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Biological conversion of marine biomass-derived renewable carbonaceous materials into functional products for advanced biorefinery application

Biological conversion of marine biomass-derived renewable carbonaceous materials into functional products for advanced biorefinery application

자료유형
학위논문
개인저자
정다운, 鄭多云
서명 / 저자사항
Biological conversion of marine biomass-derived renewable carbonaceous materials into functional products for advanced biorefinery application / Da Woon Jeong
발행사항
Seoul :   Greduate School, Korea university,   2021  
형태사항
iii, 162장 : 삽화, 도표 ; 26 cm
기타형태 저록
Biological Conversion of Marine Biomass-derived Renewable Carbonaceous Materials into Functional Products for Advanced Biorefinery Application   (DCOLL211009)000000235567  
학위논문주기
학위논문(박사)-- 고려대학교 대학원: 생명공학과, 2021. 2
학과코드
0510   6YD48   528  
일반주기
지도교수: 한성옥  
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참고문헌: 장 146-156
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비통제주제어
Enzyme Complex , Functional Products , Marine Biomass,,
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001 000046072076
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008 210102s2021 ulkad bmAC 000c eng
040 ▼a 211009 ▼c 211009 ▼d 211009
041 0 ▼a eng ▼b kor
085 0 ▼a 0510 ▼2 KDCP
090 ▼a 0510 ▼b 6YD48 ▼c 528
100 1 ▼a 정다운, ▼g 鄭多云
245 1 0 ▼a Biological conversion of marine biomass-derived renewable carbonaceous materials into functional products for advanced biorefinery application / ▼d Da Woon Jeong
260 ▼a Seoul : ▼b Greduate School, Korea university, ▼c 2021
300 ▼a iii, 162장 : ▼b 삽화, 도표 ; ▼c 26 cm
500 ▼a 지도교수: 한성옥
502 1 ▼a 학위논문(박사)-- ▼b 고려대학교 대학원: ▼c 생명공학과, ▼d 2021. 2
504 ▼a 참고문헌: 장 146-156
530 ▼a PDF 파일로도 이용가능; ▼c Requires PDF file reader(application/pdf)
653 ▼a Enzyme Complex ▼a Functional Products ▼a Marine Biomass
776 0 ▼t Biological Conversion of Marine Biomass-derived Renewable Carbonaceous Materials into Functional Products for Advanced Biorefinery Application ▼w (DCOLL211009)000000235567
900 1 0 ▼a Jeong, Da-woon, ▼e
900 1 0 ▼a 한성옥, ▼g 韓盛鈺, ▼e 지도교수
900 1 0 ▼a Han, Sung-ok, ▼e 지도교수
945 ▼a KLPA

전자정보

No. 원문명 서비스
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Biological conversion of marine biomass-derived renewable carbonaceous materials into functional products for advanced biorefinery application
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No. 소장처 청구기호 등록번호 도서상태 반납예정일 예약 서비스
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컨텐츠정보

초록

The use of marine algae has many advantages for the production of food additives compared to the use of biomaterials based on starch or lignocellulose. Among them, agars are a family of complex polysaccharides found inside some red algae's cell walls. The bacterial expansin BpEX from Bacillus pumilus was fused with the dockerin module of a cellulosome system for assembly with agarolytic complexes. The assembly of chimeric expansin caused an indicative enhancement in agarase activity. The enzymatic activities on agar substrate and natural biomass were 3.7-fold and 3.3-fold higher respectively than that of agarase as a single enzyme. To validate the effect on the agar degradation, the regulation potential of parameters related to gel rheology by bacterial expansin was experimentally investigated to indicate that the bacterial expansin lowered the gelling temperature and viscosity of agar. Also, the construction of agarolytic complexes composed of L-arabinose isomerase caused efficient isomerization activity on agar-derived sugars. Agar is a major component of the red algae and can be hydrolyzed into monomeric sugars, such as D-galactose and 3,6-anhydro-L-galactose. In a trienzymatic complex, the chimeric β-agarase (cAgaB) and anhydro-galactosidase (cAhgA) from Zobellia galactanivorans could synergistically hydrolyze natural agar substrates and L-arabinose isomerase isomerize the D-galactose into the D-tagatose. A trienzymatic complex increased the concentration of D-tagatose from agar substrate to 4.2 g/L. Compared with the monomeric enzyme, the multimeric enzyme showed a 1.4-fold increase in tagatose production. Thus, these results demonstrated the possibility of advancing more efficient strategies for utilizing agar as oligo sugar source in the biorefinery field that uses marine biomass as feedstocks. Alginate can be enzymatically degraded by alginate lyases into uronate monomers and it can be non-enzymatically converted to unsaturated monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Here we constructed alginolytic enzyme complex based on the principle of cellulosome from Clostridium cellulovorans for the synergistic effect on the alginate degradation. Compared with the monomeric enzyme, the multimeric enzyme showed a 1.85-fold increase in DEH production. Final product DEH can be used as a key substrate for the synthesis of biofuels and commodity chemicals such as bioethanol by using metabolically engineered microorganism. Alginate-derived DEH was utilized by Ralstonia eutropha as sole carbon sources to produce polyhydroxybutyrate (PHB). This biological strategies may be a new potential conversion tool to produce biodegradable PHB from natural biomass.

목차

CONTENTS
Page
LIST OF TABLES	i
LIST OF FIGURES	iii
LIST OF ABBREVIATIONS	viii

ABSTRACT	1

CHAPTER I.
GENERAL INTRODUCTION	4
1.1. Marine Biomass as the Source for Biorefinery	5
1.2. Enzyme Complex as Biological Tools	15
1.2.1. Agarolytic Complexes	16
1.2.2. Carrageenolytic Complexes	18
1.2.3Alginolytic Complexes	22
1.3. Functional Products Produced from Marine Biomass	23

CHAPTER II. 
Integration of Bacterial Expansin on Agarolytic Complexes to Enhance the Degrading Activity of Red Algae by Control of Gelling Properties
2.1. Abstract	31
2.2. Introduction	 32
2.3. Materials and methods	36
2.3.1. Bacterial Strains, Plasmids, and Media	36
2.3.2. DNA Manipulations	39
2.3.3. Overlap PCR and Plasmid Construction for Protein Expression	39
2.3.4. Protein Expression from the Recombinant Strains	42
2.3.5. Binding Assay to Insoluble Substrates	43
2.3.6. Complex Assembly and Activity Assay of Enzymes	44
2.3.7. Gel Phase Transition Analysis	45
2.4. Results	47
2.4.1. Design and Expression of Chimeric Expansin for the Assembly of Complexes	47
2.4.2. Synergic Hydrolysis of Agarolytic Complex Containing Bacterial Expansin	52
2.4.3. Gel Phase Transition of Bacterial Expansin by Hydrogen Bond Disruption	56
2.5. Discussion	62
2.6. Conclusion	64

CHAPTER III. 
A Trienzymatic Complex System for Isomerization of Agar-derived D-Galactose into D-Tagatose as a Low-calorie Sweetener 
3.1. Abstract	67
3.2. Introduction	69
3.3. Materials and methods	75
3.3.1. Reagents and Chemicals	75
3.3.2. Strains and Plasmids	78
3.3.3. Plasmid Construction	78
3.3.4 Culture medium and Conditions	80
3.3.5. Expression and Purification of Recombinant Proteins	80
3.3.6. Assembly of Enzyme Complexes for D-tagatose Production and CBM-Assisted Affinity Purification	82
3.3.7. Comparison of Hydrolytic Activity of Enzymes	83
3.3.8 L-Arabinose Isomerase Enzyme Assays	84
3.3.9. Thermostability and Reusability of the Enzyme Complexes	86
3.4. Results	87
3.4.1. Design of Chimeric L-arabinose Isomerase for the Assembly of Complexes	87
3.4.2. Synergistic Effect of the Agarolytic Complex Containing L-arabinose Isomerase	92
3.4.3. Enhanced Performances of the Agarolytic Complex Containing L-Arabinose Isomerase	98
3.4.4. Isomerization of Natural Substrates Extracted from Various Red Algae	 	102

CHAPTER IV. 
Efficient Brown Algae Degradation by Alginolytic Enzyme and Its Utilization as Carbon Source for Production of Polyhydroxybutyrate (PHB) from Ralstonia eutropha 
4.1. Abstract	110
4.2. Introduction	112
4.3. Materials and Methods	118
4.3.1. Binding Assay to Insoluble Substrates	118
4.3.2. Strains and Plasmids	118
4.3.3. Plasmid Construction	119
4.3.4. Culture Medium and Conditions	120
4.3.5. Expression and Purification of Recombinant Proteins	121
4.3.6. Assembly of Enzyme Complexes for DEH Production and CBM-utilizing Purification	123
4.3.7. Alginate Lyase Enzyme Assays	124
4.3.8. Thermostability and Reusability of the Enzyme Complexes	125
4.3.9. Culture Conditions of R. eutropha and PHB Analysis	125
4.4. Results	128
4.4.1. Design of Chimeric Alginate Lyase for the Assembly of Complexes	  	128
4.4.2. Synergistic Effect of the Alginolytic Complex	131
4.4.3. Enhanced Performances of the Alginolytic Complex	134
4.4.4. Degradation of Natural Substrates Extracted from Various Brown Algae	136
4.5. Discussion	140
4.6. Conclusion	142

REFERENCES	144
ABSTRACT IN KOREAN	153
ACKNOWLEDGEMENT	156