Assessing phytase activity–methods, definitions and pitfalls

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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, IP₆). 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 IP₆ 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 IP₆ commonly contains contamination of lower inositol phosphates, and further, the products of phytase IP₆ 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 IP₆ 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 IP₆ 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 IP₆ is degraded, and total inositol phosphatase activity, corresponding to total released phosphate during the enzymatic reaction.

Keywords

inositol phosphatase activity

inositol phosphate

phosphate analysis

phytase activity

phytate

References

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