 1:10–19 Imran M, Asad MJ, Hadri SH, Mehmood S (2012) Production and industrial applications of laccase enzyme. J Cell Mol Biol 10:1–11 Min KL, Kim YH, Kim YW, Jung HS, Hah YC (2001) Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis. Arch Biochem Biophys 392:279–286 More SS, Renuka PS, Pruthvi K, Swetha M, Malini S, Veena SM (2011) Isolation, purification, and characterization of fungal laccase from Pleurotus sp. Enzyme Res. doi:10.4061/ 2011/248735 Neifar M, Kamoun A, Jaouani A, Ellouze-Ghorbel R, EllouzeChaabouni S (2011) Application of asymetrical and hoke designs for optimization of laccase production by the white-rot fungus Fomes fomentarius in solid-state fermentation. Enzyme Res. doi:10.4061/2011/368525 Osma JF, Toca-Herrera JL, Rodriguez-Couto S (2010) Uses of laccases in the food industry. Enzyme Res. doi:10.4061/ 2010/918761 Piscitelli A, Giardina P, Lettera V, Pezzella C, Sannia G, Faraco V (2011) Induction and transcriptional regulation of laccases in fungi. Curr Genom 12:104–112 Poojary H, Mugeraya G (2012) Laccase Production by Phellinus noxius hpF17: optimization of submerged culture conditions by response surface methodology. Res Biotechnol 3:9–20 Risdianto H, Suhardi SH, Setiadi T, Kokugan T (2010) The influence of temperature on laccase production in solid state fermentation by using white rot fungus Marasmius sp. The 1st international seminar on fundamental & application ISFAChE 2010 of chemical engineering Thakker GD, Evans CS, Rao KK (1992) Purification and characterization of laccase from Monocillium indicum Saxena. Appl Microbiol Biotechnol 37:321–323 Theuerl S, Dorr N, Guggenberger G, Langer U, Kaiser K, Lamersdorf N, Buscot F (2010) Response of recalcitrant soil substances to reduced N deposition in a spruce forest soil: integrating laccase-encoding genes and lignin decomposition. FEMS Microbiol Ecol 73:166–177