臺灣博碩士論文加值系統

L-瓜胺酸在保健產業中是一種具有多種生理功能的非必需胺基酸，可利用精胺酸脫亞胺酶（L-arginine deiminase, ADI，EC 3.5.3.6）生產L-瓜胺酸。酵素生產法由於其具有溫和的條件、高收率、低成本和環境友好性，是較常被適用的一種方式。然而，ADI的低酵素活性嚴重限制了工業應用，特別是來自Lactococcus lactis subsp. cremoris TIFN6的ADI (LcADI)。另外，重組LcADI在大腸桿菌中表現時的低可溶性是嚴重的缺點。為了解決這些問題，分別應用添加融合伴侶和產生突變來增加LcADI的可溶性表現和活性。所有重組原生型、融合和突變型LcADIs均在ClearColi BL21(DE3)中表現。但是，LcADI 與ATS (the acidic tail of α-synuclein)、NCP (negative charged peptide)或EDA (E.coli 2-keto-3-deoxy-6-phosphogluconate aldose)融合後呈現出的低溶解度和表現程度，顯示這些融合伴侶不適合用於增加LcADI的可溶性表現。使用金屬親和層析純化原生型和突變型LcADIs。純化的原生型和G67V/Q306K/G308D LcADIs的比活性分別為10.11 U/ mg和22.60 U/ mg。純化的原生型和突變型酵素在60oC和pH 6.0（50 mM磷酸鈉緩衝液）下顯示最佳活性。原生型和突變型G67V/ Q306K/G308D LcADIs在5.5-7.5的pH範圍（50 mM磷酸鈉緩衝液）中穩定。與原生型LcADI相比，G67V/Q306K/G308D LcADI具有同樣的熱穩定性。這些結果顯示，突變型G67V/Q306K/G308D LcADI在生產L-瓜胺酸上將優於原生型酵素。

L-Citrulline is a non-essential amino acid with multiple physiological functions in the health care industry and can be enzymatically produced by arginine deiminase (ADI, EC 3.5.3.6). The enzymatic production is a favorable way because of its mild conditions, high yield, low cost, and environmental benignity. However, the low enzyme activity of ADI is serious constrain on industrial application, especially ADI from Lactococcus lactis subsp. cremoris TIFN6 (LcADI). In addition, the low soluble expression of recombinant LcADI in E. coli is a severe disadvantage. To address these issues, adding fusion partners and generating mutations were applied to increase the soluble expression and activity of LcADI, respectively. All recombinant wild-type, fused, and mutant LcADIs were expressed in ClearColi BL21 (DE3). However, the low solubility and expression level of the LcADI fused to ATS (the acidic tail of α-synuclein), NCP (negative charged peptide) or EDA (E.coli 2-keto-3-deoxy-6-phosphogluconate aldose) indicated that these fusion partners are not suitable for increasing soluble expression of LcADI. The wild-type and mutant LcADIs were purified using metal-affinity chromatography. The specific activities for purified wild-type and G67V/Q306K/G308D LcADIs are 10.11 U/mg and 22.60 U/mg, respectively. The purified wild-type and mutant enzymes showed the optimal activity at 60oC and pH 6.0 (50 mM sodium phosphate buffer). The wild-type and mutant G67V/Q306K/G308D LcADIs were stable in pH range of 5.5-7.5 (50 mM sodium phosphate buffer). The G67V/Q306K/G308D LcADI displays a similar thermostability with wild-type LcADI. These results indicated that mutant G67V/Q306K/G308D LcADI would be better than wild-type enzyme for the production of L-citrulline.

ACKNOWLEDGEMENTS i
ABSTRACT ii
摘要 iii
TABLE OF CONTENTS iv
FIGURE INDEX viii
TABLE INDEX ix
I. INTRODUCTION 1
1.1. Research background 1
1.2. Research objective 4
II. LITERATURE REVIEW 5
2.1. Enzyme activity 5
2.2. Arginine deiminase (ADI) 6
2.3. L-Citrulline 7
2.3.1 Production of L-citrulline 8
2.4. Protein Engineering 9
2.4.1 Rational protein design 9
2.4.2 Directed evolution 10
2.5 Polymerase incomplete primer extension 11

III. EXPERIMENTAL DESIGN 12
IV. MATERIALS AND METHODS 13
4.1. Materials 13
4.1.1 Gene and plasmid vector 13
4.1.2 Host strain 13
4.1.3 Antibiotics 13
4.1.4 DNA standard 13
4.1.5 Protein standard 13
4.1.6 Enzymes 14
4.1.7 Medium materials 14
4.1.8 Chemicals 14
4.1.9 Instrument 16
4.1.10 Reagent and medium preparation 17
4.2. Methods 22
4.2.1. Gene fusions by Polymerase incomplete primer extension (PIPE) 22
4.2.1.1. PCR 22
4.2.1.2. Restriction digestion and transformation 23
4.2.1.3. Colony PCR to select transformants 23
4.2.1.4. Sequence analysis. 24
4.2.2. Fusion for expression partner EDA 24
4.2.2.1. Target gene amplification (I-PIPE) 24
4.2.2.2. Amplification of vector pET-21b-ADI (V-PIPE) 25
4.2.2.3. Restriction enzyme cleavage 26
4.2.2.4. Transformation by electroporation. 26
4.2.3. Mutagenesis of the vector pET-21b (+)-ADI, pET-21b (+)-ADI-NCP and pET-21b (+)-ADI-ATS 27
4.2.3.1. First stage PCR (1st PCR) 27
4.2.3.2. Second stage PCR (2nd PCR) 28
4.2.3.3. Cleavage of template DNA with restriction enzyme DpnI 28
4.2.4. Transforming the plasmid containing mutant or fused ADI gene into E. Coli ClearColi BL21(DE3) 29
4.2.4.1. Preparation of competent cells 29
4.2.4.2. Electroporation 29
4.2.5. Recombinant ADI expression 30
4.2.6. Purification of recombinant ADI 30
4.2.6.1 Cell disruption 30
4.2.6.2 Ni2+ affinity chromatography 31
4.2.7. ADI expression analyzed by SDS-PAGE 31
4.2.7.1 Preparation of the gel SDS-PAGE 32
4.2.8. Activity assays of arginine deiminase 33
4.2.9. Protein concentration analysis 33
4.2.10. Enzymatic characterization of recombinant ADI 34
4.2.10.1. Optimal temperature and thermal stability 34
4.2.10.2. Optimal pH and pH stability 34
4.2.10.3. Kinetic parameters of recombinant ADI 34
V. RESULT AND DISCUSSION 35
5.1. Expression of recombinant fusion ADI-ATS, ADI-NCP and ADI-EDA 35
5.2. Selection and evaluation of mutation points of LcADI 36
5.3. Expression and purification of LcADI and its variant mutants 36
5.4. Characterization of LcADI and its mutant 37
5.4.1. Effect of pH on the activity of LcADI and its mutant 37
5.4.2. Effect of pH stability on the activity of LcADI and its mutant 37
5.4.3. Effect of temperature on the activity of LcADI and its mutant 37
5.4.4. Thermostabilities of LcADI and its mutant 38
5.4.5. Kinetic characterization of LcADI and its mutant 38
VI. CONCLUSIONS 40
References 41

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