理學碩士學位論文
Leuconostoc mesenteroides LBP-K06 에서 cyclic dipeptide 를 합성하는 효소
Cyclic dipeptide synthetase from Leuconostoc mesenteroides LBP-K06
2015 年 2 月
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cyεlic d요pePUde 를 합성하는 효소
指導敎授 姜 思 旭
이 論文올 理學碩士學位論文으로 提出함
2015 年
2月 서올大學校 大學院
生命科學部
(于 穎
于 穎의 理學碩士學位論文올 認准함
2ⓛ15
年
2月
委 員 長 副委員長
委 員 캭 七
Cyclic dipeptide synthetase from Leuconostoc mesenteroides LBP-K06
by YING YU
Advisor:
Professor Sa-Ouk Kang, Ph.D.
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science
i
ABSTRACT
This study worked with Leuconostoc mesenteroides LBP-K06, one kind of lactic acid bacteria separated from Korea traditional fermented kimchi, which had a higher antimicrobial activity than other strains, and focused on the cyclic dipeptide synthetase isolated from it. This study used ninhydrin staining method, a specific color reaction for amino acids, to determine the cyclic dipeptide synthetase in Ln. mesenteroides LBP-K06. And the activity of cyclic dipeptide synthetase was expected by the phenomenon that achromatic bands appeared on native gel after ninhydrin staining. The achromatic fragments on the native gel performed by SDS-PAGE, and the single bands on SDS gel were performed by 2D LC-MS analysis for protein identification. Analysis of the 2D LC-MS results, the ATPase family associated with various cellular activities proteins (gi:
227351295) was identified as the enzyme that synthesized cyclic dipeptides in Ln.
mesenteroides LBP-K06. In this study, polymerase chain reaction was performed to clone the gene segment of cyclic dipeptide synthetase, the pET3a vector system was used to transform the target gene segment into E. coli, and isopropyl β-D-1- thiogalactopyranoside (IPTG) was used for inducing the overproduce of recombinant cyclic dipeptide synthetase. Then the overproduced proteins were purified by ammonium sulfate and DEAE-Sepharose chromatography, and the purified proteins were added to the amino acid to determine the enzyme activity.
By ninhydrin staining method and HPLC analysis, the enzyme activity of cyclic dipeptide synthetase separated from Ln. mesenteroides LBP-K06 was confirmed.
It was observed that the cyclic dipeptide synthetase could not only catalyze between L-leucine and L-proline, but also between L-phenylalanine and L-proline to synthesize the cyclic dipeptides.
Keywords: Leuconostoc mesenteroides LBP-K06, cyclic dipeptide (CDP), cyclic dipeptide synthetase (CDPS), cis-cyclo(L-Leu-L-Pro), cis-cyclo(L-Phe-L-Pro), ninhydrin staining method, HPLC analysis
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CONTENTS
ABSTRACT ... i
CONTENTS ... iii
LIST OF FIGURES ... v
LIST OF TABLES ... vi
LIST OF ABBREVIATIONS ... vii
CHAPTER I INTRODUCTION ... 1
CHAPTER II MATERIALS AND METHODS ...9
1. Ln. mesenteroides LBP-K06 culture conditions ... 10
2. Anion exchange resin DEAE-Sepharose chromatography ... 10
3. Screening experiments of Leuconostoc CDPS ... 11
4. CDPS gene cloning for overproducing CDPS in E. coli cells ... 12
5. The recombinant CDPS protein purification ... 13
6. Electrophoretic separation of fractions and enzyme reaction ... 13
7. Enzyme activity assay by HPLC ... 14
CHAPTER III RESULTS ... 16
1. Screening of Leuconostoc CDPS ... 17
2.2D LC-MS data ... 19
3. Sequence analysis... 21
4. Culture conditions ... 25
5. Purification of recombinant CDPS... 27
6. Enzyme reaction and HPLC ... 30
CHAPTER IVDISCUSSION ... 37
CHAPTER VI REFERNCES ... 41 ABSTRACT IN KOREAN ... 45
v
LIST OF FIGURES
Scheme 3.HPLC profiles of culture filtrate of various lactic acid bacteria .... 7 Fig. 1. Ninhydrin staining for screening Leuconostoc CDPS ... 18 Fig. 2. Nucleotide and amino acid sequence of Leuconostoc CDPS
(gi：227351295) ... 23 Fig. 3. DNA gel electrophoresis of resulting PCR product... 24 Fig. 4. SDS-PAGE of E. coli transformants
for determining culture condition... 26 Fig. 5. SDS-PAGE showed Leuconostoc CDPS were precipitated
by different amounts of solid ammonium sulfate. ... 28 Fig. 6. SDS-PAGE and native-PAGE of DEAE fractions. ... 29 Fig. 7. HPLC profile for detecting enzyme activity among
leucine, proline and phenylalanine. ... 31 Fig. 8. HPLC profiles of amino acids, Tris-HCl buffer and ATP. ... 32 Fig. 9. HPLC profiles of cis-cyclo(L-Leu-L-Pro) and
cis-cyclo(L-Phe-L-Pro).. ... 33 Fig. 10. HPLC profile and native gel for detecting enzyme activity
between two amino acids.. ... 34
LIST OF TABLES
Scheme 1. Numbers of lactic acid bacteria strains isolated
from plant materials ... 3 Scheme 2. Comparison antibacterial activity of stains isolated
from plant materials ... 4 Table 1. Proteins detected from 2D LC-MS ... 20
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LIST OF ABBREVIATIONS
LAB lactic acid bacteria CDP cyclic dipeptide
CDPS cyclic dipeptide synthetase LB Luria-Bertani
IPTG isopropyl β-D-1-thiogalactopyranoside ATP adenosine triphosphate
MRS de Man, Rogosa and Sharpe PMSF phenylmethylsulfonyl fluoride DEAE diethylaminoethanol
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis BLAST basic local alignment search tool
PCR polymerase chain reaction
HPLC high performance liquid chromatography TDW triple-distilled water
C HAPTER I
I NTRODUCTION
2
Lactic acid bacteria (LAB) is Gram-positive, facultative anaerobic and non-spore forming bacterium. They can ferment sugars to form lactic acid (lactate).
Leuconostoc mesenteroides is a kind of LAB in Korean traditional fermented food kimchi. By contributing to slime mold formation and CO2 production, they change the smells and flavors of food products (Wang H-Y et al., 2013).
LAB is known to be a natural antimicrobial tool driven by substrates, which produced by fermenting of animals and plants materials (Holzapfel et al., 2001).
Antimicrobial substances produced by lactic acid bacteria against microbes have been mainly debated regarding bacteriocin-like peptides especially including bacteriocin, plantaricin and pediocin, whose major activities have been demonstrated against Gram-positive and Gram-negative bacteria regardless of unstable antimicrobial functions (Abee et al., 1995). Also, small molecules produced by lactic acid bacteria as secondary metabolites against microbes have been investigated in terms of cellular metabolism during cell growth and fermentation, such as reutericyclin, 3-phenyllactic acid, benzoic acid, methylhydantoin, benzeneacetic acid, 2-propenyl ester, mevalonolactone 2,6-diphenyl-piperidine, and cyclic dipeptides (Niku-Paavola et al., 1999; Gänzle et al., 2000; Ström K et al., 2002; Wang et al., 2012 ).
Approximately 400 strains of lactic acid bacteria were isolated from three types of Korean traditional kimchi, including Leuconostoc spp., Lactobacillus spp., Weissella spp. and Lactococcus lactis (Scheme 1, Oh E-S, 2010). Of these isolated strains, the culture filtrates of 30 isolates showed a higher antimicrobial activity than did other isolates (Scheme 2, Oh E-S, 2010).
Scheme 1. Numbers of lactic acid bacteria strains isolated from plant materials. (Oh E-S, 2010)
Strain number
Sources Mustard leaves
and stems
Stonecrop Chinese cabbage
Leuconostoc spp. 93 10 28
Lactobacillus spp. 14 8 17
Lactococcus spp. - 1 -
Weisella spp. 2 14 18
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Scheme 2. Comparison antibacterial activity of stains isolated from plant materials. (Oh E-S, 2010)
Source Strain Antagonism
test a MIC b,c Taxon confirmed by sequencing
Mustard leaves and stems
LBP-B01 ++ +++ Lb. sakei
LBP-B02 ++ ++ Ln. kimchii
LBP-B03 ++ ++ Ln. mesenteroides
LBP-B04 ++ ++ Ln. mesenteroides
LBP-B05 +++ ++ Ln. paramesenteroides
LBP-B06 +++ ++ W. cibaria
Stonecrop
LBP-S01 ++ +++ Lb. sakei
LBP-S02 +++ +++ Lb. plantarum
LBP-S03 ++ ++ Lc. lactis
LBP-S04 ++ ++ Ln. citreum
LBP-S05 ++ ++ Ln. citreum
LBP-S06 + ++ Lc. lactis
LBP-S08 ++ ++ W. hellenica
Chinese cabbage
LBP-K01 ++ +++ Lb. plantarum
LBP-K03 ++ ++ Ln. citreum
LBP-K04 ++ - Ln. citreum
LBP-K05 ++ ++ Ln. holzapfelii
LBP-K06 +++ ++ Ln. mesenteroides
LBP-K07 ++ ++ Ln. pseudomesenteroides
LBP-K08 ++ + Ln. mesenteroides
LBP-K09 ++ ++ Lb. brevis
LBP-K10 +++ +++ Lb. plantarum
LBP-K11 ++ ++ Ln. citreum
LBP-K12 +++ ++ Ln. mesenteroides
LBP-K13 ++ ++ Ln. mesenteroides
LBP-K14 ++ ++ Ln. pseudomesenteroides
LBP-K15 +++ ++ W. cibaria
LBP-K16 ++ ++ W. confusa
a. Symbol: +, <15mm; ++, <22mm; +++, >22mm (Indicator strain: B. subtilis) b. MIC: Minimum inhibitory concentration
c. Symbol: +, 1 fold; ++, 0.5 fold; +++, 0.25 fold (Indicator strain: B. subtilis)
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Cyclic dipeptides (CDPs) have been found in humans, vertebrates, invertebrates, plants, fungi and bacteria. They have significant physiological activities which plays important roles in human being lives (Zhong F et al., 2006). The relevant activities of cyclic dipeptides have been widely reported, including alcohol paralysis reduction, memory loss resistance, inhibitory effect on both the growth of virus-transformed cells and aflatoxin production (Li H-F et al., 2010). Additionally, they might function as hormone, growth promoter, and antibiotics, which can be driven by the considerable interest using them in different fields (Yin P et al., 2008).
The antifungal substances of the culture filtrates from several kinds of isolates, such as Lactobacillus sakei LBP-S01, L. lactis LBP-S06, Ln. mesenteroides LBP-K06, Lb. plantarum LBP-K10, W. cibaria LBP-K15 and W. confusa coincided with the 17 different fractions through the high performance liquid chromatography (HPLC) (Scheme 3, Kwak et al., 2013). When structural analysis by mass determinations and X-ray crystallography, several fractions were revealed to be the cyclic dipeptides as follows: cis-cyclo(L-Tyr-L-Pro), C14H16N2O3, cis-cyclo(L-Val-L-Pro), C10H16N2O2, cis-cyclo(L-Ser-L-Pro), C8H12N2O3, cis-cyclo(L-Leu-L-Pro), C11H18N2O2 and cis-cyclo(L-Phe-L-Pro), C16H16N2O2 (Kim A-H, 2014).
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culture filtrate of various lactic acid bacteria (adopted from Kwaket al., 2013).
8
Herein, based on these observations, Ln. mesenteroides LBP-K06 was selected for the experimental purposes and we therefore found an enzyme activity in culture filtrate, which catalyzes the conversion of free amino acids to the dioxo- and diaza-containing diketopiperazines by the cyclization reaction between two amino acids in the presence of adenosine triphosphate (ATP) and minor metal ions. The isolated cyclic dipeptide synthetase (CDPS) from Ln. mesenteroides LBP-K06 was identified, cloned and transformed into Escherichia coli. Using Luria-Bertani (LB) medium, Leuconostoc CDPS were overproduced in E. coli by the strong promoter induction using isopropyl β-D-1-thiogalactopyranoside (IPTG). After protein purification, we observed Leuconostoc CDPS activity, which trigger the catalysis of the conversion of amino acids to cyclic dipeptides when using various kinds of amino acids, including L-leucine, L-proline and
L-phenylalanine, respectively.
C HAPTER II
M ATERIALS A ND M ETHODS
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1. Ln. mesenteroides LBP-K06 culture conditions
Ln. mesenteroides LBP-K06 was isolated from Korean traditional side dish kimchi and cultured in modified de Man, Rogosa and Sharpe (mMRS) medium [2%
glucose, 0.5% yeast extract, 0.2% KH2PO4, 0.5% CH3COONa, 0.2%
HOC(COOH)(CH2COONH4)2, 0.01% MgSO4 and 0.0056% MnSO4.H2O] at 30 °C for 3 days without agitation.
Ln. mesenteroides LBP-K06 cells grown in mMRS broth were harvested by centrifugation (8,000 rpm, 15 min), washed once with 0.1 M Tris-hydrochloride (Tris-HCl) buffer (pH 8.0) and resuspended in the same buffer (1 : 5) (Bruce M.C et al., 1980). 50 mg/ml lysozyme was added into the cell crude extracts or filtrated culture supernatant. This reactant was incubated at 37 °C water bath for 3 h. Then 10 mg/ml phenylmethylsulfonyl fluoride (PMSF) was added into cell lysates for further disruption by sonication at appropriate frequency (24-kHz, 3 s on and 7 s off) (Li Y-X et al., 2012). The cell debris were removed by centrifugation at 12,000 rpm for 10 min.
2. Anion exchange resin DEAE-Sepharose chromatography
DEAE CL-6B column was made by packing with 45 ml of DEAE CL-6B resin into a glass-column (45 mm 200 mm). The resin was washed with 5 volumes of water and consecutively 10 volumes of 0.1 M Tris-HCl buffer (pH 8.0). Then the total proteins in cell crude extracts from Ln. mesenteroides LBP-K06 cells was loaded onto the anion exchange column, and the fractions were collected by NaCl
gradient ranging from 0 to 0.5 M (Krisna C.D-L et al., 2014), which proceeds with two bed volumes of 0.1 M Tris-HCl buffer for column washing. Additionally, the bound proteins were eluted with 0.1 M Tris-HCl buffer, containing 0 M-0.5 M NaCl linear gradient at the flow of 15 ml/min (Tara K.S et al., 2004).
3. Screening experiments of Leuconostoc CDPS
A 12% native-gel without SDS in a large lane of the fractions from DEAE CL-6B was prepared. The native-PAGE was performed at 4°C. The resulting native gel was washed twice with 0.1 M Tris-HCl buffer (pH 8.0), once for 10 minutes.
Then the native gel was soaked in 40 ml amino acid buffer [0.1 M Tris-HCl buffer (pH 8.0), containing 4 ml 0.1 M L-leucine, 4 ml 0.1 M L-proline, 4 ml 0.1 M
L-phenylalanine, 4 ml 0.5 M ATP, 0.4 ml 0.3 M MnSO4 and 0.4 ml 0.8 M MgSO4] and incubated at 37 °C for 4 h. After amino acid reaction, 10 ml amino acid buffer was removed and 30 ml 0.1 M ninhydrin solution was added for staining the gel.
Then the native gel was incubated in a dark condition at 37 °C for further 16 h.
The achromatic bands appeared on the stained native gel was considered to be the site of a protein having an activity of Leuconostoc CDPS. The achromatic bands were cut, stained with Laemmli staining solution and performed for SDS-PAGE.
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and protein sequence homology searches of GenBank were performed using the BLAST program.
4. CDPS gene cloning for overproducing CDPS in E. coli cells
A 2151 bp DNA segment from Ln. mesenteroides LBP-K06 genomic DNA encoding the ATPase family associated with various cellular activities proteins (79 kDa) without signal peptide was PCR-amplified. Its primers included the 5' end primer 5'-GGATCCTTAGTTAGTAACCGCAGTCTTCTC-3', which containing a BamHI-engineered restriction site and the 3' end primer 5'-CATATGGCTAGCAATGATTTTTTCAATGAT-3', which containing an NdeI-engineered restriction site. The 2151bp BamHI/NdeI DNA fragment of Leuconostoc CDPS was cloned into pGEM-T EASY vector (Promega) for DNA sequencing. The resulting PCR product was double digested by NdeI and BamHI, and inserted into pET3a vector (Novagen, Germany), which digested by the same enzymes, to form the expression plasmid pET3a-CDPS.
E. coli DH5a was used to propagate the plasmid pET3a-CDPS serving as the host strain. E. coli BL21 cells were transformed with pET3a-CDPS grown on LA agar plant [1% tryptone, 1% NaCl, 0.5% yeast extract, 1.7% agar powder and 50 mg/ml ampicillin] at 37 °C overnight. A fresh single colony of E. coli BL21 was inoculated in the liquid LB medium [1% tryptone, 1% NaCl, 0.5% yeast extract, and 50 mg/ml ampicillin] until the OD 600 reached to 0.3. Then 0.5 mmol/l IPTG was added to induce overproduction of recombinant CDPS at 30 °C (Kwak, 2009).
5. The recombinant CDPS protein purification
The grown cells were centrifuged at 8,000 rpm for 15 min, washed and resuspended with 50 mM Tris-HCl buffer (pH 7.0). The collected cells were disrupted by sonication (24-kHz, 3 s on and 7 s off) for 30 min. The overproduced proteins were purified by the methods of salting out and DEAE-Sepharose chromatography.
Firstly, the recombinant proteins were precipitated by ammonium sulfate (NH4)2SO4 for pre-purification. The amounts of (NH4)2SO4 were according to the saturation concentration of it. Solid ammonium sulfate was added into protein solution and fully dissolved at 4 °C for more than 2 h. Then precipitated proteins were redissolved in 50 mM Tris-HCl buffer (pH 7.0) (Krisna C.D-L et al., 2014).
Secondly, the initial purified protein solution was loaded onto DEAE CL-6B column for final purification. Column was washed by 50 mM Tris-HCl buffer (pH 7.0), bound proteins were eluted with the 50 mM Tris-HCl buffer containing 0 M-1 M NaCl and finally collected fractions (GE Healthcare, 2009).
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acetic acid solution. For native-PAGE, a standard native-gel was prepared and performed in the 4 °C cold room.
Leuconostoc CDPS was detected by amino acid reaction and ninhydrin staining.
The native gel was washed twice with 50 mM Tris-HCl buffer (pH 7.0), once for 10 minutes. Then the native gel was soaked in 40 ml amino acid buffer [50 mM Tris-HCl buffer (pH 7.0), containing 4 ml 0.5 M ATP, 0.4 ml 0.3 M MnSO4 and 0.4 ml 0.8 M MgSO4 and 4 ml any two kinds of 0.1 M amino acid solution among
L-leucine, L-proline, L-phenylalanine] and incubated at 37 °C for 4 h. After amino acid reaction, the native gels were stained with 0.1 M ninhydrin solution as the method mentioned above.
7. Enzyme activity assay by HPLC
The purified Leuconostoc CDPS was added into amino acid buffer, which was same as the buffer reacted with native gel, and the reaction solution was incubated at 37 °C water bath for 4 h. After reaction, reaction solution was shaking reacted with 5 times of methylene chloride at 25 °C for 1 day so that products were extracted by methylene chloride. The reaction solution was poured into a separate funnel for the separation of aqueous solution and organic solution. Then evaporator was used for evaporating methylene chloride at 55 °C and the rest products were dissolved in triple-distilled water (TDW) (Oh E-S, 2010).
For HPLC, Ecliose XDB-C18 ODS hypersil column (4.6×150mm, Agilent Technologies, USA) was used and column temperature was kept at 40 °C. 67%
TDW, 30% methanol and 3% acetonitrile were served as the mobile phase. 20 μl
each of samples was injected at injection volume of and detected at a wavelength of 210 nm with 0.7 ml/min flow rate (Oh E-S, 2010).
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C HAPTER III
R ESULT
1. Screening of Leuconostoc CDPS
Ln. mesenteroides LBP-K06 cells were collected and cracked by lysozyme and sonication for obtaining total proteins. For screening Leuconostoc CDPS, obtained proteins was eluted by DEAE-Sepharose chromatography and the fractions performed native-PAGE. Then the proteins on native gel were reacted with amino acid buffer and stained by ninhydrin (Fig. 1). The achromatic bands appeared on native gel showed the enzyme activity of Leuconostoc CDPS.
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Fig. 1. Ninhydrin staining for screening Leuconostoc CDPS. The proteins on native gel were reacted with amino acid buffer [0.1 M Tris-HCl buffer (pH 8.0), containing 4 ml 0.1 M L-leucine, 4 ml 0.1 M L-proline, 4 ml 0.1 M
L-phenylalanine, 4 ml 0.5 M ATP, 0.4 ml 0.3 M MnSO4 and 0.4 ml 0.8 M MgSO4] and stained with ninhydrin. There were 4 teams of achromatic bands on native gel showed the activity of Leuconostoc CDPS.
2. 2D LC-MS data
The achromatic bands appeared on the stained native gel was cut and preformed SDS-PAGE. The resulting gel slices after SDS-PAGE were extracted by the in-gel digestion method and further analyzed by 2D LC-MS. After 2D LC-MS, it showed that there were amounts of different proteins existed in different bands.
According to the content scores and repeatability, 16 kinds of main proteins were discovered on the gel slices (Table. 1).
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Table 1. Proteins detected from 2D LC-MS. AccessionScoreDescription gi22735129569.71-851.15ATPase family associated with various cellular activities (AAA) [Leuconostoc mesenteroides subsp. cremoris ATCC 19254] gi491037850419.35-799.69ATP-dependent Clp protease ATP-binding protein [Leuconostoc citreum] gi29611105148.01-789.62ATP-dependent proteinase ATP-binding subunit [Leuconostoc kimchii IMSNU 11154] gi51787171848.00-533.09ATP-dependent Clp protease ATP-binding protein, partial [Leuconostoc carnosum] gi22735311832.66-309.5chaperonin GroL [Leuconostoc mesenteroides subsp. cremoris ATCC 19254] gi49768830533.46-298.72ATPase with chaperone activity, ATP-binding subunit, partial [Leuconostoc lactis] gi40771782081.78-251.16chaperonin GroEL [Leuconostoc carnosum JB16] gi8616950963.27-165.92HSP70 [Leuconostoc fallax KCTC 3537] gi86169497100.67-142.06HSP70 [Leuconostoc mesenteroides subsp. dextranicum] gi38133590837.72-127.51glyceraldehyde-3-phosphate dehydrogenase [Leuconostoc mesenteroides subsp. mesenteroides J18] gi49769030513.43-117.60ATP-dependent proteinase ATP-binding subunit, partial [Leuconostoc lactis] gi8616952138.61-94.38HSP70 [Weissella confusa] gi8616950757.02-90.84HSP70 [Leuconostoc pseudomesenteroides KCTC 3652] gi8616951733.97-85.47HSP70 [Weissella paramesenteroides] gi8616950160.96-79.66HSP70 [Leuconostoc citreum] gi49796428311.95-33.97pyruvate kinase, partial [Leuconostoc pseudomesenteroides]
3. Sequence analysis
By analyzing the data of 2D LC-MS, the ATPase family associated with various cellular activities proteins (79 kDa) was identified as Leuconostoc CDPS.
Nucleotide and amino acid sequence homology searches of GenBank were performed using the BLAST program (Fig. 2). According to nucleotide sequence, 5’ and 3’ end primer were designed and used for polymerase chain reaction (PCR).
In PCR, the reaction mixture were subjected to 30 cycles under the following condition: denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2 min. Then resulting PCR product was performed by DNA gel electrophoresis for size determination (Fig. 3).
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1 ATGGCTAGCAATGATTTTTTCAATGATCCATTTGGATCAGACATGAATGACATTTTTAAT M A S N D F F N D P F G S D M N D I F N 20
61 AACATGATGGGCAATATGAACGGCATGAACTCTGAGAACCGTCGTTATTTGATTAACGGT N M M G N M N G M N S E N R R Y L I N G 40
121 CGCGAGGTTACGCCGGAAGAGTTTGTTCAATATCGACAGACTGGGAAACTACCACAAGGT R E V T P E E F V Q Y R Q T G K L P Q G 60
181 CTTCAAGCAGCCAATGCTACAAACGGTCAGCCAGTAGCACAACAAGCTGGGCAACCTGGA L Q A A N A T N G Q P V A Q Q A G Q P G 80
241 CAAGTAAAGCAGGAAGGAATGCTGGCAAAGCTTGGACGTAACTTGACGAAAGAGGCTCGC Q V K Q E G M L A K L G R N L T K E A R 100
301 GACGGTTTGCTTGATCCAGTCATTGGACGTAACAAAGAGATTCAAGAAACGGCTGAAATT D G L L D P V I G R N K E I Q E T A E I 120
361 TTAGGACGCCGTACAAAGAACAACCCAGTGCTAGTTGGCGATGCTGGTGTTGGTAAGACA L G R R T K N N P V L V G D A G V G K T 140
421 GCAGTGGTTGAAGGTTTGGCACAAGCGATTGTTAATGGGGATGTCCCAGCAGCAATTAAG A V V E G L A Q A I V N G D V P A A I K 160
481 GACAAGGAGATTTACAGTATCGATATCTCTAGCCTTGAAGCTGGTACTCAATTCCGTGGT D K E I Y S I D I S S L E A G T Q F R G 180
541 GCTTTTGAGGAAAATATCCAGAACCTTTTGAAAGAAGTTAAACAAGCAGGCAACGTTATC A F E E N I Q N L L K E V K Q A G N V I 200
601 TTGTTCTTTGATGAGATTCATCAAATCTTGGGAGCTGGTTCAACCGGTGGCGAAGATGGC L F F D E I H Q I L G A G S T G G E D G 220
661 GGTAAAGGACTAGCTGATATTATCAAGCCAGCTTTGAGTCGTGGTGAAATTTCATTAATT G K G L A D I I K P A L S R G E I S L I 240
721 GGTGCGACGACGCAAGATGAATATCGTAACACGATTATGAAGAACGCTGCACTAGCTCGG G A T T Q D E Y R N T I M K N A A L A R 260
781 CGTTTTAATGAAGTGACTGTGAATGCACCATCACCTAAGGATACGCTTGAAATCTTGAAG R F N E V T V N A P S P K D T L E I L K 280
841 GGGATTGCGAAGTTGTATGAAAAGCATCACCATGTTTCATTGCCTGAAAACGTATTAAAG G I A K L Y E K H H H V S L P E N V L K 300
901 GCAGCAGTTGACTATGCAGTACAGTATATTCCACAACGTTCATTACCAGATAAGGCGATA A A V D Y A V Q Y I P Q R S L P D K A I 320
961 GATTTGCTTGACATGACAGCGGCTCACCTGTCAGCTAAGAACCCAGTAACAGATAAGGTT D L L D M T A A H L S A K N P V T D K V 340
1021 AGTTTAGAGAAGCAGATGAGCGATGCTAAAGCAAAGCAAGAAAAGGCTGTTGCTGATGAA S L E K Q M S D A K A K Q E K A V A D E 360
1081 GACTATGAGGCCGCTCTAAAGTACAAGAACCAGATTGCTGATTTGGAAAAGAAAGTTGGT D Y E A A L K Y K N Q I A D L E K K V G 380
1141 AGCGCTGACGAAGCTAAAAAGGTTGAGGCAACGCCTAATGATGTGGCCGAATCAGTAGAA S A D E A K K V E A T P N D V A E S V E 400
1201 CGTTTGACTGGTATTCCTGTGGCACAGATGGGTGCGTCAGATATCGAACGTTTGAAGACA R L T G I P V A Q M G A S D I E R L K T 420
1261 ATTGGTGATCGTTTGGCTGGTAAAGTTATTGGTCAAGATGAAGCCGTAAGTATGGTCGCT I G D R L A G K V I G Q D E A V S M V A 440
– continued
1321 AAAGCCATTCGTCGTAACCGCGCGGGATTTGATGAAGGAAACCGACCAATTGGTTCGTTC K A I R R N R A G F D E G N R P I G S F 460
1381 TTGTTCGTCGGTCCAACTGGTGTTGGTAAGACGGAGTTGGCAAAGCAACTAGCTTTGGAC L F V G P T G V G K T E L A K Q L A L D 480
1441 ATGTTTGGTAGCAAAGATAACATTGTTCGTTTGGATATGAGTGAGTATTCGGATTTGACA M F G S K D N I V R L D M S E Y S D L T 500
1501 GCGGTATCTAAGTTAATTGGTACGACCGCTGGGTATGTTGGCTATGATGATAATAGCAAC A V S K L I G T T A G Y V G Y D D N S N 520
1561 ACACTAACTGAAAAAGTTCGCCGTAATCCTTATTCAATTATTCTCCTTGATGAAATTGAA T L T E K V R R N P Y S I I L L D E I E 540
1621 AAGGCCAATCCACAAGTAGTAACGTTGTTATTACAAGTGATGGACGATGGTCGCTTAACA K A N P Q V V T L L L Q V M D D G R L T 560
1681 GACGGTCAGGGAAACGTCGTTAACTTCAAGAATACGGTCATTATTGCAACATCTAACGCT D G Q G N V V N F K N T V I I A T S N A 580
1741 GGATTTGGACACAATTCGAGTGATGCAGATCAGGATTTGATGGCTAAGTTGGCGCCATAC G F G H N S S D A D Q D L M A K L A P Y 600
1801 TTCCGTCCAGAGTTCTTGAACCGTTTTAACGGTGTGATCGAATTTAGTACGTTGACTAAG F R P E F L N R F N G V I E F S T L T K 620
1861 GATGATTTGAAGCAAATTGTTGATTTGATGTTAGATGATGTCAATAAGACATTGGCTAAG D D L K Q I V D L M L D D V N K T L A K 640
1921 AAAGACTTGACATTACATGTGTCTGACGAAGTTAAGGATCACTTGATTGAAGATGGTTAT K D L T L H V S D E V K D H L I E D G Y 660
1981 GATGAAGCCTTAGGTGCACGACCATTGCGTCGAGTAATTGAGCAACAAATTCGTGACCAA D E A L G A R P L R R V I E Q Q I R D Q 680
2041 GTAACGGACTTCTATTTGGATAATCCAGGTGAAAAATCCTTAAGCGCAGACTTGGTTGAT V T D F Y L D N P G E K S L S A D L V D 700
2101 GGCAAAGTTGTCATCTCGGCTGTTGTTGAGAAGACTGCGGTTACTAACTAA
G K V V I S A V V E K T A V T N * 717
Fig. 2. Nucleotide and amino acid sequence of Leuconostoc CDPS (gi： 227351295). Leuconostoc CDPS is encoded by 2151bp nucleotides and 717 amino acids. The nucleotides and amino acids are numbered on the left and right side, respectively. And the termination codon is indicated with an asterisk.
24
Fig. 3. DNA gel electrophoresis of resulting PCR product. 2151 bp DNA segment encoding the Leuconostoc CDPS was amplified by PCR and showed on the DNA gel. The size of gene segment was determined by DNA marker.
4. Culture conditions
The E. coli transformants were shaking cultured in 37 °C incubator. The incubation time was changed from 0 h to 5 h, and the amount of IPTG was changed from 30 μl to 50 μl. According to the result of SDS-PAGE, it was found that recombinant CDPS were most produced when E. coli cells were cultured with IPTG for 4 h (Fig. 4).
26
Fig. 4. SDS-PAGE of E. coli transformants for determining culture condition.
E. coli transformants were shaking cultured in LB medium containing ampicillin.
(A) E. coli cells were cultured for 0 h, 1 h, 2 h, 3 h, 4 h and 5 h after adding 30 μl IPTG. The amounts of Leuconostoc CDPS increased along time and stopped the increase after 4 h. (B) E. coli cells were cultured for 4 h after adding the amount of 30 μl, 40 μl and 50 μl IPTG. There were no changes between the amounts of Leuconostoc CDPS overproduced with different amounts of IPTG.
5. Purification of recombinant CDPS
Firstly, ammonium sulfate was used to precipitate recombinant CDPS according to the saturation concentration of (NH4)2SO4. The precipitated proteins were redissolved into the appropriate amount of 50 mM Tris-HCl buffer (pH 7.0). By SDS-PAGE, I found that the recombinant CDPS could be almost precipitated at 0-30% (NH4)2SO4 (Fig. 5). Secondly, preliminary purified proteins were redissolved into buffer and further purified by DEAE-Sepharose chromatography.
The fractions of DEAE-Sepharose chromatography performed SDS-PAGE and native-PAGE (Fig. 6). Then the proteins on native gel were reacted with 40 ml amino acid buffer [50 mM Tris-HCl buffer (pH 7.0), containing 4 ml 0.1 M
L-leucine, 4 ml 0.1 M L-proline, 4 ml 0.1 M L-phenylalanine, 4 ml 0.5 M ATP, 0.4 ml 0.3 M MnSO4 and 0.4 ml 0.8 M MgSO4] at 37 °C for 4 h. 10 ml amino acid buffer was discarded and 30 ml 0.1 M ninhydrin was added. The native gel was stained for further 16 h in dark (Fig. 6- B).
28
Fig. 5. SDS-PAGE showed Leuconostoc CDPS were precipitated by different amounts of solid ammonium sulfate. According to the saturation concentration of (NH4)2SO4, solid ammonium sulfate was add to protein solution. The precipitated proteins were redissolved into buffer for SDS-PAGE. Samples of each column was as follows; M: protein marker, 1: all protein solution, 2: proteins precipitated by 0-20% (NH4)2SO4, 3: proteins precipitated by 20-30% (NH4)2SO4, 4: proteins precipitated by 30-40% (NH4)2SO4, 5: proteins precipitated by 40-50%
(NH4)2SO4, 6: proteins precipitated by 50-60% (NH4)2SO4, 7: proteins precipitated by 60-70% (NH4)2SO4, 8: proteins precipitated by 70-80%
(NH4)2SO4, 9: proteins precipitated by 80-100% (NH4)2SO4.
Fig. 6. SDS-PAGE and native-PAGE of DEAE fractions. (A) In DEAE-Sepharose chromatography, bound proteins were eluted with 50 mM Tris-HCl buffer (pH 7.0), containing 0 M -1 M NaCl. Depending on the different concentrations of NaCl, Leuconostoc CDPS were eluted. (B) The same samples as (A) were performed by native-PAGE. And the proteins on native gel were reacted with amino acid buffer [50 mM Tris-HCl buffer (pH 7.0), containing 4 ml 0.1 M
L-leucine, 4 ml 0.1 M L-proline, 4 ml 0.1 M L-phenylalanine, 4 ml 0.5 M ATP, 0.4 ml 0.3 M MnSO4 and 0.4 ml 0.8 M MgSO4] and stained with ninhydrin. After amino acid reaction and ninhydrin staining, there were achromatic bands appeared
30
6. Enzyme reaction and HPLC
The purified recombinant CDPS reacted with amino acid buffer including
L-leucine, L-proline and L-phenylalanine at 37 °C for 4 h to complete the peptides cyclization. Then methylene chloride (CH2Cl2) was use to extract cyclic dipeptides from the reaction solution. After distillation of methylene chloride, the distillates were analyzed by HPLC. In the 210 nm wavelength, 67% TDW, 30%
methanol and 3% acetonitrile were served as the mobile phase and passed through the column at a rate of 0.7 ml/min (Fig. 7). At the same condition, the amino acid solutions (L-leucine, L-proline and L-phenylalanine), Tris-HCl buffer and ATP were perform HPLC for analysis (Fig. 8). In addition, two pure compounds cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) were also analyzed by HPLC in the same condition (Fig. 9).
Apart from those above, recombinant CDPS reacted with any two amino acids among L-leucine, L-proline and L-phenylalanine. We checked the results by ninhydrin staining and HPLC (Fig. 10), and the method was the same as that mentioned above.
31
cting enzyme activity among L-leucine, L-proline andL-phenylalanine.20 μl purified acid buffer [50 mM Tris-HCl buffer (pH 7.0), containing 2 ml 0.1 M L-leucine, 2 ml 0.1 M nylalanine, 2 ml 0.5 M ATP, 0.2 ml 0.3 M MnSO4 and 0.2 ml 0.8 M MgSO4] for enzyme d that the amount of amino acids significantly reduced and there was a new peak appeared on.
32
Fig. 8. HPLC profiles of amino acids, Tris-HCl buffer and ATP. The solutions used in the experiment, including 0.1 M L-leucine, 0.1 M L-proline, 0.1 M
L-phenylalanine, 50 mM Tris-HCL buffer and 0.5 M ATP were respectively analyzed by HPLC at a wavelength of 210 nm with 0.7 ml/min flow rate.
34
Fig. 10.HPLC profile and native gel for detecting enzyme activity between two amino acids. (A) L-leucine and L-prolinewerecatalyzed byLeuconostocCDPS. Amino acid buffer was 20 ml50mM Tris-HClbuffer (pH7.0), containing 2 ml 0.1 M L-leucine, 2 ml 0.1 M L-proline, 2 ml 0.5 M ATP, 0.2 ml 0.3 M MnSO4 and 0.2 ml 0.8 M MgSO4.
35
native gel for detecting enzyme activity between two amino acids. (B) L-proline and d byLeuconostoc CDPS. Amino acid buffer was 20 ml 50 mM Tris-HCl buffer (pH7.0), , 2 ml 0.1 M L-phenylalanine, 2 ml 0.5 M ATP, 0.2 ml 0.3 M MnSO4 and 0.2 ml 0.8 M
36
Fig. 10. HPLC profile and native gel for detecting enzyme activity between two amino acids. (C) L-leucine and L-phenylalanine were catalyzed byLeuconostoc CDPS. Amino acid buffer was 20 ml 50 mM Tris-HCl buffer (pH7.0), containing 2 ml 0.1 M L-leucine, 2 ml 0.1 M L-phenylalanine, 2 ml 0.5 M ATP, 0.2 ml 0.3 M MnSO4 and 0.2 ml 0.8 M MgSO4.
C HAPTER IV
D ISCUSSION
38
The isolated Ln. mesenteroides LBP-K06 from Korean traditional food kimchi produces various fermentation end-products, which are also used for their own use during the normal growth. Based on previous studies, the compounds inhibited bacteria and fungi in culture filtrates were investigated that most of the components was determined as cyclic dipeptides (Kwak et al., 2013). So we inferred that lactic acid bacteria could encode some proteins that could cyclize the amino acids to cyclic dipeptides.
Firstly, we made screening experiments of Leuconostoc CDPS by ninhydrin staining method. We got different achromatic bands from native gel showing the enzyme activity (Fig. 1). By analyzing the results of preliminary experiments, we assumed that the gene segment of the ATPase family associated with various cellular activities proteins encodes Leuconostoc CDPS, which has the activity of dipeptide cyclization. We searched the protein sequence by performing the BLAST program and designed 5’ and 3’ primers according the gene sequence (Fig. 2). Then we isolated this gene segment from Ln. mesenteroides LBP-K06 and amplified it by PCR. DNA gel electrophoresis was used for defining the size of target gene (Fig. 3).
Secondly, we transformed this target gene into E. coli and overproduces the Leuconostoc CDPS in E. coli cells. For the recombinant E. coli cells culture, we changed the amounts of IPTG and cells culture time in order to get the most effective culture conditions (Fig. 4).
Thirdly, we collected amounts of recombinant E. coli cells and cracked cells by sonication to get all proteins expressed in E. coli. Then we added solid ammonium sulfate to protein solution to precipitate proteins for preliminary purification. By SDS-PAGE, we discovered that target proteins were almost precipitate at 0-30%
ammonium sulfate (Fig. 5). For further purification, the preliminary purified
recombinant CDPS were redissolved into buffer and further purified by DEAE-Sepharose chromatography with NaCl gradient. We performed SDS-PAGE and native-PAGE for checking size and activity of recombinant CDPS (Fig. 6). On the native gel, there were achromatic bands appeared after amino acid reacion and ninhydrin staining. It certified that the recombinant CDPS maintained the enzyme activity when overproduced in E. coli. In addition, Leuconostoc CDPS catalyzed
L-leucine, L-proline and L-phenylalanine. The reaction solution was extracted by methylene chloride and analyzed by HPLC (Fig. 7). Reacted with Leuconostoc CDPS, the substrates of amino acid buffer were reduced significantly.
At the same time, the solutions used in the amino acid reaction were analyzed by HPLC (Fig. 8). Based on the HPLC profiles of them, the time of theses substrates appeared on the profiles were all before 5 min. And the pure compound cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) were also analyzed by HPLC at the same condition (Fig. 9). We found that the peaks of these two cyclic dipeptids appeared between 5 min and 15 min.
Finally, the purified recombinant CDPS were performed amino acid reaction with two amino acids among L-leucine, L-proline and L-phenylalanine. The enzyme activity of Leuconostoc CDPS was confirmed by ninhydrin staining method and HPLC analysis (Fig. 10). When L-leucine and L-proline reacted, achromatic bands
40
protein bands appeared on the stained native gel but achromatic phenomenon was not obvious. On HPLC profile, the content of substrates was essentially unchanged (Fig. 10- C).
Through this experiment, we can conclude that Ln. mesenteroides LBP-K06 cells can express an enzyme that cyclizing L-leucine, L-proline and L-phenylalanine. The gene segment of the ATPase family associated with various cellular activities proteins was supposed as the gene of Leuconostoc CDPS, which is encoded by 2151 bp nucleotides and 717 amino acids. After constructing the recombinant system, recombinant CDPS was overproduced in E. coli and maintained its enzyme activity. Recombinant CDPS were most produced when E. coli cells were cultured for 4 h. Recombinant CDPS could be purified by DEAE-Sepharose chromatography after ammonium sulfate precipitation in Tris-HCl buffer (pH 7.0).
Through ninhydrin staining method and HPLC analysis, the enzyme activity of Leuconostoc CDPS was confirmed. Based on the results above, we also can discovered that Leuconostoc CDPS plays different impacts among L-leucine,
L-proline and L-phenylalanine. By comparison, the catalytic ability between
L-leucine and L-proline is slightly higher than that between L-proline and
L-phenylalanine; while there is almost no catalytic ability between L-leucine and
L-phenylalanine. Leuconostoc CDPS seems to be an enzyme to complete L-proline and L-phenylalanine synthesis, and also seems to have the activity between
L-proline and L-leucine.
C HAPTER V
R EFRENCES
42
Holzapfel WH, Haberer P, Geisen R, Björkroth J, and Schillinger U. (2001) Taxonomy and important features of probiotic microorganisms in food and nutrition. Am J Clin Nutr 73,365S-73S.
Abee T, Krockel L, and Hill C. (1995) Bacteriocins: modes of action and potentials in food preservation and control of food poisoning. Int J Food Microbiol 28,169-85.
Niku-Paavola ML, Laitila A, Mattila-Sandholm, and Haikara A. (1999) New types of antimicrobial compounds produced by Lactobacillus plantarum. J Appl Microbiol 86,29-35.
Gänzle MG, Höltzel A, Walter J, Jung G, and Hammes WP. (2000) Characterization of reutericyclin produced by Lactobacillus reuteri LTH2584.
Appl Environ Microbiol 66,4325-33.
Ström K, Sjögren J, Broberg A, and Schnürer J. (2002) Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid.
Appl Environ Microbiol 68,4322-7.
Wang H, Yan Y, Wang J, Zhang H, and Qi W. (2012) Production and characterization of antifungal compounds produced by Lactobacillus plantarum IMAU10014. PloS One 7,e29452.
Zhong F, Jiang X-H, Tian M-Q, Bai W, Ma Y-P, and Dang M-Z. (2006) Research advances in bioactive cyclic dipeptides. Journal of Hainan Normal University (Natural Science) 19-4,352-8.
Li H-F, Ye Y-H, and Guo J-H. (2010) Isolation and identification of cyclic dipeptides from Bacillus subtilis 7Ze3. Jiangsu Agricultural Sciences 2010-2,107-9.
Yin P, Hu M-L, and Hu L-C. (2008) Synthesis, structural characterization and anticarcinogenic activity of a new Gly–Gly dipeptide derivative: Methyl
2-(2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)acetate.
Journal of Molecular Structure 882,75-9.
Wang H-Y, Wen C-F, Chiu Y-H, Lee I-N, Kao H-Y, Lee I-C and Ho W-H.
(2013) Leuconostoc Mesenteroides growth in food products: prediction and sensitivity analysis by adaptive-network-based fuzzy inference systems. Plos One 8,e64995.
Kim A-H. (2014) Antifungal substances produced by Lactobacillus plantarum LBP-K10. M.S. Thesis.
Kang H-K, Seo M-Y, Seo E-S, Kim-D, Chung S-Y, Atsuo K, Donal F.D, and John F.R. (2005) Cloning and expression of levansucrase from Leuconostoc mesenteroides B-512 FMC in Escherichia coli. Biochimica et Biophysica Acta 1727,5–15.
Bruce M.C and Alfred G. (1980) Method for the lysis of gram-positive, asporogenous bacteria with lysozyme. Applied and Environmental Microbiology 39(1), 153-8.
Li Y-X and Qing L-K. (2012) Optimization of gram-positive bacillus cracking and screening of high peptidase strains. Food and Fermentation Industries 292,89-93.
Krisna C.D-L and Sandra B.G. (2014) Using ion exchange chromatography to purify a recombinantly expressed protein. Methods in Enzymology 541,95-103.
Tara K.S, Renee C, Priya R.G, and Michael W.C. (2004) Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose
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Krisna C.D-L and Sandra B.G. (2014) Salting out of proteins using ammonium sulfate precipitation. Methods in Enzymology 541,85-94.
GE Healthcare. (2009) Simple purification of other recombinant or native proteins. Recombinant Protein Purification Handbook-Principles and Methods,205-210.
국 문 초 록
본 연구는 한국 전통 발효 식품 김치에서 유산균을 동정하여 그 중 항균 활성이 높은 균주들 중 Leuconostoc mesenteroides LBP-K06을 재료 로 하여 싸이클릭디펩티드 생합성 효소 (cyclic dipeptide synthetase)를 분 리하고자 하였다. 아미노산에 특이적인 발색 반응을 이용한 ninhydrin staining방법으로 Ln. mesenteroides LBP-K06에서 싸이클릭디펩티드 생합성 효소를 확인하고자 하였고 효소활성의 경우 native gel에 싸이클릭디펩티 드가 합성되는 곳에서 achromatic band를 나타낼 것으로 예측하였다. 이 활성으로 gel 상에서 획득한 achromatic 절편으로 SDS-PAGE를 수행하였 고 single band를 확보한 뒤 단백질 동정을 위한 2D LC-MS 분석을 실시 하였다. 2D LC-MS 결과를 통해 Ln. mesenteroides LBP-K06에서 싸이클릭 디펩티드를 합성 시키는 효소는 the ATPase family associated with various cellular activities proteins (gi: 227351295)임을 확인하였다. 확보한 싸이클릭 디펩티드 생합성 효소는 polymerase chain reaction으로 싸이클릭디펩티드 합성 효소의 클로닝을 수행하였고 이를 pET3a vector 시스템을 이용하여
46
에서와 같은 방법으로 분리된 단백질과 외부에서 첨가한 아미노산과의 효소 활성은 ninhydrin staining과 HPLC analysis방법으로 확인하였으며, 싸이클릭디펩티드 생합성효소는 L-leucine과 L-proline 사이와
L-phenylalanine과 L-proline을 합성에 필요한 효소의 기질로 사용하였을 때에 싸이클릭디펩티드 생합성 활성이 보임을 관찰하였다.
Keywords: Leuconostoc mesenteroides LBP-K06, cyclic dipeptide, cyclic dipeptide synthetase, cis-cyclo(L-Leu-L-Pro), cis-cyclo(L-Phe-L-Pro), ninhydrin staining method, HPLC analysis
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理學碩士學位論文
Leuconostoc mesenteroides LBP-K06 에서 cyclic dipeptide 를 합성하는 효소
Cyclic dipeptide synthetase from Leuconostoc mesenteroides LBP-K06
2015 年 2 月
서울大學校 大學院
生命科學部
于 穎
ιℓ
"ε
oκoJtυ
ε
"ηℓΞℓκι
"Io'㎐
Ξ
I」BP■ K06
에서
cyεlic d요pePUde 를 합성하는 효소
指導敎授 姜 思 旭
이 論文올 理學碩士學位論文으로 提出함
2015 年
2月 서올大學校 大學院
生命科學部
(于 穎
于 穎의 理學碩士學位論文올 認准함
2ⓛ15
年
2月
委 員 長
副委員長
Cyclic dipeptide synthetase from Leuconostoc mesenteroides LBP-K06
by YING YU
Advisor:
Professor Sa-Ouk Kang, Ph.D.
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science
February, 2015
School of Biological Sciences Graduate School
Seoul Nat
