Production of recombinant D-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application
D-Allulose 3-epimerase (DAEase) is the key enzyme catalyzing D-fructose to catalyze into D-allulose, a rare sugar in foods, which has lately drawn increasing worldwide attention owing to its possible health advantages and application as a substitute sucrose. This work focused on the development of a...
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| Format: | Article |
| Language: | English |
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Public Library of Science
2025
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| Online Access: | http://psasir.upm.edu.my/id/eprint/120688/ http://psasir.upm.edu.my/id/eprint/120688/1/120688.pdf |
| _version_ | 1848868217238323200 |
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| author | Lischer, Kenny Laksmi, Fina Amreta Nugraha, Yudhi Az-Zahra, Fauziah Herawan, David Juanssilfero, Ario Betha Wibowo, Des Saputro Ramadhan, Kharisma Panji Nuryana, Isa Ali, Mohd Shukuri Mohamad |
| author_facet | Lischer, Kenny Laksmi, Fina Amreta Nugraha, Yudhi Az-Zahra, Fauziah Herawan, David Juanssilfero, Ario Betha Wibowo, Des Saputro Ramadhan, Kharisma Panji Nuryana, Isa Ali, Mohd Shukuri Mohamad |
| author_sort | Lischer, Kenny |
| building | UPM Institutional Repository |
| collection | Online Access |
| description | D-Allulose 3-epimerase (DAEase) is the key enzyme catalyzing D-fructose to catalyze into D-allulose, a rare sugar in foods, which has lately drawn increasing worldwide attention owing to its possible health advantages and application as a substitute sucrose. This work focused on the development of an economical, scalable production method of DAEase by using the Escherichia coli BL21 star™ (DE3) as host expression. The research work aims to optimize the production of the enzyme through an auto-induction strategy in chemically defined media by using lactose as a natural inducer, thereby overcoming various limitations of conventional IPTG induction methods. The optimal concentration of lactose, glucose, and glycerol for maximum expression of DAEase was determined to be 1.5%, 0.125%, and 1.5%, respectively. Fermentor-scale optimization yielded a maximum amount of this enzyme with 5% inoculant, 300 rpm agitation, and 2 vvm aeration. Purification by affinity and anion exchange chromatography resulted in a sevenfold increase in specific activity with an overall yield of 12% and 43 mg of pure recombinant DAEase per liter of culture. Enzyme assays confirmed that DAEase had catalytic activity in the conversion of D-fructose to D-allulose, which was further confirmed by HPLC analysis. This optimized auto-induction-based strategy demonstrated its potential for large-scale production of DAEase in a cost-effective manner with enhanced reproducibility to meet industrial demands for synthesizing D-allulose. |
| first_indexed | 2025-11-15T14:48:53Z |
| format | Article |
| id | upm-120688 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T14:48:53Z |
| publishDate | 2025 |
| publisher | Public Library of Science |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | upm-1206882025-10-08T05:08:36Z http://psasir.upm.edu.my/id/eprint/120688/ Production of recombinant D-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application Lischer, Kenny Laksmi, Fina Amreta Nugraha, Yudhi Az-Zahra, Fauziah Herawan, David Juanssilfero, Ario Betha Wibowo, Des Saputro Ramadhan, Kharisma Panji Nuryana, Isa Ali, Mohd Shukuri Mohamad D-Allulose 3-epimerase (DAEase) is the key enzyme catalyzing D-fructose to catalyze into D-allulose, a rare sugar in foods, which has lately drawn increasing worldwide attention owing to its possible health advantages and application as a substitute sucrose. This work focused on the development of an economical, scalable production method of DAEase by using the Escherichia coli BL21 star™ (DE3) as host expression. The research work aims to optimize the production of the enzyme through an auto-induction strategy in chemically defined media by using lactose as a natural inducer, thereby overcoming various limitations of conventional IPTG induction methods. The optimal concentration of lactose, glucose, and glycerol for maximum expression of DAEase was determined to be 1.5%, 0.125%, and 1.5%, respectively. Fermentor-scale optimization yielded a maximum amount of this enzyme with 5% inoculant, 300 rpm agitation, and 2 vvm aeration. Purification by affinity and anion exchange chromatography resulted in a sevenfold increase in specific activity with an overall yield of 12% and 43 mg of pure recombinant DAEase per liter of culture. Enzyme assays confirmed that DAEase had catalytic activity in the conversion of D-fructose to D-allulose, which was further confirmed by HPLC analysis. This optimized auto-induction-based strategy demonstrated its potential for large-scale production of DAEase in a cost-effective manner with enhanced reproducibility to meet industrial demands for synthesizing D-allulose. Public Library of Science 2025 Article PeerReviewed text en http://psasir.upm.edu.my/id/eprint/120688/1/120688.pdf Lischer, Kenny and Laksmi, Fina Amreta and Nugraha, Yudhi and Az-Zahra, Fauziah and Herawan, David and Juanssilfero, Ario Betha and Wibowo, Des Saputro and Ramadhan, Kharisma Panji and Nuryana, Isa and Ali, Mohd Shukuri Mohamad (2025) Production of recombinant D-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application. PLOS ONE, 20 (7). art. no. e0327420. pp. 1-18. ISSN 1932-6203 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0327420 10.1371/journal.pone.0327420 |
| spellingShingle | Lischer, Kenny Laksmi, Fina Amreta Nugraha, Yudhi Az-Zahra, Fauziah Herawan, David Juanssilfero, Ario Betha Wibowo, Des Saputro Ramadhan, Kharisma Panji Nuryana, Isa Ali, Mohd Shukuri Mohamad Production of recombinant D-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application |
| title | Production of recombinant D-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application |
| title_full | Production of recombinant D-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application |
| title_fullStr | Production of recombinant D-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application |
| title_full_unstemmed | Production of recombinant D-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application |
| title_short | Production of recombinant D-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application |
| title_sort | production of recombinant d-allulose 3-epimerase utilizing an auto-induction approach in fermentor cultures suitable for industrial application |
| url | http://psasir.upm.edu.my/id/eprint/120688/ http://psasir.upm.edu.my/id/eprint/120688/ http://psasir.upm.edu.my/id/eprint/120688/ http://psasir.upm.edu.my/id/eprint/120688/1/120688.pdf |