臺灣博碩士論文加值系統

於本研究中利用饋料批次醱酵培養重組Escherichia coli DH5�� 以生產含有核酸水解酶(nuclease domain)與免疫蛋白(immunity protein, Im7)之coliciny Im7 complex。由於核酸水解酶為細胞毒素，所以當IPTG進行誘導表現時，必須藉由coliciny Im7 complex 的生成以降低細胞毒素的毒性。利用Luria-Bertani (LB) medium 進行批次醱酵培養，所獲得的coliciny Im7 complex比活性(specific biomass activity)、單位體積活性(volumetric nuclease activity) 與產率活性(productivity of nuclease activity)分別為763 U/g cell、4223 U/l 與384 U/l/h。
於研究中利用2III3-1 部分因子實驗設計獲得之semi-synthetic medium (SS medium) 可以獲得最大的細胞產量。採用SS medium 進行批次醱酵培養可以增進coliciny Im7 complex 之產量。因此，於SS medium中進行限制葡萄糖濃度(1 g/l glucose)之控制策略，並且於饋料過程添加酵母抽出物(yeast extract)可以coliciny Im7 complex比活性(specific biomass activity)、單位體積活性(volumetric nuclease activity) 與產率活性(productivity of nuclease activity)分別為24852 U/g cell、618187 U/l 與41212 U/l/h。

In this work, we developed a fed-batch culture to enhance the production of the coliciny Im7 complex, which consisted of nuclease domain and immunity protein (Im7) of colicin E7 using a recombinant Escherichia coli DH5��. Since the nuclease activity is cytotoxic, the cell biomass may degrade after isopropyl-��-D-thiogalacto- pyranoside
(IPTG) induction for gene expression. Therefore, it is beneficial to reduce the cytotoxicity of nuclease domain with coliciny Im7 complex. When Luria-Bertani (LB) medium is added to the batch culture, the specific biomass, volumetric and productivity of nuclease activity of coliciny Im7 complex were measured to be 763 U/g cell, 4223 U/l and 384 U/l/h.
We successfully developed a semi-synthetic medium (SS medium) to obtain maximum biomass by 2III3-1 fractional factorial design. SS medium could be utilized to increase coliciny Im7 complex production. By using a SS medium, along with the glucose-limited control strategy (at 1 g/l of glucose concentration), and adding yeast extract (YE) at post-induction stage, increased the specific biomass, volumetric and productivity of nuclease activity were increased to be 24852 U/g cell, 618187 U/l and 41212 U/l/h, respectively.

ENGLISH ABSTRACT……….……………….…………………………I
CHINESE ABSTRACT…..……..……..………………..…………….…II
TABLES OF CONTENTS….…………......…….…………..………….III
LIST OF FIGURES………………….......………………..…………….VI
LIST OF TABLES……………….......…………………….……………IX
CHAPTER
I Introduction.………………………...….................………….….1
1.1 Introduction of colicin………..……..……………….…1
1.2 E-type colicins….....……………………………………4
1.3 Interactions of colicin E7 and immunity protein…….....8
1.4 Function of nuclease domain of colicin E7……….........9
1.5 Measurement of nuclease activity……………………..13
1.6 Recombinant protein production by Escherichia coli...15
1.7 The object for the experiment…………………………18
II Materials and Methods…………….…..……………..…………19 2.1 Strains...…….…………..……………………………..19
2.2 Medium Preparation and 2III3-1 fractional factorial design…………………………………………………19
2.3 Seed cultivation……………………………………….23
2.4 Batch and fed-batch fermentation……………………..23
2.5 Analytical methods for fermentation process…………27
2.6 Protein Harvesting…………………………………….28
2.7 Purification of colicin E7 nuclease……………………28
2.8 Nuclease activity assay……...………………………...29
III Results and Discussion.…..……....……………………………..31
3.1 Batch cultivation with LB medium…..…......................31
3.2 Effect of the yeast extract on fed-batch cultivation with
LB medium……………………………………………33
3.3 Medium improvement for colicin E7 nuclease
production………………………………………….…37
3.4 Glucose-limited and YE effects on fed-batch cultivation
with SS medium………………………………………48
IV Conclusions……….........................................................…….....53
REFERENCES…........…………………….…………………...….……54
APPENDIXES..........................................................................................64
Appendix 1. The display scheme of Advantech Genie Software using in
fermentation program…….......................................…..65
Appendix 2. The standard curve of dry cell weight and OD600nm….....70
Appendix 3. The Gompertz model analysis of Run 1..........................71
Appendix 4. The Gompertz model analysis of Run 2………………..72
Appendix 5. The Gompertz model analysis of Run 3………………..73
Appendix 6. The Gompertz model analysis of Run 4………………..74
Appendix 7. The standard curve of standard Calf thymus DNA
concentration and fluorescent unit…..…………………75 Appendix 8. The colicin E7 nuclease activity assay in batch
cultivation with LB medium……..…………………….76
Appendix 9. The colicin E7 nuclease activity assay in fed-batch
cultivation (glucose: YE = 1:0) with LB medium……..77
Appendix 10. The colicin E7 nuclease activity assay in fed-batch
cultivation (glucose: YE = 1:1) with LB medium……78
Appendix 11. The colicin E7 nuclease activity assay in fed-batch
cultivation with SS medium………………………….79
Appendix 12. The colicin E7 nuclease activity assay in fed-batch
cultivation (glucose: YE = 1:1) with SS medium…….80







LIST OF FIGURES
Figure 1.1 Mechanism of cell killing by E-type nuclease colicins…….7
Figure 2.1 The process of fed-batch fermentation……………………26
Figure 3.1 Time courses of cell growth and production of colicin E7 nuclease by recombinant E. coli with LB medium in batch cultivation……………..………………...………………32
Figure 3.2 Time courses of cell growth and production of colicin E7
nuclease by recombinant E. coli with LB medium in fed-batch cultivation. The ratio of glucose to yeast extract in the feeding solution was 1:0.....…………………..…35
Figure 3.3 Time courses of cell growth and production of colicin E7
nuclease by recombinant E. coli with LB medium in fed-batch cultivation. The ratio of glucose to yeast extract in the feeding solution was 1:1...………………………36
Figure 3.4 The growth curve and acetate accumulation of 2III3-1 fractional factorial design…………………..………….40
Figure 3.5 Main effect plot of carbohydrate (glucose concentration)
with response of maximum dry cell weight……...…….42
Figure 3.6 Time courses of cell growth and production of colicin E7 nuclease by recombinant E. coli with SS medium in batch cultivation………………………………………….......47
Figure 3.7 Time courses of cell growth and production of colicin E7 nuclease by recombinant E. coli with SS medium in fed-batch cultivation. The ratio of glucose to yeast extract in the feeding solution was 1:1……………….……......50
Figure 3.8 SDS-PAGE analysis of purified colicin E7 nuclease.....51


ENGLISH ABSTRACT……….……………….…………………………I
CHINESE ABSTRACT…..……..……..………………..…………….…II
TABLES OF CONTENTS….…………......…….…………..………….III
LIST OF FIGURES………………….......………………..…………….VI
LIST OF TABLES……………….......…………………….……………IX
CHAPTER
I Introduction.………………………...….................………….….1
1.1 Introduction of colicin………..……..……………….…1
1.2 E-type colicins….....……………………………………4
1.3 Interactions of colicin E7 and immunity protein…….....8
1.4 Function of nuclease domain of colicin E7……….........9
1.5 Measurement of nuclease activity……………………..13
1.6 Recombinant protein production by Escherichia coli...15
1.7 The object for the experiment…………………………18
II Materials and Methods…………….…..……………..…………19 2.1 Strains...…….…………..……………………………..19
2.2 Medium Preparation and 2III3-1 fractional factorial design…………………………………………………19
2.3 Seed cultivation……………………………………….23
2.4 Batch and fed-batch fermentation……………………..23
2.5 Analytical methods for fermentation process…………27
2.6 Protein Harvesting…………………………………….28
2.7 Purification of colicin E7 nuclease……………………28
2.8 Nuclease activity assay……...………………………...29
III Results and Discussion.…..……....……………………………..31
3.1 Batch cultivation with LB medium…..…......................31
3.2 Effect of the yeast extract on fed-batch cultivation with
LB medium……………………………………………33
3.3 Medium improvement for colicin E7 nuclease
production………………………………………….…37
3.4 Glucose-limited and YE effects on fed-batch cultivation
with SS medium………………………………………48
IV Conclusions……….........................................................…….....53
REFERENCES…........…………………….…………………...….……54
APPENDIXES..........................................................................................64
Appendix 1. The display scheme of Advantech Genie Software using in
fermentation program…….......................................…..65
Appendix 2. The standard curve of dry cell weight and OD600nm….....70
Appendix 3. The Gompertz model analysis of Run 1..........................71
Appendix 4. The Gompertz model analysis of Run 2………………..72
Appendix 5. The Gompertz model analysis of Run 3………………..73
Appendix 6. The Gompertz model analysis of Run 4………………..74
Appendix 7. The standard curve of standard Calf thymus DNA
concentration and fluorescent unit…..…………………75 Appendix 8. The colicin E7 nuclease activity assay in batch
cultivation with LB medium……..…………………….76
Appendix 9. The colicin E7 nuclease activity assay in fed-batch
cultivation (glucose: YE = 1:0) with LB medium……..77
Appendix 10. The colicin E7 nuclease activity assay in fed-batch
cultivation (glucose: YE = 1:1) with LB medium……78
Appendix 11. The colicin E7 nuclease activity assay in fed-batch
cultivation with SS medium………………………….79
Appendix 12. The colicin E7 nuclease activity assay in fed-batch
cultivation (glucose: YE = 1:1) with SS medium…….80







LIST OF FIGURES
Figure 1.1 Mechanism of cell killing by E-type nuclease colicins…….7
Figure 2.1 The process of fed-batch fermentation……………………26
Figure 3.1 Time courses of cell growth and production of colicin E7 nuclease by recombinant E. coli with LB medium in batch cultivation……………..………………...………………32
Figure 3.2 Time courses of cell growth and production of colicin E7
nuclease by recombinant E. coli with LB medium in fed-batch cultivation. The ratio of glucose to yeast extract in the feeding solution was 1:0.....…………………..…35
Figure 3.3 Time courses of cell growth and production of colicin E7
nuclease by recombinant E. coli with LB medium in fed-batch cultivation. The ratio of glucose to yeast extract in the feeding solution was 1:1...………………………36
Figure 3.4 The growth curve and acetate accumulation of 2III3-1 fractional factorial design…………………..………….40
Figure 3.5 Main effect plot of carbohydrate (glucose concentration)
with response of maximum dry cell weight……...…….42
Figure 3.6 Time courses of cell growth and production of colicin E7 nuclease by recombinant E. coli with SS medium in batch cultivation………………………………………….......47
Figure 3.7 Time courses of cell growth and production of colicin E7 nuclease by recombinant E. coli with SS medium in fed-batch cultivation. The ratio of glucose to yeast extract in the feeding solution was 1:1……………….……......50
Figure 3.8 SDS-PAGE analysis of purified colicin E7 nuclease.....51
















LIST OF TABLES
Table 1.1 Types of colicins………………...………………..................3
Table 1.2 Examples of nonspecific endonucleases participating in cell
defense……………………………………………………..12
Table 1.3 Methods for measuring nucleic acid concentrations……....14
Table 2.1 The three factors selected for the 2III3-1 fractional factorial
design………………………………………………………21
Table 2.2 The 2III3-1 fractional factorial design with the defining relation I=ABC………………………………………….....22
Table 3.1 The results of the 2III3-1 fractional factorial design.………...39
Table 3.2 Analysis of variance with response of maximum dry cell weight…..………………………...………………………41
Table 3.3 Analysis of variance with response of maximum cell growth
rate………………………………………….……………...43
Table 3.4 Analysis of variance with response of the lag time of cell
growth...................................................................................44
Table 3.5 Analysis of variance with response of acetate accumulation
……………………………………………………………..45
Table 3.6 Medium composition of LB medium and SS medium….....46
Table 3.7 Cell growth and nuclease activity of coliciny Im7 complex in batch and fed-batch culture with different mediums and weight ratio of glucose to yeast extract in the feeding stream………………………………………………………52














LIST OF TABLES
Table 1.1 Types of colicins………………...………………..................3
Table 1.2 Examples of nonspecific endonucleases participating in cell
defense……………………………………………………..12
Table 1.3 Methods for measuring nucleic acid concentrations……....14
Table 2.1 The three factors selected for the 2III3-1 fractional factorial
design………………………………………………………21
Table 2.2 The 2III3-1 fractional factorial design with the defining relation I=ABC………………………………………….....22
Table 3.1 The results of the 2III3-1 fractional factorial design.………...39
Table 3.2 Analysis of variance with response of maximum dry cell weight…..………………………...………………………41
Table 3.3 Analysis of variance with response of maximum cell growth
rate………………………………………….……………...43
Table 3.4 Analysis of variance with response of the lag time of cell
growth...................................................................................44
Table 3.5 Analysis of variance with response of acetate accumulation
……………………………………………………………..45
Table 3.6 Medium composition of LB medium and SS medium….....46
Table 3.7 Cell growth and nuclease activity of coliciny Im7 complex in batch and fed-batch culture with different mediums and weight ratio of glucose to yeast extract in the feeding stream………………………………………………………52

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