Screening of Eucalyptus Wood Endophytes for Laccase Activity

Process Biochemistry 51 (2016) 589–598

Contents lists available at ScienceDirect

Process Biochemistry

jo urnal homepage: www.elsevier.com/locate/procbio

Screening of eucalyptus wood endophytes for laccase activity

a,∗ a b b

Úrsula Fillat , Raquel Martín-Sampedro , David Macaya-Sanz , Juan A. Martín ,

a c a

David Ibarra , María J. Martínez , María E. Eugenio

INIA-CIFOR, Forestry Products Department, Cellulose and Paper Laboratories, 28040 Madrid, Spain

Department of Natural Systems and Resources, School of Forest Engineers, Technical University of Madrid, 28040 Madrid, Spain

Centro de Investigaciones Biológicas (CIB), CSIC, Environmental Biology Department, 28040 Madrid, Spain

a r a

t i c l e i n f o b s t r a c t

Article history: New lignin-degrading microorganisms and their enzymes can contribute to more efﬁcient and environ-

Received 3 November 2015

mentally sound use of lignocellulosic biomass in future bioreﬁneries. In view of this, 127 endophytic fungi

Received in revised form 22 January 2016

were isolated from eucalyptus trees. Strains were tested for their ability to produce ligninolytic enzymes

Accepted 12 February 2016

in agar-plate medium containing ABTS, and 21 showed positive ABTS-oxidation. Positive strains in liquid

Available online 19 February 2016

medium confirmed that five produced laccase but no peroxidase activity. These strains were identified

as Hormonema sp. and Pringsheimia smilacis, of the family Dothioraceae, Ulocladium sp. (Pleosporaceae)

Keywords:

and Neofusicoccum luteum and Neofusicoccum australe (Botryosphaeriaceae). Laccase production by fungi

Endophytic fungi

of the family Dothioraceae is described here for the ﬁrst time.

Eucalyptus wood

Laccases To increase laccase production from these fungi, copper sulphate and ethanol were assayed as inducers.

Ligninolytic enzymes An increase in laccase activity of 85% was found for Hormonema sp. and N. luteum and 95% for N. australe.

◦

Screening No increase was obtained for P. smilacis. A maximum temperature of 40 C is recommended for possible

biotechnological applications with these four strains at pH 2 for N. luteum and N. australe, pH 4 for

Hormonema sp. and pH 4–5 for P. smilacis.

1. Introduction lignocellulose-degrading enzymes comprising peroxidases, oxi-

dases and reductases and low molecular mass compounds that

Wood is the most abundant biopolymer in nature and consists of mediate the action of these enzymes [2]. This ability makes white-

three main polymeric components: cellulose, hemicelluloses, and rot fungi useful for a wide range of biotechnological applications in

lignin [1]. Lignin gives plants mechanical resistance and, due to its industrial uses of lignocellulosic biomass [2]. The main oxidoreduc-

chemical complexity, very few organisms are capable of degrading tases studied in biotechnological applications are laccases, which

it [2]. Removal of lignin is a key factor in the pulp and paper industry act on phenol and aromatic compounds through the reduction

and the bioreﬁneries of the future for producing value-added chem- of molecular oxygen to water [3]. Basidiomycota is recognised as

icals, materials and biofuels based on cellulose and hemicelluloses. the most signiﬁcant phylum of white-rot fungi that secretes these

In the development of bioreﬁneries, biotechnology could play an enzymes [4]. However, some species in Ascomycota, such as Myce-

important role by providing efﬁcient and ecofriendly biocatalysts liophthora thermophila, also produce laccases of major interest for

for lignin degradation [3]. industry because of their high thermal stability and activity at alka-

Bacteria and fungi, notably wood-decaying fungi and, in par- line pH [5].

ticular, white-rot fungi, are among a number of microorganisms Most studies of wood-decaying fungi are based on advanced

that are capable of efﬁciently depolymerising and mineralising stages of wood degradation. However, some endophytic fungi could

lignin [2]. These fungi have developed a complex system of be involved in triggering the development of early stages of wood

decay. In nature, endophytes inhabit asymptomatic plant tissues,

living in symbiosis with their hosts. These fungi represent an enor-

∗ mous fungal diversity of as yet unknown magnitude and ecological

Corresponding author.

functions [6,7]. The discovery of their taxonomy and functions

E-mail addresses: ﬁ[email protected], yurs [email protected] (Ú. Fillat),

is a great challenge that would fall within the scope of recent

[email protected] (R. Martín-Sampedro), [email protected]

(D. Macaya-Sanz), [email protected] (J.A. Martín), [email protected] initiatives which propose the study of the diversity and effects

(D. Ibarra), [email protected] (M.J. Martínez), [email protected]

of microbial ecosystems in a uniﬁed, coordinated and interdisci-

(M.E. Eugenio).

http://dx.doi.org/10.1016/j.procbio.2016.02.006

590 Ú. Fillat et al. / Process Biochemistry 51 (2016) 589–598

plinary way [8,9]. After the death of the plant or any of its organs, 2.2. Primary ligninolytic activity screening in solid medium

some endophytes seem to move from a dormant state to become

◦

key primary colonisers involved in the degradation of plant tis- Extracellular enzymatic activities were ﬁrstly assayed at 22 C

sues [10]. In this phase, these fungi would have an advantage for three days in Petri dishes with a basal solid medium

over other competing saprophytes, having gained the niche before (KH2PO4, 1 g/L; C4H12N2O6, 0.5 g/L; CuSO4·5H2O, 0.001 g/L;

decomposition begins [11]. As a result of co-evolutionary processes, MgSO4·7H2O, 0.5 g/L; Fe2(SO4)3, 0.001 g/L; CaCl2·2H2O, 0.01 g/L;

certain endophytes have developed complex enzymatic systems MnSO4·H2O, 0.001 g/L; and yeast extract, 0.01 g/L) containing glu-

and metabolites [12,13], becoming highly specialised in degrading cose, 4 g/L; agar, 16 g/L; and 2,2 -azino-bis(3-ethylbenzothiazoline-

the wood polymers of their particular host species, especially dur- 6-sulphonic acid) (ABTS, from Roche Applied Sciences) [17]. ABTS

ing the initial stages of wood decomposition. Interestingly, other is a very sensitive substrate that allows rapid screening of fungal

specialised microorganisms, including dark septate endophytes strains producing extracellular ABTS-oxidising enzymes (such as

and arbuscular mycorrhizal fungi, modulate lignin biosynthesis in laccases and peroxidases) by means of color reaction (dark green

living plants (mainly in above-ground tissues) through their inter- color in solid media) [18]. Pycnoporus sanguineus and Trametes sp.

action with plant roots, and this way they can alter plant resistance I–62, both basidiomycete white-rot fungi obtained from the IJM col-

to pest and pathogens [14]. Other studies have emphasized the con- lection (Instituto Jaime Ferrán de Microbiología—CIB, CSIC), were

ditioning effect of environment on the outcome of plant-endophyte used as positive controls for wood-rot fungi. Plates were inocu-

interactions, and this way the non-static nature of these interac- lated with agar disks of active mycelia previously cultured in malt

tions [15,16]. However, little is known about the temporal and extract agar.

spatial variations in endophyte communities in large, long-lived

forest trees.

2.3. Identiﬁcation of fungal strains

It is necessary to further explore the role of wood-inhabiting

fungi in wood biodegradation and study their ligninolytic enzymes,

Strains showing laccase activity in solid medium were identiﬁed

as they could be an important alternative for degrading lignin or

by their speciﬁc Internal Transcribed Spacer regions (ITS1 and ITS2)

other recalcitrant compounds that cause environmental problems.

of ribosomal DNA isolated following the method described by Cenis

Technological application of these fungi in industrial processes,

[19], with minor modiﬁcations. A Polymerase Chain Reaction (PCR)

either before or in combination with secondary saprophytes, could

was then performed to amplify ITS regions. The thermal protocol

improve current technological performance. ◦ ◦

for ampliﬁcation was 4 min at 94 C, 30 cycles of 30 s at 94 C, 45 s

The aim of this study was to evaluate the production of ligni- ◦ ◦

at 55 C, and 1 min at 72 C, followed by a ﬁnal extension of 5 min

nolytic enzymes in liquid medium secreted by endophytic fungi ◦

at 72 C. In a ﬁnal volume of 25 ␮L, the PCR mix included 5–10 ng

isolated from eucalyptus wood as an alternative to the more

DNA template, 0.625 units of Taq (Bioline, London, UK), and a ﬁnal

studied wood-rotting fungal species. To reach this goal, after a

concentration of 0.2 ␮M of each primer [ITS1-F [8] and ITS4 [9]],

preliminary screening on solid medium containing ABTS, strains

0.2 mM each dNTP, and 1 mM MgCl2.

showing ABTS oxidation were identiﬁed and production of ligni-

After checking for positive ampliﬁcation in agarose gel (2%) with

nolytic enzymes, laccases and peroxidases in liquid medium was ®

SYBR Safe staining (Life Technologies, Carlsbad, CA, USA), PCR

analyzed. To increase laccase production from the selected fungi, ◦

product was puriﬁed by enzymatic incubation (20 min at 37 C fol-

the medium was supplemented with copper sulphate and ethanol ◦

lowed by 15 min at 85 C for enzyme deactivation). Enzymes used

as inducers. The characterization (stability and optimum pH and

were Exonuclease I (Exo I; Thermo Scientiﬁc, Waltham, MA, USA)

temperature) of the laccases secreted by these fungi were also

and Calf Intestine Phosphatase alkaline (CIP; AppliChem, Darm-

studied for different biotechnological applications.

stadt, Germany). Final concentrations were 1.4 units/␮L for Exo I

and 0.14 units/␮L for CIP. PCR puriﬁed products were delivered to

external facilities (Secugen, Madrid, Spain) for Sanger sequencing

in both directions.

For each strain, two sequences were obtained. These were

2. Materials and methods

aligned to achieve one consensus sequence for each strain. The

BLAST (MEGAblast algorithm) online programme, by GenBank

2.1. Fungal isolation

(NCBI, USA), was used to ﬁnd the most similar sequence (highest

bit score) within this database.

A total of 127 strains of endophytic fungi were isolated from

Eucalyptus globulus trees in ﬁve regions of Spain: Cantabria (coded

CA at the beginning of the strain name), Asturias (AS), Seville, (SE),

2.4. Secondary ligninolytic activity screening in liquid medium

Extremadura (EX) and Toledo (TO).

For endophyte isolation, four twigs approximately 2 cm in diam-

Only strains showing positive ABTS oxidation in solid medium

eter were collected from 1–3 trees from each sampling location.

were cultivated in liquid medium for 21

◦

After surface sterilisation with 70% ethanol and 4% bleach and

days at 22 C for ligninolytic enzyme determination. Pre-inocula

removal of the outermost bark, ﬁve explants per twig were cultured

were prepared by homogenising the mycelium from three-day-old

in the following media: Malt Extract Agar (MEA), Potato Dextrose

fungal cultures in MEA into modiﬁed Kirk medium (sacarose, 10 g/L;

Agar, Rose Bengal Chloramphenicol Agar, Yeast Extract Agar, and

KH2PO4, 2 g/L; aspargine, 1 g/L; tiamine·HCl, 1 mg/L; MgSO4·7H2O,

Sapwood Agar (coded M, P, R, Y, and S in the penultimate position

0.5 g/L; veratryl alcohol, 57 ␮L/L; CaCl2·2H2O, 0.1 g/L; CuSO4,

of the strain name). The ﬁrst four media were prepared follow-

0.27 mM; wheat straw, 2 g/L; and trace elements: nitriloacetic acid,

ing the manufacturer’s instructions (Cultimed, Panreac, Barcelona,

1.5 g/L; MnSO4·H2O, 0.5 g/L; NaCl, 1 g/L; FeSO4·7H2O, 0.1 g/L; CoCl2,

Spain). Sapwood agar was prepared by mixing 50 g ground, dried

0.1 g/L; ZnSO4·7H2O, 0.1 g/L; CuSO4·5H2O, 0.01 g/L; AlKSO4·12H2O,

and autoclaved eucalyptus twigs with 7.5 g agar in 500 mL water.

0.01 g/L; H3BO3, 0.01 g/L; NaMoO4·2H2O, 0.01 g/L). After three days,

Explants were axial twig slices approximately 2 mm thick, includ-

4 mL pre-inoculum was inoculated in 500 mL Erlenmeyer ﬂasks

ing phloem and xylem tissue. After two weeks of incubation in a

containing 200 mL of this medium. Culture supernatants were peri-

◦

dark chamber at 25 C, growing fungal strains were transferred to

odically taken from two replicate ﬂasks to measure enzymatic

separate plates containing MEA.

activities. Mycelium was separated from the fungal culture by

Ú. Fillat et al. / Process Biochemistry 51 (2016) 589–598 591

centrifugation at 13,000 rpm for 10 min and enzyme activity was recently isolated strains. However, some studies used strains iso-

checked in the supernatant. lated from decayed wood in a speciﬁc geographical area, such as

Laccase activity was assayed by oxidation of ABTS forests in Zimbabwe [22], Tunisia [23] and Spain [24]. Literature

and monitored via absorbance increase at 436 nm describing ligninolytic enzyme production by endophytes is scarce.

␧ ·+ −1 −1

( ABTS = 29300 M cm ). The reaction mixture contained In solid medium, ligninolytic activity has previously been reported

5 mM ABTS and 100 mM sodium tartrate buffer at pH 4, and for endophytic strains of the family Xylariaceae using Poly R-478

enzyme activity was determined at room temperature for 1 min. as indicator [25] and of the genus Botryosphaeria using ABTS [26].

Peroxidase activity was assayed as laccase activity in the presence Ligninolytic activities in fungi isolated from healthy wood samples

of 0.1 mM H2O2. Enzymatic activity was expressed as international from Chilean tree species were found using Poly R-478 [27]. To the

units (IU), deﬁned as the amount of enzyme needed to oxidise best of our knowledge, this is the ﬁrst screening study to use a large

1 ␮ mol of substrate per minute. amount of endophytic strains isolated from eucalyptus trees.

Stability at room temperature was determined by incubating the A total of 127 strains of endophytic fungi isolated from E. glob-

◦

cultures for 4 h at 25 C. Laccase activity was measured as described ulus trees in ﬁve regions of Spain were screened on solid medium

above. for their ability to oxidise ABTS, a substrate generally used for rapid

screening of extracellular ligninolytic enzyme production [18].

A positive reaction, determined as dark green rings around the

2.5. Laccase production

mycelial colonies due to oxidation of ABTS to its cation radical

·+

(ABTS ), appeared with some isolated fungi, suggesting production

Strains that showed a positive ligninolytic activity in Kirk’s

of extracellular oxidoreductases such as laccases and peroxidases.

liquid medium were cultivated in various liquid cultures for lac-

Among all the strains of endophytic fungi assayed, 21 fungal strains

case production. Pre-inocula were prepared by homogenising the

were able to oxidise ABTS to varying degrees (Table 1). Eight strains

mycelium from three-day-old fungal cultures in MEA into Kirk’s

produced high ABTS oxidation after 48 h, where the [diameter

liquid medium and after three days, 4 mL of this culture was

of green halo/diameter of colony] ratio was higher than 1.5, and

used to inoculate 200 mL modiﬁed Czapek Dox medium (KH2PO4,

ﬁve strains produced medium oxidation (diameter halo/diameter

1 g/L; C4H12N2O6, 2 g/L; yeast extract, 1 g/L; MgSO4·7H2O, 0.5 g/L;

colony ratio 1.5–1.2). The 21 strains selected to continue the study

KCl, 0.5 g/L; and 1 mL of a mineral solution with trace elements:

were those able to oxidise ABTS during the ﬁrst three days of incu-

Na2B4O7, 0.092 mg/L; MnSO4·H2O, 0.1 mg/L; (NH4)6Mo7O2·4H2O,

bation (diameter halo/diameter colony ratio ≥1). However, the

0.01 mg/L; FeSO4·7H2O, 0.5 mg/L; ZnSO4·7H2O, 0.1 mg/L; and

green halos were smaller than those obtained with P. sanguineus

CuSO4·5H2O, 0.01 mg/L) contained in 500 mL Erlenmeyer ﬂasks.

and Trametes sp. I–62 strains (diameter halo/diameter colony ratio

Two concentrations of CuSO4 (0.15 or 0.30 mM) and 1% (v/v)

approx. 2 at 48 h) used as a model of white-rot fungi able to secrete

ethanol were tested as laccase inducers in the modiﬁed Czapek Dox

a high level of laccases [28,29].

medium. Fungal endophytes were grown using an orbital shaker at

◦

120 rpm, 22 C, for 30 days. Two culture supernatants were peri-

3.2. Identiﬁcation of fungal strains

odically taken from two replicate ﬂasks to measure enzymatic

activities. Before measurement, mycelium was separated from the

Endophytic fungi represent huge fungal diversity [6]. Often iso-

culture liquid by centrifugation at 13,000 rpm for 10 min. Enzy-

lates cannot be identiﬁed and are putatively assigned to a new

matic activities were analyzed as described in Section 3.4.

taxonomic class. Therefore isolation and genetic characterisation

of fungal communities associated with forest species is of great

2.6. Laccase stability and optimum pH and temperature studies

interest when estimating their diversity, interactions and possible

technological applications.

For these studies, fungal cultures enriched in laccase were

Endophyte strains were identiﬁed by sequencing their internal

obtained by ﬁltration from experiments assayed in Section 3.5 on

transcribed spacer regions of ribosomal DNA and contrasting these

the day of maximum laccase activity. Optimum pH was investigated

with the fungal ITS sequences deposited at GenBank. Accessions

in pH range 2–8 at room temperature in duplicate. The activity of

with best matches were retained (Table 1) and used for puta-

each fungal culture at varying pH was measured as described in Sec-

tive taxonomic identiﬁcation. It should be noted that this method

tion 3.4 using different buffers (sodium phosphate 100 mM for pH 7

cannot always provide a precise identiﬁcation (i.e. high identity

and 8 and sodium citrate 100 mM for pH range 2–6). To determine

percentage), given that the GenBank database, though extensive,

optimum temperature, laccase activity was measured as described

still lacks information about many fungal groups. In this study,

in Section 3.4, in 100 mM sodium citrate buffer at optimum pH and

◦ strains are referred to by their most similar identiﬁed accession

a range of temperatures from 22 to 80 C in duplicate.

but their taxonomic status could later be revised (especially where

Thermal stability was determined by incubating fungal cultures

the identity percentage is moderate; see Table 1) when taxonomic

in 100 mM sodium citrate buffer at the optimum pH previously

◦ advances in forest endophytes have been completed.

obtained for each strain and at temperatures from 40 to 70 C for 4 h.

Selected fungal strains showing positive ABTS-oxidation in solid

Laccase stability towards pH 2–4 or 5 (depending on optimum pH)

◦ ◦ medium were identiﬁed and classiﬁed as ascomycetes of the classes

was studied in samples incubated at 40 C and 50 C for 4 h. Laccase

Dothideomycetes (genera Neofusicoccum, Ulocladium, Lophiostoma,

activity in both studies was measured as described in Section 3.4

Pringsheimia, Hormonema, Dothiorella, Pyrenochaeta and Conio-

from two replicate tubes.

thyrium) and Sordariomycetes (genus Leiosphaerella) (Table 1). Four

These results were expressed as residual activity (%) related to

strains belonged to genera Phaeomoniella and Tumularia and were

the maximum laccase activity measured in each study.

of uncertain families, given that their taxonomic status has not been

determined. The genus Phaeomoniella belongs to the class Euro-

3. Results and discussion tiomycetes and Tumularia is assigned to the phylum Ascomycota in

an incertae sedis status.

3.1. Primary screening: solid media Interestingly, most of the selected strains (15 out of 21) belonged

to the class Dothideomycetes, namely to the orders of Pleosporales

Most studies on screening fungi for ligninolytic enzymes have (families Lophiostomataceae, Montagnulaceae, Coniothyriaceae

been conducted using culture collection strains [20,21] rather than and Pleosporaceae), Botryosphariales (family Botryosphaeriaceae)

592 Ú. Fillat et al. / Process Biochemistry 51 (2016) 589–598

Table 1 Strain.

a b c

Strains ABTS oxidation Sequence length Most similar identiﬁed accession Score (bits) Identity (%) Accession family Accession species

AS1M1 +++ 486 AJ244257 832 98 Dothioraceae Pringsheimia smilacis

AS2S4 + 486 JQ044435 839 98 Incertae sedis Phaeomoniella niveniae

AS3R2 +++ 415 EU770246 767 100 Lophiostomataceae Lophiostoma corticola

AS3R3 +++ 531 AF182378 870 98 Dothioraceae Hormonema sp.

CA1R5 + 493 JX496118 911 100 Montagnulaceae Paraconiothyrium variabile

CA3M1# +++ 361 JX271823 667 100 Botryosphaeriaceae Neofusicoccum luteum

CA3R3# +++ 485 JX271823 887 99 Botryosphaeriaceae Neofusicoccum luteum

EX2S6 +++ 430 GQ153128 612 93 Incertae sedis Phaeomoniella effusa

EX3M2 +++ 406 JX982411 750 100 Pleosporaceae Ulocladium sp.

EX3S1 ++ 338 JF440976 442 91 Amphisphaeriaceae Leiosphaerella praeclara

EX3S2 ++ 443 JF440976 564 90 Amphisphaeriaceae Leiosphaerella praeclara

SE1M1 + 490 KF702388 905 100 Botryosphaeriaceae Neofusicoccum australe

TO1M1§ ++ 881 JQ411416 1574 99 Botryosphaeriaceae Dothiorella sarmentorum

TO1Y1§ + 853 JQ411416 1520 99 Botryosphaeriaceae Dothiorella sarmentorum

TO2M1§ ++ 896 JN693506 1594 98 Botryosphaeriaceae Dothiorella iberica

TO2M2 + 508 GU062248 911 99 Pleosporaceae Pyrenochaeta cava

TO2R2 +++ 467 AF182378 767 100 Dothioraceae Hormonema sp.

TO2Y1 ++ 536 KF251209 900 97 Coniothyriaceae Coniothyrium carteri

TO3M2 + 529 JQ044435 894 97 Incertae sedis Phaeomoniella niveniae

TO3M4 + 795 HQ115698 905 99 Pleosporaceae Pyrenochaeta cava

TO3P1 + 516 AY265337 795 95 Incertae sedis Tumularia aquatica

In bold strains producing laccase in liquid medium.

Strains sharing symbols (#,*,§) possessed identical ITS sequences but differed in length.

+++ (high oxidation), ++ (medium oxidation) and + (low oxidation).

Bit score and identity percentage between the strain sequence and the most similar identiﬁed accession in GenBank.

Other accessions share the same bit score and identity percentage, and were also identiﬁed as the same taxon.

a) N. luteum b) N. australe

0.30 0.30 ) ) L 0.25 L

m 0.25 m / / U U ( 0.20 ( 0.20 y y t t i i v v i i t 0.15 t 0.15 c c a a e 0.10 e 0.10 s s a a c c c 0.05 c 0.05 a a L

0.00 L 0.00

0 5 10 15 20 25 30 0 5 10 15 20 25 30

Days Days c) Hormonema sp. d) P. similacis

0.30 0.30 ) ) L 0.25 L m 0.25 / m / U U (

0.20 (

y 0.20 t y i t i v i v t 0.15 i t

c 0.15 c a a

e 0.10 e

s 0.10 s a a c c

c 0.05

c 0.05 a a L

0.00 L 0.00

0 5 10 15 20 25 30

Days Days

Cu 0.15mM

Cu 0.30mM

Cu 0.15mM; 1%EtOH

Cu 0.30mM; 1% EtOH

Fig. 1. Laccase production in modiﬁed Czapek Dox medium supplemented with two CuSO4 concentrations (0.15 and 0.30 mM) and with or without 1% (v/v) ethanol for (a)

N. luteum, (b) N. australe, (c) Hormonema sp. and (d) P. smilacis. Error bars represent enzymatic activities from two replicate ﬂasks.

Ú. Fillat et al. / Process Biochemistry 51 (2016) 589–598 593

a) b)

Fig. 2. Optimum pH (a) and temperature (b) of N. luteum, N. australe, Hormonema sp. and P. smilacis. Error bars represent laccase activities in duplicates.

and Dothideales (family Dothioraceae). A few were of the class activity in fungi of the family Dothioraceae (strains Pringsheimia

Sordariomycetes (genus Leiosphaerella) and Eurotiomycetes (genus smilacis (AS1M1) and Hormonema sp. (AS3R3)).

Phaeomoniella). The predominance of Dothideomycetes in this In contrast, no peroxidase activity was found in any of the fungal

selection could be because (i) they are more abundant in endo- strains. The literature has described various patterns of ligninolytic

phytic ﬂora, (ii) they are more easily isolable than the others or enzymes in basidiomycetes [40], including those that produce only

(iii) they produce more oxidoreductase activity. However, this pre- lignin peroxidase (LiP) and manganese peroxidase (MnP); or LiP,

dominance was not observed in the endophytic composition of MnP and laccase [41,42]; or laccase and either LiP or MnP [43,44];

eucalyptus twigs (unpublished data). As we preserved and iden- MnP and versatile peroxidase [45] or only laccase [34,46]. Some

tiﬁed only strains with ABTS oxidative activity, we cannot know if ascomycetes have also been reported to degrade lignin [32] and

the abundance of Dothideomycetes is due to the ease of isolation some produce only laccase, without either LiP or MnP activities

of this class or to a higher oxidoreductase capability. Positive ABTS- [34,47]. Nevertheless, synthesis and secretion of these enzymes is

oxidising enzymes produced by fungi from the family Dothioraceae often induced by limited carbon or nitrogen levels. Moreover, per-

are described here for the ﬁrst time. oxidase production, such as LiP and MnP, is generally optimum at

high oxygen tension but is repressed by agitation in submerged

liquid [48], whereas laccase activity is often enhanced by agitation

3.3. Secondary screening: liquid cultures

or the presence of aromatic compounds or organic solvents [23]. It

is therefore possible that the culture conditions used in this study

The 21 selected fungal strains with ability to oxidise ABTS on

could explain the lack of peroxidase activities in the endophytic

solid medium were screened in modiﬁed Kirk’s liquid medium to

fungi cultures.

quantify laccase secretion and peroxidase activities. Among the

The role of these ﬁve strains in lignin degradation has been also

strains studied, only Neofusicoccum luteum, Neofusicoccum aus-

studied during targeting speciﬁcally cellulose-based industrial pro-

trale, Pringsheimia smilacis, Hormonema sp. and Ulocladium sp.

cesses applications. They were used as biological pre-treatment

were able to produce laccase activity, reaching maximums of

to enhance the enzymatic sacchariﬁcation [49] and chemical and

0.01–0.04 U/mL by the ﬁfth day in all cases. These ascomycete

mechanical pulping of eucalypt wood [50], proving the high poten-

strains belong to the class Dothideomycetes, as mentioned above.

tial of endophytic fungi in these biotechnological applications

Ascomycetes have largely been ignored as being responsible for

[49,50].

the biodegradation of lignocellulosic materials, although some

show a clear ability to degrade lignin [17,30,31]. Laccase activ-

ity in liquid medium was described for some endophytic fungi 3.4. Laccase production

in earlier studies [32]. These include members of the class Sor-

dariomycetes, such as some fungi of the family Xylariaceae [25], Laccase production is inﬂuenced by several factors, including

Fusarium proliferatum, of the family Nectriaceae [32], and Podospora growth medium composition, pH, C/N ratio, presence of inducers,

anserina, of the family Lasiosphaeriaceae. Similarly, members of temperature and aeration rate [20]. Therefore, a good strategy to

the class Dothideomycetes, such as Monotospora sp., of the family increase laccase production would be to supplement the medium

Hysteriaceae [33], and some species of the family Botryosphaeri- with laccase inducers. Laccase induction by copper is widespread

aceae, have been reported to produce laccase activity [26,34,35], among fungi and essential for the catalytic activity of the enzyme

consistent with Botryosphaeriaceae strains N. australe (SE1M1) [51]. According to the literature, the amount of copper required to

and N. luteum (CA3R3) tested in the present study. Three lac- enhance laccase production varies greatly among fungi, from 0.1 to

cases from endophytes have been puriﬁed and characterised: 1 mM [37,52]. Ethanol has also been studied as a growth substrate

Cladosporium cladosporioides, of the family Davidiellaceae [36], of fungi for laccase production from 0.1 to 5% (v/v) [52–54].

Paraconiothyrium variabile, of the family Montagnulaceae[37] and Fig. 1 shows laccase production for N. luteum, N. australe, P. smi-

Trichoderma harzianum, of the family Hypocreaceae [38]. In the lacis, and Hormonema sp. using modiﬁed Czapek Dox medium with

family Pleosporaceae, we detected laccase production in the strain CuSO4 and ethanol as inducers. Ulocladium sp. was excluded from

Ulocladium sp. (EX3M2), which was also reported in related strains the study because laccase produced by this strain was quickly deac-

[39]. However, as far as we know, this is the ﬁrst report of laccase tivated in 2 h (loss of 36% laccase activity at room temperature and

594 Ú. Fillat et al. / Process Biochemistry 51 (2016) 589–598

a) b)

c) d)

Fig. 3. Laccase stability related to temperature in samples incubated at optimum pH for (a) N. luteum, (b) N. australe, (c) Hormonema sp. and (d) P. smilacis. Error bars represent

laccase activities in duplicates.

◦

70% activity at 40 C). As seen in Fig. 1a, the highest laccase produc- mization using multiple micronutrients and genetic modiﬁcation

tion was achieved by N. luteum, at 0.28 U/mL after 20 days in the [57]. Namely, to increase laccase production in our experiments,

presence of 1% (v/v) ethanol and CuSO4 0.30 mM. N. australe and a further study taking into account other inducers and different

Hormonema sp. showed maximum laccase activity (0.14 U/mL) in carbon and nitrogen sources, pH and operational conditions will

the presence of 1% (v/v) ethanol and 0.30 mM CuSO4 (at day 15 and be necessary to complete the characterisation of these enzymes

30, respectively; Fig. 1b and c). This improvement in laccase produc- and analyze their potential for industrial applications. However, it’s

tion in some strains due to the presence of ethanol was previously worth reminding that laccase overexpression in an active form in

observed in cultures of white-rot basidiomycetes Trametes ver- heterologous systems is difﬁcult but laccase genes have been suc-

sicolor, Pycnoporus cinnabarinus and Ganoderma ludicum [52–54]. cessfully cloned into ﬁlamentous fungi and yeasts that led to very

However, in the case of P. smilacis, laccase production was not promising results [57].

improved by addition of ethanol (maximum activity 0.04 U/mL after

48 h without ethanol, Fig. 1d) and was even lower at longer times.

3.5. Laccase stability and optimum pH and temperature

This effect was reported in earlier studies; less than 40% activity

was observed in P. sanguineus when 4.3% (v/v) ethanol was added

Initial laccase activity for each fungal culture, measured at pH

to the medium [55] and Trametes sp. I-62 showed a 60% activity

4 and room temperature, was 0.25, 0.15, 0.15 and 0.05 U/mL for

decrease in the same conditions [28]. The inﬂuence of inducers

N. luteum, N. australe, Hormonema sp. and P. smilacis, respectively.

on laccase activity therefore depends strongly on the fungi stud-

Fig. 2 shows the laccase activity in liquid culture of the fungi at

ied. No peroxidase activity was found in any of the samples in the

different pH and temperatures. Maximum activity for laccase from

media and conditions tested. Laccase production found at opti-

Hormonema sp., N. australe and N. luteum was obtained at pH 2

mum culture conditions in other fungi such as white-rot fungi P.

(Fig. 2a). For N. luteum, activity decreased abruptly at pH higher than

cinnabarinus and G. ludicum was 9 U/mL and 74 U/mL, respectively

3 (3% residual activity at pH 3). For N. australe and Hormonema sp.,

[52,56]. Other endophytic fungi such as C. cladosporioides, Mono-

enzymatic activity decreased signiﬁcantly at pH higher than 4 (67%

tospora sp. and Botryosphaeria sp. produced 3 U/mL, 13 U/mL and

and 41% relative activity at pH 4, respectively). P. smilacis showed

7 U/mL, respectively [33,34,36]. Laccase activities measured in liq-

optimum activity at pH 4, decreased 80% at pH 7 and was inactive

uid cultures of endophytic fungi studied were lower than those

at pH 8.

obtained by white-rot fungi in previous studies [28,57]. Different

The catalytic performance of laccases is greatly inﬂuenced by

actions could be performed to efﬁciently produce this enzyme at an

their activity and stability at different pH and temperature con-

industrial scale. These methods include fermentation media opti-

ditions. The pH activity proﬁles of laccases are often bell shaped

Ú. Fillat et al. / Process Biochemistry 51 (2016) 589–598 595 a) N. luteum b) N. aus trale

100 100 ) ) % % 80 80 ( ( y y t t i i v v i i 60 60 t t c c a a l l a a 40 40 u u d d i i s s

e 20

e 20 R R

0 0

0 1 2 3 4 0 1 2 3 4

time (h) time (h)

c) H ormonema sp. d) P. smilacis

100 100 ) ) % % 80 80 ( ( y y t t i i v v i i 60 60 t t c c a a l l a a 40 40 u u d d i i s s

e 20

e 20 R R

0 0

0 1 2 3 4 0 1 2 3 4 time (h) time (h)

pH2 pH3 pH4 pH5

◦

Fig. 4. Laccase stability related to pH in samples incubated at 40 C for (a) N. luteum, (b) N. australe, (c) Hormonema sp. and (d) P. smilacis. Error bars represent laccase activities

from two replicate tubes.

◦

when measured with phenolic substrates. Several laccases show other fungi. At 80 C, Hormonema sp. reached maximum activity.

maximum activity measured with ABTS at a pH range of 4–6, such According to the literature, the optimum temperature of laccases

◦

as ascomycete Trichoderma harzianum [38], or even at higher pH usually ranges from 30–60 C [36,52,58] but higher temperatures

◦

ranges of 4–9, e.g., basidiomycetes P. cinnabarinus, Trametes villosa (70 C) have been reported for G. lucidum [60].

and ascomycete M. thermophila [5]. Other basidiomycete laccases Enzymatic thermostability was studied in fungal cultures incu-

show optimum activity at pH 3, e.g., P. sanguineus [55] and T. bated at the optimum pH obtained. Residual enzymatic activity was

versicolor [58], as do some ascomycete fungal laccases, such as related to the maximum laccase activity measured in this study.

Paraconiothyrium variabile [37], C. cladosporioides [36] and F. pro- Maxima activities were 1.5 U/mL at pH 2 for N. luteum, 0.12 U/mL

liferatum [32]. The ﬁnal two are also described as endophytes. Like at pH 2 for N. australe, 0.15 U/mL at pH 3 for Hormonema sp. and

most fungi studied, P. smilacis shows maximum activity at pH 3–4, 0.05 U/mL for P. smilacis at pH 4. Different behavior was observed

but an unusually low optimum pH was found for endophytic cul- for each strain. Deactivation was not observed in N. luteum at

◦

tures from Hormonema sp., N. australe and N. luteum. Nevertheless, 40 C. A rapid increase in enzymatic activity was observed for this

low optimum pH around 2.5 were previously observed for basid- strain during the ﬁrst hours, reaching maximum enzymatic activ-

iomycete laccases from Ganoderma fornicatum, G. lucidum [52] and ity at 4 h (1.6 U/mL corresponds to 100% residual activity, Fig. 3a).

ascomycete Mauginiella sp. [59]. Optimum pH in the acidic range Slow deactivation was observed at this temperature after 2 h. Total

suggests that activity is physiologically more signiﬁcant. Fungi enzymatic deactivation was observed in samples incubated at tem-

◦

growing in acidic environments come into contact with various peratures above 60 C during the ﬁrst hour. This behavior was

acidic plant phenols or pesticides, and the lower the optimum pH observed by Farnet et al. [61] and Xu et al. [62], who found that

of the laccase is, the more effective oxidation of toxic compounds pre-incubation of laccases from M. thermophila, Scytalidium ther-

◦

will be [51]. mophilum and Myrothecium verrucaria at 40–50 C greatly increased

◦

Optimum temperature was studied in the range of 22–80 C. laccase activity. As seen in Fig. 3b, ABTS-oxidising activity produced

The higher the temperature, the greater laccase activity was for by N. australe was the least stable of all strains (50% activity loss

◦ ◦ ◦

the four strains (∼25% higher at 60 C than activities measured at at 40 C and 80% at 50 C in 30 min) and totally deactivated above

◦

room temperature, Fig. 2b). P. smilacis shows an abrupt decrease 60 C in the ﬁrst 30 min. Hormonema sp. was slightly more stable

◦

of laccase activity at 80 C. This effect was not observed for the than N. australe, although similar deactivation was obtained, in this

596 Ú. Fillat et al. / Process Biochemistry 51 (2016) 589–598 a) N. luteum b) N . australe

100 100 ) ) % % 80 80 ( ( y y t t i i v v i i 60 60 t t c c a a l l

a 40 a 40 u u d d i i s s

e 20

e 20 R R

0 0

0 1 2 3 4 01234

time (h) time (h)

c) H ormonema sp. d) P. smilacis

100 100 ) ) % 80 % 80 ( ( y y t t i i v v i 60 i 60 t t c c a a l l

a 40 a 40 u u d d i i s s

e 20

e 20 R R

0 0

0 1 2 3 4 0 1 2 3 4 tim e (h) time (h) pH2 pH3 pH4 pH5

◦

Fig. 5. Laccase stability related to pH in samples incubated at 50 C for (a) N. luteum, (b) N. australe (c) Hormonema sp. and (d) P. smilacis. Error bars represent laccase activities

from two replicate tubes.

◦

case in 1 h (Fig. 3c). Laccase activity in P. smilacis was more stable strain N. australe incubated at pH 3 and 4 at 40 C compared to

◦

than in Hormonema sp. and N. australe at 40 C (only 50% deacti- the culture incubated at pH 2 at the beginning of the treatment.

◦

vation was observed in 4 h), although at temperatures above 50 C After 30 min of incubation, similar results were obtained for these

the enzyme was highly deactivated. It has been reported that tem- three pHs. Fig. 4c shows that residual activity in the culture from

perature stability of laccases varies considerably depending on the Hormonema sp. was higher in samples incubated at pH 4 than at

◦

fungus. In general, laccases are stable at 30–50 C and rapidly lose pH 2 and 3 (up to 50% after 4 h). A similar effect was observed

◦

activity at temperatures above 60 C [5,36–38]. For example, lac- with culture from P. smilacis (Fig. 4d), but the differences were

cases from endophytic ascomycete Paraconiothyrium variabile and greater. Residual activity was higher (80% in the ﬁrst hour) in sam-

basidiomycetes P. cinnabarinus and T. villosa retained 50%, 80% and ples incubated at pH 5 and 4 than in samples incubated at pH 3 or 2.

◦

95% of their initial activity at 50 C after 1 h, respectively [5,38] and Similar results regarding enzymatic activity were obtained for the

◦ ◦

laccase from T. harzianum retained 70% after 1 h at 55 C [38]. four strains when fungal cultures were incubated at 50 C (Fig. 5)

◦

Regarding pH stability, a pH range of 2–4 was studied for Hor- compared to cultures incubated at 40 C, but a higher loss of activ-

monema sp., N. australe and N. luteum and pH 5 for P. smilacis, based ity was observed in N. australe, Hormonema sp. and P. smilacis. No

◦

on previous results for optimum pH (Fig. 2a). Fungal cultures were deactivation was observed for N. luteum at pH 4 and 50 C.

◦ ◦ ◦

incubated at 40 C and 50 C taking into account earlier results for Based on these results, a maximum temperature of 40 C is rec-

optimum temperature (Fig. 2b) and thermostability (Fig. 3). Fig. 4 ommended for possible biotreatments with these four strains. pH

shows residual laccase activity measured at various time intervals 2 is suggested for N. luteum and N. australe and pH 4 or 5 for P. smi-

◦

for samples incubated at 40 C at different pH. Residual activity was lacis. Even though optimum pH for Hormonema sp. was pH 2 and

related to the maximum laccase activity measured in this study 3, pH 4 would be a better choice for biotreatments at times longer

(1.57 U/mL, 0.12 U/mL, 0.15 U/mL and 0.05 U/mL for N. luteum, N. than 30 min.

australe, Hormonema sp. and P. smilacis, respectively). As seen in

Fig. 4a, the increase in enzymatic activity observed in N. luteum ◦

4. Conclusions

when the fungal culture was incubated at pH 2 and 40 C was also

observed at pH 3 to a lesser extent. This effect was not produced

In a search for new enzymes that could be applied in indus-

at pH 4, but laccase activity was maintained for 4 h. Lower laccase

trial transformation of lignocellulosic biomass, several endophytic

activity was measured (less than 60%; Fig. 4b) in cultures from the

fungi able to produce ligninolytic enzymes were assayed in this

Ú. Fillat et al. / Process Biochemistry 51 (2016) 589–598 597

study. From 127 strains isolated from eucalyptus trees in ﬁve areas [19] J. Cenis, Rapid extraction of fungal DNA for PCR ampliﬁcation, Nucleic Acids

Res. 20 (1992) 2380.

of Spain, 21 produced varying degrees of oxidative activity in ABTS

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containing solid media. Five strains were able to produce laccases

for their ability to produce lignin-modifying enzymes and decolorize

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ascomycetes in the class Dothideomycetes. Laccase production by

manganese peroxidases: a comparison between the speciﬁc activities of the

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Acknowledgement endophytes for lignocellulolytic enzyme production and wood

biodegradation, Int. Biodeterior. Biodegrad. 57 (2006) 129–135.

[28] R. Martín-Sampedro, J. Miranda, J.C. Villar, M.E. Eugenio, Laccase from

The authors wish to thank the Spanish Ministry of Economy

Trametes sp. I-62: production, characterization, and application as a new

and Competitiveness (MINECO) for funding this study via Projects laccase for Eucalyptus globulus kraft pulp biobleaching, Ind. Eng. Chem. Res. 52

(2013) 15533–15540.

CTQ 2011-28503-C02-01, CTQ2011-28503-C02-02 and CTQ2013-

[29] M.E. Eugenio, J.M. Carbajo, J.A. Martín, A.E. González, J.C. Villar, Laccase

47158-R and Programme PTA 2011-4857-I.

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