UNIVERSITI PUTRA MALAYSIA

A SIMPLIFIED AND SELECTIVE TECHNIQUE FOR THE DIRECT RECOVERY OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE

FROM UNCLARIFIED YEAST FEEDSTOCK

CHOW YEN MEI.

FK 2005 27

A SIMPLIFIED AND SELECTIVE TECHNIQUE FOR THE DIRECT RECOVERY OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM

UNCLARIFIED YEAST FEEDSTOCK

BY

CHOW YEN ME1

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia in Fulfilment of the Requirements for the Degree of Master of Science

September 2005

Specially dedicated

To LORD my GOD "My success is the glory oSyour name"

To my lovely mother

Chan Tai Thai "My success is only for you"

To my lovely friend

Michael Ling Tung Lien "Thanks for your spiritual support and caring"

To my siblings

Yuen Kuan & Lip Hor "Thanks for your lovely caring and support"

To my dear friends "Thanks for your support"

Abstract of thesis presented to Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science

A SIMPLIFIED AND SELECTIVE TECHNIQUE FOR THE DIRECT RECOVERY OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM

UNCLARIFIED YEAST FEEDSTOCK

CHOW YEN ME1

September 2005

Chairman : Ling Tau Chuan, PhD

Faculty : Engineering

The development of a simplified and rapid technique for the selective recovery of

proteins from Bakers' yeast was undertaken. Purification of Glucose-6-phosphate

dehydrogenase (G6PDH) from Saccharomyces cerevisiae was chosen demonstrate of

this principle. Cell disruption is a mandatory first step in the recovery of intracellular

products. The influence of the operational parameters of Dyno bead mill on the

release of G6PDH and proteins study were studied, and demonstrated that 45 ~hr- '

flow rate, 85% (v/v) bead volume, 10 ms" tip speed are optimum condition for

protein released. The comparative study on expanded beds ion-exchange and affinity

adsorbents for the purification of G6PDH from crude feed-stock was conducted. The

use of Streamline DEAE (p-1.2 @-I) and UpFront Cibacron Blue 3GA (p-1.5

gmL,-') in adsorption of G6PDH from Bakers' yeast is adapted in this study.

Hydrodynamic performance testing indicated that UpFront adsorbent providing a

more stable fluidized bed than Streamline adsorbent does. Due to consisting higher

density, higher flow rate (225 to 450 cmhf') and biomass concentration (up to 30%

W/V) could be applied on expanded UpFront adsorbent bed. In contrast, Streamline

adsorbent only able to afford a range of flow rate, from 164.2 to 248.3 cmhr-' and

fmHJsTM(WAN SULTAN ABOUL SAMAp t.Nwmm PUTRA W V W

biomass concentration up to 20% wlv. For dye affinity system, there is a light

reduction on dynamic binding capacity of BSA (1 1.1% to 27.8%) only as compare

with ion-exchange system (43.1% to 68.6%) when the adsorption was conducted in

the presence of intact yeast cells. The adsorption characteristics of the afiinity system

were not greatly altered in the presence of cells in contrast to the results fiom a less

selective ion-exchange system. It was demonstrated that dye aflhity chromatography

had provided a higher purification factor (3.9 to 8.2) with as compared with ion-

exchange chromatography (2.7 to 4.1) in G6PDH recovery.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

SUATU TEKNIK YANG MUDAH DAN MEMILIH BAG1 PROSES PEMULIHAN TERUS GLUCOSE-6-PHOSPHATE DEHYDROGENASE

DARIPADA BEKALAN YIS YANG TIDAK DIJELASKAN

Oleh

CHOW YEN ME1

Mei 2005

Pengerusi : Ling Tau Chuan, PhD

Fakulti : Kejuruteraan

Pembinaan suatu teknik yang mudah dan pantas untuk memulihkan protein daripada

yis telah diusahakan. Pemulihan Glucose-6-phosphate dehydrogenase (G6PDH)

daripada Saccharomyces cerevisiae telah dipilih sebagai tunjukan pada dasamya.

Pemecahan sel microorganisma merupakan Iangkah pertama yang diperlukan dalam

proses pemulihan enzim dalaman. Parameter operasi yang mempengaruhi proses

pemecahan untuk melepaskan enzim (G6PDH) dan protein dalam loji Dyno telah

dikaji, dan menunjukan bahawa kadar pengaliran, 45 ~hr- ' , isipadu m a d , 85% (vh),

kelajuan penghasut, 10 ms-' merupakan parameter operasi yang optimum bagi

pelepasan protein. Kajian perbandingan dalam penggunaan penjerap "ion-exchange"

dan "affinity" dalarn proses penulenan G6PDH daripada bekalan aslinya telah

dijalankan. Penggunaan penjerap Streamline DEAE (p-1.2 gn~- ' ) and UpFront

Cibacron Blue 3GA (p-1.5 g m ~ - ' ) dalam proses penjerapan G6PDH daripada yis

telah disesuaikan dalam kajian hi. Pemeriksaan perlaksanaan hidrodinamik telah

menunjukkan bahawa penjerap UpFront dapat memberikan lapisan mengembang

yang lebih mantap jika dibanding dengan penjerap Streamline. Oleh sebab penjerap

UpFront mengandungi ketumpatan yang leblh tinggi, dan seterusnya membolehkan

kadar pengaliran (225 hingga 450 cmhr-I) dan bio-jisim (30% wlv) yang lebih tinggi

dapat disesuaikan dalam twus lapisan mengembang. Sebaliknya, penjerap

Streamline hanya dapat mengatasi kadar pengaliran, dari 164.2 hingga 248.3 cmhr-'

dan bio-jisim sebanyak 20% wlv sahaja. Bagi sistem "dye aflhity", kapasiti

penjerapan dalam lapisan mengembang hanya mengalami pengurangan yang sedikit

sahaja (1 1 -1 % hingga 27.8%) jika dibanding dengan sistem "ion-exchange" (43.1%

hingga 68.6%) apabila penjempan dijalankan dalam keadaan kehadiran keseluruhan

sel yis (tanpa pemecahan). Ciri-ciri penjerapan bagi sistem "afiinity" hanya

mengalami perbezaan yang minimum dan ia arnat berbeza daripada sistem "ion-

exchange" yang kurang memilih. Kromatografi "dye affinity" telah menunjukkan

faktor penulenan yang lebih tinggi (3.9 hingga 8.2) jika dibandingkan kromatografi

"ion-exchange" (2.7 hingga 4.1) dalam proses pemulihan G6PDH.

vii

ACKNOWLEDGEMENTS

I wish to express my gratitude and appreciation to my Supervisory Committee: Dr.

Ling Tau Chuan, Prof. Dr. Arbakariya Ariff and Dr. Tey Beng Ti for their patience,

constructive suggestions and encouragement throughout the years of my master

study. I would like to express my thanks to members and staffs in Unit Fermentation

Laboratory, Institute Bioscience, UPM.

My special thanks go to all my friends, especially Hii Siew Ling, Lim Chin Ming,

Tan Yan Peng and Liew Shiao Tsui. You all had given me a strong spirit to go

through the years. With special recognition to: my sweet mom, sister, brother and

Ling Tung Lien. You did a great job of raising me along the years.

. . . Vlll

I certify that an Examination Committee met on 1 2 ~ September 2005 to conduct the final examination of Chow Yen Mei on her Master of Science thesis entitled "A Simplified and Selective Technique for the Direct Recovery of Glucose-6- phosphate dehydrogenase from Unclarified Yeast Feedstock" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 198 1. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

COSCAN LICALI, PhD Professor Faculty of Engineering Universiti Putra Malaysia (Chairman)

JOHARI ENDON, PhD Associate Professor Faculty of Engineering Universiti Putra Malaysia (Internal Examiner)

BADLISHAH SHAM BAHARIN Associate Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Internal Examiner)

SHALIZA IBRAHIM, PhD Associate Professor Faculty of Engineering Universiti Malaya (External Examiner)

Professor/Deputy ~ e k School of Graduate Studies Universiti Putra Malaysia

Date: 2 5 OCT 2005

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirements for the degree of Master of Science. The members of the Supervisory Committee are as follows:

LING TAU CHUAN, PhD Lecturer Faculty of Engineering Universiti Putra Malaysia (Chairman)

ARBAKARIYA ARIFF, PhD Professor Faculty of Biotechnology and Bio-Molecular Science Universiti Putra Malaysia (Member)

TEY BENG TI, PhD Lecturer Faculty of Engineering Universiti Putra Malaysia (Member)

AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia

Date: 17 NOV 2005

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

Date: 3 / 10 / 05

TABLE OF CONTENTS

Page

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABRREVIATIONS

CHAPTER

INTRODUCTION Aim of Study

LITERATURE REVIEW 2.1 Intracellular Product Release and Recovery

2.1.1 Dyno Bead mill disruption 2.2 Expanded Bed Adsorption (EBA)

2.2.1 Principles of EBA 2.2.2 Theoretical consideration

2.2.2.1 Fluidization 2.2.2.2 Bed expansion 2.2.2.3 Axial dispersion 2.2.2.4 Number of theoretical plates

2.3 Ion Exchange Chromatography 2.3.1 Interaction of proteins between ion-exchanger 2.3.2 Elution of adsorbed proteins

2.4 Dye Affiity Chromatography 2.4.1 Chemical structure of dye ligands 2.4.2 Interactions between dye-ligands and proteins 2.4.3 Ligand immobilization 2.4.4 Adsorption and elution

... 111

iv vi ... Vll l

ix xi xiv xvi xx

CELLS DISRUPTION-HORIZONTAL DYNO BEAD MILL 3.1 Materials and methods 34 3.2 Results and Discussion 38

3.2.1 Effect of number of passages 3 8 3.2.2 Effect of feed flow rate 42 3.2.3 Effect of agitator tip speed 43 3.2.4 Effect of biomass concentration 45 3.2.5 Effect of bead loading volume 47 3.2.6 G6PDH Specific activity 49

3.3 Conclusion 50

xii

HYDRODYNAMIC PERFORMANCE OF STREAMLINE AND UPFRONT ADSORBENT 4.1 Materials and methods 4.2 Results and Discussion

4.2.1 Fluidization properties of Streamline DEAE 4.2.2 Fluidization properties of UpFront adsorbent 4.2.3 Degree of expansion of Streamline DEAE 4.2.4 Degree of expansion of UpFront adsorbent 4.2.5 Number of the theoretical plates of expanded

Streamline DEAE bed 4.2.6 Number of the theoretical plates of expanded

UpFront adsorbent bed 4.3 Conclusion

ION EXCHANGE CHROMATOGRAPHY 5.1 Materials and methods 76 5.2 Results and Discussion 80

5.2.1 Batch Binding 80 5.2.1.1 Effect of cells upon binding capacity 80

5.2.2 Expanded Bed Adsorption 84 5 -2.2.1 Breakthrough curve of BSA 84 5.2.2.2 Recovery of G6PDH from Bakers' yeast 87

by expanded bed Streamline DEAE 5.3 Conclusion 90

DYE AFFINITY CHROMATOGRAPHY 6.1 Materials and methods 6.2 Results and Discussion

6.2.1 Cibacron Blue 3GA Immobilization 6.2.1.1 Determination of CB concentration of

UpFront Cibacron Blue 3GA 6.2.2 Batch Binding 99

6.2.2.1 Effect of CB coupling 99 6.2.2.2 Effect of ionic strength 102 6.2.2.3 Effect of pH 105

6.2.3 Expanded Bed Adsorption 107 6.2.3.1 Breakthrough curve of BSA 107 6.2.3.2 Recovery of G6PDH from Bakers' yeast 1 10

by expanded bed UpFront Cibacron Blue 3GA

6.3 Conclusion

CONCLUSION

REFERENCES APPENDICES BIODATA OF THE AUTHOR PUBLICATION

.a .

Xlll

LIST OF TABLES

Table

1 Functional groups used on ion exchangers.

2 Total protein released and degree of disruption against a range of flow rate was studied. The disruption was conducted with a number of passes disruptions, biomass concentration of 30% wlv, bead concentration of 75% (v/v) and an agitator tip speed of 10.0 ms-' .

3 Calculated value of first-order rate constant, k fiom Equation (1) against different agitator tip speed, fiom 8.0 to 14.0 ms-' was obtained. The disruption was conducted with a flow rate of 45.0 Lhr-', biomass concentration of 30% wlv and bead loading of 75% (v/v).

4 Excretion of soluble protein, after disruption with Dyno-mill with a number of 6 passes disruptions, a flow rate of 45.0 Lhr- and bead concentration of 75% (v/v) and biomass concentration of 30% wlv was studied. A various range of agitator tip speed of 8.0 ms-', 10.0 ms-' and 14.0 ms-' had been used.

5 Calculated value of first-order rate constant, k fiom Equation (1) against different biomass concentration fiom 10.0 to 50% w/v was obtained. The disruption was conducted with a flow rate of 45.0 ~hr-' , bead concentration of 75% (v/v) and an agitator tip speed of 10.0 ms-'.

6 Total protein released and degree of disruption against a range of biomass concentration was studied. The disruption was conducted with a number of 6 passes disruptions, a flow rate of 45.0 Lhr-', bead concentration of 75% (v/v) and an agitator tip speed of 10.0 ms-'.

7 Calculated value of first-order rate constant, k f?om Equation (1) against different bead load volume, fiom 75 to 85% (vlv) was obtained. The disruption was conducted with a flow rate of 45.0 Lhr-', biomass concentration of 30% wlv and an agitator impeller tip speed of 10.0 ms-'.

8 Different bead concentration, 1050 rnL (75% (vlv)) and 1190 mL (85% (dv)), was used to determine the excretion of total protein and degree of disruption after disruption of Saccharomyces cerevisiae using Dyno-mill. The experiments were conducted under a number of 6 passes disru tions, a flow rate of 45.0 ~hr - ' , ? an agitator tip speed of 10.0 ms' , and biomass concentration of 30% w/v.

Page

23

43

xiv

9 G6PDH specific activity and total protein released against 6 passes of disruptions at agitator tip speed of 10.0 ms-' and 14.0 ms-'. The test was carried out with 85% (vlv) of zirconia beads, a flow rate of 45.0 ~ h r - ' and a range of biomass concentration of 10 to 30% wlv.

10 Fluidization properties of Streamline DEAE in a 20 mm diameter FastLine column.

11 Fluidization properties of UpFront adsorbent in a 20 mm diameter FastLine column.

12 Biomass content tested on Streamline DEAE and UpFront cross- linked agarose (1 5 cm sedimented bed height) expanded in a FastLine20 column (20 rnm i.d., 75 cm tube height), applying a Various flow rate.

13 Equilibrium adsorption of BSA to Streamline DEAE in a range of S. cerevisiae cells concentration. The data obtained was well fitted to Langrnuir adsorption isotherms. The maximum capacity of the adsorbent (q,) and the dissociation constant (&) of the adsorbent- protein were determined using Equation (5.1) & (5.2).

14 Purification of G6PDH from 5 to 20% wlv yeast cell disruptate using expanded bed ion-exchange adsorption. 54 mL of Streamline DEAE which is corresponding to 15 cm of settlement height in FastLine20 contactor was used. The running process was conducted at flow velocity, 164.2 to 248.3 cmhr". The enzyme was desorbed from the bed by three stages elution scheme using a flow velocity of 210 cmhf'. The product yield was calculated by using formula in Section 5.1.7 based on the value of recovered G6PDH in second stage elution.

15 Purification of G6PDH from 5 to 30% wlv yeast cell disruptate using expanded bed dye affinity adsorption. 50 mL of UpFront Cibacron Blue 3GA which is corresponding to 15 cm of settlement bed height in FastLine20 contactor was used. The running process was conducted at flow velocity, 225 to 450 cmhr-'. The enzyme was desorbed from the bed by one stage elution scheme using a flow velocity of 418 cmhr-'. The product yield was calculated by using formula in Section 5.1.7.

16 The comparison of purification of G6PDH &om Bakers' yeast cell disruptate using expanded bed dye a e t y and ion-exchange adsorption. Both systems utilized 50 mL and 54 mL adsorbent respectively which are corresponding to 15 cm of settlement bed height in FastLine20 column.

LIST OF FIGURES

Page

2 1

Figure

Calculation of the mean residence time and the variance using the RTD.

Illustration of the ion-exchange as the present of negatively charged protein adsorbs to an anion exchanger. Five positively charged ions (e.g. ~ ~ r i s + ) associated with the protein molecule are displaced, together with five negative ions (Cl-) from the exchanger.

Structure of Cibacron Blue 3GA molecule.

Coupling of triazinyl dyes to the matrix bearing hydroxyl groups.

Released of soluble protein (+), and degree of disruption (m) after disruption of Saccharomyce cerevisiae with the Dyno-mill as a function of the number of passes. Disruption operation was conducted with biomass concentration of 30% wlv, bead load volume of 75% (vlv), an agitator tip speed of 10.0 ms" and a flow rate of 45.0 ~hr- ' .

Experimental data from Figure 5 was replotted by using Equation (1) to identi@ the fitting of model. The disruption was conducted under parameters: flow rate, 45.0 ~hr-'; biomass concentration, 30% wlv; zirconia bead load volume, 75% (vlv); agitator tip speed, 10.0 ms".

Morphology of yeast cells (a) Intact cells before pass through Dyno-mill chamber, (b) Disrupted cell after 6 passes through Dyno-mill. (Magnification, X100)

Relative expansion at different flow velocities (50 to 350 cmhr-') of Streamline DEAE matrix with applied different biomass concentration, 0% (+), 5% (m), 10% (A), 15% ( 0 ) (wlv) wet weight per volume in Tris-HC1 buffer in a FastLine20 column.

Experimental expansion data fiom Figure 10 replotted as log(U) versus log(&) to determine the slope (n- index) and y-intercept (Ut).

Relative voidage at different flow velocities of Streamline DEAE Matrix with applied different biomass concentration, 13% (+), 5% (o), 10% (A), 15% (0) (wlv) wet weight per volume in Tris-HC1 buffer in a FastLine20 column.

xvi

11 Relative expansion at different flow velocities (1 15 to1000 cmh-') of UpFront adsorbent with applied different biomass concentration, 0% (o), 5% (+), 10% (m), 15% (A), 30% (0 ) (wh) wet weight per volume in Tris-HC1 buffer in a FastLine20 column.

Experimental expansion data fiom Figure 13 replotted as logU versus log& to determine the slope (n- index) and y-intercept (UJ.

Relative voidage at different flow velocities (1 15-1000 cmh-I) of UpFront adsorbent with applied different biomass concentration, 0% (+), 5% (n), 10% (A), 15% (0) (wlv) wet weight per volume in Tris-HCI buffer in a FastLine20 column.

UV-signal recording during the test procedure for the determination of the number of theoretical plates for first run (S t red ine DEAE).

UV-signal recording during the test procedure for the determination of the number of theoretical plates for second run (Streamline DEAE).

UV-signal recording during the test procedure for the determination of the number of theoretical plates for first run (UpFront adsorbent).

UV-signal recording during the test procedure for the determination of the number of theoretical plates for second run (UpFront adsorbent).

The net charge of protein as a function of pH.

The adsorption of BSA onto Streamline DEAE in the presence of yeast cells. Adsorption isotherms were measured based on mass balance. The adsorption of BSA (0 to 80 mgm~-') onto Streamline DEAE in 10 mM Tris-HC1 buffer. The concentration of cells present in various experiment were: O%(A), 5%(+), lo'%(.) and IS%(.).

Binding of S. cerevisiae on Streamline DEAE (a) and (b) as a control. The adsorption of cells to the solid phase decreased the available charged groups for protein binding and subsequently decreased the equilibrium capacity for the target protein. An inspection of sample of the adsorbent under an optical microscope with magnification X40.

21 Effect of the presence of yeast cells on expanded bed adsorption of BSA onto Streamline DEAE. The feedstocks (3 mgrn~-' BSA) comprising 5% (A), 10% (m), 15 % (+) equivalent wet cell weights at pH 7.5 were studied herein. Streamline DEAE (54 mL

xvii

settled volume, corresponding to 15 cm settled bed height) was loaded into the contactor and equilibrated with Tris buffer in expanded bed mode. Samples were taken from the effluent outlet at regular intervals and the feedstock application was continued until a desired adsorbent saturation state (GIG -- 1) was reached. Samples were centrifiged (at 10,000 rpm) and assayed for BSA concentration and expressed as CICo. The dynamic binding capacity of BSA at C/C,= 0.1 for various biomass concentration are 50.97 mgI3~~mL- ' DEAE (5% wlv), 28.84 mg BSA mL-' DEAE (10% wlv), 16.0 mg BSA m~-' DEAE (15% wlv).

Purification of G6PDH from bakers' yeast cell disruptate on an expanded bed of Streamline DEAE. The chromatographic protocol used to puri@ G6PDH £tom a feedstock of disrupted 15% w/v yeast cells broken by Dyno bead mill. Samples collected during chromatography were analyzed for total protein content (+) and G6PDH activity (m). The eluents were 0.05 M NaCl, 0.5 M NaCl and 1.0 M NaCl in 50 mM Tris-HC1. The various stages of the procedure were performed at a constant flow-rate.

23 2.71 prnolm~-' adsorbent Cibacron Blue 3GA was successfid 98 immobilized on UpFront adsorbent (a), compare with the blank UpFront adsorbent (b), under magnification x20.

24 Immobilized Cibacron Blue 3GA on Upfiont adsorbent was studied. A range of concentration of dye fiom 0 to 56.92 pmolmL- ' was used.

25 The up take of BSA onto UpFront CB 3GA with different CB 100 coupling densities in 20 mM Tris-HC1, 0.05 M NaC1, pH 7.5, (+) was studied. The initial BSA concentration is 1.0 mgmL-'.

26 The up take of G6PDH onto UpFront CB 3GA with different CB 101 coupling densities in 20 mM Tris-HCl, 0.05 M NaCl, pH 7.5 was studied, (+). 5% wlv of yeast disruptate had been used in this study.

27 The adsorption of BSA onto immobilized CB 3GA adsorbent was 103 carried out. Adsorption isotherms were measured based on mass balance. The adsorption of BSA onto immobilized CB 3GA adsorbent in 0 to 40 mgmL-' BSA with 20 m M Tris-HC1 buffer, 0.05 M NaCl, pH 7.5, (+), and 50 mM Tris-HC10.05 M NaC1, pH 7.5, (m) were studied.

28 The up take of G6PDH profile to UpFront CB 3GA with different 104 CB coupling densities in 50 rnM Tris-HC1,0.05 M NaCI, pH 7.5 (m) and 20 mM Tris HCl, 0.05 M NaCI, pH 7.5, (4) were studied. 5% wlv of yeast disruptate had been used in this study.

xviii

29 The up take of BSA profile to UpFront Cibacron Blue 3GA with 106 different pH values in 50 mM Tris-HC1,0.05 M NaCI. The initial concentration of BSA is 1.0 mgm~- ' .

30 Binding of BSA onto CB 3GA UpFront is unaffected by the 108 presence of biomass. An inspection of sample of the adsorbent under an optical microscope with magnification X20, (a) and (b) as a control.

31 Effect of the presence of yeast cells on expanded bed adsorption 109 of BSA onto UpFront CB 3GA. The feedstocks (3 mgmL-' BSA) comprising 5% wlv (o), 10% wlv (m), 15 % wlv (A), 30% wlv (+) equivalent wet cell weights at pH 7.5 were studied herein. UpFront CB 3GA (50 mL settled volume, corresponding to 15 cm settled bed height) was loaded into the contactor and equilibrated with 50 mM Tris-HC1 in expanded bed mode. The feedstock was loaded into column with range of flow rate from 225 to 450 cmhr- 1 . Samples were taken fiom the effluent outlet at regular intervals and the feedstock application was continued until a desired adsorbent saturation state (CICo = 1) was reached. Samples were centrifuged (at 10,000 rpm) and assayed for BSA concentration and expressed as CICo. The dynamic binding capacity of BSA at C/C,=O. 1 for various biomass concentration are 9.0 mg BSA m ~ " adsorbent (5% wlv), 8.0 mg BSA mL" adsorbent (10% wlv), 7.5 mg BSA m ~ - ' adsorbent (15% wlv), and 6.5 mg BSA m ~ " adsorbent (30% wlv).

32 Purification of G6PDH fiom bakers' yeast cell disruptate on an expanded bed of UpFront Cibacron Blue 3GA. The chromatographic protocol used to purie G6PDH fiom a feedstock of disrupted 15% wlv yeast cells broken by Dyno bead mill. Samples collected during chromatography were analysed for total protein content (+) and G6PDH activity (m). The eluent was 1.0 M NaCl in 50 rnM Tris-HC1. The elution was conducted at flow rate of 4 1 8 cmhfl.

33 The selectivity of dye affinity chromatography as compared with ion-exchange chromatography.

xix

LIST OF ABBREVIATIONS

AMP

ATP

BS A

CB

CIP

cs

c,

DEAE

DNA

EBA

G6PDH

HETP

HCl

HEPES

MES

MOPS

NaCl

Na2C03

NaOH

NaHC03

NAD+

NADP

NADPH

Adenine Monophosphate

Adenine Triphophate

Bovine Serum Albumin

Cibacron Blue

Clean-in-place

Adsorbed mg/mL of BSA per g of adsorbent at equilibrium

BSA concentration at equilibrium, mgm~- '

Diethylaminoethyl

Deoxyribonucleic Acid

Expanded bed adsorption

Glucose-6-phosphate dehydrogenase

Height equivalent to a theoretical plate

Hydroxide chloride

N-2-Hydroxyethylpiperazine-N'-2-ethaneslfonic acid

2-(N-Morpholino) ethanesulfonic acid

3-(N-Morpholino) propanesulfonic acid

Sodium chloride

Sodium Carbonate

Sodium Hydroxide

Sodium Bicarbonate

Ion Nicotinanide Adenine Dinucleotide

Nicotinanide Adenine Dinucleotide Phosphate

Nicotinanide Adenine Dinucleotide Phosphate Dehydrogenase

Wet weight per volume

voiume per volume

CHAPTER 1

INTRODUCTION

The success in simplifLing industrial practice with the latest biotechnology is the

ultimate responsibility of the bio-process and bio-chemical engineering profession.

The advances in recovery, separation, and purification techniques used in

downstream processing may play a crucial role in the development of large scale

biotechnology. Downstream operations are crucial stages in terms of maintaining

product characteristics and activity aiming at hi# yield and purity and cost saving.

A dominant cost element in the production of the biological origin can be

downstream operations. This is particularly true when the application of the product

demands a very high level of product purity. For an example the cost ratio between

fermentation and product recovery is approximately 60:40 fbr older antibiotics

produced by fermentation. For newer antibiotics, third and fourth generation, the

ratio is reversed to 40:60 fermentation to recovery. For recombinant DNA

fermentation products such as therapeutic proteins the downstream purification

accounts for 80 to 90% of the process costs (Dwyer, 1984). Downstream process unit

operations included primary separation, product purification and product isolation.

Disruption of the outer envelope of microbial cells is an essential step in the recovery

of microbiaI products such as intracellular products. Mechanical methods are

generally applicable for cell disruption, while the non-mechanical methods may be

very effective but are restricted to special cases (Gaver & Huyghebaert, 1990). From

an iodustrial applications point of view, only cell disruption technology based on

mechanical technique has its potential use (Chisti & Moo-Young, 1986). Probably

because of the high capital, operating costs and complexity of separation processes

(especially disintegrated by chemical mean) for large scale recoveIy of intracellular

products, non-mechanical methods lost its industrial potential use. Cell disruption in

bead mill is considered as one of the most efficient techniques for the physical cell

disruption (Darbyshire, 1981). A continuous protein production process involving

disruption of 10% dry weight bakers' yeast and brewers' yeast in a 5 liter nominal

capacity bead mill has been reported (Hedenskog & Morgan, 1973). A wide range of

bacteria (E.coli, Bacillus sphaericus, Lactobacrllus conJirses, Brevibacterium

ammmoniagenes and Bacillus subtilis) and fungus (S. cerevisiae, S. carlsbergensis,

C. boidinii, C. utilzs) have been disrupted in bead mills (Chisti & Moo-Young, 1986).

The traditional primary purification of the target molecule has been addressed by

adsorption chromatography using a conventional packed bed of adsorbent. Before

being further purified by traditional packed bed chromatography, centrifbgation and

microfiltration are needed, in order to obtain a particle fi-ee solution. However,

microfiltration has its drawbacks. The flux of liquid per unit membrane area is o h

dramatically decreased, even though microfiltration could provide a particle free

solution. During the filtration process, fouling of the microfiltration membrane is

another critical problem that significantly adds to the operational cost. Normally, the

combined use of centrifugation and microfitration may result in long process time

and cause significant additional costs for equipment maintenance. It also brought in

significant product loss due to product deterioration, especially the intracellular

products. Consequently, direct adsorption from crude feed-stocks potentially offers

significant reduction of process time and costs compared to traditional processes

(Chase, 1994; Chase & Draeger, 1992).

Expanded bed adsorption (EBA) is a technique which was created to circumvent all

the drawbacks of conventional downstream processing. The process steps of

clarification, concentration and initial purification can combine into one unit

operation by using EBA technique. This leads to providing increased process

economy due to a decreased number of process steps, increased yield, shorter overall

process time (Suding & Tomusiak, 1993), reduced labor cost (Batt et al., 1995) and

reduced running cost and capital expenditure (Schmidt et al., 1993). Further more,

EBA technique is not only limited at laboratory process scale, it is available for

scale-up and potentially offer industrial scale process.

Expanded bed procedures are becoming increasingly popular in bio-separation as a

way of avoiding the need for clarification techniques such as centrifugation and

filtration (Chang et al., 1995; McCreath et a]., 1995). One step unit operation of

capture target molecules fi-om crude feed-stock may reduce products degradation and

avoiding bio-product handling problems. Expanded bed adsorption has postulated to

be a versatile tool that can be applied on cells commonly used source materials.

S u c c e s s ~ y processing by expanded bed adsorption has been reported for E. coli

homogenate (Daniels et al., 1996; Ollivier et al., 1996), E. coli lysate (Daniels et a/.,

1996; Johansson et al., 1996), yeast cell homogenate (Chang et a/., 1995; Chang &

Chase, 1996), secreted products fi-om yeast (Blomqvist et al., 1996; Gellissen et al.,

1996; Zurek et al., 1996), whole hybridoma fermentation broth (Born et al., 1996;

Lutkemeyer et al., 1996), myeloma cell culture (Jagersten et al., 1996), whole

mammalian cell culture broth (Beck et al., 1996; Zapata et al., 1996) milk, and

animal tissue extracts (Garg et al., 1996).

The present work is focused on the development of a simplified and rapid technique

for the selective recovery of intracellular enzyme fiom bakers' yeast. G6PDH was

chosen as reference enzyme due to its high level present in Bakers' yeast and

commercial value. The Dyno bead mill had been used in this work for effective

release of G6PDH fiom yeast. Ion exchange and dye aflinity chromatography has

been applied in this study. The matrices are Streamline DEAE and UpFront Cibacron

Blue 3GA. UpFront Fastline20 was used as a contactor to recover glucose 6-

phophate dehydrogenase (G6PDH) fiom Bakers' yeast homogenate. The

performance of an anion exchanger, Streamline DEAE, (p-1.2 g n ~ - ' ) was studied

and compared with UpFront adsorbent (p-1.5 g n ~ - ' ) immobilized with Cibacron

Blue 3GA. The applicability and practicability of an innovative contactor

characterized with mechanized stining flow distribution was explored.