Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase
Lytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes that play a pivotal role in the enzymatic conversion of the most recalcitrant polysaccharides, such as cellulose and chitin. Hence, protein engineering is highly required to enhance their catalytic efficiencies. To this effect,...
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MDPI AG
2023-05-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/10/8963 |
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| author | Miesho Hadush Berhe Xiangfei Song Lishan Yao |
| author_facet | Miesho Hadush Berhe Xiangfei Song Lishan Yao |
| author_sort | Miesho Hadush Berhe |
| collection | DOAJ |
| description | Lytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes that play a pivotal role in the enzymatic conversion of the most recalcitrant polysaccharides, such as cellulose and chitin. Hence, protein engineering is highly required to enhance their catalytic efficiencies. To this effect, we optimized the protein sequence encoding for an LPMO from <i>Bacillus amyloliquefaciens</i> (<i>Ba</i>LPMO10A) using the sequence consensus method. Enzyme activity was determined using the chromogenic substrate 2,6-Dimethoxyphenol (2,6-DMP). Compared with the wild type (WT), the variants exhibit up to a 93.7% increase in activity against 2,6-DMP. We also showed that <i>Ba</i>LPMO10A can hydrolyze <i>p</i>-nitrophenyl-β-D-cellobioside (PNPC), carboxymethylcellulose (CMC), and phosphoric acid-swollen cellulose (PASC). In addition to this, we investigated the degradation potential of <i>Ba</i>LPMO10A against various substrates such as PASC, filter paper (FP), and Avicel, in synergy with the commercial cellulase, and it showed up to 2.7-, 2.0- and 1.9-fold increases in production with the substrates PASC, FP, and Avicel, respectively, compared to cellulase alone. Moreover, we examined the thermostability of <i>Ba</i>LPMO10A. The mutants exhibited enhanced thermostability with an apparent melting temperature increase of up to 7.5 °C compared to the WT. The engineered <i>Ba</i>LPMO10A with higher activity and thermal stability provides a better tool for cellulose depolymerization. |
| first_indexed | 2024-03-11T03:40:03Z |
| format | Article |
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| language | English |
| last_indexed | 2024-03-11T03:40:03Z |
| publishDate | 2023-05-01 |
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| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-dd08ed31353f4c52be7f72ca1e5bd79e2023-11-18T01:45:02ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-05-012410896310.3390/ijms24108963Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide MonooxygenaseMiesho Hadush Berhe0Xiangfei Song1Lishan Yao2Qingdao New Energy Shandong Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, ChinaQingdao New Energy Shandong Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, ChinaQingdao New Energy Shandong Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, ChinaLytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes that play a pivotal role in the enzymatic conversion of the most recalcitrant polysaccharides, such as cellulose and chitin. Hence, protein engineering is highly required to enhance their catalytic efficiencies. To this effect, we optimized the protein sequence encoding for an LPMO from <i>Bacillus amyloliquefaciens</i> (<i>Ba</i>LPMO10A) using the sequence consensus method. Enzyme activity was determined using the chromogenic substrate 2,6-Dimethoxyphenol (2,6-DMP). Compared with the wild type (WT), the variants exhibit up to a 93.7% increase in activity against 2,6-DMP. We also showed that <i>Ba</i>LPMO10A can hydrolyze <i>p</i>-nitrophenyl-β-D-cellobioside (PNPC), carboxymethylcellulose (CMC), and phosphoric acid-swollen cellulose (PASC). In addition to this, we investigated the degradation potential of <i>Ba</i>LPMO10A against various substrates such as PASC, filter paper (FP), and Avicel, in synergy with the commercial cellulase, and it showed up to 2.7-, 2.0- and 1.9-fold increases in production with the substrates PASC, FP, and Avicel, respectively, compared to cellulase alone. Moreover, we examined the thermostability of <i>Ba</i>LPMO10A. The mutants exhibited enhanced thermostability with an apparent melting temperature increase of up to 7.5 °C compared to the WT. The engineered <i>Ba</i>LPMO10A with higher activity and thermal stability provides a better tool for cellulose depolymerization.https://www.mdpi.com/1422-0067/24/10/8963lytic polysaccharide monooxygenases (LPMOs)protein engineeringcellulosedepolymerizationenzyme activity and stabilitysynergism |
| spellingShingle | Miesho Hadush Berhe Xiangfei Song Lishan Yao Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase International Journal of Molecular Sciences lytic polysaccharide monooxygenases (LPMOs) protein engineering cellulose depolymerization enzyme activity and stability synergism |
| title | Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase |
| title_full | Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase |
| title_fullStr | Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase |
| title_full_unstemmed | Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase |
| title_short | Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase |
| title_sort | improving the enzymatic activity and stability of a lytic polysaccharide monooxygenase |
| topic | lytic polysaccharide monooxygenases (LPMOs) protein engineering cellulose depolymerization enzyme activity and stability synergism |
| url | https://www.mdpi.com/1422-0067/24/10/8963 |
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