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Comparison of different microbial laccases as tools for industrial uses
Accepted Manuscript Title: Comparison of different microbial laccases as tools for industrial uses Author: Fabio Tonin Roberta Melis Arno Cordes Antonio Sanchez-Amat Loredano Pollegioni Elena Rosini PII: DOI: Reference:
S1871-6784(16)00010-8 http://dx.doi.org/doi:10.1016/j.nbt.2016.01.007 NBT 854
To appear in: Received date: Revised date: Accepted date:
28-9-2015 18-12-2015 22-1-2016
Please cite this article as: Tonin, F., Melis, R., Cordes, A., Sanchez-Amat, A., Pollegioni, L., Rosini, E.,Comparison of different microbial laccases as tools for industrial uses, New Biotechnology (2016), http://dx.doi.org/10.1016/j.nbt.2016.01.007 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.
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Highlights Laccases are used as lignin-degrading enzymes
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Biochemical properties of ten laccases under identical conditions were investigated
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Bacterial laccases retained high activity in DMSO and Tween-80
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Properties of recombinant B. licheniformis laccase satisfy requirements for industrial uses
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Comparison of different microbial laccases as tools for industrial uses
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Fabio Tonina, Roberta Melisa, Arno Cordesb, Antonio Sanchez-Amatc, Loredano Pollegionia,d and
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Elena Rosinia,d
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a
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Dunant 3, 21100 Varese, Italy
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b
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Università degli Studi dell’Insubria, Milano, Italy
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Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell’Insubria, via J.H.
ASA Spezialenzyme GmbH, Am Exer 19c, D-38302 Wolfenbüttel, Germany
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Department of Genetics and Microbiology, University of Murcia, Murcia, Spain
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Correspondence: Loredano Pollegioni, Dipartimento di Biotecnologie e Scienze della Vita, Università
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degli studi dell’Insubria, via J.H. Dunant 3, 21100 Varese, Italy, Fax: +39 0332421500; Tel.: +39
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0332421506; E-mail address: [email protected]
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The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and
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Abbreviations: ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); 2,6-DMP, 2,6-
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dimethoxyphenol; DMSO, dimethyl sulfoxide; BALL, laccase from Bacillus licheniformis; OB1,
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laccase variant from basidiomycete PM1; MmPPO, polyphenol oxidase from Marinomonas
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mediterranea MMB-1
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Abstract
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Laccases from different sources are employed in a number of biotechnological processes, each
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characterized by specific reaction constraints and thus requiring an enzyme with suitable properties. In
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order to avoid the bias generated by different assay methodologies, in this work we investigated the
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main properties of ten (commercial and recombinant) laccases from fungi and bacteria under identical
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conditions. As a general rule, the optimal activity was apparent at pH 3-4 and was lost at pH ≥ 7.0 (all
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laccases were stable at pH ≥ 7.0); enzymes active at neutral pH values were also identified. For all
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tested laccases, activity increased with temperature up to 80 °C and stability was good at 25 °C.
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Interestingly, laccases insensitive to high salt concentration were identified, this favoring their use in
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treating waste waters. Indeed, bacterial laccases retained a significant activity in the presence of DMSO
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(up to 40% final concentration) and of surfactants, suggesting that they can be applied in lignin
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degradation processes requiring solvents. The available laccases are versatile and satisfy requirements
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related to different processes. Notably, the recombinant laccase from Bacillus licheniformis favorably
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compares with the tested enzymes, indicating that it is well suited for different biotechnological
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applications.
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Keywords: Ligninolytic enzymes; Lignin valorization; Enzymatic depolymerization; Green
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biotechnology
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Introduction
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Laccases (benzenediol:oxygen oxidoreductases; EC 1.10.3.2), sometimes also referred to as polyphenol
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oxidases, belong to the group of blue multi-copper oxidases along with ceruloplasmin, ascorbate
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oxidase, bilirubin oxidase, and various manganese oxidases. Laccase is attracting great scientific
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interest because of its very basic requirements - it only uses oxygen from the air and releases water as
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the sole by-product - and great catalytic capabilities, rendering it one of the ‘‘greenest’’ enzymes [1-3].
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Laccase couples the four-electron reduction of dioxygen to water with the oxidation of a broad range of
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substrates (including phenols, polyphenols, arylamines, anilines, hydroxyindols, and thiols). Its
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substrate promiscuity can be even further expanded by using redox mediators, i.e., diffusible electron
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carriers from natural or synthetic sources [4-6].
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Laccases are widely distributed among fungi, higher plants and bacteria. Fungal laccases contain four
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Cu ions, organized into two copper clusters: one situated in a mononuclear T1 site close to the protein
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surface and three others buried at a trinuclear site, consisting of a mononuclear T2 site and a binuclear
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T3 site [7,8]. In the resting form of the enzyme, the four copper ions are in the +2 oxidation state [7, 9].
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The catalytic cycle begins by sequestering one electron at a time from the reducing substrate and
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transferring it to the trinuclear T2/T3 copper cluster through a highly conserved His-Cys-His pathway,
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where four electrons reduce dioxygen to two molecules of water.
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Laccases are commonly classified into two main groups: “low-medium” and “high” redox potential
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laccases according to their redox potentials at the T1 site, ranging from +430 mV in bacterial and plant
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laccases to +790 mV in some fungal laccases: the latter are typically secreted by ligninolytic
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basidiomycetes and are the most important for biotechnological applications [1,10].
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The potential uses of laccases are numerous due to their low catalytic requirements and oxidative
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versatility. In fact, these enzymes catalyze a wide spectrum of reactions, which include cross-linking of
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monomers, degradation of polymers, ring cleavage, and oxyfunctionalization of aromatic compounds
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[4,5,8]. Laccases are used in several industrial sectors, including the textile (for bleaching) and dye
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industry, for effluent treatment and bioremediation, in the food industry (for beverage processing and
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baking), and in forest-product industry, where lignin removal is the main goal (paper manufacture) or
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where lignin polymerization is the main objective (e.g., manufacture of fiberboards and synthesis of
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novel materials) [6]. Furthermore, fungal laccases have been recently employed to improve the
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conversion of plant biomass in integrated lignocellulose biorefineries, in organic synthesis (i.e., for the
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enzymatic conversion of chemical intermediates and the synthesis of pigments and antioxidants
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through dimerization of phenolic and nonphenolic acids), in oxidative transformation of environmental
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pollutants (e.g., herbicides), and in bioelectrocatalysis for detecting phenolic pollutants [1,11,12].
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Moreover, in the presence of redox mediators, laccases can transform compounds with higher redox
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potentials as well as complex polymers such as lignin, avoiding limits due to steric hindrance [13,14].
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In the past several years, a number of laccases from different sources [15] were isolated and
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characterized. These enzymes have been assayed on different compounds using different experimental
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conditions, however, making it difficult to compare their characteristics: this limit is further hampered
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by the fact that various isoenzymes, which differ in biochemical properties, are produced by the same
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strain. A major challenge of laccase use is that the catalytic activity and stability are highly dependent
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on experimental variables such as pH, temperature, ionic strength, and co-solvents. In this study we
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evaluated the main biochemical properties of a number of commercial and recombinant laccases under
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identical experimental conditions, with the final goal to compare their features in order to identify the
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best biocatalyst for specific applications.
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Materials and methods
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Reagents
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The laccases from Rhus vernicifera, Trametes versicolor, and Pleurotus ostreatus were purchased from
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Sigma-Aldrich (L2157, S38429 and S75117, respectively) (Sigma-Aldrich, Milano, Italy). LAC
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enzymes were supplied by ASA Spezialenzyme GmbH as lyophylized powder: LAC C from Trametes
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versicolor (2.500 U/g on ABTS, 400 U/g on catechol as substrate) and LAC 4 from Trametes sp. (50
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U/g on ABTS as substrate) are extracellular enzymes isolated from the supernatant of fermentation
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broth; LAC 3 from Myceliophthora thermophila (Mt, 2.200 U/g on ABTS, 3.000 U/g on catechol as
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substrate) is expressed in Aspergillus niger as extracellular enzyme and is isolated from supernatant of
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the fermentation broth; LAC A from Agaricus bisporus (Ab or LAC A, 2.200 U/g on ABTS, 6.000 U/g
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on catechol as substrate) is extracted and purified from the stems of the fruit body; LAC 5 from
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Thielavia sp. (1.100 U/g on ABTS as substrate) is an extracellular enzyme isolated from the
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supernatant of the fermentation broth.
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The total protein concentration was quantified using the biuret method; purity was judged by SDS-
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PAGE and native-PAGE analyses (see Supplementary Table S1). All LAC enzymes gave a single band
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by zymogram analysis with the only exception of LAC 5 that produced a smear containing at least two
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different forms. All chemicals were of analytical grade (Sigma-Aldrich).
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Design and cloning of cDNA encoding for BALL laccase
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The synthetic cDNA encoding the laccase from Bacillus licheniformis (BALL) [16] was designed by in
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silico back translation of the amino acid sequence reported in the GenBank database (Accession no.
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GU972589.1). In order to facilitate subcloning into the pET24b(+) plasmid (Merck Millipore,
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Vimodrone, Italy), sequences corresponding to NdeI (CATATG) and XhoI (CTCGAG) restriction sites
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were added at the 5’- and 3’-ends of the cDNA, respectively. The codon usage of the synthetic cDNA
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was optimized for expression in Escherichia coli and produced by GeneArt (Life Technologies,
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Monza, Italy) (Accession no. KR348913). BALL cDNA was inserted in the pET24b(+) vector using 6 Page 6 of 35
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the NdeI and XhoI sites, resulting in a 6.9-kb (pET24-BALL) construct. Six codons (encoding for six
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additional histidines) were added to the 3’-end of the BALL gene during the subcloning process.
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BALL expression and purification
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The pET24-BALL plasmid was transferred to the host BL21(DE3) E. coli strain (Merck Millipore,
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Vimodrone, Italy). Cells were grown at 37 °C in Terrific broth (TB) medium. Protein expression was
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induced at an OD600nm ≈ 1.6 by adding 0.25 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 2
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mM CuSO4; the cells were grown further at 18 °C for 18 h. In order to enhance copper incorporation,
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eighteen hours after adding IPTG the cells were incubated at different temperatures for further 6 h
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without shaking. Cells were harvested by centrifugation (8000xg for 10 min at 4 °C) and lysed by
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sonication (4 cycles of 30 s each, with 30 s interval on ice). The crude extract was heated at 75 °C for
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15 min and centrifuged at 39,000xg for 30 min at 4 °C.
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The enzyme was purified using a HiTrap chelating affinity column (1 mL) previously loaded with
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metal ions (1 mL of 100 mM NiCl2) and equilibrated with 50 mM Tris-HCl buffer (pH 7.0) containing
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1 M NaCl and 5% glycerol. The column was washed with this buffer until the absorbance value at 280
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nm was that of the buffer. Then, the bound protein was eluted with 50 mM Tris-HCl buffer (pH 7.0)
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containing 500 mM imidazole and 10% glycerol [17]. The fractions containing laccase activity were
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dialyzed against 50 mM sodium acetate, pH 5.0. The amount of protein was estimated by absorbance at
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280 nm using a molar extinction coefficient of 84,739 M-1 cm-1 [18].
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Design and cloning of cDNA encoding for OB1 laccase
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The synthetic cDNA encoding the mutated α-factor prepro-leader and the OB1 variant of laccase from
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basidiomycete PM1 [19] was designed by in silico back translation of the amino acid sequence reported
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in the GenBank database (Accession no. CAA78144.1) and was optimized for expression in 7 Page 7 of 35
Saccharomyces cerevisiae. In order to facilitate the subcloning into the pEMBL and pVTU plasmids
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(inducible or constitutive expression plasmid, respectively, both carrying the URA3 gene for
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auxotrophy selection in S. cerevisiae) [20,21], XbaI (TCTAGA) and HindIII (AAGCTT) restriction
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sites were added at the 5’- and 3’- ends of the cDNA, respectively. Synthetic cDNA was produced by
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GeneArt (Life Technologies) after optimizing the coding nucleotide sequence for expression in S.
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cerevisiae (Accession no. KR348914). OB1 cDNA was inserted in the pEMBL and pVTU vectors
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using the XbaI and HindIII sites, giving a 10.6-kb and 8.7-kb constructs, respectively.
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OB1 expression and purification
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The pEMBL- and pVTU-OB1 plasmids were transferred to the host S. cerevisiae strain X4004 by
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employing the lithium acetate method [22]. Cells were grown at 30 °C in minimal medium (0.68%
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yeast nitrogen base, YNB, 2% glucose, 67 mM potassium phosphate, pH 6.0) for 3 days. An aliquot of
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cells was removed and inoculated into a final volume of 50 mL of minimal medium in a 500-mL flask
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starting from an OD600nm ≈ 0.25. Incubation proceeded until OD600nm ≈ 1 was reached (6 to 8 h) and
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then a 50-mL preculture was inoculated in a 2-L baffled flask containing 450 mL of expression
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medium (10 g/L yeast extract, 21 g/L peptone, 2 mM CuSO4, 6% ethanol, 67 mM potassium
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phosphate, pH 6.0, and 2.2% glucose for pVTU-OB1 plasmid or galactose for the pEMBL-inducible
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expression plasmid). For protein expression trials, cells were harvested at different times of growth at
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30 °C, and proteins in the supernatant were precipitated at 75% saturation of ammonium sulfate. Pellets
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obtained after centrifugation were dissolved in 50 mM sodium acetate, pH 5.0, and ammonium sulfate
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was removed by extensive dialysis against the same buffer. The sample was then concentrated using an
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Amicon ultra apparatus equipped with a 30-kDa membrane.
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MmPPO expression 8 Page 8 of 35
A single colony of Marinomonas mediterranea MMB-1 (ATCC 700492) was inoculated in Marine
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Medium 2216 (Becton Dickinson Italia, Milano, Italy) and grown at 25 °C, with shaking for 24 h. Cells
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were harvested by centrifugation for 6 min at 4000xg and 4 °C. Pellets were resuspended into 100 mM
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sodium pyrophosphate, pH 7.0, and lysed by sonication: the membrane fraction corresponded to the
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supernatant following centrifugation at 10000xg for 4 min at 4 °C. The soluble (crude extract) fraction
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was instead obtained following cell lysis by centrifugation at 40000xg, for 45 min at 4 °C: on this
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sample the effect of several detergents (0.01, 0.1 or 1% SDS, 1% Triton X100, 0.2 or 2% Tween-20) on
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the solubility of MmPPO was studied by assaying the enzymatic activity on 2,6-DMP [23]. To remove
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the excess detergent, the soluble fraction was incubated for 1 h in an ice-water-bath and cleared by
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centrifugation at 10000xg for 3 min at 4 °C. For activity assays - on different substrates, pH, and
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temperature values, see Results section - the crude extract obtained using 1% SDS was used.
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Activity and kinetic measurements
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Kinetic parameters were determined at 25 °C using different concentrations of ABTS (2,2′-azino-bis(3-
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ethylbenzothiazoline-6-sulphonic acid) (2–1,000 μM), catechol (2–50,000 μM), or 2,6-DMP (2,6-
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dimethoxyphenol) (2–1,000 μM), respectively. The absorbance coefficients were as follows: ε420nm =
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36,000 M−1·cm−1 for ABTS, ε410nm = 2,211 M−1·cm−1 for catechol, and ε468nm = 49,600 M−1·cm−1 for
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2,6-DMP. One unit of activity was defined as the amount of enzyme that consumed 1 μmol substrate
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per minute at 25 °C and pH 5.0. The v0 vs. [S] results were fitted to a classical Michaelis-Menten
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equation or modified to account for a substrate inhibition effect [24].
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The pH dependence of the activity of laccase towards ABTS (or 2,6-DMP for BALL and MmPPO) was
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determined using a multicomponent buffer [25]: 15 mM Tris, 15 mM sodium carbonate, 15 mM
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phosphoric acid, and 250 mM potassium chloride adjusted to the appropriate pH with HCl or KOH in
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the pH range 3.0-9.0. The stability was assessed by measuring the residual enzymatic activity after
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incubating the enzyme preparations at different final protein concentrations, depending on their specific
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activity (OB1, Th - LAC 5 and MmPPO, 20 µg/mL; Mt, 30 µg/mL; Tv - Sigma and BALL, 50 µg/mL;
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Ab, 100 µg/mL; Po, 150 µg/mL; Tv - LAC C, 250 µg/mL; Tv - LAC 4, 800 µg/mL) at 25 °C for 24
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hours at different pH values.
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The temperature dependence of laccase activity was investigated by measuring ABTS (or 2,6-DMP for
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MmPPO) oxidation at temperatures ranging from 15 to 80 °C. Enzyme stability was assessed at 25 °C
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and 37 °C by incubating the enzyme solution at different final concentrations (see above) in 50 mM
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sodium acetate buffer, pH 5.0. At different times, samples were taken and residual activity was
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assayed.
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The effect of 1, 10, 100 or 1000 mM NaCl, 0.5, 1 or 5% Tween-80, 10 or 50% dimethyl sulfoxide
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(DMSO) on laccase activity was determined by adding these compounds to the assay solution.
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Native-PAGE electrophoresis
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The zymogram analysis was performed after electrophoretic separation by native-PAGE using a 10%
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acrylamide-resolving gel. The band of the enzyme was visualized by incubating the gel in the staining
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solution (50 mM sodium acetate buffer, pH 5.0, 1 mM ABTS, 7% acetic acid) until color developed.
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Results and discussion
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Expression and purification of the BALL laccase
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Recombinant laccase from Bacillus licheniformis (BALL) [16] was produced using E. coli BL21(DE3)
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host cells transformed with the pET24-BALL plasmid, grown at 37 °C in TB medium, adding IPTG
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and CuSO4 at an OD600nm ≈ 1.6, and collecting cells after another 18 h of incubation at 18 °C. To
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simplify subsequent purification steps, the supernatant was heated: while most of the E. coli proteins
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precipitated, no loss of laccase activity in the solution was observed. Recombinant BALL expressed 10 Page 10 of 35
using pET24-BALL plasmid contains a His-tag at the C-terminus; accordingly, it was purified by
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HiTrap chelating affinity chromatography. By employing a single chromatographic step, recombinant
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BALL was isolated as a single band at ≈ 60 kDa, with a > 90% purity as judged by SDS-PAGE
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analysis (not shown): ≈ 20 mg of purified BALL/L fermentation broth was thus obtained. The specific
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activity of purified BALL laccase with 0.5 mM ABTS as substrate was 10 U/mg protein, a value
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similar to that reported in [26].
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The change in the aeration conditions from shaking (aerobic cultures) to static conditions (microaerobic
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cultures) resulted in a further 2-fold increase in the specific activity (≈ 23 U/mg protein) reaching a
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volumetric yield of 418 U/L.
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Expression and purification of the OB1 variant of laccase from Basidiomycete PM1
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The synthetic gene encoding for the OB1 laccase variant from basidiomycete PM1 was designed by
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Mate et al. [19] to contain the α-PM1 signal sequence constituted by the prepro-leader of the yeast α-
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factor mating pheromone coupled to the mature PM1 native signal sequence. The pEMBL-OB1
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plasmid was transferred to the host S. cerevisiae strain X4004: owing to the presence of the signal
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peptide, OB1 is secreted into culture broth. As shown by activity staining on native-PAGE in Fig. S1,
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the maximal activity was detected for the supernatant collected after 48 h: under these conditions,
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recombinant OB1 was expressed in S. cerevisiae fermentation broth with a yield of 3 U/L culture.
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In order to increase OB1 laccase expression, trials were performed employing a simplified factorial
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design approach [27]. As summarized in Table S2, the highest production, in terms of enzymatic
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activity on ABTS as substrate, was apparent by growing cells transformed with the plasmid pVTU-
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OB1 at 30 °C in the presence of 2 mM Cu2+. Under these conditions, the scale-up process to 500 mL of
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fermentation broth reached an expression level of 140 U/L culture and 45 mg/L fermentation broth vs.
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8 mg/L at 144 hours of growth [19]. OB1 laccase was isolated from concentrated broth culture as a
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single band in SDS-PAGE analysis (at ≈ 53 kDa, not shown), showing a specific activity with 0.5 mM
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ABTS of 3.2 U/mg protein. Notably, the recombinant enzyme did not require any further purification
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procedure since it is sufficiently pure for our purposes (> 75% purity).
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Expression of MmPPO laccase
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Polyphenol oxidase from M. mediterranea (MmPPO) was selected for our analysis since its properties
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are known to differ from fungal counterparts, because of activity and elevated redox potential at neutral
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pH, high substrate selectivity and tolerance to halide ions [28].
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Marinomonas mediterranea MMB-1 was grown at 25 °C with shaking and the cells were collected
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after 24 h [29]. The enzymatic activity was detected in the membrane fraction: the highest activity in
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the soluble fraction was achieved by adding 1% SDS to the lysis buffer, which made it possible to fully
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recover activity in the membrane fraction. MmPPO preparation was only partially purified (≈ 5%
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purity as judged by SDS-PAGE) showing a yield of 34 U/L culture with ABTS. Since PPO is the only
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laccase activity synthesized by M. mediterranea [29], this preparation was used for all activity assays
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reported below.
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Dependence on pH
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An important parameter for the use of laccases is the pH-dependence of their activity. For phenolic
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substrates, the pH dependence of laccase activity is known to be bell-shaped: the activity increase with
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the pH is attributed to the pH dependence decrease of the redox potential of the phenol groups, whereas
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the decrease in activity with pH has been ascribed to the increase in hydroxide concentration and thus
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in trinuclear cluster inhibition [19,30,31]. In general, the laccase activity decreases with pH owing to
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the inhibition by the hydroxide anion, which is presumed to bind on the T2/T3 trinuclear site to
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interfere with electron transfer from T1 to T2/T3 sites [32]. 12 Page 12 of 35
For all tested laccases, the maximum of the bell-shaped curve occurs at acidic pH values (see Fig. 1).
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Interestingly, a detectable enzymatic activity at pH 7.0 was apparent with laccase from A. bisporus
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(Ab), M. thermophila (Mt), MmPPO (assayed on 2,6-DMP) and BALL laccases: notably, ≈ 50% of the
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enzymatic activity measured at pH 5.0 was retained by the bacterial enzymes at neutral pH. The
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enzymatic activity of bacterial BALL laccase was the one less affected by pH change in the range 3-7,
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while the OB1 laccase from basidiomycete PM1 showed the higher increase in enzymatic activity at
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acidic pH values, up to 2-fold at pH 3.0 in comparison to the value measured at pH 5.0 (see Fig. 1B,C).
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The pH effect on the enzymatic activity of BALL laccase is substrate-dependent: on 2,6-DMP as
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substrate, the enzymatic activity was detectable at pH ≥ 4.0, showing the maximum of the bell-shaped
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curve at neutral pH (see Fig. 1C). In bacterial laccases, differently from the fungal counterparts, this
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effect is most likely due to the lack of the Glu or Asp residue close to the T1 copper site that stabilizes
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the phenoxy radical product at low pH and are therefore dependent for activity on the deprotonation of
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the phenolic group at pH ≥ pKa of the phenolic compound itself. The dependence of the pH activity
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profile on the substrate used was also reported for laccases from T. versicolor and Streptomyces
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cyaneus [33], OB1 [19], laccase from B. clausii [34], and a small laccase from Streptomyces coelicolor
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(i.e., SLAC) [35].
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Concerning the stability of the different laccases in the 3-9 pH range following incubation for 24 h at
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25 °C, all the enzymes showed a higher residual activity at pH ≥ 5.0, as reported in Fig. 2, a behavior
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previously reported [33] and attributed to slow conformational changes. With the only exception being
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the bacterial BALL enzyme, all other tested laccases possess a good stability at pH 5.0: the highest
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stability at basic pH values was apparent for laccase from Pleorotus ostreatus (Po) (see Fig. 2B).
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Accordingly, pH = 5 was chosen for further assays as the optimal compromise between enzyme
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activity and stability and also because it allows a higher solubility of lignin samples than at more acidic
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values.
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In comparison to our results, M. thermophila laccase was reported to show the highest activity on
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ABTS at pH 3.0 and the highest stability at pH 6.0 [36]. Anyway, a deep investigation on this enzyme
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reported a pH profile of the initial activity on ABTS corresponding to the one we reported in Fig. 1: the
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profile at increasing pH values depended on both a decrease in kcat and increase in Km values [30].
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Even the pH-dependence profile of Po activity reported by [37] significantly differed from the one we
292
observed (relative activity was > 40% of the maximal value only within the pH range 5-6: such a
293
discrepancy probably depended on the single-component buffer employed and the use of
294
syringaldazine as substrate. Indeed, also the pH profile of MmPPO on 2,6-DMP was slightly different
295
than the one previously reported since the optimum was at pH 4.0 in our results vs. 5.0 as reported in
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[28].
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Dependence on temperature
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As shown in Fig. 3, all laccases are quite thermophilic, showing an optimum temperature at around 70-
300
80 °C. In this range, laccase from M. thermophila expressed in A. niger (Mt) and OB1 expressed in S.
301
cerevisiae showed a 2-fold increase in enzymatic activity compared to the value measured at 25 °C.
302
Concerning the stability, all laccases tested after incubation for 24 h showed a ≥ 85% and ≥ 65%
303
residual activity at 25 °C or 37 °C, the only exceptions being the ones from M. thermophila and from
304
Thielavia sp., which showed a ≈ 20% residual activity after incubation at 37 °C (not shown). For sake
305
of comparison, 25 °C was chosen as the reference temperature.
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Kinetic properties
308
The kinetic parameters of recombinant BALL and OB1 laccases and MmPPO were determined on the
309
nonphenolic ABTS and the phenolic catechol and 2,6-DMP substrates and then compared to the values
310
of commercial laccases (Table 1). Laccase from the tree Rhus vernicifera was the first laccase to be 14 Page 14 of 35
uncovered, in 1883 by Yoshida [38]: it is now commercially available. Owing to its moderately low
312
specific activity on the substrates tested (1.4 U/mg on ABTS, 0.1 U/mg on 2,6-DMP, and 1.2 U/mg on
313
catechol) and high cost, it was not taken into consideration for further characterization.
314
For all tested laccases, the dependence of the reaction rate on the substrate concentration followed a
315
Michaelis-Menten kinetics. The bacterial BALL showed the highest specific activity and catalytic
316
efficiency on ABTS while the recombinant OB1 from basidiomycete PM-1 showed the highest affinity
317
for this compound. In contrast, commercial laccases showed higher kinetic parameters on catechol and
318
2,6-DMP. On the previous substrate, the highest maximal activity was apparent for commercial laccase
319
from the fungus Agaricus bisporus (Ab) and highest apparent substrate affinity and kinetic efficiency
320
was observed for MmPPO (Table 1). On 2,6-DMP as substrate, the highest maximal activity and
321
kinetic efficiency were determined for commercial T. versicolor laccase from Sigma, and all laccases
322
from Trametes as well as the one from M. thermophila showed low Km (ranging in the 6- to 16 µM
323
range). Notably, recombinant bacterial BALL laccase showed a high activity on all tested compounds,
324
but Km for catechol and 2,6-DMP was in the millimolar range (see Table 1).
325
Seemingly large discrepancies were evident comparing our results and those previously determined for
326
Trametes laccase from Sigma [39]: the higher maximal activity on ABTS (38,900 vs 11.5 s-1) and on
327
2,6-DMP (24,800 vs 7.6 s-1) largely arises from the different assay temperature (40 vs. 25 °C used in
328
our work) as well as from the different pH used with the previous substrate (3.5 vs 5.0). Notably, Km
329
values determined in [39] show a < 2-fold change in comparison to our results. Analogously, the
330
kinetic properties of P. ostreatus laccase isoenzymes PoxA1, PoxA2 and PoxC on ABTS at pH 3.0 and
331
25 °C (kcat in the 260-5830 s-1 range) [40] significantly differ from the values we determined at pH 5.0
332
(Table 1). Indeed, while the kcat values for 2,6-DMP at pH 5.0 (= 1.7-4.1 s-1) were similar to the one we
333
determined, a 10-fold higher Km was reported (0.23-2.1 mM).
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334 15 Page 15 of 35
Effect of sodium chloride, DMSO, and Tween-80 concentrations on laccase activity
336
An additional factor influencing laccase activity is the presence of halide ions. Different laccases have
337
been reported to be inhibited by halides, thus affecting their possible applications in decolorizing dye
338
effluents containing high concentrations of halide ions [30]. Notably, a common mechanism underlies
339
inhibition of the laccase activity by the hydroxide anion and chloride [32,41]. The chloride is not likely
340
to reach the type 2 copper because of its size, and thus to inhibit the oxygen reduction; the decrease in
341
enzymatic activity was proposed to be due to the electron transfer inhibition from the T1 site to T2/T3
342
trinuclear sites [32,41]. Accordingly, the effect of sodium chloride concentration on the activity of the
343
available laccases was investigated. Interestingly, as shown in Fig. 4, the activity of recombinant
344
bacterial BALL laccase was not affected by the chloride ion concentration in solution (up to 1 M).
345
Moreover, a large part of the enzymatic activity in the presence of 1 M sodium chloride was also
346
maintained for laccase from ascomycota Thielavia sp. (Th - LAC 5), the basidiomycetes A. bisporus
347
(Ab), M. thermophila (Mt), PM-1 (OB1), and MmPPO (i.e., Th - LAC 5 and MmPPO retained a 80%
348
of enzymatic activity, see Fig. 4B and C). The residual activity of MmPPO at 1 M NaCl was higher
349
than the value (30% residual activity) previously reported in [28]. IC50 values ≤ 100 mM were apparent
350
for Tv and Po laccases.
351
In order to verify the potential for using laccase in processes requiring solvents, the effect of DMSO on
352
enzymatic activity was investigated (see Fig. 5). Noteworthy, it has been shown that DMSO is the most
353
active organic compound used to solubilize lignin, a prerequisite for its further industrial valorization
354
[42,43], but also reported to be a strong inhibitor of laccases [44]. In the presence of 30% DMSO (at
355
which the maximal solubility of organosolv lignin was reached, data not shown) the bacterial BALL
356
laccase and MmPPO retained ≈ 55% and ≈ 100% of the activity assayed in buffer only; the previous
357
recombinant enzyme was also the only laccase that showed significant activity in the presence of 40%
358
DMSO (up to 35% of the value assayed in the absence of DMSO, see Fig. 5C). Interestingly, when the
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16 Page 16 of 35
activity was assayed with 2,6-DMP as substrate, ≈ 60% of enzymatic activity was retained by BALL
360
laccase in up to 50% DMSO in the reaction mixture (Fig. 5C). Moreover, the residual activity of BALL
361
laccase after incubation at 25 °C for 24 h in the presence of 30% DMSO is ≈ 50% of its initial activity:
362
this represents an interesting feature for industrial applications requiring DMSO treatments. Our results
363
on Mt laccase once more differ from the report by [36] reporting that full activity was maintained up to
364
30% DMSO (and that 82% of the initial value was apparent at 50% DMSO) following incubation at 30
365
°C for 24 hours: the rationale for this discrepancy is not evident.
366
The effectiveness of enzymatic hydrolysis of lignocellulosic biomass is significantly enhanced if lignin
367
is modified before adding the biocatalyst. Here, different surfactants are known to increase the removal
368
of lignin and reduce nonproductive enzyme binding on the biomass surface, and, in particular, Tween-
369
80 was reported to increase enzymatic hydrolysis yields [45]. Accordingly, the enzymatic activity of
370
the different laccases in the presence of increasing Tween-80 concentrations was investigated. As
371
shown in Fig. 6, the enzymatic activity of laccases is not significantly affected in up to 5% (vol/vol) of
372
surfactant in the reaction medium, the only exception being the one from A. bisporus (Ab) (Fig. 6B).
373
Interestingly, laccase from the ascomycota Thielavia sp. (Th - LAC 5) showed higher enzymatic
374
activity in the presence of the 1% (vol/vol) surfactant (Fig. 6B) and the recombinant one from M.
375
thermophila exhibited a 2-fold increased activity in the presence of 5% (vol/vol) Tween-80 (Fig. 6A).
376
On this side, the insensitivity on detergent concentration of laccases from Trametes well correlates with
377
the observed beneficial effect of the nonionic surfactant Triton X100 on folding and stabilization [46].
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378 379
Conclusions
380
Laccases are important biocatalysts in a number of applications (see Introduction). In the past several
381
years, laccases were isolated from a variety of sources and strongly differed in terms of dependence of
17 Page 17 of 35
the enzymatic activity on pH. E.g., the optimal value with syringaldazine as substrate spans from pH 4
383
for P. cinnaribus laccase to pH 9 for R. vernicifera laccase [35].
384
For the sake of comparison, we now investigated the pH dependence of the enzymatic reaction of ten
385
laccases under identical conditions (Fig. 1): as a general rule, the optimum was at pH 3-4 and the
386
activity was lost at pH ≥ 7.0. The main exceptions were bacterial BALL and MmPPO, A. bisporus
387
(Ab) laccase and the recombinant one from M. thermophila, which were active at neutral pH values.
388
The pH range was limited by protein instability at pH ≤ 3.0 (Fig. 2) while all tested enzymes were
389
stable at pH ≥ 7.0. Notably, BALL laccase showed a pH-dependence of its catalytic activity that was
390
different with ABTS vs. 2,6-DMP as substrate (Fig. 1C). Concerning the influence of temperature,
391
activity for all tested laccases increased with temperature up to 80 °C, reaching a ≈ 1.5-2.0-fold higher
392
activity than the value determined at 20 °C (Fig. 3). The laccase from M. thermophila and the one from
393
the ascomycete Thielavia (Th – LAC 5) showed the lowest stability when incubated for 24 h at 37 °C.
394
Furthermore, many of the investigated laccases are not sensitive to sodium chloride thus favoring their
395
use in treating municipal and industrial waste water (e.g., textile waste waters contain from 0.4 to 16
396
g/L of NaCl) and sea waters. Activity of T. versicolor laccase was previously reported to be strongly
397
affected to sodium chloride [32]: our results confirmed these findings (Fig. 4). Interestingly, bacterial
398
laccases (BALL and MmPPO) retain significant activity in the presence of DMSO (up to 40% final
399
concentration), showing that they could be applied in lignin-degradation processes requiring solvents.
400
Moreover, the presence of surfactants useful in modifying lignin before adding enzyme to the
401
hydrolysis reaction does not affect the enzymatic activity of most laccases under investigation to a
402
significant extent.
403
Taken together, our comparison of ten laccases under identical conditions indicates that the bacterial
404
laccase from B. licheniformis compares favorably with the tested enzymes since it: i) shows a high
405
activity with both phenolic and nonphenolic substrates; ii) shows a good activity in a broad pH range,
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18 Page 18 of 35
even at values ≥ 7.0; iii) possesses good activity at high temperature and good stability for 24 h at 37
407
°C; and iv) is not significantly affected by sodium chloride and Tween-80. These properties and the
408
improved expression yield in E. coli as reported in this work (≈ 20 mg protein/L fermentation broth and
409
≈ 440 U/L) demonstrate that BALL is a well-suited laccase for a number of biotechnological
410
applications. Furthermore, the different laccases show peculiar properties (i.e., high stability at basic
411
pH values for P. ostreatus laccase or at 80 °C for M. thermophila and OB1, or at 30% DMSO for
412
MmPPO, etc.) that make them suitable for specific applications: the versatility of the available laccases
413
makes it possible to satisfy requirements related to different processes.
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414
Acknowledgements
416
This work was done as part of the ValorPlus project that has received funding from the European
417
Union's Seventh Framework Programme for research, technological development and demonstration
418
under grant agreement no FP7-KBBE-2013-7-613802 to AC and LP. This work was supported by
419
Biorefill grant (Project ID 42611813) to LP. FT is a PhD student of the “Dottorato in Biotecnologie,
420
Bioscienze e Tecnologie Chirurgiche” at Università degli studi dell’Insubria.
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TABLE 1 Kinetic parameters of different laccases (from Trametes species, fungi, and bacteria) on canonical laccase substrates. The
Enzyme
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activity was assayed at pH 5.0 and 25 °C
ABTS
Catechol
Km (µM)
Vmax/Km
Vmax (U/mg)
Km (µM)
Vmax/Km
Vmax (U/mg)
Km (µM)
Vmax/Km
Sigma
13.1 ± 0.3
32 ± 3
0.42 ± 0.05
3.4 ± 0.1
410 ± 53
(8.3 ± 1.3)·10-3
8.6 ± 0.2
16.2 ± 1.7
0.53 ± 0.07
LAC C
5.6 ± 0.5
41 ± 9
0.14 ± 0.03
1.40 ± 0.03
334 ± 34
(4.2 ± 0.4)·10-3
1.20 ± 0.03
8.5 ± 1.1
0.14 ± 0.02
LAC 4
0.10 ± 0.01
55 ± 4
(0.20 ± 0.01)·10-2
0.030 ± 0.001
491 ± 45
(1.0 ± 0.3)·10-3
0.030 ± 0.001
14.5 ± 2.6
(2.1 ± 0.4)·10-3
M. thermophila (Mt)
15.5 ± 0.5
99 ± 2
0.16 ± 0.01
5.7 ± 0.4
2480 ± 207
(2.3 ± 0.3)·10-3
2.20 ± 0.06
6.0 ± 1.1
0.40 ± 0.04
P. ostreatus (Po)
7.9 ± 0.2
28 ± 2
0.28 ± 0.03
2.7 ± 0.1
770 ± 174
(3.5 ± 0.8)·10-3
2.9 ± 0.1
27.2 ± 1.4
0.11 ± 0.01
A. bisporus (Ab)
10.9 ± 0.7
602 ± 110
(2.0 ± 0.3)·10-2
11.0 ± 1.8
8900 ± 800
(1.2 ± 0.2)·10-3
0.90 ± 0.09
6700 ± 705
(0.10 ± 0.02)·10-3
OB1
3.2 ± 0.3
9±1
0.36 ± 0.07
0.89 ± 0.02
2400 ± 237
(0.4 ± 0.1)·10-3
0.76 ± 0.02
48 ± 6
0.015 ± 0.002
Thielavia (LAC 5)
8.0 ± 0.3
15 ± 3
0.53 ± 0.10
2.80 ± 0.05
2010 ± 108
(1.40 ± 0.06)·10-3
3.00 ± 0.03
20.0 ± 1.1
0.15 ± 0.08
BALL
23.0 ± 0.2
14 ± 2
1.62 ± 0.25
3.9 ± 0.2
1870 ± 351
(2.1 ± 0.5)·10-3
3.9 ± 0.3
2600 ± 157
(1.5 ± 0.2)·10-3
MmPPO
3.4 ± 1.0
1840 ± 230
(1.8 ± 0.6)·10-3
3.2 ± 0.2
11.2 ± 3.7
0.29 ± 0.07
1.6 ± 0.2
172 ± 30
(9.3 ± 0.2)·10-3
Ac
ce pt
Other fungi:
ed
Trametes:
M an
Vmax (U/mg)
2,6-DMP
Bacteria:
26 Page 26 of 35
FIGURE 1 Effect of pH on the enzymatic activity of various laccases at 25 °C. Enzymes from: (A) M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial sources:
ip t
BALL and MmPPO (the latter two enzymes were assayed on ABTS (black bars) or 2,6-DMP (gray
cr
bars) as substrates). The value at pH 5.0 is taken as 100%.
us
FIGURE 2 Effect of pH on the enzymatic stability of various laccases; residual activity was assayed after 24 h of incubation by measuring ABTS (or 2,6-DMP for MmPPO) oxidation, at 25 °C. Enzymes
an
from (A) fungal sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5);
M
(C) bacterial sources: BALL and MmPPO. The activity value at time = 0 at each pH value is taken as
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FIGURE 3 Effect of temperature on the enzymatic activity of various laccases, determined by
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measuring ABTS (or 2,6-DMP for MmPPO) oxidation, at pH 5.0. Enzymes from (A) fungal sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial sources: BALL and MmPPO. The value at 25 °C is taken as 100%.
FIGURE 4 Effect of NaCl on the enzymatic activity of various laccases, determined by measuring ABTS (black bars) or 2,6-DMP (gray bars) oxidation, at pH 5.0, 25 °C. Enzymes from (A) fungal sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial sources: BALL and MmPPO. The value in absence of NaCl is taken as 100%. 27 Page 27 of 35
FIGURE 5 Effect of DMSO on the enzymatic activity of various laccases, determined by measuring ABTS (black bars) or 2,6-DMP (gray bars) oxidation, at pH 5.0, 25 °C. Enzymes from: (A) fungal
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sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial
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sources: BALL and MmPPO. The value in absence of DMSO is taken as 100%.
FIGURE 6 Effect of Tween-80 on the enzymatic activity of various laccases, determined by measuring
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ABTS (black bars) or 2,6-DMP (gray bars) oxidation, at pH 5.0, 25 °C. Enzymes from: (A) fungal sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B)
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fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial
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sources: BALL and MmPPO. The value in absence of Tween-80 is taken as 100%.
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S1871-6784(16)00010-8 http://dx.doi.org/doi:10.1016/j.nbt.2016.01.007 NBT 854
To appear in: Received date: Revised date: Accepted date:
28-9-2015 18-12-2015 22-1-2016
Please cite this article as: Tonin, F., Melis, R., Cordes, A., Sanchez-Amat, A., Pollegioni, L., Rosini, E.,Comparison of different microbial laccases as tools for industrial uses, New Biotechnology (2016), http://dx.doi.org/10.1016/j.nbt.2016.01.007 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.
1
Highlights Laccases are used as lignin-degrading enzymes
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Biochemical properties of ten laccases under identical conditions were investigated
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Bacterial laccases retained high activity in DMSO and Tween-80
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Properties of recombinant B. licheniformis laccase satisfy requirements for industrial uses
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Comparison of different microbial laccases as tools for industrial uses
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Fabio Tonina, Roberta Melisa, Arno Cordesb, Antonio Sanchez-Amatc, Loredano Pollegionia,d and
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Elena Rosinia,d
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a
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Dunant 3, 21100 Varese, Italy
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b
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Università degli Studi dell’Insubria, Milano, Italy
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Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell’Insubria, via J.H.
ASA Spezialenzyme GmbH, Am Exer 19c, D-38302 Wolfenbüttel, Germany
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Department of Genetics and Microbiology, University of Murcia, Murcia, Spain
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Correspondence: Loredano Pollegioni, Dipartimento di Biotecnologie e Scienze della Vita, Università
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degli studi dell’Insubria, via J.H. Dunant 3, 21100 Varese, Italy, Fax: +39 0332421500; Tel.: +39
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0332421506; E-mail address: [email protected]
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The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and
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Abbreviations: ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); 2,6-DMP, 2,6-
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dimethoxyphenol; DMSO, dimethyl sulfoxide; BALL, laccase from Bacillus licheniformis; OB1,
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laccase variant from basidiomycete PM1; MmPPO, polyphenol oxidase from Marinomonas
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mediterranea MMB-1
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Abstract
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Laccases from different sources are employed in a number of biotechnological processes, each
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characterized by specific reaction constraints and thus requiring an enzyme with suitable properties. In
30
order to avoid the bias generated by different assay methodologies, in this work we investigated the
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main properties of ten (commercial and recombinant) laccases from fungi and bacteria under identical
32
conditions. As a general rule, the optimal activity was apparent at pH 3-4 and was lost at pH ≥ 7.0 (all
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laccases were stable at pH ≥ 7.0); enzymes active at neutral pH values were also identified. For all
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tested laccases, activity increased with temperature up to 80 °C and stability was good at 25 °C.
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Interestingly, laccases insensitive to high salt concentration were identified, this favoring their use in
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treating waste waters. Indeed, bacterial laccases retained a significant activity in the presence of DMSO
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(up to 40% final concentration) and of surfactants, suggesting that they can be applied in lignin
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degradation processes requiring solvents. The available laccases are versatile and satisfy requirements
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related to different processes. Notably, the recombinant laccase from Bacillus licheniformis favorably
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compares with the tested enzymes, indicating that it is well suited for different biotechnological
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applications.
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Keywords: Ligninolytic enzymes; Lignin valorization; Enzymatic depolymerization; Green
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biotechnology
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Introduction
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Laccases (benzenediol:oxygen oxidoreductases; EC 1.10.3.2), sometimes also referred to as polyphenol
50
oxidases, belong to the group of blue multi-copper oxidases along with ceruloplasmin, ascorbate
51
oxidase, bilirubin oxidase, and various manganese oxidases. Laccase is attracting great scientific
52
interest because of its very basic requirements - it only uses oxygen from the air and releases water as
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the sole by-product - and great catalytic capabilities, rendering it one of the ‘‘greenest’’ enzymes [1-3].
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Laccase couples the four-electron reduction of dioxygen to water with the oxidation of a broad range of
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substrates (including phenols, polyphenols, arylamines, anilines, hydroxyindols, and thiols). Its
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substrate promiscuity can be even further expanded by using redox mediators, i.e., diffusible electron
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carriers from natural or synthetic sources [4-6].
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Laccases are widely distributed among fungi, higher plants and bacteria. Fungal laccases contain four
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Cu ions, organized into two copper clusters: one situated in a mononuclear T1 site close to the protein
60
surface and three others buried at a trinuclear site, consisting of a mononuclear T2 site and a binuclear
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T3 site [7,8]. In the resting form of the enzyme, the four copper ions are in the +2 oxidation state [7, 9].
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The catalytic cycle begins by sequestering one electron at a time from the reducing substrate and
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transferring it to the trinuclear T2/T3 copper cluster through a highly conserved His-Cys-His pathway,
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where four electrons reduce dioxygen to two molecules of water.
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Laccases are commonly classified into two main groups: “low-medium” and “high” redox potential
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laccases according to their redox potentials at the T1 site, ranging from +430 mV in bacterial and plant
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laccases to +790 mV in some fungal laccases: the latter are typically secreted by ligninolytic
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basidiomycetes and are the most important for biotechnological applications [1,10].
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The potential uses of laccases are numerous due to their low catalytic requirements and oxidative
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versatility. In fact, these enzymes catalyze a wide spectrum of reactions, which include cross-linking of
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monomers, degradation of polymers, ring cleavage, and oxyfunctionalization of aromatic compounds
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[4,5,8]. Laccases are used in several industrial sectors, including the textile (for bleaching) and dye
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industry, for effluent treatment and bioremediation, in the food industry (for beverage processing and
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baking), and in forest-product industry, where lignin removal is the main goal (paper manufacture) or
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where lignin polymerization is the main objective (e.g., manufacture of fiberboards and synthesis of
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novel materials) [6]. Furthermore, fungal laccases have been recently employed to improve the
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conversion of plant biomass in integrated lignocellulose biorefineries, in organic synthesis (i.e., for the
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enzymatic conversion of chemical intermediates and the synthesis of pigments and antioxidants
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through dimerization of phenolic and nonphenolic acids), in oxidative transformation of environmental
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pollutants (e.g., herbicides), and in bioelectrocatalysis for detecting phenolic pollutants [1,11,12].
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Moreover, in the presence of redox mediators, laccases can transform compounds with higher redox
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potentials as well as complex polymers such as lignin, avoiding limits due to steric hindrance [13,14].
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In the past several years, a number of laccases from different sources [15] were isolated and
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characterized. These enzymes have been assayed on different compounds using different experimental
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conditions, however, making it difficult to compare their characteristics: this limit is further hampered
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by the fact that various isoenzymes, which differ in biochemical properties, are produced by the same
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strain. A major challenge of laccase use is that the catalytic activity and stability are highly dependent
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on experimental variables such as pH, temperature, ionic strength, and co-solvents. In this study we
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evaluated the main biochemical properties of a number of commercial and recombinant laccases under
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identical experimental conditions, with the final goal to compare their features in order to identify the
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best biocatalyst for specific applications.
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Materials and methods
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Reagents
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The laccases from Rhus vernicifera, Trametes versicolor, and Pleurotus ostreatus were purchased from
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Sigma-Aldrich (L2157, S38429 and S75117, respectively) (Sigma-Aldrich, Milano, Italy). LAC
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enzymes were supplied by ASA Spezialenzyme GmbH as lyophylized powder: LAC C from Trametes
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versicolor (2.500 U/g on ABTS, 400 U/g on catechol as substrate) and LAC 4 from Trametes sp. (50
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U/g on ABTS as substrate) are extracellular enzymes isolated from the supernatant of fermentation
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broth; LAC 3 from Myceliophthora thermophila (Mt, 2.200 U/g on ABTS, 3.000 U/g on catechol as
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substrate) is expressed in Aspergillus niger as extracellular enzyme and is isolated from supernatant of
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the fermentation broth; LAC A from Agaricus bisporus (Ab or LAC A, 2.200 U/g on ABTS, 6.000 U/g
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on catechol as substrate) is extracted and purified from the stems of the fruit body; LAC 5 from
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Thielavia sp. (1.100 U/g on ABTS as substrate) is an extracellular enzyme isolated from the
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supernatant of the fermentation broth.
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The total protein concentration was quantified using the biuret method; purity was judged by SDS-
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PAGE and native-PAGE analyses (see Supplementary Table S1). All LAC enzymes gave a single band
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by zymogram analysis with the only exception of LAC 5 that produced a smear containing at least two
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different forms. All chemicals were of analytical grade (Sigma-Aldrich).
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Design and cloning of cDNA encoding for BALL laccase
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The synthetic cDNA encoding the laccase from Bacillus licheniformis (BALL) [16] was designed by in
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silico back translation of the amino acid sequence reported in the GenBank database (Accession no.
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GU972589.1). In order to facilitate subcloning into the pET24b(+) plasmid (Merck Millipore,
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Vimodrone, Italy), sequences corresponding to NdeI (CATATG) and XhoI (CTCGAG) restriction sites
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were added at the 5’- and 3’-ends of the cDNA, respectively. The codon usage of the synthetic cDNA
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was optimized for expression in Escherichia coli and produced by GeneArt (Life Technologies,
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Monza, Italy) (Accession no. KR348913). BALL cDNA was inserted in the pET24b(+) vector using 6 Page 6 of 35
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the NdeI and XhoI sites, resulting in a 6.9-kb (pET24-BALL) construct. Six codons (encoding for six
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additional histidines) were added to the 3’-end of the BALL gene during the subcloning process.
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BALL expression and purification
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The pET24-BALL plasmid was transferred to the host BL21(DE3) E. coli strain (Merck Millipore,
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Vimodrone, Italy). Cells were grown at 37 °C in Terrific broth (TB) medium. Protein expression was
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induced at an OD600nm ≈ 1.6 by adding 0.25 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 2
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mM CuSO4; the cells were grown further at 18 °C for 18 h. In order to enhance copper incorporation,
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eighteen hours after adding IPTG the cells were incubated at different temperatures for further 6 h
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without shaking. Cells were harvested by centrifugation (8000xg for 10 min at 4 °C) and lysed by
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sonication (4 cycles of 30 s each, with 30 s interval on ice). The crude extract was heated at 75 °C for
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15 min and centrifuged at 39,000xg for 30 min at 4 °C.
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The enzyme was purified using a HiTrap chelating affinity column (1 mL) previously loaded with
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metal ions (1 mL of 100 mM NiCl2) and equilibrated with 50 mM Tris-HCl buffer (pH 7.0) containing
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1 M NaCl and 5% glycerol. The column was washed with this buffer until the absorbance value at 280
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nm was that of the buffer. Then, the bound protein was eluted with 50 mM Tris-HCl buffer (pH 7.0)
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containing 500 mM imidazole and 10% glycerol [17]. The fractions containing laccase activity were
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dialyzed against 50 mM sodium acetate, pH 5.0. The amount of protein was estimated by absorbance at
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280 nm using a molar extinction coefficient of 84,739 M-1 cm-1 [18].
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Design and cloning of cDNA encoding for OB1 laccase
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The synthetic cDNA encoding the mutated α-factor prepro-leader and the OB1 variant of laccase from
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basidiomycete PM1 [19] was designed by in silico back translation of the amino acid sequence reported
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in the GenBank database (Accession no. CAA78144.1) and was optimized for expression in 7 Page 7 of 35
Saccharomyces cerevisiae. In order to facilitate the subcloning into the pEMBL and pVTU plasmids
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(inducible or constitutive expression plasmid, respectively, both carrying the URA3 gene for
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auxotrophy selection in S. cerevisiae) [20,21], XbaI (TCTAGA) and HindIII (AAGCTT) restriction
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sites were added at the 5’- and 3’- ends of the cDNA, respectively. Synthetic cDNA was produced by
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GeneArt (Life Technologies) after optimizing the coding nucleotide sequence for expression in S.
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cerevisiae (Accession no. KR348914). OB1 cDNA was inserted in the pEMBL and pVTU vectors
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using the XbaI and HindIII sites, giving a 10.6-kb and 8.7-kb constructs, respectively.
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OB1 expression and purification
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The pEMBL- and pVTU-OB1 plasmids were transferred to the host S. cerevisiae strain X4004 by
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employing the lithium acetate method [22]. Cells were grown at 30 °C in minimal medium (0.68%
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yeast nitrogen base, YNB, 2% glucose, 67 mM potassium phosphate, pH 6.0) for 3 days. An aliquot of
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cells was removed and inoculated into a final volume of 50 mL of minimal medium in a 500-mL flask
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starting from an OD600nm ≈ 0.25. Incubation proceeded until OD600nm ≈ 1 was reached (6 to 8 h) and
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then a 50-mL preculture was inoculated in a 2-L baffled flask containing 450 mL of expression
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medium (10 g/L yeast extract, 21 g/L peptone, 2 mM CuSO4, 6% ethanol, 67 mM potassium
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phosphate, pH 6.0, and 2.2% glucose for pVTU-OB1 plasmid or galactose for the pEMBL-inducible
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expression plasmid). For protein expression trials, cells were harvested at different times of growth at
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30 °C, and proteins in the supernatant were precipitated at 75% saturation of ammonium sulfate. Pellets
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obtained after centrifugation were dissolved in 50 mM sodium acetate, pH 5.0, and ammonium sulfate
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was removed by extensive dialysis against the same buffer. The sample was then concentrated using an
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Amicon ultra apparatus equipped with a 30-kDa membrane.
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MmPPO expression 8 Page 8 of 35
A single colony of Marinomonas mediterranea MMB-1 (ATCC 700492) was inoculated in Marine
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Medium 2216 (Becton Dickinson Italia, Milano, Italy) and grown at 25 °C, with shaking for 24 h. Cells
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were harvested by centrifugation for 6 min at 4000xg and 4 °C. Pellets were resuspended into 100 mM
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sodium pyrophosphate, pH 7.0, and lysed by sonication: the membrane fraction corresponded to the
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supernatant following centrifugation at 10000xg for 4 min at 4 °C. The soluble (crude extract) fraction
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was instead obtained following cell lysis by centrifugation at 40000xg, for 45 min at 4 °C: on this
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sample the effect of several detergents (0.01, 0.1 or 1% SDS, 1% Triton X100, 0.2 or 2% Tween-20) on
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the solubility of MmPPO was studied by assaying the enzymatic activity on 2,6-DMP [23]. To remove
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the excess detergent, the soluble fraction was incubated for 1 h in an ice-water-bath and cleared by
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centrifugation at 10000xg for 3 min at 4 °C. For activity assays - on different substrates, pH, and
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temperature values, see Results section - the crude extract obtained using 1% SDS was used.
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Activity and kinetic measurements
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Kinetic parameters were determined at 25 °C using different concentrations of ABTS (2,2′-azino-bis(3-
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ethylbenzothiazoline-6-sulphonic acid) (2–1,000 μM), catechol (2–50,000 μM), or 2,6-DMP (2,6-
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dimethoxyphenol) (2–1,000 μM), respectively. The absorbance coefficients were as follows: ε420nm =
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36,000 M−1·cm−1 for ABTS, ε410nm = 2,211 M−1·cm−1 for catechol, and ε468nm = 49,600 M−1·cm−1 for
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2,6-DMP. One unit of activity was defined as the amount of enzyme that consumed 1 μmol substrate
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per minute at 25 °C and pH 5.0. The v0 vs. [S] results were fitted to a classical Michaelis-Menten
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equation or modified to account for a substrate inhibition effect [24].
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The pH dependence of the activity of laccase towards ABTS (or 2,6-DMP for BALL and MmPPO) was
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determined using a multicomponent buffer [25]: 15 mM Tris, 15 mM sodium carbonate, 15 mM
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phosphoric acid, and 250 mM potassium chloride adjusted to the appropriate pH with HCl or KOH in
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the pH range 3.0-9.0. The stability was assessed by measuring the residual enzymatic activity after
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incubating the enzyme preparations at different final protein concentrations, depending on their specific
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activity (OB1, Th - LAC 5 and MmPPO, 20 µg/mL; Mt, 30 µg/mL; Tv - Sigma and BALL, 50 µg/mL;
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Ab, 100 µg/mL; Po, 150 µg/mL; Tv - LAC C, 250 µg/mL; Tv - LAC 4, 800 µg/mL) at 25 °C for 24
194
hours at different pH values.
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The temperature dependence of laccase activity was investigated by measuring ABTS (or 2,6-DMP for
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MmPPO) oxidation at temperatures ranging from 15 to 80 °C. Enzyme stability was assessed at 25 °C
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and 37 °C by incubating the enzyme solution at different final concentrations (see above) in 50 mM
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sodium acetate buffer, pH 5.0. At different times, samples were taken and residual activity was
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assayed.
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The effect of 1, 10, 100 or 1000 mM NaCl, 0.5, 1 or 5% Tween-80, 10 or 50% dimethyl sulfoxide
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(DMSO) on laccase activity was determined by adding these compounds to the assay solution.
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Native-PAGE electrophoresis
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The zymogram analysis was performed after electrophoretic separation by native-PAGE using a 10%
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acrylamide-resolving gel. The band of the enzyme was visualized by incubating the gel in the staining
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solution (50 mM sodium acetate buffer, pH 5.0, 1 mM ABTS, 7% acetic acid) until color developed.
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Results and discussion
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Expression and purification of the BALL laccase
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Recombinant laccase from Bacillus licheniformis (BALL) [16] was produced using E. coli BL21(DE3)
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host cells transformed with the pET24-BALL plasmid, grown at 37 °C in TB medium, adding IPTG
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and CuSO4 at an OD600nm ≈ 1.6, and collecting cells after another 18 h of incubation at 18 °C. To
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simplify subsequent purification steps, the supernatant was heated: while most of the E. coli proteins
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precipitated, no loss of laccase activity in the solution was observed. Recombinant BALL expressed 10 Page 10 of 35
using pET24-BALL plasmid contains a His-tag at the C-terminus; accordingly, it was purified by
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HiTrap chelating affinity chromatography. By employing a single chromatographic step, recombinant
217
BALL was isolated as a single band at ≈ 60 kDa, with a > 90% purity as judged by SDS-PAGE
218
analysis (not shown): ≈ 20 mg of purified BALL/L fermentation broth was thus obtained. The specific
219
activity of purified BALL laccase with 0.5 mM ABTS as substrate was 10 U/mg protein, a value
220
similar to that reported in [26].
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The change in the aeration conditions from shaking (aerobic cultures) to static conditions (microaerobic
222
cultures) resulted in a further 2-fold increase in the specific activity (≈ 23 U/mg protein) reaching a
223
volumetric yield of 418 U/L.
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Expression and purification of the OB1 variant of laccase from Basidiomycete PM1
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The synthetic gene encoding for the OB1 laccase variant from basidiomycete PM1 was designed by
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Mate et al. [19] to contain the α-PM1 signal sequence constituted by the prepro-leader of the yeast α-
228
factor mating pheromone coupled to the mature PM1 native signal sequence. The pEMBL-OB1
229
plasmid was transferred to the host S. cerevisiae strain X4004: owing to the presence of the signal
230
peptide, OB1 is secreted into culture broth. As shown by activity staining on native-PAGE in Fig. S1,
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the maximal activity was detected for the supernatant collected after 48 h: under these conditions,
232
recombinant OB1 was expressed in S. cerevisiae fermentation broth with a yield of 3 U/L culture.
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In order to increase OB1 laccase expression, trials were performed employing a simplified factorial
234
design approach [27]. As summarized in Table S2, the highest production, in terms of enzymatic
235
activity on ABTS as substrate, was apparent by growing cells transformed with the plasmid pVTU-
236
OB1 at 30 °C in the presence of 2 mM Cu2+. Under these conditions, the scale-up process to 500 mL of
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fermentation broth reached an expression level of 140 U/L culture and 45 mg/L fermentation broth vs.
238
8 mg/L at 144 hours of growth [19]. OB1 laccase was isolated from concentrated broth culture as a
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single band in SDS-PAGE analysis (at ≈ 53 kDa, not shown), showing a specific activity with 0.5 mM
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ABTS of 3.2 U/mg protein. Notably, the recombinant enzyme did not require any further purification
241
procedure since it is sufficiently pure for our purposes (> 75% purity).
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Expression of MmPPO laccase
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Polyphenol oxidase from M. mediterranea (MmPPO) was selected for our analysis since its properties
245
are known to differ from fungal counterparts, because of activity and elevated redox potential at neutral
246
pH, high substrate selectivity and tolerance to halide ions [28].
247
Marinomonas mediterranea MMB-1 was grown at 25 °C with shaking and the cells were collected
248
after 24 h [29]. The enzymatic activity was detected in the membrane fraction: the highest activity in
249
the soluble fraction was achieved by adding 1% SDS to the lysis buffer, which made it possible to fully
250
recover activity in the membrane fraction. MmPPO preparation was only partially purified (≈ 5%
251
purity as judged by SDS-PAGE) showing a yield of 34 U/L culture with ABTS. Since PPO is the only
252
laccase activity synthesized by M. mediterranea [29], this preparation was used for all activity assays
253
reported below.
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Dependence on pH
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An important parameter for the use of laccases is the pH-dependence of their activity. For phenolic
257
substrates, the pH dependence of laccase activity is known to be bell-shaped: the activity increase with
258
the pH is attributed to the pH dependence decrease of the redox potential of the phenol groups, whereas
259
the decrease in activity with pH has been ascribed to the increase in hydroxide concentration and thus
260
in trinuclear cluster inhibition [19,30,31]. In general, the laccase activity decreases with pH owing to
261
the inhibition by the hydroxide anion, which is presumed to bind on the T2/T3 trinuclear site to
262
interfere with electron transfer from T1 to T2/T3 sites [32]. 12 Page 12 of 35
For all tested laccases, the maximum of the bell-shaped curve occurs at acidic pH values (see Fig. 1).
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Interestingly, a detectable enzymatic activity at pH 7.0 was apparent with laccase from A. bisporus
265
(Ab), M. thermophila (Mt), MmPPO (assayed on 2,6-DMP) and BALL laccases: notably, ≈ 50% of the
266
enzymatic activity measured at pH 5.0 was retained by the bacterial enzymes at neutral pH. The
267
enzymatic activity of bacterial BALL laccase was the one less affected by pH change in the range 3-7,
268
while the OB1 laccase from basidiomycete PM1 showed the higher increase in enzymatic activity at
269
acidic pH values, up to 2-fold at pH 3.0 in comparison to the value measured at pH 5.0 (see Fig. 1B,C).
270
The pH effect on the enzymatic activity of BALL laccase is substrate-dependent: on 2,6-DMP as
271
substrate, the enzymatic activity was detectable at pH ≥ 4.0, showing the maximum of the bell-shaped
272
curve at neutral pH (see Fig. 1C). In bacterial laccases, differently from the fungal counterparts, this
273
effect is most likely due to the lack of the Glu or Asp residue close to the T1 copper site that stabilizes
274
the phenoxy radical product at low pH and are therefore dependent for activity on the deprotonation of
275
the phenolic group at pH ≥ pKa of the phenolic compound itself. The dependence of the pH activity
276
profile on the substrate used was also reported for laccases from T. versicolor and Streptomyces
277
cyaneus [33], OB1 [19], laccase from B. clausii [34], and a small laccase from Streptomyces coelicolor
278
(i.e., SLAC) [35].
279
Concerning the stability of the different laccases in the 3-9 pH range following incubation for 24 h at
280
25 °C, all the enzymes showed a higher residual activity at pH ≥ 5.0, as reported in Fig. 2, a behavior
281
previously reported [33] and attributed to slow conformational changes. With the only exception being
282
the bacterial BALL enzyme, all other tested laccases possess a good stability at pH 5.0: the highest
283
stability at basic pH values was apparent for laccase from Pleorotus ostreatus (Po) (see Fig. 2B).
284
Accordingly, pH = 5 was chosen for further assays as the optimal compromise between enzyme
285
activity and stability and also because it allows a higher solubility of lignin samples than at more acidic
286
values.
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In comparison to our results, M. thermophila laccase was reported to show the highest activity on
288
ABTS at pH 3.0 and the highest stability at pH 6.0 [36]. Anyway, a deep investigation on this enzyme
289
reported a pH profile of the initial activity on ABTS corresponding to the one we reported in Fig. 1: the
290
profile at increasing pH values depended on both a decrease in kcat and increase in Km values [30].
291
Even the pH-dependence profile of Po activity reported by [37] significantly differed from the one we
292
observed (relative activity was > 40% of the maximal value only within the pH range 5-6: such a
293
discrepancy probably depended on the single-component buffer employed and the use of
294
syringaldazine as substrate. Indeed, also the pH profile of MmPPO on 2,6-DMP was slightly different
295
than the one previously reported since the optimum was at pH 4.0 in our results vs. 5.0 as reported in
296
[28].
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M
297
Dependence on temperature
299
As shown in Fig. 3, all laccases are quite thermophilic, showing an optimum temperature at around 70-
300
80 °C. In this range, laccase from M. thermophila expressed in A. niger (Mt) and OB1 expressed in S.
301
cerevisiae showed a 2-fold increase in enzymatic activity compared to the value measured at 25 °C.
302
Concerning the stability, all laccases tested after incubation for 24 h showed a ≥ 85% and ≥ 65%
303
residual activity at 25 °C or 37 °C, the only exceptions being the ones from M. thermophila and from
304
Thielavia sp., which showed a ≈ 20% residual activity after incubation at 37 °C (not shown). For sake
305
of comparison, 25 °C was chosen as the reference temperature.
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306 307
Kinetic properties
308
The kinetic parameters of recombinant BALL and OB1 laccases and MmPPO were determined on the
309
nonphenolic ABTS and the phenolic catechol and 2,6-DMP substrates and then compared to the values
310
of commercial laccases (Table 1). Laccase from the tree Rhus vernicifera was the first laccase to be 14 Page 14 of 35
uncovered, in 1883 by Yoshida [38]: it is now commercially available. Owing to its moderately low
312
specific activity on the substrates tested (1.4 U/mg on ABTS, 0.1 U/mg on 2,6-DMP, and 1.2 U/mg on
313
catechol) and high cost, it was not taken into consideration for further characterization.
314
For all tested laccases, the dependence of the reaction rate on the substrate concentration followed a
315
Michaelis-Menten kinetics. The bacterial BALL showed the highest specific activity and catalytic
316
efficiency on ABTS while the recombinant OB1 from basidiomycete PM-1 showed the highest affinity
317
for this compound. In contrast, commercial laccases showed higher kinetic parameters on catechol and
318
2,6-DMP. On the previous substrate, the highest maximal activity was apparent for commercial laccase
319
from the fungus Agaricus bisporus (Ab) and highest apparent substrate affinity and kinetic efficiency
320
was observed for MmPPO (Table 1). On 2,6-DMP as substrate, the highest maximal activity and
321
kinetic efficiency were determined for commercial T. versicolor laccase from Sigma, and all laccases
322
from Trametes as well as the one from M. thermophila showed low Km (ranging in the 6- to 16 µM
323
range). Notably, recombinant bacterial BALL laccase showed a high activity on all tested compounds,
324
but Km for catechol and 2,6-DMP was in the millimolar range (see Table 1).
325
Seemingly large discrepancies were evident comparing our results and those previously determined for
326
Trametes laccase from Sigma [39]: the higher maximal activity on ABTS (38,900 vs 11.5 s-1) and on
327
2,6-DMP (24,800 vs 7.6 s-1) largely arises from the different assay temperature (40 vs. 25 °C used in
328
our work) as well as from the different pH used with the previous substrate (3.5 vs 5.0). Notably, Km
329
values determined in [39] show a < 2-fold change in comparison to our results. Analogously, the
330
kinetic properties of P. ostreatus laccase isoenzymes PoxA1, PoxA2 and PoxC on ABTS at pH 3.0 and
331
25 °C (kcat in the 260-5830 s-1 range) [40] significantly differ from the values we determined at pH 5.0
332
(Table 1). Indeed, while the kcat values for 2,6-DMP at pH 5.0 (= 1.7-4.1 s-1) were similar to the one we
333
determined, a 10-fold higher Km was reported (0.23-2.1 mM).
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334 15 Page 15 of 35
Effect of sodium chloride, DMSO, and Tween-80 concentrations on laccase activity
336
An additional factor influencing laccase activity is the presence of halide ions. Different laccases have
337
been reported to be inhibited by halides, thus affecting their possible applications in decolorizing dye
338
effluents containing high concentrations of halide ions [30]. Notably, a common mechanism underlies
339
inhibition of the laccase activity by the hydroxide anion and chloride [32,41]. The chloride is not likely
340
to reach the type 2 copper because of its size, and thus to inhibit the oxygen reduction; the decrease in
341
enzymatic activity was proposed to be due to the electron transfer inhibition from the T1 site to T2/T3
342
trinuclear sites [32,41]. Accordingly, the effect of sodium chloride concentration on the activity of the
343
available laccases was investigated. Interestingly, as shown in Fig. 4, the activity of recombinant
344
bacterial BALL laccase was not affected by the chloride ion concentration in solution (up to 1 M).
345
Moreover, a large part of the enzymatic activity in the presence of 1 M sodium chloride was also
346
maintained for laccase from ascomycota Thielavia sp. (Th - LAC 5), the basidiomycetes A. bisporus
347
(Ab), M. thermophila (Mt), PM-1 (OB1), and MmPPO (i.e., Th - LAC 5 and MmPPO retained a 80%
348
of enzymatic activity, see Fig. 4B and C). The residual activity of MmPPO at 1 M NaCl was higher
349
than the value (30% residual activity) previously reported in [28]. IC50 values ≤ 100 mM were apparent
350
for Tv and Po laccases.
351
In order to verify the potential for using laccase in processes requiring solvents, the effect of DMSO on
352
enzymatic activity was investigated (see Fig. 5). Noteworthy, it has been shown that DMSO is the most
353
active organic compound used to solubilize lignin, a prerequisite for its further industrial valorization
354
[42,43], but also reported to be a strong inhibitor of laccases [44]. In the presence of 30% DMSO (at
355
which the maximal solubility of organosolv lignin was reached, data not shown) the bacterial BALL
356
laccase and MmPPO retained ≈ 55% and ≈ 100% of the activity assayed in buffer only; the previous
357
recombinant enzyme was also the only laccase that showed significant activity in the presence of 40%
358
DMSO (up to 35% of the value assayed in the absence of DMSO, see Fig. 5C). Interestingly, when the
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16 Page 16 of 35
activity was assayed with 2,6-DMP as substrate, ≈ 60% of enzymatic activity was retained by BALL
360
laccase in up to 50% DMSO in the reaction mixture (Fig. 5C). Moreover, the residual activity of BALL
361
laccase after incubation at 25 °C for 24 h in the presence of 30% DMSO is ≈ 50% of its initial activity:
362
this represents an interesting feature for industrial applications requiring DMSO treatments. Our results
363
on Mt laccase once more differ from the report by [36] reporting that full activity was maintained up to
364
30% DMSO (and that 82% of the initial value was apparent at 50% DMSO) following incubation at 30
365
°C for 24 hours: the rationale for this discrepancy is not evident.
366
The effectiveness of enzymatic hydrolysis of lignocellulosic biomass is significantly enhanced if lignin
367
is modified before adding the biocatalyst. Here, different surfactants are known to increase the removal
368
of lignin and reduce nonproductive enzyme binding on the biomass surface, and, in particular, Tween-
369
80 was reported to increase enzymatic hydrolysis yields [45]. Accordingly, the enzymatic activity of
370
the different laccases in the presence of increasing Tween-80 concentrations was investigated. As
371
shown in Fig. 6, the enzymatic activity of laccases is not significantly affected in up to 5% (vol/vol) of
372
surfactant in the reaction medium, the only exception being the one from A. bisporus (Ab) (Fig. 6B).
373
Interestingly, laccase from the ascomycota Thielavia sp. (Th - LAC 5) showed higher enzymatic
374
activity in the presence of the 1% (vol/vol) surfactant (Fig. 6B) and the recombinant one from M.
375
thermophila exhibited a 2-fold increased activity in the presence of 5% (vol/vol) Tween-80 (Fig. 6A).
376
On this side, the insensitivity on detergent concentration of laccases from Trametes well correlates with
377
the observed beneficial effect of the nonionic surfactant Triton X100 on folding and stabilization [46].
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378 379
Conclusions
380
Laccases are important biocatalysts in a number of applications (see Introduction). In the past several
381
years, laccases were isolated from a variety of sources and strongly differed in terms of dependence of
17 Page 17 of 35
the enzymatic activity on pH. E.g., the optimal value with syringaldazine as substrate spans from pH 4
383
for P. cinnaribus laccase to pH 9 for R. vernicifera laccase [35].
384
For the sake of comparison, we now investigated the pH dependence of the enzymatic reaction of ten
385
laccases under identical conditions (Fig. 1): as a general rule, the optimum was at pH 3-4 and the
386
activity was lost at pH ≥ 7.0. The main exceptions were bacterial BALL and MmPPO, A. bisporus
387
(Ab) laccase and the recombinant one from M. thermophila, which were active at neutral pH values.
388
The pH range was limited by protein instability at pH ≤ 3.0 (Fig. 2) while all tested enzymes were
389
stable at pH ≥ 7.0. Notably, BALL laccase showed a pH-dependence of its catalytic activity that was
390
different with ABTS vs. 2,6-DMP as substrate (Fig. 1C). Concerning the influence of temperature,
391
activity for all tested laccases increased with temperature up to 80 °C, reaching a ≈ 1.5-2.0-fold higher
392
activity than the value determined at 20 °C (Fig. 3). The laccase from M. thermophila and the one from
393
the ascomycete Thielavia (Th – LAC 5) showed the lowest stability when incubated for 24 h at 37 °C.
394
Furthermore, many of the investigated laccases are not sensitive to sodium chloride thus favoring their
395
use in treating municipal and industrial waste water (e.g., textile waste waters contain from 0.4 to 16
396
g/L of NaCl) and sea waters. Activity of T. versicolor laccase was previously reported to be strongly
397
affected to sodium chloride [32]: our results confirmed these findings (Fig. 4). Interestingly, bacterial
398
laccases (BALL and MmPPO) retain significant activity in the presence of DMSO (up to 40% final
399
concentration), showing that they could be applied in lignin-degradation processes requiring solvents.
400
Moreover, the presence of surfactants useful in modifying lignin before adding enzyme to the
401
hydrolysis reaction does not affect the enzymatic activity of most laccases under investigation to a
402
significant extent.
403
Taken together, our comparison of ten laccases under identical conditions indicates that the bacterial
404
laccase from B. licheniformis compares favorably with the tested enzymes since it: i) shows a high
405
activity with both phenolic and nonphenolic substrates; ii) shows a good activity in a broad pH range,
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18 Page 18 of 35
even at values ≥ 7.0; iii) possesses good activity at high temperature and good stability for 24 h at 37
407
°C; and iv) is not significantly affected by sodium chloride and Tween-80. These properties and the
408
improved expression yield in E. coli as reported in this work (≈ 20 mg protein/L fermentation broth and
409
≈ 440 U/L) demonstrate that BALL is a well-suited laccase for a number of biotechnological
410
applications. Furthermore, the different laccases show peculiar properties (i.e., high stability at basic
411
pH values for P. ostreatus laccase or at 80 °C for M. thermophila and OB1, or at 30% DMSO for
412
MmPPO, etc.) that make them suitable for specific applications: the versatility of the available laccases
413
makes it possible to satisfy requirements related to different processes.
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414
Acknowledgements
416
This work was done as part of the ValorPlus project that has received funding from the European
417
Union's Seventh Framework Programme for research, technological development and demonstration
418
under grant agreement no FP7-KBBE-2013-7-613802 to AC and LP. This work was supported by
419
Biorefill grant (Project ID 42611813) to LP. FT is a PhD student of the “Dottorato in Biotecnologie,
420
Bioscienze e Tecnologie Chirurgiche” at Università degli studi dell’Insubria.
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TABLE 1 Kinetic parameters of different laccases (from Trametes species, fungi, and bacteria) on canonical laccase substrates. The
Enzyme
us
activity was assayed at pH 5.0 and 25 °C
ABTS
Catechol
Km (µM)
Vmax/Km
Vmax (U/mg)
Km (µM)
Vmax/Km
Vmax (U/mg)
Km (µM)
Vmax/Km
Sigma
13.1 ± 0.3
32 ± 3
0.42 ± 0.05
3.4 ± 0.1
410 ± 53
(8.3 ± 1.3)·10-3
8.6 ± 0.2
16.2 ± 1.7
0.53 ± 0.07
LAC C
5.6 ± 0.5
41 ± 9
0.14 ± 0.03
1.40 ± 0.03
334 ± 34
(4.2 ± 0.4)·10-3
1.20 ± 0.03
8.5 ± 1.1
0.14 ± 0.02
LAC 4
0.10 ± 0.01
55 ± 4
(0.20 ± 0.01)·10-2
0.030 ± 0.001
491 ± 45
(1.0 ± 0.3)·10-3
0.030 ± 0.001
14.5 ± 2.6
(2.1 ± 0.4)·10-3
M. thermophila (Mt)
15.5 ± 0.5
99 ± 2
0.16 ± 0.01
5.7 ± 0.4
2480 ± 207
(2.3 ± 0.3)·10-3
2.20 ± 0.06
6.0 ± 1.1
0.40 ± 0.04
P. ostreatus (Po)
7.9 ± 0.2
28 ± 2
0.28 ± 0.03
2.7 ± 0.1
770 ± 174
(3.5 ± 0.8)·10-3
2.9 ± 0.1
27.2 ± 1.4
0.11 ± 0.01
A. bisporus (Ab)
10.9 ± 0.7
602 ± 110
(2.0 ± 0.3)·10-2
11.0 ± 1.8
8900 ± 800
(1.2 ± 0.2)·10-3
0.90 ± 0.09
6700 ± 705
(0.10 ± 0.02)·10-3
OB1
3.2 ± 0.3
9±1
0.36 ± 0.07
0.89 ± 0.02
2400 ± 237
(0.4 ± 0.1)·10-3
0.76 ± 0.02
48 ± 6
0.015 ± 0.002
Thielavia (LAC 5)
8.0 ± 0.3
15 ± 3
0.53 ± 0.10
2.80 ± 0.05
2010 ± 108
(1.40 ± 0.06)·10-3
3.00 ± 0.03
20.0 ± 1.1
0.15 ± 0.08
BALL
23.0 ± 0.2
14 ± 2
1.62 ± 0.25
3.9 ± 0.2
1870 ± 351
(2.1 ± 0.5)·10-3
3.9 ± 0.3
2600 ± 157
(1.5 ± 0.2)·10-3
MmPPO
3.4 ± 1.0
1840 ± 230
(1.8 ± 0.6)·10-3
3.2 ± 0.2
11.2 ± 3.7
0.29 ± 0.07
1.6 ± 0.2
172 ± 30
(9.3 ± 0.2)·10-3
Ac
ce pt
Other fungi:
ed
Trametes:
M an
Vmax (U/mg)
2,6-DMP
Bacteria:
26 Page 26 of 35
FIGURE 1 Effect of pH on the enzymatic activity of various laccases at 25 °C. Enzymes from: (A) M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial sources:
ip t
BALL and MmPPO (the latter two enzymes were assayed on ABTS (black bars) or 2,6-DMP (gray
cr
bars) as substrates). The value at pH 5.0 is taken as 100%.
us
FIGURE 2 Effect of pH on the enzymatic stability of various laccases; residual activity was assayed after 24 h of incubation by measuring ABTS (or 2,6-DMP for MmPPO) oxidation, at 25 °C. Enzymes
an
from (A) fungal sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5);
M
(C) bacterial sources: BALL and MmPPO. The activity value at time = 0 at each pH value is taken as
te
d
100%.
FIGURE 3 Effect of temperature on the enzymatic activity of various laccases, determined by
Ac ce p
measuring ABTS (or 2,6-DMP for MmPPO) oxidation, at pH 5.0. Enzymes from (A) fungal sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial sources: BALL and MmPPO. The value at 25 °C is taken as 100%.
FIGURE 4 Effect of NaCl on the enzymatic activity of various laccases, determined by measuring ABTS (black bars) or 2,6-DMP (gray bars) oxidation, at pH 5.0, 25 °C. Enzymes from (A) fungal sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial sources: BALL and MmPPO. The value in absence of NaCl is taken as 100%. 27 Page 27 of 35
FIGURE 5 Effect of DMSO on the enzymatic activity of various laccases, determined by measuring ABTS (black bars) or 2,6-DMP (gray bars) oxidation, at pH 5.0, 25 °C. Enzymes from: (A) fungal
ip t
sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B) fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial
us
cr
sources: BALL and MmPPO. The value in absence of DMSO is taken as 100%.
FIGURE 6 Effect of Tween-80 on the enzymatic activity of various laccases, determined by measuring
an
ABTS (black bars) or 2,6-DMP (gray bars) oxidation, at pH 5.0, 25 °C. Enzymes from: (A) fungal sources: M. thermophila (Mt) and Trametes species: T. versicolor (Tv, Sigma), LAC C and LAC 4; (B)
M
fungal sources: P. ostreatus (Po), A. bisporus (Ab), OB1 and Thielavia sp. (LAC 5); (C) bacterial
Ac ce p
te
d
sources: BALL and MmPPO. The value in absence of Tween-80 is taken as 100%.
28 Page 28 of 35
Ac ce p
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Graphical Abstract
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Figure
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Figure
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Figure
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Figure
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Figure
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Figure
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