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Domain shuffling of cyclodextrin glucanotransferases for tailored product specificity and thermal stability
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Titel: |
Domain shuffling of cyclodextrin glucanotransferases for tailored product specificity and thermal stability |
In: | FEBS Open Bio, 9, 2019, 2, S. 384-395 |
veröffentlicht: |
Wiley
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Umfang: | 384-395 |
ISSN: |
2211-5463 |
DOI: | 10.1002/2211-5463.12588 |
Zusammenfassung: | <jats:p>Cyclodextrin glucanotransferases (<jats:styled-content style="fixed-case">CGT</jats:styled-content>ases) convert α‐1,4‐glucans to cyclic oligosaccharides (cyclodextrins, <jats:styled-content style="fixed-case">CD</jats:styled-content>), which have found applications in the food and the pharmaceutical industries. In this study, we used two <jats:styled-content style="fixed-case">CGT</jats:styled-content>ases with different cyclization activities, product specificities, and <jats:styled-content style="fixed-case">pH</jats:styled-content> and temperature optima to construct chimeric variants for the synthesis of large‐ring <jats:styled-content style="fixed-case">CD</jats:styled-content>. We used (a) a synthetic thermostable <jats:styled-content style="fixed-case">CGT</jats:styled-content>ase mainly forming α‐ and β‐<jats:styled-content style="fixed-case">CD</jats:styled-content> (<jats:styled-content style="fixed-case">CD</jats:styled-content>6 and <jats:styled-content style="fixed-case">CD</jats:styled-content>7) derived from <jats:italic>Geobacillus stearothermophilus </jats:italic><jats:styled-content style="fixed-case">ET</jats:styled-content>1/<jats:styled-content style="fixed-case">NO</jats:styled-content>2 (GeoT), and (b) a <jats:styled-content style="fixed-case">CGT</jats:styled-content>ase with lower cyclization activity from the alkaliphilic <jats:italic>Bacillus</jats:italic> sp. G825‐6, which mainly synthesizes γ‐<jats:styled-content style="fixed-case">CD</jats:styled-content> (<jats:styled-content style="fixed-case">CD</jats:styled-content>8). The A1, B, A2, and <jats:styled-content style="fixed-case">CDE</jats:styled-content> domains of the G825‐6 <jats:styled-content style="fixed-case">CGT</jats:styled-content>ase were replaced with corresponding GeoT <jats:styled-content style="fixed-case">CGT</jats:styled-content>ase domains by utilizing a megaprimer cloning approach. A comparison of the optimum temperature and <jats:styled-content style="fixed-case">pH</jats:styled-content>, thermal stability, and <jats:styled-content style="fixed-case">CD</jats:styled-content> products synthesized by the variants revealed that the B domain had a major impact on the cyclization activity, thermal stability, and product specificity of the constructed chimera. Complete suppression of the synthesis of <jats:styled-content style="fixed-case">CD</jats:styled-content>6 was observed with the variants GeoT‐A1/B and GeoT‐A1/A2/<jats:styled-content style="fixed-case">CDE</jats:styled-content>. The variant GeoT‐A1/A2/<jats:styled-content style="fixed-case">CDE</jats:styled-content> showed the desired enzyme properties for large‐ring <jats:styled-content style="fixed-case">CD</jats:styled-content> synthesis. Its melting temperature was 9 °C higher compared to the G825‐6 <jats:styled-content style="fixed-case">CGT</jats:styled-content>ase and it synthesized up to 3.3 g·L<jats:sup>−1</jats:sup> <jats:styled-content style="fixed-case">CD</jats:styled-content>9 to <jats:styled-content style="fixed-case">CD</jats:styled-content>12, corresponding to a 1.8‐ and 2.3‐fold increase compared to GeoT and G825‐6 <jats:styled-content style="fixed-case">CGT</jats:styled-content>ase, respectively. In conclusion, GeoT‐A1/A2/<jats:styled-content style="fixed-case">CDE</jats:styled-content> may be a candidate for the further development of <jats:styled-content style="fixed-case">CGT</jats:styled-content>ases specifically forming larger CD.</jats:p> |
Format: | E-Article |
Quelle: | Wiley (CrossRef) |
Sprache: | Englisch |