Development and Evaluation of an HPLC Method for Accurate Determinations of Enzyme Activities of Naringinase Complex

• Published in: Journal of agricultural and food chemistry 2013-10, Vol.61 (42), p.10026-10032
• Main Authors: Ni, Hui, Xiao, An-Feng, Wang, Ya Qi, Chen, Feng, Cai, Hui-Nong, Su, Wen-Jun
• Format: Article
• Language: English
• Subjects: alpha-L-rhamnosidase; beta-glucosidase; beta-Glucosidase - chemistry; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; enzyme activity; Enzyme Assays - methods; Flavanones - chemistry; Fundamental and applied biological sciences. Psychology; high performance liquid chromatography; Hydrolysis; Kinetics; Multienzyme Complexes - chemistry; naringenin; naringin; naringinase; Phlorhizin - analogs & derivatives; Phlorhizin - chemistry; solubility
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf402711h

An HPLC method that can separate naringin, prunin, and naringenin was used to help accurately measure the activities of naringinase and its subunits (α-l-rhamnosidase and β-d-glucosidase). The activities of the naringinase and β-d-glucosidase were determined through an indirect calculation of the naringenin concentration to avoid interference from its poor solubility. The measured enzymatic activities of the naringinase complex, α-l-rhamnosidase, and β-d-glucosidase were the as same as their theoretical activities when the substrates’ (i.e., naringin or prunin) concentrations were 200 μg/mL, and the enzyme concentrations were within the range of 0.06–0.43, 0.067–0.53, and 0.15–1.13 U/mL, respectively. The β-d-glucosidase had a much higher V max than either naringinase or α-l-rhamnosidase, implying the hydrolysis of naringin to prunin was the limiting step of the enzyme reaction. The reliability of the method was finally validated through the repeatability test, indicating its feasibility for the determinations of the naringinase complex.