EFFECT OF PH AND TEMPERATURE ON FRUCTOSYLTRANSFERASE (FTase) PRODUCTION FROM CANDIDA KRUSEI MOHD NAZARNI BIN CHE ISA Thesis submitted to the Faculty of Chemical and Natural Resource Engineering in fulfillment of the requirements for the award of the Degree of Bachelor of Chemical Engineering in Biotechnology Faculty of Chemical and Natural Resources Engineering UNIVERSITI MALAYSIA PAHANG FEBRUARY 2013
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EFFECT OF PH AND TEMPERATURE ON FRUCTOSYLTRANSFERASE (FTase)
PRODUCTION FROM CANDIDA KRUSEI
MOHD NAZARNI BIN CHE ISA
Thesis submitted to the Faculty of Chemical and Natural Resource Engineering in
fulfillment of the requirements for the award of the Degree of Bachelor of Chemical
Engineering in Biotechnology
Faculty of Chemical and Natural Resources Engineering
UNIVERSITI MALAYSIA PAHANG
FEBRUARY 2013
v
EFFECT OF PH AND TEMPERATURE ON FRUCTOSYLTRANSFERASE
(FTase) PRODUCTION FROM CANDIDA KRUSEI
ABSTRACT
The relationship between consumer, food and health become one of important
issues because of increasingly demand on healthier food. Based on functional
properties of fructosyltransferases (FOS), the consumer demands have been
increasing in order to response on that. In Malaysia, it is reported that the number of
diabetic patients is increasing daily and it is one of the fatal diseases. A significant
driving force in the „functional food‟ market place is the quest by consumers to
optimize their health through food. The enzyme source synthesis production can be
divided into two classes which are from microorganism and plant. Where by plant,
the production is restricted from any sources .In view of that, production of crude
enzyme from micro fungi is studied in order to obtain high yield of FOS. This study
is carried out using sucrose as a substrate in the fermentation process to produce
crude enzyme from micro fungi, namely Candida krusei. In this study, an
investigation on pH and temperature of enzyme was analysing ranging from 3.0-8.0
and 25-55oC,
respectively. The crude enzyme was analysed using UV-Vis U-1800
Spectrophotometer at wavelength of 540 nm. The protein concentration was
determined using UV-Vis U-1800 Spectrophotometer at wavelength of 750 nm. By
using an extracellular enzyme which is fructosyltansferase (FTase) from selected
micro fungi, the food-grade FOS can be produced commercially from sucrose. As
result indicates that the highest pH and temperature for activity of FTase from
Candida krusei is pH5.5 and temperature 55oC. The highest enzyme activity
(148.501 IU/mL) was figured out at pH 5.5, 55oC and the specific activity was
observes as 10.638 IU/mg. As a conclusion, this research was done to produce other
alternative way of producing crude FTase using selected microfungi that are less
harmfull to use in a production of FOS in food health industries.
vi
KESAN DARI PENGGUNAAN PH DAN SUHU TERHADAP PENGHASILAN
FRUKTOSILTRANSFERASE (FTase) DARI CANDIDA KRUSEI
ABSTRAK
Hubungan yang terjalin antara pengguna, makanan dan juga kesihatan
menjadi salah satu isu yang penting kerana meningkatnya permintaan terhadap
makanan yang sihat. Berdasarkan kepada ciri-ciri dan fungsi yang ada pada
fruktosiltransferase (FOS), permintaan pengguna terhadapnya telah meningkat. Di
Malaysia, telah dilaporkan bahawa bilangan pesakit kencing manis semakin
meningkat setiap hari dan ia merupakan salah satu penyakit yang membawa maut.
Satu daya penggerak penting dalam pasaran “makanan berfungsi” adalah usaha yang
dilakukan oleh pengguna untuk mengoptimumkan kesihatan mereka melalui
pemilihan makanan yang betul. Sumber penghasilan sintesis enzim boleh
dibahagikan kepada dua kelas iaitu mikroorganisma dan juga tumbuhan. Jika dari
tumbuhan, pengeluaran enzim adalah terhad dari mana-mana sumber. Sehubungan
itu, pengeluaran enzim mentah dari kulat mikro telah dikaji dalam usaha
mendapatkan penghasilan yang tggi dalam FOS. Kajian ini telah dijalankan
menggunakan sukrosa sebagai substrat dalam proses penapaian untuk menghasilkan
enzim mentah dari kulat mikro, iaitu Candida krusei. Dalam kajian ini, siasatan telah
dijalankan keatas kesan pH dan suhu enzim yang telah dianalisis masing-masing
antara 3.0-8.0 dan 25-55ºC. Enzim mentah dan kepekatan protein telah dianalisa
dengan menggunakan UV-Vis U-1800 Spektrofotometer pada panjang gelombang
540 nm dan 750 nm. Dengan menggunakan enzim extracellular fruktosiltransferase
(FTase) daripada kulat mikro yang dipilih, gred makanan berasaskan FOS boleh
dihasilkan secara komersial daripada sukrosa. Hasil keputusan menunjukkan bahawa
pH dan suhu paling tinggi untuk aktiviti FTase enzim dari Candida krusei adalah
pada pH 5.5 dan suhu 55ºC. Aktiviti enzim yang tertinggi (148.501 IU/mL) telah
dicatatkan pada pH 5.5, 55ºC dan aktiviti tertentu telah diperhatikan pada nilai
10.638 IU/mg. Sebagai kesimpulannya, kajian ini dilakukan untuk menghasilkan
cara alternative lain dalam menghasilakn FTase mentah menggunakan mikrofungi
terpilih yang kurang berbahaya untuk digunakan dalam penghasilan FOS dalam
industri makanan kesihatan.
vii
TABLE OF CONTENTS
Page
SUPERVISOR DECLARATION i
STUDENT DECLARATION ii
ACKNOWLEDGMENT iv
ABSTRACT v
ABSTRAK vi
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF SYMBOLS xiii
LIST OF ABBREVIATIONS xiv
CHAPTER 1 INTRODUCTION
1.1 Background of Study 1-3
1.2 Problem Statement 4-5
1.3 Research Objective 5
1.4 Scope of Study 5-6
1.5 Rationale and Significance 6-7
CHAPTER 2 LITERATURE REVIEW
2.1 Introduction 8-9
2.2 Sucrose 9
2.3 Oligosaccharides 10
2.4 Fructooligosaccharides (FOSs) 10-11
2.4.1 Physical and Chemical Properties 11-12
2.4.2 Functional Properties of FOS 12-13
2.4.3 Application of FOS 13-14
2.5 Fructosyltransferases (FTase) 14-15
2.5.1 Mechanism of Fructosyltransferase (FTase) 15-17
viii
2.5.2 Bacterial FTase 17-19
2.5.3 Fungal FTase 19-20
2.5.4 Fermentative Methods of FTase Production 21
2.6 Features of Candida krusei 22
2.6.1 Application of Candida krusei 22-23
2.7 Characterization of enzyme 24
2.7.1 The effect of pH 24-25
2.7.2 The effect of Temperature 25-26
CHAPTER 3 METHODOLOGY
3.1 Chemical and equipment 27-28
3.2 Overview of methodology 29
3.3 Culture method
3.3.1 Agar plate Culture 30
3.3.2 Inoculum preparation 30-31
3.3.3 Fermentation of Candida krusei 31-32
3.4 Data analysis
3.4.1 Detrmination of FTase activity 33-34
3.4.2 Determination of sucrose consumption 34-35
3.4.3 Determination of biomass 35
3.4.4 Determination of protein concentration 35-36
3.4.5 Determination of specific activity 36
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Glucose Calibration Curve 38-39
4.2 Growth profile 39-41
4.3 Effect of incubation time on enzyme activity 42-43
ix
4.4 Effect of temperature on enzyme activity 43-45
4.5 Effect of pH on enzyme activity 45-47
4.6 Determination of protein concentration 47-49
4.7 Specific activity of enzyme 49-50
CHAPTER 5 CONCLUSION
5.1 Conclusion 51
5.2 Recommendation 52-53
REFERENCES 54-56
APPENDICES
Appendix A 57-59
Appendix B 60-63
x
LIST OF TABLES
Page
Table 2.1 Physical and chemical properties of FOS 12
Table 2.2 Application of FOS 13-14
Table 2.3 List of microorganism able to produce FTase enzyme 17-18