ENZYMATIC DEINKING USING THERMOSTABLE AND … deinking using thermostable .. (24... · deinking of Mi xed Ofiice Wastepaper (MOW) using 50% enzyme of total reaction volume shows enhancement

ENZYMATIC DEINKING USING THERMOSTABLE AND ALKALIPHILIC ENDOGLUCANASE OF

INDIGENOUS BACTERIA

Dayang Syabreeny Bt Abang Mustafa

QP 5199

Bacbelor of Science witb Honours (Resource Biotechnology)

E46 2010 0273 2010

Pnat Khldmat Maklomat Akademlllt TNIVERSm MALAVSIA SARAWAllt

ENZYMATIC DEINKING USING THERMOSTABLE AND ALKALIPIDLIC ENDOGLUCANASE OF

INDIGENOUS BACTERIA

Dayang Syahreeny Bt Abang Mustafa (18286)

This report is submitted in partial fulfillment of the requirements for

the degree of Bachelor of Science with Honors in

Resource Biotechnology

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ACKNOWLEDGMENTS

Thanks to Mak and Abah for everything

Dr Awang Ahmad Sallehin George Lan Hidayah and Fraser Your guidance and support are much appreciated

Family of Molecular Genetics Laboratory and K7C28G Your presences are my strength

usaf Khldmat Mlkluat AkJdetJIlk UNlVERSm MALAYSIA S WAk

Table of Contents

Page

ACKNOWLEDGEMENT

TABLE OF CONTENT ii

LIST OF TABLES VI

LIST OF FIGURES VB

LIST OF ABBREVIATIONS viii

ABSTRACT IX

ABSTRAK IX

10 INTRODUCTION

20 LITERATURE REVIEW

21 Endoglucanase 3

22 Extremophiles 4

23 Conventional Deinking 6

24 Enzymatic Deinking 7

30 MATERIALS AND METHODS

31 Screening and Isolation ofEndoglucanase Producing Bacteria 9

311 Sampling 9

312 Culturing of the Sample 9

313 Screening and Isolation 9

32 Biochemical and Molecular Identification of Isolates 10

10321 Biochemical Test

3211 Gram Staining 10

ii

3212 Citrate Test 10

3213 Sulfate-Indole-Motility (SIM) Test 11

3214 Triple Sugar Ion (TSI) Test 11

3215 Catalase Test 12

3216 Methyl Red Test 12

3217 Voges Proskauer Test 12

3218 Oxidase Test 12

322 Molecular Identification 13

3221 Total DNA Extraction 13

3222 PCR Amplification 14

3223 Genomic DNA and PCR Product Analysis 14

3224 Genomic DNA Gel DNA Recovery 14

3225 Sequencing 14

3226 Sequence Analysis 14

33 Optimization of Endoglucanase Production 15

331 Enzyme Assay 15

332 Protein Determination 15

333 Fermentation Experimental Design 16


3341 Time Course of Incubation 16

3342 Temperature 16

173343 pH

3344 Inoculum Percentage 17

iii

t

17335 Acetone Precipitation

17336 Native PAGE

18337 SDS PAGE and Molecular Mass Detennination

1934 Kinetic Studies of Crude Endoglucanase

19341 Effect ofSubtrate Concentration

193411 Detennination of Vmax and Km

3412 Determination of turnover value Kcal 19

2035 Biodeinking Trial on MOW (Mixed Office Waste)

20351 Endoglucanase Enzyme Production

20352 Enzymatic Deinking of MOW

203521 MOW Pulping

203522 Enzymatic Deinking

203523 Washing

203524 Handsheet Preparation

40 RESULTS AND DISCUSSION

2141 Screening and Isolation of Endoglucanase Producing Bacteria

21411 Bacterial Isolation

21412 Screening of Endoglucanase Producer

2442 Biochemical Test and Molecular Identification of Isolates

24421 Biochemical Test Analysis

27422 Molecular Analysis

274221 Genomic DNA Extraction

284222 Polymerase Chain Reaction (PCR)

iv

j

294223 Sequence Analysis

30

35

43 Optimization of Enzyme Endoglucanase Production

44 Native PAGE

45 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS 36 PAGE) and Molecular Mass Determination

46 Kinetic Studies of the Crude Endoglucanase 38

47 Biodeinking Trial on MOW (Mixed Office Waste) Analysis 39

4250 CONCLUSION AND RECOMMENDATIONS

44REFERENCES

49APPENDIX A

50APPENDIXB

51APPENDIXC

53APPENDIXD

54APPENDIX E

v

LIST OF TABLES

Table Page

Table 1 Results of different combination colours ofTSI Test indicating 11 different characteristics

Table 2 Numbers of Isolates from Different Location 21

Table 3 Endoglucanase Degrading Potential Index among Biggest Halo 22 Producing Isolates

Table 4 Endoglucanase Activity among Biggest Halo Producing Isolates 23

Table 5 Biochemical Tests Results of Isolate 8 24

vi

LIST OF FIGURES

Figure Page

Figure 1 Congo Red Screening 22

Figure 2 Gram Staining 25

Figure 3 Simmon Citrate Test 25

Figure 4 Triple Sugar Ion (TSI) Test 25

Figure 5 Methyl Red Test 25

Figure 6 Sulfate-Indole-Motility (SIM) Test 25

Figure 7 Total genomic DNA extraction of Isolate 8 27

Figure 8 PCR products of bacterial isolate using PA forward and PH reverse 28 primer

Figure 9 Effects of time course of incubation on endoglucanase production 30

Figure 10 Effects of temperature on endoglucanase production 31

Figure 11 Effects of initial pH on crude endoglucanase production 32

Figure 12 Effects of different percentage of inoculum for endoglucanase 33 production

Figure 13 Native PAGE of crude enzyme of Klebsiella spp 35

Figure 14 Coomasie Brilliant Blue R-250 staining of SDS PAGE gel of crude 36 endoglucanase of Klebsiella spp

Figure 15 Enzyme Concentrations on Initial Velocity 38

Figure 16 Lineweaver-Burk Plot 38

Figure 17 Enzymatic Deinking using 100 enzyme of total volume reaction at 39 11 consistency


vii

LIST OF ABBREVIATIONS

16S rONA 16S Ribosomal Deoxyribonucleic Acid

16S rRNA 16S Ribosomal Ribonucleic Acid

BSA Bovine Serum Albumin

CMC Carboxymethylcellulose

DNA Deoxyribonucleic Acid

DNS Dinitrosalicyclic Acid

dNTPs Deoxyribonucleotide triphosphate

LB Luria Bertani

MSM Minimal salt media

MOW Mixed Office Wastepaper

00 Optical Density

PAGE Polyacrylamide Gel Electrophoresis

PCR Polymerase Chain Reaction

SDS Sodium Dodecyl Sulfate

SIM Sulfite-Indole-Motility Test

TEMED Tetramethylethylenediamine

TSI Triple Sugar Ion

vw Volume Weight (concentration)

wv Weight Volume (concentration)

viii

Enzymatic Deinking using Thermostable and Alkaliphilic Endoglucanase of Indigenous Bacteria

Dayang Syahreeny Bt Abang Mustafa

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

The best endoglucanase producer has been successfully isolated and identified from Paku Hot Springs based on halo fonnation during Congo red screening on minimal salt media containing 1 (wv) of carboxymethyIceIlulose Molecular approach targeting the conserved region of 16S rRNA bacterial strain performed was unable to further confinn initial morphological and biochemical tests results of the isolate which was identified as Klebsiella spp An optimal condition for endoglucanase secretion by Klebsiella spp was determined successful at pH 72 and 37degC after 9 hours of incubation using 5 of inoculums The crude endogucanase activity was determined using ONS method and prolein content was determined via Bradford method Native PAGE and SOS PAGE were performed to detennine crude endoglucanase enzymatic activity and molecular weight of crude endoglucanase respectively Km

VNX Kbullbull values of crude endoglucanase were 07765 mgml 06242 unitslml and 16 SmiddotI respectively Enzymatic deinking of Mi xed Ofiice Wastepaper (MOW) using 50 enzyme of total reaction volume shows enhancement in brightness but deinking ability was not determined

Key words Thermophilic CMC endoglucanase deinking mixed office wastepaper (MOW)

ABSTRAK

Satu bakleria terbaik yang menghasilkan enzim endoglukanase dari Kolam Air Panas Paku telah berjaya dipencilkan dan dikenalpasti berdasarkan pembentukan halo kelika penyaringan Congo red di alas agar Minimal Salt Media (MSM) Pendekalan molekul mensasarkan kawasan jujukan 16s rRNA yang dipulihara tidak berjaya untuk megesahkon kepulusan ujikaji biokimia sebelumnya yang lelah dikenalpasti sebagai Klebsiella spp Keadaan optimum untuk penghasilan enzim endoglukanase telah dicirikan pada pH 72 dan 31C selepas 9 jam fermenlasi dengan menggunakan 5 inokulum Aktivii enzim menlah ditentukan melalui kaedah DNS dan kandungan protein ditentukan melailli kaedah Bradford Native PAGE dan SDS PAGE dilakukan unluk menentukan aktiviti enzimatik dan berat molekul bagi endoglukanasementah Nilai Km dan VmaT bagi endoglukanase mentah adalah 07765 mgml dan 06242 unitsm manakala nilai Keal adalah 16 SmiddotI Penghilangan dakwat ke alas kerlas campuran pejabat menggunakan 50 enzim daripada jumlah keseluruhan isipadu untuk tindakbalas menunjukkan peningkalan dari segi kecerahan manakalan kebolehan menghilangkan dakwat tidak ditentukan

Kata kunci Termofilik CMC endoglukanase penghilangan dakwat kertas eampuran pejabat

ix

CHAPTER 1

INTRODUCTION

High grade recycled paper consists mainly of magazine waste (OMG) mixed office waste

(MOW) and old newspaper (ONP) (Soni el aI 2008) Recycling of paper requires deinking

stage which is the removal of the printing ink from used paper to obtain brighter pulp

(Mohandass amp Raghukumar 2005) Two types of printing methods are usually carried out

impact and non-impact ink Impact ink is easier to be removed or dispersed during deinking as it

does not fuse into paper On the contrary photocopying inkjet and laser printing used nonshy

impact ink which results in fused ink into the paper making it non-dispersible which render

deinking process (Mohandass amp Raghukumar 2005) Mix office waste (MOW) is favored to

produce newsprint and as well as high grade paper because it has longer and brighter fibers when

compared to old newspaper (ONP) Thennoplastic copolymer inks that present in MOW (Soni

el aI 2008) caused conventional chemical deinking process expensive as it barely remove by

dewatering di persian additional floatation and washing process (Vyas amp Lachke 2003) On

the other hand conventional deinking requires large amount of chemicals such as sodium

hydroxide hydrogen peroxide chelating agents sodium silicates and other collector chemicals

which are easi ly available and cheap which have high potential for environmental damage (Soni

el al 2008) Enzymatic deinking is proposed as alternative method of various harsh chemicals

used during disintegration which favors ink detachment from fibers without discharge of

pollutants thus contributing to environmental compatibility (Soni el al 2008)

1

Cellulose is embedded in a network of hemicelluloses and lignin requiring an alkaline

pretreatment to become accessible to enzyme action Most celluloses alkaline pretreatment is

perfonned at high temperatures hyperthermophilic cellulases should be the best candidate

catalysts for cellulose degradation (Vieille amp Zeikus 2001) Therefore alkaline and thermostable

enzyme should replace the conventional process which takes places in high pH and high

temperature Indigenous thermophilic microorganisms can be obtained from local hot springs

around Malaysia They can survive harsh environment (Schiraldi amp Rosa 2002) such as high

temperature of hot spring area Thus there is high potential that thermophilic bacterial strain

producing thermostable endoglucanase enzymes is present and can be characterized In this

research a thermophilic endoglucanase with medium enzymatic activity was successfully

isolated from Paku Hot Springs Lundu Sarawak

The objectives of this research were to isolate indigenous thermostable and alkaliphile

bacteria with cellulolytic capability and investigate biodeinking of mixed office paper potential

by using the endoglucanase produced by the bacteria isolates which consequently can lead to

mass production for the application in paper recycling industry or other industries In order to

achieve this aim the specific objectives of this project are as follows

I To isolate and identify thermophilic bacterial strains that produces extracellular

endoglunacase to be used in enzymatic deinking on mixed office paper

II To characterize endoglucanase enzyme from the isolates this suits the optimal

temperature and pH of the deinking process

IJ To perfOlm trial enzymatic deinking using endoglucanase of indigenous bacteria isolates

2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase

Endoglucanases often classified as endo-acting cellulases which cleave P-1 A-glycosidic bonds

internally only and appear to have cleft-shaped open active sites (Maki el aI 2009) However

there is suggestion that some cellulases display both modes of action endo- and exo- which

changed the classification cellobiohydrolases (exoglucanases) are active on the crystalline

regions of cellulose whereas endoglucanases are active on soluble amorphous region of the

cellulose crystal (Maki et al 2009) On the other hand Turner et al (2007) suggested that

endoglucanases ([Ee 3214] are classified under 12 different glycosyl hydrolase families with

both inverting and retaining reaction mechanisms and with different folds which catalyse

random cleavage of internal bonds in the cellulose chain while cellobiohydrolases

(exoglucanases) (EC 321 91 G H 5 7 [retaining] and 6 9 [inverting]) attack the chain ends

releasing cellobiose Glucosidases (EC 32121 GH 1 3 [retaining] and 9 [inverting]) are only

active on cello-oligosaccharides and cellobiose releasing glucose

A high degree of synergy between exoglucanases and endoglucanases required for the

efficient hydrolysis of cellulose crystals (Ogel 2001) Endoglucanase can be either cell-bound or

extracellular and only a few endoglucanase producers produces significant quantities of free

enzyme that abl to completely hydrolyze crystalline cellulose even though large number of

microbes were reported capable to degrade cellulose (Koomnok 2005) Bacterial cellulase

system lack FPase activity (Ariffin el aI 2008) and the main activity is p-glucanase (endo-1 A-Pshy

glucanase EC 3214) which randomly hydrolyses internal l4-p-bonds in cellulose and does not

cl e crystalline cellulose (Liu el ai 2008) Besides bacterial cellulases are often more

3

effective catalyst and may also be less inhibited by feedback inhibition which is the presence of

material that has already been hydrolyzed (Ariffin et al 2008) Different effects on fibers

properties are obtained too when cellulases with different cellobiohydrolases and endoglucanases

activities were used Individual endoglucanase treatment is considered necessary for

modification of the pulp fiber as it does not result in the decrease of average fiber length or

coarseness Endoglucanase and cellobiohydrolase synergetic effect could lead to cross breakage

which causes decreasing in paper strength In addition Vyas and Lachke (2003) found that pure

endoglucanase were responsible in most of success deinking after treating laser-printed waste

paper individually and combination of purified endoglucanase from Gloeophyllum sepiarium and

Gloeophyllum trabeum

22 Extremophiles

Extremophiles are microbes that can live and reproduce in extreme environments (Schiraldi amp

Rosa 2002) High and low temperatures defined as thennophiles and psychrophiles

respectively high and low pH values defined as alkaliphiles and acidophiles respectively high

salt concentrations defined as halophiles and high pressure defined as barophiles are the

common four parameters used to classify extremophiles There has been a great deal of interest

in the biotechnological potential of their extremozymes

Thennophilics cellulases brought advantages In industrial applications as higher

processing temperature can be employed for offering accelerated reaction rates Increase

solubility and reduced contamination (Ng et al 2009) Thennostable ability also gives a longer

h If-life to the enzyme (Ibrahim amp El-diwany 2007) Thennophilic can be classified into

m philes (50-60degC) extreme thennophiles (60-80degC) and hyperthennophiles (80- [ lOOC)

4

PoSit Khidmat MlkJum t Akademfk UNlVERSm MALAYSIA SAMWAK

Thermophilic microorganisms are widely distributed among genera Bacillus Clostridium

Thermoanaerobacter Thennus Thennotoga and Aquifex (Ng et al 2009) while

hyperthermophilic species are dominated by the Archaea (Turner et aI 2007)

Themostability is inherented by their cell components such as membranes and enzymes

Principal among tlUs are the ether linked lipids from archaeal cell membranes (Hoist et aI

1997) Adaptation is apparently genetically encoded and as revealed by biophysical and

structural studies it includes sequence modification addition of salt bridges increased

hydrophobic interactions additional ion pairing and hydrogen bonding improved core packing

and shortening of loops (Eichler 2001) This confers higher thennal stability rigidity and

resistance to chemical denaturation (Eichler 2001)

Meanwhile alkaliphiles are the organisms that have optimal growth around pH 10

Alkaliphiles diver ely occur from aerobic and anaerobic archaea and prokaryotes as well as

eukaryotic fungi (Jones et aI 1994) widening their industrial application According to

Krulwich (2006) facultative alkaliphile is applied to species and strains that able to grow at both

pH 65 - 75 and above 95 whereas that grow only at pH 95 is termed as obligate a lka~iphiles

Soda lakes desert soils and alkaline springs are alkaline environments where pH can be

consistently about pH 10 (Magan 2007) Jones et al (1998) reviewed that alkaline hot springs is

interesting alkaliphiles source but are ihsufficiently buffered to support extraordinary high pH

value which contra)t to naturally occurring soda lakes which are widely distributed and stable

alkaline environments (Krulwich 2006) The possession of flagella modifications of cell walls

and membranes and biochemical activity including respiration and oxidative phosphorylation

enable the adaptation and growth of alkaliphiles in alkaline environment (Magan 2007)

5

23 Conventional Deinking

reviewed by Pelach e al (2003) a conventional deinking process starts with disintegration

of recycled paper and followed by the addition of chemicals in a strong alkaline medium in order

to promote de-fibering and ink particle detachment Finally mild alkaline of washing or flotation

technologies allow ink removal from the suspension

Sodium hydroxide hydrogen peroxide chelating agents and sodium silicates are among the

chemicals used during conventional deinking Sodium hydroxide adjusts the pulp pH as

alkalinity resulted in soften ing the paper fibers by saponification or hydrolysis while hydrogen

peroxide acts as bleaching agent in the fiber treatment process and counterbalance darkening due

to alkalinity above pH 102 Unfortunately hydrogen peroxide is very sensitive to pUlping

environment due to existence of heavy metals ions found in inks These require other chemicals

to stabilize the environment in which hydrogen peroxide works chelating agent and sodium

silicate

Sodium silicate is favored as it is less harmful than chelating agent such as

ethylenediaminetetracetic acid (EDTA) However former problems of stability coating of fibers

and harshness of paper due to silicates formation lead to the use of latter which can cause heavyshy

metal poisoning in rivers streams and severely affect aquatic life when disposed off Various

chemical usages of conventional deinking impose environmental problem and health risks and to

overcome this highly cost waste water treatment needed to be applied (Lee et al 2006)

6


Deinking is a removal ink process According to Biermann (1993) deinkinkin can be

divided into 4 steps repulping with the associated ink removal from the fiber cleaning to

remove the ink from the stock separation of residual ink contaminants from the fiber stock and

bleaching Enzymatic deinking is the alternative for chemical deinking which exploit

microorganisms enzyme for deinking process and it was suggested that the enzyme hydrolyzed

the fiber-ink regions directly which splitting the cellulose fiber and ink particles apart According

to Soni el al (2008) microbial enzyme such as cellulase xylanase esterases and lipase play

important role in biological deinking Enzymes rarely require toxic metal ions for functionality

which can be advantageous as industrial catalysts hence creating the possibility to use more

environmentally friendly processing (Turner et al 2007) Removal of small fibers from the

surface of ink particles is resulted by the action of cellulase binding and altering the fiber

surface This alters the relative hydrophobicity of the toner particles and reduces hydrodynamic

drag that facilitates toners removal during floatation step (Soni et al 2008)

It was found that laccase treatment reduced the kappa number and increases the

brightness of waste photocopy paper but treatments with laccase alone or as the mediator is

unsuitable for deinking of aged prints or for bleaching of deinked pulp unprinted recycling

paper and groundwood (Widsten amp Kandelbauer 2008) Zhang et al (2008) reported that three

commercial ceJlulases enzymes were able to detach a significant amount of ink from the old

recycled newsprint or magazine but cellulase deinking was less efficient than alkaline deinking

chemistry CeJlulases tested were unable to deink aged ONPOMG and a poor deinkability was

also observed by using either sulphite or alkaline chemistry However combining enzymes with

suI te chemistry significa ntly enhanced sulphite deinking and provided a potential strategy to

achieve effective deinking of aged newsprint at neutral pH In 2009 a combination of cellulase

hemicellulase and laccase-violuric acid system was developed and higher brightness higher

tearing index better physical properties of deinked pulp was produced However enzymatic

deinking has not clearly defined but most researchers agree that a combination of mechanical

action and synergistic deinking effect of surfactant is vital for process viability (Sykes et al

1996) In addition they have been proven to be cost effective and more efficient than

con entional deinking Thus alternate option for replacing some of the deinking chemicals by

enzymatic deinking is sui table and being considered (Soni et al 2008)

8

CHAPTER 3

MATERIALS AND METHODS


11 Sampling

Samples ofwater and ediments were collected from Matangs drains and Paku Hot Springs The

pH was checked using pH paper and the samples were brought back to the laboratory for further

analysis

312 Culturing of the Sample

The samples pH was checked and pH adjustment to pH75 was done using 1 M NaOH The

amount of water sediments and shredded Mixed Office Wastepaper (MOW) were 150 mL 50

mL and 10 gram respectively The samples were cultured in 250 mL flask for 1-2 weeks at

55degC at 120 rpm

313 Screening and Isolation

The cultures were filtered using filter paper under sterile condition and each filtrate were

subjected to standard serial dilution prior plated onto Luria Broth (LB) agar containing 1 (wv)

carboxymethylcellulose (CMC) and incubated at 55degC for an overnight Each colony identified

was streaked two times to obtain single colony Endoglucanase producers were identified by

Congo red test using (MSM) + 1 CMC agar plates which grown 3 days for endoglucanase

secretion The plates was flooded with 02 Congo red solution and left for 1 hour Congo red

lution was poured off and washed with 10 M NaCl for 15 minutes Yellow halos indicated

co ies that hydrolyzed CMC where Congo red was absent

9

Then

Biochemical and Molecular Identification of Isolates

Biochemical Test

11 Gram Staining

drop of distilled water was placed on microscope slide and a suspension of test culture was

placed onto the distilled water The slide was dry fixed by passing the slide repeatedly on flame

lide was allowed to cool The slide was flooded with crystal violet for 60 seconds and

ashed with distilled water Iodine was applied for 60 seconds and washed with distilled water

1 the slide was rinsed with 70 ethanol until there was no purple wash out Safranin was

flooded onto the slide for 60 seconds and rinse with distilled water The slide was allowed to dry

and viewed under microscope using oil immersion at 100x magnification A Gram-negative

bacterium was identified in pink colour and Gram-positive organisms remained purple colour

3212 Citrate Test

colony of Isolate 8 was streaked onto Simmon Citrates medium and incubated at 37degC for 48

hours The growth was accompanied by pH change of the medium which was indicated by the

medium color initial green colour to deep blue indicating positive result

10

fate-Indole-Motility (SIM) Test

culture of Isolate 8 was stabbed into SIM media that prepared in screw-capped test tube

culture was incubated for 24 hours at 37degC Motility was indicated by diffuse turbidity of

ulture surrounding the puncture line while immotility took place only along puncture line

Hydrogen sulfide (H2S) formation was shown by blackening of mjcrobial growth areas Indole

was performed by covering a layer of KOYACS Indole Reagent Indole production caused

reagent layer becoming purple-pink in colour

214 Triple Sugar Ion (TSI) Test

Pure culture of Isolate 8 was streaked on the surface and stabbed into TSI media The culture was

incubated up to 48 hours at 37degC Degradation of sugar accompanied by acid production are

detect by the pH indicator phenol red which changed from red-orange to yellow while

alkalinization shown by deep red Thiosulfate was reduced to hydrogen sulfide with an iron salt

to give black iron sulfide

Table 1 Resul ts of different combination colours of TSI Test indicating di fferent characteristics

Butt Colour Slant Colour Results Yellow Red Glucose fermented Yellow Yellow Glucose fermented also lactose and or sucrose Red Red No action on glucose lactose and sucrose Black precipitation along puncture Hydrogen sulfide production

11

~talllle t t was performed to detect the presence of catalase in the colonies of Isolate 8

erial colonies were taken on glass slides and one drop of H202 (3) was added Appearance

(gas bubbles indicated the presence of catalase (MacFaddin 1980)

116 Methyl Red Test

o tubes containing MR-VP was inoculated with pure culture of Isolate 8 and incubated for 48

hours at 3rc Then five drops of methyl red indicator was added into the first tube Positive

IeSUlt as red indicating pH below 6 and negative was yellow indicating no acid production

bull217 Voges Proskauer Test

Three milimeter of BARRITs solution and 1 ml 40 potassium hydroxide solution was added

1010 S ond tube described in Section 3216 Positive reaction was indicated by pink colour

(after frequent shaking) approximately 20 minutes beginning at the surface and become more

intense within 2 hours

rap of oxidase reagent containing 1 of tetramethyl-para-phenylenediamine dihydrochloride

as spotted on a piece of filter paper in a Petri dish Sterile wooden stick was used to rub a

colon of Isolate 8 onto the spot of spotted oxidase reagent Positive test was indicated by rapid

appearance of a purple color and negative result indicated by no colour changes

12

olecular Identification

__11ar identification was done to futher identify the bacteria up to genera level

as incubated in 5 ml Luria Bertani (LB) media an overnight prior genomic DNA

lion Approximately 15 ml of bacterial culture was transferred into Eppendorf tube and

1 maximum for 30 seconds Supernatant was removed and cell pellet was resuspended in

JLI TE buffer and mixed well by vortexing Then 30 III of 10 (wv ) SDS 5 III 20 mglml

inase K and 100 III of 5 M aCI solution was added and mixed well 80 III of CTABlNaCI

mixed into the mixture and incubated for 60 minutes at 65degC Next an equal volume of

oroformllsoamyl alcohol (24 1) was added vortexed gently and centrifuged for 5 minutes to

t the phases Clear supernatant was transferred into new Eppendorf tube and re-extracted

ng PhenolChlorofonnilsoamyl alcohol by 5 minutes centrifugation Clear supernatant was

ferred into new Eppendorf tube and 06 volume of isopropanol was added The mixture was

Iy mixed by vortexing it up and down until white DNA precipitate appeared Then DNA

pelleted by 30 seconds centrifugation Supernatant was removed and DNA pellet was

ith 200 fll 70 ethanol and centrifuged for 30 seconds at maximum speed 15000 rpm

8U1penllBtant was removed and DNA pellet was air dried Lastly air-dried DNA pellet was

dissol ed in 100 fll ofTE buffer and stored at -20degC for further analysis

13

Pnat Khldmat Maklomat Akademlllt TNIVERSm MALAVSIA SARAWAllt

ENZYMATIC DEINKING USING THERMOSTABLE AND ALKALIPIDLIC ENDOGLUCANASE OF

INDIGENOUS BACTERIA

Dayang Syahreeny Bt Abang Mustafa (18286)

This report is submitted in partial fulfillment of the requirements for

the degree of Bachelor of Science with Honors in

Resource Biotechnology

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ACKNOWLEDGMENTS





Table of Contents

Page

ACKNOWLEDGEMENT

TABLE OF CONTENT ii

LIST OF TABLES VI

LIST OF FIGURES VB


ABSTRACT IX

ABSTRAK IX

10 INTRODUCTION


21 Endoglucanase 3

22 Extremophiles 4





311 Sampling 9






ii













3225 Sequencing 14



331 Enzyme Assay 15





3342 Temperature 16

173343 pH


iii

t


17336 Native PAGE









203521 MOW Pulping


203523 Washing











iv

j


30

35


44 Native PAGE





44REFERENCES

49APPENDIX A

50APPENDIXB

51APPENDIXC

53APPENDIXD

54APPENDIX E

v

LIST OF TABLES

Table Page






vi

LIST OF FIGURES

Figure Page



















vii









LB Luria Bertani



00 Optical Density









viii





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13

ACKNOWLEDGMENTS





Table of Contents

Page

ACKNOWLEDGEMENT

TABLE OF CONTENT ii

LIST OF TABLES VI

LIST OF FIGURES VB


ABSTRACT IX

ABSTRAK IX

10 INTRODUCTION


21 Endoglucanase 3

22 Extremophiles 4





311 Sampling 9






ii













3225 Sequencing 14



331 Enzyme Assay 15





3342 Temperature 16

173343 pH


iii

t


17336 Native PAGE









203521 MOW Pulping


203523 Washing











iv

j


30

35


44 Native PAGE





44REFERENCES

49APPENDIX A

50APPENDIXB

51APPENDIXC

53APPENDIXD

54APPENDIX E

v

LIST OF TABLES

Table Page






vi

LIST OF FIGURES

Figure Page



















vii









LB Luria Bertani



00 Optical Density









viii





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13


Table of Contents

Page

ACKNOWLEDGEMENT

TABLE OF CONTENT ii

LIST OF TABLES VI

LIST OF FIGURES VB


ABSTRACT IX

ABSTRAK IX

10 INTRODUCTION


21 Endoglucanase 3

22 Extremophiles 4





311 Sampling 9






ii













3225 Sequencing 14



331 Enzyme Assay 15





3342 Temperature 16

173343 pH


iii

t


17336 Native PAGE









203521 MOW Pulping


203523 Washing











iv

j


30

35


44 Native PAGE





44REFERENCES

49APPENDIX A

50APPENDIXB

51APPENDIXC

53APPENDIXD

54APPENDIX E

v

LIST OF TABLES

Table Page






vi

LIST OF FIGURES

Figure Page



















vii









LB Luria Bertani



00 Optical Density









viii





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13













3225 Sequencing 14



331 Enzyme Assay 15





3342 Temperature 16

173343 pH


iii

t


17336 Native PAGE









203521 MOW Pulping


203523 Washing











iv

j


30

35


44 Native PAGE





44REFERENCES

49APPENDIX A

50APPENDIXB

51APPENDIXC

53APPENDIXD

54APPENDIX E

v

LIST OF TABLES

Table Page






vi

LIST OF FIGURES

Figure Page



















vii









LB Luria Bertani



00 Optical Density









viii





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13

t


17336 Native PAGE









203521 MOW Pulping


203523 Washing











iv

j


30

35


44 Native PAGE





44REFERENCES

49APPENDIX A

50APPENDIXB

51APPENDIXC

53APPENDIXD

54APPENDIX E

v

LIST OF TABLES

Table Page






vi

LIST OF FIGURES

Figure Page



















vii









LB Luria Bertani



00 Optical Density









viii





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13


30

35


44 Native PAGE





44REFERENCES

49APPENDIX A

50APPENDIXB

51APPENDIXC

53APPENDIXD

54APPENDIX E

v

LIST OF TABLES

Table Page






vi

LIST OF FIGURES

Figure Page



















vii









LB Luria Bertani



00 Optical Density









viii





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13

LIST OF TABLES

Table Page






vi

LIST OF FIGURES

Figure Page



















vii









LB Luria Bertani



00 Optical Density









viii





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13

LIST OF FIGURES

Figure Page



















vii









LB Luria Bertani



00 Optical Density









viii





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13









LB Luria Bertani



00 Optical Density









viii





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13





ABSTRACT




ABSTRAK



ix

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13

CHAPTER 1

INTRODUCTION



















1






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13






















2

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13

CHAPTER 2

LITERATURE REVIEW

21 Endoglucanase





















3











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13











22 Extremophiles












4
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13
























5













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13













silicate







6
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13
































8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13









8

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13

CHAPTER 3



11 Sampling



analysis





55degC at 120 rpm










9

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13

Then


Biochemical Test

11 Gram Staining









3212 Citrate Test




10
















11




116 Methyl Red Test













12


















13
















11




116 Methyl Red Test













12


















13




116 Methyl Red Test













12


















13


















13

Documents

ENZYMATIC DEINKING USING THERMOSTABLE AND … deinking using thermostable .. (24... · deinking of Mi xed Ofiice Wastepaper (MOW) using 50% enzyme of total reaction volume shows enhancement