A New Rapid and Quantitative Assay to Determine the Phytase Activity of Feed.

The fortification of animal feed with enzymes in order to optimize feed utilization has become a standard for the meat production industry. A method for measuring levels of active enzymes that can be carried out quickly would ensure that feed has been supplemented with the appropriate amount of enzyme. Phytase is the most widely used feed enzyme and is routinely quantified with an activity assay in a limited number of specialized laboratories. As an alternative, we report here the development of a rapid and easy method to perform a quantitative assay for the phytase from Citrobacter braakii. The method is suitable for use at local sites with a minimum lab setup and will reduce delays and potential interferences due to improper sample storage and shipment. The new assay is based on a lateral flow immunoassay that utilizes magnetic immune-chromatographic test (MICT) technology to quantify the phytase content of a feed extract. After extraction of the phytase from the feed, the sample is simply diluted and added to a reaction tube containing a specific anti-phytase antibody coupled to superparamagnetic particles. The mixture is then applied on an assay cassette, where the formed particle-antibody-phytase complexes are captured by immobilized antibodies on a nitro-cellulose strip housed in a cassette. The cassette is placed in the MICT reader that measures the magnetic signal of the captured particles. Using the calibration information stored in the cassette barcode, the signal is converted to a phytase concentration, given as phytase activity (FYT) per kilogram of feed. The accuracy and robustness of the assay compared to the ISO phytase activity assay were demonstrated through a large validation study including real feed samples from different compositions and origins. The MICT assay is the first quantitative assay for feed enzymes that is fast, reliable, and simple to use outside of a specialized reference laboratory and that is suitable for use in place of the current ISO assay.

Degradation of phytate by the 6-phytase from Hafnia alvei: a combined structural and solution study.

Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), ß-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.

In vitro and in vivo degradation of myo-inositol hexakisphosphate by a phytase from Citrobacter braakii.

Phytases (EC 3.1.3) are widely used in animal feed to increase the availability of phosphorus and decrease the anti nutritive effect of myo-inositol hexakisphosphate (InsP6). The aim of this work was to investigate the stereospecific degradation of InsP6 in vitro and in vivo by a phytase from Citrobacter braakii (C. braakii), and to study gastric survival of the phytase as well as the site of action in the gastrointestinal tract. The in vitro results showed that the C. braakii phytase belongs to the group of 6-phytases (EC 3.1.3.26). However, in approximately one out of 10 instances the phytase initiated hydrolysis at the D-3 (L-1) position, demonstrating that phytase specificity is not unambiguous. Following the main degradation pathway, InsP6 was degraded by stepwise removal of the phosphate groups on positions 6/1/5. The stereospecificity was found to be similar under in vitro and in vivo conditions. The phytase was found to be stable in the gastric environment and to be active in the stomach and possibly also in the proximal small intestine. While InsP4 was accumulated under in vitro conditions this was not the case in vivo, where both InsP5 and InsP4 were seen to be hydrolysed in the small intestine, possibly as a combined action of the C. braakii phytase and endogenous phosphatases present in the mucosa. The ability of the C. braakii phytase to focus its activity on degrading InsP6 to InsP4 is believed to be a favourable complement to the endogenous phosphatases.

The degradation of phytate by microbial and wheat phytases is dependent on the phytate matrix and the phytase origin.

BACKGROUND: Phytases increase utilization of phytate phosphorus in feed. Since wheat is rich in endogenous phytase activity it was examined whether wheat phytases could improve phytate degradation compared to microbial phytases. Moreover, it was investigated whether enzymatic degradation of phytate is influenced by the matrix surrounding it. Phytate degradation was defined as the decrease in the sum of InsP6 + InsP5. RESULTS: Endogenous wheat phytase effectively degraded wheat Ins6 + InsP5 at pH 4 and pH 5, while this was not true for a recombinant wheat phytase or phytase extracted from wheat bran. Only microbial phytases were able to degrade InsP6 + InsP5 in the entire pH range from 3 to 5, which is relevant for feed applications. A microbial phytase was efficient towards InsP6 + InsP5 in different phytate samples, whereas the ability to degrade InsP6 + InsP5 in the different phytate samples ranged from 12% to 70% for the recombinant wheat phytase. CONCLUSION: Wheat phytase appeared to have an interesting potential. However, the wheat phytases studied could not improve phytate degradation compared to microbial phytases. The ability to degrade phytate in different phytate samples varied greatly for some phytases, indicating that phytase efficacy may be affected by the phytate matrix.