PRODUCTION, PURIFICATION AND CHARACTERIZATION

OF DYE DEGRADING ENZ1'ME PRODUCED BY

Marasmius sp.

Fatin Syahirah Binti Awang Shapie (39510)

Bachelor of Science with Honours

(Resource Biotechnology)

2015

PRODUCTION, PURIFICATION AND CHARACTERIZATION OF DYE

DEGRADING ENZYME PRODUCED BY Marasmius sp.

Fatin Syahirah binti Awang Shapie (39510)

This thesis is submitted in partial fulfillment of the requirement for the

Degree of Bachelor of Science with Honours

(Resource Biotechnology)

Supervisor: Assoc. Prof. Dr. Awang Ahmad Sallehin Awang Husaini

Co-Supervisor: Dr. Azham Zulkhamain

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2015

ACKNOWLEDGEMENT

Praise to Allah the Almighty and the great Merciful, for giving me strength and guidance

to complete my Final Year Project. I would like to express my gratitude to my supervisor,

Assoc. Prof. Dr. Awang Ahmad Sallehin for his moral support and encouragement

throughout my fmal year project.

I would like to thank the postgraduate students from Molecular Genetic Laboratory for

guiding and assisting me while completing my project. Sincere appreciation to all my

laboratory mates that provide moral support and helping me out in completing this

project.

Special thanks to my parents that never forget to reminds me of my purpose in studying

so that i did not lose my focus. Thank you for the endless prayer.

DECLARATION OF ORIGINAL WORK

I hereby declare that this thesis is based on my original work except for

quotation and citation, which have been duty acknowledged. I also declare that it

has not been previously or concurrently submitted for any other degree at

UNIMAS or other institutions.

39510,

Fatin Syahirah binti Awang Shapie,

Resource Biotechnology Programme

Department ofMolecular Biology

Faculty of Resource Science and Technology

University Malaysia Sarawak

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TABLE OF CONTENT

ACKNOWLEDGEMENT

DECLARA TION OF WORK

TABLE OF CONTENT

LIST OF ABBREVIATIONS

LIST OF TABLES

LIST OF FIGURES

ABSTRACTIABSTRAK

1.0 INTRODUCTION

2.0 LITERATURE REVIEW

2.1 Endophytic fungus

2.1 .1 Marasmius sp.

2.2 Synthetic dye

2.2.1 Remazol Brilliant Blue R (RBBR) dye

2.3 Dye degrading enzymes

2.3.1 Laccase

2.4 Microbial dye decolourisation

2.5 Submerged fermentation (SmF)

3.0 MATERIALS AND METHODS

3.1 Organism

3.2 Chemicals

3.3 Production ofdye degrading enzymes

3.4 Protein determination

3.5 Laccase enzyme assay

3.6 Purification ofenzymes

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3.6.1 Preparation of resin 11

3.6.2 Buffer selection 11

3.6.3 Gradient elution 11

3.7 SDS-PAGE (Laemmli) Buffer System 12

3.7.1 Coomassie Brilliant Blue staining 12

3.7.2 ABTS staining 13

3.7.3 RBBR dye staining 13

3.8 Characterization ofenzymes

3.8.1 Effects of pH 13

3.8.2 Effects of temperature 14

4.0 RESULT AND DISCUSSION

4.1 Production ofdye degrading enzymes 15

4.2 Protein determination 16

4.3 Laccase assay 17

4.4 Purification of laccase 19

4.5 SDS-PAGE 22

4.6 Characterization ofenzyme

4.6.1 Effects of pH 24

4.6.2 Effects oftemperatur 25

5.0 CONCLUSION 27

REFERENCES 28

APPENDIX A 31

APPENDIXB 34

APPENDIX C 35

APPENDIX D 36

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ABTS

ANOVA

BSA

CV

LiP

LMEs

mm

MnP

MnS04

NaCl

NaOH

NO

N02

RBBR

rpm

Vrnax

List of Abbreviations

2,2' -azino-bis(3-ethylbenzthiazo line-6-sulphonic acid)

Analysis of variance

Bovine serum albumin

Coefficient of variation

Lignin peroxidase

Lignin mineralizing enzymes

minutes

Manganese peroxidase

Magnesium sulphate

Sodium chloride

Sodium hydroxide

Nitric oxide

Nitrous oxide

Remazol Brilliant Blue R

rotation per minutes

Maximum velocity

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List of Tables

Table 1 Summary of laccase enzyme purification 21

Table 2 Components for preparation ofGlucose Minimal Medium (GMM) 31

Table 3 Preparation of 12% resolving gel and 5% stacking gel 39

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List of Figures

Figure 1 Oxidation of phenolic subunits of lignin by laccase 7

Figure 2 Graph ofcomparison of total protein (mg) in different stage of 16

purification

Figure 3 Graph ofcomparison of laccase enzyme activity in different stage of 18

purification

Figure 4 Graph of comparison of laccase specific activity in different stage of 18

purification

Figure 5 Graph of laccase enzyme activity in purification of laccase 20

Figure 6 The color of reaction mixture oflaccase enzyme assays according to their 20

gradient elution in purification of laccase.

Figure 7 (Left) Gel stained by Comassie Brilliant Blue, (middle) Gel stained by 23

ABTS solution, (right) Gel stained by 2% RBBR dye stock solution

Figure 8 Graph ofeffect ofpH on activity and stability of laccase produced by 25

Marasmius sp.

Figure 9 Graph ofeffect of temperature on activity and stability of laccase 26

produced by Maras~s sp.

Figure 10 Protein Standard Curve 34

Production, Purification and Characterization of Dye Degrading Enzyme Produced by Endophytic Fungus, Marasmius sp.

Fatin Syahirah binti Awang Shapie (39510)

Resource Biotechnology

vii

Faculty ofResource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

The presence ofcarcinogenic compound in synthetic dye has raised various concerns as it cause harmful effects toward environment and also health. Furthermore, the synthetic dyes are not easily degraded by aerobic bacteria. The purpose of this research project is to determine and characterize the dye degrading enzymes produced by an endophytic fungus, Marasmius sp. that are responsible in the degradation of synthethic dyes. The presence of laccase was conflfmed by the oxidation of ABTS in the enzyme assay. The total lac case activity of crude enzyme was recorded as 259.45 U with specific activity of 5 765.56 mg/ml. Laccase was purified by open column chromatography and salted out with 70% ammonium sulphate thus the enzyme was purified up to 1.074 fold with specific activity of 6 189.38 mg/ml and 76.34% yield. The laccase enzyme activity was optimum and stable in pH 4 and at 25°C. As a conclusion, Marasmius sp. produced laccase as the dye degrading enzymes.

Keyword~: Endophytic fungi, Enzyme characterization, Laccase, Purification.

ABSTRAK

Kehadiran bahan yang mengakibatkan kanser dalam pewarna timan telah menimbulkan pelbagai kerisauan terhadap kesan-kesan pewarna timan kepada alam sekitar dan juga kesihatan manusia. Tambahan pula, pewarna tiruan ini tidak dapat dihapuskan oleh bakteria aerobik. Tujuan kajian ini adalah untuk membuktikan kehadiran enzim yang boleh mendegradasikan pewama timan oleh kulat spesis Marasmius dan mengkaji circiri utama enzim tersebut. Kehadiran laccasse dapat dilihat melalui pengoksidaan ABTS dalam asai enzim. Keselumhan hasil laccase daripada enzim asli adalah 259.45 U dengan keselumhan hasil spesijik 5 765.56 mg/ml manakala laccase yang telah melalui penulenan oleh kromatografi kolum terbuka dan di pekatkan dengan 70% ammonium sulfat mempunyai sebanyak 1.074 kali ganda penulenan dengan keseluruhan hasil spesijik untuk laccase sebanyak 6189.38 mg/ml and hasil sebanyak 76.34%. Keseluruhan aktiviti bagi enzim laccase adalah paling optimum pada pH 4 dan suhu 25°C. Kesimpulannya, kulat spesis Ml/IhlSmius menghasilkan enzim laccase yang mampu medegradasikan pewarna timan.

Kata kunci: Kulat endofitik, Ciri-ciri enzim, laccase, penulenan.

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1.0 INTRODUCTION

Endophytic fungi can be found worldwide as it is a highly diverse group of fungi and can

normally found within tissues of plants showing a commensalism relation as endophytes

remain latent in the host tissues until a favourable condition arise that causes the

endophytes to become pathogenic (Hyde, 2010).

Marasmius sp. produced enzyme such as lignin peroxidase (LiP), manganese

peroxidase (MnP) and laccase that degrade many aromatic compound due to their non

specific activity. These enzymes are inducible under specific conditions of low nutrient

nitrogen, high oxygen tension, presence of the inducer veratryl alcohol and static

cultivation.

According to Banat et. al. (1996), these enzymes were reported as main enzyme

involve in dye decolorization. Lignin peroxidase (LiP), mangnese peroxidase (MnP) and

laccase are the major lignin mineralizing enzymes (LMEs) involved in lignin and

xenobiotic degradation by endophytic fungus. Lignin peroxidases (LiPs) are capable of

mineralizing a variety of recalcitrant aromatic compounds (Shrivastava et al. 2005),

manganese peroxidases (MnPs) are extracellular glycoproteins and laccases are N

glycosylated extracellular blue multicopper oxidases (Wells et at., 2006).

The lignin modifying enzymes (LME), that are MnP, LiP and laccases, are

directly involved in the degradation of not only lignin in their natural lignocellulosic

substrates but also various xenobiotic compounds including synthetic dyes. Peroxidases

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and laccase are oxidative enzymes, which do not need any other cel1ular components to

work; they have broad substrate specificity and are able to transform a wide range of

toxic compounds. These enzymes, which are widely distributed in nature and have been

studied for many years because of their potential use as biocatalysts in pulp and paper

bleaching, wastewater treatment, soil remediation, on-site waste destruction and medical

diagnostics (Erkurt et at., 2010).

Synthetic dyes are not easily degraded by aerobic bacteria and can form

mutagenic compound when exposed to environment and some may become mutagenic

(Banat et at., 1996). Most widely researched fungi in producing dye degrading enzymes

are ligninolytic fungi. The activation of the ligninolytic system is affected by some

gradients, such as nitrogen, carbon, metal ions and sulfur in media and these ligninolytic

conditions could significantly affect the production of enzyme (Mirsha & Kumar, 2007;

Liu et. at., 2009).

This project aimed to provide information about the dye degrading enzymes from

endophytic fungus, Marasmius sp. Therefore, the specific objective of this research are:

1. To determine the production oflaccase under standard condition ... ii. To purify the laccase enzyme

lll. To determine the effect of pH and temperature on lac case enzyme activity

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2.0 LITERATURE REVIEW

2.1 Endophytic fungus

Endophyte is any organism that lives inside another plant and it can either be parasitic or

symbiotic while fungi are eukaryotes with complex cell structure and abilities to make

tissues and organ (Moore et aI., 2011). They play an important role in forest ecosystem as

they help to break down little layer of forest floor while some of them are involved in

lignin degradation as they can degrade the lignin and some also can degrade cellulose

(Mooreet. aI., 2011).

2.1.2 Marasmius sp.

Marasmius is the most well-known genus under Tricholomataceae family, it mostly can

be found in tropics and are often small mushrooms that can be found on the ground (as

shown in Figure 1) as they grow on forest floor, dead living wood and also on other plant

tissues (Mehrotra & Aneja, 1990). This genus could dry out but later revived when

moistened and continue producing it spores (Mehrotra & Aneja, 1990).

According to Gerhard et ~(2013), there are antibiotics and anti-carcinogenic

properties found in the filaments of Marasmius sp. Besides that, Marasmius sp. also

produce A oxidase isoenzyme, dimeric peroxidase, thermostablexylanase, laccase related

enzymes and also aromatic peroxygenase cytochrome P450 other than the main enzymes

which are laccasse, lignin peroxidase and manganese peroxidase (Gerhard et. aI., 2013).

A research done by Ratanachomsri (2005) found that thermostablexylanase from

Marasmius sp. can be used to hydrolyse xylan and cellulose at high temperature as it has

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dual specificity towards both substrates thus makes it effective to be extensively use in

industry. Cytochrome P450 is the heme-thiolate protein that involves in fungal

denitrification by reducing nitric oxide (NO) to nitrous oxide (N02) (Poole, 2008).

2.2 Synthetic dye

Synthetic dyes are commonly used in industries and noticeably used in textile industry

(Ahmad et. ai., 2014). The wide use of synthetic dyes contributes to water pollution as

the dye effiuents are directly discharge into streams (Ahmad et. ai., 2014). Synthetic dyes

are classified according to their chemical structures (Stolz, 2001).Synthetic dyes are

complex compounds thus making it hard for anaerobic bacteria to degrade them as they

are very recalcitrant (Ahmad et. ai., 2014). The inability to degrade the dyes may lead to

various harmful effects as it can form mutagenic compound thus become carcinogenic

(Stolz, 2001; Pavko, 2011).

There are several ways of degrading synthetic dyes in which are by physical,

chemical, electrochemical or biological method (Pavko, 2011). Physical method involve

the adsorption, sedimentation, fl

White-rot fungi and ligninolytic fungi are the widely researched fungi that can

produce dye degrading enzymes that can be used in degrading the synthetic dyes.

However recent study by Husaini et at. (2013) shows that endophytic fungi isolated from

senduduk plant (Melastomamalabathricium) also produced the dye degrading enzymes

which are laccase, lignin peroxidase and manganese peroxidase.

2.2.1 Remazol Brilliant Blue R (RBBR) dye

Remazol Brilliant Blue R dye is one of the dyes used in textile industry as it is easy to

use, consume low energy also has high solubility of water (Ahmad et. al., 2014). RBBR

also commonly referred as Reactive Blue 19 is a type of anthraquinone dye (Sigma

Aldrich, 2014). The molecular weight ofRBBR is 626.54 g/mol with chemical formula

OfC22Hl6N2Na2011S3 (Sigma-Aldrich, 2014).

2.3 Dye degrading enzymes

Dye degrading enzymes is im}W1ftant in degrading the synthetic dyes that nowadays are

frequently used in industries (Khalid et ai., 2010). Laccase, lignin peroxidase and

manganese peroxidase are known as dye degrading enzymes. According to Rodriguez et

at. (1998), laccase is the main enzyme involve in dye decolourization as it has the highest

decolourization capability compared to lignin peroxidase and manganese peroxidase.

However, the production of laccase is differs in different organisms as it depends on the

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amino acid sequences and also other enzymatic systems In the orgamsm such as

peroxidases or cytochrome P450.

2.3.1 Laccase

Laccases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase) are the blue multi-copper

oxidases which are diametric or tetrameric glycoproteins (More et. aI., 2011; Irshad et.

al., 2011). Laccase also contain four copper atoms in their redox sites that catalyze

oxidation of variation of substrates such monophenols, diphenols, polyphenols, amino

phenols, methoxyphenols, aromatic amines and ascorbates (Madhavi & Lele, 2009).

Hydrogen peroxide is absent in oxidation of substrate and possess wider spectrum than

peroxidises (lrshad et. aI., 2011).

According to Madhavi and Lele (2009), laccase has been studied since back in

nineteenth century in which firstly described by Yoshida in 1883 when he discovered

laccase in Japanese lacquer tree, Rhus vernici/era. Following that, in 1896, Bertrand and

Laborde proved that laccase is a fungal enzyme. The presence of laccase has been

identified in various plant, fun~nd bacteria however most of the studied fungi was

isolated from higher fungi such as ascomycetes, deuteromycetes and basidiomycetes

fungi (Madhavi & Lele, 2009). Laccase also plays different role in different species such

as lignin degradation in white-rot fungi, morphogenesis, sporulating and resting

structures in basidiomycetes.

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Besides that, laccase also considered as polyphenol oxidases due to its ability to

oxidize aromatic compound. Laccase disrupt the phenolic subunits of lignin, leading to

Ca oxidation, C~ cleavage, and aryl-alkyl cleavage (as shown in Figure 2). The

mechanism of laccase reaction is by coupling the phenoxy radicals produced from

oxidation of lignin phenolic groups with wide range of substrates such as non-phenolic

subunits of lignin such as 2,2' -azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)

that are widely used as substrate in laccase enzyme assay (Madhavi & Lele, 2009).

lignin lignin I I6 ~wL~reS An_'

amount of chromophore a dye carries in its chemical structure therefore detennine the

resistance of the dye towards decolourization (Stolz, 2001).

2.5 Submerged fermentation

The fermentation condition where free flowing liquid substrates were utilized and the

bioactive compounds needed were supplied by the fermentation broth (Subramaniyam &

Vimala, 2004).

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3.0 MATERIALS AND METHODS

3.1 Organisms

Endophytic fungus, Marasmius sp. was obtained from Molecular Genetic Laboratory,

FRST, UNlMAS fungal collection and sub cultured in every 2 weeks in malt extract agar

(MEA) plates at room temperature. The malt extract agar preparation protocol can be

referred in Append ix A.

3.2 Chemicals

Remazol Brilliant Blue R (RBBR) dye, citrate-NaOH, 2,2' -azino-bis(3

ethylbenzthiazoline-6-sulphonic acid) (ABTS), sodium acetate, bovine serum albumin

and other chemicals and reagents that were obtained from Molecular Genetic Laboratory,

FRST, UNIMAS.

3.3 Production of dye degrading enzymes

Fresh 2 mm in diameter tissues ofMarasmius sp. fungal fiuiting bodies were aseptically

cultured in the Petri dished containing malt extract agar (MEA) and incubated at room

temperature for 7 days for production of mycelia mats.

A number of20 agar plugs (5 mm diameter) from 7 day old mycelium mats were

inoculated in 250 ml conical flasks containing 99.5 mL of glucose minimal medium

(GMM) and 0.5 ml of 2% RBBR stock solution making the final solution of 100 ml and

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then covered by aluminium foil and parafilm. Subsequently, the flasks are incubated on a

rotary shaker at 150 rpm at room temperature for 10 days. All of the steps were carried

out in duplicate and control.

The cells were removed by centrifugation (6000 rpm, 10 min) and the

supernatants were harvested as crude enzyme preparation for the subsequent enzyme

assays (Mtui & Masalu, 2008).

3.4 Protein determination

The Bradford (1976) method was used in measuring the protein concentration by using

bovine serum albumin (BSA) as the standard.

3.5 Laccase enzyme assay

Laccase activities were determined by oxidation of ABTS method. The non-phenolic dye

ABTS were oxidised by laccase to a more stable cation radical and the concentration of

cation radical can be detected b~e intensity of blue-green color correlated to enzyme

activity and was read at 420 nm. The assay mixture used contain 0.5 mM ABTS, O. lM

sodium acetate (pH 4.5). Oxidations of ABTS were monitored by determining the

increase in A420. The reaction mixtures were incubated for 5 minutes at room

temperature and the absorbance are read at 420 nm using spectrophotometer (More et al.,

2011). Laccase enzyme activity unit is CV= 36,000M-1 cm-1 (Li et al., 2008).

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3.6 Purification of enzymes

Purification of enzymes were carried out by using open column chromatography and

AmberLite IRA-400, strongly basic gel-type resin to bind impurities and letting the target

protein pass the column.

3.6.1 Preparation of resin

The resins were supplied in chloride form thus it needs to be swell up by adding sodium

phosphate buffer and let the mixture for an hour before removing the buffer. The resins

were then incubated overnight at room temperature.

3.6.2 Buffer selection

10 mM sodium phosphate buffer (start buffer A) was used and 10 mM sodium phosphate

buffer and sodium chloride buffer (elution buffer B) was used as elution buffer.

3.6.3 Gradient elution

Gradient elution was carried out by using 10 times of the column volume and performed

by increasing the salt solution (buffer B) from 0% and gradually until it reach lOO% and

collect 2-5 ml fractions throughout this step for each gradient.

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3.7 SDS-PAGE (Laemmli) Buffer System

SDS-PAGE analysis was used for protein purity control and relative molecular mass.

CoJlected fractions were separated by SDS-PAGE, bands of protein were observed.

Single band indicates high purity of enzymes. The stock solutions and buffers preparation

protocol were prepared according to Bio-Rad Mini-PROTEAN® Tetra Cell Instruction

Manual.

Non-denaturing SDS-PAGE was carried out using 12% resolving gel and 5%

stacking gel with the absence of heat and ~-mercaptoethanol and was performed under

constant voltage of lOOV. The gels were then stained using Coomassie brilliant blue

staining method, ABTS staining and lastly RBBR dye staining.

3.7.1 Coomassie brilliant blue staining

The first gel were stained with 0.1 % Comassie Blue R250 III 10% acetic acid, 50%

methanol and 40% H20 to visualize the band of interest and exposed to 10% acetic acid,

50% methanol (stain and destain). The gel was exposed in staining solution overnight and

with shaking. After that, the Itwas destained by soaking in 10% acetic acid, 50%

methanol and 40% H20 at least two changes of solvent or until the bands were observed

clearly.

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3.7.2 ABTS staining

The second gel were stained by using 300 mM ABTS solution for 5 minutes and then the

laccase bands were visualized as green bands in the white (colourless) gel background.

3.7.3 RBBR dye staining

The third gel were stained by 2% RBBR dye solution for awhile and then destained by

using acetate buffer until halozone was observed thus showing the presence of dye

degrading enzyme.

3.8 Characterization of enzymes

3.8.1 Effects of pH

Optimum pH was determined by performing enzymatic assays at different pH (3, 4, 5, 6,

7). The pH level was adjusted by using following buffers: 0.1 M citrate buffer (pH 3-5),

0.1 M phosphate buffer (pH 6-8) and 0.1 M carbonate buffer (pH 9). The effect of pH on

the enzymes stability are determined by incubating the purified enzymes at 4°C in

different pH levels for 24 hours and determine the residual activity.

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3.8.2 Effects of temperature

Optimum temperature was determined by performing enzymatic assays at different

temperatures (25°C, 30°C, 35°C, 40°C, 45°C). The stability of purified enzymes at

various temperatures was investigated by pre incubating the purified enzymes at different

temperatures for 1 hour and determines the residual activity.

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