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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
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
protein markers that ran alongside using the gel viewing 2.5
NaCl (M )
A28 0nm
0.5
1.5
0.3
underneath [4, 12-14]. It also appeared that when the pH of the Laccase activity
900
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
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
sajor-caju
A280nm
0.30 40
30
separated the enzymes into three distinct peaks (Figs. 2 and 3). 20
738
observed from a single laccase peak after Sephacryl S-200
1 2 3 4 M
column chromatography. kDa
200.0 Myosin
The isolation resulted in a yield of almost 19% of the total 14.4 Lysozyme
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
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]
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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.
kinetic parameters, inhibitors and inducers, and to determine its [12] C. Eggert, U. Temp, and K.-E. L. Eriksson, "The Ligninolytic System of
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application of the enzyme in areas of agriculture, medicine and 1151-1158, 1996.
environment, such as bioremediation of many industrial wastes [13] A. Lomascolo, E. Record, I. Herpoel-Gimbert, M. Delattre, J. L. Robert,
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[14] G. V. Reddy, P. Ravindra Babu, P. Komaraiah, K. R. R. M. Roy, and I.
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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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