Enzyme and Microbial Technology 53 (2013) 33–37
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Laccases from Aureobasidium pullulans夽 Joseph O. Rich a,∗ , Timothy D. Leathers a,∗ , Amber M. Anderson a , Kenneth M. Bischoff a , Pennapa Manitchotpisit b a Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA b Biochemistry Unit, Department of Medical Sciences, Faculty of Science, Rangsit University, Patumthani 12000, Thailand
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Article history: Received 25 January 2013 Received in revised form 19 March 2013 Accepted 20 March 2013 Keywords: Aureobasidium pullulans Glycosylation Laccase Thermostability Trametes versicolor Pycnoporus cinnabarinus
a b s t r a c t Laccases are polyphenol oxidases (EC 18.104.22.168) that have numerous industrial and bioremediation applications. Laccases are well known as lignin-degrading enzymes, but these enzymes can play numerous other roles in fungi. In this study, 41 strains of the fungus Aureobasidium pullulans were examined for laccase production. Enzymes from A. pullulans were distinct from those from lignin-degrading fungi and associated with pigment production. Laccases from strains in phylogenetic clade 5, which produced a dark vinaceous pigment, exhibited a temperature optimum of 50–60 ◦ C and were stable for an hour at 50 ◦ C, unlike enzymes from the lignin-degrading fungi Trametes versicolor and Pycnoporus cinnabarinus. Laccase puriﬁed from A. pullulans strain NRRL 50381, a representative of clade 5, was glycosylated but had a molecular weight of 60–70 kDa after Endo H treatment. Laccase puriﬁed from strain NRRL Y-2568, which produced a dark olivaceous pigment, was also glycosylated, but had a molecular weight of greater than 100 kDa after Endo H treatment. Published by Elsevier Inc.
1. Introduction Laccases are polyphenol oxidases (EC 22.214.171.124) containing four copper atoms in their active sites, and are the largest subclass of blue multicopper oxidases . Laccases are well known as a component of fungal enzyme systems for lignin degradation. However, these enzymes can play numerous other roles in fungi, such as in host-pathogen interactions, sporulation, and morphogenesis . Laccases have broad substrate speciﬁcities, which can be further extended through the use of mediator systems [3,4]. Potential industrial applications include the degradation of textile dyes and toxic materials [5–9]. Laccases have been well studied in white-rot fungi. However, these enzymes are widely distributed in nature, and studies have sought to identify new microbial sources of laccase with novel properties [10,11]. Aureobasidium pullulans is considered to be a ﬁlamentous ascomycete in class Dothideomycetes, subclass Dothideomycetidae [12,13]. A. pullulans is well known as the source of the
夽 Mention of any trade names or commercial products in this publication is solely for the purpose of providing speciﬁc information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. ∗ Corresponding authors. Tel.: +1 309 681 6620; fax: +1 309 681 6040. E-mail addresses: [email protected]
(J.O. Rich), [email protected]
(T.D. Leathers). 0141-0229/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.enzmictec.2013.03.015
commercial polysaccharide, pullulan . It can also produce degradative enzymes, including xylanase [15,16]. A. pullulans is sometimes associated with the deterioration of painted wood [17,18], and certain strains can grow on lignin-related aromatic compounds [19–21]. However, little has been reported on laccase production by A. pullulans [22,23]. In the current study, 41 strains of A. pullulans were examined for their capacity to produce laccase.
2. Methods 2.1. Organisms and growth conditions A. pullulans strains used in this study were obtained from the ARS Culture Collection, Peoria, IL (NRRL strains). Control strains of the lignin-degrading fungi Trametes versicolor (strain ATCC 11235) and Pycnoporus cinnabarinus (ATCC 200478) were purchased from the American Type Culture Collection. Strains were cultured on potato dextrose agar plates at 28 ◦ C for 2 days. Preinocula were grown in 30 mL of malt extract broth in 300 mL ﬂasks, incubated at 28 ◦ C, 130 rpm (2 in displacement) for 3 days. Preinocula of T. versicolor and P. cinnabarinus were composed of mycelia clumps and required homogenization for 30 s with a PowerGen 700 homogenizer (Fisher Scientiﬁc). Preinocula were used to inoculate laccase induction cultures at 5% (v/v). Laccase induction medium contained the following per liter: 20 g glucose, 2.5 g l-asparagine, 0.05 mg thiamine–HCl, 1 g KH2 PO4 , 0.1 g Na2 HPO4 ·2H2 O, 0.5 g MgSO4 ·7H2 O, 0.01 g CaCl2 , 0.01 g FeSO4 ·7H2 O, 0.001 g MnSO4 ·4H2 O, 0.001 g ZnSO4 ·7H2 O, and 0.002 g CuSO4 ·5H2 O . Laccase induction cultures were 25 mL, grown in triplicate 125 mL ﬂasks at 30 ◦ C, 130 rpm. After two days, cultures were induced by the addition of 2,5-xylidine to a ﬁnal concentration of 200 M . After an additional day, cultures were assayed for laccase activity. Time course studies showed that one day after induction produced maximal activities (data not shown).
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2.2. Laccase activity assay
duplicate lanes of non-denaturing gels by staining with 2 mM ABTS in McIlvaine buffer (pH 4.4) for 30 min on a rocker shaker at room temperature .
One mL samples taken from induced cultures were clariﬁed by centrifugation for 5 min at 15,871 × g and assayed for laccase activity by measurement of the enzymatic oxidation of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, Fluka, Switzerland). Reaction kinetics were followed at 420 nm for 5 min using a Molecular Devices SpectraMax M5 plate reader. Reactions contained 30 L culture supernatant, 60 L McIlvaine buffer (pH 4.4), and 10 L ABTS (13 mM) in McIlvaine buffer (pH 4.4) at 30 ◦ C. At pH 4.4, 100 mL of McIlvaine buffer contains 44.1 mL of 0.2 M Na2 HPO4 and 55.9 mL of 0.1 M citric acid . Enzyme activity was expressed in units/mL (1 U = 1 mol product formed/min) and as speciﬁc activity (U/mg protein). Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA), based on the Bradford dye-binding method . To conﬁrm that laccase activity was enzymatic, duplicate samples were boiled for 15 min to serve as negative controls.
2.4. Enzyme puriﬁcation by medium pressure liquid chromatography Enzyme samples were puriﬁed using a medium pressure liquid chromatography system (Biologic Duoﬂow, Bio-Rad, Hercules, CA). Cell-free culture supernatants were applied to a Superose 6 10/300 GL gel ﬁltration column (GE Healthcare, Piscataway, NJ) equilibrated with McIlvaine buffer, pH 5.0. Alternatively, samples were applied to a 5.0 mL High Q anion exchange column (Bio-Rad) equilibrated in 20 mM sodium phosphate buffer, pH 6.5, and bound protein was eluted with an increasing gradient of 0.0–1.0 M NaCl in the same buffer. Puriﬁed laccases were treated with endoglycosidase H (Endo H, New England Biolabs, Ipswich, MA) according to manufacturer’s instructions.
3. Results and discussion
2.3. Polyacrylamide gel electrophoresis and zymogram analysis Samples were denatured by boiling for 5 min at 95 ◦ C in 2× SDS sample buffer (4.0% (w/v) SDS, 20% (v/v) glycerol, 0.005% (w/v) bromophenol blue, 0.126 M Tris–HCl pH 6.8 and 5.0% (v/v) ␤-mercaptoethanol) and applied to an SDS-PAGE gel (5.0% stacking, 10% resolving). After electrophoresis at 100 V for approximately 1 h, the gel was stained with Coomassie Brilliant Blue. Molecular masses of bands were estimated using the Bio-Rad Precision Plus All Blue Protein Standards (BioRad, #161-0373) and Kodak 1D analysis software. SDS-PAGE gels of puriﬁed laccases were stained with SYPRO Ruby protein gel stain (Invitrogen, Grand Island, NY) and used Bio-Rad Precision Plus Unstained Protein Standards (Bio-Rad, #161-0363). Non-denaturing polyacrylamide gel electrophoresis was performed under similar conditions, except that SDS was omitted, ␤-mercaptoethanol was excluded from the sample buffer, and samples were not boiled. Zymograms were prepared from
3.1. Strains of A. pullulans used in this study In a preliminary study, laccase production was observed in certain A. pullulans strains that also produce a dark vinaceous pigment . In some fungi, laccase production appears to be associated with pigment formation rather than lignin degradation . For the current study, 41 strains of A. pullulans were chosen based on their capacity to form pigment (Table 1). We recently completed a multilocus molecular phylogeny of A. pullulans . Interestingly, certain phenotypic traits, including pigment production, were
Table 1 Laccase production by strains of Aureobasidium pullulans. Cladea
Maximal laccase (mU/mL)
Speciﬁc activity (U/mg protein)
Color of cultureb
NRRL 58530 NRRL 58533 NRRL 58537 NRRL 58555 NRRL 58556 NRRL 58519 NRRL 58532 NRRL 58548 NRRL 50381 NRRL 58546 NRRL 58549 NRRL 58523 NRRL 58524 NRRL 58526 NRRL 58536 NRRL 58550 NRRL 58552 NRRL Y-2311 NRRL Y-2311-1 NRRL Y-6754a NRRL Y-12971 NRRL Y-12972 NRRL YB-4026 NRRL YB-4588 NRRL 58515 NRRL 58517 NRRL 58518 NRRL 58520 NRRL 58535 NRRL 58553 NRRL Y-12973 NRRL Y-12974 NRRL 58525 NRRL 58527 NRRL 58528 NRRL 58529 NRRL 58538 NRRL 58012 NRRL 62033 NRRL 62036 NRRL Y-2568
CU 17 CU 20 CU 24 CU 44 CU 45 CU 6 CU 19 CU 36 RSU 12 CU 33 CU 37 CU 10 CU 11 CU 13 CU 23 CU 38 CU 40
<0.01 <0.01 0.5 ± 0.3 <0.01 <0.01 18 ± 8.6 20 ± 3.4 39 ± 1.5 43 ± 1.3 <0.01 <0.01 6.5 ± 1.7 4.6 ± 0.1 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.1 ± 0.2 1.0 ± 0.2 <0.01 0.3 ± 0.0 <0.01 0.1 ± 0.2 <0.01 2.0 ± 0.5 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 1.5 ± 0.2 <0.01 110 ± 3.2
n/a n/a 0.04 ± 0.03 n/a n/a 1.2 ± 0.2 1.6 ± 0.5 3.6 ± 0.2 3.5 ± 0.2 n/a n/a 0.6 ± 0.1 0.4 ± 0.1 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a 0.0 ± 0.01 0.07 ± 0.01 n/a 0.03 ± 0.0 n/a 0.01 ± 0.01 n/a 0.2 ± 0.1 n/a n/a n/a n/a n/a n/a 0.1 ± 0.02 n/a 4.4 ± 0.1
Cream Cream Cream Cream Cream Vinaceous Vinaceous Vinaceous Vinaceous Cream Cream Gray Gray Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream Dark olivaceous
13 14 NDc a b c
ATCC 62921 ATCC 62922
CU 2 CU 4 CU 5 CU 7 CU 22 CU 41
CU 12 CU 14 CU 15 CU 16 CU 25 CBS 584.75 RSU 11 RSU 15
Phylogenetic clade according to the system of Manitchotpisit et al. . Grown on laccase induction medium. Not determined.
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3.2. Production of laccase by A. pullulans In laccase induction medium, all strains in phylogenetic clade 5 produced vinaceous pigments (Table 1). These strains also produced from 18 to 43 mU laccase/mL. Two strains in color variant clade 8 produced gray cultures in laccase medium, along with 4.6–6.5 mU laccase/mL. Strain NRRL Y-2568, of undetermined phylogenetic clade, produced dark olivaceous to black cultures, with laccase activity of 110 mU/mL (Table 1). The remaining 34 strains produced cream-colored cultures on laccase medium, with no more than 2.0 mU laccase/mL. Twenty-seven of these strains produced less than 0.01 mU laccase/mL. Thus, there appears to be an association between pigment production and laccase production in A. pullulans. This suggests that laccase activity in this organism may be involved in pigment production rather than lignin degradation. By comparison, the well-known lignin-degrading fungi T. versicolor (strain ATCC 11235) and P. cinnabarinus (ATCC 200478) produced 800–1500 mU laccase/mL under the same conditions. 3.3. Temperature optima and stability of laccases produced by A. pullulans Laccase from A. pullulans strain NRRL Y-2568 exhibited a temperature optimum of 35–45 ◦ C, while strains from clade 5 showed optima of 50–60 ◦ C (Fig. 1A). Optimal temperatures for fungal laccases vary greatly, but are often in this range . Laccases from strains in clade 5 were further compared with those from the lignindegrading fungi T. versicolor and P. cinnabarinus for temperature stability (Fig. 1B). Laccases from A. pullulans retained at least 90% of maximal activities for 1 h at 50 ◦ C, while those from T. versicolor and P. cinnabarinus lost 90% of their maximal activities under these conditions. Thus, laccases from A. pullulans appear to be relatively thermostable. 3.4. SDS-PAGE and zymograms of laccases produced by A. pullulans SDS-PAGE of laccases from A. pullulans shows diffuse, high molecular weight protein species of greater than 100 kDa (Fig. 2A). By comparison, lignin-degrading fungi T. versicolor and P. cinnabarinus produced distinct species of approximately 60–70 kDa.
Laccase activity (% maximum)
NRRL 58519 NRRL 58532 NRRL NRR LY Y-2568 2568
100 80 60 40 20 0 20
Temperature ( C) 140
Laccase activity (% maximum)
characteristic of particular phylogenetic clades. Strains in clade 1 generally produce cream-colored cultures in pullulan production medium (PM). However, they can produce olivaceous to black pigments in malt extract medium (ME) or on prolonged culture in yeast malt extract medium (YM). Strains in phylogenetic clade 5 produce a dark vinaceous pigment and were previously reported to produce laccase . Members of clade 6 produce olivaceous cultures in PM and black pigments on YM . Clade 8 includes strains originally described as “color variants” that can produce brilliant pigments of red, yellow or orange . We recently discovered that these strains produce color rings on exposure to diurnal cycles of light and darkness . Strains in clade 9 produce olivaceous to black pigments in both ME and PM . Strains in clade 10 also have been described as color variants . Strains in clade 11 are generally low pigment producers, included here as negative controls. They never produce pigment on YM and seldom do on ME, although a few strains produce olivaceous cultures in PM . Strains in clade 14 have been observed to produce red pigments, and these may also be color variants . Strain NRRL Y-2568, of undetermined phylogenetic clade, was included in this study because it produces dark pigment on YM. Strain NRRL 58012 (CBS 584.75), a member of clade 13, was included as the exneotype strain for the species (the exneotype is the strain designated to replace the lost type strain).
100 + 6
2 80 4 6
NRRL 58519 NRRL 58548 ATCC 11235 ATCC 200478
+ 32 34 36 38 40 42 44 46 48 50
60 40 20 0 0
Time (min) Fig. 1. Temperature optima (A) and stability (B) of crude laccases in cell-free culture supernatants of representative Aureobasidium pullulans strains NRRL 58519, NRRL 58532, NRRL 58548, and NRRL Y-2568. For temperature optima, laccase activities were measured at the indicated temperatures under otherwise standard assay conditions. For temperature stability, supernatants were incubated at 50 ◦ C for the indicated time and remaining laccase activities were measured under standard assay conditions. Temperature stability is also compared with laccases from Trametes versicolor strain ATCC 11235 and Pycnoporus cinnabarinus strain ATCC 200478.
Zymogram activity gels suggested that laccase activity from A. pullulans was associated with these diffuse high molecular weight proteins (Fig. 2B and C). The diffuse nature of these protein bands suggested that laccases from A. pullulans were modiﬁed by glycosylation. Most laccases from lignin-degrading fungi are glycosylated in the range of 10–20%, although this varies greatly [31,32]. 3.5. Puriﬁcation of laccases produced by A. pullulans Crude laccase from A. pullulans strain NRRL 50381 was puriﬁed by gel ﬁltration using a Superose 6 column (Fig. 3). Laccase activity eluted in fractions 20–56, separated from most of the protein and contaminating pigment. The speciﬁc activity of puriﬁed laccase was approximately 1.2 U/mg protein. Attempts to similarly purify laccase from A. pullulans strain NRRL Y-2568 using a Superose column were not successful (data not shown). Consequently, this enzyme was puriﬁed using a High Q anion exchange column (Supplementary Fig. 1) and the laccase eluted in fractions 14–30, separated from most of the protein and contaminating pigment. The speciﬁc activity was approximately 0.06 U/mg protein. Some residual laccase activity remained in the bulk of the remaining protein (about 0.01 U/mg protein in fraction 4). By comparison, the
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Fig. 2. SDS-PAGE (A), Native-PAGE (B), and ABTS zymogram (C) of crude extracellular proteins produced by Aureobasidium pullulans and control strains on laccase induction medium. (M) protein marker; (1) Trametes versicolor strain ATCC 11235; (2) Pycnoporus cinnabarinus strain ATCC 200478; (3) A. pullulans strain NRRL 58523; (4) A. pullulans strain NRRL 58548; (5) A. pullulans strain NRRL 58532; (6) A. pullulans strain NRRL 50381; (7) A. pullulans strain NRRL 58519.
speciﬁc activities of crude laccase from the lignin-degrading fungi T. versicolor (strain ATCC 11235) and P. cinnabarinus (ATCC 200478) produced under the same conditions were 330 U and 74 U/mg protein, respectively. Reported speciﬁc activities of puriﬁed laccases from the lignin-degrading fungi Trametes hirsuta (Coriolus hirsutus), Pleurotus ostreatus, P. cinnabarinus, and Chaetomium thermophilum (C. thermophilium) range from 37 to 610 U/mg [33–36]. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enzmictec. 2013.03.015. Puriﬁed laccase from A. pullulans strains NRRL 50381 (fractions 30–41) and NRRL Y-2568 (fractions 20–21) were concentrated by ultraﬁltration (Nanosep 30K Omega low protein binding spin cells) and treated with Endo H (Fig. 4). Untreated laccases from A. pullulans strains NRRL Y-2568 and NRRL 50381 were both greater than 100 kDa. After treatment with Endo H, laccase from NRRL 50381 was 60–70 kDa, while that from NRRL Y-2568 was of reduced molecular weight, but still greater than 100 kDa. The molecular weights of laccases from lignin-degrading fungi vary greatly, but are often in the 60–70 kDa range . After Endo H treatment, laccases from A. pullulans remained active but fell to 50–60% of
Fractions 1 2 3 4 5 6 7 8 9 10 15 20 25 30 35 40 45 50 55 58 60 62 64
10 8 6 4 2 0 -2
Absorbance (280 nm)
Fig. 4. SDS-PAGE of puriﬁed laccases from Aureobasidium pullulans before and after treatment with Endo H. (M) Bio-Rad Precision Plus protein standards; (1) A. pullulans strain NRRL 50381 before Endo H; (2) A. pullulans strain NRRL 50381after Endo H; (3) A. pullulans strain NRRL Y-2568 before Endo H; (4) A. pullulans strain NRRL Y-2568 after Endo H.
their original activities. Glycosylation may be important in enzyme secretion, and often enhances enzyme activity and stability [37,38]. 4. Conclusions
Volume (mL) Fig. 3. Puriﬁcation of laccase from Aureobasidium pullulans strain NRRL 50381 using a Superose 6 gel ﬁltration column.
In a survey of 41 strains of A. pullulans, laccase production was exclusively associated with pigment production. A. pullulans produces at least two types of laccase. Strain NRRL 50381, a representative of phylogenetic clade 5, produced laccase with a molecular weight of 60–70 kDa after Endo H treatment and temperature optimum of 50–60 ◦ C. Laccase from strains in clade 5 were stable for an hour at 50 ◦ C, unlike those from the lignin-degrading fungi T. versicolor and P. cinnabarinus. Strain NRRL Y-2568, of undetermined phylogenetic clade, produced laccase with a molecular weight of greater than 100 kDa after Endo H treatment and a temperature optimum of 35–45 ◦ C. Both laccases were glycosylated and exhibited lower speciﬁc activities than those from T. versicolor and P. cinnabarinus. Thus, laccases from A. pullulans differ from
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