Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains, Reagents and Media
2.2. Construction of pHBM905BDM Expression Vectors for Free and Surface Displayed CDA Expression
2.3. Confirmation of the Number of Copies of the Recombinant Strains Using qPCR
2.4. Expression of CDA and CDA-AGα1 Fusion Proteins
2.5. High Density Fermentation of Recombinant Strains
2.6. Enzymatic Characterization of Free and Displayed CDA
2.7. Determination of Kinetic Parameters
2.8. TLC Examination
2.9. Assessment of Displayed CDA Using Immunofluorescence and Fluorescence Microscopy
3. Results and Discussion
3.1. Cloning and Identification of the CDA Gene
3.2. Construction of Recombinant Strains
3.3. High Density Fermentation Using the PCDA4 Strain
3.4. CDA Characterization
3.5. Large-Scale Hydrolysis and Analysis of Chitosan Oligosaccharides with TLC
3.6. Construction and Expression of a CDA Surface Display System in P. pastoris
3.7. Detection of Displayed CDA Using Immunofluorescence and Fluorescence Microscopy
3.8. High Cell Density Fermentation of Strains Expressing Recombinant Surface Displayed CDA
3.9. Displayed CDA Characterization on the P. pastoris Cell Surface
3.10. Recycling of Displayed CDA and Conversion of Chitosan into Different Chitooligosaccharides
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
Abbreviations
CDA | cold-adapted chitosanase |
GlcN | 2-amino-2-deoxy-D-glucose |
GAPDH | glyceraldehyde-3-phosphate dehydrogenase |
MD | minimal dextrose |
BMGY | buffered minimal glycerol |
BMMY | buffered minimal methanol |
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Strain | Final Cell Density (g/L) | Protein Concentration (mg/mL) | Enzyme Activity (U/mL) |
---|---|---|---|
PCDA1 | 46.2 ± 1.22 | 0.029 ± 0.002 | 66.8 ± 5 |
PCDA2 | 43.1 ± 1.31 | 0.048 ± 0.015 | 125.6 ± 6 |
PCDA3 | 43.4 ± 1.18 | 0.075 ± 0.046 | 195.1 ± 2.5 |
PCDA4 | 42.2 ± 1.15 | 0.121 ± 0.096 | 258.5 ± 2.1 |
Metal Ion | Concentration | Relative Activity (%) |
---|---|---|
No addition | 0 mM | 100 |
Fe2+ | 5 mM | 43.9 |
Cu2+ | 5 mM | 35.9 |
Mg2+ | 5 mM | 121.2 |
Ca2+ | 5 mM | 115.1 |
Mn2+ | 5 mM | 98.5 |
Co2+ | 5 mM | 21.5 |
Cr2+ | 5 mM | 11.4 |
Zn2+ | 5 mM | 8.9 |
Fe3+ | 5 mM | 6.5 |
Strain | Final Cell Density (g/L) | Enzyme Activity (U/g) |
---|---|---|
PCDA1- AGα1 | 58.5 ± 2.21 | 29012.2 ± 3.21 |
PCDA2-AGα1 | 57.3 ± 2.13 | 50810.8 ± 3.12 |
PCDA3-AGα1 | 59.9 ± 2.21 | 64112.1 ± 3.25 |
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Peng, Y.; Wang, Y.; Liu, X.; Zhou, R.; Liao, X.; Min, Y.; Ma, L.; Wang, Y.; Rao, B. Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation. Molecules 2022, 27, 800. https://doi.org/10.3390/molecules27030800
Peng Y, Wang Y, Liu X, Zhou R, Liao X, Min Y, Ma L, Wang Y, Rao B. Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation. Molecules. 2022; 27(3):800. https://doi.org/10.3390/molecules27030800
Chicago/Turabian StylePeng, Yanhong, Yaping Wang, Xiaoyan Liu, Ronghua Zhou, Xianqing Liao, Yong Min, Lixin Ma, Ying Wang, and Ben Rao. 2022. "Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation" Molecules 27, no. 3: 800. https://doi.org/10.3390/molecules27030800