Biochemical characterization of a novel hyperthermophilic α-l-rhamnosidase from Thermotoga petrophila and its application in production of icaritin from epimedin C with a thermostable β-glucosidase

Xie, Jingcong; Zhang, Shanshan; Tong, Xinyi; Wu, Tao; Pei, Jianjun; Zhao, Linguo

doi:10.1016/j.procbio.2020.03.019

Cited by 22 publications

(22 citation statements)

References 46 publications

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“…The molecular mass of the enzyme from P. laurentii ZJU-L07 was similar to those of α-L-rhamnosidases from Bacillus sp. GL1 (100.0 kDa) [ 27 ], Bifidobacterium dentium (100.0 kDa) [ 28 ], Thermotoga petrophila (101.7 kDa) [ 23 ], Aspergillus nidulans (102.0 kDa) [ 29 ], and Penicillium griseoroseum MTCC-9224 (97.0 kDa) [ 30 ].…”

Section: Resultsmentioning

confidence: 99%

“…When epimedin C was used as a nature substrate to determine the kinetic parameters of α-L-rhamnosidase, the values of K m and V max were 3.28 mM and 0.01 μmol·mg −1 ·min −1 , respectively ( Figure 3 g). K m of α-L-rhamnosidase with epimedin C as the substrate was higher than those of α-L-rhamnosidase from A. nidulans and T. petrophila [ 14 , 23 ].…”

Section: Resultsmentioning

confidence: 99%

“…Different sources of α-L-rhamnosidase have different abilities to hydrolyze different glycosidic bonds [ 15 ]. It was reported that α-L-rhamnosidase from B. thetaiotaomicron [ 13 ], A. nidulans [ 14 ], and T. petrophila [ 23 ] could convert epimedin C into icariin, but it was rarely reported that α-L-rhamnosidase from yeast could hydrolyze epimedin C. In this study, α-L-rhamnosidase from P. laurentii ZJU-L07 could hydrolyze the rhamnoside-α-1,2-rhamnoside glycosidic bond and transform epimedin C into icariin. Moreover, α-L-rhamnosidase from P. laurentii ZJU-L07 could also hydrolyze the glucoside-α-1,2-rhamnoside glycosidic bond and transform neohesperidin and naringin into hesperetin-7-O-glucoside and prunin, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…The flavonoids were determined by HPLC analysis using the modified methods described previously [ 13 , 16 , 20 , 23 ]. The epimedin C and icariin were analyzed using an HPLC system (Angilent1100) and a C18 column with acetonitrile (solvent A) and water (solvent B).…”

Section: Methodsmentioning

confidence: 99%

“…The sample volume injected was 10 μL and the column temperature was 30 °C. The flow rate was 1 mL·min −1 and the absorbance was determined at 270 nm [ 13 , 23 ]. For the elution program, the following proportions of solvent A were used: 0–15 min, 22%A–28%A; 15–30 min, 28%A–29%A; 30–56 min, 29%A–100%A.…”

Section: Methodsmentioning

confidence: 99%

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Purification and Characterization of a Novel α-L-Rhamnosidase from Papiliotrema laurentii ZJU-L07 and Its Application in Production of Icariin from Epimedin C

Lou

Liu

et al. 2022

JoF

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Icariin is the most effective bioactive compound in Herba Epimedii. To enhance the content of icariin in the epimedium water extract, a novel strain, Papiliotrema laurentii 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−1 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 Ca2+, Fe2+, Cu2+, and Mg2+, especially Fe2+. The kinetic parameters of Km and Vmax were 1.38 mM and 24.64 μmol·mg−1·min−1 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 Km and Vmax were 3.28 mM and 0.01 μmol·mg−1·min−1, 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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Purification and Characterization of a Novel α-L-Rhamnosidase from Papiliotrema laurentii ZJU-L07 and Its Application in Production of Icariin from Epimedin C

Lou

Liu

et al. 2022

JoF

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Aspergillus oryzae α‐l‐rhamnosidase: Crystal structure and insight into the substrate specificity

Makabe,

Ishida,

Kanezaki

et al. 2023

Proteins

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The subsequent biochemical and structural investigations of the purified recombinant α‐l‐rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild‐type was higher toward hesperidin and narirutin, where the l‐rhamnose residue was α‐1,6‐linked to β‐d‐glucoside, than toward neohesperidin and naringin with an α‐1,2‐linkage to β‐d‐glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C. rAoRhaA kinetic analysis indicated that Km values for neohesperidin, naringin, and rutin were lower compared to those for hesperidin and narirutin. kcat values for hesperidin and narirutin were higher than those for neohesperidin, naringin, and rutin. High catalytic efficiency (kcat/Km) toward hesperidin and narirutin was a result of a considerably high kcat value, while Km values for hesperidin and narirutin were higher than those for naringin, neohesperidin, and rutin. The crystal structure of rAoRhaA revealed that the catalytic domain was represented by an (α/α)6‐barrel with the active site located in a deep cleft and two β‐sheet domains were also present in the N‐ and C‐terminal sites of the catalytic domain. Additionally, five asparagine‐attached N‐acetylglucosamine molecules were observed. The catalytic residues of AoRhaA were suggested to be Asp254 and Glu524, and their catalytic roles were confirmed by mutational studies of D254N and E524Q variants, which lost their activity completely. Notably, three aspartic acids (Asp117, Asp249, and Asp261) located at the catalytic pocket were replaced with asparagine. D117N variant showed reduced activity. D249N and D261N variants activities drastically decreased.

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α-l-rhamnosidase: production, properties, and applications

Pan

Zhang

et al. 2023

World J Microbiol Biotechnol

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Biochemical characterization of a novel hyperthermophilic α-l-rhamnosidase from Thermotoga petrophila and its application in production of icaritin from epimedin C with a thermostable β-glucosidase

Cited by 22 publications

References 46 publications

Purification and Characterization of a Novel α-L-Rhamnosidase from Papiliotrema laurentii ZJU-L07 and Its Application in Production of Icariin from Epimedin C

Purification and Characterization of a Novel α-L-Rhamnosidase from Papiliotrema laurentii ZJU-L07 and Its Application in Production of Icariin from Epimedin C

Aspergillus oryzae α‐l‐rhamnosidase: Crystal structure and insight into the substrate specificity

α-l-rhamnosidase: production, properties, and applications

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