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2010 International Conference on Science and Social Research (CSSR 2010), December 5 - 7, 2010, Kuala Lumpur, Malaysia

Isolation and Purification of Laccase from Rice Straw


Fermented with Pleurotus sajor-caju

*Nurfariza Bahrin, **Pat M Lee, and *Kamsani Ngalib


* Microbiology Program, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM) Shah Alam, Malaysia
**Materials Technology Program, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM) Shah Alam, Malaysia
e-mail: drpatlee@salam.uitm.edu.my
patmlee.1@gmail.com

Abstract Rice crop is abundant in Malaysia. With the high in the polyphenol oxidase group. Laccase is a group of multi-
demand of food sources, the amount of rice straw left in the copper proteins (four copper atoms in the active site, as Cu2+ in
plantation field increases over time. This highly lignocellulosic the resting enzyme) that are distributed among different
material is a promising substrate to produce laccase- a versatile binding sites, with each copper ion appearing to play an
enzyme of low specificity that can function well in several important role in the catalytic mechanism [2, 3]. In nature,
important areas such as bioremediation, industry, agriculture laccase exist as a natural lignin degrader produced by most
and medicine. Utilizing the rice straw in a solid-state white-rot fungi.
fermentation process using Pleurotus sajor-caju, laccase was
produced and subjected to isolation and purification processes. Screening for the best incubation period for the SSF
The optimum laccase production was studied from 0 to 21 days. process has found that laccase is always secreted by fungal
The highest laccase activity of 224.93 U/mg was obtained from mycelium at the beginning of colonization to degrade the lignin
the crude extract after 9 days of fermentation with Pleurotus barrier of its substrates tissues. As laccase is an extracellular
sajor-caju. It was concentrated by ultrafiltration and purified enzyme, the purification procedures are generally attainable
using DEAE-Sepharose anion exchange chromatography. The than intracellular enzymes and laccases generally exhibit a
peak fraction obtained was then loaded into Sephacryl S200-HR considerable level of stability in the extracellular environment.
gel filtration chromatography. Laccase was purified by 43-fold to In addition to the generally low substrate specificity, laccase
a specific activity of 19335 U/mg, with an overall protein recovery has other properties that make this enzyme potentially useful
of 18.6%. It appeared as a single band on SDS-PAGE with an
for biotechnological applications. These include the fact that
apparent molecular weight of 53 kDa.
laccase, unlike other peroxidases (lignin peroxidase and
KeywordsLaccase; Enzyme; Solid-state Fermentation; manganese peroxidase), only require oxygen, which is usually
Pleurotus sajor-caju; Rice straw present in its environment. The inducible expression of the
enzyme in most fungal species also contributes to the easy
applicability in most biotechnological processes.
I. INTRODUCTION
Compared to submerged fermentation, solid-state
Utilization of agro-industrial residues as substrates in solid- fermentation was proven to produce higher yield, less
state fermentation (SSF) provides an alternative avenue and contamination, less effluent, easier to isolate and purify [4-6].
value-addition to these otherwise under- or non-utilized The production of laccase in Pleurotus species occurs during
residues. Rice crop residues which are mainly lignocellulosic the early stage of colonization and sometimes even before the
are very suitable fermentation substrates. Most edible fungi, fruit-body formation [7]. Therefore, its production is faster than
such as Pleurotus sajor-caju, are employed because of the well any other lignocellulolytic enzyme which makes it favorable
known ability to break down the cellulose-lignin complex and for this research. The objectives of this study were to isolate
liberate digestible compounds. Lignocellulose degradation by and purify laccase from solid-state fermented rice straw using
fermentation is mediated by extracellular enzymes comprising Pleurotus sajor-caju. The purified laccase would have
lignin modifying enzymes (mainly laccase, lignin peroxidase biotechnological applications.
and manganese peroxidase), cellulase and xylanase for
cellulose and hemicellulose degradation [1]. Laccase is
II. METHODOLOGY
preferred to be thoroughly investigated compared to the other
lignocellulolytic enzymes (cellulase and xylanase) due to its
A. Screening of the Optimum Incubation Period
particularly high enzyme activity, shorter incubation time for
secretion and therefore, making it cost effective towards Fermentation substrate: Rice straw (variety MR 219) was
commercialization. obtained from MARDI Tanjung Karang. They were field dried
and cut into pieces before being ground through a 1 mm sieve
Laccase (EC 1.10.3.2), systematically known as grinder (IKA, Germany).
benzenediol:oxygen oxidoreductase is also known as urishiol
oxidase, and p-diphenol oxidase. It is one of the many enzymes

This research was funded in part by IRDC, UiTM (code ST 756)

978-1-4244-8986-2/10/$26.00 2010 IEEE 736


Inoculum: Spawn in the form of wheat grain koji of Concentration of enzyme: Culture fluid of 1000 ml was
Pleurotus sajor-caju was procured from a commercial centrifuged at 5000 rpm for 30 minutes using Biofuge Primo
mushroom planter, C&C Farm in Semenyih, Selangor. The (Heraeus-Sorvall, Germany) to obtain the supernatant prior to
mycelia were recultured on yeast-PDA Petri dish and later ultrafiltration. Enzyme concentration was done using the
transferred to a steriled boiled wheat grain koji in 250 ml tangential flow filter cassette; Vivaflow 50 (Sartorius,
conical flask for spawning. The koji served as the fermentation Germany) with 10 kDa molecular weight cut-off membrane.
inoculum once the mycelia have ran over the whole wheat The procedure was carried out via connection of the cassette to
grain. Masterflex peristaltic pump (Cole-Palmer, USA) at a
constant flow rate of 200 ml/min. The concentrated enzyme
Optimization of fermentation period: Twenty two flasks solution served as the starting material for the purification of
were prepared for the screening experiment, which was carried laccase.
out for 0-21 days. The ground rice straw was filled into 250 ml
conical flasks for an amount of 20 g each. Then, 60 ml mixed Anion-exchange chromatography: An empty column with
mineral solution consisting of 2 g/L KH2PO4 and 0.5 g/L dimension of 1.6 x 20 cm was packed with DEAE-Sepharose
MgSO4 was added to each flask. All flask are then sterilized by Fast Flow matrix (GE-Healthcare, Sweden) and equilibrated
autoclaving at 121C for 20 minutes before being left to cool with three column volumes of 20 mM sodium phosphate
down to room temperature (27C). They were then inoculated buffer, pH 6.0. An amount of 10 ml concentrated supernatant
with approximately 2 g of the koji spawn. Each day, one flask was loaded into the column and eluted by a linear gradient
was taken out and stored at 4C for determination of activity, consisting three column volumes of equilibration buffer and
protein concentration and pH. five column volumes of 1.0 M NaCl [10]. The linear gradient
was formed by GM-1 Gradient Mixer (GE-Healthcare,
B. Enzyme Extraction Sweden) connected to Masterflex (Cole-Palmer, USA)
The fermented substrates were extracted with deionized peristaltic pump pre-set with a 5 ml/min flow rate. Eluates were
water according to 1: 4 w/v ratios for 24 hours on an orbital collected using Retriever 500 (ISCO, USA) fraction
shaker at 250 rpm. After the shaking procedure was completed collector at 75 drops per fraction (approximately 6 ml per
and the spent wastes were fully soaked with the water extract, a tube). Protein peaks for each fraction were detected by
pH meter (Hanna Instrument, Romania) was used to determine absorbance at 280 nm in spectrophotometer using bovine
the pH of fermented rice straw. The extracts were then filtered serum albumin as standard. Fractions that formed a distinct
through Whatman No. 4 filter paper using vacuum separation peak were assayed for laccase activity and one with the highest
force. The filtrate was then subjected to a centrifugation at specific activity was stored at 4C for the next column run.
5000 rpm for 30 minutes to remove any residual substrates and Gel-filtration chromatography: Fractions containing the
mycelia. The supernatant was recovered and stored at 4C. highest laccase activity from the ion exchange column was
loaded into a Sephacryl S-200 High Resolution column (GE-
C. Enzyme and Protein Assay Healthcare, Sweden) with a dimension of 1.2 x 50 cm pre-
For the determination of laccase activity, the total reaction equilibrated with 20 mM sodium phosphate buffer (pH 6.0).
volume of 1.5 ml contained 0.25 ml sample (supernatant), 1.1 The enzyme was eluted with the same buffer at a flow rate of 2
ml potassium phosphate buffer (100 mM, pH 6.5) and 0.15 ml ml/min and collected as 3 ml/tube fractions. Again, protein
(0.2 mM) syringaldazine in absolute methanol as substrate. peaks for each fraction were detected by absorbance at 280 nm
Samples in triplicate were measured at 530 nm against reaction so as the enzyme activity. The top of the activity peak which
blank, which contained 0.25 ml deionised water to replace the contained the highest specific activity was concentrated with
sample using a continuous spectrophotometry method in centrifugal filter units, Vivaspin 20 diafiltration tube
Lambda 35 UV-Vis spectrophotometer (Perkin Elmer) [8]. (Sartorius, Germany) with 10 kDa molecular weight cut-off
Laccase activity was calculated as follows (1): membrane at 3000 rpm. The retained sample was used for
molecular weight estimation.

Activity (U/ml) = (Anm/min sample - Anm/min blank) x df E. Molecular Weight Determination


SV (1) Molecular weight of the purified laccase was determined by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The procedure was done using a 10%
Protein concentration was determined by Lowry method acrylamide NuPAGE Pre-Cast Gel System in a Novex Xcell
using the Folin-Cicolteau phenol reagent and bovine serum SureLock mini cell (Invitrogen, Germany) as described by the
albumin as standard for calibration [9]. manufacturers protocol for standard molecular weight
determination [11].
D. Enzyme Isolation and Purification The lyophilized samples were dissolved in a minimum
Optimized fermentation: Another batch of fermentation was amount of distilled water. SDS-PAGE was performed to
set up for 25 flasks for 9 days in the same manner as the determine sample purity and to estimate the molecular weight
screening stage. All of the fermented waste was then weighed of the purified laccase. The apparent molecular weight of the
and immersed in deionized water for enzyme extraction. laccase was determined by calibration against broad range
molecular weight markers, See Mark 12 unstained standard

737
(Invitrogen, Germany), which contained the proteins myosin An isocratic equilibration and elution using 20 mM sodium
(200 kDa), -galactosidase (116.3 kDa), phosphorylase B (97.4 phosphate buffer at pH 6.0 resulted in a single distinct peak.
kDa), bovine serum albumin (66.3 kDa), glutamic anhydrase This peak contained the highest laccase activity which was
(55.4 kDa), lactate dehydrogenase (36.5 kDa), carbonic eluted in fraction 7, after being washed with 21 ml buffer as
anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme seen from the elution profile in Fig. 4.
(14.4 kDa), aprotinin (6 kDa) and insulin; B chain (3.5 kDa),
insulin; A chain (2.5 kDa).
A280nm
A280nm
The gel was later stained with Simply Blue Coomasie Safe
4.5 1
Concentration of NaCl

Concentration of NaCl
Stain (Invitrogen, Germany) and digital image of the migrated 4.0 0.9

bands was captured using AlphaImager HP (Alpha Innotech, 3.5


0.8

USA). The molecular weight of the purified laccase was 3.0


0.7

determined by calculating the relative mobility of standard 0.6

protein markers that ran alongside using the gel viewing 2.5

NaCl (M )
A28 0nm
0.5

systems AlphaView Q Software. 2.0


0.4

1.5
0.3

III. RESULTS AND DISCUSSION 1.0


0.2

The specific activity of laccase enzyme in the culture 0.5 0.1

extract was at a maximum peak on the 9th day of fermentation 0.0 0

with 224.93 U/mg and started to decline drastically after the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40


Fraction

10th day of incubation as shown by Fig. 1. Laccase secretion is


Figure 2. Profile of gradient elution for the anion exchange
usually the highest during the 7th to the 10th day of cultivation, chromatography of laccase using DEAE-Sepharose
a stage for the mycelia growth and colonization. It was
understood that its secretion was essential to break the lignin
barrier and providing an access to the structural carbohydrate 4.5
A280nm
1000

underneath [4, 12-14]. It also appeared that when the pH of the Laccase activity
900

substrate was 5.72, the specific activity reached its maximum


4.0

with the temperature being kept constant at 27C during the


800
3.5

incubation period. 3.0


700

600

Activity (U/ml)
2.5
A280nm

500

2.0
400
250 6.5
1.5
Specific activity 300

pH 1.0
200
200 6
0.5 100

0.0 0
Specific act (U/mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
150 5.5 Fraction

Figure 3. Profile for A280nm and laccase activity of the


pH

100 5 DEAE-Sepharose eluates

50 4.5
0.60 A280nm 80

Laccase activity
70
0.50
0 4
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
60
Incubation period (Day)
0.40
50

Figure 1. Fermentation profile of laccase production by Pleurotus


Activity (U/ml)

sajor-caju
A280nm

0.30 40

30

DEAE-Sepharose anion exchange chromatography (IEX) 0.20

separated the enzymes into three distinct peaks (Figs. 2 and 3). 20

The second peak, which is between fractions 1416, contained 0.10


10

more than 90% of the total enzyme activity. From a


concentrated sample with a specific activity of 860 U/mg, the
0.00 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Fraction

eluted fraction has a specific activity of 6659 U/mg. The other


two peaks were found to have high protein content, but without Figure 4. Profile of Sephacryl S-200 gel filtration
chromatography of laccase DEAE-Sepharose peak fraction
any enzymatic activity. Fraction 15, eluted at almost 0.5 M
NaCl, showed the highest activity peak (Fig. 2). Further The purification techniques employed in this study, i.e. the
purification of fraction 15 was performed by loading the ultrafiltration, anion exchange and gel filtration column
fraction into Sephacryl S-200 gel filtration column. chromatography has successfully purified the enzyme as

738
observed from a single laccase peak after Sephacryl S-200
1 2 3 4 M
column chromatography. kDa
200.0 Myosin

Laccase is likely to be a high molecular weight protein as it 116.3 -galactosidase


was eluted earlier during the chromatographic run. The peak 97.4
66.3
Phosphorylase B
BSA
has a specific activity of 19335 U/mg, showing more than 43- 53 kDa
55.4 Glutamic anhydrase

fold purification compared to the culture liquid at the end of the


36.5 Lactate dehydrogenase
purification procedures. The purification steps and relevant 31.0 Carbonic anhydrase
details such as laccase activity, specific activity, yield
percentage and purification fold are shown in Table I. 21.5 Trypsin inhibitor

The isolation resulted in a yield of almost 19% of the total 14.4 Lysozyme

units of enzyme activity initially present in the culture filtrate.


The dark-brown color of the concentrated enzyme produced 6.0 Aprotinin

after the ultrafiltration procedure was removed after the DEAE- 3.5
2.5
Insulin, B chain
Insulin, A chain
Sepharose chromatography yielding a colorless solution with
36.3% of the initial enzyme activity. Figure 5. Digital image of the purified laccase on SDS-PAGE.
(L-R): Lanes 1-3:GFC (~7 g), 4: IEX (~65 g),M: Mark 12
Fig. 5 shows that the laccase was purified to its apparent unstained MW marker.
homogeneity [15] which appeared as a distinct band (lane 1-3)
on SDS-PAGE and has a molecular weight of 53 kDa. Table II
shows some references on several purified laccase properties. IV. CONCLUSION
The result obtained from SDS-PAGE indicates that the isolated
Value added product, laccase, was successfully isolated and
enzyme is in the range similar to those described for other
purified from the solid-state fermentation of rice straw, an
fungal laccases in which most of them are monomeric proteins
abundant agricultural waste in Malaysia, using Pleurotus sajor-
with molecular mass ranging between 40 to 70 kDa [15-24].
caju. The apparent molecular weight of laccase purified by
Further work will be conducted to study the biochemical
anion exchange and gel filtration chromatography was 53 kDa.
properties of the purified laccase.

TABLE I. PURIFICATION TABLE SUMMARIZING LACCASE ISOLATION FROM THE FERMENTED WASTE OF PLEUROTUS
SAJOR-CAJU

Purification step Volume (ml) Total protein (mg) Total activity (U) Specific activity (U /mg) Yield (%) Fold
Culture liquid 700 3024 1,346,800 445.37 100 1

Ultrafiltration (10 kDa filter) 105 2627 2,260,020 860.27 167.8 1.93

DEAE-Sepharose 70 73.31 488,201 6659.31 36.3 14.95

Sephacryl S200 120 12.92 249,888 19,335.19 18.6 43.41

TABLE II. PROPERTIES OF SEVERAL LACCASES ISOLATED FROM THE FERMENTATION WITH FUNGUS OF THE GENUS PLEUROTUS
Optimum
Molecular Optimum
Species Assay substrate temperature Yield (%) Fold References
weight (kDa) pH
(C)
Pleurotus sajor-caju 61 ABTS 5.0 40 53 10.3 [15]

Pleurotus sajor-caju 65 Syringaldazine 5.5-6.5 30 NA NA [16]

Pleurotus sajor-caju IV 55 ABTS 2.1 45 4.9 152 [17]


Pleurotus sajor-caju ABTS 3.5
59 35 28.6 22.65 [18]
(recombinant) syringaldazine 6.5
Pleurotus eryngii lac I: 65 30 42
ABTS 4.5 55 [19]
(isoenzymes) lac II: 61 26.3 43
Pleurotus ostreatus 55 Guaiacol 6.5 50 23 1500 [20]
POXA1: 61 45-65 23 85
Pleurotus ostreatus Syringaldazine 6.0 [21]
POXA2: 67 25-35 0.5 NA
Pleurotus ostreatus 67 Syringaldazine 5.8 50 46.5 31.7 [22]

Pleurotus ostreatus 64 Syringaldazine 6.0-6.5 30-35 10.8 42.9 [23]


Pleurotus pulmonarius
46 Syringaldazine 6.2-6.5 50 49 5.9 [24]
Lcc2

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It is of great interest to study the biochemical properties of [11] U. K. Laemmli, "Cleavage of Structural Proteins During Assembly of
the purified laccase such as thermal and pH stabilities, enzyme the Head of Bacteriophage T4," Nature, vol. 227, pp. 680-685, 1970.
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ACKNOWLEDGMENT Lignolytic and Cellulolytic Enzymes by Solid Substrate Fermentation
Using Two Pleurotus Species (P. ostreatus and P. sajor-caju)," Process
The authors would like to thank MOSTI for the scholarship Biochem, vol. 38, pp. 1457-1462, 2003.
awarded to N.B., Faculty of Applied Sciences, UiTM for the [15] K. Murugesan, M. Arulmani, I.-H. Nam, Y.-M. Kim, Y.-S. Chang, and
research facilities and a research grant to the late Assoc. Prof. P. T. Kalaichelvan, "Purification and Characterization of Laccase
Dr James Vadiveloo for the preliminary studies of this research Produced by a White Rot Fungus Pleurotus sajor-caju under Submerged
by the Institute of Research, Development and Culture Condition and Its Potential in Decolorization of Azo Dyes,"
Commercialization (IRDC), UiTM as well as MARDI Tanjung Appl Microbiol Biotechnol, vol. 72, pp. 939-946, 2006.
Karang for the rice straw. [16] A. Salis, M. Pisano, M. Monduzzi, V. Solinas, and E. Sanjust, "Laccase
from Pleurotus sajor-caju on Functionalised SBA-15 Mesoporous
Silica: Immobilisation and Use for the Oxidation of Phenolic
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