Simultaneous Improvement of Final Product-Tolerance and Thermostability of GH39 Xylosidase for Prebiotic Production by Directed Evolution
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains, Plasmids, and Biochemical Reagents
2.2. Construction of Mutant Library
2.3. High-Throughput Screening of Mutant Library
2.4. Expression, Purification and Tolerance Assays of Wild Type and Variants
2.5. Construction and Characteristics of Single Site Variants
2.6. Sequence Alignment, Homology Modeling, and Structural Analysis
2.7. Statistical Analysis
3. Results and Discussion
3.1. Construction of the Random Mutagenesis Library by Error-Prone PCR
3.2. Protein Expression, Purification, and Characterization of Mutants
3.3. Xylose and Ethanol Tolerance Analysis of Single Site Mutants K16R, L94I and K262N
3.4. Protein Structure Modeling and Analysis of Intramolecular Interactions in the Wild Type and the Variants
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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Source | Family | Xylose Tolerance | Ethanol Tolerance | Optimum Temperature/°C | Optimum pH | Thermostability/1 min | Reference |
---|---|---|---|---|---|---|---|
Metagenomic DNA of soil (Xyl21) | 39 | 111% a (0.3 M xylose), 74% (0.6 M xylose), 53% (0.9 M xylose), 20% (2.1 M xylose) | 176% (5%, v/v), 105% (15%, v/v), 20% (20%, v/v) | 45 | 5.5 | 50 °C, 84%, 55 °C, 39% | This study |
Metagenomic DNA of soil (Xyl21-A16) | 39 | 130% (0.3 M xylose), 89% (0.6 M xylose), 62% (0.9 M xylose), 20% (2.1 M xylose) | 190% (5%, v/v), 156% (15%, v/v), 63% (20%, v/v) | 45 | 5.5 | 50 °C, 93%, 55 °C, 68% | This study |
Dictyoglomus thermophilum | 39 | 60% (3 M xylose) | 100% (10%, v/v) | 75 | 6.0 | 85 °C, 90% | [16] |
Geobacillus thermodenitrificans NG80-2 | 39 | 50% (400 mM xylose) | 85% (5%, v/v) | 60 | 5.5 | 75 °C, 100% | [17] |
Sphingobacterium sp. | 43 | 100%, 54%, 249%, 91% (20 mM glucose, xylose, arabinose, galactose) | 26% (10%, v/v) | 20 | 8.0 | 30 min, 30 °C, 90% | [18] |
Geobacillus thermodenitrificans NG80-2 | 43 | 50% (600 mM xylose) | 79% (5%, v/v) | 65 | 6.0 | 75 °C, 90% | [17] |
Geobacillus thermodenitrificans NG80-2 | 52 | 50% (300 mM xylose) | 81% (5%, v/v) | 65 | 6.0 | 75 °C, 100% | [17] |
Yak rumen metagenome 3A | 3 | 4% (5 mM glucose), 82% (5 mM xylose) | 112.7% (5%, v/v) | 40 | 7.0 | 50 °C, 75%, 55 °C, 10% | [19] |
Yak rumen metagenome 3B | 3 | 54% (5 mM glucose), 97% (5 mM xylose) | 317% (5%, v/v) | 40 | 7.0 | 50 °C, 1% | [19] |
Colletotrichum graminicola | 3 | 94%, 29%, 95%, 84%, 85% (50 mM glucose, xylose, galactose, mannose, arabinose) | 55% (5%, v/v) | 65 | 4.5 | 70 °C, 78%, 75 °C, 5% | [20] |
Pichia membranifaciens | - | 50% (200 mM glucose) | 100% (20%, v/v) | 35 | 6.0 | 50 °C, 35%, 60 °C, 3% | [21] |
Aspergillus niger CCRC31494 | - | 100% (30 mM galactose, mannose, xylose), Ki = 543 mM (glucose) | 68% (40%, v/v) | 55 | 5.0 | 60 °C, 90% | [22] |
Aureobasidium pullulans CBS 135684 | - | 100% (70 mM glucose, arabinose, mannose), Ki = 18.2 mM (xylose) | 84% (30%, v/v) | 70 | 6.0 | 6 h, 70 °C, 50% | [23] |
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Zhang, Z.; Zhang, Z.; Yu, Z.; Chen, S.; Zhang, M.; Zhang, T.; Luo, X.; Zhao, J.; Li, Z. Simultaneous Improvement of Final Product-Tolerance and Thermostability of GH39 Xylosidase for Prebiotic Production by Directed Evolution. Foods 2022, 11, 3039. https://doi.org/10.3390/foods11193039
Zhang Z, Zhang Z, Yu Z, Chen S, Zhang M, Zhang T, Luo X, Zhao J, Li Z. Simultaneous Improvement of Final Product-Tolerance and Thermostability of GH39 Xylosidase for Prebiotic Production by Directed Evolution. Foods. 2022; 11(19):3039. https://doi.org/10.3390/foods11193039
Chicago/Turabian StyleZhang, Zirui, Zhengjie Zhang, Zhao Yu, Shiheng Chen, Mengwei Zhang, Tongcun Zhang, Xuegang Luo, Junqi Zhao, and Zhongyuan Li. 2022. "Simultaneous Improvement of Final Product-Tolerance and Thermostability of GH39 Xylosidase for Prebiotic Production by Directed Evolution" Foods 11, no. 19: 3039. https://doi.org/10.3390/foods11193039