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Biochemical and molecular characterization of laccase isoforms produced by the white-rot fungus Trametes versicolor under submerged culture conditions
Accepted Manuscript Title: Biochemical and molecular characterization of laccase isoforms produced by the white-rot fungus Trametes versicolor under submerged culture conditions. Author: Brandt Bertrand Fernando Mart´ınez-Morales Raunel Tinoco-Valencia Sonia Rojas Lourdes Acosta-Urdapilleta Mar´ıa R. Trejo-Hern´andez PII: DOI: Reference:
S1381-1177(15)30088-6 http://dx.doi.org/doi:10.1016/j.molcatb.2015.10.009 MOLCAB 3259
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
13-5-2015 9-10-2015 10-10-2015
Please cite this article as: Brandt Bertrand, Fernando Mart´inez-Morales, Raunel Tinoco-Valencia, Sonia Rojas, Lourdes Acosta-Urdapilleta, Mar´ia R.Trejo-Hern´andez, Biochemical and molecular characterization of laccase isoforms produced by the white-rot fungus Trametes versicolor under submerged culture conditions., Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.10.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biochemical and molecular characterization of laccase isoforms produced by the white-rot fungus Trametes versicolor under submerged culture conditions. Brandt Bertrand1, Fernando Martínez-Morales1, Raunel Tinoco-Valencia2, Sonia Rojas2, Lourdes Acosta-Urdapilleta3, María R. Trejo-Hernández1*
1 Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos. Avenida Universidad 1001, Chamilpa CP 62209 Cuernavaca, Morelos, México 2 Instituto de Biotecnología, Universidad Nacional Autónoma de México. Avenida Universidad 2001, Chamilpa CP 62210 Cuernavaca, Morelos, México 3 Centro de Investigaciones Biológicas, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Chamilpa CP 62209 Cuernavaca, Morelos, México
Corresponding Author: María R. Trejo-Hernández. Laboratorio de Biotecnología
Ambiental. Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Cuernavaca, Morelos, CP 62209. E-mail address: [email protected] Phone +52-777-3297057; Fax +52-777-3297030
Graphical abstract Purification and biochemical characterization of laccase heterodimeric complex
Zymography
Isoelectric Focusing
pI 3.5 4.5 Heterodimeric laccase complex
lcc2
4.8 5.2
Laccase monomers
lcc1
C
B
A
5.8
lcc2
Purification and Characterization
Research highlights:
Laccase band lcc2 (100 kDa) was resolved into 5 distinct isoforms by IEF.
Lcc2 laccase band is a heterodimeric complex.
The heterodimer complex may have had an influence of its catalyzing capacity.
Isoform diversity was validated by the molecular characterization of 5 alleles.
Structure-function assumptions suggest biochemical differences of laccase alleles.
Abstract Many fungi produce several laccase isoenzymes endowed with different catalytic properties, however, the physiological significance of this multiplicity is still unclear. Multiple laccase isoforms produced by the same organism imply a greater flexibility and/or adaptation to constantly changing environments. In this study, we explore and discuss the laccase isoform diversity of Trametes versicolor under submerged culture conditions. Oak sawdust was used as a natural, abundant and cheap source of laccase inducers. Two laccase bands (lcc1 and lcc2) were detected by SDS-PAGE zymography. Lcc2 exhibited an apparent molecular mass of 100 kDa, and was purified to its characteristic blue color for biochemical characterization. Lcc2 displayed high affinities towards all the substrates used in this study (0.01 ± 0.0043, 0.5 ± 0.053, 2.23 ± 0.53 and 3.77 ± 0.85 mM, for ABTS, DMP, GUA and SYR, respectively). Isoelectric focusing analysis of this band revealed the presence of five distinct laccase isoforms. The suggested existence of different laccase heterodimers in lcc2 may have had an influence on its catalyzing capacity. Laccase isoform diversity was corroborated by molecular characterization of five allelic mRNA sequences. Multiple alignment analysis, 3-Dimensional mapping and theoretical sequence-structurefunction assumptions suggests that the newly characterized alleles would most likely display diversity in their biochemical behavior if functionally expressed.
Key words: Biochemical characterization, Laccase isoform diversity, Trametes versicolor, Molecular characterization
1 Introduction Laccases (benzenediol: oxygen oxidoreductase, E.C. 1.10.3.2) are a class of proteins that belong to the multicopper oxidase family [1]. Laccases catalyze the oxidation of a wide range of substances including aromatic compounds by the removal of electrons with the concomitant reduction of molecular oxygen to water, and thus, there is great interest for their application in many biotechnological processes [2, 3]. The industrial applications for laccases include pulp and paper, environmental and food industries [4]. Therefore, numerous studies on biochemical characterization have been carried out to increase laccase production with high redox potential and high stability.
Laccase distribution is widespread and have been isolated from fungi, insects, bacteria and plants, with diverse biological functions including morphogenesis, fungal plantpathogen/host interactions, stress defense, lignin degradation and soil organic matter recycling [5]. Among fungi, the basidiomycetes, e.g., Agaricus bisporus, Pleurotus ostreatus, Trametes versicolor, Phanerochaete chrysosporium and Coprinus cinereus, produce different laccase isoforms that catalyze the oxidation of a broad spectrum of substrates, including toxic xenobiotic compounds [6]. As a result, fungal laccases have been proposed as biocatalysts that can be used in bioconversion processes in an environmentally friendly way [7].
The production of numerous laccase isoforms by a single fungus is not rare and has been demonstrated by the addition of inducers, and these inducers have been proven to affect the expression of laccase genes differentially at the transcriptional level [6, 8]. A wide range of
molecules derived from lignin can be used as laccase inducers. Phenolic acids and metal ions can also serve as specific laccase inducers [9]. Fungal growth phase and nutritional conditions, e.g., the nitrogen or carbon sources, may affect the laccase isoform profile. These inducers and factors affect productivity and the relative amounts of the different laccase isoforms secreted. Laccase profiles and concentrations obtained are very important because the different isoforms possess particular catalytic properties. In this study, a laccase band (lcc2) with an apparent molecular mass of approximately 100120 kDa was purified to its characteristic blue color and biochemically characterized. Lcc2 displayed a similar pH profile to a previously characterized laccase band, however, presented very high affinities towards all substrates used in this work. This band was resolved into five distinct laccase isoforms by isoelectric focusing, with pI values ranging from 3.5-5.8. Given the apparent molecular mass of lcc2, and the theoretical molecular mass of the individual laccase isoforms, we hypothesized that this band may be in fact composed of different heterodimers. The suggested heterodimeric structure may have had an influence on its catalyzing capacity displayed after biochemical characterization. Additionally, the natural laccase isoform diversity was corroborated by isolating, identifying and characterizing five mRNA allelic sequences. Multiple alignment analysis, 3-Dimensional mapping and theoretical sequence-structure-function assumptions suggests that the newly characterized alleles, although very similar in their amino acid sequences, would most likely display diversity in their biochemical behavior, if functionally expressed.
2 Materials and Methods 2.1 Organism, strain preservation and growth conditions 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. T. versicolor HEMIM-9 was maintained in darkness at room temperature on potato dextrose agar (PDA) plates with periodic transfers. An inoculum was obtained from a fully colonized Petri dish (100 x 15 mm) with the actively growing mycelium.
2.2 Lignocellulosic media for fungal cultures Refined white wheat flour medium: White wheat flour (1 %) was dissolved completely in a 100 mM sodium phosphate buffer (pH 6), this medium was used as the control culture medium. The culture media for production of laccase were constituted of lignocellulosic substrates (oak sawdust and Bran flakes cereal). Oak sawdust medium: was prepared by adding solid oak sawdust (0.2 and 0.5 %) to the refined white wheat flour medium. Bran flakes medium: 20 g Bran flakes cereal was boiled at 95 °C for 20 min in 1 l of sodium phosphate buffer solution (pH 6). After cooling down, the mixture was filtered through filter paper (Whatman 1) to remove solids. The filtrate was used as the culture media for fungal growth and laccase production. All media were sterilized at 121 °C for 15 min. Inoculum preparation: T. versicolor HEMIM-9 was grown in a Petri dish for 7 days in darkness at 29 °C. Once fully grown, the fungus was homogenized, and 10 ml was used to
inoculate the respective media. Laccase production was quantified every 24 h until the maximum activity was attained. 2.3 Crude laccase extract preparation Laccase production was measured until the volumetric activity was maximal. The fungal culture was centrifuged at 10 000 x g for 10 min to remove the biomass. The supernatant was filtered using Whatman 1 filter paper and then concentrated by lyophilization. The crude extract was dialyzed by ultrafiltration (Amicon Millipore Ultrafiltration Disc) using a membrane with a 10 kDa cutoff at 10 psi.
2.4 Laccase purification The concentrated sample was loaded into the anion exchange column. The column was packed with a dimethylaminoethyl cellulose resin (DE52 Whatman), coupled with a BioRad pump (Econo Gradient Pump). The column was activated with a 20 mM NaCl solution. The crude concentrated laccase extract was loaded with a 20 mM Tris buffer solution pH 5, and the flow rate adjusted to 3 mlmin⁻¹. The column was eluted with a 0-60 % NaCl gradient. Laccase activity of the fractions collected was measured using 2, 2´azino-bis (3-ethylbenzothiazoline-6 sulphonic acid) (ABTS) and protein concentration was quantified by the Bradford method [10]. Zymograms were used to reveal the progress of the purification process. Subsequently, a cation exchange column packed with a CM sepharose resin (Sigma Fastflow) was used. A hydrophobic interaction column (butyl HIC support from BioRad) was also prepared. The sample to be loaded was precipitated previously with 30 % ammonium sulfate, and was then centrifuged for 20 min at 10 000 x g. A size
exclusion chromatography column was prepared using Sephadex 200 (GE Healthcare Life Sciences), with a particle size of 13 µm and a separation range of 10-600 kDa for globular proteins. The fractions with laccase activity were pooled together and concentrated by ultrafiltration.
2.5 Protein electrophoresis Proteins were electrophoresed in a MINI-PROTEAN 3 electrophoresis cell (Bio-Rad) using a 12 % SDS-PAGE gel as described by Laemmli [11]. The Page Ruler pre-stained protein ladder plus (Fermentas) was used as the molecular mass marker standard. Zymograms were used for visualization of the laccase activity profiles. Samples were ran at 150 V for 1 h; zymograms were washed with a 100 mM sodium acetate buffer solution (pH 5) before revealing to remove SDS. Bands of laccase activity were detected by reaction with 1 mM 2, 6-dimethoxyphenol (DMP) dissolved in the same buffer. For denatured gels, laccase samples were mixed with 5 µl of the loading buffer (containing β-Mercaptoethanol and SDS) and by heating at 96 ºC for 5 min in a dry bath. After electrophoresis, the gels were stained with Coomassie blue dye R-250.
2.6 Enzyme assays 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 µl aliquots of enzyme in a total volume of 1 ml. The oxidation of ABTS was determined at 436 nm (ε=29,300 M-1 cm-1) using a
UV/Vis spectrophotometer (DU 640 Beckman). Enzyme activities were expressed in international units (U). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 µmole of oxidized ABTS per min.
2.7 Enzymatic characterization 2.7.1 Kinetic parameters (apparent Km and Vmax) - effect of the substrate concentration. The kinetics constants were determined using the linear equations of Michaelis-Menten, developers of the Lineweaver-Burk and Langmuir. The parameters were determined using ABTS (λ=436 nm, ɛ= 29300), DMP (λ=468 nm, ɛ= 27500), Syringaldazine (SYR) (λ=530 nm, ɛ= 64000) and Guaiacol (GUA) (λ=470 nm, ɛ= 26600), using concentrations of 0.0025 to 5 mM of each substrate.
2.7.2 Effect of pH on enzymatic activity The effect of pH on enzyme activity was determined for the purified laccase band. The activity was determined using 1 mM of ABTS, DMP, GUA and SYR. Different buffer solutions were prepared 100 mM (acetate buffer pH 3-5 and phosphate buffer pH 6-7).
2.7.3 Effect of temperature on laccase activity In order to determine the laccase temperature profile, the substrate with the most affinity for laccase activity was used. The reactions were monitored spectrophotometrically
(Beckman model DU-650) at different temperatures (25-70 °C). The substrate was incubated for 5 min at the indicated temperature, and the enzyme was then added to start the reaction. The enzyme thermal stability was realized at the optimum pH and temperature for laccase activity, under the best conditions for the selected substrate. The incubation periods varied from 5 to 120 min.
2.8.1 RNA isolation and cDNA synthesis Total RNA was isolated from T. versicolor HEMIM-9 after 10 days of mycelial growth. Fresh samples of mycelium were harvested, washed with a 100 mM phosphate buffer pH 6, frozen with liquid nitrogen and crushed to a fine dust. Total RNA was obtained from 100 mg of the frozen biomass using 1ml of Trizol reagent (Life technologies). First strand cDNA synthesis was carried out using the reverse transcriptase Superscript II (Invitrogen).
2.8.2 PCR amplification of laccase sequences The cDNA obtained was used as the template for the amplification of laccase sequences. Laccase sequences isolated from T. versicolor and deposited in the GeneBank (National Center for Biotechnology Information, NCBI, http://www.ncbi.nlm.nih.gov) were used to design primers for amplifying complete laccase sequences for the freshly synthesized cDNA. PCR was realized using the foward primer (Lf) 5′-atgggtctgcagcgattc-3′ and the reverse primer (Lr) 5′-tcactggttagcctcgctc-3′ to amplify the complete laccase sequences including the signal peptide. DNA amplification was realized using standard PCR procedures 94°C, 55°C and 72°C for 30 cycles. The expected laccase PCR products
were purified using the DNA Gel Recovery Kit (Zymoresearch), cloned into the TOPO 2.1 PCR cloning vector (Invitrogen) and sequenced.
2.8.3 Characterization of derived protein sequences ORF sequences of laccase cDNA obtained from T. veriscolor HEMIM-9 were translated into amino acid sequences using the Expasy (Expert Protien Analysis System) translate tool, a proteomics server of Bioinformatics. Deduced amino acid sequences were subjected to motif analysis and verification of the presence of laccase signatures. Laccase sequences obtained from the GeneBank (NCBI) with high similarities to the recently identified sequences were used for alignment analysis with the CLUSTAL W2 multiple alignment program of the European Molecular Biology Network. Putative N-terminal signal peptides were identified using the Expasy SignalP server (http://www.cbs.dtu.dk/service/signalP). The theoretical isoelectric focusing point (pI) and molecular mass (kDa) were calculated for full-length sequences, with removed signal peptides using the ExPASy proteomics server (http://web.expasy.org/compute_pi/). Putative N-glycosylation sites were identified using the Expasy GlycoMod tool server (http://web.expasy.org/glycomod/).
3 Results and discussion 3.1 Laccase production Laccases are very attractive biomolecules and are considered to be green enzymes due to their high oxidizing power, and the fact that they only require oxygen and produce water as the by-product. As a result of the limitations faced, many strategies have been proposed to
enhance laccase production and higher catalytic activity; these include the use of abundant, cheap and effective natural sources of laccase inducers [9]. Ligninolytic enzyme production by the wood rotting fungi is a phenomenon that combine the interaction between the physiology of fungi and the composition of essential media used for cultivation [12]. Laccase production by ligninolytic fungi has been comprehensively investigated due to the ability of these microorganisms to grow on economic substrates, secretion of enzymes and their admirable capacity to oxidize xenobiotic compounds [1314]. Organic wastes from the agriculture, forest, and food industries contain lignin and cellulose and/or hemi cellulose, that in-turn acts as inducers of ligninolytic activity. Additionally, most of them have a high sugar content, which makes the processes more economic [15]. The use of these types of wastes not only provides an alternative source of substrates, but also aids in solving environmental pollution problems [16-17]. The two main strategies for laccase production are solid-state fermentation (SSF) and submerged or liquid fermentation (SF). Studies of SSF claim higher yields, simpler techniques, reduced energy requirements, low wastewater output and improved product recovery. However, there are several major problems in the development of SSF on an industrial scale, including the mass and heat transfer limitations and difficult solids handling inherent in the process as run in existing reactors and the lack of kinetic and design data on various fermentation processes [18].
3.1.1 Laccase production in lignocellulosic media (oak sawdust and Bran flakes cereal) In order to produce sufficient amounts of laccase, various lignocellulosic substrates were tested using submerged fermentation. On comparing the tendencies of T. versicolor to synthesize laccase with the given media, laccase production in the presence of oak sawdust used as a natural inducer was 0.8 Uml⁻¹ after 2 days, demonstrating the potential of oak sawdust as a feasible source for laccase production. The laccase production of T. versicolor using different growth media is presented in figure 1. An increase of the volumetric activity was observed when T. versicolor was grown in the three lignocellulosic media, compared with control medium that contained only the refined wheat flour. A higher volumetric activity was observed with 0.5 % of oak sawdust (0.8 Uml-1) at 48 h. A lower volumetric activity was observed after 60 h with bran flakes and 0.2 % oak sawdust medium, 0.2 and 0.6 Uml⁻¹, respectively. After studying laccase production with the different growth media, 0.5 % oak media was used for enzyme production, purification and biochemical characterization.
3.2 Laccase purification Two laccase bands were detected by zymography after being revealed with DMP in a 100 mM acetate buffer, pH 5. Laccase bands denominated lcc1 and lcc2 presented apparent molecular masses of 55 and 100-120 kDa, respectively (Figure 2A). In this study, only the heavier band (lcc2) was isolated and characterized. Lcc2 was purified and biochemically characterized because laccase isoforms with approximately 100 kDa and higher have been studied to a lesser extent. The zymograms of laccase activity of each purification step is
presented (Figure 2). Lcc1 and lcc2 were denominated as such for simple differentiation between the heavier and lighter laccase bands as observed in native SDS gels. The purification processes was carried out by using different chromatographic columns in order to isolate the lcc2 band. A summary of purification processes is presented in Table 1. The initial specific activity was 6 Umg⁻¹. The crude extract was concentrated and purified with different chromatography columns. After the purification process the lcc2 specific activity and yield was 208 Umg⁻¹ and 3714 %, respectively. Anionic exchange column chromatography was used to separate lcc1 from lcc2. The first laccase band to be separated was lcc2 obtained using 20 mM Tris buffer solution pH 5, and the second (lcc1) was obtained with an elution gradient of a 0-30 % NaCl solution. Lcc2 was obtained first, because it presumably presented less net negative charge and had less affinity towards the resin. Lcc1 was obtained after, because it probably presented a greater net negative charge, and became more tightly attached to the column. The other columns were used to remove any other contaminating proteins, which may have been present. At the end of the purification process, 4 ml of lcc2 was obtained with its characteristic blue color, caused by the copper atom in its T1 binding site.
3.3 Biochemical characterization of purified lcc2 3.3.1 The effect of pH on lcc2 activity Lcc2 pH profile was determined for 4 different laccase substrates. Several reports by different authors that laccase production and biochemical characteristics may vary
depending of the fermentation strategy (solid-state fermentation or submerged fermentation). However, the pH profile of the lcc2 band purified in this study (under submerged culture conditions) was very similar to the previously purified lcc2, partially purified under solid-state fermentation condition reported by Martínez-Morales et al [19] (Figure 3). The highest enzyme activity registered was 243.86 ± 22.98 Umg⁻¹ at pH 3 for ABTS, with a constant decrease in activity and increasing pH. With respect to SYR, the optimum pH was 5.5 and presented 135 ± 14.38 Umg⁻¹. Lcc2 showed maximum activity for DMP at pH 3 with 123 ± 0.49 Umg⁻¹. GUA also revealed an optimum pH of 3.5 with a specific activity of 35 Umg⁻¹.
3.3.2 Kinetic studies After laccase production and purification, the determination of the kinetic constants Km and Vmax permitted the determination of the affinity of the enzyme for the different substrates. The values of Km and Vmax obtained for the purified lcc2 are shown in Table 2. Although the pH profile of the lcc2 band previously produced by solid-state fermentation, and the lcc2 band produced under submerged culture conditions (present study) were very similar, the kinetic parameter values were very different. The Km values reported for lcc2 produced under submerged culture conditions were much lower than the previously characterized lcc2 produced under solid-state fermentation [19]. The highest affinities recorded for lcc2 were towards ABTS and DMP, 0.01 ± 0.0043 mM and 0.5 ± 0.05 mM, respectively. Although SYR presented higher activity than 2, 6-DMP and GUA after pH profiling, lcc2 showed higher affinity towards these substrates (see figure 3 and table 2). This can be explained by
the fact that, for pH profiles of Km and Kcat in general, the affinity for substrates does not reach its maximum at the optimal pH. Apparently, the pH-induced change at the active site in laccases does not affect the substrate affinity in the same way as it affects the rate. Presumably, the laccases regulate the affinity through the substrate channel and the reaction rate through the T1 site [20]. Baldrian [5] reported a Km values (using ABTS) for T. versicolor and Trametes sp. AH282A of 37 and 25 µM, respectively. The affinity towards DMP by laccase produced by the same species were 15 and 25 µM, respectively. Mun-Jung et al [21] reported a value of 12.8 µM for ABTS. The Km values determined in this study for the other two substrates GUA and DMP were relatively high (2.23 ± 0.53 and 3.77 ± 0.85 mM), compared to 420 µM for GUA as reported by Baldrian [5]. Minussi et al [22] determined the affinity towards SYR for two laccase isoforms (L1 and L2) with Km values of 28.6 and 5 µM, respectively. Still, the Km values reported in this study qualifies lcc2 as attractive for application in any field where laccases are essential.
3.3.3 Temperature profile and thermal stability study of lcc2 Protein thermostability is of both fundamental and industrial importance. Thermostable enzymes allow high process temperatures with associated higher reaction rates and less risk of microbial contamination [23]. ABTS was used as the substrate for temperature profiling of the laccase band examined. Laccase activity was measured in a range of 25-70 °C with the highest activity observed at 50 °C (Figure 4). Thermal stability of lcc2 at its optimum temperature was also analyzed (Figure 5). The enzyme was incubated at 50 °C with varying
incubation periods of 5-120 min. Graphical representation showed that after 20 min lcc2 lost 60 % of its initial activity. A previous report by Christensen and Kepp [23] suggested that the glycosylation effect was consistent with the reduction of thermal motion across all physiological and near-physiological states of T. versicolor laccase TvLα. However, laccase band lcc2 (presenting 30% glycosylation) in this present study was not very stable at 50°C, its optimum temperature for ABTS oxidation. Laccases can exhibit different levels of glycosylation, generally between 10 and 30 % [5]. Glycosylation plays an important role in secretion, proteolytic stability, copper retention capacity and thermal stability [9]. Laccase heterologous expression in yeast species such as S. cerevisiae has been shown to present hyperglycosylation. Hypergycosylation affects expression yield and ultimately affect laccase activity [24]. Among other factors that affect expression levels and misfolded proteins are differences in codon usage, missing chaperones, and other posttranslational modifications [25].
3.3.4 Isoelectric focusing of lcc2 In an attempt to determine the pI value of the purified laccase band, isoelectric focusing of the purified lcc2 band was carried out. Lcc2 was comprised of five individual isoforms with pI values ranging from 3.5-5.8, and not a single isoform, as previously perceived from the zymogram analysis (Figure 6). These findings indicate that lcc2 could in fact be an enzyme complex, presumably hetero dimeric in nature. In general, the mature laccase protein is a holoenzyme, and in its active form could be monomeric, dimeric or tetrameric with four atoms of copper for each monomer [3, 26].
Laccases present molecular masses between 60 and 100 kDa [9]. In this study, lcc2, one of the laccase bands isolated from T. versicolor, had an apparent 100-120 kDa molecular mass (Figure 2). However, after denaturing lcc2 we observed the appearance of protein bands with molecular masses in the range of 55-66 kDa (gel not shown). This phenomenon has been previously observed by our group [19]. These observations indicate that lcc2 could in fact be an enzyme complex, presumably dimeric in nature. Moldes et al [16] and Antoroni et al [27] isolated a laccase bands that were resolved by isoelectric focusing into five distinct isoforms. This phenomena has also been observed in other fungal species [28-29]. To confirm whether the laccase multiplicity observed in the pI gels were not due to differential glycosylation lcc2 was subjected to deglycosylation. The intensity of the laccase bands was slightly affected, however, the number of isoforms in the pI profile was unchanged (supplementary data 1). The presence of several laccase genes in the genome of one fungus is not rare. In fact, the complete genome of T. versicolor ATCC20869 is currently available, and genome analysis reveals the presence of eight laccase genes (http://genome.fungalgenomics.ca). The estimated pI of laccases isoforms detected in this study using isoelectric focusing were consistent with the theoretical pI calculated for the laccases from the genome of T. versicolor ATCC20869. Many fungi produce several laccase isoenzymes endowed with different catalytic properties, however, the physiological significance of this multiplicity is still unknown [30]. The results of the kinetic parameters after biochemical characterization of lcc2 may be due to the collective performance of these isoforms detected by isoelectric focusing. The combined activities of the various laccase isoforms could confer an advantage in terms of extended pH oxidizing range. This is particularly interesting since
various isoforms with different affinities would be working together as a “cocktail of isoforms”. So, we hypothesized that a wide variety of isoforms produced by the same organism, as in the case of T. versicolor, acts as a “diverse kit or toolbox” that confers greater flexibility and/or adaptation to constantly changing environments. Multiple isoforms with different biochemical properties could mean that the organism producing these enzymes has access to a wider range of carbon and nitrogen sources, as possible substrates for growth and development. Thus, creating a more options for survival in terms of nutrition and defense mechanisms, for example. Theoretical data generated from computational studies, indicate that laccase isoform variation may arise from trade-offs between proficiency and stability, since stable proteins may be selected more in variable environments, as the enzymes are often excreted. In fact, several sites near copper or substrate that illustrate such a trade-offs, have been identified. Thus, it is conceivable that, under rough conditions in the environment, the more stable isoforms may be used despite their lower proficiency [31].
3.4 Molecular corroboration 3.4.1 Laccase identification and molecular characterization Five mRNA laccase sequences were isolated, successfully identified and molecularly characterized using bioinformatics (Table 3). All the protein sequences presented the 4 highly conserved regions (L1-L4) of copper binding sites shared among the amino acid residue sequences that conforms the 3 domain holo laccase structure. However, these sequences presented variation in all parameters analyzed, with the exception of the signal peptide.
3.4.2 Molecular characterization There are different methods for studying laccase diversity in basidiomycetes. The phenomenon of laccase isoforms can be studied at the genome level or at the transcription level. The regulation of laccases may be better studied at the genomic level, where laccase sequences contain introns and are regulated by numerous control elements. The study of intron positions and protein structure help to understand the origin and evolution of basidiomycete laccases [32]. On the other hand laccase isoform diversity at the protein level may better be studied at the transcription (mRNA) and translation level; where the final mRNA products are already processed and do not contain introns, for example. In fact, there are over a hundred mRNA sequences reported in the GeneBank (NCBI). Laccase mRNA sequences of actively expressing genes were isolated due to the fact that the purification of individual laccase isoforms produced (and detected by isoelectric focusing) by the fungus was not achieved. Thus, we focused directly on the laccase isoform diversity at the transcription level, and so decided to “fish” the laccase mRNA sequences already processed and ready for translation, for subsequent molecular characterization. Molecular characterization of the five newly identified sequences coding for laccase enzymes suggests that they may be allelic variants, due to the fact that, all of them originates from the same gene (Laccase-2, Trave2p4_000509) in the genome of T. versicolor (http://genome.fungalgenomics.ca/) (Table.3). The sequences characterized in this current study share 95-99% similitude in nucleotide and amino acid sequences. The capacity to isolate numerous, distinct sequences from the same gene suggests that many
more laccase sequences codifying for more laccase alleles may be isolated from the other genes present in the genome of T. versicolor. Diversity of laccase genes can come about by frequent codon changes, (synonym and non-synonym). Synonymous codon change may be reflected in alleles, with up to total difference of 12 % in the codons in a given pair of alleles [1]. Studies on phylogenetic reconstructions indicate that sequence diversity between fungal laccases is moderate and that the isoforms described to date originates from the same common ancestor [33]. Several laccase genes have been isolated and characterized recently, and in some cases, these sequences are part of multigene families, coding for more than one non-allelic variant [34]. At least three main laccase subfamilies (A, B and C) are present in Polyporales and Agaricales [32]. The nomenclature of laccase classification proposed by Necochea et al [33] is α, β, δ and γ. Further analysis revealed that the five newly identified laccase sequences reported in this study would be classified as group α members (supplementary data 2). This information was in accordance to data generated from a phylogenetic analysis of the five alleles isolated in this study, in relationship with 27 other laccase sequences reported for T. versicolor and other basidiomycetes. The phylogenetic tree was generated using the Phylogeny fr. software [35].
3.4.3 Multiple alignment analysis, 3-Dimensional mapping and theoretical sequencestructure-function assumptions Laccases typically bind four copper molecules in two highly conserved copper binding centres. The mononuclear centre T1 with one copper atom (type-1 Cu, blue by a maximum absorbance at around 600 nm) is the primary acceptor of electrons from the reducing substrate. The trinuclear cluster T2/T3 is composed of a binding site T2 for one copper
atom of type-2 with weak absorbance in the visible spectrum and a binding site T3 for two coupled copper atoms of type-3 characterized by an absorbance at around 330 nm. The trinuclear cluster acts in dioxygen binding and reduces the molecular oxygen upon receipt of four electrons forwarded from the mononuclear centre T1 [1]. Ligands for copper binding at the T1 centre are two histidines and one cysteine and ligands at the T2/T3 cluster are eight histidines [36]. These 12 amino acids are spread over 4 highly conserved amino acid regions referred to as laccase signature sequences L1-L4. These regions not only include residues involved in copper binding, but also non-copper ligating residues responsible for conformational functions [27]. To speculate whether alleles KR492185, KR492187, KR492188, KR492189 and KR492186 could present any theoretical difference of importance or interest, we compared the newly isolated sequences to the reported laccase sequence gi 2833233 by multiple alignment analysis (supplementary data 3). Laccase gi 2833233(Q12718 NCBI or 1GYC PDB) shared a 95-99% similarity with the sequences reported in this study. Moreover, laccase 1GYC is the only laccase structure available with its full copper complement. After multiple alignment analysis using CLUSTALW, the differences detected using color representation in the program pyMOL were mapped (Figure 7 A-G). 1GYC was isolated from T. veriscolor and was determined by X ray diffraction at a resolution of 1.9A [36]. Theoretical differences of interest included differences around the copper binding sites, differences (substitutions or deletions) in the amino acid residues like hydrophobic or hydrophilic changes and differences in putative glycosylation sites. One of the most interesting differences was presented in allele KR492189. KR492189 presented 1560 bp or 519 amino acid residues with a theoretical molecular mass of 53.6
kDa and a theoretical pI value of 5.8 (Table 3). KR492189 shared a 98% similarity with the amino acid sequence of laccase 1GYC. Multiple alignment analysis revealed that the HSH highly conserved T3 copper binding site was different for amino acid residue 131 (Supplementary data 3) of the KR492189 isoform (HSR) (Figure 7 B-C). The sites T1, T2, T3 and T4 HWH, HSH, HGH and HCH respectively, are copper union sites highly conserved that form part of the laccase active site. This difference could be attributed to an event in the transcription of the mRNA sequence or a PCR error, since this variation was not detected at the genomic level. In these regions, all the histidine and cysteine residues are critical for coordination of the copper atoms [37]. This change could possibly result in significant differences in terms of redox potential and catalytic capacities resulting from important differences in copper coordination. In a study carried out by Autore et al [38] on the molecular determinants of peculiar properties of a Pleurotus ostreatus laccase by sitedirected mutagenesis, molecular dynamics simulations allowed them to demonstrate that introducing Arg 205 mutation in a highly conserved region perturbs the structural local environment in POXA1bD205R, leading to a large rearrangement of the structure. The same study showed that a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein. In a similar study conducted by Koschorreck et al [24], the structural basis of observed different activities of four laccases was investigated. Amino acid 164 (leucine) and amino acid 265 (phenylalanine) of lac β were replaced by valine and alanine, respectively. Laccase activity was strongly reduced by these single mutations, as well as both mutations together. Although these amino acid residues are not conserved, their positions indicated
that they contributed to the binding and stabilization of bulky hydrophobic aromatic compounds like PAHs in the substrate-binding site. Substrate specificity is usually defined by the geometry and chemical nature of the substrate binding pocket. Such a pocket or crevice provides a suitable environment for the binding of the substrate (s) and their emerging intermediates [36].The replacement of a histidine residue by arginine could result in different spatial conformations in the active site area. The rest of the differences in amino acid residue composition were mainly located on the surface. Laccase allele KR492188 shared a 98% similarity with laccase 1GYC also revealed interesting peculiarities. Its sequence revealed that it was the smallest isoform detected with 1389 pb or 462 amino acids and an estimated theoretical molecular mass of 47.9 kDa and a pI of 6. A total of 58 amino acid residues seems to have been deleted (359-417) (Supplementary data 3), probably as a result of a post transcriptional modification such as differential splicing; and thus the resulting shorter amino acid chain (Figure 7D). This apparent elimination of a number of amino acids did not occur near to or involve the conserved regions in the protein sequence. However, after mapping the differences, the active site region seems to be theoretically very different from that of laccase 1GYC. It seems as if this isoform may present structural differences in the overall structure of the active site´s crevice. Shao et al [30] reported an increase in thermal stability at higher temperatures of a laccase from Shigella dysenteriae after adding a deletion of an α helix domain corresponding to Leu 351 to Gly 378. The structure of this isoform may favor the oxidation of different substrate species when compared than the other isoforms detected, if functionally active. Current efforts are being under taken, to isolate other sequences
originating from the other laccase genes, and, functionally express allelic laccase isoforms using heterologous systems.
4 Conclusions In this study, oak sawdust was used for the production of laccase of T. versicolor. One of the two bands detected by zymography (lcc2) was purified to its characteristic blue color and was characterized. Its kinetic parameters were attractive in terms of its catalytic capacities which makes it a very promising candidate for use in many industrial applications. Our findings demonstrate that lcc2 has potential for application in the treatment of water contaminated with phenols over a wide pH range. Lcc2 was resolved showing a total of 5 laccase individual bands. This suggests the existence of multimeric structures composed of different laccase isoforms that may confer oxidation advantages. This is evident due to the diversity of laccase isoforms apparently working collectively. Multiple alignment analysis, 3-Dimensional mapping and theoretical sequence-structurefunction assumptions suggest that these isoforms would most likely display differences in their biochemical behavior if they were functionally expressed.
5 Acknowledgements BB acknowledges the fellowship of the PROMEP project 103.5/07/2674 and CONACYT (Mexico), CVU 349655.
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Figure 1. Laccase production of T. versicolor in different lignocellulosic liquid media. Control (X), 2% Bran flakes ( ), 0.2 % oak sawdust ( ) and 0.5 % oak sawdust ( ).
Figure 2. A) Separation of the two laccase bands (lcc1 and lcc2) with the anionic column. Lane 1 = the crude laccase extract, lane 2= 1st run, lane 3= 2nd run and lane 4= column elution with 30 % NaCl. B) Purification of lcc2 with different columns. Lane 1= crude laccase extract, lane 2= anionic exchange chromatography, lane 3= hydrophobic interaction chromatography and lane 4= size exclusion chromatography. Both gels were revealed with 1 mM DMP in a 100 mM acetate buffer, pH 5.
Figure 3. Effect of pH on specific Activity for lcc2 with 4 different substrates, SYR (X), ABTS ( ), DMP ( ) and GUA ( ).
Figure 4. Temperature profile of the purified lcc2 with ABTS. Figure 5. Thermal stability of the purified lcc2 with ABTS at the optimum temperature of 50 ° C. Figure 6. Isoelectric focusing analysis of the previously purified lcc2 isoform. A) Zymogram of crude laccase extract produced by T. versicolor in liquid oak medium B) lcc2 after isoelectric focusing, the sample was revealed with 1 mM DMP dissolved in a 100 mM acetate buffer solution. The pI standard marker, Amersham Isoelectric Focusing Calibration Kit Broad pI (pH 3–10) was stained with Coomassie and the lcc2 sample was revealed with 1 mM DMP dissolved in a 100 mM acetate buffer solution.
Figure 7. 3-Dimensional mapping for pinpointing differences in deduced amino acid arrangements after multiple alignment analysis. Laccase gi 2833233(Q12718 NCBI or 1GYC PDB) shared a 95-99% similarity with the sequences reported in this study. Thus, it was used as the model for mapping. (A)- Laccase gi 2833233(Q12718 NCBI or 1GYC PDB). (B and C)- Laccase isoform KR492189, in blue the HSH highly conserved T3 copper binding site was different for amino acid residue 131 of the KR492189 isoform (HSR). (D and E)- Laccase isoform KR492188 the smallest isoform detected where 58 (color magenta) amino acid residues seems to have been deleted (359-417). (F)-Laccase KR492185. (G)-Laccase KR492186.
Figure 1 Volumetric activity (Uml⁻¹)
1.0 0.8 0.6 0.4 0.2 0.0 0
12
24 36 Time (h)
48
Figure 2 A)
Lcc2
Lcc1
B)
60
Figure 3
Specific activity( Umg⁻¹)
300 250 200 150 100 50 0 3
4
5 pH
6
Specific activity (Umg⁻¹)
Figure 4 300 250 200 150 100 50 0 25 30 35 40 45 50 55 60 65 70 Temperature (⁰C)
7
Relative activity (%)
Figure 5 100 90 80 70 60 50 40 30 20 10 0 0
20
40
60 80 Time (min)
100
120
Figure 6
B
A pI 3.5 4.5 lcc2
4.8 5.2 5.8
lcc1
lcc2
Figure 7 B
C
D
G E
G
F
Table 1. Summary of purification processes of laccase lcc2
Purification step
Sample vol (l)
Volumetric activity (U ml-1)
Specific activity (Umg⁻¹)
Yield (%)
PF
Crude extract
4.3
0.8
6
100
-
Anionic C
0.2
0.5
7
130
1
Cationic C
0.1
11
37
658
7
Hydrophobic C
0.006
197
163
2916
520
Molecular ex. C
0.004
700
208
3714
663
Anionic C= Anionic column chromatography, Cationic C= Cationic column chromatography, Hydrophobic C= Hydrophobic column chromatography, Molecular ex. C= Molecular exclusion chromatography. PF= Purification factor.
Table 2. Characteristics of the purified lcc2 laccase isoform produced by T. versicolor HEMIM-9: Kinetic constants and optimum pH for different laccase substrates. Substrate
Optimum pH 3
Km (mM) 0.01 ± 0.0043
Vmax (µmolmin¯¹) 216 ± 11.63
ABTS DMP
3
0.50 ± 0.053
102.77 ± 0.50
GUA
3.5
2.23 ± 0.53
26.03 ± 2.70
3.77 ± 0.85
32.70 ± 4.57
SYN
5.5
Table 3. Molecular characterization of the laccase isoforms isolated from T. versicolor HEMIM-9.
Accession number KR492185
Length (pb) 1560
Length (a.a) 519
Mol.Mass pI (kDa) 53.6 5.8
Signal Putative Peptide glycos. 1-20 14
KR492189
1560
519
53.6
5.8
1-20
14
KR492186
1560
519
53.9
5.9
1-20
14
KR492187
1557
518
53.5
6.1
1-20
12
KR492188
1389
462
47.9
6.0
1-20
13
S1381-1177(15)30088-6 http://dx.doi.org/doi:10.1016/j.molcatb.2015.10.009 MOLCAB 3259
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
13-5-2015 9-10-2015 10-10-2015
Please cite this article as: Brandt Bertrand, Fernando Mart´inez-Morales, Raunel Tinoco-Valencia, Sonia Rojas, Lourdes Acosta-Urdapilleta, Mar´ia R.Trejo-Hern´andez, Biochemical and molecular characterization of laccase isoforms produced by the white-rot fungus Trametes versicolor under submerged culture conditions., Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.10.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biochemical and molecular characterization of laccase isoforms produced by the white-rot fungus Trametes versicolor under submerged culture conditions. Brandt Bertrand1, Fernando Martínez-Morales1, Raunel Tinoco-Valencia2, Sonia Rojas2, Lourdes Acosta-Urdapilleta3, María R. Trejo-Hernández1*
1 Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos. Avenida Universidad 1001, Chamilpa CP 62209 Cuernavaca, Morelos, México 2 Instituto de Biotecnología, Universidad Nacional Autónoma de México. Avenida Universidad 2001, Chamilpa CP 62210 Cuernavaca, Morelos, México 3 Centro de Investigaciones Biológicas, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Chamilpa CP 62209 Cuernavaca, Morelos, México
Corresponding Author: María R. Trejo-Hernández. Laboratorio de Biotecnología
Ambiental. Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Cuernavaca, Morelos, CP 62209. E-mail address: [email protected] Phone +52-777-3297057; Fax +52-777-3297030
Graphical abstract Purification and biochemical characterization of laccase heterodimeric complex
Zymography
Isoelectric Focusing
pI 3.5 4.5 Heterodimeric laccase complex
lcc2
4.8 5.2
Laccase monomers
lcc1
C
B
A
5.8
lcc2
Purification and Characterization
Research highlights:
Laccase band lcc2 (100 kDa) was resolved into 5 distinct isoforms by IEF.
Lcc2 laccase band is a heterodimeric complex.
The heterodimer complex may have had an influence of its catalyzing capacity.
Isoform diversity was validated by the molecular characterization of 5 alleles.
Structure-function assumptions suggest biochemical differences of laccase alleles.
Abstract Many fungi produce several laccase isoenzymes endowed with different catalytic properties, however, the physiological significance of this multiplicity is still unclear. Multiple laccase isoforms produced by the same organism imply a greater flexibility and/or adaptation to constantly changing environments. In this study, we explore and discuss the laccase isoform diversity of Trametes versicolor under submerged culture conditions. Oak sawdust was used as a natural, abundant and cheap source of laccase inducers. Two laccase bands (lcc1 and lcc2) were detected by SDS-PAGE zymography. Lcc2 exhibited an apparent molecular mass of 100 kDa, and was purified to its characteristic blue color for biochemical characterization. Lcc2 displayed high affinities towards all the substrates used in this study (0.01 ± 0.0043, 0.5 ± 0.053, 2.23 ± 0.53 and 3.77 ± 0.85 mM, for ABTS, DMP, GUA and SYR, respectively). Isoelectric focusing analysis of this band revealed the presence of five distinct laccase isoforms. The suggested existence of different laccase heterodimers in lcc2 may have had an influence on its catalyzing capacity. Laccase isoform diversity was corroborated by molecular characterization of five allelic mRNA sequences. Multiple alignment analysis, 3-Dimensional mapping and theoretical sequence-structurefunction assumptions suggests that the newly characterized alleles would most likely display diversity in their biochemical behavior if functionally expressed.
Key words: Biochemical characterization, Laccase isoform diversity, Trametes versicolor, Molecular characterization
1 Introduction Laccases (benzenediol: oxygen oxidoreductase, E.C. 1.10.3.2) are a class of proteins that belong to the multicopper oxidase family [1]. Laccases catalyze the oxidation of a wide range of substances including aromatic compounds by the removal of electrons with the concomitant reduction of molecular oxygen to water, and thus, there is great interest for their application in many biotechnological processes [2, 3]. The industrial applications for laccases include pulp and paper, environmental and food industries [4]. Therefore, numerous studies on biochemical characterization have been carried out to increase laccase production with high redox potential and high stability.
Laccase distribution is widespread and have been isolated from fungi, insects, bacteria and plants, with diverse biological functions including morphogenesis, fungal plantpathogen/host interactions, stress defense, lignin degradation and soil organic matter recycling [5]. Among fungi, the basidiomycetes, e.g., Agaricus bisporus, Pleurotus ostreatus, Trametes versicolor, Phanerochaete chrysosporium and Coprinus cinereus, produce different laccase isoforms that catalyze the oxidation of a broad spectrum of substrates, including toxic xenobiotic compounds [6]. As a result, fungal laccases have been proposed as biocatalysts that can be used in bioconversion processes in an environmentally friendly way [7].
The production of numerous laccase isoforms by a single fungus is not rare and has been demonstrated by the addition of inducers, and these inducers have been proven to affect the expression of laccase genes differentially at the transcriptional level [6, 8]. A wide range of
molecules derived from lignin can be used as laccase inducers. Phenolic acids and metal ions can also serve as specific laccase inducers [9]. Fungal growth phase and nutritional conditions, e.g., the nitrogen or carbon sources, may affect the laccase isoform profile. These inducers and factors affect productivity and the relative amounts of the different laccase isoforms secreted. Laccase profiles and concentrations obtained are very important because the different isoforms possess particular catalytic properties. In this study, a laccase band (lcc2) with an apparent molecular mass of approximately 100120 kDa was purified to its characteristic blue color and biochemically characterized. Lcc2 displayed a similar pH profile to a previously characterized laccase band, however, presented very high affinities towards all substrates used in this work. This band was resolved into five distinct laccase isoforms by isoelectric focusing, with pI values ranging from 3.5-5.8. Given the apparent molecular mass of lcc2, and the theoretical molecular mass of the individual laccase isoforms, we hypothesized that this band may be in fact composed of different heterodimers. The suggested heterodimeric structure may have had an influence on its catalyzing capacity displayed after biochemical characterization. Additionally, the natural laccase isoform diversity was corroborated by isolating, identifying and characterizing five mRNA allelic sequences. Multiple alignment analysis, 3-Dimensional mapping and theoretical sequence-structure-function assumptions suggests that the newly characterized alleles, although very similar in their amino acid sequences, would most likely display diversity in their biochemical behavior, if functionally expressed.
2 Materials and Methods 2.1 Organism, strain preservation and growth conditions 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. T. versicolor HEMIM-9 was maintained in darkness at room temperature on potato dextrose agar (PDA) plates with periodic transfers. An inoculum was obtained from a fully colonized Petri dish (100 x 15 mm) with the actively growing mycelium.
2.2 Lignocellulosic media for fungal cultures Refined white wheat flour medium: White wheat flour (1 %) was dissolved completely in a 100 mM sodium phosphate buffer (pH 6), this medium was used as the control culture medium. The culture media for production of laccase were constituted of lignocellulosic substrates (oak sawdust and Bran flakes cereal). Oak sawdust medium: was prepared by adding solid oak sawdust (0.2 and 0.5 %) to the refined white wheat flour medium. Bran flakes medium: 20 g Bran flakes cereal was boiled at 95 °C for 20 min in 1 l of sodium phosphate buffer solution (pH 6). After cooling down, the mixture was filtered through filter paper (Whatman 1) to remove solids. The filtrate was used as the culture media for fungal growth and laccase production. All media were sterilized at 121 °C for 15 min. Inoculum preparation: T. versicolor HEMIM-9 was grown in a Petri dish for 7 days in darkness at 29 °C. Once fully grown, the fungus was homogenized, and 10 ml was used to
inoculate the respective media. Laccase production was quantified every 24 h until the maximum activity was attained. 2.3 Crude laccase extract preparation Laccase production was measured until the volumetric activity was maximal. The fungal culture was centrifuged at 10 000 x g for 10 min to remove the biomass. The supernatant was filtered using Whatman 1 filter paper and then concentrated by lyophilization. The crude extract was dialyzed by ultrafiltration (Amicon Millipore Ultrafiltration Disc) using a membrane with a 10 kDa cutoff at 10 psi.
2.4 Laccase purification The concentrated sample was loaded into the anion exchange column. The column was packed with a dimethylaminoethyl cellulose resin (DE52 Whatman), coupled with a BioRad pump (Econo Gradient Pump). The column was activated with a 20 mM NaCl solution. The crude concentrated laccase extract was loaded with a 20 mM Tris buffer solution pH 5, and the flow rate adjusted to 3 mlmin⁻¹. The column was eluted with a 0-60 % NaCl gradient. Laccase activity of the fractions collected was measured using 2, 2´azino-bis (3-ethylbenzothiazoline-6 sulphonic acid) (ABTS) and protein concentration was quantified by the Bradford method [10]. Zymograms were used to reveal the progress of the purification process. Subsequently, a cation exchange column packed with a CM sepharose resin (Sigma Fastflow) was used. A hydrophobic interaction column (butyl HIC support from BioRad) was also prepared. The sample to be loaded was precipitated previously with 30 % ammonium sulfate, and was then centrifuged for 20 min at 10 000 x g. A size
exclusion chromatography column was prepared using Sephadex 200 (GE Healthcare Life Sciences), with a particle size of 13 µm and a separation range of 10-600 kDa for globular proteins. The fractions with laccase activity were pooled together and concentrated by ultrafiltration.
2.5 Protein electrophoresis Proteins were electrophoresed in a MINI-PROTEAN 3 electrophoresis cell (Bio-Rad) using a 12 % SDS-PAGE gel as described by Laemmli [11]. The Page Ruler pre-stained protein ladder plus (Fermentas) was used as the molecular mass marker standard. Zymograms were used for visualization of the laccase activity profiles. Samples were ran at 150 V for 1 h; zymograms were washed with a 100 mM sodium acetate buffer solution (pH 5) before revealing to remove SDS. Bands of laccase activity were detected by reaction with 1 mM 2, 6-dimethoxyphenol (DMP) dissolved in the same buffer. For denatured gels, laccase samples were mixed with 5 µl of the loading buffer (containing β-Mercaptoethanol and SDS) and by heating at 96 ºC for 5 min in a dry bath. After electrophoresis, the gels were stained with Coomassie blue dye R-250.
2.6 Enzyme assays 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 µl aliquots of enzyme in a total volume of 1 ml. The oxidation of ABTS was determined at 436 nm (ε=29,300 M-1 cm-1) using a
UV/Vis spectrophotometer (DU 640 Beckman). Enzyme activities were expressed in international units (U). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 µmole of oxidized ABTS per min.
2.7 Enzymatic characterization 2.7.1 Kinetic parameters (apparent Km and Vmax) - effect of the substrate concentration. The kinetics constants were determined using the linear equations of Michaelis-Menten, developers of the Lineweaver-Burk and Langmuir. The parameters were determined using ABTS (λ=436 nm, ɛ= 29300), DMP (λ=468 nm, ɛ= 27500), Syringaldazine (SYR) (λ=530 nm, ɛ= 64000) and Guaiacol (GUA) (λ=470 nm, ɛ= 26600), using concentrations of 0.0025 to 5 mM of each substrate.
2.7.2 Effect of pH on enzymatic activity The effect of pH on enzyme activity was determined for the purified laccase band. The activity was determined using 1 mM of ABTS, DMP, GUA and SYR. Different buffer solutions were prepared 100 mM (acetate buffer pH 3-5 and phosphate buffer pH 6-7).
2.7.3 Effect of temperature on laccase activity In order to determine the laccase temperature profile, the substrate with the most affinity for laccase activity was used. The reactions were monitored spectrophotometrically
(Beckman model DU-650) at different temperatures (25-70 °C). The substrate was incubated for 5 min at the indicated temperature, and the enzyme was then added to start the reaction. The enzyme thermal stability was realized at the optimum pH and temperature for laccase activity, under the best conditions for the selected substrate. The incubation periods varied from 5 to 120 min.
2.8.1 RNA isolation and cDNA synthesis Total RNA was isolated from T. versicolor HEMIM-9 after 10 days of mycelial growth. Fresh samples of mycelium were harvested, washed with a 100 mM phosphate buffer pH 6, frozen with liquid nitrogen and crushed to a fine dust. Total RNA was obtained from 100 mg of the frozen biomass using 1ml of Trizol reagent (Life technologies). First strand cDNA synthesis was carried out using the reverse transcriptase Superscript II (Invitrogen).
2.8.2 PCR amplification of laccase sequences The cDNA obtained was used as the template for the amplification of laccase sequences. Laccase sequences isolated from T. versicolor and deposited in the GeneBank (National Center for Biotechnology Information, NCBI, http://www.ncbi.nlm.nih.gov) were used to design primers for amplifying complete laccase sequences for the freshly synthesized cDNA. PCR was realized using the foward primer (Lf) 5′-atgggtctgcagcgattc-3′ and the reverse primer (Lr) 5′-tcactggttagcctcgctc-3′ to amplify the complete laccase sequences including the signal peptide. DNA amplification was realized using standard PCR procedures 94°C, 55°C and 72°C for 30 cycles. The expected laccase PCR products
were purified using the DNA Gel Recovery Kit (Zymoresearch), cloned into the TOPO 2.1 PCR cloning vector (Invitrogen) and sequenced.
2.8.3 Characterization of derived protein sequences ORF sequences of laccase cDNA obtained from T. veriscolor HEMIM-9 were translated into amino acid sequences using the Expasy (Expert Protien Analysis System) translate tool, a proteomics server of Bioinformatics. Deduced amino acid sequences were subjected to motif analysis and verification of the presence of laccase signatures. Laccase sequences obtained from the GeneBank (NCBI) with high similarities to the recently identified sequences were used for alignment analysis with the CLUSTAL W2 multiple alignment program of the European Molecular Biology Network. Putative N-terminal signal peptides were identified using the Expasy SignalP server (http://www.cbs.dtu.dk/service/signalP). The theoretical isoelectric focusing point (pI) and molecular mass (kDa) were calculated for full-length sequences, with removed signal peptides using the ExPASy proteomics server (http://web.expasy.org/compute_pi/). Putative N-glycosylation sites were identified using the Expasy GlycoMod tool server (http://web.expasy.org/glycomod/).
3 Results and discussion 3.1 Laccase production Laccases are very attractive biomolecules and are considered to be green enzymes due to their high oxidizing power, and the fact that they only require oxygen and produce water as the by-product. As a result of the limitations faced, many strategies have been proposed to
enhance laccase production and higher catalytic activity; these include the use of abundant, cheap and effective natural sources of laccase inducers [9]. Ligninolytic enzyme production by the wood rotting fungi is a phenomenon that combine the interaction between the physiology of fungi and the composition of essential media used for cultivation [12]. Laccase production by ligninolytic fungi has been comprehensively investigated due to the ability of these microorganisms to grow on economic substrates, secretion of enzymes and their admirable capacity to oxidize xenobiotic compounds [1314]. Organic wastes from the agriculture, forest, and food industries contain lignin and cellulose and/or hemi cellulose, that in-turn acts as inducers of ligninolytic activity. Additionally, most of them have a high sugar content, which makes the processes more economic [15]. The use of these types of wastes not only provides an alternative source of substrates, but also aids in solving environmental pollution problems [16-17]. The two main strategies for laccase production are solid-state fermentation (SSF) and submerged or liquid fermentation (SF). Studies of SSF claim higher yields, simpler techniques, reduced energy requirements, low wastewater output and improved product recovery. However, there are several major problems in the development of SSF on an industrial scale, including the mass and heat transfer limitations and difficult solids handling inherent in the process as run in existing reactors and the lack of kinetic and design data on various fermentation processes [18].
3.1.1 Laccase production in lignocellulosic media (oak sawdust and Bran flakes cereal) In order to produce sufficient amounts of laccase, various lignocellulosic substrates were tested using submerged fermentation. On comparing the tendencies of T. versicolor to synthesize laccase with the given media, laccase production in the presence of oak sawdust used as a natural inducer was 0.8 Uml⁻¹ after 2 days, demonstrating the potential of oak sawdust as a feasible source for laccase production. The laccase production of T. versicolor using different growth media is presented in figure 1. An increase of the volumetric activity was observed when T. versicolor was grown in the three lignocellulosic media, compared with control medium that contained only the refined wheat flour. A higher volumetric activity was observed with 0.5 % of oak sawdust (0.8 Uml-1) at 48 h. A lower volumetric activity was observed after 60 h with bran flakes and 0.2 % oak sawdust medium, 0.2 and 0.6 Uml⁻¹, respectively. After studying laccase production with the different growth media, 0.5 % oak media was used for enzyme production, purification and biochemical characterization.
3.2 Laccase purification Two laccase bands were detected by zymography after being revealed with DMP in a 100 mM acetate buffer, pH 5. Laccase bands denominated lcc1 and lcc2 presented apparent molecular masses of 55 and 100-120 kDa, respectively (Figure 2A). In this study, only the heavier band (lcc2) was isolated and characterized. Lcc2 was purified and biochemically characterized because laccase isoforms with approximately 100 kDa and higher have been studied to a lesser extent. The zymograms of laccase activity of each purification step is
presented (Figure 2). Lcc1 and lcc2 were denominated as such for simple differentiation between the heavier and lighter laccase bands as observed in native SDS gels. The purification processes was carried out by using different chromatographic columns in order to isolate the lcc2 band. A summary of purification processes is presented in Table 1. The initial specific activity was 6 Umg⁻¹. The crude extract was concentrated and purified with different chromatography columns. After the purification process the lcc2 specific activity and yield was 208 Umg⁻¹ and 3714 %, respectively. Anionic exchange column chromatography was used to separate lcc1 from lcc2. The first laccase band to be separated was lcc2 obtained using 20 mM Tris buffer solution pH 5, and the second (lcc1) was obtained with an elution gradient of a 0-30 % NaCl solution. Lcc2 was obtained first, because it presumably presented less net negative charge and had less affinity towards the resin. Lcc1 was obtained after, because it probably presented a greater net negative charge, and became more tightly attached to the column. The other columns were used to remove any other contaminating proteins, which may have been present. At the end of the purification process, 4 ml of lcc2 was obtained with its characteristic blue color, caused by the copper atom in its T1 binding site.
3.3 Biochemical characterization of purified lcc2 3.3.1 The effect of pH on lcc2 activity Lcc2 pH profile was determined for 4 different laccase substrates. Several reports by different authors that laccase production and biochemical characteristics may vary
depending of the fermentation strategy (solid-state fermentation or submerged fermentation). However, the pH profile of the lcc2 band purified in this study (under submerged culture conditions) was very similar to the previously purified lcc2, partially purified under solid-state fermentation condition reported by Martínez-Morales et al [19] (Figure 3). The highest enzyme activity registered was 243.86 ± 22.98 Umg⁻¹ at pH 3 for ABTS, with a constant decrease in activity and increasing pH. With respect to SYR, the optimum pH was 5.5 and presented 135 ± 14.38 Umg⁻¹. Lcc2 showed maximum activity for DMP at pH 3 with 123 ± 0.49 Umg⁻¹. GUA also revealed an optimum pH of 3.5 with a specific activity of 35 Umg⁻¹.
3.3.2 Kinetic studies After laccase production and purification, the determination of the kinetic constants Km and Vmax permitted the determination of the affinity of the enzyme for the different substrates. The values of Km and Vmax obtained for the purified lcc2 are shown in Table 2. Although the pH profile of the lcc2 band previously produced by solid-state fermentation, and the lcc2 band produced under submerged culture conditions (present study) were very similar, the kinetic parameter values were very different. The Km values reported for lcc2 produced under submerged culture conditions were much lower than the previously characterized lcc2 produced under solid-state fermentation [19]. The highest affinities recorded for lcc2 were towards ABTS and DMP, 0.01 ± 0.0043 mM and 0.5 ± 0.05 mM, respectively. Although SYR presented higher activity than 2, 6-DMP and GUA after pH profiling, lcc2 showed higher affinity towards these substrates (see figure 3 and table 2). This can be explained by
the fact that, for pH profiles of Km and Kcat in general, the affinity for substrates does not reach its maximum at the optimal pH. Apparently, the pH-induced change at the active site in laccases does not affect the substrate affinity in the same way as it affects the rate. Presumably, the laccases regulate the affinity through the substrate channel and the reaction rate through the T1 site [20]. Baldrian [5] reported a Km values (using ABTS) for T. versicolor and Trametes sp. AH282A of 37 and 25 µM, respectively. The affinity towards DMP by laccase produced by the same species were 15 and 25 µM, respectively. Mun-Jung et al [21] reported a value of 12.8 µM for ABTS. The Km values determined in this study for the other two substrates GUA and DMP were relatively high (2.23 ± 0.53 and 3.77 ± 0.85 mM), compared to 420 µM for GUA as reported by Baldrian [5]. Minussi et al [22] determined the affinity towards SYR for two laccase isoforms (L1 and L2) with Km values of 28.6 and 5 µM, respectively. Still, the Km values reported in this study qualifies lcc2 as attractive for application in any field where laccases are essential.
3.3.3 Temperature profile and thermal stability study of lcc2 Protein thermostability is of both fundamental and industrial importance. Thermostable enzymes allow high process temperatures with associated higher reaction rates and less risk of microbial contamination [23]. ABTS was used as the substrate for temperature profiling of the laccase band examined. Laccase activity was measured in a range of 25-70 °C with the highest activity observed at 50 °C (Figure 4). Thermal stability of lcc2 at its optimum temperature was also analyzed (Figure 5). The enzyme was incubated at 50 °C with varying
incubation periods of 5-120 min. Graphical representation showed that after 20 min lcc2 lost 60 % of its initial activity. A previous report by Christensen and Kepp [23] suggested that the glycosylation effect was consistent with the reduction of thermal motion across all physiological and near-physiological states of T. versicolor laccase TvLα. However, laccase band lcc2 (presenting 30% glycosylation) in this present study was not very stable at 50°C, its optimum temperature for ABTS oxidation. Laccases can exhibit different levels of glycosylation, generally between 10 and 30 % [5]. Glycosylation plays an important role in secretion, proteolytic stability, copper retention capacity and thermal stability [9]. Laccase heterologous expression in yeast species such as S. cerevisiae has been shown to present hyperglycosylation. Hypergycosylation affects expression yield and ultimately affect laccase activity [24]. Among other factors that affect expression levels and misfolded proteins are differences in codon usage, missing chaperones, and other posttranslational modifications [25].
3.3.4 Isoelectric focusing of lcc2 In an attempt to determine the pI value of the purified laccase band, isoelectric focusing of the purified lcc2 band was carried out. Lcc2 was comprised of five individual isoforms with pI values ranging from 3.5-5.8, and not a single isoform, as previously perceived from the zymogram analysis (Figure 6). These findings indicate that lcc2 could in fact be an enzyme complex, presumably hetero dimeric in nature. In general, the mature laccase protein is a holoenzyme, and in its active form could be monomeric, dimeric or tetrameric with four atoms of copper for each monomer [3, 26].
Laccases present molecular masses between 60 and 100 kDa [9]. In this study, lcc2, one of the laccase bands isolated from T. versicolor, had an apparent 100-120 kDa molecular mass (Figure 2). However, after denaturing lcc2 we observed the appearance of protein bands with molecular masses in the range of 55-66 kDa (gel not shown). This phenomenon has been previously observed by our group [19]. These observations indicate that lcc2 could in fact be an enzyme complex, presumably dimeric in nature. Moldes et al [16] and Antoroni et al [27] isolated a laccase bands that were resolved by isoelectric focusing into five distinct isoforms. This phenomena has also been observed in other fungal species [28-29]. To confirm whether the laccase multiplicity observed in the pI gels were not due to differential glycosylation lcc2 was subjected to deglycosylation. The intensity of the laccase bands was slightly affected, however, the number of isoforms in the pI profile was unchanged (supplementary data 1). The presence of several laccase genes in the genome of one fungus is not rare. In fact, the complete genome of T. versicolor ATCC20869 is currently available, and genome analysis reveals the presence of eight laccase genes (http://genome.fungalgenomics.ca). The estimated pI of laccases isoforms detected in this study using isoelectric focusing were consistent with the theoretical pI calculated for the laccases from the genome of T. versicolor ATCC20869. Many fungi produce several laccase isoenzymes endowed with different catalytic properties, however, the physiological significance of this multiplicity is still unknown [30]. The results of the kinetic parameters after biochemical characterization of lcc2 may be due to the collective performance of these isoforms detected by isoelectric focusing. The combined activities of the various laccase isoforms could confer an advantage in terms of extended pH oxidizing range. This is particularly interesting since
various isoforms with different affinities would be working together as a “cocktail of isoforms”. So, we hypothesized that a wide variety of isoforms produced by the same organism, as in the case of T. versicolor, acts as a “diverse kit or toolbox” that confers greater flexibility and/or adaptation to constantly changing environments. Multiple isoforms with different biochemical properties could mean that the organism producing these enzymes has access to a wider range of carbon and nitrogen sources, as possible substrates for growth and development. Thus, creating a more options for survival in terms of nutrition and defense mechanisms, for example. Theoretical data generated from computational studies, indicate that laccase isoform variation may arise from trade-offs between proficiency and stability, since stable proteins may be selected more in variable environments, as the enzymes are often excreted. In fact, several sites near copper or substrate that illustrate such a trade-offs, have been identified. Thus, it is conceivable that, under rough conditions in the environment, the more stable isoforms may be used despite their lower proficiency [31].
3.4 Molecular corroboration 3.4.1 Laccase identification and molecular characterization Five mRNA laccase sequences were isolated, successfully identified and molecularly characterized using bioinformatics (Table 3). All the protein sequences presented the 4 highly conserved regions (L1-L4) of copper binding sites shared among the amino acid residue sequences that conforms the 3 domain holo laccase structure. However, these sequences presented variation in all parameters analyzed, with the exception of the signal peptide.
3.4.2 Molecular characterization There are different methods for studying laccase diversity in basidiomycetes. The phenomenon of laccase isoforms can be studied at the genome level or at the transcription level. The regulation of laccases may be better studied at the genomic level, where laccase sequences contain introns and are regulated by numerous control elements. The study of intron positions and protein structure help to understand the origin and evolution of basidiomycete laccases [32]. On the other hand laccase isoform diversity at the protein level may better be studied at the transcription (mRNA) and translation level; where the final mRNA products are already processed and do not contain introns, for example. In fact, there are over a hundred mRNA sequences reported in the GeneBank (NCBI). Laccase mRNA sequences of actively expressing genes were isolated due to the fact that the purification of individual laccase isoforms produced (and detected by isoelectric focusing) by the fungus was not achieved. Thus, we focused directly on the laccase isoform diversity at the transcription level, and so decided to “fish” the laccase mRNA sequences already processed and ready for translation, for subsequent molecular characterization. Molecular characterization of the five newly identified sequences coding for laccase enzymes suggests that they may be allelic variants, due to the fact that, all of them originates from the same gene (Laccase-2, Trave2p4_000509) in the genome of T. versicolor (http://genome.fungalgenomics.ca/) (Table.3). The sequences characterized in this current study share 95-99% similitude in nucleotide and amino acid sequences. The capacity to isolate numerous, distinct sequences from the same gene suggests that many
more laccase sequences codifying for more laccase alleles may be isolated from the other genes present in the genome of T. versicolor. Diversity of laccase genes can come about by frequent codon changes, (synonym and non-synonym). Synonymous codon change may be reflected in alleles, with up to total difference of 12 % in the codons in a given pair of alleles [1]. Studies on phylogenetic reconstructions indicate that sequence diversity between fungal laccases is moderate and that the isoforms described to date originates from the same common ancestor [33]. Several laccase genes have been isolated and characterized recently, and in some cases, these sequences are part of multigene families, coding for more than one non-allelic variant [34]. At least three main laccase subfamilies (A, B and C) are present in Polyporales and Agaricales [32]. The nomenclature of laccase classification proposed by Necochea et al [33] is α, β, δ and γ. Further analysis revealed that the five newly identified laccase sequences reported in this study would be classified as group α members (supplementary data 2). This information was in accordance to data generated from a phylogenetic analysis of the five alleles isolated in this study, in relationship with 27 other laccase sequences reported for T. versicolor and other basidiomycetes. The phylogenetic tree was generated using the Phylogeny fr. software [35].
3.4.3 Multiple alignment analysis, 3-Dimensional mapping and theoretical sequencestructure-function assumptions Laccases typically bind four copper molecules in two highly conserved copper binding centres. The mononuclear centre T1 with one copper atom (type-1 Cu, blue by a maximum absorbance at around 600 nm) is the primary acceptor of electrons from the reducing substrate. The trinuclear cluster T2/T3 is composed of a binding site T2 for one copper
atom of type-2 with weak absorbance in the visible spectrum and a binding site T3 for two coupled copper atoms of type-3 characterized by an absorbance at around 330 nm. The trinuclear cluster acts in dioxygen binding and reduces the molecular oxygen upon receipt of four electrons forwarded from the mononuclear centre T1 [1]. Ligands for copper binding at the T1 centre are two histidines and one cysteine and ligands at the T2/T3 cluster are eight histidines [36]. These 12 amino acids are spread over 4 highly conserved amino acid regions referred to as laccase signature sequences L1-L4. These regions not only include residues involved in copper binding, but also non-copper ligating residues responsible for conformational functions [27]. To speculate whether alleles KR492185, KR492187, KR492188, KR492189 and KR492186 could present any theoretical difference of importance or interest, we compared the newly isolated sequences to the reported laccase sequence gi 2833233 by multiple alignment analysis (supplementary data 3). Laccase gi 2833233(Q12718 NCBI or 1GYC PDB) shared a 95-99% similarity with the sequences reported in this study. Moreover, laccase 1GYC is the only laccase structure available with its full copper complement. After multiple alignment analysis using CLUSTALW, the differences detected using color representation in the program pyMOL were mapped (Figure 7 A-G). 1GYC was isolated from T. veriscolor and was determined by X ray diffraction at a resolution of 1.9A [36]. Theoretical differences of interest included differences around the copper binding sites, differences (substitutions or deletions) in the amino acid residues like hydrophobic or hydrophilic changes and differences in putative glycosylation sites. One of the most interesting differences was presented in allele KR492189. KR492189 presented 1560 bp or 519 amino acid residues with a theoretical molecular mass of 53.6
kDa and a theoretical pI value of 5.8 (Table 3). KR492189 shared a 98% similarity with the amino acid sequence of laccase 1GYC. Multiple alignment analysis revealed that the HSH highly conserved T3 copper binding site was different for amino acid residue 131 (Supplementary data 3) of the KR492189 isoform (HSR) (Figure 7 B-C). The sites T1, T2, T3 and T4 HWH, HSH, HGH and HCH respectively, are copper union sites highly conserved that form part of the laccase active site. This difference could be attributed to an event in the transcription of the mRNA sequence or a PCR error, since this variation was not detected at the genomic level. In these regions, all the histidine and cysteine residues are critical for coordination of the copper atoms [37]. This change could possibly result in significant differences in terms of redox potential and catalytic capacities resulting from important differences in copper coordination. In a study carried out by Autore et al [38] on the molecular determinants of peculiar properties of a Pleurotus ostreatus laccase by sitedirected mutagenesis, molecular dynamics simulations allowed them to demonstrate that introducing Arg 205 mutation in a highly conserved region perturbs the structural local environment in POXA1bD205R, leading to a large rearrangement of the structure. The same study showed that a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein. In a similar study conducted by Koschorreck et al [24], the structural basis of observed different activities of four laccases was investigated. Amino acid 164 (leucine) and amino acid 265 (phenylalanine) of lac β were replaced by valine and alanine, respectively. Laccase activity was strongly reduced by these single mutations, as well as both mutations together. Although these amino acid residues are not conserved, their positions indicated
that they contributed to the binding and stabilization of bulky hydrophobic aromatic compounds like PAHs in the substrate-binding site. Substrate specificity is usually defined by the geometry and chemical nature of the substrate binding pocket. Such a pocket or crevice provides a suitable environment for the binding of the substrate (s) and their emerging intermediates [36].The replacement of a histidine residue by arginine could result in different spatial conformations in the active site area. The rest of the differences in amino acid residue composition were mainly located on the surface. Laccase allele KR492188 shared a 98% similarity with laccase 1GYC also revealed interesting peculiarities. Its sequence revealed that it was the smallest isoform detected with 1389 pb or 462 amino acids and an estimated theoretical molecular mass of 47.9 kDa and a pI of 6. A total of 58 amino acid residues seems to have been deleted (359-417) (Supplementary data 3), probably as a result of a post transcriptional modification such as differential splicing; and thus the resulting shorter amino acid chain (Figure 7D). This apparent elimination of a number of amino acids did not occur near to or involve the conserved regions in the protein sequence. However, after mapping the differences, the active site region seems to be theoretically very different from that of laccase 1GYC. It seems as if this isoform may present structural differences in the overall structure of the active site´s crevice. Shao et al [30] reported an increase in thermal stability at higher temperatures of a laccase from Shigella dysenteriae after adding a deletion of an α helix domain corresponding to Leu 351 to Gly 378. The structure of this isoform may favor the oxidation of different substrate species when compared than the other isoforms detected, if functionally active. Current efforts are being under taken, to isolate other sequences
originating from the other laccase genes, and, functionally express allelic laccase isoforms using heterologous systems.
4 Conclusions In this study, oak sawdust was used for the production of laccase of T. versicolor. One of the two bands detected by zymography (lcc2) was purified to its characteristic blue color and was characterized. Its kinetic parameters were attractive in terms of its catalytic capacities which makes it a very promising candidate for use in many industrial applications. Our findings demonstrate that lcc2 has potential for application in the treatment of water contaminated with phenols over a wide pH range. Lcc2 was resolved showing a total of 5 laccase individual bands. This suggests the existence of multimeric structures composed of different laccase isoforms that may confer oxidation advantages. This is evident due to the diversity of laccase isoforms apparently working collectively. Multiple alignment analysis, 3-Dimensional mapping and theoretical sequence-structurefunction assumptions suggest that these isoforms would most likely display differences in their biochemical behavior if they were functionally expressed.
5 Acknowledgements BB acknowledges the fellowship of the PROMEP project 103.5/07/2674 and CONACYT (Mexico), CVU 349655.
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[36] K. Piontek, M. Antorini, T. Choinowski, J. Biol. Chem. 277 (2002), 3766337669. [37] D.S. Yaver, F. Xu, E.J. Golightly, K.M. Brown, S.H. Brown, M.W. Rey, P. Schneider, T. Halkier, K. Mondorf, H. Dalbøge, Appl. Environ. Microbiol. 62 (1996) 834-841. [38] F. Autore, C. Del Vecchio, F. Fraternali, P. Giardina, G. Sannia, V. Faraco, Enzyme Microb. Technol. 45 (2009) 507-513.
Figure 1. Laccase production of T. versicolor in different lignocellulosic liquid media. Control (X), 2% Bran flakes ( ), 0.2 % oak sawdust ( ) and 0.5 % oak sawdust ( ).
Figure 2. A) Separation of the two laccase bands (lcc1 and lcc2) with the anionic column. Lane 1 = the crude laccase extract, lane 2= 1st run, lane 3= 2nd run and lane 4= column elution with 30 % NaCl. B) Purification of lcc2 with different columns. Lane 1= crude laccase extract, lane 2= anionic exchange chromatography, lane 3= hydrophobic interaction chromatography and lane 4= size exclusion chromatography. Both gels were revealed with 1 mM DMP in a 100 mM acetate buffer, pH 5.
Figure 3. Effect of pH on specific Activity for lcc2 with 4 different substrates, SYR (X), ABTS ( ), DMP ( ) and GUA ( ).
Figure 4. Temperature profile of the purified lcc2 with ABTS. Figure 5. Thermal stability of the purified lcc2 with ABTS at the optimum temperature of 50 ° C. Figure 6. Isoelectric focusing analysis of the previously purified lcc2 isoform. A) Zymogram of crude laccase extract produced by T. versicolor in liquid oak medium B) lcc2 after isoelectric focusing, the sample was revealed with 1 mM DMP dissolved in a 100 mM acetate buffer solution. The pI standard marker, Amersham Isoelectric Focusing Calibration Kit Broad pI (pH 3–10) was stained with Coomassie and the lcc2 sample was revealed with 1 mM DMP dissolved in a 100 mM acetate buffer solution.
Figure 7. 3-Dimensional mapping for pinpointing differences in deduced amino acid arrangements after multiple alignment analysis. Laccase gi 2833233(Q12718 NCBI or 1GYC PDB) shared a 95-99% similarity with the sequences reported in this study. Thus, it was used as the model for mapping. (A)- Laccase gi 2833233(Q12718 NCBI or 1GYC PDB). (B and C)- Laccase isoform KR492189, in blue the HSH highly conserved T3 copper binding site was different for amino acid residue 131 of the KR492189 isoform (HSR). (D and E)- Laccase isoform KR492188 the smallest isoform detected where 58 (color magenta) amino acid residues seems to have been deleted (359-417). (F)-Laccase KR492185. (G)-Laccase KR492186.
Figure 1 Volumetric activity (Uml⁻¹)
1.0 0.8 0.6 0.4 0.2 0.0 0
12
24 36 Time (h)
48
Figure 2 A)
Lcc2
Lcc1
B)
60
Figure 3
Specific activity( Umg⁻¹)
300 250 200 150 100 50 0 3
4
5 pH
6
Specific activity (Umg⁻¹)
Figure 4 300 250 200 150 100 50 0 25 30 35 40 45 50 55 60 65 70 Temperature (⁰C)
7
Relative activity (%)
Figure 5 100 90 80 70 60 50 40 30 20 10 0 0
20
40
60 80 Time (min)
100
120
Figure 6
B
A pI 3.5 4.5 lcc2
4.8 5.2 5.8
lcc1
lcc2
Figure 7 B
C
D
G E
G
F
Table 1. Summary of purification processes of laccase lcc2
Purification step
Sample vol (l)
Volumetric activity (U ml-1)
Specific activity (Umg⁻¹)
Yield (%)
PF
Crude extract
4.3
0.8
6
100
-
Anionic C
0.2
0.5
7
130
1
Cationic C
0.1
11
37
658
7
Hydrophobic C
0.006
197
163
2916
520
Molecular ex. C
0.004
700
208
3714
663
Anionic C= Anionic column chromatography, Cationic C= Cationic column chromatography, Hydrophobic C= Hydrophobic column chromatography, Molecular ex. C= Molecular exclusion chromatography. PF= Purification factor.
Table 2. Characteristics of the purified lcc2 laccase isoform produced by T. versicolor HEMIM-9: Kinetic constants and optimum pH for different laccase substrates. Substrate
Optimum pH 3
Km (mM) 0.01 ± 0.0043
Vmax (µmolmin¯¹) 216 ± 11.63
ABTS DMP
3
0.50 ± 0.053
102.77 ± 0.50
GUA
3.5
2.23 ± 0.53
26.03 ± 2.70
3.77 ± 0.85
32.70 ± 4.57
SYN
5.5
Table 3. Molecular characterization of the laccase isoforms isolated from T. versicolor HEMIM-9.
Accession number KR492185
Length (pb) 1560
Length (a.a) 519
Mol.Mass pI (kDa) 53.6 5.8
Signal Putative Peptide glycos. 1-20 14
KR492189
1560
519
53.6
5.8
1-20
14
KR492186
1560
519
53.9
5.9
1-20
14
KR492187
1557
518
53.5
6.1
1-20
12
KR492188
1389
462
47.9
6.0
1-20
13