Purification and Characterization of a Novel α-L-Rhamnosidase from <i>Papiliotrema laurentii</i> ZJU-L07 and Its Application in Production of Icariin from Epimedin C
Icariin is the most effective bioactive compound in Herba Epimedii. To enhance the content of icariin in the epimedium water extract, a novel strain, <i>Papiliotrema laurentii</i> ZJU-L07, producing an intracellular α-L-rhamnosidase was isolated from the soil and mutagenized. The specifi...
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MDPI AG
2022-06-01
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| Online Access: | https://www.mdpi.com/2309-608X/8/6/644 |
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| author | Hanghang Lou Xiayu Liu Siyu Liu Qihe Chen |
| author_facet | Hanghang Lou Xiayu Liu Siyu Liu Qihe Chen |
| author_sort | Hanghang Lou |
| collection | DOAJ |
| description | Icariin is the most effective bioactive compound in Herba Epimedii. To enhance the content of icariin in the epimedium water extract, a novel strain, <i>Papiliotrema laurentii</i> ZJU-L07, producing an intracellular α-L-rhamnosidase was isolated from the soil and mutagenized. The specific activity of α-L-rhamnosidase was 29.89 U·mg<sup>−1</sup> through purification, and the molecular mass of the enzyme was 100 kDa, as assayed by SDS-PAGE. The characterization of the purified enzyme was determined. The optimal temperature and pH were 55 °C and 7.0, respectively. The enzyme was stable in the pH range 5.5–9.0 for 2 h over 80% and the temperature range 30–40 °C for 2 h more than 70%. The enzyme activity was inhibited by Ca<sup>2+</sup>, Fe<sup>2+</sup>, Cu<sup>2+</sup>, and Mg<sup>2+</sup>, especially Fe<sup>2+</sup>. The kinetic parameters of <i>K<sub>m</sub></i> and <i>V<sub>max</sub></i> were 1.38 mM and 24.64 μmol·mg<sup>−1</sup>·min<sup>−1</sup> using pNPR as the substrate, respectively. When epimedin C was used as a nature substrate to determine the kinetic parameters of α-L-rhamnosidase, the values of <i>K<sub>m</sub></i> and <i>V<sub>max</sub></i> were 3.28 mM and 0.01 μmol·mg<sup>−1</sup>·min<sup>−1</sup>, respectively. The conditions of enzymatic hydrolysis were optimized through single factor experiments and response surface methodology. The icariin yield increased from 61% to over 83% after optimization. The enzymatic hydrolysis method could be used for the industrialized production of icariin. At the same time, this enzyme could also cleave the α-1,2 glycosidic linkage between glucoside and rhamnoside in naringin and neohesperidin, which could be applicable in other biotechnological processes. |
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| language | English |
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| spelling | doaj.art-88378bb771014a7b8fa127790b746dee2023-11-23T17:25:31ZengMDPI AGJournal of Fungi2309-608X2022-06-018664410.3390/jof8060644Purification and Characterization of a Novel α-L-Rhamnosidase from <i>Papiliotrema laurentii</i> ZJU-L07 and Its Application in Production of Icariin from Epimedin CHanghang Lou0Xiayu Liu1Siyu Liu2Qihe Chen3Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, ChinaDepartment of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, ChinaDepartment of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, ChinaDepartment of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, ChinaIcariin is the most effective bioactive compound in Herba Epimedii. To enhance the content of icariin in the epimedium water extract, a novel strain, <i>Papiliotrema laurentii</i> ZJU-L07, producing an intracellular α-L-rhamnosidase was isolated from the soil and mutagenized. The specific activity of α-L-rhamnosidase was 29.89 U·mg<sup>−1</sup> through purification, and the molecular mass of the enzyme was 100 kDa, as assayed by SDS-PAGE. The characterization of the purified enzyme was determined. The optimal temperature and pH were 55 °C and 7.0, respectively. The enzyme was stable in the pH range 5.5–9.0 for 2 h over 80% and the temperature range 30–40 °C for 2 h more than 70%. The enzyme activity was inhibited by Ca<sup>2+</sup>, Fe<sup>2+</sup>, Cu<sup>2+</sup>, and Mg<sup>2+</sup>, especially Fe<sup>2+</sup>. The kinetic parameters of <i>K<sub>m</sub></i> and <i>V<sub>max</sub></i> were 1.38 mM and 24.64 μmol·mg<sup>−1</sup>·min<sup>−1</sup> using pNPR as the substrate, respectively. When epimedin C was used as a nature substrate to determine the kinetic parameters of α-L-rhamnosidase, the values of <i>K<sub>m</sub></i> and <i>V<sub>max</sub></i> were 3.28 mM and 0.01 μmol·mg<sup>−1</sup>·min<sup>−1</sup>, respectively. The conditions of enzymatic hydrolysis were optimized through single factor experiments and response surface methodology. The icariin yield increased from 61% to over 83% after optimization. The enzymatic hydrolysis method could be used for the industrialized production of icariin. At the same time, this enzyme could also cleave the α-1,2 glycosidic linkage between glucoside and rhamnoside in naringin and neohesperidin, which could be applicable in other biotechnological processes.https://www.mdpi.com/2309-608X/8/6/644<i>Papiliotrema laurentii</i> ZJU-L07epimedin Cicariinα-L-rhamnosidaseenzyme characteristicsoptimization |
| spellingShingle | Hanghang Lou Xiayu Liu Siyu Liu Qihe Chen Purification and Characterization of a Novel α-L-Rhamnosidase from <i>Papiliotrema laurentii</i> ZJU-L07 and Its Application in Production of Icariin from Epimedin C Journal of Fungi <i>Papiliotrema laurentii</i> ZJU-L07 epimedin C icariin α-L-rhamnosidase enzyme characteristics optimization |
| title | Purification and Characterization of a Novel α-L-Rhamnosidase from <i>Papiliotrema laurentii</i> ZJU-L07 and Its Application in Production of Icariin from Epimedin C |
| title_full | Purification and Characterization of a Novel α-L-Rhamnosidase from <i>Papiliotrema laurentii</i> ZJU-L07 and Its Application in Production of Icariin from Epimedin C |
| title_fullStr | Purification and Characterization of a Novel α-L-Rhamnosidase from <i>Papiliotrema laurentii</i> ZJU-L07 and Its Application in Production of Icariin from Epimedin C |
| title_full_unstemmed | Purification and Characterization of a Novel α-L-Rhamnosidase from <i>Papiliotrema laurentii</i> ZJU-L07 and Its Application in Production of Icariin from Epimedin C |
| title_short | Purification and Characterization of a Novel α-L-Rhamnosidase from <i>Papiliotrema laurentii</i> ZJU-L07 and Its Application in Production of Icariin from Epimedin C |
| title_sort | purification and characterization of a novel α l rhamnosidase from i papiliotrema laurentii i zju l07 and its application in production of icariin from epimedin c |
| topic | <i>Papiliotrema laurentii</i> ZJU-L07 epimedin C icariin α-L-rhamnosidase enzyme characteristics optimization |
| url | https://www.mdpi.com/2309-608X/8/6/644 |
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