[보고서]미생물 유래 바이오폴리머의 단위 생산공정 개발 및 기능 연구

윤병대

미생물 유래 바이오폴리머의 단위 생산공정 개발 및 기능 연구
Characterization of microbial biopolymers and development of unit process for their mass production 원문보기

보고서 정보
주관연구기관	한국생명공학연구원 생물산업공정연구센터
연구책임자	윤병대
참여연구자	김민수 , 박찬선 , 안극현 , 김차영
보고서유형	1단계보고서
발행국가	대한민국
언어	한국어
발행년월	2011-12
주관부처	미래창조과학부 KA
과제관리전문기관	한국생명공학연구원 Korea Research Institute of Bioscience and Biotechnology
등록번호	TRKO201300020766
DB 구축일자	2013-09-21
키워드	바이오폴리머,면역활성,대량생산공정Biopolymer,immuno-stimulation,Mass production

초록 ▼

Ⅲ. 연구개발의 내용 및 범위
1. 생물 유래 유용 바이오폴리머의 발굴 및 특성 분석
- 버섯류 및 미생물 유래 바이오폴리머의 발굴
- 발굴된 폴리머의 생화학적 특성 분석
- 버섯류 유래 바이오폴리머의 특성 및 생산 공정 개발
2. 바이오폴리머의 구조 분석 및 저분자화 기술
- 생물 유래 바이오폴리머의 평균점도 분자량 규명
- FT-IR, NMR 등을 이용한 구조 분석
- 물리 화학적 방법에 의한 베타글루칸의 저분자화 기술 확보
3. 순수 베타글루칸을 생산하는 Aureobasidium pullulans 균주 개발
- pul 합성 유전자 파쇄에 의한 순수 베타글루칸 생산 균주 개발
- 야생형 및 변이주에 의한 베타글루칸 생산 kinetic paramer 규명
4. 베타글루칸의 수지상 세포 활성 및 신호전달
- Mutz-3와 dectin-1 발현으로부터 muDC 분화
- muDC cell에서 베타글루칸에 의한 TNF-α 활성 증진효과 확인
5. Aureobasidium pullulans 균주에 의한 베타글루칸 생산 공정 개발
- 반응표면분석법 및 6-sigma 기법을 이용한 베타글루칸 대량생산 기술 개발
- 500 L 및 5 ton 규모의 scale-up 공정 확립

Abstract ▼

Ⅲ. Contents and ranges of R&D
1. Screening and characterization analysis of biopolymers
- Screening, purufying the biopolymers from microorganisms, mushroom, and oat.
- Characterization of their physical and chemical properties.
- Development of production process and characterization from biopolymer origined mushroom.
2. Analysis of structure and development of low molecular techniques in biopolymer
- Investigation of viscosity-average molecular weight from biopolymer origined microorganisms.
- Analysis of structure using FT-IR, NMR, and et al.
- Development of low molecular techniques by physical and chemical methods.
3. Development of A. pullulans for pure β-glucan production
- Development of A. pullulans for pure β-glucan production by disruption of pul synthetase.
- Analysis of kinetic parameter for β-glucan production in wild and mutant type
4. Activity at dendritic cell of β-glucan and signal transduction
- Differentiation of muDC from expression of Mutz-3 and dectin-1
- Identification of improving effect about TNF-α activity of by β-glucan in muDC cell
5. Development of β-glucan mass production process by A. pullulans
- Development of β-glucan mass production process using RSM and 6-sigma
- Establishment of scale-up process at 500 L and 5 ton scale

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 초록 ... 4
요 약 문 ... 6
SUMMARY ... 9
CONTENTS ... 12
목 차 ... 13
제 1 장 연구개발과제의 개요 ... 14
제 1 절 연구의 필요성 ... 14
제 2 장 국내외 기술개발 현황 ... 21
제 1절 국내의 기술개발 현황 ... 21
제 2절 국외의 기술개발 현황 ... 23
제 3 장 연구개발수행 내용 및 결과 ... 25
제 1절 베타 글루칸 생산 미생물의 확보 및 물성 연구 ... 25
1. 연구개발수행 내용 ... 25
2. 연구개발 수행결과 ... 28
제 2 절 기능성 다당류 생산균주의 다양성 확보 ... 33
1. 연구개발수행 내용 ... 33
2. 연구개발 수행결과 ... 36
2. 꽃송이버섯 실용화 연구 ... 48
3. 차가버섯 다당류 산업화 연구 ... 51
제 3 절 베타 1-3 글루칸의 수지상 세포 활성과 신호전달에 미치는 영향에 관한 연구 ... 69
1. 연구개발 수행 내용 ... 69
2. 연구개발수행 결과 ... 76
제 4 절 A. pullulans IMS-822 KCTC11179BP의 pullulan 합성 유전자 제거를 통한 순수 베타글루칸의 생산 ... 95
1. 연구개발수행 내용 ... 95
2. 연구개발수행 결과 ... 102
제 5 절. A. pullulans wild과 mutant type의 β-glucan 생산성 확인 ... 112
1. 연구개발수행 내용 ... 112
2. 연구개발수행 결과 ... 115
제 5절. A. pullulans 균주의 배양 조건 최적화(RSM) ... 117
1. 연구개발수행 내용 ... 117
2. 연구개발수행 결과 ... 122
제 6절. Six sigma 기법을 이용한 A. pullulans NP1221의 대량생산 공정 개발 ... 134
1. 연구개발수행 내용 ... 134
2. 연구개발수행 결과 ... 136
제 4 장 목표달성도 및 관련분야에의 기여도 ... 147
제 1 절 목표달성도 ... 147
제 2 절 관련분야의 기여도 ... 148
제 5 장 연구개발결과의 활용계획 ... 149
제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 150
제 7 장 참고문헌 ... 152

표/그림 (87)

표 Intrinsic viscosity of the exo-polysaccharide at the 0.65 M NaCl solution.
표 Component determination of the exo-polysaccharide by HPLC analysis. The peak at 8.856 min represents that of glucose.
표 Infrared spectra of exo-polysaccharide produced by A. pullulans IMS-822.
표 Gamma-ray 조사에 따른 베타-글루칸의 저분자화.
표 Ultrasonication에 의한 베타-글루칸의 저분자화
표 초고압액화처리장치에 의한 베타-글루칸의 저분자화.
표 Extraction and purification scheme of and immuno-stimulating olysaccharides from cultured mycelia of mushroom strains
표 The effect of crude extracts isolated from Lentinus edodes (10003; 10005),Pleurotus ostreatus (10015; 10016). and Pleurotus eryngii (10017; 10018; 10019) on the nitrite evolution of RAW264.7 cells and on the proliferation of splenic cells
표 The effect of crude extracts isolated from Lyophyllum cinerascens (10032),Hypsizigus marmoreus (10060), and Flammulina velutipes (10041; 10042; 10043)on the nitrite evolution of RAW264.7 cells and on the proliferation of splenic cells
표 The effect of crude extracts isolated from Agaricus bisporus (10050; 10051; 10052),Agrocybe aegerita (10070) on the nitrite evolution of RAW264.7 cells and on the proliferation of splenic cells
표 The effect of crude polysaccharide isolated from Cordyceps militaris GRANSTERand other edible mushrooms on the nitrite evolution of RAW264.7 cells and on the proliferation of splenic cells. Cordyceps militaris, C. militaris GRANSTER.
표 The effect of crude polysaccharide isolated from Flammulina velutipes 10042,Inonotus obliquus 11044, and Hypsizigus marmoreus 10060 on the nitrite evolution of RAW264.7 cells and on the proliferation of splenic cells.
표 The effect of crude polysaccharide isolated from Pholiota nameko 10080, Inonotusradiatus 11054, Ganoderma lucidum 11120, and Amillaria mellea 11150 on the nitrate evolution of RAW264.7 cells and on the proliferation of splenic cells.
표 The effect of crude polysaccharide isolated from Cordyceps militaris 12002, Cordyceps scarabaeicola 12020, and Paecilomyces cicada 12052 on the nitrite evolution of RAW264.7 cells and on the proliferation of splenic cells.
표 The effect of crude extracts isolated from Paecilomyces cicada 12053, Beauveria bassiana 12061, and Cordyceps opioglossoides 12070 on the nitrite evolution of RAW264.7 cells and on the proliferation of splenic cells.
표 Extraction and purification scheme of and immuno-stimulating polysaccharides from fruitbody of Sparassis crispa. DEAE-Sephadex A25(Cl-) was used in anion exchange chromatography.
표 HPAEC-PAD chromatogram on neutal sugar of hydrolysed purified polysaccharides from fruitbody of Sparassis crispa.
표 The effect of soluble polysaccahride isolated from fruit body of Sparassis crispa on the nitrite evolution of RAW264.7 cells and on the proliferation of splenic cells.
표 Isolation scheme of water-soluble exo-polysaccharide from culture broth of Inonotus obliquus 11040.
표 Batch fermentation patterns of Inonotus obliquus 11040 in a 500 l pilot vessels.The changes of dissolved oxygen (DO), pH and glucose concentration are also shown. The seed culture was conducted for 144 hr in 50 liter fermentor and the cultural broth in log phase was transferred to 500 liter scale. This culture was conducted for 312 hours.
표 The effect of polysaccharides of Inonotus obliquus 11040 on the nitrite evolutionof RAW264.7 cells and on the proliferation of splenic cells.
표 Mycelial dry weight and exo-polysaccharide production depending on carbon source. (YMB, YM Broth)
표 Mycelial dry weight and exo-polysaccharide production depending on nitrogen source. (YMB, YM Broth)
표 Effect of organic acid addition on mycelial dry weight and exo-polysaccharide production. (Control, YM Broth)
표 Effect of various substrates and their concentration on mycelial dry weight and exo-polysaccharide production.
표 Time profiles of mycelial growth and exopolysaccharides production in 5 liter batch fermentation. A. BC(YMB); B. BC(MYMB); C. STR(MYMB)
표 Time profiles of mycelial growth and exopolysaccharides production in 30 liter batch fermentation. A. YMB; B. MYMB(modified YM broth)
표 Time profiles of mycelial growth and exo-polysaccharides production and its mitogenic specific activities in 300 liter batch fermentation.
표 Comparative analysis of mycelial and exo-polysaccharide weight produced usingvarious scale of bioreactor.
표 Specific primer sequence for PCR.
표 Solution and buffer composition for SDS-PAGE and transfer.
표 Expression of dendritic cell surface marker. muDC was differentiated by GM-CSF, IL-4, TNF-α for 48hr.
표 Difference of cell morphology between Mutz-3(Top) and muDC(48hr, Bottom).
표 Dectin-1 is expressed in muDC, but not in Mutz-3 (Top; FACS analysis, Bottom; fluorescent microscopy).
표 CD1a, CD11c and Dectin-1 expression in muDC.
표 TNF-α secretion by zymosan in muDC. Top; secretion of TNF-α was dependent to zymosan concentration(6hr incubation). Bottom; TNF-α increase and decrease by zymosan(100㎍/ml) was dependent to time variation.
표 Dectin-1 silenced muDC by Dectin-1 specific siRNA, analyzed by FACS(Top) and RT-PCR(Bottom).
표 TNF-α secretion by zymosan in Dectin-1 silenced muDC.
표 Screening of co-stimulation factor for TNF-α secretion with zymosan.
표 TNF-α secretion was dependent to time variation by PZYc in muDC.
표 Morphologic changes of muDC by PMA, zymosan and PZYc. Differentiated muDC for 48hr(A), stimulated muDC with PMA(100ng/ml) was attached on the plate, and extend many side branch(B), stimulated muDC with zymosan(100㎍/ml) was containing the suspension condition, and small dark point is zymosan partial(C), stimulated muDC with PZYc was attached on the plate, however, so weakly attached than only PMA stimulated muDC(D).
표 Various beta 1-3 glucans effects on muDC.
표 Inhibition of specific signal protein- PKC(Top), MEK (middle), NF-kB(Bottom).
표 Inhibition of specific signal protein- ERK(Top), PI3K (Bottom).
표 Phosphosrylation of MEK1/2, and its stability by PMA, zymosan and PZYc.
표 Phosphosrylation of ERK1/2, and its stability by PMA, zymosan and PZYc.
표 Phosphorylation and degradation of IkB.
표 Differences phosphorylation pattern of IKK-subunits (85kDa; α-subunit, 87kDa; β-subunit).
표 Translocation of p50/p65 to nuclear from cytosol by PZYc in muDC.
표 Strains and plasmid list.
표 Primers used for PCR amplification of DNA segments.The underline in primers was the overlapped sequences in each PCR product.
표 Antibiotic resistant test of Aureobasidium sp.
표 PCR amplification and identification pullulan synthetase gene in A. pullulans(partial sequence - 1,816 bp).
표 Construction of TH cassette.M, 1 kb plus ladder marker (Fermentas Co.)
표 Construction of hygromycin resistance cassette.
표 Construction of the pKBR103 recombinant plasmid. M, 1 kb plus ladder marker (Fermentas Co.); 2, Amplification of the template the recombinant cassette using pKBR103 as the template 3, Cloning confirmed by XbaI; 4, Cloning confirmed by XbaI and HindIII
표 Confirmation of double crossover in A. pullulans by PCR. M, 1 kb plus ladder marker (Fermentas Co.); 5, PCR confirmation of double crossover in A..
표 Comparison of the morphological characters between the wild type and mutant strain.
표 After vacuum filtration, liberated carbohydrates were determined by HPLC analysis.
표 Change of fermentation products in A. pullulans and A. pullulans NP1221. Change of sucrose concentration, DCW and total exopolysaccharides in 35L batch cultures of NP1221 (150 rpm, closed circle; 250 rpm, open circle; 350 rpm, closed triangle; 500 rpm, open triangle).
표 Comparison of kinetic parameters in A. pullulans and A. pullulans NP 1221. Yx/suc (g·g-1), Biomass yield; Yglucan/suc (g·g-1), β-glucan yield; Yglucan/x (g·g-1), β-glucan concentration; P, highest β-glucan concentration μ (h-1), Specific growth rate; qsuc (g·g-1 h-1), Specific substrate uptake rate; qglucan (g·g-1 h-1), specific β-glucan production rate
표 Code and experimental conditions for the central composite design.
표 The actual constitution of culture medium for RSM.
표 β-glucan concentration and DCW by carbon source in NP1221.a) β-glucan concentration, b) DCW
표 β-glucan concentration and DCW by nitrogen source in NP1221.a) β-glucan concentration, b) DCW
표 β-glucan concentration and DCW by concentration in ascorbic acid in NP1221.a) β-glucan concentration, b) DCW
표 Central composite design and experimental results.
표 Polynomial equations calculated by RSM program for β-glucan concentration.
표 Result of ANOVA(Analysis of variance) for RSM. a) ANOVA for response of dependent variables, b) R-square value for response of dependent variables c) Eigenvalues by RSM
표 Stationary point by canonical analysis and predicted response value.
표 Critical value by canonical analysis.
표 Contour plots of β-glucan concentration.
표 Response surface plots for β-glucan concentration.
표 β-glucan concentration and DCW by variation of culture temperature in NP1221.a) β-glucan concentration, b) DCW
표 β-glucan concentration and DCW by variation of initial pH in NP1221.a) β-glucan concentration, b) DCW
표 Effect of agitation speed on the products of expolysaccharides.Change of sucrose concentration, dry cell weight (DCW) and total exopolysaccharides in 5L batch cultures of NP1221 (150 rpm, closed circle; 250 rpm, open circle; 350 rpm, closed triangle; 500 rpm, open triangle).
표 β-glucan 생산성 증진을 위한 SWOT 분석
표 Multi-volting법을 통한 생산성 증진을 위한 CTQ Y's의 선정
표 Process capability analysis of present level
표 CTQ-Y의 도출 및 성과지표 결정
표 Characteristic Diagram for selection of X's factor
표 X-Y Matrix for selection of X's factor
표 Analysis of plan and data collection plan
표 Probability plot of initial and result value
표 Equal variances analysis of initial and result value
표 2 sample -T test result of initial and result value
표 One-way ANOVA of initial and result values

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미생물 유래 바이오폴리머의 단위 생산공정 개발 및 기능 연구
Characterization of microbial biopolymers and development of unit process for their mass production 원문보기