A Novel Sucrose Isomerase Producing Isomaltulose from <i>Raoultella terrigena</i>
Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene <i>pal</i>-2 from <i>...
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MDPI AG
2021-06-01
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| author | Li Liu Shuhuai Yu Wei Zhao |
| author_facet | Li Liu Shuhuai Yu Wei Zhao |
| author_sort | Li Liu |
| collection | DOAJ |
| description | Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene <i>pal</i>-2 from <i>Raoultella terrigena</i> was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the <i>E. coli</i> system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (<i>K<sub>m</sub></i>) and maximum reaction rate (<i>V<sub>max</sub></i>) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the <i>Raoultella</i> genus and its mutants showed a potential to be used for the production of isomaltulose in the industry. |
| first_indexed | 2024-03-10T10:24:31Z |
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| institution | Directory Open Access Journal |
| issn | 2076-3417 |
| language | English |
| last_indexed | 2024-03-10T10:24:31Z |
| publishDate | 2021-06-01 |
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| record_format | Article |
| series | Applied Sciences |
| spelling | doaj.art-7db5e1d0dd2548b99c11ddc7eb63a4592023-11-22T00:07:28ZengMDPI AGApplied Sciences2076-34172021-06-011112552110.3390/app11125521A Novel Sucrose Isomerase Producing Isomaltulose from <i>Raoultella terrigena</i>Li Liu0Shuhuai Yu1Wei Zhao2State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, ChinaState Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, ChinaState Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, ChinaIsomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene <i>pal</i>-2 from <i>Raoultella terrigena</i> was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the <i>E. coli</i> system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (<i>K<sub>m</sub></i>) and maximum reaction rate (<i>V<sub>max</sub></i>) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the <i>Raoultella</i> genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.https://www.mdpi.com/2076-3417/11/12/5521sucrose isomeraseisomaltuloseenzymology propertiesmolecular modification |
| spellingShingle | Li Liu Shuhuai Yu Wei Zhao A Novel Sucrose Isomerase Producing Isomaltulose from <i>Raoultella terrigena</i> Applied Sciences sucrose isomerase isomaltulose enzymology properties molecular modification |
| title | A Novel Sucrose Isomerase Producing Isomaltulose from <i>Raoultella terrigena</i> |
| title_full | A Novel Sucrose Isomerase Producing Isomaltulose from <i>Raoultella terrigena</i> |
| title_fullStr | A Novel Sucrose Isomerase Producing Isomaltulose from <i>Raoultella terrigena</i> |
| title_full_unstemmed | A Novel Sucrose Isomerase Producing Isomaltulose from <i>Raoultella terrigena</i> |
| title_short | A Novel Sucrose Isomerase Producing Isomaltulose from <i>Raoultella terrigena</i> |
| title_sort | novel sucrose isomerase producing isomaltulose from i raoultella terrigena i |
| topic | sucrose isomerase isomaltulose enzymology properties molecular modification |
| url | https://www.mdpi.com/2076-3417/11/12/5521 |
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