Intraspecific Diversity in the Production and Characterization of Laccase within Ganoderma lucidum

Abstract

Comparison of the potential for laccase and Mn-oxidizing peroxidases synthesis by ten strains of Ganoderma lucidum, originating from different worldwide areas, during solid-state fermentation of selected plant raw materials was the aim of this study. The great intraspecific variability in the production of analyzed enzymes as well as the dependence of the enzyme activity on plant raw materials were reported. The strain HAI 957 was the best laccase producer in the presence of corn stem, as a unique carbon source (129.46 U/L). The highest level of Mn-dependent peroxidase activity was noted after wheat straw fermentation by G. lucidum HAI 246 (78.64 U/L), while the maximal versatile peroxidase production (59.72 U/L) was observed in strain HAI 957 in the medium with oak sawdust.

Full text

Intraspecific Diversity within Ganoderma lucidum
in the Production of Laccase and Mn-Oxidizing
Peroxidases During Plant Residues Fermentation
Jasmina Simonić&Jelena Vukojević&Mirjana Stajić&
Jasmina Glamočlija
Received: 3 June 2009 /Accepted: 25 October 2009 /
Published online: 28 November 2009
#Humana Press 2009
Abstract Comparison of the potential for laccase and Mn-oxidizing peroxidases synthesis
by ten strains of Ganoderma lucidum, originating from different worldwide areas, during
solid-state fermentation of selected plant raw materials was the aim of this study. The great
intraspecific variability in the production of analyzed enzymes as well as the dependence of
the enzyme activity on plant raw materials were reported. The strain HAI 957 was the best
laccase producer in the presence of corn stem, as a unique carbon source (129.46 U/L). The
highest level of Mn-dependent peroxidase activity was noted after wheat straw fermentation
by G. lucidum HAI 246 (78.64 U/L), while the maximal versatile peroxidase production
(59.72 U/L) was observed in strain HAI 957 in the medium with oak sawdust.
Keywords Ganoderma lucidum .Laccase .Mn-dependent peroxidase .Versatile
peroxidase .Plant raw materials
Introduction
Ganoderma lucidum (W. Curt.: Fr.) P. Karst. is a medicinal mushroom species that belongs
to the group of white-rot fungi. It produces extracellular ligninolytic enzymes: laccase
(Lac), lignin peroxidases, and Mn-oxidizing peroxidases [Mn-depending peroxidase (MnP)
and versatile peroxidase (VP)], which participate in modification and degradation of lignin
and structurally similar aromatic compounds into low-molecular-weight components [1–4].
Due to the mentioned facts, G. lucidum could be a participant in various biotechnological
processes, among which biotransformation of raw plant materials takes important place.
Lignocellulose is the major component of plant biomass, which represents the most
abundant renewable organic resource. Thus, world annual generation of agricultural
Appl Biochem Biotechnol (2010) 162:408–415
DOI 10.1007/s12010-009-8833-3
J. Simonić(*):J. Vukojević:M. Stajić
Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia
e-mail: simonicj@bio.bg.ac.rs
J. Glamočlija
Institute for Biological Research “Siniša Stanković”, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia

lignocellulose residues is considerable, 123×10
6
tons per year [5]. Since approximately half
of that amount is used neither for food and feed nor for textile and paper production, the
plant raw materials present as significant environmental pollutant, which could be
bioconverted to several valuable products [6].
Physiological demands for ligninolytic enzyme production vary among white-rot
species, even among strains of a species [7]. The factors that significantly affect enzyme
production are cultivation type (submerged or solid state), carbon and nitrogen sources and
concentrations, presence or absence of different inducers, medium pH, temperature,
agitation, cultivation period, etc. [8–10]. According to the results of numerous studies,
various agricultural and forestry residues present better substrates for enzyme production
than glucose or other simple saccharides [9,11–13]. These residues contain significant
amount of soluble carbohydrates and inducers of enzyme synthesis [14–17] and therefore
appear as prospective substrates for bioconversion into fungal biomass and ligninolytic
enzymes.
The aim of this study was the research of intraspecific diversity within G. lucidum sensu
lato based on the production of Lac, MnP, and VP under conditions of solid-state
fermentation of selected plant raw materials.
Material and Methods
Organisms and Cultivation Conditions
Ten G. lucidum strains, collected from different worldwide areas, were objects of this study
(Table 1). The cultures were obtained from the culture collection of the Institute of
Evolution, University of Haifa (HAI), Israel and from the National Agricultural Research
Foundation–Institute of Kalamata (Ik), Greece and preserved on malt agar medium in the
culture collection of the Institute of Botany, Faculty of Biology, University of Belgrade.
The inoculum preparation was composed of few steps: (1) inoculation of 100 mL of
synthetic medium (glucose, 10.0 g/L; NH
4
NO
3
,2.0g/L;K
2
HPO
4
,1.0g/L;NaH
2
PO
4
×
H
2
O, 0.4 g/L; MgSO
4
×7H
2
O, 0.5 g/L; yeast extract, 2.0 g/L; pH6.5) with 25 mycelial
discs (Ø 0.5 cm, from 7-day-old culture from malt agar); (2) incubation at room
temperature (22±2 °C), on a rotary shaker (160 rpm), for 7 days; (3) washing of obtained
Table 1 Investigated Ganoderma lucidum strains and their origin.
Scientific name of species Strain code Origin of strain
G. lucidum (Curt.: Fr.) Karst. HAI 447 Israel, Tel Aviv, park, on Quercus spp.
HAI 109 CCBA (922)
HAI 158 China
HAI 246 USA, New York, on deciduous tree
HAI 611 Ukraine, Kiev, A.S. Buchalo (922)
HAI 626 Germany, Stuttgart, Botanical garden
HAI 957 China
Ik-1 Greece
Ik-2 Czech Republic
Ik-3 USA
Appl Biochem Biotechnol (2010) 162:408–415 409

biomass (three times) by sterile distilled water (dH
2
O); (4) biomass homogenization with
100 mL of sterile dH
2
O in laboratory blender.
Analyzed plant residues were wheat straw, corn stem, oak sawdust, and grapevine
sawdust. The length of plant particles was about 0.5 cm. Solid-state fermentation was
carried out at 25°C in 100-mL flasks containing 2 g of analyzed plant residue soaked with
10 mL of the synthetic medium modified by the presence of NH
4
NO
3
in one of two tested
nitrogen concentrations (10 and 20 mM) and pH5.0. In this way, prepared substrates were
inoculated with 3 mL of the homogenized inoculum. Samples were harvested after 7 days
of cultivation, and the enzymes were extracted by stirring of samples with 50 mL of dH
2
O
for 10 min at 4°C. The obtained extracts were separated by centrifugation (4 °C, 3,000 rpm,
15 min), and the supernatants were further used for measurements of the Lac and Mn-
oxidizing peroxidase activity as well as total protein content.
Three repeats for each analyzed strain, plant residue, and nitrogen concentration were
prepared in order to decrease the statistical error.
Enzyme Activity Assays
Lac activity was assayed spectrophotometrically using 50 mM ABTS (ε
436
=29,300 M
−1
cm
−1
)
as a substrate in 0.1 M phosphate buffer (pH6.0). The reaction mixture contained buffer,
ABTS, and sample (V
tot
=1 mL).
Mn-oxidizing peroxidase activities were determined with 3 mM phenol red (ε
610
=
22000 M
−1
cm
−1
) as a substrate, in a buffer with the following content: succinic acid disodium
salt, albumin from bovine serum, and DL-lactic acid sodium salt (pH 4.5). The reaction mixture
(V
tot
=1 mL) contained buffer, sample, 2 mM H
2
O
2
, and phenol red, with or without 2 mM
MnSO
4
(for MnP and VP, respectively). Reaction was stopped with 2 M NaOH.
Enzymatic activity of 1 U is defined as the amount of enzyme that transforms 1 μmol of
substrate per minute. An UV-160 A Spectrophotometer (Shimadzu) was used for these assays.
Determination of Total Proteins
The amount of total proteins was performed by means of a standard curve obtained from
solutions containing bovine serum albumin at known concentrations (0.00, 0.01, 0.02, 0.03,
0.04, 0.05, 0.06, and 0.07 mg/mL), Bradford’s reagent (0.2 mL), and sufficient water to
complete a final volume of 1 mL. The mixture contained 0.80 mL of the sample and
0.20 mL of Bradford’s reagent, and absorbance was measured at 595 nm after reaction at
room temperature for 5 min. Total protein content is shown in milligram per milliliter [4].
Results
The tested G. lucidum strains produced Lac, MnP, and VP in all selected plant raw materials
and both nitrogen concentrations, after 7 days of solid-state cultivation. The results have
documented the great intraspecific diversity in the production of the analyzed enzymes
within G. lucidum, under the same cultivation conditions (Figs. 1,2, and 3).
Laccase Production
The best Lac producer was strain HAI 957 in the all selected substrates, except in grapevine
sawdust medium, where the strain Ik-1 was the best producer (Fig. 1). In strain HAI 957,
410 Appl Biochem Biotechnol (2010) 162:408–415

the maximum Lac activity (129.46 U/L) was noted in the corn stem medium with nitrogen
concentration of 20 mM, while at nitrogen concentration of 10 mM, it was about threefold
lower (44.90 U/L). Wheat straw was also a good substrate for Lac synthesis at nitrogen
concentration of 10 mM (129.01 U/L), while at 20 mM nitrogen concentration, Lac activity
Fig. 2 Effect of selected plant raw materials and nitrogen concentrations on Mn-dependent peroxidase activity.
Gray bar 10 mM nitrogen concentration, white bar 20 mM nitrogen concentration, circle specific activity (data
represent mean value of activities of three different samples. Variations are given as standard errors)
Fig. 1 Effect of selected plant raw materials and nitrogen concentrations on Lac activity. Gray bar 10 mM
nitrogen concentration, white bar 20 mM nitrogen concentration, circle specific activity (data represent mean
value of activities of three different samples. Variations are given as standard errors)
Appl Biochem Biotechnol (2010) 162:408–415 411

was even fourfold lower (32.20 U/L). Strain Ik-1, which was the best producer in 20 mM
nitrogen-enriched grapevine sawdust medium (110.01 U/L), was also a good producer
during cultivation in the other three substrates (Fig. 1). The weakest producers were
strains HAI 158, in oak sawdust and wheat straw media (1.45 and 10.40 U/L,
respectively), and HAI 626, in grapevine sawdust and corn stem substrates (5.17 and
13.40 U/L, respectively). In these cases, the influence of nitrogen concentration was
minor (Fig. 1).
According to the specific Lac activity, corn stem was the best substrate, where strain
HAI 957 achieved the maximum value (3.74 U/mg), while oak sawdust was the worst
medium, with the obtained activity of 0.08 U/mg, in HAI 158 (Fig. 1).
Mn-Dependent Peroxidase Production
The highest level of MnP activity was noted in the strains HAI 246 (78.64 U/L) and Ik-1
(78.35 U/L) after cultivation in 20 mM nitrogen-enriched wheat straw and 10 mM nitrogen-
enriched grapevine sawdust substrate, respectively. MnP production in both strains was
lower at another tested nitrogen concentration (Fig. 2). Low MnP production was noted in
strain HAI 957 only during cultivation in corn stem (9.26 U/L) and grapevine sawdust
substrates (5.80 U/L), while the lowest activity was obtained in strain HAI 611 in corn stem
medium enriched with 20 mM nitrogen (2.28 U/L).
All analyzed substrates, except corn stem, were good for the enzyme production, while
wheat straw was the optimal one, where activity level did not fall below 32.50 U/L.
However, specific MnP activity had the highest value in strain Ik-3 after grapevine sawdust
fermentation (11.40 U/mg), while the lowest value was noted in HAI 447 and HAI 626 in
the same substrate (0.1 U/mg; Fig. 2).
Fig. 3 Effect of selected plant raw materials and nitrogen concentrations on versatile peroxidase activity.
Gray bar 10 mM nitrogen concentration, white bar 20 mM nitrogen concentration, circle specific activity
(data represent mean value of activities of three different samples. Variations are given as standard errors)
412 Appl Biochem Biotechnol (2010) 162:408–415

Versatile Peroxidase Production
G. lucidum HAI 957 was emphasized by high values of VP activity at both nitrogen
concentrations during oak sawdust fermentation (58.12 and 59.72 U/L, respectively).
Significant activity level was noted in strain HAI 109 after cultivation in grapevine
sawdust (43.92 U/L) and oak sawdust (37.90 U/L) at the nitrogen concentration of
10 mM. Other tested strains, in all analyzed substrates, showed lower activity (Fig. 3).
Strain HAI 447 was generally a bad VP producer, with activity level ranging from 1.93 to
7.96 U/L. However, it should be emphasized that strain HAI 109 during cultivation in
corn stem substrate, independent on nitrogen concentration, has not synthesized this
enzyme. The same result was reported in strain HAI 611 in the same medium with
nitrogen concentration of 10 mM.
The best substrate for VP production was oak sawdust; good ones were also grapevine
sawdust and wheat straw, while the worst one was corn stem, as in the case of MnP. The total
protein production was highest in Ik-1 during wheat straw fermentation (0.166 mg/mL) and
lowest in HAI 626 in grapevine sawdust medium, which was reflected on specific VP activity
(Fig. 3).
Discussion
This study showed possibility of usage of enzyme production ability as taxonomic character
for strains separation within G. lucidum sensu lato. Contribution of the study is also finding
the optimal strain degrader of different plant raw materials, which are potential
environmental pollutants.
The previous studies have investigated the ability of enzyme production by different
mushroom genera. By studying production of MnP in four species of the genus Pleurotus,
Camarero et al. [18] obtained significant interspecific differences in rate of wheat straw
lignin mineralization during solid-state fermentation. Lignin degradation by Pleurotus
pulmonarius was the highest, lignin degradation by Pleurotus floridanus was the lowest,
while that by Pleurotus ostreatus and Pleurotus sajor-caju was between the two mentioned.
The inter- and intraspecific diversity in Lac and MnP production during solid-state
fermentation of grapevine sawdust and Mandarin peels were also reported by Stajićet al.
[7] and Elisashvili et al. [19]. Thus, significant interspecific differences in MnP production
were noted between Coriolus hirsutus and Coriolus pubescens, as well as Pleurotus
salignus and P. ostreatus, while the intraspecific diversity was noted for Lac activity
between two strains of Cerrena maxima and two strains of P. ostreatus. Silva et al. [4], by
evaluating Lac and MnP production by four Brazilian strains of G. lucidum during
submerged fermentation of wheat bran, showed presence of intraspecies diversity. The
differences in Lac production by Brazilian strains were much higher (ranged from 0.581 to
even 49,519 U/L) than among the strains used in this study (from 1.45 to 129.46 U/L). MnP
production was obtained in all analyzed strains of this study, which was not the case with
Brazilian strains. The same result was reported by Songulashvili et al. [10] within
Ganoderma aplanatum and Trametes versicolor during Mandarin peel submerged
fermentation, as well as by Elisashvili et al. [20]inLentinus edodes and P. ostreatus
during solid-state fermentation of wheat straw. G. aplanatum and T. versicolor strains were
good Lac producers with high level of intraspecific diversity (190–27,380 U/L and 17,140–
20,360 U/L, respectively), while Lentinula edodes and P. ostreatus strains were much
weaker producers, but intraspecific diversities were notable.
Appl Biochem Biotechnol (2010) 162:408–415 413

Although numerous studies of different white-rot fungi species showed significant
participation of Mn-oxidizing peroxidases in lignin degradation, the presence and characterization
of VP in G. lucidum was mentioned recently for the first time [Stajićet al., unpublished data].
VP production, as well as production of other two tested enzymes, was different among studied
G. lucidum strains, which is in accordance with the results of Stajićet al. [7] that demonstrated,
for the first time, the presence of intraspecific diversity within the genus Pleurotus.
Effect of composition of plant raw materials on production of selected ligninolytic
enzymes was the object of numerous studies. D’Souza et al. [2] reported interesting and
unique results on the effect of substrate type on Lac and MnP production by selected G.
lucidum strain. Lac activity was fourfold higher in cultures with high nitrogen content than
in culture with low nitrogen concentration. It is in accordance with the results of this study
because corn stem, which was the optimum substrate for Lac production, is much richer in
nitrogen amount (7.9%) than grapevine sawdust (0.7–0.8%) and wheat straw (0.53%) [15–
17]. Significant differences in Lac production in Ganoderma adspersum during submerged
cultivation depending on the nature and composition of plant raw materials were also
observed by Songulashvili et al. [9]. The maximum Lac synthesis was noted during
cultivation in Mandarin peel medium (34,000 U/L), while it was significantly lower in
wheat or corn bran medium (600 and 700 U/L, respectively). In Phlebia floridensis,
sugarcane bagasse-enriched medium was a better substrate for Lac production than wheat
and rice straws [21]. The results of Fenice et al. [22] once again confirmed the influence of
medium composition on Lac production. These authors showed that Lac production was
stimulated by solid-state cultivation of Panus tigrinus in olive mill waste medium due to
significant content of phenols in the substrate. Songulashvili et al. [10] also showed effect
of numerous plant residues on MnP production in G. lucidum HAI 447. In that strain, MnP
synthesis was absent during submerged fermentation of corn bran, kiwi fruits, and banana
peels, while soy bran, Mandarin peels, and specially wheat bran were good substrates.
According to obtained results, ligninolytic enzyme production could be used as
taxonomic character. However, generally, these results have much higher importance for
biotechnology because, recently, special attention has been given in finding the best
producer of ligninolytic enzymes for degradation of various agricultural and food industry
residues, which can often be serious environmental pollutants. The low-molecular-weight
degradation products are easily absorbed by fungi, better digested by animals, and could be
used in further processing such as in producing food of high nutrition value (mushroom
fruiting bodies), feeds, and basic commodities for different industrial purposes.
Acknowledgment The authors thank Prof. Dr. Solomon P. Wasser, from the Institute of Evolution
University of Haifa (Israel) and Prof. Dr. George Zervakis from Agricultural University of Athens,
Department of Agricultural Biotechnology (Greece), for supplying cultures of G. lucidum. This study has
been carried out within project no. 143041, which is financially supported by the Ministry of Science and
Technological Development of Serbia.
References
1. D'Souza, T. M., Boominathan, K., & Reddy, C. A. (1996). Applied and Environmental Microbiology, 62,
3739–3744.
2. D'Souza, T. M., Merritt, C. S., & Reddy, C. A. (1999). Applied and Environmental Microbiology, 65,
5307–5313.
3. Varela, E., Martinez, A. T., & Martinez, M. J. (2000). Journal of Biotechnology, 83, 245–251.
414 Appl Biochem Biotechnol (2010) 162:408–415

4. Silva, C. M. M. S., Melo, I. S., & Oliveira, P. R. (2005). Enzyme and Microbial Technology, 37, 324–
329.
5. Villas-Bôas, S. G., Esposito, E., & Mitchell, D. A. (2002). Animal Feed Science and Technology, 98,1–
12.
6. Sánchez, C. (2009). Biotechnology Advances, 27, 185–194.
7. Stajić, M., Persky, L., Cohen, E., Hadar, Y., Brčeski, I., Wasser, S. P., et al. (2004). Applied Biochemistry
and Biotechnology, 117, 155–164.
8. Stajić, M., Persky, L., Friesem, D., Hadar, Y., Wasser, S. P., Nevo, E., et al. (2006). Enzyme Microbial
Technology, 38,65–73.
9. Songulashvili, G., Elisashvili, V., Wasser, S. P., Nevo, E., & Hadar, Y. (2006). Biotechnology Letters, 28,
1425–1429.
10. Songulashvili, G., Elisashvili, V., Wasser, S. P., Nevo, E., & Hadar, Y. (2007). Enzyme Microbial
Technology, 41,57–61.
11. Maltseva, O. V., Niku-Paavola, M. L., Leontievsky, A. A., Myasoedova, N. M., & Golovleva, L. A.
(1991). Biotechnology and Applied Biochemistry, 13, 291–302.
12. Kapich, A. N., Prior, B. A., Botha, A., Galkin, S., Lundell, T., & Hatakka, A. (2004). Enzyme Microbial
Technology, 34, 187–195.
13. Moldes, D., Lorenzo, M., & Sanroman, M. A. (2004). Biotechnology Letters, 26, 327–330.
14. Nishimura, K., & Matsuyama, R. (1989). In J. R. Piggott, R. Sharp & R. E. B. Duncan (Eds.), The
science and technology of whiskies (pp. 235–263). Harlow: Longman Scientific and Technical.
15. Mckean, W. T., & Jacobs, R. S. (1997). Retrieved from http://cwc.org/paper/pa971rpt.pdf.
16. Tsiklauri, D. N., Khardziani, Sh T, Kachlishvili, T. E., & Elisashvili, I. V. (1999). Applied Biochemistry
and Microbiology, 35, 291–295.
17. Oleskowicz-Popiel, P., Lisiecki, P., Holm-Nielsen, J. B., Thomsen, A. B., & Thomsen, M. H. (2008).
Bioresource Technology, 99, 5327–5334.
18. Camarero, S., Bőckle, B., Martinez, M. J., & Martinez, A. T. (1996). Applied and Environmental
Microbiology, 62, 1070–1072.
19. Elisashvili, V., Kachlishvili, E., Chichua, D., Bakradze, M., Kokhreidze, N., & Kvesitadze, G. (2001).
Advances in Food Sciences, 23,117–123.
20. Elisashvili, V., Penninckx, M., Kachlishvili, E., Tsiklauri, N., Metreveli, E., Kharziani, T., et al. (2008).
Bioresource Technology, 99, 457–462.
21. Arora, D. S., & Gill, P. K. (2000). Bioresource Technology, 73, 283–285.
22. Fenice, M., Sermanni, G. G., Federici, F., & D'Annibale, A. (2003). Journal of Biotechnology, 100,77–
85.
Appl Biochem Biotechnol (2010) 162:408–415 415