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Flow cytometric analysis of Salmonella enterica serotype Typhimurium inactivated with supercritical carbon dioxide and Overexpression and characterization of a recombinant agarase, Aga50D, from Saccharophagus degradans 2-40

  • 김희택 (대학원 식품과학과 식품생물공학전공)

초록/요약

Supercritical carbon dioxide (SC-CO2) treatment is non-thermal process to sterilize and inactivate microorganisms efficiently. Because it can achieve microbial inactivation at relatively low temperatures, foods and bioactive materials could be applied safely without deterioration of them. However, in spite of its applicability to industries, it was not exactly determined how changes of cell physiology caused by treatment with SC-CO2 because most investigations using SC-CO2 only depended on plating methods to date. In this study, we observed changes of physiology state of Salmonella enterica serotype Typhimurium by SC-CO2 treatment using flow cytometry. Then, we compared the flow cytometric data with the survival rate obtained by the plating method to reveal possible mechanisms. The results showed that the treatment of SC-CO2 affected microbial physiology in various aspects, including the efflux pump and membrane integrity. While efflux pump activities of S. typhimurium were easily lost by SC-CO2 treatment irrelevant to its survival rate, membrane integrity had strong relationship with cell death. Agarose is one of cell wall components in red algae. It is linear polymer composed of alternating subunits of 3-O-linked β-D-galactopyranose and 4-O-linked 3, 6-anhydro-α-L-galactopyranose. Many bacteria, especially marine bacteria, produce agarases to metabolize agarose as their carbon source. Agarases are divided into α-agarases (EC 3.2.1.158), β-agarases (EC 3.2.1.81) and α-neoagaro-oligosacchairde hydrolase (EC 3.2.1.159) according to its mode of action against substrate. Alpha-agarases and beta-agarases cleave α- and β- linkages to produce agarooligosaccharides and neoagarooligosaccharides, respectively. Finally, these oligosaccharides are converted to monomeric sugar such as 3, 6-anhydro-L-galactose and D-galactose by α-neoagarooligosacchairde hydrolase. Saccharophagus degradans 2-40 is a marine bacterium to utilize agar as a sole carbon source and it has five potential agarase genes (agaA, B, C, D, and E) reported by complete genome sequence. Among them, two agarase genes, such as agaA and agaD have been neither characterized nor expressed yet. In this study, we have cloned agaD gene and expressed successfully in Escherichia coli. Its molecular size was around 75kDa. Its product was mostly neoagarobiose, which was determined using LC/MS and NMR, and optimum reaction condition was at 37°C and pH 7.0. Its Km, Vmax, Kcat and Kcat/Km values were 41.89 mg/ml (4.19 х 10-3 M), 17.86 U/mg, 22.32 s-1 and 5.33 х 103 M-1s-1 , respectively.

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초록/요약

초임계 이산화탄소 처리 방법은 비열처리 방법으로 효과적으로 미생물을 살균할 수 있다. 미생물 살균과정이 비교적 낮은 온도에서 수행되기 때문에 식품 및 생물소재들의 품질저하를 일으키지 않고 안전하게 적용할 수 있다. 그러나 이러한 산업적 유용성에도 불구하고 대부분의 연구가 평판법에 의존하고 있기 때문에 초임계 이산화탄소 처리에 의해 미생물의 생리학적 상태가 어떻게 변화하는지에 대한 연구가 미흡한 실정이다. 따라서 이번 연구에서 유세포 분석기를 이용하여 초임계 이산화탄소에 의한 Salmonella enterica serotype Typhimurium의 생리적 변화를 측정하였다. 측정한 결과를 평판법에 의해서 구한 결과와 비교하여 잠재적 살균 기작을 밝혔다. 그 결과, 초임계 이산화탄소 처리는 미생물의 efflux pump와 membrane integrity의 변화를 유발하였다. Efflux pump system은 생존율과는 관계없이 초임계 이산화탄소 처리에 의해 쉽게 손상되었지만 membrane integrity는 미생물 사멸과 강한 상관 관계가 있었다. 아가로오즈는 홍조류의 세포벽 구성성분 중의 하나이며 3-O-linked β-D-galactopyranose와 4-O-linked 3, 6-anhydro-α-L-galactopyranose이 교대로 결합된 직쇄형 중합체이다. 많은 미생물들과 특히 해양 미생물들은 탄소원으로서 아가로오즈를 대사하기 위해 아가레이즈를 생산한다. 아가레이즈는 기질에 대한 반응 방식에 따라 알파-아가레이즈 (EC 3.2.1.158), 베타-아가레이즈 (EC 3.2.1.81)와 알파-네오아가로올리고사카라이드 가수분해효소 (EC 3.2.1.159)로 구분된다. 알파-아가레이즈와 베타-아가레이즈는 각각 알파와 베타 결합을 분해하여 아가로올리고사카라이드와 네오아가로올리고사카라이드를 형성한다. 이것들은 최종적으로 알파-네오아가로올리사카라이드 가수분해효소에 의해 3, 6-anhydro-galactose와 D-galactose와 같은 단당으로 분해된다. Saccharophagus degradans 2-40 또한 탄소원으로 아가를 이용할 수 있는 해양미생물이며 complete genome sequence에 의해 다섯 가지의 잠재적 아가레이즈 유전자인 aga50A, 16B, 86C, 50D, 86E가 존재한다고 보고 되었다. 보고된 유전자 중 aga50A와 aga50D는 아직까지 발현과 특성이 정확하게 규명되지 않았다. 따라서 이번 연구를 통해 aga50D의 특성을 규명하기 위해 이를 클로닝하여 Escherichia coli에 성공적으로 발현하였다. Aga50D의 분자량은 75kDa이었고 LC/MS와 NMR을 통해 분석한 결과 산물의 대부분이 네오아가로바이오즈이었고 최적 활성은 37도와 pH 7이었다. Km, Vmax, Kcat, Kcat/Km는 각각 41.89mg/ml(4.19 х 10-3 M), 17.86U/mg, 22.32s-1, 5.33 х 103M-1s-1 이었다.

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목차

LIST OF FIGURES………………………………………………….…………..IV
LIST OF TABLES ……………………………………………………………….X
ABSTRACT OF THE THESIS ………………………………………………....XI
CHAPTER 1 Introduction ………………………………………………………1
1.1 Suprecritical carbon dioxide (SC-CO2) ...………………… 2
1.2 Flow cytometry ....………………………....……………… 5
1.3 Properties of agarase ...........................................................10
1.4 Research objectives .............................................................14
References ………………………………………………………17

CHAPTER 2 Flow cytometric analysis of Salmonella enterica serotype
Typhimurium inactivated with supercritical carbon dioxide……..25
Abstract ……………………….………………………………...26
2.1 Introduction …………………………………………….…28
2.2 Materials and Methods .…………………………………....32
2.2.1 Bacterial strain and cultivation conditions .……....32
2.2.2 Preparation of cell suspension …………................33
2.2.3 Supercritical CO2 treatment ……………………....33
2.2.4 Viable cell counting ...............…...……….….……34
2.2.5 Fluorescent dyes ..…………………........................35
2.2.6 Staining of cells with fluorescent dyes …………..36
2.2.7 Flow cytometric analysis of SC-CO2 treated cells
…………………………………………………….36
2.3 Results and Discussion …………………….……………..37
2.3.1 Flow cytometry of efflux pump activity …………37
2.3.2 Flow cytometric analysis of the membrane integrity
…………………………………………………….42
2.4 Discussion ………………………………………………....46
2.5 Conclusions .………….…………………………………....49
References …………………………………………………..…..50

CHAPTER 3 Overexpression and characterization of a recombinant agarase, Aga50D, from Saccharophagus degradans strain 2-40.……….57
Abstract …………………………………………………..…….58
3.1 Introduction …………………………………….….……..60
3.2 Materials and Methods ………………………….….…….64
3.2.1 Cloning of the aga50D from the marine bacterium S. degradans 2-40 .…………………………..…....64
3.2.2 Overexpression and purification of recombinant agarase ……………………………………………67
3.2.3 Enzyme assay……………………………………..68
3.2.4 Properties of AgaD ……………………………….68
3.2.5 Kinetic characterization of AgaD. ………………..69
3.2.6 Chromatographic analysis of the products of AgaD ……………………………………………………69
3.2.7 Liquid chromatography-tandem mass spectrometry
……………………………………………………………70
3.2.8 13C NMR spectroscopy……………………………..72
3.3 Results and Discussion …………………………………...73
3.3.1 Overexpression of recombinant agarase …………73
3.3.2 Effects of temperature and pH on the activity of the
recombinant agarase…………………………...…75
3.3.3 Effects of salt and metal ion on the activity of the
recombinant agarase ……………………………..78
3.3.4 Kinetic parameter of recombinant agarase ....…….78
3.3.5 Mode of action of recombinant agarase ………….82
3.3.6 Determination of the product by recombinant
agarase reaction …………………………………..87
3.4 Discussion ………………………………………………..91
3.5 Conclusions ………………………………………………97
References …………………………….………………………..98

CHAPTER 4 Conclusions ……………………………………………………109
SUMMARY IN KOREAN ………………………………………….………..112

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