방선균 유래의 락케이즈 Sv1의 클로닝, 발현을 통한 양모 염색 균주의 개발 및 자가 치유가능 PU 생성을 위한 폴리올 사슬 연장체의 합성

학위논문 (석사) -- 서울대학교 대학원 : 공과대학 화학생물공학부, 2020. 8. 김병기.Polyurethane (PU) has 6 % of the total global polymer market resulted from versatile applications. The development of biodegradable and sustainable material is essential to solve the serious environmental problem in petrochemical polyol and demand of biological content increase. Aromatic monomers in PU are usually made from petroleum, exerting serious toxicity in human neural system as well as depriving of fossil resources. In this research, we provided byproduct derived from lignin decomposition as aromatic chain extenders, which can replace 1,4-butanediol. Lignocellulos biomass monomer, vanillin-based polyol chain extender gives excellent mechanical properties to the PU elastomer.1 Herein, we expanded perspectives into developing environmental PU elastomer with self-healing property. We reported enzymatic and chemical synthesis methods of new eugenol based compound, dieugenol-based hexaol. Dieugenol based polyurethane has 6.3 Mpa of tensile strength and 0.016 Mpa of Youngs modulus, and -75.6 ℃ of glass transition temperature. Eugenol based polyurethane has reversible urethane bond after heat treatment for 3 hours at 150 ℃, the PU elastomer had the self-healing efficiency of 84.7 %. Recently, bio-inspired dyeing methods using oxidative enzymes such as laccase, peroxidase have been used to enhance the color depth of fabrics. Here, a novel and eco-friendly dyeing method using recombinant Streptomyces glaucescens was investigated, which has a great potential for application in textile processes. Especially, we used both melanin- and laccase-secreting cells instead of purified laccase for applying this system to cost effective industrial-grade biocatalysts for coloration. At first, we evaluated a performance of laccase secretion as well as a dye production in the supernatant of liquid cultures of the transformed cells. Dosages of inductor and induction time were optimized for effective laccase secretion and dye production. Afterwards, cell supernatants containing laccase and melanin were exploited for coloration of wool fabrics. The properties of colored wool fabrics were evaluated in terms of color depth and color fastness. Also, binding of the melanin with wool fabric was verified with Fourier transform infrared spectroscopy, surface morphology, and color fastness test. These results would provide an inspiration in extending research activities on this one-pot cellular system and in replacing isolated enzyme system.락케이즈는 미생물에서부터 고등생물까지 광범위하게 발견되고 라디칼 산화환 반응을 촉매한다. 라케이즈는 목질계 리그닌 바이오매스로부터 다양한 페놀류 단량체의 생합성에 중요한 역할을 하는 엔자임이다. 그러나, 많은 연구에서 버섯 유래 락케이즈의 최적 pH가 산성이어서 실질적인 적용이 어렵다고 보고되어 있다. 본 연구논문은 Streptomyces viridosporus 유래의 락케이즈 Sv1을 대장균 및 방선균에서 발현하여 신규 물질의 생합성 및 염색 균주의 개발에 적용하였다. 대장균에서 발현된 Sv1 효소는 다양한 유기용매에서 뛰어난 안정성을 보였고, 넓은 pH 범위에서 높은 활성을 보여주었다. 이를 바탕으로, 친환경적이고 효율적인 염색 미생물 균주를 개발하기 위해 R2YE 복합 배지에서 멜라닌을 다량 분비하는 S.glaucescens 균주에 외래 Sv1 유전자를 도입한 균주를 제작하였다. 적외선분광법과 전자 현미경 분석법을 통해 Sv1이 염료를 양모에 공유 결합으로 염착시킴을 확인할 수 있었다. 결과적으로 이러한 염색 균주를 사용하여 색차를 5 배 증진 시키고 색상 견뢰도를 한 단계 향상시킬 수 있었다. 락케이즈의 다른 사용예로서는, 자가치유 가능 폴리우레탄의 사슬 연장제로 사용 가능한 Dieugenol-based hexaol의 합성 과정 중간체인 에폭시 유지놀 이량체를 sv1을 이용하여 합성하였다. Sv1은 에폭시 유지놀의 82 % 을 이량체화 시킬 수 있었다. 그러나, 에폭사이드의 고리열림반응 효율이 낮아서 이후로는 화학적인 변형을 통해 Dieugenol-based hexaol을 합성하였고 핵자기공명분석법과 기체 크로마토그래피 질량 분석법을 통해 생성됨을 확인하였다. Dieugenol-based hexaol을 도입한 폴리우레탄은 84.7 %의 우수한 자가치유특성을 띰을 확인할 수 있었다.CHAPTER ⅠCloning, expression, and purification of a small laccase Sv1 from Streptomyces viridosporus 11 1.1. INTRODUCTION 12 1.2. MATERIALS AND METHODS 14 1.2.1. Chemicals and materials 14 1.2.2. Protein expression 14 1.2.3. Laccase activity assay 15 1.3. RESULTS AND DISCUSSION 16 1.3.1. Soluble expression and purification of laccase 16 1.3.2. Optimal conditions for laccase activity 16 3.5.1. Effect of pH. 16 3.5.2. Effect of temperature 17 3.5.3. Effect of organic solvents 20 1.4. CONCLUSION 23 CHAPTER ⅡLaccase catalyzed dimerization of eugenol oxide for potential polyol extender of robust, self-healable polyurethane elastomer 24 2.1. INTRODUCTION 25 2.2. MATERIALS AND METHODS 30 2.2.1. Chemicals and materials 30 2.2.2. Synthesis of dieugenol-based hexaol 31 2.2.3. Synthesis of polyurethane film 35 2.2.4. Characterization of polyurethane film 36 2.3. RESULTS AND DISCUSSION 37 2.3.1. Preparation of dieugenol-based hexaol 37 2.3.2. Chemical identification of polyurethane films 40 2.3.3. Properties of eugenol-based polyurethane elastomer 41 2.3.3.1. Self-healabiling property of the polyurethane elastomer 41 2.3.3.2. Other physical properties of the polyurethane elastomer. 45 2.4. CONCLUSION 47 CHAPTER Ⅲ Laccase mediated one-pot dyeing of wool fabrics using recombinant Streptomyces gluacescens 50 3.1. INTRODUCTION 51 3.2. MATERIALS AND METHODS 53 3.2.1. Cell culture conditions 53 3.2.2. Plasmid and strains construction 54 3.2.3. Protein expression 56 3.2.4. Coloration and color measurement 56 3.2.5. Dye pigment and colored wool fabric analysis 57 3.3. RESULTS AND DISCUSSION 58 3.3.1. Overexpression and secretion of laccase using a heterologous system in Streptomyces glaucescens 59 3.3.2. Enhanced colorization of wool fabrics by the transformant 62 3.3.3. Effect of Sv1 on structure of the dye pigment and the colored wool fabrics 67 3.5.2. FTIR analysis of dyed wool fabrics 68 3.5.3. Surface morphology of dyed wool fabrics 69 3.4. CONCLUSION 70 APPENDIX Site-specific o-methylation of EGCG using Bacillus o-methyltransferases 79 S1. INTRODUCTION 80 S2. MATERIALS AND METHODS 82 S2.1. Cell culture conditions 82 S2.2. Plasmid construction 83 S2.3. Expression and purification of the enzymes 83 S2.4. Determination of the enzyme activity 84 S2.5. Purification and NMR spectroscopy of o-methylated EGCGs 86 S3. RESULTS AND DISCUSSION 87 S3.1. Expression and purification of recombinant o-methyltransferases from Bacillus in E.coli system 87 S3.2. Enzyme activity and regiospecificity on EGCG 88 S3.3. Optimization of EGCG3"me production 93 S4. CONCLUSION 98 4. REFERENCES 99 국문초록 106Maste