Assessing phytase activity

Abstract

Phytases are nutritionally important for increased bioavailability of dietary minerals and phosphate for monogastric animals including humans. Release of minerals and phosphate is accomplished by the enzymatic stepwise degradation of phytate (inositol hexaphosphate, IP 6 ). Activity determinations of phytase is often based on analysis of total released phosphate (P i ), but phytase activity in its purest form represents released product per time from IP 6 only. Microbial and plant preparations often also contain mixtures of phosphatases and organic phosphate compounds; hence some released phosphate in enzymatic assays may originate from non-phytase phosphatases degrading non-phytate molecules. Moreover, even purified enzyme extracts assessed via P i release may result in errors, since commercial IP 6 commonly contains contamination of lower inositol phosphates, and further, the products of phytase IP 6 hydrolysis are also substrates for the phytase. These facts motivate a quantitative comparative study. We compared enzyme activity determination in phytase assay samples at four different time points, based on analyzing the substrate IP 6 versus the product P i using different selected methods. The calculated activities varied substantially. For example, at 15 min into enzymatic assay, variations from 152 mU/ml (by IP 6 analysis on HPIC) to 275-586 mU/ml (by P i analysis using several methods) was detected. Our work emphasizes the importance of defining the type of activity assessed, showing that phytase activity based on released P i may yield false positive results and/or overestimations. We propose to differentiate between phytase activity, being the activity by which IP 6 is degraded, and total inositol phosphatase activity, corresponding to total released phosphate during the enzymatic reaction.

Effect of malting period on physicochemical properties, minerals, and phytic acid of finger millet (Eleusine coracana) flour varieties

“…Principally, phytase reduces the hexa form of phytic acid (IP6, myo ‐inositol 1,2,3,4,5,6‐hexakisphosphate) into lower forms such as IP5, IP4, IP3, IP2, IP1, and myo ‐inositol (Agte, Gokhale, & Chiplonkar, ). In the process, lower phytic acids IP5, IP4, and IP3 are simultaneously being formed as products from the dissociation and or reconfiguration of other higher inositol phosphates already present like IP5 and IP4 (Qvirist, Carlsson, & Andlid, ). This sequence of enzymatic reaction in turn increases the amount of inorganic phosphate and lower phytic acids, which co‐elute as total phytic acid content.…”

“…The increase in the mineral content of the cereal grains observed after 24 hr of malting could be due to an accumulative effect resulting from the dissociation and or reconfiguration of phytate complexes and other mineral binding components of the grain during malting, and as well as the presence of mineral enhancers like ascorbic acids (Krishnan et al, 2012;Mamiro et al, 2001;Sripriya et al, 1997) & Chiplonkar, 1997). In the process, lower phytic acids IP5, IP4, and IP3 are simultaneously being formed as products from the dissociation and or reconfiguration of other higher inositol phosphates already present like IP5 and IP4 (Qvirist, Carlsson, & Andlid, 2015). This sequence of enzymatic reaction in turn increases the amount of inorganic phosphate and lower phytic acids, which co-elute as total phytic acid content.…”

Isolation, Identification and Characterization of Yeasts from Fermented Goat Milk of the Yaghnob Valley in Tajikistan

“…The incubation was performed for 24 h at 30°C with stirring. After incubation, cells were pelleted by centrifugation at 5000 × g, and the cell-free supernatant was used for assay of phytase activity as previously described (Qvirist et al, 2015). The assay samples were analyzed for IP 6 concentration using HPIC as previously described (Carlsson et al, 2001), and compared with the phytase positive reference strain Pichia kudriavzevii TY13 from previous work (Qvirist et al, 2015).…”

Microbial production of phytases for combating environmental phosphate pollution and other diverse applications

“…Microbial preparations for the analysis contain mixtures of phosphatases and organic phosphate compounds; hence some released phosphate in enzymatic assays may originate from nonphytase phosphatases degrading nonphytate molecules. Moreover, even purified enzyme extracts assessed via Pi release may result in errors, as commercial IP6 commonly contains contamination of other inositol phosphates, and furthermore the products of phytase IP6 hydrolysis by phytase can serve as substrates for additional hydrolysis by the phytase (Qvirist et al, 2015). Phytase activity determinations may seem straightforward, but can be deceptive and involve a number of uncertainties.…”