Characterization of β-1,3-1,4-glucanase produced from halotolerant marine Bacillus sp. and improvement of enzymatic properties using site-directed mutagenesis
- Alternative Title
- 해양 유래 내염성 Bacillus가 생산하는 β-1,3-1,4-glucanase의 생화학적 특성 및 부위 지정 돌연변이를 이용한 열 안정성과 염 안정성 개선에 관한 연구
- Abstract
- The aim of this study was to analyze the complete genome sequence of the halotolerant bacterium isolated from a traditional Korean food, i.e., salt-fermented fish (Jeotgal), and to investigate the biochemical characterization of the β-1,3-1,4-glucanase produced by this bacterium.
Pacific Biosciences (PacBio) RSІІ sequencing was used for complete genome sequencing. Additional analyzes were performed using the Hierarchical Genome Assembly Process (HGAP) version 2.3 and the Rapid Annotation using Subsystem Technology (RAST) server. Complete genome sequence of Bacillus sp. SJ-10 has a single circular chromosome of 4,041,649 bp with a guanine-cytosine content of 46.39%. The genome sequence contains 3,582 protein-coding genes and contains commercially useful enzymes such as protease, beta-glucosidase and beta-glucanese.
Gene cloning was performed to investigate the biochemical properties of β-1,3-1,4-glucanase, an enzyme produced by this strain. This enzyme showed the highest activity when β-D-glucan of barley was used as a substrate. The optimal temperature and pH of the purified enzyme towards barley b-glucan were 50 °C and pH 6, respectively. More than 80 % of activity was retained at temperatures of 30–70 °C and pH values of 4–9, which differed from all other bacterial β-1,3-1,4-glucanase.
It was found that 48th amino acids of β-1,3-1,4-glucanase were different with other reported glucanase sequences. It was a lysine, while the other glucanase was a glutamine residue, a hydrophilic amino acid. Analysis of 3D-structure prediction and amino acid residue interactions showed that the 48th amino acid residue is located in the first beta sheet of the substrate binding site and will contribute to the stability of glucanase. Thus, the 48th amino acid residue was substituted with a hydrophobic amino acid residue using a site directed mutagenesis method. The catalytic efficiency, thermostability, and halostability of the recombinant proteins in which the 48th amino acid residues were substituted with alanine and leucine, respectively, were increased.
These results can be applied to industrial fields requiring high catalytic activity at high temperature and salt conditions, and can be used as basic data for enzyme modification and protein engineering research.
- Issued Date
- 2019
- Awarded Date
- 2019. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 생물공학과
- Advisor
- 공인수
- Table Of Contents
- 1. Chapter1 1
1.1. Properties of β-glucan 2
1.2. Properties of β-1,3-1,4-glucanase 3
1.3. Purpose of this study 4
References 5
2. Chapter2 6
2.1. Introduction 8
2.2. Materials and methods 9
2.2.1. Bacterial isolation and general characteristics 9
2.2.2. Genome Sequencing, assembly and annotation 9
2.3. Results and discussion 11
2.4. Conclusion 18
References 15
3. Chapter3 28
3.1. Introduction 30
3.2. Material and methods 32
3.2.1. Bacterial strains, plasmid, media, and growth conditions 32
3.2.2. Sequencing of β-1,3-1,4-glucanase from Bacillus sp. SJ-10 32
3.2.3. Synthesis of the gene and construction of the expression vector 33
3.2.4. Overexpression and purification of β-1,3-1,4-glucanase 33
3.2.5. β-1,3-1,4-glucanase activity assay 33
3.2.6. Determination of enzyme degradation products by thin-layer chromatography 34
3.3. Results and discussion 35
3.3.1. Analysis of the nucleotide and amino acid sequences of bg1314 35
3.3.2. Purification of β-1,3-1,4-glucanase 35
3.3.3. Substrate specificity 37
3.3.4. Enzyme assay and stability test 39
3.3.5. TLC analysis 44
3.4. Conclusion 45
References 47
4. Chapter4 51
4.1. Introduction 53
4.2. Materials and methods 55
4.2.1. Bacterial strains and growth conditions 55
4.2.2. Molecular modeling, docking and calculation of intramolecular interactions 55
4.2.3. Site-directed mutagenesis 56
4.2.4. Expression and purification 56
4.2.5. β-glucanase activity 57
4.2.6. Kinetic analysis 57
4.2.7. Binding affinity experiment 57
4.2.8. Thermostability and thermodynamic parameters 58
4.2.9. Effects of salt on β-glucanase activity and halostability 59
4.3. Results and discussion 60
4.3.1. Amino acid sequence analysis of β-1,3-1,4-glucanase 60
4.3.2. Homology modeling of Bg1314 62
4.3.3. Choosing the amino acid residues for site-directed mutagenesis 64
4.3.4. Effects of the mutations on kinetic parameters 65
4.3.5. Effects of the mutations on the thermostability and thermodynamics 69
4.3.6. Effects of the mutations on the salt 72
4.4. Conclusion 78
References 79
Abstract (in Korean) 83
Acknowledgements 85
- Degree
- Doctor
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