Flexible and expeditious assay for quantitative monitoring of alpha-amylase and amyloglucosidase activities.

The monitoring of the activity of alpha-amylase and amyloglucosidase is an important tool for studying their role in the hydrolysis of starch. Here we introduced an improved method capable to measure the activity of alpha-amylase and amyloglucosidase from different sources based on a quantitative starch-iodine assay. The developments of the assay sought the consistent preparation of the reagents, the rescale of the assay and the adjustment of the sensitivity. This was complemented by a glucose yield assay for amyloglucosidase that allowed a secondary source of information when insoluble starches were studied. The proposed method showed high precision in long-term use (RSD < 6.3%). Furthermore, all experimental conditions can be adapted according to the equipment available at each laboratory, transforming this method in a broadband analytical tool for screening alpha-amylase and amyloglucosidase activities. •Tailorable assay based on the starch-iodine staining for the determination of alpha-amylase and amyloglucosidase activities.•Enhanced consistence of reagent preparation.•High intra-day and inter-day reproducibility.

The intensification of amyloglucosidase-based saccharification by ultrasound.

The present report studied the role of ultrasound (US) energy in the amyloglucosidase-based starch hydrolysis using two complementary approaches: (i) in the activity of six commercially-available amyloglucosidases (using soluble starch as substrate), and (ii) in the hydrolysis of four pure starches from different botanical sources. This corresponds to the first systematic evaluation of the role of US in starch hydrolysis mediated by amyloglucosidase, being a consequence of our previous report that assessed the effect of US in the activity of alpha-amylase (LWT - Food Science and Technology 84 (2017) 674-685). Regarding amlyloglucosidases, three enzymes obtained from Aspergillus niger (AN1-AN3), and Spirizyme Achieve (SPA), Spirizyme Fuel (SPF) and Spirizyme Ultra (SPU) were submitted to a Box-Behnken experimental design in order to establish the optimum conditions for their maximum activity. In the presence of US, we found both inactivation and activation, ranging from -88% (AN3) to 699% (SPA). The US promoted the enzyme activity when combined with lower temperatures (40-60 °C), with a marked effect in Spirizyme enzymes. Based on the optimum conditions established by the experimental design, we also evaluated the role of US in the glucose yield resulting from the hydrolysis of pure starches (corn, rice, potato, wheat). In this case, US led to higher glucose yields in all conditions tested. The enhancement factors observed ranged from 1.2 (AN1, rice starch) to 65 (SPA, potato starch) times. We compared these findings with previous reports, which highlighted the role of US in intensifying amyloglucosidase-based saccharification in mild conditions, by simultaneously influencing both enzyme and substrate. Hence, US power has to be fine-tuned for each particular enzyme in order to maximize process intensification.