[보고서]전통발효식품(소재)의 단백질체 연구 및 기능성·품질증진을 위한 미생물유전체 연구

김정상

전통발효식품(소재)의 단백질체 연구 및 기능성·품질증진을 위한 미생물유전체 연구
Proteomic Analyses of Traditional Fermented Foods and Foodstuffs, and Genomic study for Microorganisms to Promote Functionality and Quality 원문보기

보고서 정보
주관연구기관	경북대학교 KyungPook National University
연구책임자	김정상
참여연구자	김정환 , 박천석 , 임진규
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2010-05
과제시작연도	2010
주관부처	교육과학기술부 Ministry of Education and Science Technology(MEST)
등록번호	TRKO202100005426
과제고유번호	1345101130
사업명	미래기반기술개발
DB 구축일자	2021-07-17
키워드	청국장.된장.메주.바실러스.아스퍼질러스.유전체.단백질체.이소플라본.cheonggukjang.doenjang.meju.Bacillus.Aspergillus.genome.proteome.isoflavones.

초록 ▼

본 세부과제에서는 1단계 연구에서 확립된 유전체 및 단백질체 방법을 이용하여 전통청국장과 메주로부터 우수 균주를 분리동정한 후, 건강기능성과 관련이 깊은 유전자를 클로닝하고 다른 숙주에서의 발현을 시도하였으며, 각 균주들의 콩 발효특성을 연구하여 다음과 같은 주요 결과를 도출하였다.
1. Bacillus 균주에서 polyglutamate(PGA) 생산 4.5 kb pgsBCA 유전자 클로닝 및 유전자 도입에 의한 대장균과 B. subtilis에서 발현
2. 혈전용해효소 유전자 bpr86-1 클로닝 및 B. subtilis에서 생산
3. B. cereus 신속 동정 RAPD-PCR 및 B. subtilis 그룹 종들 구별 RAPD-PCR 방법 개발
4. 전통청국장에서 분리한 Bacillus 균주별 isoflavones대사특성 및 청국장의 건강기능성 규명
5. Bacillus subtilis CH97 와 Bacillus amyloliquefaciens 균주의 secretome 분석완료
6. 전통식품유래 Aspergillus 균주로부터 polyketide 생합성 유전자의 다양성 분석 및 확인
7. 전통식품유래 Aspergillus oryzae 균주로부터 기능성 당으로 그 활용도가 높은 arabinose를 생산하는데 이용할 수 있는 arabinofuranosidase와 기능성 식품소재로 이용이 가능한 ferulate를 생산하는데 사용되는 feruloyl esterase를 cloning하고 효모에서 발현
8. Aspergillus oryzae strains별 beta-glucosidase 활성과 콩 발효과정중 isoflavones 대사 및 항산화 활성에 미치는 영향 평가하여 최적 발효기간 설정
9. Aspergillus oryzae 의 세포질 단백질과 secretome분석하여 부분적인 protein map 완성
10. 단백질분해효소 저항성 콩단백질과 상호작용하는 장점막단백질의 동정을 통한 소화생리학적 새로운 기능에 대한 가능성 제시.

(출처 : 보고서 요약서 3p)

Abstract ▼

This study was conducted to screen the high quality microorganisms suitable for manufacturing traditional fermented soy foods such as cheonggukjang, soy sauce, and soy paste (doenjang). The genomic and proteomic technologies were utilized for reliable identification of the microorganism. Various strains in Bacillus and Aspergillus family which were isolated from traditional cheonggukjang and meju, respectively, were tested for their capability to produce soy products with health benefits including antioxidant activity.
Bacilli, major microorganisms found in traditional cheonggukjang, confer important health-related benefits to fermented soybeans. The production of γ-PGA is one example and fibrinolysisis is another. Genes responsible for the γ-PGA production were cloned from B. amyloliquefaciens CH86-1, a strain isolated from traditional cheonggukjang through this study, and the genetic organization was elucidated. A 4.5 kb fragment encompassing pgsBCA was introduced into B. subtilis and E. coli. Production of protein(s) was observed by SDS-PAGE and the presence of transcript was confirmed by slot blot analysis. More detailed studies are required for transforming a PGA-nonproducer into an efficient producer. Fibrinolytic enzymes were studied more in detail and bpr86-1 gene encoding bacillopeptidase F was cloned from B. amyloiliquefaciens CH86-1. These genes will be useful for the construction of strains for fermented soy products with enhanced health benefits.
A rapid and reliable RAPD-PCR method were also developed for the detection of Bacillus cereus, a common contaminant, from fermented soyfoods. All B. cereus strains produced 0.91 kb and 0.5 kb when S-30 was used as a single primer. A RAPD-PCR protocol was also developed to distinguish B. subtilis strains from closely related B. amyloliquefaciens and B. licheniformis strains. Accurate identification of a Bacillus species is helpful for the quality control of fermented foods.
Meanwhile, five Aspergillus oryzae strains were selected from Korean traditional meju manufactured in Sunchang Yeastopia by ITS-RFLP method. The absence of aflatoxin biosynthesis by five selected Aspergillus oryzae strains was confirmed by using RT-PCR. In addition, 8 β-glucosidase genes (ABG 1, 7, 9, 13, 21, 22, 30, and 31) from one of selected Asp oryzae strain were cloned and expressed in Saccharomyces cerevisiae . The β-glucosidase expressed on the yeast could hydrolyze various isoflavones in soybean. Although five strains of Aspergillus oryzae expressed different levels of the enzymes, they did not have significant effect on the profile of isoflavone derivatives during soybean fermentation. However, isoflavone profile of soybean was greatly altered depending upon soybean variety and fermentation period, with the highest concentration of free isoflavones at 4-5th day after fermentation in high isoflavone variety (Aga 3). The antioxidant activity of fermented soy was proportional to the free isoflavone content.
Two biotechnologically important enzymes, arabinofuranosidase and feruloyl esterase, were cloned and expressed in Pichia pastoris and their properties were characterized. This research also undertook the proteome analysis of Bacillus and Aspergillus strains isolated from the traditionally prepared cheonggukjang and meju, proteome analysis of the intestinal mucosa from the rats fed with cheonggukjang, and observed the interaction of the pepsin-resistant soy proteins with the intestinal mucosa membrane proteins as well. Proteomes of Bacillus subtilis and B. amyloliquefaciens were separated on 2-D gels and identified by peptide mass fingerprinting. Due to the variations in genomic sequences, the identified proteins were overlapped less than 10%. The phenomenon is also true between same species. The proteome profiles obtained from the bacillus strains could be used to classify the microorganisms isolated throughout this project. In case of Aspergillus, 2-D proteome profiles agree each other very well within the same species (over 90%), such as between Aspergillus oryzae strains. However, proteome profiles between A. oryzae matched only by 50% and A. awamori only overlapped by less than 20%. This also suggests that the Aspergillus strains identified by genomic sequence data should be complemented by proteomic data for accurate identification and classification.
Proteomic analysis of the mucosa of the small intestine from the rats fed with cheonggukjang and soy showed differential expression of cytoskeletal proteins, particularly, tropomyosin, which cause the increase of the length of microvilli of the small intestine. This phenomenon could be caused by the interaction of the pepsin-resistant soy proteins with intestinal surface. The pepsin-resistant soy proteins were found to interact with a 32 kDa protein on the mucosa membrane. The interaction of soy proteins with small intestinal mucosa need to be further investigated.
In conclusion, we made major breakthrough in understanding microorganisms involved in manufacturing Korean traditional fermented soy foods. The information generated from this study should be exploited for improving manufacturing process of traditional fermented soy products.

(출처 : SUMMARY 9p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
요 약 문 ... 4
S U M M A R Y ... 9
C O N T E N T S ... 11
목차 ... 12
제 1 장 연구개발과제의 개요 ... 14
1절 연구개발의 목적 ... 14
2절 연구개발의 필요성 ... 14
3절 연구개발의 범위 ... 17
제 2 장 국내외 기술개발 현황 ... 19
1절 국내외 연구현황 ... 19
1. Bacillus와 Aspergillus의 유전체 연구 ... 19
2. Bacillus와 Aspergillus의 단백질체 연구 ... 21
3. Bacillus와 Aspergillus에 의한 대사체 연구 ... 22
2절 본 연구팀의 연구결과가 국내·외 기술개발현황에서 차지하는 위치 ... 22
제 3 장 연구개발수행 내용 및 결과 ... 23
1절 연구방법 ... 23
1. Bacillus amyloliquefaciens CH86-1에서 기능성 소재 생산관련 유전자 확보 ... 23
2. PGA 생성 유전자 클로닝 ... 24
3. 혈전용해효소 bpr86-1 클로닝 및 발현 ... 27
4. B. subtilis 그룹 균들간 구별을 위한 RAPD-PCR 방법 개발 ... 29
5. B. cereus 균주 신속한 검출을 위한 RAPD-PCR 방법 개발 ... 30
6. Bacillus 균주별 청국장 제조과정중 isoflavone profile 분석 및 항산화활성 평가 ... 31
7. 전통식품유래 곰팡이 이차대사산물 관련 유전체 연구 ... 33
8. 전통식품 유래 곰팡이의 isoflavonoid 대사 관련 유전체 연구 ... 34
9. 전통식품유래 곰팡이의 기능성 식품소재 생산관련 유전체 연구 ... 36
10. 전통 청국장에서 분리한 Bacillus 균주의 Proteome 분석 ... 40
11. 청국장 급여 쥐의 장점막 단백질체 변화 ... 40
12. 청국장 급여 쥐의 장점막의 구조 변화 분석 ... 40
13. 장점막 단백질과 콩단백질과의 상호 작용 ... 41
2절 연구결과 ... 43
1. PGA 생성 유전자 클로닝 및 발현 ... 43
2. 혈전용해효소 bpr86-1 클로닝 및 발현 ... 52
3. 중요 Bacillus 신속 동정을 위한 RAPD-PCR 방법 개발 ... 57
4. B. subtilis 그룹 균들간 구별을 위한 RAPD-PCR 방법 개발 ... 58
4. B. cereus 균주 신속한 검출을 위한 RAPD-PCR 방법 개발 ... 61
5. 표준 청국장 제조과정중 isoflavones 대사체 및 항산화 활성의 변화 ... 64
6. 전통식품유래 곰팡이 이차대사산물 관련 유전체 연구 ... 74
7. 전통식품 유래 곰팡이의 isoflavonoid 대사 관련 유전체 연구 ... 84
8. 전통식품유래 곰팡이의 기능성 식품소재 생산관련 유전체 연구 ... 109
9. 전통 청국장에서 분리한 Bacillus 균주의 Proteome 분석 ... 117
10. 청국장 급여 쥐의 장점막 단백질체 변화 ... 125
11. 청국장 급여 쥐의 장점막의 구조 변화 분석 ... 133
12. 장점막 단백질과 콩단백질과의 상호 작용 ... 135
13. Aspergillus 균주의 proteome 분석 및 정확한 동정과 관리를 위한 분석결과의 활용 ... 143
제 4 장 목표달성도 및 관련분야에의 기여도 ... 152
제 5 장 연구개발결과의 활용계획 ... 154
제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 155
제 7 장 참고문헌 ... 156
끝페이지 ... 160

표/그림 (123)

표 PGA 구조 및 전통청국장 (Moon, HS 등. Natural and edible biopolymer poly-Υ -glutamic acid: synthesis, production, and applications. The Chemical Record, 5: 352-366. 2005)
표 Production of γ-PGA by B. amyloliquefaciens CH86-1. Cheongukjang fermented at 24 h (top left), 36 h (top right), 48 h (bottom left) and 60 h (bottom right)
표 Genes responsible for γ-PGA production in Bacilli. Structural features of the PgsB, PgsC, and PgsA proteins are also shown
표 Vectors used for the cloning of pgsBCA
표 The strategy showing the construction of a pHY300PLK derivative containing both aprE3-5 and bpr86-1
표 Conditions for RAPD-PCR for Bacillus species
표 Microorganisms used for RAPD-PCR
표 HPLC conditions for analysis of isoflavones
표 Oligonucleotide sequence of primer for AFase used in this study
표 Oligonucleotide sequence of primer for FEase used in this study
표 Map of expression vector, pPICZɑ, used in this study
표 Agarose gel electrophoresis of PCR product of pgsBCA gene
표 Restriction digests for pgsBCA gene amplified by PCR. 1. XbaΙ and SalΙ digested pHY300PLK, 2. pgsBCA, 3. plasmid DNA from transfomant, 4. SalΙ digested plasmid DNA from a transformant, 5. XbaΙ and SalΙ digested plasmid DNA from a transformant
표 Restriction enzyme digests of pGAk. 1. pGAk plasmid; 2. pGAk SalI cut; 3. pGAk XbaI cut; 4. pGAk SalI and XbaI cut; 5. pET-26b(+) (5.36 kb); 6. amplified pgsBCA (3 kb)
표 Organization of cloned pgsBCA genes in pGAk23
표 Restriction digestion of pGAk23. 1. pGAk23 plasmid; 2. pGAk23, SalI cut; 3. pGAk23, XbaI cut; 4. pGAk23, SalI and XbaI cut; 5. pHY300PLK (4.8 kb); 6. pgsBCA gene (4.5kb)
표 Transformation of B. subtilis WB600 by pGAk23. 1. WB600 [pGAk23] plasmid; 2, WB600 [pGAk23] SalI cut; 3, WB600 [pGAk23] XbaI cut; 4, WB600 [pGAk23] SalI and XbaI cut; 5, pGAk23 SalI cut; 6, pGAk23 XbaI cut; 7, pGAk23 SalI and XbaI cut
표 SDS-PAGE of protein samples from B. subtilis WB600 TF
표 Slot blot of RNA samples from E. coli BL21 TFs. A. RNA sample untreated with DNase (RNase-Free); B. RNA sample treated with DNase (RNase-Free). 1. BL21 [pET-26b] induced by 0.1 mM IPTG; 2. BL21 [pGAk] induced by 0 mM IPTG; 3. BL21 [pGAk] induced by 0.1 mM IPTG; 4. BL21 [pGAk] induced by 1 mM IPTG
표 The organization of pgsBCA gens and the surrounding genes. The 5.3 kb fragment was tried to be cloned into vectors after PCR amplification. The locations of forward and reverse primer binding sites are marked as F and R. The size of each gene and the distance between genes are also shown
표 Nucleotide sequence of 5.3 kb region encompassing pgsBCA
표 Restriction cut of vectors and pgsBCA genes. (left) 1. pHY300PLK, 2. pET-26b(+), 3, pBlueScript, 4. pgs genes amplified (5.3 kb) (right) 1. pHY300PLK, 2. gel extracted pgs gens after amplification (5.3 kb), 3. pHY300PLK, SalI and BamHI cut, 4. pgs genes, SalI and BamHI cut M, size maker (Fermentas, Burlington, Canada)
표 SDS-PAGE for protein samples from E. coli BL21(DE3)[pGAk100]. Lane 1-4, soluble proteins and lane 5-8, insoluble proteins. BL21 [pET-26b(+)] at 0.1 mM IPTG(1 and 5); BL21 [pGAk100] at 0 mM IPTG (2 and 6); BL21[pGAk100] at 0.1 mM IPTG (3 and 7); BL21[pGAk100] at 1 mM IPTG (4 and 8). M, size markers
표 Slot blot results. 1. B. amyloliquefacience CH86-1; 2. E. coli DH5a[pHY300PLK]; 3. E. coli DH5a[pGAk23]; 4, B. subtilis WB600[pHY300PLK]; 5. B. subtilis WB600[pGAk23].
표 Amino acid sequence alighment of Bpr86-1 with other homologous proteins. FJ432003 (bpr86-1), CP000560 (B. amyloliquefacien FZB42), FN597644 (B. amyloliquefaciens DSM7), AL009126 (B. subtilis 168), D44498 (B. subtilis (natto) No. 16)
표 Restriction cut of pHYbpr86-1 from B. subtilis TFs. M, size marker; 1, pHYbpr86-1 plasmid; 2, pHYbpr86-1 BamHⅠ cut (9.67 kb); 3, pHYbpr86-1 BamHⅠand XbaⅠ cut(4.8 kb, 4.87 kb); 4, pHY300PLK BamⅠand XbaⅠ cut (4.87 kb); 5, bpr amplified (4.8 kb)
표 Growth of Bacillus strains harboring pHYbpr86-1. ▲, B. amyloliquefaciens CH86-1; ○, B. subtilis WB600[pHY300PLK]; ●, B. subtilis WB600[pHYbpr86-1]
표 Fibrin plate assay showing fibrinolytic activity of B. subtilis WB600[pHYbpr86-1] during growth at 37 ℃. -○-, B. subtilis WB600[pHY300PLK]; -● -, B. subtilis WB600[pHYbpr86-1]. Culture supernatant at each time point was obtained, filtered, and then spotted on a fibrin plate. Plasmin at different concentration was used as a positive control
표 SDS-PAGE (left) and fibrin zymogram (right) of culture supernatant. M, protein size markers(DokDo-MARKTM, Elpisbio, Taejeon, Korea); B. amyloliquefaciens CH86-1 at 48 h (lane 1), at 96 h (2), at 120 h (3); B. subtilis WB600[pHYbpr86-1] at 48 h (4), at 96 h (5), at 120 h (6); 7, B. subtilis WB600[pHY300PLK] at 48 h (7), at 96 h (8), at 120 h (9). 15 μg was applied and 10% acrylamide gel was used. Bands marked in the left are absent in control
표 Restriction cut of pHYbpr86-1 and pHY3-5. 1, pHY3-5, 2, pHY3-5 EcoRI cut (4.8 kb, 1.8 kb), 3, pHYbpr86-1, 4, pHYbpr86-1 BamHI cut (9.7 kb), 5, pHYbpr86-1 BamH Ⅰ, XbaⅠcut (4.8 kb, 4.9 kb), M, size maker (Fermentas, Burlington, Canada). 1% agarose gel was used
표 Confirmation of pGHk by restriction cut. 1, pHY3-5 + bpr86-1 (pGHk) plasmid; 2, pGHk BamHI cut; 3, pGHk, BamHI and XbaI cut; 4, pHY3-5 BamHI and XbaI cut (6.6 kb); 5, bpr86-1 BamHI and XbaI cut (4.9 kb); M, size maker (Fermentas, Burlington, Canada), 1% agarose gel was used
표 Restriction digestion of pGHk prepared from B. subtilis TF. M, 1 kb size maker (Fermentas); 1, pHY3-5 plasmid DNA from DH5α; 2, pGHk plasmid DNA from B. subtilis WB600; 3, #1 digested with BamHI (6.6 kb); 4, bpr86-1 gene (insert, 4.99 kb); 5, pGHk plasmid DNA (from DH5α) digested with XbaI and BamH (6.6 kb, 4.99 kb); 6, pGHk plasmid DNA (from DH5α) digested with BamHI (11.6 kb); 7, #2 digested with BamHI (11.6 kb); 8, #2 digested with XbaI and BamH (6.6 kb, 4.99 kb)
표 RAPD-PCR patterns of Bacillus strains isolated from Chunggukjang and reference strains. Lane: 1, B. subtilis168; 2, CH3-5; 3, CH3-25; 4, CH97; 5, B. amyloliquefaciens ATCC 23845; 6, CH51; 7, CH86-1; 8, B. licheniformis ATCC 27811; 9, CH3-17; M, 1 kb size ladder (MBI, Glen Burnie, USA). 2% agarose gel was used for the electrophoresis
표 RAPD-PCR patterns of Bacillus reference strains. Lane: 1, B. subtilis 168; 2, B. amyloliquefaciensATCC 23845; 3, B. amyloliquefaciens ATCC 23350; 4, B. licheniformis ATCC 4527; 5, B. licheniformis ATCC 21416; 6, B. licheniformis ATCC 27811; 7, B. licheniformis ATCC39326; 8, B. licheniformisATCC 14580; 9, B. thuringiensis ATCC 33679; 10, B. circulans ATCC 4513; M, 1kb size ladder (MBI, Glen Burnie, USA).2% agarose gel was used
표 RAPD-PCR profiles of seven B. subtilis reference strains. All seven B. subtilis reference strains showed the same pattern and 500 bp fragments were circled. Lane: 1, B. subtilis 168; 2, B. subtilis ATCC 33608; 3, B. subtilisATCC 6051A; 4, B. subtilis ATCC 9372; 5, B. subtilis ATCC 39088; 6, B. subtilisATCC 33234; 7, B. subtilis ATCC 21777; M, 1kb size ladder (MBI, Glen Burnie, USA). 2% agarose gel was used
표 RAPD-PCR of 3 strains isolated from cheonggukjang and standard strains. 1, B. subtilis 168; 2, B. cereus ATCC9634; 3, B. cereus ATCC21768; 4, B. cereus ATCC14579; 5, B. cereus ATCC31429; 6, B. cereus ATCC27348; 7, B. cereus ATCC21366; 8, B. cereus ATCC21772; 9, B. cereus ATCC12480; 10, B. subtilis 168; 11, 부산대 1; 12. 부산대 2; 13, 부산대 3; M, 1kb size ladder (MBI). 2% agarose gel이 사용되었다
표 RAPD-PCR results of standard Bacillus strains. 1, B. subtilis 168; 2, B. cereus ATCC 9634; 3, B. cereus ATCC21768; 4, B. cereus ATCC14579; 5, B. cereus ATCC31429; 6, B. cereus ATCC27348; 7, B. cereus ATCC21366; 8, B. cereus ATCC21772; 9, B. cereus ATCC12480; 10, B. subtilis 168; 11, B. licheniformis ATCC4527; 12. B. licheniformis ATCC21416; 13, B. licheniformis ATCC27811; 14, B. amyloliquefaciens ATCC23350; 15, B. amyloliquefaciens ATCC23845; M, 1kb size ladder (MBI). 2% agarose gel was used
표 Nucleotide sequences of PCR fragments. A. sequence of 0.5 kb fragment conserved in bacilli (EU669668). B. sequence of 0.91 kb fragment conserved among B. cereus reference strains (GU230872)
표 RAPD-PCR patterns of cheonggukjang spiked with B. cereus. A. 1, control (non-spiked cheonggukjang) 2-8, cheonggukjang samples spiked with B. cereus cells (cells/g cheongukjang): 2, 100 ; 3, 101 4, 102 5, 103 6, 104 ;7, 105 8, 106 , M, 1 kb size ladder (MBI Fermentas). B. Enlarged picture showing the 0.91 kb fragment
표 Isoflavone concentrations of cheonggukjang prepared with different Bacillus species (Continued)
표 Isoflavone concentrations of cheonggukjang prepared with different Bacillus species (Continued)
표 Isoflavone concentrations of cheonggukjang prepared with different Bacillus species (Continued)
표 Isoflavone concentrations of cheonggukjang prepared with different Bacillus species
표 ABTS cation decolorization activity of ethanolic extracts of cheonggukjang prepared by different Bacillus species. A, Daewon; B, Aga 3; C, A4; D, Chungja
표 FRAP levels of ethanolic extracts of cheonggukjang prepared by different Bacillus species
표 Total phenolic contents of cheonggukjang prepared by different Bacillus species
표 Multiplex PCR. M, 100 bp ladder; lane 1, A. sydowii ; lane 2, Fusarium asitaticum; lane 3, A. oryzae
표 Confirmation of mRNA related aflatoxin biosynthetic pathway by RT-PCR. Lane 1, positive control β-tubulin; lane 2, aflR; lane 3, avnA; lane 4, omtA; lane 5, vbs; lane 6, verB
표 The gene cluster of aflatoxin biosynthetic pathway. Red points represent not detected mRNA related gene cluster in Group 1 strains
표 HR KS domain amino acid alignment of fungal strains. A, Asp fumigatus; B, Group 3 (Asp. fumigatus); C, Neosartorya fischeri; D, Asp. ochraceus; E, Group 2 (Asp. oryzae); F, Penicillium citrinum; G, Asp. clavatus NRRL 1; H, Group 4 (Arthrinium sp.)
표 Neighbor-joining phylogenetic tree of HR KS domain PKS (Group 2, Asp. oryzae; Group 3, Asp. fumigatus; Group 4, Arthrinium sp.) with Asp. ochraceus polyketide synthase 1, Asp. fumigatus LovB-like polyketide synthase, Asp. clavatus NRRL1 equisetin synthetase, Neosartorya fischeri Polyketide synthase, and Penicillium citrinum Polyketide synthase
표 Gel electrophoresis of PCR products with degenerated primers for NR KS domain. M : 100 bp ladder; Lane 1, Fusarium. sp. (Group 1); Lane 2 : Asp. oryzae (Group 2); Lane 3, Asp. fumigatus (Group 3); Lane 4, Arthrium. sp. (Group 4); Lane 5, Asp. sydowii (Group 5)
표 Identification of NR KS domain sequence and homology with other polyketide synthase sequence. A) Group 2, Asp. oryzae; B) Group 3, Asp. fumigatus
표 Identification of NR KS domain sequence and homology with other polyketide synthase sequence. C) Group 4, Arthrium sp.; D) Group 5, Asp. sydowii
표 Neighbor-joining phylogenetic tree of NR KS domain PKS inferred by Neighbor-joining method. bootstrapped consensus tree in MEGA 4.0, number of replicates = 1000). Group 2, Asp. oryzae; Group 3, Asp. fumigatus; Group 4, Arthrinium sp.; Group 5, Asp. sydowii
표 Gel electrophoresis of PCR products with degenerated primers for PR KS domain. M : 100 bp ladder; Lane 1, Fusarium. sp. (Group 1); Lane 2, Asp. oryzae (Group 2); Lane 3, Asp. fumigatus (Group 3); Lane 4, Arthrium. sp (Group 4); Lane 5, Asp. sydowii (Group 5)
표 Identification of PR KS domain sequence and homology with other polyketide synthase sequence. A) Group 2, Asp. oryzae; B) Group 3, Asp. fumigatus C) Group 4 , Arthrium sp.; D) Group 5, Asp. sydowii
표 Neighbor-joining phylogenetic tree of PR KS domain PKS inferred by Neighbor-joining method (bootstrapped consensus tree in MEGA 4.0, number of replicates = 1000). Group 2, Asp. oryzae; Group 3, Asp. fumigatus; Group 4, Arthrinium sp.; Group 5, Asp. sydowii
표 Phylogenetic tree of three KS domains inferred by Neighbor-joining method (bootstrapped consensus tree in MEGA 4.0, number of replicates = 1000) (Group 2, Asp. oryzae Group 3, Asp. fumigatus Group 4, Arthrinium sp.; Group 5, Asp sydowii )
표 Phylogenetic tree of putative β-glucosidases gene, main targeted genes have a apostrophe point
표 Main targeted β-glucosidase genes in this study
표 Gel electrophoresis of Aspergillus oryzae FMB-2a genomic DNA
표 Gel electrophoresis of PCR products using primers for the exon of putative genes, β-glucosidase. Lane 1, ABG 7, 2895 bp; lane 2, ABG 9, 2601 bp; lane 3, ABG 13, 2571 bp; lane 4, ABG 30, 2295 bp
표 Plasmid map of pCTCON for yeast surface display in this study
표 FACS analysis of yeast surface displayed enzymes
표 Effect of temperature on displayed ABG 9 activity in a 50 mM sodium acetate pH 5.0 and effect of pH on displayed ABG 9 activity at 50 ℃
표 Effect of temperature on displayed ABG 13 activity in a 50 mM sodium acetate pH 5.0 and effect of pH on displayed ABG 13 activity at 50 ℃
표 Time course of displayed ABG30 acitivity by induction time
표 Effect of temperature on displayed ABG30 activity in a 50 mM sodium acetate pH 6.0 and Thermal stability of displayed ABG30 at 45, 50, 55 and 60 ℃
표 Effect of pH on displayed ABG30 activity at 50 ℃
표 Metal ion and reagent effects for ABG 30
표 Substrate specificity of displayed ABG30
표 Specific activity of various displayed β-glucosidase from Aspergillus oryzae
표 HPLC analysis of hydrolysis product of daidzin after 8 hr at 30 ℃ with displayed enzyme. (A) daidzin control; (B) EBY100; (C) ABG 9; (D) ABG 30
표 Change of isoflavone composition of soybean (Daewon variety) during fermentation with Asp oryzae NL 5
표 Change of isoflavone composition of soybean (Daewon variety) during fermentation with Asp oryzae NL 15
표 Change of isoflavone composition of soybean (Daewon variety) during fermentation with Asp oryzae NL 21
표 Change of isoflavone composition of soybean (Daewon variety) during fermentation with Asp oryzae NL 27
표 Change of isoflavone composition of soybean (Daewon variety) during fermentation with Asp oryzae NL 28
표 Change of isoflavone composition of soybean (Aga 3 variety) during fermentation with Asp oryzae NL 5
표 Change of isoflavone composition of soybean (Aga 3 variety) during fermentation with Asp oryzae NL 15
표 Change of isoflavone composition of soybean (Aga 3 variety) during fermentation with Asp oryzae NL 21
표 Change of isoflavone composition of soybean (Aga 3 variety) during fermentation with Asp oryzae NL 27
표 Change of isoflavone composition of soybean (Aga 3 variety) during fermentation with Asp oryzae NL 28
표 Ferric reducing activity (FRAP) of Aga 3 soybean fermented with Asp oryzae NL5,NL15,NL21,NL27,NL28 for various periods
표 Ferric reducing activity (FRAP) of Daewon soybean fermented with Asp oryzae NL5, NL15, NL21, NL27, NL28 for various periods
표 ABTS radical scavenging activity of Aga 3 soybean fermented with Asp oryzae NL5, NL15, NL21, NL27, NL28 for various periods
표 ABTS radical scavenging activity of Daewon soybean fermented with Asp oryzae NL5, NL15, NL21, NL27, NL28 for various periods
표 Total phenolic contents of Aga 3 and Daewon varieties of soybean fermented with various strains of Asp oryzae for 4 days
표 Detection of intracellular reactive oxygen species by DCFDA (A) and DHE (B) in hepa1c1c7 cells. Hepa1c1c7 cells were exposed to hydrogen peroxide in the absence and presence of fermented soy extract from Aga 3 or Daewon variety of soybean, followed by observation under fluorescent microscope after the addition of DCFDA or DHE, fluorescent probes
표 Score plot (A) and PCA loading (B) of isoflavone profile in cooked soybean according to fungal fermentation period
표 Phylogenetic tree of putative arabinofuranosidase genes, AO(RIB40)AF, AFase from Aspergillus oryzae RIB40; AFase from Neosartorya fischeri NRRL 181-5; TSAF, AFase from Talaromyces stipitatus ATCC 10500; AO(NL5)AF AFase from Aspergillus oryzae FMB-2a; ACAF, AFase from Aspergillus clavatus NRRL 1-3; PPAF. AFase from Penicillium purpurogenum; AUAF, AFase from Aspergillus fumigatus Af293; PCAF, AFase from Penicillium chrysogenum-6; AFAF, AFase from Aspergillus flavus NRRL3357
표 Phylogenetic tree of putative feruloyl esterase genes, AoFaeC, Aspergillus oryzae feruloyl esterase C (XP_001819091); ANFaeB, Aspergillus nidulans feruloyl esterase (XP_659376); AoFaeB, Aspergillus oryzae feruloyl esterase B (XP_001818628); ANIFaeB, Aspergillus niger feruloyl esterase(CAC83933); AoFaeNL5, Aspergillus oryzae FMB-2a feruloyl esterase; AoprbA, Aspergillus oryzae feruloyl esterase B (XP_001821143)
표 SDS-PAGE analysis. The proteins were separated by 10%SDS-PAGE gel and stained with coomassie blue. M; protein marker, 1; crude enzyme, 2; crude through, 3; eluted enzyme
표 TLC analysis of FEase reaction products. Lane 1 and 2, FEase and control reacted with methyl ferulate; Lane 3, methyl ferulate control; Lane 4 and 5, FEase and control reacted with methyl p-coumarate; Lane 6, methyl-p-coumarate control; Lane 7, ferulic acid control
표 HPLC analysis of FEase reaction products. Lane 1, Ferulic acid control; Lane 2, Methyl ferulate control; Lane 3, methyl-p-coumarate control; Lane 4, reaction products with methyl ferulate as substrate, a, ferulic acid; b, p-coumaric acid; Lane 5, reaction products with methyl-p-coumarate as substrate
표 Expression vector, pPICZa-afase used for expression in P ichia pastoris
표 Over expression of arabinofuranosidase in P ichia pastoris GS115, X33. Lane M, protein size marker; Lane 1, Negative control crude extract ; Lane 2, Crude extract ; Lane 3, Negative control crude extract Lane 4, Crude extract
표 Vector Map of express vector, pET-afase and pRARE vector in rosetta gami
표 Overexpression of arabinofuranosidase in E. coli BL21 rosetta gami. Lane M, protein size marker; Lane 1, Negative control crude extract of E. coli BL21; Lane 2, Crude extract of E. coli BL21 haboring pET-af
표 Effect of temperature on AFase activity in a 50 mM sodium citrate pH 6.0 and effect of pH on activity at 45 ℃
표 Hydroysis pattern of arabinofurnosidase on arabinan as substrate. Lane NAf, arabinan reacted with New arabinofuranosidase; Lane Ar, arabinan; Lane CAf, arabinan reacted with control arabinofuranosidase; Lane A, arabinose
표 HPEAC analysis of final hydrolysate produced from sugar beet arabinan with new arabinofuranosidase
표 2-D gel secretome of Bacillus subtilis species. The spots identified by peptide mass fingerprinting are indicated by arrows and numbers
표 2-D gel profiles of the secretomes of Bacillus amyloliquefaciens strains. The proteins idnetified by PMF are indicated by arrows and numbers
표 1-D image of four strains of Bacillus sp (A) and overlapping of extracellular proteomes of B. subtilis and B. amyloliquefaciens
표 2-D gel containing intestinal proteins from a pool of designated group of small intestinal mucosa samples stained with coomassie blue. All IEF was perfomed on an 17-cm length of IPG strip having a pH range of 3-10 (linear pH gradient). The second separation was done using a 12% SDS-PAGE gel; A. control, B. cheongkukjang, C. control, D. soy E. genistein, F. control
표 Identification of intestinal proteins specifically regulated by soy or cheonggukjang-containing diet in mouse
표 Identities of differentially expressed proteins between groups by 2-D based MALDI-TOF-MS analysis
표 Distribution of intestinal proteomes
표 Western blot of tropomyosin in intestinal surface with and without soy treatment. 동일양의 단백질을 SDS-PAGE에 분리한 후 (A), PVDF-membrane으로 옮기고 antibody로 probing하였다 (B). 단백질량을 보정하기 위해 anti-GAPDH antibody를 사용하였다
표 immunohistochemistry of tropomyosin in intestine of rat fed soy or cheonggukjang. 콩 (B)과 청국장 (C) 급여 쥐의 장점막의 microvili에 control (A) 쥐의 것보다 tropomyosin의 발현이 현저하게 증가하였다. 화살표는 tropomyosin의 발현이 특이적으로 많은 부분. 조직은 hematoxylin으로 counterstaining하였다
표 Measurement of microvilii in duodenum from rat fed samples. Vili의 길이와 장의 직경을 측정하여 control (A), 콩 (B), 청국장 (C) 급여 쥐의 소장에서의 villi 발달을 비교 하였다
표 Ligand-binding study scheme using FITC-labeled protein
표 Ligand binding on FITC-labed protein of IEC cells. 콩이나 아몬드 단백질들은 BSA 와 비교할 때 현저하게 높은 결합을 보였으며 digested 또는 undigested 단백질들이 모두 결합됨을 알 수 있다 (A). 콩과 아몬드 단백질이 소장점막 단백질 중 32 kDa 단백질에 특이적으로 결합하였다 (B). Soy protein이 결합된 affinity column을 만들어서 장점막단백질과의 결합을 이루고 콩단백질과 결합하는 장점막단백질을 분리하여 1-D SDS-PAGE로 단백질을 분리하였다(C)
표 Identification of intestinal proteins bound by soy protein as analyzed by LC-MSMS
표 Rat intestinal proteins bound by potato, egg, wheat, rice and milk. 감자 (P), 계란 (E), 밀 (W), 우유 (M), 쌀 (R), 및 BSA (B)로 couple된 Affinity column에 결합한 장점막 단백질. A와 B panel은 두 차례에 걸쳐 이루어진 실험에서 결합하는 단백질의 양상이 비슷함을 나타내고 있으며 이는 결합이 specific 한 것이었음을 의미한다
표 Cytosolic proteins of (A) A.wamori , (B) A.oryzae NL28 and (C) A. oryzae KCCM60166 on 2-DE gel with pH 4-7
표 Image analysis of cytosolic proteome of A.wamori, A.orizae NL28 and A.orizae KCCM60166 using PDQuest
표 Overlapping proteins between the identified proteins of A. oryzae strains showed significantly higher numbers than those between A.wamori and A.orizae NL28, or A. awamori and A.orizae KCCM60166
표 Funtional classification of cytosolic protiens
표 Secretome of (A) A.wamori , (B) NL28 and (C) KCCM60166 separated on 2-DE with pH 4-7
표 Major secreted proteins of A.wamori, A.orizae NL28 and A.orizae KCCM60166 identified by PMF using Maldi-TOF

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전통발효식품(소재)의 단백질체 연구 및 기능성·품질증진을 위한 미생물유전체 연구
Proteomic Analyses of Traditional Fermented Foods and Foodstuffs, and Genomic study for Microorganisms to Promote Functionality and Quality 원문보기