Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain
Abstract
:1. Introduction
2. Results
2.1. Bioinformatic Analysis of VvGH17
2.2. Biochemical Properties of VvGH17
2.3. C-terminal-Truncated Mutant
2.4. Affinity Gel Analysis of C-Terminal-Truncated Mutants of VvGH17
2.5. Affinity Gel Analysis of Uk-C and Point Mutants of VvGH17
2.6. Prediction of Uk-C Structure and Function
2.7. N-Terminal-Truncated Mutants of VvGH17
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Bioinformatic Analysis of VvGH17
4.3. Construction, Expression, and Purification of VvGH17
4.4. Construction of VvGH17 Mutants
4.5. VvGH17 Standard Activity Assay
4.6. Analysis of Hydrolysis Products by TLC and Gel Filtration Chromatography
4.7. CD Spectroscopy
4.8. Polyacrylamide Gel Electrophoresis (PAGE) Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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kcat/Km (mM−1 s−1) | kcat (s−1) | Km (mM−1) | |
---|---|---|---|
VvGH17 | 93.0 | 148 | 1.60 |
ΔC466 | 35.3 | 81.7 | 2.49 |
ΔC441 | 32.7 | 87.6 | 2.87 |
ΔC415 | 0.2 | 1.4 | 9.39 |
ΔN50 | 67.5 | 143.3 | 2.12 |
ΔN65 | 24.7 | 86.8 | 3.52 |
Primer Name | Primer Sequence (5′-3′) | Purpose |
---|---|---|
ΔC466-S | TGATGGCAAGCTTGCGGCCGCACTC | Truncation of 149 AAs from C-terminus |
ΔC466-AS | GCAAGCTTGCCATCAAACGCGCCGGC | |
ΔC441-S | GACCGGCAAGCTTGCGGCCGCACTC | Truncation of 174 AAs from C-terminus |
ΔC441-AS | GCAAGCTTGCCGGTCAGTAATGCACT | |
ΔC415-S | TGCTCCGTGAAAGCTTGCGGCCGCACTC | Truncation of 200 AAs from C-terminus |
ΔC415-AS | GCAAGCTTTCACGGAGCAAGAACGGAAT | |
ΔN50-S | CCATATGGGCAACTATCCGACAGCT | Truncation of 50 AAsf rom N-terminus |
ΔN50-AS | TAGTTGCCCATATGGCTGCCGCGCGG | |
ΔN65-S | CCATATGGGCAACGCGAATTATCCG | Truncation of 65 AAs from N-terminus |
ΔN65-AS | GCGTTGCCCATATGGCTGCCGCGCGG | |
UK-C-S | CCATATGGCCGGCGCGTTTGATGGC | Truncation of 410 AAs from N-terminus |
UK-C-AS | GCGCCGGCCATATGGCTGCCGCGCGG | |
W472A-S | ATCGCAgcgGAAGGTACCGCCTATCTG | Mutation of Trp472 to Ala472 |
W472A-AS | ACCTTCcgcTGCGATCGCTTCCCCGCC | |
W499A-S | TGGGGTgcgGGAGCGGGCGTCGTGCTC | Mutation of Trp499 to Ala499 |
W499A-AS | CGCTCCcgcACCCCAGTCTTTTGCAGT | |
W542A-S | GGCCTGgcgGGCAACAACGACCGTCCG | Mutation of Trp542 to Ala542 |
W542A-AS | GTTGCCcgcCAGGCCGGTCTGAAATCC | |
W567A-S | ACCGAAgcgACAGCCTACACGATTCCG | Mutation of Trp567 to Ala567 |
W567A-AS | GGCTGTcgcTTCGGTTGAAATGGCACG |
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Kumagai, Y.; Kishimura, H.; Lang, W.; Tagami, T.; Okuyama, M.; Kimura, A. Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain. Mar. Drugs 2022, 20, 250. https://doi.org/10.3390/md20040250
Kumagai Y, Kishimura H, Lang W, Tagami T, Okuyama M, Kimura A. Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain. Marine Drugs. 2022; 20(4):250. https://doi.org/10.3390/md20040250
Chicago/Turabian StyleKumagai, Yuya, Hideki Kishimura, Weeranuch Lang, Takayoshi Tagami, Masayuki Okuyama, and Atsuo Kimura. 2022. "Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain" Marine Drugs 20, no. 4: 250. https://doi.org/10.3390/md20040250