Aqueous micellar two phase systems as novel tools to recover pepsin like surubí proteases: effect of surfactants on enzyme activity

Abstract

Surubí is farmed in the northeast of Argentina. It is of interest to valorize the current disposal of fish processing through the use of viscera waste, source of enzymes such as Pepsin. These enzymes have been recovered mainly using conventional methods (e.g.salting out, chromatography), however the use of liquid-liquid extraction using surfactants have not been fully explored. Aqueous micellar two-phase systems (AMTPS) are an extractive method based on the ability of some surfactants to form two immiscible aqueous phases, a rich and a poor micelle phases to recover the product of interest. Thus, the objective of this work was to firstly evaluate the stability of surubí crude extracts under different concentrations of Genapol (GX080) and Tergitol (Tg7) to estimate the feasibility of their use as potential micellar extractants of surubí pepsin. Enzymatic extracts (ERPi) were recovered from stomachs homogenates using salting-out procedure and pepsin activity was estimated by acid hemoglobin method. ERPi were first incubated with various concentrations of surfactants (1, 3 and 5% GX080/Tg7) in 100mM NaCit pH 5 for different times (0, 1, 2 and 3h) and then enzymatic activity was measured. Data represent the mean ± standard deviation (SD) of at least three replications. Statistical significance was tested by one-way ANOVA and Tukey (HSD) (p<0.05) Results showed that any surfactant concentration tested affected ERPi enzymatic activity moderately and this effect was dose dependent. ERPi retained 92, 65 and 60% of its initial activity when were incubated with 1, 3 and 5% of Tg7. After incubating with GX080 (1, 3 and 5%), the ERPi exhibited 80, 70 and 60% of its initial activity, respectively. The time variable in these assays had not significant influence on enzymatic activity decrease. Pepsin, a hydrophilic protein, is expected to be preferentially distributed into the aqueous phase of AMTPS.Considering that the surfactant concentration at this phase is always below 1%, the obtained results suggest that AMTPS formed with either GX080 or Tg7 could be viable tools in the primary recovery of pepsin-like proteases from fishing waste.