Characterization of a Novel Acid-Stable Chitosanase from Lentinula edodes Suitable for Chitooligosaccharide Preparation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Polysaccharides
2.2. Strains and Plasmids
2.3. Sequence Analysis
2.4. RNA Extraction and cDNA Synthesis
2.5. Cloning, Expression and Purification of LeCho1
2.6. Hydrolytic Activity Assay
2.7. Biochemical Characterization of Recombinant LeCho1
2.8. Analysis of Hydrolysis Products
2.9. Statistical Analysis
3. Results
3.1. Sequence Analysis of LeCho1
3.2. Expression and Purification of LeCho1
3.3. Substrate Specificity of the Purified Protein
3.4. Enzymological Characterization of LeCho1
3.5. LeCho1 Is an Endo-Chitosanase
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Substrate | Specific Activity (U/mg) |
---|---|
Chitosan (DDA 70%) | 37.18 ± 1.28 c |
Chitosan (DDA 80%) | 50.22 ± 1.73 b |
Chitosan (DDA 95%) | 71.88 ± 2.48 a |
Chitin | 0 d |
Microcrystalline cellulose | 0 d |
Sodium carboxymethylcellulose | 0 d |
Metal Ions | Concentration (mM) | Relativity Activity (%) |
---|---|---|
No addition | 0 | 100 ± 2.11 c |
Na+ (NaCl) | 1 | 95.93 ± 3.09 d |
K+ (KCl) | 1 | 107.89 ± 3.48 c |
Ba2+ (BaCl2) | 1 | 108.46 ± 3.50 c |
Cu2+ (CuCl2) | 1 | 98.94 ± 2.72 c |
Ca2+ (CaCl2) | 1 | 110.25 ± 3.56 b |
Co2+ (CoCl2) | 1 | 90.32 ± 2.91 e |
Fe2+ (FeCl2) | 1 | 107.32 ± 3.46 c |
Mn2+ (MnCl2) | 1 | 156.63 ± 5.05 a |
Mg2+ (MgCl2) | 1 | 118.71 ± 3.83 b |
Ni2+ (NiCl2) | 1 | 94.87 ± 3.06 d |
Zn2+ (ZnCl2) | 1 | 95.69 ± 3.09 d |
Fe3+ (FeCl3) | 1 | 0.00 ± 0.00 h |
Al3+ (AlCl3) | 1 | 85.19 ± 2.75 f |
Cd3+ (CdCl3) | 1 | 83.32 ± 2.69 f |
EDTA | 1 | 56.17 ± 1.78 g |
10 | 0.00 ± 0.00 h |
Microorganism | Chitosanase | Family | Mw (kDa) | Optimal Temperature (°C) | Optimal pH | Metal Ions (+) | Metal Ions (−) | End Products | Reference or Source |
---|---|---|---|---|---|---|---|---|---|
Lentinula edodes | LeCho1 | GH75 | 27 | 50 | 3.0 | Mn2+ | Fe3+ | DP2–5 | This study |
Aspergillus sp. W-2 | CsnW2 | GH75 | 28 | 55 | 6.0 | Mn2+, Ca2+, Mg2+ | Cu2+, Fe2+, Zn2+, Ni2+, Ge2+ | DP2–6 | [34] |
Aspergillus fumigatus CJ22-326 | Csn75 | GH75 | 23.5 | 55–65 | 5.0–6.0 | Mn2+ | Cu2+, Mg2+ | DP2–4 | [35] |
Beauveria bassiana | BbCSN-1 | GH75 | 33 | 30 | 5.0 | Mn2+ | Cu2+, Co2+ | DP2–3 | [36] |
Streptomyces coelicolor A3(2) M145 | CsnA | GH46 | 26 | 65 | 6.0 | Mn2+, Co2+ | Fe3+, Al3+, Ni2+ | DP2–3 | [39] |
Paenibacillus elgii TKU051 | chitosanase | GH46 | 28 | 55 | 6.0 | Mn2+ | Cu2+ | DP2–3 | [23] |
Streptomyces hygroscopicus R1 | ShCsn46 | GH46 | 28 | 55 | 5.5 | Mn2+ | Cu2+, Fe2+, Al3+ | DP2–6 | [37] |
Bacillus sp. BY01 | CsnB | GH46 | 30 | 35 | 5.0 | Mn2+, Cu2+, Mg2+ | Fe3+ | DP2–3 | [18] |
Matsuebacter chitosanotabidus 3001 | ChoA | GH80 | 34 | 30–40 | 4.0 | ND | Ag+ | DP2–6 | [32] |
β-Proteobacterium KNU3 | ChoK | GH80 | 34 | 70 | 6.0 | Cu2+, Co2+ | Ca2+, Zn2+, Mg2+ | ND | [33] |
Mitsuaria sp. 141 | ChoA | GH80 | 33 | 65 | 5.5 | Fe2+ | Cu2+, Hg2+ | DP1–5 | [38] |
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Wang, Y.; Zhao, Y.; Li, J.; Zong, H.; Chen, Y.; Zhou, J.; Li, X.; Ye, X. Characterization of a Novel Acid-Stable Chitosanase from Lentinula edodes Suitable for Chitooligosaccharide Preparation. Foods 2024, 13, 3127. https://doi.org/10.3390/foods13193127
Wang Y, Zhao Y, Li J, Zong H, Chen Y, Zhou J, Li X, Ye X. Characterization of a Novel Acid-Stable Chitosanase from Lentinula edodes Suitable for Chitooligosaccharide Preparation. Foods. 2024; 13(19):3127. https://doi.org/10.3390/foods13193127
Chicago/Turabian StyleWang, Yanxin, Yujie Zhao, Jingchen Li, Haobo Zong, Ying Chen, Jinyu Zhou, Xinlian Li, and Xianfeng Ye. 2024. "Characterization of a Novel Acid-Stable Chitosanase from Lentinula edodes Suitable for Chitooligosaccharide Preparation" Foods 13, no. 19: 3127. https://doi.org/10.3390/foods13193127