Integration of stability and activity for Candida antarctica lipase B and its covalent immobilization on a modified sol-gel matrix for biodiesel production

• Main Author: 카밀라
• Other Authors: Ji Sook Hahn, Camila Flor J. Yagonia, 공과대학 화학생물공학부
• Format: Thesis
• Language: English
• Published: 서울대학교 대학원 2015
• Subjects: Candida antarctica lipase B; enzyme flexibility; enzyme stability; mutagenesis; sol-gel matrix; covalent immobilization; 660; Antarc*; Antarctica
• Online Access: http://hdl.handle.net/10371/119742

학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2015. 2. Ji Sook Hahn. Lipases play a vital role in various processes. Lipase-mediated processes can be done in mild reaction conditions requiring less energy, faster reaction rates, a cheaper starting materials can be utilized since lipases are selective and wastewater treatment/downstream processing pose no problem for lipases are biodegradable. Even though the use of lipase provides many advantages, their industrial application is hindered due to their expensive cost. In addition, lipases have low activity with long chain fatty acids as substrate and they are easily inhibited by short chain alcohol which leads to their shorter life span when applied as biocatalyst. A lipase with improved characteristics is in demand for a biochemical process to be economically feasible. In this study, structural flexibility modulation and immobilization were carried out in order to enhance the functionality of Candida antarctica lipase B (CALB). The dynamics, structure configuration and functional groups of CALB were used to obtain an enzyme that shows higher activity and robustness. The dynamics of CALB in terms of its flexibility was modulated to improve the activity and stability. Structure configuration was also considered to maximize the effect and impact of mutation. Functional groups specifically exposed lysine residues were exploited to immobilize CALB on a modified sol-gel matrix. Both stability and activity improvements were incorporated in Candida antarctica lipase B (CALB) through multiple-site mutagenesis. CALB was divided into two different regions to optimize its performance. Modulating the flexibility within the substrate-binding region and the hydrophilic solvent-affecting region enhanced the catalytic activity and organic solvent stability of CALB, respectively. Combining the mutation sites from the substrate-binding region and from the hydrophilic solvent-affecting region yielded an enzyme (V139E,A92E) with improved functionality. The use of modified sol-gel matrix to immobilize CALB was investigated. Free hydroxyl groups on the matrix surface were exploited to covalently immobilize the enzyme. Based from the results, incorporating hydrophobic sol-gel precursor (ethyltrimethoxysilane) enhanced enzyme activity. An enzyme activity of 192.02 U/g beads with 80.88% attachment was obtained. At alkaline pH, immobilization yield of enzyme increased. The attachment of enzyme on the surface of the matrix was confirmed by scanning electron microscope images. Covalently immobilized CALB on sol-gel supports yielded higher thermal stability with 2.7 times higher half-life compared with soluble enzymes at 60oC. This enzyme immobilization system retained the enzyme residual activity even for repetitive use. The resultant enzyme with enhanced functionality was covalently immobilized on the modified sol-gel matrix and its performance on biodiesel production was tested. The biodiesel production was approximately 3.7 times higher using the immobilized double mutant (V139E,A92E) enzyme compared with the immobilized wild type CALB. Optimization on the immobilized enzyme system using the double mutant (V139E,A92E) can be further investigated to further increase the biodiesel production. The technique applied in this study can also be extended to other lipases. Abstract i List of Tables vii List of Figures ix Chapter 1: Introduction 1 1.1 Backgrounds 2 1.2 Research Objectives 4 Chapter 2: Literature Review 6 2.1 Microbial Lipases 7 2.2 Functional Motions of Enzymes 10 2.3 Enzyme Immobilization 17 2.3.1 Adsorption 20 2.3.2 Covalent Immobilization 21 2.3.3 Cross-linking 22 2.3.4 Entrapment 24 2.4 Sol-Gel Material for Enzyme Immobilization 25 2.4.1 Sol-Gel Process Steps 26 2.4.2 Sol-Gel Entrapment 30 2.5 Biodiesel 33 2.5.1 Biodiesel Production Methods 34 2.5.2 Factors Affecting Biodiesel Production 39 Chapter 3: Integration of Activity and Stability for Candida antarctica lipase B through multiple-site mutagenesis 44 3.1 Introduction 45 3.2 Materials and Methods 47 3.2.1 Materials 47 3.2.2 Cloning and transformation of CALB variants 47 3.2.3 CALB expression and purification 50 3.2.4 Activity assay and stability measurements 51 3.2.5 In silico mutagenesis and flexibility analysis 52 3.3 Results and Discussion 53 3.3.1 Selection of mutation sites 53 3.3.2 Activity enhancement of the substrate-binding region 57 3.3.3 Stability enhancement of the hydrophilic solvent-affecting region 63 3.3.4 Integration of mutation sites for activity and stability enhancements 69 3.3.5 Computational analysis between activity and stability 73 3.4 Conclusion 86 Chapter 4: Immobilization of Candida antarctica lipase B on the surface of modified sol-gel matrix 87 4.1 Introduction 88 4.2 Materials and Methods 91 4.2.1 Materials 91 4.2.2 Support preparation and activation 91 4.2.3 Enzyme immobilization 92 4.2.4 Enzyme activity assay 93 4.2.5 Protein determination 94 4.2.6 Stability determination 96 4.3 Results and Discussion 96 4.3.1 Influence of sol-gel matrix size on attachment and enzyme activity 99 4.3.2 Sol-gel matrix composition 101 4.3.3 Influence of enzyme loading on activity 104 4.3.4 Influence of pH on immobilization 108 4.3.5 Stability of the immobilized CALB 110 4.3 Conclusion 113 Chapter 5: Application for functionally enhanced Candida antarctica lipase B immobilized on modified sol-gel matrix 115 5.1 Introduction 116 5.2 Materials and Methods 119 5.2.1 Materials 119 5.2.2 Production of functionally enhanced lipase 119 5.2.3 Enzyme immobilization 120 5.2.4 Biodiesel production and analysis 120 5.3 Results and Discussion 121 4.3 Conclusion 127 Chapter 6: Overall Discussion and Recommendations 128 References 133 국문초록 (Abstract in Korean) 148 Doctor