臺灣博碩士論文加值系統

本研究已從土壤裡單離出一株可利用酚做為唯一碳源的酵母菌－Candida albicans TL3；相較於其它可降解酚之微生物而言，這菌株不僅對酚具有較高的耐受性而且分解酚的速率也相對較快，它對酚的降解能力目前是第一次被發現的。基於酵素活性、層析及質譜等分析，我們推論此菌株是經由ortho-fission途徑分解酚。涉及此途徑的相關酵素－phenol hydroxylase和catechol 1,2-dioxydanase兩者皆是可誘導性酵素，當C. albicans TL3被培養在培養液分別含酚濃度是22 mM和10 mM時，可達到最大活性。另外，此菌株除了能降解甲醛樹脂工廠廢液中的酚之外，亦可對甲醛加以降解。
藉由硫酸銨沉澱、Sephadex G-75凝膠過濾和HiTrap Q Sepharose管柱層析，可從C. albicans TL3中分離出一高純度的catechol 1,2-dioxydanase（1,2-CTD）；此酵素是由相同的兩個單體所組成的，每一個單體的分子量是32,000 Da且含一個鐵離子。此真菌的1,2-CTD的pI值、最適溫度和最適酸鹼值分別為5.3~5.7、25℃和pH 8.0。由受質特異性之研究顯示，此酵素應屬於type Ι catechol 1,2-dioxydanase。這是首次有關來自真核細胞之catechol 1,2-dioxygenase的研究報導。在二維電泳膠片上，可看到此純化的1,2-CTD具有五個分子量相近但等電點稍微不同的蛋白質點；這五種異構型態的1,2-CTD可能是不同程度的轉譯後修飾作用所造成的。利用Edman降解和MALDI-TOF/TOF對經胰蛋白酶水解後的此1,2-CTD裂解之胜肽片段進行序列分析，所得的序列結果可比對到一與其具高度相同性來自於Candida albicans SC5314的假想蛋白質－CaO19_12036 (GenBank accession no. XM 717691)；我們建議此一假想蛋白質應該是一1,2-CTD。

A yeast strain isolated from soil was able to utilize phenol as the sole carbon source and was further identified as Candida albicans TL3. This microbe possesses higher tolerance on phenol (24 mM) as well as stronger activity on the rate of phenol degradation than other microorganisms at 30℃. The capability of this strain on phenol degradation is first reported herein. Based on the enzymatic, chromatographic and mass spectrometric analyses, we concluded that C. albicans TL3 follows the ortho-fission pathway on phenol degradation. The optimal activity of phenol hydroxylase and catechol 1,2-dioxygenase were found when this strain grew in culture media containing 22 mM and 10 mM phenol, respectively. In addition to phenol, C. albicans TL3 also exhibited catalytic power on degrading formaldehyde in wastewater directly obtained from phenolic resin-producing factory.
The catechol 1,2-dioxygenase (1,2-CTD) induced from Candida albicans TL3 was purified via ammonium sulfate precipitation, Sephadex G-75 gel filtration and HiTrap Q Sepharose column chromatography. The enzyme was purified to homogeneity and characterized to be a homodimer, with a molecular weight of 32,000 Da for each subunit. The investigation of this eukaryotic 1,2-CTD revealed that the iron content for each subunit, pI value, optimal temperature, and optimal pH are 1 iron/subunit, 5.3~5.7, 25℃ and pH 8.0, respectively. Substrate analysis showed that the purified enzyme belongs to the type I catechol 1, 2-dioxygenase. The study on this eukaryotic 1,2-CTD was reported for the first time. On 2-D gel analysis of the purified 1,2-CTD, five spots with approximately similar molecular weight but with different pIs were found. These spots were further analyzed by MALDI-TOF mass spectrometry. Results suggested that these spots (isotypes) were derived from the same 1,2-CTD. Peptide sequencing on fragments of 1,2-CTD by Edman degradation and MALDI-TOF/TOF analysis provide information of amino acid sequences for BLAST search, the outcome of the BLAST revealed that this eukaryotic 1,2-CTD has high identity with a hypothetical protein, CaO19_12036, from Candida albicans SC5314 (GenBank accession no. XM 717691). We, thus, suggested that the hypothetical protein should be 1,2-CTD.

Table of Contents

Abstract (Chinese)…………………………………………………………………….I
Abstract (English)…………………………………………………………………...III
Acknowledgements…………………………………………………………………..V
Table of Contents……………………………………………………………………VI
List of Tables……………………………………………………………………...…IX
List of Figures……………………………………………………………………..….X
Chapter 1 Background………………………………………………………………..1
1.1 Introduction……………………………………………………………..1
1.2 References………………………………………………………………6
Chapter 2 Experimental……………………………………………………………..15
2.1 Experimental of Materials……………………………………………..15
2.1.1 Strain………………………………………………………………15
2.1.2 Reagents…………………………………………………………...15
2.1.3 Buffers and solution……………………………………………….16
2.1.4 Equipment………………………………………………………….20
2.2 Experimental of Principles……………………………………………...22
2.2.1. Experimental of Quantitative……………………………………..22
2.2.1.1 Phenol determination………………………………………….22
2.2.1.2 Glucose determination………………………………………...22
2.2.1.3 Formaldehyde determination………………………………….22
2.2.1.4 Protein determination………………………………………….23
2.2.2 Experimental of Chromatographic separations……………………23
2.2.2.1 Ion chromatography…………………………………………...23
2.2.2.2 Reverse phase high-performance liquid chromatography…….24
2.2.2.3 Q sepharose chromatography…………………………………24
2.2.2.4 Gel filtration chromatography………………………………...25
2.2.3 Experimental of Mass Spectrometry Methods…………………….25
2.2.3.1 Gas chromatography-mass spectrometry……………………..25
2.2.3.2 Inductively Coupled Plasma Mass Spectrometry…………….26
2.2.3.3 Quadrupole-time of flight electrospray ionization-mass spectrometry and tandem mass spectrometry…………………26
2.2.3.4 MALDI-TOF-MS and MALDI-TOF/TOF-MS………………27
2.2.4 Others……………………………………………………………...28
2.2.4.1 Salting-out…………………………………………………….28
2.2.4.2 Edman sequencing……………………………………………29
2.2.4.3 Two-dimensional gel electrophoresis…………………………29
2.3 Experimental of Methods………………………………………………31
2.3.1 Phenol determination………………………………………………31
2.3.2 Formaldehye determination………………………………………..31
2.3.3 Glucose determination……………………………………………..31
2.3.4 Protein determination………………………………………………31
2.3.5 Phenol hydroxylase activity assay…………………………………31
2.3.6 Catechol 1,2-dioxygenase activity assay…………………………..32
2.3.7 In-solution digestion……………………………………………….32
2.3.8 In-gel digestion…………………………………………………….33
2.3.9 SDS-PAGE………………………………………………………....34
2.3.10 2-D PAGE…………………………………………………………34
2.3.11 Coomassie blue staining…………………………………………..35
2.4 References………………………………………………………………37
Chapter 3 An isolated Candida albicans TL3 capable of degrading phenol at large concentration………………………………………………………………46
3.1 Abstract…………………………………………………………………46
3.2 Introduction……………………………………………………………..48
3.3 Experimental……………………………………………………………50
3.3.1 Media formulation and microorganism screening…………………50
3.3.2 Cell growth and Phenol degradation……………………………….50
3.3.3 Enzyme activity assays…………………………………………….52
3.3.4 Product analysis and identification………………………………...53
3.3.5 Mass-spectrometric analysis……………………………………….54
3.4 Results and discussion………………………………………………….56
3.4.1 Identification of the isolated strain and its tolerance against phenol…
……………………………………………………………………...56
3.4.2 Cell growth and phenol degradation……………………………….57
3.4.3 Effect of temperature and nitrogen bases on the growth of C.albicans TL3………………………………………………………………..59
3.4.5 Characterization of the pathway of phenol degradation by C. albicans TL3……………………………………………………....60
3.4.6 Application to the treatment of industrial effluent………………....64
3.5 Conclusion………………………………………………………………66
3.6 References………………………………………………………………67
Chapter 4 Purification and characterization of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3…………………………………87
4.1 Abstract………………………………………………………………....87
4.2 Introduction……………………………………………………………..88
4.3 Experimental……………………………………………………………90
4.3.1 Cell culture…………………………………………………………90
4.3.2 Preparation of crude extract and enzyme purification……………..90
4.3.3 Determination of protein concentration……………………………91
4.3.4 Determination of molecular mass………………………………….91
4.3.5 Enzyme activity assays……………………………………….……92
4.3.6 Kinetic measurements………………………………………….......93
4.3.7 Iron analysis………………………………………………………..93
4.4 Results and Discussion…………………………………………………95
4.4.1 Purification of 1,2-CTD……………………………………………95
4.4.2 Characterization of 1,2-CTD………………………………………95
4.5 Conclusion……………………………………………………………...99
4.6 References……………………………………………………………..100
Chapter 5 Proteomic analysis of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3………………………………………………..…120
5.1 Abstract………………………………………………………………..120
5.2 Introduction……………………………………………………………121
5.3 Experimental…………………………………………………………..123
5.3.1 2-D gel electrophoresis…………………………………………...123
5.3.2 In-gel digestion…………………………………………………...123
5.3.3 N-terminal protein sequencing…………………………………...123
5.3.4 Peptide sequencing by MALDI-TOF…………………………….124
5.4 Results and Discussion………………………………………………..125
5.4.1 MALDI-TOF analysis of the isotypes of 1,2-CTD……………….125
5.4.2 Amino acid sequence analysis of 1,2-CTD………………………125
5.5 Conclusion…………………………………………………………….128
5.6 References…………………………………………………………….129

List of Tables

Table 2-1. Compositions of in-solution digestion……………………………………39
Table 2-2. Compositions of SDS-PAGE……………………………………………..40
Table 2-3. Compositions of 2-D PAGE………………………………………………41
Table 3-1. Capability of complete degradation of phenol by various yeasts………...72
Table 3-2. Identification of the phenol-degradation isolated strain………………….73
Table 3-3. Growth of Candida albicans TL3 on differents aromatic and related compounds（200ppm）after seven days in shake-flask…………….......74
Table 3-4. Comparison of enzyme specific activity of Candida albicans TL3……...75
Table 3-5. Effect of temperature on specific enzyme activity of Candida albicans TL3*……………………………………………………………………...76
Table 4-1. Purification of catechol-1,2-dioxygenase from C. albicans TL3……….104
Table 4-2. Substrate specificity of 1,2-CTD from C. albicans TL3………………..105
Table 4-3. The properties of 1,2-CTD from C. albicans TL3……………………...106
Table 4-4. Effects of some metal ions and compounds on the activity of 1,2-CTD from C. albicans TL3 for catechol…………………………………………...107













List of Figures

Figure 1-1. Two common phenol degradation pathways, the ortho- and meta-fission, occur in microorganisms………………………………………………..13
Figure 1-2. Aerobic catabolism of monoaromatic hydrocarbons…………………….14
Figure 2-1. Condensational reaction of phenol and 4-aminoantipyrine……………...42
Figure 2-2. A process of ESI…………………………………………………………43
Figure 2-3. Configuration used in Q-TOF ESI-MS/MS……………………………..44
Figure 2-4. A process of MALDI-TOF………………………………………………45
Figure 3-1. Time-course profiles of cell growth of Candida albicans TL3………….77
Figure 3-2. Consumption of phenol and glucose of Candida albicans TL3………....78
Figure 3-3. The kinetic parameters of phenol biodegradation catalyzed by C. albicans TL3……………………………………………………………………...79
Figure 3-4. Temperature effect on the growth of Candida albicans TL3……………80
Figure 3-5. Comparison of the growth of Candida albicans TL3 with different nitrogen bases.…………………………………………………………..81
Figure 3-6. HPLC analysis of the product of phenol is catalyzed by crude enzyme extract…………………………………………………………………...82
Figure 3-7. GC-mass analysis of the product of phenol is catalyzed by crude enzyme extract…………………………………………………………………...83
Figure 3-8. Ion-chromatographic analysis of the product(s) of catechol is catalyzed by crude enzyme extract……………………………………………………84
Figure 3-9. Electrospray ionization mass analysis (ESI) of the product of catechol is catalyzed by crude enzyme extract……………………………………...85
Figure 3-10. Growth and phenol and formaldehyde consumption of Candida albicans TL3 was cultured on waste water as a sole carbon source…………….86
Figure 4-1. Separation of catechol 1,2-dioxygenase from 50-70% (NH4)2SO4 ppt on a G-75column (2x80 cm)…………………………………..……………108
Figure 4-2. Separation of catechol 1,2-dioxygenase from the catechol 1,2-dioxygenase-containing fractions of G-75 column on a Q-sephadex column…………………………………………………………………109
Figure 4-3. Native molecular mass determination of 1,2-CTD from C. albicans TL3 by -75 column Chromatography………………………………………110
Figure 4-4. SDS-PAGE analysis of 1,2-CTD from C. albicans TL3 in various steps of purification…………………………………………………………….111
Figure 4-5. The mass spectrum of the purified 1,2-CTD from C. albicans TL3 (inset) and the deconvolution of the spectrum to give a molar mass of 31,994 atomic mass units……………………………………………………...112
Figure 4-6. ESI-MS/MS analysis of the product of catechols catalyzed by 1,2-CTD from C. albicans TL3………………………………………………….113
Figure 4-7. Kinetic property of 1,2-CTD from C. albicans TL3 for catechol……...114
Figure 4-8. Kinetic property of 1,2-CTD from C. albicans TL3 for 4-methylcatechol ……….……………………………………………..115
Figure 4-9. Optimal temperature of catechol 1,2-dioxygenase from C. albicans TL3 .………………………………………………………………...…116
Figure 4-10. Optimal pH of catechol 1,2-dioxygenase from C. albicans TL3……..117
Figure 4-11. Thermal stability of catechol 1,2-dioxygenase from C. albicans TL3..118
Figure 4-12. pH stability of catechol 1,2-duoxygenaase from C. albicans TL3……119
Figure 5-1. 2-D gel electrophoresis (pH 3–10 NL) of 1,2-CTD from C. albicans TL3.……………………………………………………………………133
Figure 5-2. 2-D gel electrophoresis (pH 4�o7 NL)of 1,2-CTD from C. albicans TL3…………………………………………………………………….134
Figure 5-3. MALDI-TOF mass spectrometry analysis of 5 1,2-CTD isotypes from C. albicans TL3 on the 2-D gel…………………………………………..135
Figure 5-4. RP-HPLC separation of fragments from trypsin-digested 1,2-CTD of C. albicans TL3…………………………………………………………..136
Figure 5-5. De novo sequences of peptide fragment with m/z 932 Da derived from 1,2-CTD from C. albicans TL3……………………………………….137
Figure 5-6. De novo sequences of peptide fragment with m/z 1199 Da derived from 1,2-CTD from C. albicans TL3……………………………………….138
Figure 5-7. Internal amino acid sequence homology of 1,2-CTD of C. albicans TL3 with hypothetical protein CaO19_12036 of C. albicans SC5314 (XP_722784 XP_431250)…………………………………………….139
Figure 5-8. Amino acid sequence alignment of 1,2-CTDs and 1,2-ClCTD………..140

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