Production, Purification, and Characterization of Alginate lyase and Agarase from Turban Shell Gut Strain Agarivorans albus YKW-34
- 발행기관 Kangnung National University Graduate School
- 지도교수 Kim, Sang Moo
- 발행년도 2008
- 학위수여년월 2008. 2
- 학위명 석사
- 학과 및 전공 Department of Marine Bioscience and Technology
- 원문페이지 82 p.
- 본문언어 영어
초록/요약
Agarivorans albus YKW-34는 소라(Turbinidae batillus cornutus)에서 분리하여 16S rRNA로 분석한 결과 Agarivorans albus로 동정되었다. 이 균주는 다시마(Laminaria japonica) 및 우무가사리(Gelidium amansii) 조체에 대하여 분해활성을 나타내었으며, 뛰어난 알긴산 분해효소(alginate lyase)와 한천 분해효소(agarase) 활성은 나타나었다. 본 연구에서는 alginate lyase 및 agarase 생산조건을 최적화한 다음 효소를 분리ㆍ정제하여 여러가지 특성을 연구하였다. A. albus YKW-34의 알긴산 분해효소 생산조건을 최적화하기 위해 주요 배양인자로서 탄소원, 질소원, 탄소 및 질소의 비율, 배양온도, pH, 그리고 배양시간의 영향을 분석하였다. Alginate lyase의 최적생산조건은 탄소원으로 Laminaria 분말 0.5%, 질소원으로 KNO3 0.2%, 배양온도는 25℃, pH는 8.0, 배양시간은 48 h 이었다. 이때 가장 높은 효소활성은 5 U/mL 이었다. A. albus YKW-34가 생산하는 alginate lyase를 PEI 침전, DEAE Sepharose FF, Phenol Sepharose, 그리고 Sephacryl S-100 크로마토그래피의 순서로 분리ㆍ정제하였을 때 정제도는 25배, 정제수율는 7%, 그리고 비할성(specific activity)은 55.93 U/mg인 alginate lyase를 생산할 수 있었다. 정제된 alginate lyase의 분자량은 60 kDa, pI는 5.5?5.7, catalytic efficiency (kcat/Km)는 alginate를 기질로 사용하였을 때 1.7 × 106 s-1M-1이었다. Alginate lyase는 alginate를 최적 기질로 사용하였을 때 40℃ 및 pH 7.8에서 최적활성을 나타내었다. 이 효소는 alginate를 M-block 및 G-block으로 분해하였다. 또한 Na+/K+은 효소의 보조인자 (cofactor)이었으며 0.1 M Na+/K+ 첨가 시 최대 효소활성을 나타내었다. β-Mercaptoethanol, DTT, SDS는 효소활성을 약 30% 이상 억제하였으며 urea, EGTA, EDTA는 효소 활성에 크게 영향을 미치지 않았다. A. albus YKW-34의 한천 분해효소 생산을 최적화하기 위해 반응표면 분석법을 사용하였다. 반응표면 분석을 위한 실험 계획은 중심합성법에 의해 계획하였으며, 주요 배양인자인 탄소원 농도, 질소원 농도 및 pH의 영향을 분석하였다. Agarase는 탄소원으로 agar 0.23%, 질소원으로 yeast extract 0.27%, pH 7.81, 배양시간 12 h이었을때 생산량은 0.87 U/ml로 최대값을 나타내었다. A. albus YKW-34가 생산하는 agarase를 DEAE Sepharose FF, Sephacryl S-100 크로마토그래피의 순서로 분리ㆍ정제하였을 경우 정제도는 10배, 정제수율은 30%이었다. 정제된 agarase의 분자량은 50 kDa이었고, N-terminal amino acid sequence는 ASLVTSFEEA이었다. 최적 pH 및 온도는 각각 pH 8.0와 40℃이었고, pH 6.0?11.0과 50℃에서 안정하였다. Agarase 효소의 agarose 분해산물는 MALDI-TOF MS과 13C NMR로 분석하였으며, A. albus YKW-34 agarase는 β-type이었고, neoagarobiose를 생산하였다. kcat/Km값은 agarose 및 neoagarotetraose을 기질로 사용 하였을 때 각각 4.04 × 103 및 8.1 × 102 s?1M?1이었다. 금속 이온, 그리고 EGTA, EDTA, SDS, urea 첨가는 효소의 활성에 크게 영향을 미치지 않았다. 다만 β-Me 및 DTT 에 의해서 약 30% 이상 효소활성이 저해되었다.
more초록/요약
A marine bacterium strain YKW-34 that degrades the cell wall of some seaweed including Laminaria japonica and Gelidium amansii was isolated from the gut of a turban shell. This strain was identified as Agarivorans albus based on 16S rRNA gene sequence analysis. This paper describes the optimization of production, purification, and characterization of an alginate lyase and an agarase produced by this strain. The effects of medium composition and culture condition on the production of alginate lyase by A. albus YKW-34 were investigated using batch shake flasks. No alginate lyase was produced in the marine broth medium. After optimization, the activity of the alginate lyase reached 5 U/mL. The optimal production conditions for alginate lyase by A. albus YKW-34 were: inoculum volume, 10%; inoculum age, 12 h; initial pH of the medium, 8.0; culture temperature, 25°C; carbon source, 0.5% Laminaria powder; nitrogen source, 0.2% KNO3. Alginate could induce alginate lyase production, but not as efficient as did Laminaria powder. The addition of fucoidan, cellulose, and glucose had negative effect on the production of alginate lyase. Other kinds of nitrogen sources such as yeast extract, beef extract, and peptone affected positively the growth of the microorganism, but negatively the alginate lyase production. In addition, the optimal harvest time was 48 h based on the time course of alginate lyase production. An alginate lyase with high specific enzyme activity was purified from A. albus YKW-34 by in order of ion exchange, hydrophobic, and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. It was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5?5.7. The optimal pH and temperature for the activity of the alginate lyase were pH 7.0 and 40℃, respectively. It was stable over pH 7.0?10.0 and at temperature below 50℃. The enzyme had substrate specificity for both poly-guluronate and poly-mannuronate units. The kcat/Km value for alginate (heterotype) was 1.7 × 106 s-1M-1. The enzyme activity was completely lost by dialysis and restored by addition of Na+ or K+. The optimal activity exhibited in 0.1 M of Na+ or K+. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT), and chelating reagents (EGTA and EDTA). Effects of medium composition and culture conditions on agarase production by A. albus YKW-34 were investigated in shake flasks. Effects of carbon and nitrogen sources and culture temperature on agarase production were evaluated by one-factor-at-a-time design. Agar, yeast extract, and 25°C were found to be most suitable for agarase production. The most important nutritional components and culture conditions influencing agarase production were selected by Plackett-Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effect on agarase production. The optimum levels of these variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy resulted in the enhancement of agarase production from 0.23 U/ml to 0.87 U/ml. The activity staining of crude agarase preparation after electrophoresis revealed the presence of an agarase with molecular mass of 50 kDa. An extracellular β-agarase, AgaA34, was purified from A. albus YKW-34 by ion exchange and gel filtration chromatographies to homogeneity with a recovery of 30% and a fold of 10. AgaA34 was composed of a single polypeptide chain with the molecular mass of 50 kDa. N-terminal amino acid sequencing revealed a sequence of ASLVTSFEEA, which exhibited a high similarity (90%) with those of agarases from glycoside hydrolase family 50. The pH and temperature optima of AgaA34 were pH 8.0 and 40°C, respectively. It was stable over pH 6.0?11.0 and at temperature below 50°C. Hydrolysis of agarose by AgaA34 produced neoagarobiose (75 mol%) and neoagarotetraose (25 mol%), whose structures were identified by MALDI-TOF MS and 13C NMR. AgaA34 cleaved both neoagarohexaose and neoagarotetraose into neoagarobiose. The kcat/Km values for agarose and neoagarotetraose were 4.04 × 103 and 8.1 × 102 s?1M?1, respectively. AgaA34 was resistant to denaturing reagents (SDS and urea). Metal ions were not required for its activity, while reducing reagents (β-Me and DTT) increased its activity by more than 30%.
more목차
Introduction = 9
1. Review of literatures = 11
1.1. Agarivorans albus = 11
1.1.1. Description of Agarivorans albus = 11
1.1.2. Enzymes from Agarivorans albus = 11
1.2. Alginate lyase = 12
1.2.1. Cell wall composition of brown algae = 12
1.2.2. Alginate = 13
1.2.3. Production and purification of alginate lyase = 14
1.2.4. Alginate oligosaccharides = 15
1.2.5. Application of alginate lyase = 15
1.3. Agarase = 16
1.3.1. Cell wall composition of red algae = 16
1.3.2. Agar = 16
1.3.3. Production and purification of agarase = 17
1.3.4. Agar-derived oligosaccharides = 19
1.3.5. Application of agarase = 19
2. Optimization of culturing condition and medium composition for the = 21
2.1. Materials and methods = 21
2.1.1. Materials = 21
2.1.2. Identification of the microorganism = 21
2.1.3. Enzyme screening = 22
2.1.4. Fermentation medium and growth condition = 22
2.1.5. Determination of the cell growth = 23
2.1.6. Determination of the enzyme activity = 23
2.1.7. Statistical analysis = 24
2.1.8. Nucleotide sequence accession number = 24
2.2 Results and discussion = 24
2.2.1. Identification of the bacteria = 24
2.2.2. Carbohydrase screening = 25
2.2.3. Optimization of carbon source = 26
2.2.4. Optimization of nitrogen source = 27
2.2.5. Optimization of C:N and inoculum volume = 28
2.2.6. Optimization of inoculum age = 29
2.2.7. Optimization of the initial acidity = 29
2.2.8. Optimization of temperature = 30
2.2.9. Time courses of cell growth and enzyme production = 31
2.3. Conclusions = 32
3. Purification and characterization of a Na+/K+ dependent alginate lyase = 33
3.1. Materials and methods = 33
3.1.1. Materials = 33
3.1.2. Bacterial strain and growth media = 33
3.1.3. Enzyme activity assay = 33
3.1.4. Protein determination = 34
3.1.5. Enzyme purification = 34
3.1.6. Electrophoresis = 35
3.1.7. Determination of isoelectric point = 35
3.1.8. Effects of temperature and pH on enzyme activity and = 35
3.1.9. Effects of metal ions on alginate lyase activity = 36
3.1.10. Effects of various reagents on alginate lyase activity = 36
3.1.11. Substrate specificity = 36
3.1.12. Kinetic parameters = 36
3.2. Results and discussion = 37
3.2.1. Purification of the alginate lyase = 37
3.2.2. Specific activity of the alginate lyase = 39
3.2.3. Determination of molecular mass and isoelectric point = 40
3.2.4. Effects of temperature and pH on alginate lyase activity = 42
3.2.5. Effects of metal ions on alginate lyase activity = 43
3.2.6. Effects of various reagents on alginate lyase activity = 45
3.2.7. Substrate specificity of the alginate lyase = 46
3.2.8. Kinetic parameters = 46
3.3. Conclusions = 47
4. Optimization of medium composition and culture conditions for = 48
4.1. Materials and Methods = 48
4.1.1 Microorganism and growth medium = 48
4.1.2. Analytical methods = 48
4.1.3. One-factor-at-a-time design = 48
4.1.4. Statistical designs = 49
4.1.5. In-situ detection of agarase = 52
4.2. Results and discussion = 52
4.2.1. Evaluation of three factors by one-factor-at-a-time design = 52
4.2.2. Screening of significant factors by Plackett-Burman design = 56
4.2.3. Optimization of significant factors by central composite = 56
4.2.4. Agarase production in optimized medium = 60
4.2.5. In-situ detection of agarase = 60
4.3. Conclusions = 61
5. Purification and characterization of a β-agarase from Agarivorans = 62
5.1. Materials and methods = 62
5.1.1. Microorganism and culture condition = 62
5.1.2. Enzyme assay = 62
5.1.3. Purification of agarase = 62
5.1.4. Electrophoresis = 63
5.1.5. N-terminal amino acid sequence = 63
5.1.6. Effects of pH and temperature on agarase stability and = 63
5.1.7. Substrate specificity and cleavage pattern = 64
5.1.8. Effects of various reagents on agarase activity = 64
5.1.9. Estimation of kinetic parameter = 64
5.1.10. Enzymatic product analysis = 65
5.2. Results and discussion = 65
5.2.1. Purification of AgaA34 = 65
5.2.2. N-terminal sequence = 68
5.2.3. Effects of pH and temperature on the activity and stability = 69
5.2.4. Substrate specificity and cleavage pattern = 70
5.2.5. Effects of various reagents on the activity of AgaA34 = 71
5.2.6. Kinetic parameter = 72
5.2.7. Enzymatic product = 73
5.3. Conclusions = 75
6. References = 76
