Cloning, purification and characterization of trehalose-6-phosphate synthase from Pleurotus tuoliensis
Pleurotus tuoliensis, a kind of valuable and favorable edible mushroom in China, is always subjected to high environmental temperature during cultivation. In our previous study with P. tuoliensis, trehalose proved to be effective for tolerating heat stress. Trehalose-6-phosphate synthase (TPS; EC2.4...
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PeerJ Inc.
2018-07-01
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| Online Access: | https://peerj.com/articles/5230.pdf |
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| author | Xiangli Wu Zhihao Hou Chenyang Huang Qiang Chen Wei Gao Jinxia Zhang |
| author_facet | Xiangli Wu Zhihao Hou Chenyang Huang Qiang Chen Wei Gao Jinxia Zhang |
| author_sort | Xiangli Wu |
| collection | DOAJ |
| description | Pleurotus tuoliensis, a kind of valuable and favorable edible mushroom in China, is always subjected to high environmental temperature during cultivation. In our previous study with P. tuoliensis, trehalose proved to be effective for tolerating heat stress. Trehalose-6-phosphate synthase (TPS; EC2.4.1.15) plays a key role in the biosynthesis of trehalose in fungi. In this study, a full-length of cDNA with 1,665 nucleotides encoding TPS (PtTPS) in P. tuoliensis was cloned. The PtTPS amino acid was aligned with other homologues and several highly conserved regions were analyzed. Thus, the TPS protein was expressed in Escherichia coli and purified by affinity chromatography to test its biochemical properties. The molecular mass of the enzyme is about 60 kDa and the optimum reaction temperature and pH is 30 °C and 7, respectively. The UDP-glucose and glucose-6-phosphate were the optimum substrates among all the tested glucosyl donors and acceptors. Metal cations like Mg2+, Co2+, Mn2+, Ni2+, K+, Ag+ stimulated PtTPS activity significantly. Metal chelators such as sodium citrate, citric acid, EDTA, EGTA and CDTA inhibited enzyme activity. Polyanions like heparin and chondroitin sulfate were shown to stimulate TPS activity. |
| first_indexed | 2024-03-09T06:47:09Z |
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| id | doaj.art-5708b4b48e9d4c52a6d61344f9f18ed7 |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T06:47:09Z |
| publishDate | 2018-07-01 |
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| spelling | doaj.art-5708b4b48e9d4c52a6d61344f9f18ed72023-12-03T10:34:39ZengPeerJ Inc.PeerJ2167-83592018-07-016e523010.7717/peerj.5230Cloning, purification and characterization of trehalose-6-phosphate synthase from Pleurotus tuoliensisXiangli Wu0Zhihao Hou1Chenyang Huang2Qiang Chen3Wei Gao4Jinxia Zhang5Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, ChinaInstitute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, ChinaInstitute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, ChinaInstitute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, ChinaInstitute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, ChinaInstitute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, ChinaPleurotus tuoliensis, a kind of valuable and favorable edible mushroom in China, is always subjected to high environmental temperature during cultivation. In our previous study with P. tuoliensis, trehalose proved to be effective for tolerating heat stress. Trehalose-6-phosphate synthase (TPS; EC2.4.1.15) plays a key role in the biosynthesis of trehalose in fungi. In this study, a full-length of cDNA with 1,665 nucleotides encoding TPS (PtTPS) in P. tuoliensis was cloned. The PtTPS amino acid was aligned with other homologues and several highly conserved regions were analyzed. Thus, the TPS protein was expressed in Escherichia coli and purified by affinity chromatography to test its biochemical properties. The molecular mass of the enzyme is about 60 kDa and the optimum reaction temperature and pH is 30 °C and 7, respectively. The UDP-glucose and glucose-6-phosphate were the optimum substrates among all the tested glucosyl donors and acceptors. Metal cations like Mg2+, Co2+, Mn2+, Ni2+, K+, Ag+ stimulated PtTPS activity significantly. Metal chelators such as sodium citrate, citric acid, EDTA, EGTA and CDTA inhibited enzyme activity. Polyanions like heparin and chondroitin sulfate were shown to stimulate TPS activity.https://peerj.com/articles/5230.pdfExpressionTrehalose-6-phosphate synthaseCloningPurificationPleurotus tuoliensisCharacterization |
| spellingShingle | Xiangli Wu Zhihao Hou Chenyang Huang Qiang Chen Wei Gao Jinxia Zhang Cloning, purification and characterization of trehalose-6-phosphate synthase from Pleurotus tuoliensis PeerJ Expression Trehalose-6-phosphate synthase Cloning Purification Pleurotus tuoliensis Characterization |
| title | Cloning, purification and characterization of trehalose-6-phosphate synthase from Pleurotus tuoliensis |
| title_full | Cloning, purification and characterization of trehalose-6-phosphate synthase from Pleurotus tuoliensis |
| title_fullStr | Cloning, purification and characterization of trehalose-6-phosphate synthase from Pleurotus tuoliensis |
| title_full_unstemmed | Cloning, purification and characterization of trehalose-6-phosphate synthase from Pleurotus tuoliensis |
| title_short | Cloning, purification and characterization of trehalose-6-phosphate synthase from Pleurotus tuoliensis |
| title_sort | cloning purification and characterization of trehalose 6 phosphate synthase from pleurotus tuoliensis |
| topic | Expression Trehalose-6-phosphate synthase Cloning Purification Pleurotus tuoliensis Characterization |
| url | https://peerj.com/articles/5230.pdf |
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