Characterization, molecular modification, catalytic mechanism, and application of colanic acid-degrading enzyme from Escherichia phage phi92 = Escherichia phage phi92에서 유래한 콜라닌산 분해효소의 특성 분석, 분자 변형, 촉매 메커니즘 및 응용

다국어 초록 (Multilingual Abstract)

Colanic acid (CA), a polyanionic heteropolysaccharide synthesized by a variety of intestinal bacteria, has garnered significant interest due to its high fucose content and diverse biological activities, including antioxidant and anti- inflammatory properties. However, the high viscosity of CA poses significant challenges for its production and industrial applications. Colanic acid-degrading enzymes (CAEs) play a critical role in the bioconversion of CA by facilitating its breakdown into smaller oligosaccharides, thereby enhancing bioavailability and expanding its potential utility across industries. Despite their importance, research on CAEs remains limited, and their hydrolytic mechanisms are insufficiently studied, which restricts further optimization and broader application of these enzymes. In this study, CAEs with the potential to hydrolyze CA were screened through sequence mining. Four CAEs were successfully expressed, among which Gp150 from Escherichia phage phi92 demonstrated the highest enzymatic activity. The enzymatic properties and cleavage pattern of Gp150 were systematically investigated, and molecular modifications were applied to enhance its catalytic efficiency and stability. Additionally, site-directed mutagenesis was employed to explore the hydrolytic mechanisms of Gp150. Finally, Gp150 was utilized to hydrolyze CA to obtain CA with different molecular weights (MWs), and oxidative stress resistance, anti-inflammatory as well as anti-photoaging activities of CA with different MWs were evaluated. The main findings of this research are outlined as follows：
(1) Using bioinformatics and molecular modeling, five potential CAE sequences were identified, four of which were successfully expressed. Among these, Gp150 from Escherichia phage phi92 demonstrated the highest hydrolytic activity on CA. The Gp150 gene comprises 2415 base pairs and encodes with 804 amino acid protein and a theoretical molecular weight of 86.82 kDa. This protein was classified as stable and hydrophilic. It contained an immunoglobulin-like domain, a pectin lyase fold, and parallel beta-helix repeats. Optimization of expression conditions revealed that the optimal parameters for recombinant Gp150 were an induction temperature of 20℃, an IPTG concentration of 0.1 mM, and an induction duration of 24 h, resulting in peak enzyme activity. Kinetic analysis indicated that Gp150 has a Vmax of 686,460 nmol/min/mg, a Km of 19.04 mg/mL, and a Kcat of 1005.44 s−1, corresponding to a Kcat/Km ratio of 52.81 mL∙s−1∙mg−1. Additionally, enzyme activity was enhanced in the presence of 2 mM Ca2+, K+, and Na+, as well as 1 mM EDTA, whereas other metal ions and SDS were found to inhibit its activity.
(2) Gp150 enzymatically degraded CA through an endo-type hydrolysis pattern, successfully producing medium- and low-molecular-weight forms (CA-M and CA-L, respectively) as well as CA oligosaccharides (CAOSs). Structural analysis revealed that CAOSs comprise two main components: CAOS-6 (hexasaccharides) and CAOS-12 (dodecasaccharides), with CAOS-6 as the repeating unit. This unit consisted of glucose (Glc), galactose (Gal), fucose (Fuc), and glucuronic acid (GlA), along with O-acetyl and pyruvate modifications. Gp150 cleaves the β-1,4 glycosidic bond between glucose and fucose. Infrared spectroscopy and scanning electron microscopy (SEM) analyses confirmed significant structural alterations in CA following enzymatic hydrolysis. Rheological studies indicated that CA viscosity increased with concentration and exhibited shear-thinning behavior, reaching its maximum at pH 7.0 and decreasing with rising temperature. As the CA concentration increased, both the storage modulus (G′) and loss modulus (G″) rose, reflecting stronger intermolecular interactions. At low concentrations, CA displayed fluid-like behavior, while higher concentrations resulted in gel-like properties.
(3) Gp150 exists as a trimeric complex featuring a right-handed parallel β-helix domain, with its N-terminal stabilized by α-helical capping structures and its C-terminal forming an eight-stranded β-sandwich. The stability of Gp150 was improved through molecular modification by truncating the N-terminus. The mutant Gp150∆203 exhibited an increase in its melting temperature (Tm) by 8.73℃ compared to the wild type (WT). The active site of Gp150 contains a unique Glu-His catalytic dyad, with residues Y360, D387, E390, N420, W421, and Q365 identified as key contributors to substrate binding. Molecular docking analysis of CAOS-6 within the C-terminal cleft indicates that residues R457 and R518 play essential roles in facilitating substrate entry and product release from the enzyme channel.
(4) At a concentration of 0.25 mg/mL, CAOSs reduced the viability of RAW 264.7 cells by 25%, while other CA with different MWs exhibited no cytotoxic effects. In the H2O2-induced damage model, pretreatment with CA of various MWs before H2O2 stimulation significantly improved cell survival rates. Specifically, CAOSs, CA-L, CA-M, and CA-H enhanced cell viability by 53.61%, 24.51%, 35.40%, and 39.12%, respectively, compared to the H2O2 treatment group. Furthermore, CA with different MWs significantly suppressed the production of LPS-induced nitric oxide (NO) and reactive oxygen species (ROS). In LPS-stimulated macrophages, CAOSs, CA-L, CA- M, and CA-H reduced TNF-α secretion by 36.18%, 25.05%, 26.66%, and 34.64%, respectively, compared to the LPS group. CAOSs and CA-H decreased IL-6 secretion by 18.74% and 22.33%, respectively, relative to the LPS group. In HaCaT cells, CA with different MWs demonstrated no toxicity at concentrations ranging from 0.25 to 5 mg/mL. Exposure to UVB irradiation significantly induced a rapid increase in intracellular ROS levels, leading to cellular death. However, treatment with CA of different MWs demonstrated substantial ROS scavenging activity. Moreover, CA with different MWs alleviated UVB-induced damage in HaCaT cells by promoting the mRNA expression of Nrf2 and NQO1. Furthermore, CA with different MWs demonstrated distinct effects on the regulation of matrix metalloproteinases (MMPs, MMP-1 and MMP-9). In addition, CA treatment effectively inhibited UVB-induced upregulation of p53 and p21 protein expression, suggesting that CA mitigates photoaging not only by modulating extracellular matrix degradation but also by suppressing key regulators of the DNA damage response pathway.
In summary, the biological activity of CA is closely associated with its molecular weight, which plays a pivotal role in determining its physiological functions and therapeutic potential. A comprehensive understanding of the hydrolytic mechanisms of CAEs will not only facilitate their rational modification and enhance catalytic efficiency but also improve the bioavailability of CA. Furthermore, in-depth investigations into the biological properties of CA, particularly its anti-inflammatory and anti-photoaging effects, highlight its strong potential for therapeutic and cosmeceutical applications.

목차 (Table of Contents)

• Chapter 1. General introduction 1
• 1.1. Overview of colanic acid (CA) 1
• 1.1.1. Sources and structure of CA 1
• 1.1.2. Biosynthesis studies of CA 4
• 1.1.3. Applications of CA 8
• 1.2. Sources and research progress on colanic acid-degrading enzymes (CAEs) 9
• 1.3. Advances in enzyme molecular modification of enzymes 10
• 1.3.1. Directed evolution 10
• 1.3.2. Rational design and semi-rational design 11
• 1.3.3. Computer-assisted protein design 12
• 1.4. Objectives of this study 14
• Chapter 2. Mining, expression, and characterization of colanic acid-degrading enzymes 22
• 2.1. Introduction 22
• 2.2. Materials and methods 23
• 2.2.1. Materials 23
• 2.2.2. Mining for proteins with high-efficiency CA degradation activity 25
• 2.2.3. Cloning, expression and purification 26
• 2.2.4. Determination of protein concentration 28
• 2.2.5. Determination of enzyme activity 30
• 2.2.6. Bioinformatics analysis of Gp150 32
• 2.2.7. Optimization of Gp150 expression conditions 32
• 2.2.8. Enzymatic characterization of Gp150 34
• 2.2.9. Statistical analysis 35
• 2.3. Results and discussion 35
• 2.3.1. CAE mining 35
• 2.3.2. Expression of CAEs and enzyme activity analysis 40
• 2.3.3. Bioinformatics analysis of Gp150 45
• 2.3.4. Optimization of induction conditions for Gp150 48
• 2.3.5. Optimal reaction conditions and stability 54
• 2.3.6. Substrate specificity and kinetic parameters of Gp150 56
• 2.3.7. Effect of metal ions and chemical reagents on Gp150 activity 59
• 2.4. Conclusions 61
• Chapter 3. Cleavage pattern and preparation of colanic acid with different molecular weights and identification of hydrolysates 65
• 3.1. Introduction 65
• 3.2. Materials and methods 66
• 3.2.1. Materials 66
• 3.2.2. Determination of hydrolysis patterns 67
• 3.2.3. Screening of conditions for preparing CA with different MWs 67
• 3.2.4. Purification of CAOSs 68
• 3.2.5. Structural analysis of CAOSs 69
• 3.2.6. Physicochemical characterization of CA and CAOSs 70
• 3.2.7. Statistical analysis 71
• 3.3. Results and discussion 72
• 3.3.1. Cleavage pattern of Gp150 72
• 3.3.2. Preparation of various MWs of CA 74
• 3.3.3. CAOSs separation 77
• 3.3.4. Structural characterization of CAOS-6 and CAOS-12 79
• 3.3.5. Physicochemical analysis of CA and CAOSs 96
• 3.4. Conclusions 107
• Chapter 4. Molecular modification and catalytic mechanism of Gp150 111
• 4.1. Introduction 111
• 4.2. Materials and methods 112
• 4.2.1. Materials 112
• 4.2.2. Construction of Gp150 mutants 113
• 4.2.3. Transformation and sequence verification of Gp150 mutants 117
• 4.2.4. Expression and purification of Gp150 mutants 117
• 4.2.5. Determination of enzymatic activity of Gp150 mutants 118
• 4.2.6. Thermal shift analysis of Gp150 mutants 118
• 4.2.7. Multiple amino acid sequence alignment of CAEs 118
• 4.2.8. Molecular docking analysis 119
• 4.2.9. Statistical analysis 119
• 4.3. Results and discussion 119
• 4.3.1. Overall structure of Gp150 119
• 4.3.2. Effect of N-terminal amino acids truncation on CAE activity and stability 122
• 4.3.3. Analysis of the catalytic cleft and key amino acid residues in Gp150 126
• 4.3.4. Residues R457 and R518 in the catalytic function of Gp150 132
• 4.4. Conclusions 134
• Chapter 5. Anti-inflammatory and anti-photoaging effects of colanic acid with different molecular weights 138
• 5.1. Introduction 138
• 5.2. Materials and methods 139
• 5.2.1. Materials 139
• 5.2.2. Cell thawing, culture and passaging 140
• 5.2.3. Cell viability assay 141
• 5.2.4. Effect of CA on H2O2-induced oxidative stress 141
• 5.2.5. Anti-inflammatory effect of CA with different MWs 144
• 5.2.6. Anti-photoaging effects of CA with different MWs 145
• 5.2.7. Statistical analysis 148
• 5.3. Results and discussion 149
• 5.3.1. Protective effects of CA with different MWs on RAW 264.7 cells against oxidative stress and LPS-induced inflammation 149
• 5.3.2. Protective and anti-photoaging effects of CA with different MWs on skin photoaging induced by UVB irradiation 167
• 5.4. Conclusions 178
• Summary 184
• 요약 186
• Acknowledgment 189