30
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
44
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 National University
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.
ii
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
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
iv
CHAPTER VI REFERNCES ... 41 ABSTRACT IN KOREAN ... 45
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
vi
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
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
1
C HAPTER I
I NTRODUCTION
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
3
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
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
LBP-K01 ++ +++ Lb. plantarum
LBP-K03 ++ ++ Ln. citreum
LBP-K04 ++ - Ln. citreum
LBP-K05 ++ ++ Ln. holzapfelii