Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase
In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specifici...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2016-01-01
|
| Series: | Scientifica |
| Online Access: | http://dx.doi.org/10.1155/2016/4902450 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832552323154444288 |
|---|---|
| author | Gülşah P. Özgün Emel B. Ordu H. Esra Tütüncü Emrah Yelboğa Richard B. Sessions Nevin Gül Karagüler |
| author_facet | Gülşah P. Özgün Emel B. Ordu H. Esra Tütüncü Emrah Yelboğa Richard B. Sessions Nevin Gül Karagüler |
| author_sort | Gülşah P. Özgün |
| collection | DOAJ |
| description | In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6×104-fold and 5×104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH. |
| format | Article |
| id | doaj-art-d26753eb03f34a189291509ebb61c5b7 |
| institution | Kabale University |
| issn | 2090-908X |
| language | English |
| publishDate | 2016-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Scientifica |
| spelling | doaj-art-d26753eb03f34a189291509ebb61c5b72025-02-03T05:58:58ZengWileyScientifica2090-908X2016-01-01201610.1155/2016/49024504902450Site Saturation Mutagenesis Applications on Candida methylica Formate DehydrogenaseGülşah P. Özgün0Emel B. Ordu1H. Esra Tütüncü2Emrah Yelboğa3Richard B. Sessions4Nevin Gül Karagüler5Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, TurkeyDepartment of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, TurkeyDepartment of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, TurkeyDepartment of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, TurkeyDepartment of Biochemistry, University of Bristol, Bristol, Avon BS8 1TD, UKDepartment of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, TurkeyIn NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6×104-fold and 5×104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH.http://dx.doi.org/10.1155/2016/4902450 |
| spellingShingle | Gülşah P. Özgün Emel B. Ordu H. Esra Tütüncü Emrah Yelboğa Richard B. Sessions Nevin Gül Karagüler Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase Scientifica |
| title | Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase |
| title_full | Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase |
| title_fullStr | Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase |
| title_full_unstemmed | Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase |
| title_short | Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase |
| title_sort | site saturation mutagenesis applications on candida methylica formate dehydrogenase |
| url | http://dx.doi.org/10.1155/2016/4902450 |
| work_keys_str_mv | AT gulsahpozgun sitesaturationmutagenesisapplicationsoncandidamethylicaformatedehydrogenase AT emelbordu sitesaturationmutagenesisapplicationsoncandidamethylicaformatedehydrogenase AT hesratutuncu sitesaturationmutagenesisapplicationsoncandidamethylicaformatedehydrogenase AT emrahyelboga sitesaturationmutagenesisapplicationsoncandidamethylicaformatedehydrogenase AT richardbsessions sitesaturationmutagenesisapplicationsoncandidamethylicaformatedehydrogenase AT nevingulkaraguler sitesaturationmutagenesisapplicationsoncandidamethylicaformatedehydrogenase |