SB of Castor Oil On Cellulase... · sell/lase lelah dikaji dengan menjalankan IIjian enzim. Apabita minyak jarak ada dalam bahall penapaian, kepekalan elanol lelah berkurang. Walal

· .

EFFECTS OF CASTOR OIL ON CELLULASE ENZYMATIC ACTIVITIES ON PURE CELLULOSE DURING SIMULTANEOUS SACCHARIFICATION

AND FERMENTATION (SSF)

Rossyuhaida Binti Mohd Zakria

SB 299 C3

Bachelor of Science with Honours R838 (Biotechnology Resource) 2012

20ll

PUSit Khidmat Maklumat Akademik" UNlVERSm I\fALAYSIA SARAWAK

EFFECTS OF CASTOR OIL ON CELLULASE ENZYMATIC ACTIVITIES ON PURE CELLULOSE DURING SIMULTANEOUS SACCHARIFICATION AND

FERMENTATION (SSF) P.KHIDMAT MAKLUMAT AKADEMIK

111111111 rOnllllllll1 1000235545

ROSSYUHAIDA BINTI MOHD ZAKRIA (24931)

This project is,submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honors

(Biotechnology)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARA W AK

2012

..,"

, ,.

Declaration

I hereby declare that this thesis entitled "Effects of Castor Oil on Cellulase Enzymatic

Activities on Pure Cellulose during Simultaneous Saccharification and Fermentation

(SSF)" is the result of my own research work and effort. It has not been submitted

anywhere for any award. Where other sources of information that have been used, they

have been acknowledged.

Signature:

Name: Rossyuhaida binti Mohd Zakria

Date:

ACKNOWLEDGEMENTS

Without the help and support of a number of people, it would not have been possible to

prepare this report. First, I would like to express my sincere appreciation and deepest

gratitude to my supervisor, Dr. Micky Vincent who gave me this golden opportunity to

carry out my FYP research in Microbiology Laboratory 4. I thank him for his patience,

valuable advice and encouragement which enabled me to successfully complete my study.

I would also like to thank my co-supervisor, Mdm. Dayang Salwani in allowing me to

analyse data and also taught me to use the software for data analysis. My sincere thanks

also goes to Mr. Azis bin Ajim and Mr. Leo Bulin for their assistance in handling the

various analytical machines and apparatus in the laboratory.

I would like to thank all the postgraduates in Microbiology laboratory 4, especially

Velnetti Linang and Christy Chan Sien Wei for their care, advices and setting aside time

from their busy schedules to help me. As well as to all my laboratory friends, who have

shared their knowledge, materials and beautiful moment throughout the year in finishing

this project.

My truthful gratitude goes to my parents, Mr. Mohd Zakria bin Ahmad and Mdm. Zaiton

binti Jaafar who have always been caring and supporting me along my study. Without their

moral support and financial assistance, it would have been impossible to finish this project.

Finally, I would like to thank everyone else who directly or indirectly contributed to my

study.

I

..

Effects of Castor Oil on Cellulase Enzymatic Activities on Pure Cellulose during Simultaneous Saccharification and Fermentation (SSF)

Rossyuhaida binti Mohd Zakria

Resource Biotechnology Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Castor oil from castor (Ricinus communis) seed can be used as ethanol absorbent during simultaneous saccharification

and fermentation (SSF). This study was performed to investigate the effect of castor oil on cellulase enzymatic activity on pure cellulose during SSF. In this experiment, SSFs were performed in a 150 ml working volume in media bottles after

being aseptically inoculated with Saccharomyces cerevisiae. Carbomethyl cellulose (CMC) of 5% (w/v) and 10% (w/v) were used as feedstocks with 25 FPUlg CMC and 50 FPU/g CMC. Then, 15 ml castor oil was added into the

fermentation broths. The fermentation broths were incubated at 37°C and agitated at 120 rpm under anaerobic conditions

for 5 days. Data were obtained by High-Performance Liquid Chromatography (HPLC) and the respective graph were plotted. Cellulase activities were then investigated by performing enzyme assay. When castor oil was present in the

fermentation broth, the ethanol concentration was reduced. However, both cellobiose and glucose were also reduced suggesting better utilization while having access ethanol absorbent into the castor oil layer. From this study, the data

suggest that the effect of castor oil towards S. cerevisiae was not significant. Furthermore, cellulase enzymatic activities

became better at 25 FPUlg CMC in the presence of castor oil. This result showed that castor oil can be selectively used as ethanol absorbent in the fermentation broth during SSF to increase ethanol yield.

Key words: Castor oil, Simultaneous saccharification and fermentation (SSF), High-Performance Liquid

Chromatography (HPLC), Filter paper unit (FPU), Carbomethyl cellulose (CMC).

ABSTRAK

Minyak Jarak daripada biji jarak (~ com nlllnis i boleh diiadikan sebagai penyerap elanol sewaklll proses sakarifikasi dan fermenlasi serenlak (SFS). Kajian ini lelah dijalankan unlllk mengkaji kesan minyak jarak ke alas

aklivili enzim pada selulosa IlIlen semasa SFS. Dalam eksperimen ini, SFS lelah dilakukan di dalam isipadll 150 1711 dalam bOlol selepas secara aseplik dimasllkkan kullllr Saccharomyces cerevjsiae. Sebanyak 5% dan 10% sellilosa karbomelhyl (SKM) lelalt digllnakan sebagai bahan menlah berserla dengan 25 FPUlg CMC dan 50 FPUlg CMC.

Kemlldian, 15 ml minyak jarak lelah dimasukkan ke da/am bahan fermenlasi. Bahan fnmenlasi lelah dilakukan pada sllhll 37°C dan menggunakan kelajllan 120 rpm di ball'ah keadaan anaerobik selama 5 hari. Dala lelah diambit

menggl/nakan mesin Kromatograji Cecair Berpreslasi Tinggi (KCBT) dan grafyang berkailan lelah diplolkan. Aklivili sell/lase lelah dikaji dengan menjalankan IIjian enzim. Apabita minyak jarak ada dalam bahall penapaian, kepekalan elanol lelah berkurang. Walal/ bagaimanaplln, kedua-dlla selobiose dan glukos juga lelah berkurang mencadangkall

penggrmaan yang lebih baik di samping mempl/Ilyai akses elallol mellyerap ke dalam lapisan minyak jarak. Daripada kajian ini, dala menrrnjl/kkan bahawa kesan minyak jarak lerhadap pembesaran s... cerevisiae jl/ga adalah tidak begi/II

kelara. Tambahan pilla, aklivili enzim selulase menjadi lebih baik pada ml/alan enzim 25 FPUlg CMC dengan adanya minyakjarak. KepI/II/san ini lelah menunjllkkan bahawa minyakjarak boleh digllnakan sebagai pellyerap elanol dalam

bahan pellapaian sewaklll SSF rlllil/k meningkalkan "asi! elanol.

Kala kllnci: Minyakjarak. Sakarifikasi danfermenlasi serenlak (SFS). Kromalograji Cecair Berpreslasi Tinggi (KCBT),

Filler Paper Unil (FPU), Se/ulosa karbomelhyl (SKM).

II.. ....

Pusat Khidmat Maklumat Akademik, . UNlVERSm MALAYSIA SARAWAK

TABLE OF CONTENTS

ACKNOWLEDGEMENTS I

ABSTRACT II

TABLE OF CONTENTS III

LIST OF ABBREVIATIONS v

LIST OF FIGURES VI

CHAPTER 1: INTRODUCTION 1

CHAPTER 2: LITERATURE REVIEW 4

2.1 Castor Bean Plant and Castor Oil 4

2.2 Ethanol and Bioethanol 6

2.3 Simultaneous Saccharification and Fermentation (SSF) 6

2.4 Saccharomyces cerevisiae 7

2.5 Cellulase 8

2.6 High-Performance Liquid Chromatography (HPLC) 9

2.7 , Filter Paper Unit (FPU) 11

CHAPTER 3: MATERIALS AND METHOD 12

3.1 Materials 12

3.2 Microorganisms Preparation 13

3.3 Enzyme Activity Assay - Filter Paper Unit (FPU) 13

3.4 Simultaneous Saccharification and Fermentation (SSF) 15

3.5 Samples Collection 16

3.6 Preparation of Agar Plate 16

III

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3.7 Enumeration of Yeast (Spread Plate Method) 17

3.8 High-Perfonnance Liquid Chromatography (HPLC) Analysis 18

3.9 Enzyme Assay 18

CHAPTER 4: RESULTS 19

4.1 Filter Paper Unit (FPU) 19

4.2 Enumeration ofYeast 20

4.3 High-Perfonnance Liquid Chromatography (HPLC) Analysis 23

4.3.1 Ethanol Yield 24

4.3.2 Glucose Concentration 28

4.3.3 Cellobiose Concentration 32

4.3.4 Acetic Acid Concentration 35

4.3.5 Lactic Acid Concentration 38

4.3 Enzyme Assay 41

CHAPTER 5: DISCUSSION 45

CONCLUSION 48

REFERENCES 49

APPENDIX

IV

· ' LIST OF ABBREVIATIONS

SSF Simultaneous saccharification and fermentation

YM Yeast malt

LB Luria broth

FPU Filter paper unit

HPLC High Performance Liquid Chromatography

DNS 3,5-Dinitrosalicylic acid

CMC Carbomethyl cellulose

IUPAC Union ofPure and Applied Chemistry

v

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LIST OF FIGURES

Figure 2.1 Equation showing the constituion of castor oil. 5

Figure 2.2 S. cerevisiae viewed under light mIcroscope (Magnification 8 1000x).

Figure 2.3 Structural model of cellulase enzyme. 9

Figure 2.4 HPLC instruments in Unimas. 10

Figure 3.1 Fermentation broth under anaerobic condition at 0 h. 16

Figure 3.2 Yeast colonies grow on agar after 24 h of incubation. 17

Figure 3.3 Colour formation after diluted in water. 18

Figure 4.1 Glucose liberated against enzyme concentration 19

Figure 4.2 S. cerevisiae growth curve in the fermentation broth of 5% CMC 21 loading and 25 FPU/g CMC.




Figure 4.6 Time course of ethanol production at 5% CMC loading and 25 24 FPU/gCMC.



Figure 4.9 Time course of ethanol production at 10% CMC loading and 50 27 FPU/g CMC.

Figure 4.10 Time course of glucose concentration in the fermentation broth at 28 5% CMC loading and 25 FPU/g CMC.

Figure 4. 11 Time course of glucose concentration in the fermentation broth at 29 10% CMC loading and 25 FPU/g CMC.

VI

· . Figure 4.12

Figure 4.13

Figure 4.14

Figure 4.15

Figure 4.16

Figure 4.17

Figure 4.18

Figure 4. 19

Figure 4.20

Figure 4.21

Figure 4.22

Figure 4.23

Figure 4.24

Figure 4.25

Figure 4.26

Figure 4.27

Figure 4.28

Time course of glucose concentration in the fennentation broth at 5% CMC loading and 50 FPU/g CMC.

30

Time course of glucose concentration in the fermentation broth at 10% CMC loading and 50 FPU/g CMC.

31

Time course of cellobiose concentration in the fermentation broth at 5% CMC loading and 25 FPU/g CMC.

32


32


33


33

Time course of acetic acid concentration in the fermentation broth at 5% CMC loading and 25 FPU/g CMC.

35


35


36


36

Time course of lactic acid concentration in the fermentation broth at 5% CMC loading and 25 FPU/g CMC.

38


38


39


39

Time course of cellulase activity in the fermentation broth at 5% CMC loading and 25 FPU/g CMC.

41


42


43

VII

44

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Figure 4.29 Time course of cellulase activity in the fermentation broth at 10% CMC loading and 50 FPU/g CMC.

VIII

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CHAPTER 1

INTRODUCTION

The global interest for an alternative, non petroleum-based fuel has increased due to the

inevitable depletion of mineral reserve and the increasing emission of greenhouse gas

effects on the earth. Currently, bioethanol is the prime alternative to fossil fuel as it bums

cleaner and reduces net carbon dioxide emission. This is because fermentation-derived

ethanol is already a part of global carbon cycle and it is produced from renewable

resources such as lignocellulosic biomass (Krishna and Chowdary, 2000; Varga et al.,

2004' Ohgren et al., 2007).

Lignocellulosic biomass is a very promising feedstock for the production of bioethanol

because of its abundance and availability at low cost throughout the year (Wright et aI.,

1988). Lignocellulose includes wood, paper, grass, and solid municipal waste that consist

of three main components namely, cellulose, hemicellulose and lignin. Out of three

components, only cellulose and hemicellulose can be converted into simpler sugar and

fermented into ethanol by fermenting organisms (Ohgren et al., 2007).

Kadar et al. (2003) has reported that the process to convert lignocellulose into ethanol can

be done in several ways. The technologies available to produce ethanol are direct microbial

conversion (DMC), separate hydrolysis and fermentation (SHF), simultaneous

saccharification and co-fermentation (SSCF), and simultaneous saccharification and

fermentation (SSF) process (Wright et aI., 1988; Karimi et al. , 2006). Among these four

processes SSF are proven to be the most efficient way to produce ethanol (Spindler et al.,

1988; Alkasrawi et al., 2003; Karimi et al., 2006).

I t·

SSF integrates saccharification and fennentation process at the same time within a single

vessel as described first by Takagi et at. (1977). SSF can increase the yield and

saccharification rate due to the presence of both enzyme and fennenting organism which

will reduce the accumulation of sugar within a vessel (Ballesteros et at., 2003). Besides

that, SSF can lower the production cost as only one fermentor is used and reduce the

enzyme loading as it reduces the end-product inhibition of enzyme (Spindler et at., 1988).

Not only those, the presence of ethanol in the fennentation broth can reduce or eliminate

unwanted microorganisms (Ballesteros et aI., 2003). The usual fennenting microorganism

used in this process is yeast, particularly Saccharomyces cerevisiae due to its excellent

fennenting capacity, high tolerance to ethanol and capacity to grow rapidly under

anaerobic conditions (Viara et at., 2011).

Presently, industrial bioconversions of lignocellulose into bioethano] are highly expensive

and inefficient. It requires the application of high temperature and acidic or basic

conditions to break down lignin to allow enzymatic hydrolysis of target polysaccharides

(Maki el at., 2009). Another challenge that needs to be addressed is the accumulation of

the ethanol in the fennentation broth that inhibits further conversion. Therefore, in this

study, castor oil will be used as a selective ethanol separator from the fennentation broth.

Castor oil is a natural oil derived from castor seeds. It is highly viscous and dissolves

easily in alcohol, ether, glacial acetic acid, chloroform, carbon sulfide and benzene

(Forero, 2008). However, there are limited articles that document ethanol absorption by

castor oil and the effects of castor oil towards cellulase activity. Hence, this study will be

carried out to detennine the effect of castor oil on cellulase enzymatic activities on pure

cellulose during SSF.

2

, ,

The objectives of this study are to:

1. investigate the effect of castor oil on the rate of carbomethyl cellulose (CMC)

hydrolysis by cellulase.

2. study the effect of castor oil on the growth and fermentation of Saccharomyces

cerevisiae.

3. detennine the effect of castor oil on ethanol production.

3

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CHAPTER 2

LITERATURE REVIEW

2.1 Castor Bean Plant and Castor Oil

Castor bean plant or Ricinus communis belongs to the Euphorbiaceae family. It is

commonly known as castor oil plant or Palma christi. This plant originates from Eastern

Africa and is found growing on a wide scale of tropical and subtropical countries of the

world (Chakrabarti and Ahmad, 2008). It grows wildly and can tolerate long periods of

drought. It is well adapted to arid conditions and can be cultivated as commercial crop on

marginal lands and coastal sandy under warm climates (Forero, 2008; Chakrabarti and

Ahmad,2008).

Castor bean plant is a tall, non hardy and fast growing suckering perennial shrub which can

grow up to 12 m in height. It has stout, hollow branches that are dull pale green or red

while older branches and trunks tum grayish. The fruits are produced in typical clusters,

with each pod containing well developed oil bearing seed (Chakrabarti and Ahmad, 2008).

When ripe the fruit explodes violently and may disperse the seeds a distance of several

meters.

Castor oil which is also known as ricinus oil, phorboyl and tangantangan oil, is natural oil

derived from the seeds of the castor bean (Richard, 1992). The seed contains

approximately 46% oil by weight. This oil is highly viscous and ranges from pale yellow to

colorless. It has a soft, faint odor and a highly unpleasant taste (Chakrabarti and Ahmad,

2008). The boiling point of castor oil is 313 °C (595 OF) and a density of 961 kglm3. It is a

triglyceride of fatty acids that contains approximately 87% ricinoleic, 7% oleic, 3%

4

Pusat Khidmat MakJumat Akademik · UNJVERSm MALAYSIA SARAWAK .

'

linoleic, 2% palmitic, 1 % stearic and trace amount of dihydroxystearic (Richard, 1992).

Ricinoleic acid, a monounsaturated, 18 carbon fatty acid has a hydroxyl functional group

on the twelfth carbon which causes ricinoleic acid and castor oil to be unusually polar and

hence making it valuable as chemical feedstock (Chakrabarti and Ahmad, 2008).

CH-O-C(O)-R + CH-O-C(O)-R

CH2-0-C(O)-R'

R' = other fatty acid derivatives

Figure 2.1: Fonnula showing the constitution of castor oil

There are three functional groups that contribute to the unique chemistry of castor oil

which are the carboxyl group, the single bond of unsaturation and the hydroxyl group. The

carboxyl group can provide a wide range of esterifications. The single point of un saturation

can be altered, epoxidated or vulcanized. The hydroxyl group can be acetylated or

alkalyted, and may be removed by dehydration to increase the unsaturation of the

compound to give a semi-drying oil. The presence of hydroxyl group on castor oil adds

extra stability to the oil by preventing the formation of hydroperoxides and soluble in

alcohols in any proportion (Ogunniyi, 2006).

5

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2.2 Ethanol and Bioethanol

Ethanol is a versatile compound with a chemical formula of CH3CH20H. Its other name is

ethyl alcohol because it contains hydroxyl functional group from the alcohol family. It can

be used as a solvent, germicide, antifreeze, beverage and fuel. At room temperature,

ethanol is a volatile, flammable, clear and colorless liquid. When · diluted with the right

proportion of water, it gives out sweet and pleasant odour (Balat et at., 2008).

Bioethanol is a liquid fuel produced from sources other than mineral reserves such as oil,

coal and gas. It can be used to replace gasoline or blended with gasoline for transportation

fuels (Varga et at., 2004; Ferreira et at., 2010). According to Balat et at. (2008), bioethanol

bums cleaner than gasoline and thus reduces polluting gases such as aromatic

hydrocarbons and carbon monoxide. Moreover, the use of ethanol as an additive to

gasoline will also extend the shelf life of gasoline (Varga et at., 2004).

Bioethanol can be produced from lignocellulosic biomass. The production of bioethanol

employs the saccharification process which converts cellulose into simple sugar and

fermentation which converts the simple sugar into ethanol (Zhang et at., 2009; Ferreira et

at., 2010).

2.3 Simultaneous Saccharification and Fermentation (SSF)

Saccharification is a process of converting cellulose into simpler sugar whereas

fermentation is a process of converting sugar into ethanol. Therefore, simultaneous

saccharification and fermentation (SSF) is a process that combines enzymatic hydrolysis of

cellulose with simultaneous fermentation of its derived sugar to ethanol (Takagi et at.,

1977; Ballesteros et at., 2004; Ferreira et ai., 2010).

6

· ,. Compared to separate hydrolysis and fennentation (SHF), SSF is perfonned in the single

vessel for the entire process. Hence, this technique becomes more favorable to SHF as it

can reduce the enzyme loading and production cost (Kadar et ai., 2003; Ferreira et ai.,

201 0). Furthennore, the presence of ethanol will cause the medium to be less vulnerable to

invasion by undesired microorganisms (Ballesteros et ai., 2004).

2.4 Saccharomyces cerevisiae

Saccharomyces cerevisiae is a simple, unicellular fungus from the yeast family which is

commonly known as brewer's yeast or baker's yeast. It is spherical or ellipsoidal which

reproduces asexually by budding or division (Bamforth, 2005). Its size can vary, typically

measuring from 3 to 8 11m in diameter (Figure 2.2). It is facultative anaerobe which

requires a reduced carbon source to proliferate.

According to Pretorious (2003), S. cerevisiae was the first microorganism to be

domesticated by people for the production of food such as bread in ancient Rome in 100

BC and beverages such as beer in Assyria in 7000 Be. It was also the first to be observed

microscopically by Antonie van Leeuwenhoek in 1632. Later in 1818, Erxleben articulated

the view that yeast was a living organism and responsible for fennentation (Bamforth,

2005).

S. cerevisiae has always been regarded as a key microorganism in food fennentation and

beverages preparation because it produces ethanol and carbon dioxide as byproducts of the

fermentation of sugars. Many researchers have shown that it is the most suitable

microorganism for ethanol production for thousands of years. This is because it can grow

on a variety ofsugars with high substrate and ethanol tolerance (Chandel et aI., 2011).

7

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Figure 2.2: S. cerevisiae viewed under light microscope (Magnification IOOO x).

2.S Cellulase

Cellulase is a modular enzyme which consists of independently folding, structurally and

functionally discrete units called domains or modules. A typical free cellulase is composed

of a carbohydrate binding domain (CBO) at the C-tenninal joined by a short poly-linker

region to the catalytic domain at the N-tenninal. CBD is the most common accessory

module of cellulase which delivers its resident catalytic to crystalline cellulose for efficient

hydrolysis (MaId et aI., 2009).

According to Krishna and Chowdary (2000), there are three major types of cellulase which

are endoglucanases, exoglucanases and ~-glucosidases. The combination action of

endoglucanases and exoglucanases randomly hydrolyzes ~-l ,4-glucosidic linkages such as

CMC into cellobiose and oligosaccharides. ~-glucosidase acts on splitting-off cellobiose

Wlits from the non-reducing end of the chain. It hydrolyzes cellobiose and oligosaccharides

to glucose (Sinegani and Emtiazi, 2006). During enzymatic hydrolysis, cellulose is

8

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degraded by the arrangement of cellulase activities to reducing sugar that can be fermented

by bacteria or fungi to ethanol.

Cellulase activity is inhibited by cellobiose and to a lesser extent by glucose. To overcome

this problem, SSF is used where reducing sugar produced from saccharifications are

simultaneously fermented to ethanol, greatly reducing the end-product inhibition to the

hydrolysis (Brethauer and Wyman, 2009).

Figure 2.3: Structural model of cellulase enzyme (Garsoux et af., 2004).

2.6 High-Performance Liquid Chromatography (HPLC)

Chromatography is derived from two Greek words which are "chromos" stands for colour

and "grafe" stands for writing (Scott, 1994). It is a separation process in which the sample

mixture is distributed between two phases in the chromatographic column (Meyer, 2010).

One phase is stationary while the other passes through the chromatographic column which

is the mobile phase. The stationary phase is either a solid, porous, surface active material in

9

a small particle fonn or a thin film of liquid coated on a solid support or column wall

whereas the mobile phase is a liquid.

A typical HPLC system (Figure 2.4) is made up of several main components which are

solvent reservoir, transfer line with frit, high-pressure pump, sample injection device,

column, detector, and data acquisition, usually together with data evaluation (Meyer,

2010). The most important part is the column which is the smallest component in HPLC. It

is a simple tube, a few centimeters long and a few millimeters in diameter, packed with

particulate material through which the mobile phase penneates (Scott, 1994). A mixer and

a controller are needed when working with more than one solvent. Computer can be used

to control the whole system if it is used to acquire data which makes HPLC easier and

practical (Scott, 1994; Meyer, 2010).

When perfonning HPLC analysis, the mobile phase transports the eluted compounds to the

detector and recorded as Gaussian (bell-shaped) curves. The signals are known as peaks

and the whole entity is the chromatogram. It gives direct information on the qualitative and

quantitative of sample mixtures (Meyer, 2010).

Figure 2.4: A Shimadzu HPLC instruments in UNIMAS.

10

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2.7 Filter Paper Unit (FPU)

FPU is a specific enzyme activity assay which is perfonned using the protocol described

by the official National Renewable Energy Laboratory (NREL) procedure (Adney and

Baker, 2008). This method is based on the International Union of Pure and Applied

Chemistry (lUPAC) guidelines to detennine cellulase activity in tenns of "filter-paper

units" (FPU) per gram (FPU/g) of an original enzyme preparation (Ghose, 1987).

The detection of glycosidic bond cleavage by this method involves the parallel and

identical treatment of three categories of experimental tubes (assay mixtures, blanks and

controls, and glucose standard). The substrate used is a 50 mg Whatman No. 1 filter paper

strip (1.0 x 6.0 cm).

For quantitative results, the enzyme preparations must be compared on the basis of

significant and equal conversion. According to Adney and Baker (1996), the value of 2.0

mgofreducing sugar as glucose from 50 mg of filter paper (4% conversion) in 60 minutes

has been designated as the intercept for calculating filter paper cellulase units (FPU) by

IUPAC.

11

CHAPTER 3

MATERIALS AND METHODS

3.1 Materials

The materials used in this study are listed below:

• Castor oil (Aldrich Chemical Co., Inc, India)

• Carbomethyl cellulose (CMC) powder (Nacalai Tesque, Inc., kyoto, Japan)

• Liquid cellulase enzyme (Genencor International, B.Y., Netherlands)

• 1 M Citrate buffer

• Citric acid monohydrate

• Deionised water

• NaOH - add until pH equals 4.3

• DNS solution

• Distilled water

• 3,5 Dinitrosalicylic acid

• Sodium hydroxide

• Rochelle salts ( odium potassium tartrate)

• Phenol (melt at 50°C)

• Sodium metabisulfite

• 1 X YP medium

• Yeast extract

• Peptone

• Saccharomyces cerevisiae culture (ATCC 24859)

210 g

750 ml

50 to 60 g

1416 ml

10.6 g

19.8 g

306 g

7.6 ml

8.3 g

10 gil

20 gil

12

i·

• LB broth (Laboratorios Conda, S.A, Madrid, Spain)

• YM broth (Becton, Dickinson & company, France)

• Bacteriological agar (Wako Pure Chemical Industries Ltd, Osaka, Japan)

• PBS buffer

• Sterile distilled water

3.1 Microorganisms Preparation

s. cerevisiae was prepared by growing cultures overnight in 100 m1 sterile LB broth at 32

°c with constant agitation at 120 rpm. Cells were harvested via centrifugation at room

temperature in two 50 ml conical centrifuge tubes for 3 min, at 7000 rpm in a centrifuge.

3.3 Enzyme Activity Assay - Filter Paper Unit (FPU)

1. Enzyme assay tubes:

A rolled filter paper strip was placed into each 13 x 100 mm test tube. Then, 1.0 ml 0.05 M

Na-citrate, pH 4.8 was added to the tube, the buffer should saturate the filter paper strip.

Tubes were equilibrated with buffer and substrate to 50°C. Next, 0.5 ml enzyme diluted

appropriately in citrate buffer was added. At least two dilutions must be made of each

enzyme sample, with one dilution releasing slightly more than 2.0 mg of glucose (absolute

lIIlOunt) and one slightly less than 2.0 mg of glucose. This was followed by incubating at

°C for exactly 60 min. At the end of the incubation period, each assay tube was

ved from the 50°C bath and the enzyme reaction was stopped by immediately adding . .

13

Documents

SB of Castor Oil On Cellulase... · sell/lase lelah dikaji dengan menjalankan IIjian enzim. Apabita minyak jarak ada dalam bahall penapaian, kepekalan elanol lelah berkurang. Walal