Comparative Potential of Chitinase and Chitosanase from the Strain Bacillus thuringiensis B-387 for the Production of Antifungal Chitosan Oligomers
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials and Reagents
2.2. Methods
2.2.1. Bacterial Strain, Its Maintenance, and Cultivation Media
2.2.2. Purification of Chitinase/Chitosanase and Protein Measurement
2.2.3. Enzyme Activity Assay
2.2.4. Analysis of Biochemical and Catalytic Properties of the Enzymes
2.2.5. TLC- and MS-HPLC-Analyses of Chitin/Chitosan Breakdown Products by Chitinase and Chitosanase
2.2.6. Partial Depolymerization of Chitosan by Chitinase/Chitosanase and Molecular Mass Characteristics of Low-Molecular Weight Chitosan’s Fractions
2.2.7. NMR Analysis of Chitosan Oligomers’ Structure
2.2.8. Comparative Antifungal Assay of the Oligomers Generated by Partial Hydrolysis of Chitosan by Chitinase and Chitosanase
2.2.9. Microscopic Examination of the COS Effect on Fungal Hyphae Morphology
2.3. Statistical Analysis
3. Results
3.1. Bacterial Growth and Chitinase and Chitosanase Production
3.2. Purification Specifics and Main Characteristics of Chitinase and Chitosanase
3.3. Comparative Biochemical and Catalytic Characteristics of Purified Chitinase and Chitosanase
3.4. HPLC-MS Analysis of the COS Generated During Extensive Hydrolysis of Chitosan by Chitinase and Chitosanase
3.5. Molecular-Mass Characteristics, Solubility, and Antifungal Potential of the Oligomers Generated at Partial Depolymerization of Chitosan by Chitinase and Chitosanase
3.6. NMR Spectral Analysis of the Partially Hydrolyzed Chitosan Oligomers Produced by Chitinase and Chitosanase
3.7. Antifungal Effect of Oligomers Generated by Chitosan Hydrolysis Using Chitosanase and Chitinase
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Cultivation Period | LB Broth | ||||
---|---|---|---|---|---|
Growth Characteristics | Protein in CS a, mg/mL | Protease, AU b/mL | pH of CS | ||
OD600 | ×107 CFU/mL | ||||
0 h | 0.05 ± 0.01 | 1.77 ± 0.76 c | – | 0 | 6.81 |
24 h | 6.01 ± 0.14 | 66 ± 8 | 6.87 ± 0.35 | 0.093 ± 0.010 | 8.01 |
48 h | 5.09 ± 0.30 | 14.8 ± 3 | 8.87 ± 0.86 | 0.587 ± 0.057 | 8.70 |
72 h | 3.16 ± 0.10 | 3.8 ± 0.60 | 8.93 ± 0.75 | 0.394 ± 0.029 | 8.92 |
96 h | 2.47 ± 0.10 | 0.02 ± 0.004 | 9.38 ± 0.60 | 0.115 ± 0.007 | 9.02 |
The medium with 1% (w/v) of colloidal chitin (CCM) | |||||
0 h | – d | 1.77 ± 0.76 c | – | 0 | 6.53 |
24 h | – | 56 ± 15 | 5.66 ± 0.37 | 0.139 ± 0.012 | 5.90 |
48 h | – | 12 ± 2 | 5.35 ± 0.45 | 0.165 ± 0.014 | 8.30 |
72 h | – | 0.10 ± 0.02 | 3.73 ± 0.42 | 0.078 ± 0.008 | 8.80 |
96 h | – | 0.05 ± 0.001 | 3.85 ± 0.38 | 0.089 ± 0.014 | 8.99 |
Step of Purification | Total Enzyme Activity, U | Total Protein, mg | Specific Activity, U/mg | Purification Degree, -Fold | Enzyme Yield, % | |||||
---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | |
CF a, (96 h) | 1619.68 | 119.25 | 4722.3 | 1040 | 0.343 | 0.064 | 1 | 1 | 100 | 100 |
VivaFlow 200 module (30 kDa) | 931.32 | 107.46 | 822.72 | 870.96 | 1.132 | 0.123 | 3.3 | 1.9 | 58 | 90 |
Affinity adsorption b | 730.48 | 69.20 | 204.79 | 441.94 | 3.567 | 0.157 | 10.4 | 2.5 | 45 | 58 |
Phenyl-Sepharose CL 4B | — | 34.79 | — | 22.79 | — | 1.527 | — | 24 | — | 29 |
CM-Sepharose Fast Flow | 147.39 | — | 4.57 | — | 32.252 | — | 94 | — | 9 | — |
Characteristics | Chitosanase | Chitinase |
---|---|---|
Temperature optimum, °C | 55 | 55 |
Thermal stability, °C a | 60 | 60 |
Optimal pH | 6.5 | 6.5 |
pH stability b | 6–10 | 4–9 |
pI | 7.5–8 | ND |
Molecular weight | 40 | 73 |
KM, mg/mL (toward chitosan DD 85%) | 0.22 | >5.4 |
Vmax (toward chitosan DD 85%), μM × mL−1 × mg−1 | 56.52 | 0.124 |
Vmax (toward colloidal chitin), μM × mL−1 × mg−1 | ND c | 14.1 |
Action mechanism to a specific substrate | Endo- d | Exo- e |
Final products of chitosan (DD 85%) hydrolysis | (GlcN)2-4 f | (GlcN) 2-4 f |
Major products of colloidal chitin hydrolysis | NA g | (GlcNAc)2 and GlcNAc d |
Catalytic activity, nkat (chitosan DD 85%) | 942.19 | ND a |
kcat, s−1 | 5.84 × 103 | ND a |
Inhibition by metal cations and detergents | 10 mM Zn2+, Hg+, Cd2+, Ag+ Fe+2 and 10 mM SDS | 1–10 mM Hg+, Cd2+, Ag+, Fe+2, and 10 mM SDS |
Activation by metal cations and surfactants | 10 mM tween-80 | 10 mM tween-80 |
No. | Main Ion Peaks, m/z (Proposed Adducts and Charges) | COS Product | Retention Time, min | Rel. Content a, % |
---|---|---|---|---|
Chitosan DD 85% degradation products by chitosanase 40 kDa b | ||||
1 | 363.14 (Na+), 341.14+, 342.17 (H+) | (GlcN)2 | 5.47 | 30 |
2 | 502.23, 503.23 (H+), 524.2 (Na+) | (GlcN)3 | 6.29 | 54.2 |
3 | 332.152+, 332.652+, 333.15 (2H2+) 663.29+, 663.79+, 664.29 (H+) | (GlcN)4 | 7.30 | 15 |
4 | 412.692+ | (GlcN)5 | 7.30 | 0.1 |
5 | 433.692+, 705.3+, 705.8+ | GlcNAc-(GlcN)4 | 7.30 | 0.7 |
Chitosan DD 85% degradation products by chitinase 73 kDa c | ||||
1 | 341.15+, 342.15 (H+), 363.13 (Na+) | (GlcN)2 | 5.65 | 12.2 |
2 | 502.22+, 503.22 (H+), 524.2 (Na+) | (GlcN)3 | 5.70 | 12.1 |
3 | 544.2+, 545.22 (H+), 566.21(Na+) | (GlcN)2-GlcNAc | 5.70 | 1.6 |
4 | 663.29+, 663.79+, 664.28 (H+), 663.31+, 332.142+, 332.652+, 333.15 (H+) | (GlcN)4 | 6.80 | 7 |
5 | 412.682+, 413.182+, 413.68 (2H2+), 824.41+, 824.37+ | (GlcN)5 | 7.81 | 19.3 |
6 | 433.682+, 435.18 (2H2+) | (GlcN)4-GlcNAc | 7.81 | 2.7 |
7 | 493.212+, 493.712+, 494.21 (2H2+), 493.232+, 493.732+, 494.23 (2H2+) | (GlcN)6 | 7.98 | 19.2 |
8 | 514.222+, 514.722+, 515.21 (2H2+), 514.242+, 514.742+ | GlcNAc-(GlcN)5 | 8.00 | 6 |
9 | 573.772+, 574.272+, 574.77 (2H2+) | (GlcN)7 | 8.68 | 13.6 |
10 | 594.782+ | (GlcN)6-GlcNAc | 10.01 | 0.4 |
11 | 654.302+, 654.802+, 655.31 (2H2+) | (GlcN)8 | 10.05 | 3.4 |
12 | 755.852+ | (GlcN)8-GlcNAc | 10.57 | 0.1 |
13 | 734.832+, 735.332+ | (GlcN)9 | 11.16 | 0.2 |
Chitosan DD 50% degradation products by chitinase 73 kDa d | ||||
1 | 222.10+, 222.23+, 222.32+, 222.39+, 222.55+, 223.10 (H+) | GlcNAc | 4.11 | 12 |
2 | 204.09 (Na+) | GlcN | 4.09 | 7.7 |
3 | 343.18 (2H2+), 341.16+, 363.14 (Na+) | (GlcN)2 | 4.30 | 5.4 |
4 | 443.2+, 444.19 (H+), 447.17 (2H2+) | (GlcNAc)2 | 4.35 | 4.8 |
5 | 405.15 (Na+), 383.17+, 383.35+, 383.55+, 383.76+, 384.17 (H+), 406.16 (H+ Na+), 405.15 (Na+) | GlcN-GlcNAc | 4.92 | 22.7 |
6 | 502.23+, 503.24 (H+), 524.22 (Na+) | (GlcN)3 | 6.24 | 8.4 |
7 | 544.24+, 545.24 (H+), 566.23 (Na+) | (GlcN)2-GlcNAc | 6.25 | 12.9 |
8 | 608.22+ | GlcNAc-GlcN-GlcNAc (presumably) | 6.25 | 0.7 |
9 | 663.27+ | (GlcN)4 | 7.16 | 0.8 |
10 | 705.28+, 706.28 (H+) | (GlcN)3-GlcNAc | 7.17 | 0.8 |
11 | 747.30+, 748.27 (H+) | GlcNAc-(GlcN)2-GlcNAc | 7.20 | 0.8 |
12 | 433.692+, 434.20 (2H2+), 434.7 (2H2+) | (GlcN)4-GlcNAc | 7.67 | 22.5 |
13 | 412.692+ | (GlcN)5 | 7.92 | 0.4 |
Incubation Time and Temperature | Fraction | Mn, a kDa | Mw, b kDa | PDI c | Solubility d, g/L | Yield, % | Inhibition of B. sorokiniana, ED50, mg/mL e |
---|---|---|---|---|---|---|---|
– | Initial polymeric chitosan | 128.5 | 369.2 | 2.87 | – | – | 0.10 |
Depolymerization by chitosanase (~2.5–11 U/g substrate) | |||||||
1 h at 50 °C | The precipitated fraction after adding 0.5 M NaOH | 22.5 | 66.5 | 2.96 | 48.1 | 48.1 | 0.29 |
2 h at 50 °C | 23.25 | 45.34 | 1.95 | 57.7 | 29.8 | 0.45 | |
2 h at 50 °C | Supernatant after adding 0.5 M NaOH | 1.96 | 2.19 | 1.12 | 63.0 | 8 | 2.50 |
4 h at 50 °C | Total hydrolysate | 1.92 | 2.41 | 1.26 | 64.9 | 68 | 1.29 |
Depolymerization by chitinase (~8–14 U/g substrate) | |||||||
1 h at 50 °C | The precipitated fraction after adding 0.5 M NaOH | 27.43 | 71.74 | 2.62 | 24.4 | 43.4 | 0.69 |
1 h at 50 °C | Supernatant after adding 0.5 M NaOH | 2.52 | 2.89 | 1.15 | 42.8 | <6 | 3.00 |
24 h at 37 °C f | Total hydrolysate | 7.32 | 15.38 | 2.10 | 21.6 | 73 | 1.22 |
Tested Fungal Strain | MIC, μg × mL−1 | ||||
---|---|---|---|---|---|
Chitosanase Products/Hydrolysis Time | Chitinase Products/Hydrolysis Time | ||||
2 h | 4 h | 1 h | 24 h | ||
45.3 kDa a | 2.19 kDa b | 2.4 kDa c | 71.7 kDa a | 15.4 kDa c | |
A. alternata VKM F-3047 | 200 ± 15 | 2250 ± 250 | 750 ± 55 | 310 ± 30 | 575 ± 50 |
B. sorokiniana IB G-12 | 240 ± 20 | 1850 ± 200 | 675 ± 45 | 320 ± 35 | 590 ± 55 |
F. culmorum VKM F-844 | <21 | 1520 ± 140 | 230 ± 20 | 38 ± 5 | 200 ± 20 |
F. gibbosum VKM F-848 | <19 | NI d | 665 ± 55 | 55 ± 5 | 540 ± 45 |
F. graminearum VKM F-1668 | <20 | NI | 485 ± 35 | 90 ± 10 | 390 ± 35 |
F. oxysporum VKM F-137 | <21 | 2500 ± 200 | 150 ± 20 | 75 ± 5 | 145 ± 10 |
F. solani VKM F-142 | <20 | 2500 ± 170 | 295 ± 25 | 65 ± 10 | 280 ± 30 |
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Aktuganov, G.; Lobov, A.; Galimzianova, N.; Gilvanova, E.; Kuzmina, L.; Milman, P.; Ryabova, A.; Melentiev, A.; Chetverikov, S.; Starikov, S.; et al. Comparative Potential of Chitinase and Chitosanase from the Strain Bacillus thuringiensis B-387 for the Production of Antifungal Chitosan Oligomers. BioTech 2025, 14, 35. https://doi.org/10.3390/biotech14020035
Aktuganov G, Lobov A, Galimzianova N, Gilvanova E, Kuzmina L, Milman P, Ryabova A, Melentiev A, Chetverikov S, Starikov S, et al. Comparative Potential of Chitinase and Chitosanase from the Strain Bacillus thuringiensis B-387 for the Production of Antifungal Chitosan Oligomers. BioTech. 2025; 14(2):35. https://doi.org/10.3390/biotech14020035
Chicago/Turabian StyleAktuganov, Gleb, Alexander Lobov, Nailya Galimzianova, Elena Gilvanova, Lyudmila Kuzmina, Polina Milman, Alena Ryabova, Alexander Melentiev, Sergey Chetverikov, Sergey Starikov, and et al. 2025. "Comparative Potential of Chitinase and Chitosanase from the Strain Bacillus thuringiensis B-387 for the Production of Antifungal Chitosan Oligomers" BioTech 14, no. 2: 35. https://doi.org/10.3390/biotech14020035
APA StyleAktuganov, G., Lobov, A., Galimzianova, N., Gilvanova, E., Kuzmina, L., Milman, P., Ryabova, A., Melentiev, A., Chetverikov, S., Starikov, S., & Lopatin, S. (2025). Comparative Potential of Chitinase and Chitosanase from the Strain Bacillus thuringiensis B-387 for the Production of Antifungal Chitosan Oligomers. BioTech, 14(2), 35. https://doi.org/10.3390/biotech14020035