Acceleration of enzymatic hydrolysis of protein-bound selenium by focused microwave energy

Abstract

This paper demonstrates for the first time that enzymatic hydrolysis of protein-bound selenium can be significantly accelerated by microwave energy. Extraction of selenomethionine from selenised yeast, used as the model sample, was performed using two-step enzymatic hydrolysis with protease/lipase/driselase in a focused microwave reactor. HPLC-ICP-MS with species-specific isotope dilution analysis (IDA) was used for species quantitation. To investigate enzymatic hydrolysis assisted by microwave energy the effect of parameters such as microwave power and extraction time on the extraction efficiency of SeMet from the Se-yeast CRM SELM-1 was investigated. The main chemical variables affecting enzymatic extraction of SeMet, such as pH, enzyme type, sample to enzyme mass ratio and sample to solvent mass ratio were set at the conditions used for conventional enzymatic hydrolysis (in a hybridisation oven). Using a power of 60 W and two consecutive extraction cycles, each of 15 min (total extraction time of 30 min) at 37 °C, the recovery of SeMet obtained by the proposed methodology and calculated based on the certified value of 3389 ± 173 mg kg⁻¹SeMet was 99.6 ± 1.9% (n = 3). The major advantage of the newly proposed method is the dramatic reduction of extraction time in comparison with that of conventional enzymatic hydrolysis, which requires a two-step extraction, each cycle of approximately 20 h. This was achieved without compromising extraction efficiency. Application of the proposed method to the accurate quantification of SeMet in pharmaceutical yeast tablets with a total Se concentration of approximately 300 mg kg⁻¹ was also addressed. The recovery of SeMet, which was calculated based on the mean of results [561.5 mg kg⁻¹ (n = 11, with a standard deviation of 44.3 mg kg⁻¹)] obtained for the yeast tablets in the international intercomparison CCQM-P86,¹ was found to be 100.1 ± 3.3%.