The gel weakening prevention of Alaska pollock(Theragra chalcogramma) surimi-based product by protease inhinitors
- 발행기관 Kangnung National University Graduate School
- 지도교수 Kim, Sang Moo
- 발행년도 2008
- 학위수여년월 2008. 2
- 학위명 박사
- 학과 및 전공 Department of Marine Bioscience and Technology
- 원문페이지 xi, 94 p.
- 본문언어 영어
초록/요약
명태 (Theragra chalcogramma) surimi 제품의 gel 약화현상을 방지하기 위하여 재조합 연어 cystatin (recombinant chum salmon cystatin, RC)의 대량생산을 수행하였 다. RC발현 벡터인 pYES2/NT_C를 효모 (S. cerevisiae YPH 499) 에 발현시킨 다음 affinity chromatography로 RC를 정제하였다. Papain 및 cathepsin L에 대한 RC의 저 해활성 (specific inhibitory activity)은 각각 7.45 및 10.24 U/mg 이었다. RC는 pH 5.0?7.0 및 65℃이하에서 안정하였다. RC의 대량생산을위해 재조합효모는 플라스 크 및 발효조를 이용하여 최적화 연구를 수행하였다. 플라스크에서 RC 생산 최 적화연구는 반응표면분석 (response surface methodology)을 이용하여 배지 pH, 유 도시간 (inducing time), 그리고 유도자 (the amount of inducing assistant)를 변수로 설 정하여 수행하였다. 배지 pH는 RC의 생산량에 현저한 영향을 나타내었다 (P < 0.05). 반응표면 분석에 의한 RC 생산의 최대값은 배지는 pH 5.7, 유도시간은 6.7 h 및 유도물질 농도는 5.6 g/L 에서 0.57 U/mL 이었다. 발효조에서 RC 생산을 최 대화하기 위하여 교반속도와 통기량을 변화하였을 경우의 성장률, 포도당과 반 유당의 대사 및 용존 산소량을 측정하였다. RC 생산의 최대값은 0.57 U/mL 이었 으며, 최적 배양조건은 교반속도 350 rpm, 통기량 1.0 vvm 이었다. RC를 alcohol로 정제하였을 경우의 수율 및 purity 는 각각 66.1% 및 1.86 folds 이었다. 명태 surimi 제품의 gel 약화를 억제하기 위하여 무지개송어 (Oncorhynchus mykiss) 혈장단백질(rainbow trout plasma, RTP) 및 연어 (Oncorhynchus keta) 혈장단 백질 (chum salmon plasma, CSP)의 저해효과에 관한 연구를 수행하였다. RTP와 CSP의 단백질함량은 60%정도 이었다. RTP와 CSP는 cysteine 및 serine계 단백분 해효소인 papain과 trypsin를 효과적으로 억제하였다. CSP를 전기영동하여 papain 에 대한 억제활성 염색 (inhibitory activity staining) 방법으로 cysteine계 단백분해효 소저해제 (CPI)를 확인하였다. 이 CSP CPI를 affinity chromatography로 정제하였으 며, 수율은 0.94%, purity는 30.36 folds 이었다. CSP CPI를 전기영동 및 Sephacryl S- 100 gel filtration chromatography로 측정한 분자량은 70 kDa 이었다. 전기영동후 PAS 염색 결과로 CSP CPI는 당단백질 이었으며 kininogen로 분류되었다. CSP CPI 의 최적 pH와 온도는 7.0 및 20?40℃ 이었으며, pH 6.0?9.0, 온도 50℃이하 범위에 서 안정하였다. CSP CPI의 저해형태는 비길항저해 (non-competitive)이었으며, 저해 상수 (Ki value)는 105 nM 이었다. 명태 surimi 제품의 gel 약화를 억제하기 위하여 재조합 RC의 저해효과를 연구 하였다. Surimi gel의 약화를 억제하는 RC의 최적용량은 TCA-soluble peptides의 함 량으로 표시하였을 때 100 μg/g 이었다. RC의 첨가량이 높을수록 gel의 파괴강도 (breaking force), 파괴변형력 (deformation) 및 백색도 (whiteness)가 증가하였고, 압 착드립 (expressible drip)은 감소하였다 (P < 0.05). 전기영동결과를 분석하였을 때 RC는 surimi의 myosin heavy chain (MHC) 분해를 억재하였으며, 이로 인하여 명태 surimi 제품의 단백분해효소 작용에 의한 품질열화를 억제하였다. RC의 surimi gel 억제를 위한 최적첨가량은 100 μg/g 이었다. 명태 surimi 제품의 gel 약화를 억제 하기 위한 RTP와 CSP 첨가효과에 관한 연구를 수행하였다. RTP와 CSP는 0.75 mg/g 까지 첨가랑이 증가할수록 surimi gel의 파괴강도, 파괴변형력 및 백색도가 증가하였고, 압착드립은 감소하였다 (P < 0.05). 그렇지만, RTP와 CSP의 첨가량이 0.75 mg/g 을 초과하였을 경우에는 (0.75?1.50 mg/g), gel의 파괴강도, 파괴변형력 및 백색도는 감소하였고, 압착드립은 증가하였다 (P < 0.05). RTP 첨가량 0.75 mg/g 까지에는 gel의 단백질 용해도 (protein solubility)는 감소하였고, RTP 첨가량이 증 가할수록 (0.75?1.50 mg/g) gel의 단백질 용해도는 증가하였다 (P < 0.05). 그러나, CSP인 경우에는 0.25 mg/g 이상 첨가할수록 gel의 단백질 용해도에는 현저한 변 화가 없었다 (P < 0.05). RTP와 CSP의 첨가량 (0?1.50 mg/g) 이 높을수록 TCAsoluble peptides의 함량이 증가하였다 (P < 0.05). RTP와 CSP의 첨가량이 높을수록 MHC의 분해를 보다 더 억제하였기 때문에, RTP와 CSP는 단백분해효소저해제의 일종인 것으로 판단되었다. RTP와 CSP의 gel 약화를 억제하기 위한 최적용량은 0.75 mg/g 이었다. 10 kDa 투석막으로 정제한 RTP를 명태 surimi에 첨가하였을 경 우, 분자량 10 kDa 이하의 성분은 gel의 특성에 대한 현저한 영향은 없었다 (P < 0.05).
more초록/요약
Surimi is a minced and washed fish muscle consisting of salt-soluble myofibrillar proteins. The endogenous proteases in surimi cause an irreversible destruction of the gel structure of surimi and result in decreasing the quality of surimi-based product and its market value. Protease inhibitors have been used to prevent the degradation of surimi gel. Chum salmon cystatin was a specific cysteine protease inhibitor. It was overexpressed for commercial application in Alaska pollock (Theragra chalcogramma) surimi to prevent the gel weakening. The recombinant chum salmon cystatin (RC) was overexpressed in Saccharomyces cerevisiae YPH 499 incorporating pYES2NT/C vector. After overexpression, RC was purified by His-select nickel affinity chromatography. The specific inhibitory activities of RC against papain and cathepsin L were 7.45 and 10.24 U/mg, respectively. RC was stable over pH 5.0?7.0 and at temperature below 65℃. The culture condition and its scale-up for RC production by S. cerevisiae YPH 499 were optimized. In the shake flask, the culture condition was optimized using response surface methodology. Three independent variables of medium pH, inducing time, and the amount of inducing assistant were analyzed to get the optimal condition for RC production. The results were fitted to a second-order polynomial equation in which the determination coefficient (R2) was 0.904 and medium pH was considered statistically significant (P < 0.05). The highest amount of RC production in shake flask, 0.57 U/mL, was obtained at 5.7 of medium pH, 6.7 h of inducing time, and 5.6 g/L of inducing assistant. Thereafter, based on the results of shake flask, the effects of agitation and aeration rates on RC production by S. cerevisiae YPH 499 were determined for scale-up in fermentor. Growth rate, metabolism of glucose and galactose, and dissolved oxygen in fermentor were determined to explain the effects of agitation and aeration rates. The highest RC production in fermentor, 0.56 U/mL, was obtained at 350 rpm of agitation rate and 1.0 vvm of aeration rate. For the purification of RC on a large scale, two steps of alcohol precipitation were performed, in which 15% alcohol was added to remove impurities and then 45% alcohol was added to precipitate RC. RC was partially purified with a yield of 66.1% and purification ratio of 1.86 folds. Rainbow trout (Oncorhynchus mykiss) plasma (RTP) and chum salmon (Oncorhynchus keta) plasma (CSP) were also applied to prevent the gel weakening of Alaska pollock surimi. Protein was the main composition in RTP and CSP, which occupied around 60% of the total weight of plasma. RTP and CSP effectively inhibited papain and trypsin. A cysteine protease inhibitor (CPI) in CSP was detected by inhibitory activity staining against papain under non-reducing condition. The CPI was purified from CSP by affinity chromatography with a yield of 0.94% and purification ratio of 30.36 folds, respectively. CSP CPI had a Mw of 70 kDa based on the results of SDS-PAGE and Sephacryl S-100 gel filtration chromatography. CSP CPI was a glycoprotein based on the Periodic Acid?Schiff staining of SDS-PAGE gel and classified as a kininogen. CSP CPI was stable in the pH range of 6.0? 9.0 with maximal stability at pH 7.0. CSP CPI was thermostable at temperatures below 50℃ and exhibited maximal activity at temperatures of 20?40℃. CSP CPI was determined to be a non-competitive inhibitor against papain, with an inhibitor constant (Ki) of 105 nM. RC was used to prevent the gel weakening of Alaska pollock surimi. RC at 100 μg/g showed the highest inhibitory activity against the autolysis of surimi based on the analysis of trichloroacetic acid (TCA)-soluble peptides. As the concentration of RC increased, both the breaking force and deformation of modori gel significantly increased (P < 0.05). The addition of RC resulted in less expressible drip, which coincided with the increase of whiteness. Based on the result of SDS? PAGE, most myosin heavy chain (MHC) of surimi was retained as the addition of RC increased. Therefore, RC prevented the degradation of proteins in Alaska pollock surimi better than did egg white (EW). Thus, RC could be applied to Alaska pollock surimi to prevent the gel weakening and RC at 100 μg/g was the optimalconcentration. The effects of RTP and CSP on the gelation of Alaska pollock surimi were determined. For modori gel, the breaking force, deformation, whiteness, and water holding capacity increased as RTP and CSP concentrations (0?0.75 mg/g) increased, while decreased at higher concentrations of RTP and CSP (0.75?1.50 mg/g) (P < 0.05). Protein solubility of modori gel in the mixture of SDS, urea, and β-mercaptoethanol decreased as the addition amount of RTP increased up to 0.75 mg/g, whereas increased at higher concentration of RTP (0.75?1.50 mg/g) (P < 0.05). In contrast, protein solubility of modori gel changed slightly after CSP concentration was higher than 0.25 mg/g (P < 0.05). The contents of TCA-soluble peptide decreased as RTP and CSP concentrations (0?1.50 mg/g) increased (P < 0.05). Based on the result of SDS?PAGE, most MHC of surimi was not degraded when RTP and CSP were added. Thus, both RTP and CSP were supposed to act as protease inhibitors in the gelation of Alaska pollock surimi. RTP and CSP at 0.75 mg/g was the optimal concentration to prevent the gel weakening of Alaska pollock surimi. Compounds with Mws less than 10 kDa in RTP had no significant effect on the gelation of Alaska pollock surimi based on the result of the dialyzed RTP.
more목차
I. Introduction = 1
II. Review of literatures = 3
III. Overexpression of recombinant chum salmon cystatin = 14
1. Materials and methods = 14
1.1. Organism = 14
1.2. Instruments and substances = 14
1.3. Preparation and construction of expression plasmid = 15
1.4. Transformation and selection of the productive clones = 15
1.5. Plasmid stability = 16
1.6. Cultivation of recombinant yeast and expression of recombinant = 16
1.7. Purification of recombinant chum salmon cystatin using affinity = 17
1.8. Analysis = 17
1.8.1. Biomass concentration = 17
1.8.2. Protein concentration = 18
1.8.3. Concentration of recombinant chum salmon cystatin = 18
1.8.4. Characterization of recombinant chum salmon cystatin = 18
1.8.4.1. Electrophoresis = 18
1.8.4.2. Inhibitory activity assay = 19
1.8.4.3. pH and thermal stability of recombinant chum salmon = 19
1.9. Optimization of expression of recombinant chum salmon cystatin = 20
1.9.1. Experimental design = 20
1.9.2. Cultivation in shake flask = 20
1.10. Optimization of expression of recombinant chum salmon = 20
1.10.1. One factor at a time design = 20
1.10.2. Cultivation in fermentor = 21
1.11. Purification of recombinant chum salmon cystatin using alcohol = 21
2. Results and discussion = 22
2.1. Preparation of open reading frame encoding chum salmon = 22
2.2. Transformation and selection of the productive clones = 23
2.3. Plasmid stability = 24
2.4 Cultivation of recombinant yeast and expression of recombinant = 25
2.5. Purification of recombinant chum salmon cystatin using affinity = 26
2.6. Characterization of recombinant chum salmon cystatin = 28
2.6.1. Electrophoresis = 28
2.6.2. Inhibitory activity of recombinant chum salmon cystatin = 28
2.6.3. pH and thermal stability of recombinant chum salmon cystatin = 29
2.7. Optimization of expression of recombinant chum salmon cystatin = 31
2.7.1. Fitting the model = 31
2.7.2. Effect of medium pH, inducing time, and the amount of = 35
2.8. Optimization of expression of recombinant chum salmon cystatin = 38
2.8.1. Effect of agitation rate on the production of recombinant chum = 38
2.8.2. Effect of aeration rate on the production of recombinant chum = 40
2.9. Purification of recombinant chum salmon cystatin using alcohol = 42
IV. Properties of fish plasmas and cysteine protease inhibitor = 44
1. Materials and methods = 44
1.1. Substances = 44
1.2. Instruments = 44
1.3. Preparation of fish plasma = 44
1.4. Proximate composition = 45
1.5. Protease inhibitory activity = 45
1.6. Preparation of CNBr?apain?epharose = 45
1.7. SDS-substrate gel and staining for inhibitory proteins = 46
1.8. Purification of a cysteine protease inhibitor from chum salmon = 46
1.9. Determination of molecular weight = 47
1.10. Periodic acid?chiff staining for the cysteine protease inhibitor = 47
1.11. Inhibitory activity of the cysteine protease inhibitor = 47
1.12. pH stability, and thermal stability and activity of the cysteine = 48
1.13. Protein concentration = 48
1.14. Kinetics = 48
2. Results and discussion = 49
2.1. Proximate composition of fish plasma = 49
2.2. Protease inhibition = 51
2.3. Identification of inhibitory proteins = 53
2.4. Purification of the cysteine protease inhibitor from chum salmon = 54
2.5. Electrophoresis = 56
2.6. Periodic acid?chiff staining = 57
2.7. Inhibitory activity of the cysteine protease inhibitor = 58
2.8. pH stability, and thermal stability and activity of CSP CPI = 58
2.9. Inhibition type of the cysteine protease inhibitor = 60
V. Application of fishery origin protease inhibitors to Alaska = 62
1. Materials and methods = 62
1.1. Substances = 62
1.2. Instruments = 62
1.3. Inhibitory activity of recombinant chum salmon cystatin against = 62
1.4. Surimi gel preparation = 63
1.5. Texture analysis = 63
1.6. Whiteness measurement = 63
1.7. Determination of expressible drip = 64
1.8. Determination of protein solubility = 64
1.9. Determination of TCA-soluble peptide content = 64
1.10. Protein pattern = 65
1.11. Statistical analysis = 65
2. Results and discussion = 66
2.1. Application of recombinant chum salmon cystatin = 66
2.1.1. Inhibitory activity of recombinant chum salmon cystatin = 66
2.1.2. Effect of recombinant chum salmon cystatin on textural = 67
2.1.3. Effect of recombinant chum salmon cystatin on whiteness and = 69
2.1.4. Effect of recombinant chum salmon cystatin on the protein = 70
2.2. Application of rainbow trout plasma = 71
2.2.1. Effect of rainbow trout plasma on textural properties of modori = 71
2.2.2. Effect of rainbow trout plasma on whiteness and expressible = 73
2.2.3. Effect of rainbow trout plasma on protein solubility and = 75
2.2.4. Effect of rainbow trout plasma on protein pattern of modori gel = 77
2.2.5. Effect of the dialyzed rainbow trout plasma on textural = 78
2.3. Application of chum salmon plasma = 79
2.3.1. Effect of chum salmon plasma on textural properties of modori = 80
2.3.2. Effect of chum salmon plasma on whiteness and expressible = 81
2.3.3. Effect of chum salmon plasma on protein solubility and = 82
2.3.4. Effect of chum salmon plasma on protein pattern of modori gel = 83
VI. Conclusions = 85
VII. References = 86
Appendix. Abbreviation = 94
