SEPARATION OF FRUCTOSYLTRANSFERASE USING ...

SEPARATION OF FRUCTOSYLTRANSFERASE USING ULTRAFILTRATION MEMBRANE: EFFECT OF pH AND IONIC STRENGTH ON FLUX AND REJECTION

MOHD KHAIRUL AFIZAN BIN HARUN

A thesis submitted in fulfillment of the requirement for the award of the degree of Bachelor of Chemical Engineering

Faculty of Chemical and Natural Resources Engineering Universiti Malaysia Pahang

APRIL 2009

I declare that this thesis entitled “Separation of Fructosyltransferase Using Ultrafiltration Membrane: Effect of pH and Ionic Strength on Flux and Rejection” is the result of my own research except as cited in the references. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.

Signature

: ..................................................

Name of Candidate

: MOHD KHAIRUL AFIZAN BIN HARUN

Date

: 2 APRIL 2009

iii

Special Dedication of This Grateful Feeling to My

Beloved parents; Hj. Harun bin Jusoh & Hajjah Aminah binti Hj. Othman

Loving brother and sisters; Nor Ashikin Hj. Harun Samseema Hj Harun Suraya Hj. Harun Mohd Reduan Hj. Harun Suzana Hj. Harun Siti Khatijah Hj. Harun Noor Nabila Huda Hj. Harun Nor Bashirah Hj. Harun

iv

ACKNOWLEDGEMENT

I would to express gratitude to all who gave me the possibility to complete this Undergraduate Research Project (PSM). I want to thank the first and foremost, my sincere appreciation to my Undergraduate Research Project supervisor, Dr Mimi Sakinah Binti Abdul Munaim, for guiding and encouraging me throughout this experiment. Thanks a lot for giving me a professional training, advice and suggestion to bring this Undergraduate Research Project to its final form. Without her support and interest, this PSM would not have been the same as presented here.

I am grateful to the staff of Technical Unit, Faculty of Chemical & Natural Resources Engineering of Universiti Malaysia Pahang as Mr. Zainal bin Gimban and En. Abd Razak bin Abd Hamid for their cheerfulness and professionalism in handling their work.

Special appreciation for Miss Kamariah bt Mat Peah from Faculty of Civil & Earth Resources as her interest to help for using Total Orgnanic Carbon.

In particular, my sincere thankful is also extends to all my colleagues as Nor Diyana binti Abu Bakar Sidek and others masters student who have provided assistance at various occasions. Their views and tips are useful indeed. Unfortunately, it is not possible to list all of them in this limited space.

And last, but not least I thank my mother’s and other family members for their continuous support while completing this PSM.

v

ABSTRACT

There are various methods to separate between macro molecule and non dissolved particle in chemical process. One of the methods is separation process using ultrafiltarion membrane. In filtration process, the macromolecules such as enzyme will be retained on the membrane surface. This experiment is study about fouling characteristic occur in an industry. The fouled membrane surface problem gives the high cost operation and reduces the quality of production. Therefore, the main objectives for this experiment are to determine the effect of pH and ionic strength on membrane flux and rejection during fructosyltransferase (FTase) separation. The 50 kDa molecular weight cut off (MWCO) of ultrafiltration membrane was used during this experiment. Cross flow filtration was used to run this experiment in the lab scale. Total organic carbon (TOC) was used to analysis the concentration of sample. Potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) buffer solution range pH 5 to pH 8 was applied to find the effect of pH and various molarities of NaCl (0.5M to 2.0M) was used to find the effect of ionic strength in ultrafiltration membrane. The experimental result shows that the optimum pH and ionic strength was 8.0 and 0.5M, respectively, in order to separate the FTase solution using ultrafiltration membrane.

vi

ABSTRAK

Pelbagai langkah dan teknik digunakan bagi pemisahan antara molekul macro dan bahan tidak terlarut dalam prosess kimia. Salah satu kaedah yang digunakan ialah proses pemisahan dengan menggunakan penapis ultra. Dalam proses pengasingan atau penapisan ini, molekul macro seperti enzim akan tertahan di permukaan penapis. Dalam eksperimen ini, kajian dijalankan bagi mengenal pasti ciri – ciri bahan yang menyebabkan berlaku penyumbatan penapis dalam industri kerana ia akan memberi kesan negatif seperti peningkatan kos operasi dan mengurangkan kualiti produk. Objektif utama kajian ini adalah bagi mengenal pasti, kesan pH dan kekuatan ionik kepada fluks dan bahan tertahan dengan menggunakan enzim fructosyltransferase (FTase). Saiz penapis yang digunakan dalam eksperimen ini ialah 50 kDa. Manakala penapis aliran songsang yang digunakan adalah berskala kecil. Total organic content (TOC) digunakan bagi menganalisa kepekatan sampel. Kalium dihodrogen Phospahte (KH2PO4) dan diKalium hydrogen phosphate (K2HPO4) digunakan sebagai larutan penimbal dengan skala antara pH 5 sehingga pH 8, manakala 0.5M sehingga 2.0M Natrium Klorida (NaCl) digunakan bagi menganalisa kekuatan ionik larutan tersebut. Keputusan daripada kajian ini menunjukan, pH 8 dan 0.5M merupakan larutan yang optimum untuk digunakan dalam proses penapisan dengan menggunakan penapis ultra.

TABLE OF CONTENTS

CHAPTER

1

2

TITLE

PAGE

TITLE PAGE

i

DECLARATION

ii

ACKNOWLEDGEMENT

iii

ABSTRACT

v

ABSTRAK

vi

LIST OF TABLES

xi

LIST OF FIGURES

xvi

INTRODUCTION 1.1 Background of Study

1

1.2 Problem Statement

2

1.3 Objective

3

1.4 Scope of Study

3

1.5 Significant of Study

4

LITERATURE REVIEW 2.1 Enzyme of Fructosyltransferase

6

2.2 Definition of Membrane

10

2.2.1 Driving force in membrane separation process

10

2.2.2 Transmembrane pressure

10

2.3 Membrane Structure

11

2.3.1 Porous Membrane

11

2.3.2 Non-Porous Membrane

12

2.3.3 Carrier Membrane

12

2.4 Membrane Type

13

2.5 Flowsheet

14

CHAPTER

TITLE 2.6 Process Operation

15

2.7 Membrane Module Type and Their Characteristic

16

2.7.1 Plate and Frame

16

2.7.2 Spiral – Wound Module

17

2.7.3 Hollow Fiber, Capillary and Tubular

18

2.8 Unltrafiltration Membrane

19

2.8.1 Asymmetric Membrane

19

2.8.2 Porous Membrane

19

2.8.3 Types of Flow Ultrafiltration Process

20

2.8.4 Protein Separation Mechanisme

20

2.8.5 Factor Affecting Ultrafiltration Membrane

21

2.8.5.1 Temperature

21

2.8.5.2 Ratio of Concentration

21

2.8.5.3 Viscosity and Volume Flow Rate

21

2.9 Fouling

22

2.9.1 Definition of Fouling

22

2.9.2 Particles, Biofouling and Scaling

23

2.9.3 Predict Fouling

23

2.9.4 Membrane Fouling Control

24

2.9.4.1 Silt Density Index (SDI) 2.10 Limitations

3

PAGE

24 25

METHODOLOGY 3.1 Overall Methodology

27

3.2 List of Apparatus

27

3.2.1 Ultrafiltrtion System

29

3.2.2 Membrane Type

30

3.3 List of Chemical

30

3.4 Preparation of Solution

31

3.4.1 Preparation of Buffer Solution

31

CHAPTER

TITLE

PAGE

3.5 Separation of FTase using Ultrafiltration Membrane

32

for Effect of pH 3.6 Flux Analysis

33

3.7 Protein Rejection Analysis

34

3.8 Total Organic Carbon (TOC) Analysis

34

3.9 Separation of FTase using Ultrafiltration Membrane

35

for Inoic Strength

4

RESULT AND DISCUSSION 4.1 Effect of pH on Flux during Membrane Separation 4.1.1 FTase Flux at pH 5 using Ultrafiltration

37 37

Membrane 4.1.2 FTase Flux at pH 6 using Ultrafiltration

38


39


41

Membrane 4.1.5 Overall Flux Analysis during FTase Separation

42

at Different pH Solution 4.2 Effect of pH on Membrane Rejection 4.2.1 Rejection Analysis of FTase at Different pH

45 45

Solution 4.3 Effect of Ionic Strength on Membrane Flux 4.3.1 Flux Decline during FTase Separation at 0.5 M

47 47

NaCl 4.3.2 Flux Decline during FTase Separation at 1.0 M

48

NaCl 4.3.3 Flux Decline during FTase Separation at 1.5 M NaCl

50

CHAPTER

TITLE 4.3.4 Flux Decline during FTase Separation at 2.0 M

PAGE 51

NaCl 4.3.5 Overall Inonic Strength Analysis during FTase

53

Separation.

5

CONCLUSIONS AND RECOMENDATION 5.1 Conclusions

56

5.2 Recommendation

57

REFERENCES

58

APPENDIX A

61

APPENDIX B

74

APPENDIX C

95

xi

LIST OF TABLES

TABLE NO.

TITLE

PAGE

2.1

Membrane Materials for Various Applications

13

2.2

Membrane Separation Processes with its Various

15

Characteristics 2.3

Plate and Frame

16

2.4

Spiral –Wound Modules

17

2.5

Hollow-Fiber, Capillary and Tubular

18

3.1

Preparation of Buffer Solution

31

3.2

Recommendation Cleanign Conditions

33

4.1

% of Rejection at Different pH Solution

46

A.1

Flux Decline during FTAse Separation at pH 5

62

A.2


63

A.3


64

A.4


65

A.5

Volume of Flux for every pH

66

A.6

Flux for every pH

67

A.7

Flux Volume for 0.5M NaCl

68

A.8


69

A.9


71

A.10


71

A.11

Volume of Flux for every Mole

72

A.12

Flux for every Mole

73

Table from TOC

74

B

xvi

LIST OF FIGURES

FIGURES NO.

TITLE

PAGE

1.1

KvickTM Lab Cross-Flow System Units

4

2.1

Process flowsheet of industrial production of FTase.

9

2.2

Porous Membrane (separation of smaller species)

11

2.3

Non-porous membrane

12

2.4

Carriers membrane

13

2.5

Parallel Flow

14

2.6

Series Flow

14

2.7

Two Stage Flow

14

2.8

Plate and Frame Schematic

16

2.9

Spiral-Wound Schematic

17

2.10

Bore Feed Schematic

18

2.11

Shell Feed Schematic

19

2.12

Rtotal in membrane

23

3.1

Methodology for Effect of pH

27

3.2

Methodology for Effect of Ionic Strength

28

3.3

Cross flow Ultrafiltration Membrane

29

3.4

Polyethersulfone membrane

30

3.5

Total Organic Carbon

34

4.1

Flux during FTase separation at pH 5

37

4.2

Volume during FTase separation at pH 5

37

4.3


38

4.4


39

4.5


40

4.6


40

4.7


41

ix FIGURES NO.

TITLE

PAGE

4.8


42

4.9

Overall Flux Analysis during FTase separation at

43

different pH 4.10

Overall Volume Analysis during FTase separation at

43

different pH 4.11

Flux on 10 minute on different pH

44

4.12

Volume on 10 minute on different pH

44

4.13

Analysis of Rejection at Different pH Solution

46

4.14

Ionic Strength during FTase separation at 0.5M

47

4.15

Volume of Flux during FTase separation at 0.5M

48

4.16


49

4.17


49

4.18


50

4.19


51

4.20


52

4.21


52

4.22

Overall analysis of Flux during FTase separation at

54

different mole solution. 4.23

Overall analysis of volume during FTase separation at

54

different mole solution. 4.24

Flux on 10 minute at different mole

55

4.25

Volume on 10 minutes at different mole

55

C.1

Cross Flow Filtration

96

C.2

Apparatus of Experiment

96

C.3

Apparatus of Experiment

97

C.4

Sample of Experiment

97

C.5

Sample of Experiment

98

C.6

Total Organic Carbon

98

CHAPTER 1

INTRODUCTION

1.1

Background of Study

Excellent water quality produced by membrane filtration has made this advanced technology a promising process in providing better drinking water for water supply. Membrane filtration processes involving microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) in potable water production have increased rapidly for the past decade and would potentially replace the conventional treatment process trains which consist of ozonation–precipitation– coagulation– flocculation–chlorination–gravel filtration (Clever et al., 2000)

Recently, membrane separation involves partially separating a feed containing a mixture of two or more components by use of a semi permeable barrier (the membrane) through which one or more of the species moves faster than another or other species. As shown in Figure 1.1, the basic process of the membrane separation involves a feed mixture separated into a retentate (part of the feed that does not pass through the membrane, or retained) and a permeate (part of the feed that passes through the membrane). Although the majority of time the feed, retentate, and permeate are usually liquid or gas, they may also be solid. The optional sweep is a liquid or gas, used to help remove the permeate. (Ali et al., 2003)

2 Fructosyltransferase

is

an

enzyme

transforming

sucrose

into

fructooligosaccharides (FOS). FOS is fructose oligomers with a terminal glucosyl unit and with a general formula GFn, where typical values of n are 2–4. FOS is classified as prebiotics and has numerous beneficial properties for human health (Yun, 1996).

They are widely utilized in food and pharmaceutical industries. Although FTase was found in many higher plants and microorganisms, the most important industrial sources are strains of Aspergillus niger, Aspergillus japonicus and Aureobasidium pullulans (Yun, 1996).

In spite of the utilization of FTase in the industrial production of FOS and numerous scientific investigations, the only commercially available source of FTase is Pectinex SP-L, a pectinolytic and cellulolytic preparation designated for fruit juice processing.

1.2

Problem Statement

Many bioproducts are enzyms and there is a great demand for their separation. Conventional techniques such as precipitation, crystallization and centrifugation can suffer from poor selectivity of separation. The high-resolution separation techniques such as chromatography, affinity separation and electrophoresis have a very low throughput and produce small quantities of very pure proteins; to produce larger amounts of proteins using these methods is expensive. (Yunos and Field, 2007)

One of the critical issues in the development of effective whey ultrafiltration processes is the decline in system performance due to enzyme fouling, which limits the economic efficiency of the processing operation. Membrane fouling is generally characterized as a reduction of permeate flux through the membrane as a result of increased flow resistance due to pore blocking and cake formation. Several approaches have been proposed to reduce such membrane fouling and to improve the

3 membrane cleaning efficiency. Such methods include intermittent back flushing, flow pulsation and electrical field inducement. (Muthukumaran et al., 2007)

1.3

Objectives

The objectives of this research are:

a) To determine the effect of pH on membrane flux and rejection during Fructosyltransferase separation. b) To determine the effect of ionic strength on membrane flux and rejection during Fructosyltransferase separation. c) To determine the optimum condition of pH and ionic strength for Fructosyltransferase separation.

1.4

Scope of Study

There are few purposes doing this research. The purposes are:

i.

The membrane will be used is which have 50kDA number of molecular cut off.

ii.

The protein that is used is Fructosyltransferase (FTase)

iii.

KvickTM Lab Cross-Flow System Unit was used in order to separate the solution of DI water and FTase.

iv.

The FTase solution will be prepared in sample which is pH 5 to pH 8.

v.

The buffer solution will be prepared around 0.5M to 2.0M

4 vi.

Total Organic Carbon will be used to measured the carbon in feed and permeate

Figure 1.1 KvickTM Lab Cross-Flow System Units

1.5

Significant of Study

By doing this research, it is hoped can add values of FTase and membrane ultrafiltration. The main problem to solve in this experiment is to produce maximum the production of FTase using ultrafiltartion membrane. If the common industry used the others membrane rane like chromatography, affinity separation and electrophoresis to produce the FTase, this experiment hope get better result if using the ultrafiltration membrane system.

5 Normally during the separation process between FTase and solution, FTase fouling will occur, this because the molecular weight of FTase not suitable with the pore size of membrane. The important thing here is use the different value of molecular weight and pore size.

This research also suggests using the continuous system. Hence it can reduce the cost of operation. The price which is use as a raw material to produce fructoligoscaride (FOS) is expensive; the continuous system is preferable due to this problem.

CHAPTER 2

LITERATURE REVIEW

2.1 Enzyme of Fructosyltransferase

Fructosyltransferase (FTase) is an enzyme that catalyzes the transformation of sucrose into fructooligosaccharides (FOS), which are important prebiotic compounds having a broad application in food and pharmaceutical industries. Fructosyltransferase catalyzes the transfer of fructosyl moieties where a donor or acceptor of these moieties can be sucrose or fructooligosaccharides. In the industrial production of fructooligosaccharides, the cells with the FTase activity are produced by aerobic cultivation of fungi such as Aspergillus niger, Aspergillus japonicas, or Aureobasidium pullulans. They are applied for the biocatalytic process in immobilized form. (Vankova, Antosova, and Polakovic, 2005)

In our laboratory, we have dealt with the development and optimization of the process of cultivation of the cells of A. pullulans with the FTase activity. The increasing interest in prebiotic compounds opens also possibilities for small-scale use of FTase. Isolated enzyme could be a suitable form for such purposes. For that reason, we have also recently dealt with the downstream processing of FTase from the broth

7 obtained at the cultivation of A. pullulans. The obtained data can be used for the design of the production process of FTase and analysis of its economic efficiency. (Vankova, Antosova, and Polakovic, 2005)

The overall production of FTase depended strongly on the initial sucrose concentration. This effect was the most notable where the production of FTase was stopped after two days. The relative increment of the total FTase activity between the 2nd and 4th day was much lower in comparison with that between the 1st and 2nd day. Such a drop of the enzyme production rate was not observed in the cultivations with the initial sucrose concentration where the total enzyme activity reached the value in the fourth day. (Antosova et al., 2002). Suppression of FTase production by increasing sucrose concentration was observed, which is contrary to the results found the largest amount of enzyme produced of sucrose after two cultivation days. (Hayashi et al., 1991)

The FTase activity of cells represented approximately 60 to 70 % of the total activity since the second cultivation day and the ratio of activities of cells and activities in cultivation medium was 1.3 to 1.6 independently of the sucrose concentration. The ratio of the cell to cultivation medium activities depends on the content of magnesium sulfate in the production medium. The addition of magnesium sulfate to the medium at the content of 0.2 % increased this ratio to the value of about 1.2 which was almost constant during the entire cultivation period. From this point of view, the value of the ratio of 1.3:1.6 obtained by us at 0.05 % MgSO4 is noteworthy (Hayashi et al., 1991).

The specific cell activity with respect to dry cell mass is a crucial factor for the control of a cultivation run if whole cells, either free or immobilized, are used as biocatalysts. Its value reached the maximum already in the rest day at S0 = 50 g dm−3 or in the second day at S0 = 200 g dm−3 and 350 g dm−3. The maximum value of 8860 U g−1 was reached again in the cultivation with initial sucrose concentration of 350 g dm−3. As it has been mentioned above, the initial sucrose concentration influenced the

8 amount of produced FTase whereas the cell mass produced after four cultivation days was unelected. This result suggests that the FTase production was promoted by high sucrose concentrations. Although other authors used different activity assay conditions and the absolute values are not fully comparable, the FTase activities of AP CCY 27-1-1194 are of the same order of magnitude as those published for highly active production strains, which suggests a potential of our strain for industrial production of fructosyltransferase (Hayashi et al., 1991).

The design and scheduling of industrial biotechnological process is often simplified by specialized computer-aided software such as Aspen Batch Plus or SuperPro-Designer. These were applied in several studies of scale-up, optimal plant design, and analysis of investment and operating costs of pilot and industrial production of proteins. The examples include the production of insulin, tissue plasminogen activator, β-galactosidase, heparinase, or growth hormone. (Vankova, Antosova and Polakovic, 2005)

FTase of A. pullulans occurs in the periplasmic space of cells and so the part of the enzyme is easily released to the cultivation medium. Therefore, the recovery of the enzyme was considered from both the harvested cells and cultivation medium. (Vankova, Antosova and Polakovic, 2005)

9

Figure 2.1 Process flowsheet of industrial production of FTase.

10 2.2 Definition of Membrane

Membrane can be define as a thin barrier which is allow passage of particle with a certain size, particular physical or chemical properties (Ghosh, 2003). A membrane can be dividing into types which are cell membrane and synthetic membrane. The cell membrane is a semi permeable lipid bilayer which can be found in all cells (Ghosh, 2003). Meanwhile, the synthetic membrane is a membrane that being prepared for separation task in laboratory and industry. Their active part, which permits selective transport of material, usually consists of polymer or ceramics, seldom glass or material (Ghosh, 2003). Membrane can be prepare in variety forms like flat sheets, tubes, capillary and hollow fibres. Membrane is built in membrane modules like plate and frame, spiral-wound module, hollow fibre module or tube-inshell module (Ghosh, 2006).

2.2.1 Driving force in membrane separation process

Different driving force does include in membrane separation process. Some of this are being applied when to transport solute and solvent molecules through membranes. The forces include transmembrane pressure, concentration or electrochemical gradient, osmotic pressure and electric field (Ghosh, 2003)

2.2.2 Transmembrane pressure

The transmembrane pressure is the main applied driving force (Ghosh, 2003). Due to this applied driving force, the bulk liquid medium which is the solvent is forced through the pores. The solvent molecules carry the solute molecules towards the membrane and in certain case through membrane. Solute molecules might be fully

11 transmitted, partially transmitted or totally retained (or rejected) by membrane (Ghosh, 2003).

2.3 Membrane Structure

Because the membrane must allow certain constituents to pass through, they must have a high permeability to certain types of molecules. Membrane structures consist of the following three basic types:

2.3.1 Porous Membranes

Porous membranes are used in microfiltration and ultrafiltration. The dimension of the pores (0.1~10um) mainly determines the separation characteristics. High selectivity can be obtained when the size of the solute is large relative to the pore size in the membrane. Microporous membranes are similar to porous membranes and differ in regards to pore dimension (50~500 Angstrom).

Figure 2.2 Porous Membrane (separation of smaller species)