Characterization of Paenibacillus sp. GKG Endo-β-1, 3-Glucanase, a Member of Family 81 Glycoside Hydrolases
Abstract
:1. Introduction
2. Materials and Methods
2.1. Substrates and Chemical Reagents
2.2. Substrate Preparation for Screening of Microorganisms
2.3. Isolation of Bacteria
2.4. Identification of Isolates
2.5. Production of Hydrolytic Enzymes
2.6. Filter Aided Protein Sample Preparation (FASP) for Mass Spectrometry Analysis
2.7. Liquid Chromatography and Mass Spectrometry
2.8. Data Processing and Protein Identification
2.9. Cloning of GH81 Gene
2.10. Expression and Purification of Recombinant Proteins
2.11. Enzyme Assay
2.12. Characterization of Biochemical Properties
2.13. Determination of Substrate Specificity
2.14. Analysis of Hydrolysis Products
2.15. Nucleotide Sequence Accession Number
3. Results and Discussion
3.1. Identification of PsLam81A Coding Gene
3.2. Cloning, Expression and Purification of Recombinant PsLam81A and PsLam81AΔCBM56 Enzymes
3.3. Effect of pH and Temperature on the Activity of PsLam81A and PsLam81AΔCBM56
3.4. Substate Specificity of PsLam81A and PsLam81AΔCBM56
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Substrate | Glycosidic Bond | Reference Number | Source |
---|---|---|---|
Laminaribiose | β-1,3 | O-LAM2 | Megazyme |
Laminaritriose | β-1,3 | O-LAM3 | Megazyme |
Laminaritetraose | β-1,3 | O-LAM4 | Megazyme |
Laminaripentaose | β-1,3 | O-LAM5 | Megazyme |
Laminarihexaose | β-1,3 | O-LAM6 | Megazyme |
Laminarin | β-1,3; β-1,6 | L9634 | Sigma-Aldrich |
Carboxymethyl cellulose | β-1,4 | 419281 | Sigma-Aldrich |
Microcrystalline cellulose | β-1,4 | 435236 | Sigma-Aldrich |
Curdlan | β-1,3 | P-CURDL | Megazyme |
Pachyman | β-1,3 | P-PACHY | Megazyme |
Pustulan | β-1,6 | YP15423 | Carbosynth |
Barley β-glucan | β-1,3; β-1,4 | P-BGBL | Megazyme |
Yeast β-glucan | β-1,3; β-1,6 | P-BGYST | Megazyme |
Sugar beet β-glucan | β-1,2 | P-BGLU12 | Megazyme |
Substrate | U/mg | U × 109/mol | ||
---|---|---|---|---|
PsLam81A | PsLam81AΔCBM56 | PsLam81A | PsLam81AΔCBM56 | |
Laminarin | 108.8 ± 1.7 | 125.0 ± 2.8 | 10.1 ± 0.2 | 10.1 ± 0.2 |
Yeast β-glucan | 47.7 ± 1.6 | 64.1 ± 2.4 | 4.4 ± 0.1 | 5.1 ± 0.2 |
Curdlan | 48.9 ± 4.4 | 23.5 ± 1.2 | 4.5 ± 0.4 | 1.9 ± 0.1 |
Pachyman | 26.6 ± 2.0 | 8.7 ± 0.7 | 2.5 ± 0.2 | 0.7 ± 0.1 |
Other 1 | ND | ND | ND | ND |
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Plakys, G.; Gasparavičiūtė, R.; Vaitekūnas, J.; Rutkienė, R.; Meškys, R. Characterization of Paenibacillus sp. GKG Endo-β-1, 3-Glucanase, a Member of Family 81 Glycoside Hydrolases. Microorganisms 2022, 10, 1930. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10101930
Plakys G, Gasparavičiūtė R, Vaitekūnas J, Rutkienė R, Meškys R. Characterization of Paenibacillus sp. GKG Endo-β-1, 3-Glucanase, a Member of Family 81 Glycoside Hydrolases. Microorganisms. 2022; 10(10):1930. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10101930
Chicago/Turabian StylePlakys, Gediminas, Renata Gasparavičiūtė, Justas Vaitekūnas, Rasa Rutkienė, and Rolandas Meškys. 2022. "Characterization of Paenibacillus sp. GKG Endo-β-1, 3-Glucanase, a Member of Family 81 Glycoside Hydrolases" Microorganisms 10, no. 10: 1930. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10101930