Evaluation of HPSEC‐ELSD method for precise measurement of β‐agarase activity

Abstract

β-agarase activity was monitored by traditional reducing sugar content methods: Somogyi-Nelson's arsenomolybdate, Miller's dinitrosalicylic acid and Kidby and Davidson's ferricyanide methods, as well as by high-performance size exclusion chromatography coupled with a refractive index detector and an evaporative light scattering detector (ELSD). Calibration curves were established separately for each method to measure the amounts of the neoagaro-oligosaccharides (NAOS) in the reaction mixtures, which are the products from 1-10 units (U) of β-agarase cleavage activity on agarose. Product quantities from each monitoring method were compared with the isolated NAOS products. The graphs plotted by agarase activity unit and product concentration clearly displayed that the ELSD method closely followed the results of the isolated products. The percentage deviation of results measured by the five methods away from those of the isolated NAOS product mixture amounted to -13.1-35.1, -21.1-25.5, -27.1-23.81, 6.1-24.3 and 16.2-22.8%, respectively. When the loss during product isolation, about 15-17%, was taken into account, the high precision of the ELSD method was confirmed. HPSEC-ELSD methods also accurately measured the enzyme kinetics as well as enabling partial identification of oligosaccharides assembled in the NAOS product mixture. This study established the HPSEC-ELSD system as an alternative method for monitoring agarase activity.