Identification and Characterization of a Highly Active Hyaluronan Lyase from Enterobacter asburiae
<p>Typical time course of the growth of <span class="html-italic">Enterobacter</span> sp. CGJ001 in a shake flask. Enzyme activity (red, U/mL) and cell growth density (black, OD<sub>600</sub> values) were measured regularly. Values represent the mean of three replicates ± SD.</p> "> Figure 2
<p>The phylogenetic tree of strain <span class="html-italic">Enterobacter</span> sp. CGJ001 was constructed based on the analysis of 16S rDNA sequences, and the phylogenetic tree was generated by MEGA X using the neighbor–joining method.</p> "> Figure 3
<p>Whole-genome analysis of <span class="html-italic">E. asburiae</span> CGJ001: (<b>a</b>) Chromosome genome pattern of <span class="html-italic">E. asburiae</span> CGJ001. The circle diagram shows seven kinds of information, from outside to inside: The first circle is the genomic position information, the second circle is the GC content information, the third circle is the coding genes on the plus strand (red marks), the fourth circle is the coding genes on the minus strand (green marks), the fifth circle is the ncRNA information on the plus strand (blue marks), and the sixth circle is the ncRNA information on the minus strand (purple marks). The seventh circle is marked with information on long genomic repeats (orange marks). (<b>b</b>) KEGG metabolic pathway secondary classification <span class="html-italic">of E. asburiae</span> CGJ001. KEGG classified the biological metabolic pathways into 6 categories, and each category was systematically divided into secondary classifications. The number of genes in each metabolic pathway in the secondary classification was counted. (<b>c</b>) COG functional classification statistics <span class="html-italic">of E. asburiae</span> CGJ001. (<b>d</b>) GO functional classification statistics <span class="html-italic">of E. asburiae</span> CGJ001.</p> "> Figure 4
<p>The analysis of plasmid A: (<b>a</b>) Plasmid A genome pattern of <span class="html-italic">E. asburiae</span> CGJ001. (<b>b</b>) The PUL<sub>HA</sub> was anticipated in plasmid A of strain <span class="html-italic">E. asburiae</span> CGJ001.</p> "> Figure 5
<p>The expression of <span class="html-italic">hylEP0006</span>: (<b>a</b>) Colony PCR validation of the recombinant strain. Lane M: DNA marker; 1–10: PCR amplification bands of positive clones (the DNA fragment size of <span class="html-italic">hylEP0006</span>: 2379 bp). (<b>b</b>) SDS-PAGE of purified recombinant HylEP0006. Lane M, unstained protein molecular weight marker; lane <span class="html-italic">Hyl</span>, purified HylEP0006.</p> "> Figure 6
<p>The effects of temperature and pH on the enzymatic activity of HylEP0006: (<b>a</b>) Effect of temperature. The enzymatic activity of HylEP0006 was measured at 25–50 °C. HylEP0006 had the highest specific activity at 40 °C, equivalent to 100%. (<b>b</b>) Thermal stability of HylEP0006. The cells were incubated at different temperatures (30–50 °C) for 2 h, and the residual activity of HylEP0006 was determined at 40 °C. The initial specific activities of HylEP0006 were all set to be 100%. (<b>c</b>) Effect of pH. The enzymatic activity of HylEP0006 was assessed in a 50 mM buffer solution, comprising Na<sub>2</sub>HPO<sub>4</sub>-Citrate buffer (pH 3.0–5.0), NaH<sub>2</sub>PO<sub>4</sub>-Na<sub>2</sub>HPO<sub>4</sub> buffer (pH 6.0–8.0), and Glycine-NaOH buffer (pH 9.0–10.0). In NaH<sub>2</sub>PO<sub>4</sub>-Na<sub>2</sub>HPO<sub>4</sub> buffer (pH 7.0), HylEP0006 exhibited its maximum specific activity, which was recorded as being equivalent to 100%. (<b>d</b>) pH stability of HylEP0006. The residual activity of HylEP0006 was assessed at a temperature of 40 °C by subjecting it to incubation in the aforementioned buffer (with pH ranging from 3.0 to 9.0) for 2 h, maintaining the incubation temperature at 30 °C. The initial specific activities of HylEP0006 were all set to be 100%. Values represent the mean of three replicates ± SD.</p> "> Figure 7
<p>Enzymatic kinetic study of HylEP0006: (<b>a</b>) HylEP0006 degradation process of different concentrations of substrate figure. Values represent the mean of three replicates ± SD. (<b>b</b>) Lineweaver–Burk double reciprocal plot of HylEP0006.</p> "> Figure 8
<p>The analysis of HA digested by HylEP0006: (<b>a</b>) The final product of HA digested with HylEP0006 at 40 °C was analyzed by HPLC using a YMC-Pack Polyamine II column. (<b>b</b>) Analysis of the end product resulting from the digestion of HA with HylEP0006 using electrospray ionization mass spectrometry (ESI-MS).</p> "> Figure 9
<p>The analysis of HA4 degraded products by HylEP0006: (<b>a</b>) The product of HA4 degraded with HylEP0006 at 40 °C was analyzed by HPLC. (<b>b</b>) Scheme diagram of the cleavage of oligosaccharide HA4 substrates by HylEP0006.</p> "> Figure 10
<p>The analysis of HA10 degraded products by HylEP0006: (<b>a</b>) The product of HA10 degraded with HylEP0006 at 40 °C was analyzed by HPLC using a YMC-Pack Polyamine II column. (<b>b</b>) The oligosaccharide content of each product component during the initial 6 h degradation period of HA10 at 40 °C was analyzed by HPLC. Values represent the mean of three replicates ± SD. (<b>c</b>) Scheme diagram of the cleavage of oligosaccharide HA10 substrates by HylEP0006.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Isolation and Identification of Enterobacter sp. CGJ001
2.2. Whole-Genome Analysis of E. asburiae CGJ001 and Prediction of PULHA
2.3. Prediction of PULHA
2.4. Heterogenous Expression of hylEP0006
2.5. Biochemical Properties Characterization of HylEP0006
2.5.1. The Effects of Temperature and pH on Enzymatic Activity
2.5.2. Substrate Specificity of HylEP0006
2.5.3. Effects of Metal Ions on HylEP0006
2.5.4. Kinetic Constants of HylEP0006
2.6. Analysis of Final Degradation Product
2.7. Exploration of the Degradation Behavior of HylEP0006
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Methods
4.2.1. Isolation of Hyaluronate Lyase-Producing Bacteria
4.2.2. Enzyme Activity Assays
4.2.3. Identification of Strain Enterobacter sp. CGJ001
4.2.4. Genome Sequencing and Sequence Analysis
4.2.5. Sequence Analysis of HylEP0006
4.2.6. Molecular Cloning, Protein Expression, and Purification
4.2.7. Characterization of HylEP0006
4.2.8. Analysis of Kinetic Parameters
4.2.9. Analysis of Degradation Products of HylEP0006
4.2.10. The Degradation Behavior Exploration of HylEP0006
4.2.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Feature | Chromosome | Plasmid A |
---|---|---|
Size (bp) | 4,610,415 | 265,602 |
G + C content (%) | 55.91 | 48.33 |
Total genes | 4425 | 505 |
Protein-coding genes | 4193 | 505 |
rRNA | 25 | 0 |
tRNA | 82 | 0 |
Repeat genes | 200 | 0 |
Polysaccharide Species | Relative Enzyme Activity (%) |
---|---|
Hyaluronic acid | 100.00 ± 0.71 |
Chondroitin sulfate | 6.33 ± 1.57 |
Heparin | 0.88 ± 0.15 |
Sodium alginate | 0.43 ± 0.21 |
Chitosan | ND |
Metal Ions | Relative Enzyme Activity (%) | Metal Ions | Relative Enzyme Activity (%) | ||
---|---|---|---|---|---|
1 mmol/L | 10 mmol/L | 1 mmol/L | 10 mmol/L | ||
Control | 100.00 ± 2.51 | 100.00 ± 6.50 | Ba2+ | 92.06 ± 7.04 | 71.16 ± 2.54 |
Ca2+ | 117.52 ± 1.80 | 143.43 ± 4.11 | Zn2+ | 48.42 ± 0.26 | 12.48 ± 2.38 |
Mg2+ | 94.54 ± 3.15 | 152.22 ± 7.40 | K+ | 97.36 ± 2.57 | 74.96 ± 1.82 |
Li+ | 105.52 ± 0.77 | 65.93 ± 2.54 | Al3+ | 7.39 ± 1.80 | 9.86 ± 7.40 |
Cu2+ | 38.93 ± 0.58 | ND | Fe3+ | 75.96 ± 1.67 | ND |
Source | Molecular Mass | Substrate Spectrum | Optimal Temperature (°C) | Optimal pH | pH Stability | Assay Method | Specific Activity (U/mg) |
---|---|---|---|---|---|---|---|
E. asburiae CGJ001 (This study) | 87.88 kDa | HA | 40 | 7 | 6–9 | Reducing sugar method (DNS termination) | 9.50 × 105 |
Bacillus sp. A50 [21] | 123 kDa | HA, CS | 44 | 6.5 | 5–6 | Turbidimetric method | 1.02 × 106 |
Bacillus niacin JAM F8 [16] | 120 kDa | HA, CS | 45 | 6 | 6–11 | Ultraviolet method | 136.7 |
Brevibacterium halotolerans DC1 [22] | 41 kDa | HA, CS, DS, dermatan | 37 | 7 | 5–9 | Turbidimetric method | 26.37 |
Arthrobacter globiformis A152 [23] | 73.7 kDa | HA, CS | 42 | 6 | 5–7 | Ultraviolet method | 297.2 |
Streptococcus pyogenes bacteriophage H4489A [24] | 40 kDa | HA | 37 | 5.5 | 4–7 | Elson–Morgan-like method | 9.62 |
Paenibacillus aquistagni SH-7-A [25] | 110 kDa | HA | 40 | 6 | 5–7 | Ultraviolet method | 1.18 × 104 |
Bacillus sp. CQMU-D [26] | 126.2 kDa | HA, CS | 40 | 7 | 7–10 | Ultraviolet method | - |
Thermasporomyces copostie DSM22891 [27] | 90 kDa | HA | 70 | 5.93 | 6.1–10.9 | Ultraviolet method | 10.91 |
Yersinia sp. 298 [28] | 115.4 kDa | HA, CS | 40 | 7.5 | 6.0–11.0 | Ultraviolet method | 11.19 |
Escherichia sp. A99 [29] | 86.7 kDa | HA, CS | 40 | 6 | 5.5–6.6 | Ultraviolet method | 376.32 |
Homo sapiens [30] | 48.3 kDa | HA | - | 3.5–4.0 | - | Elson–Morgan-like method | 6.8 |
Bos grunniens [31] | 55 kDa | HA, CS and DS | 37 | 3.8 | - | Elson–Morgan-like method | 20.4 |
Lachesis muta rhombeata [32] | 60 kDa | - | 37 | 6 | - | Turbidimetric method | - |
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Zhang, L.; Jiang, J.; Liu, W.; Wang, L.; Yao, Z.; Li, H.; Gong, J.; Kang, C.; Liu, L.; Xu, Z.; et al. Identification and Characterization of a Highly Active Hyaluronan Lyase from Enterobacter asburiae. Mar. Drugs 2024, 22, 399. https://doi.org/10.3390/md22090399
Zhang L, Jiang J, Liu W, Wang L, Yao Z, Li H, Gong J, Kang C, Liu L, Xu Z, et al. Identification and Characterization of a Highly Active Hyaluronan Lyase from Enterobacter asburiae. Marine Drugs. 2024; 22(9):399. https://doi.org/10.3390/md22090399
Chicago/Turabian StyleZhang, Linjing, Jiayu Jiang, Wei Liu, Lianlong Wang, Zhiyuan Yao, Heng Li, Jinsong Gong, Chuanli Kang, Lei Liu, Zhenghong Xu, and et al. 2024. "Identification and Characterization of a Highly Active Hyaluronan Lyase from Enterobacter asburiae" Marine Drugs 22, no. 9: 399. https://doi.org/10.3390/md22090399