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Susceptibility of phytase to proteolysis by CIBENZA®EP150

Adding CIBENZA® EP150 to feeds containing phytase activity should not affect the levels of phytase activity.

Phytase is an enzyme that catalyzes the release of inorganic phosphate from phytic acid. Like all proteins, phytase is susceptible to degradation by proteases. Several experiments were conducted to probe the effect of a protease, CIBENZA® EP150 feed additive, on the activity of a phytase. Testing in the presence or absence of feed was performed at acidic pH,conditions favoring phytase activity, but not CIBENZA® EP150 activity. The experiments demonstrated that there was little or no effect on phytase activity in the presence of CIBENZA® EP150.

Methodology

The sources of phytase used originated from either fungal or bacterial sources (5,000 U/g) and the source of protease was CIBENZA® EP150 (min. 600.000 U/g). In the absence of feed, the standard Novus International phytase method (Method 260140-2, Assay of Phytase Activity) was used to detect phytase activity. For the experiments in which feed was present, phytase activity was determined using the International Standards Organization method 30024 (Animal Feeding Stuffs – Determination of Phytase Activity). For all experiments, phytase and CIBENZA® EP150 were used at their recommended inclusion rates, 0.5 U/g feed and 300 U/g feed, respectively. In the absence of feed, the enzyme(s) were added directly to phytase assay buffer and allowed to incubate for 10 min at 37°C prior to initiating the phytase assay.For experiments conducted in the presence of feed, a standard corn/soy diet containing 59.5 percent corn and 34.7 percent soybean meal was used. Four sets of feed samples were prepared in which 1 kg of diet was mixed with the appropriate amount of the following enzymes: 1) phytase only, 2) phytase and protease, 3) protease only and 4) no enzymes. Each feed sample was mixed manually to generate a homogenous mixture. Three independent samples were prepared by extracting the enzymes from 50 g of feed with 500 mL of water for 45 min at room temperature. A portion of each preparation was then assayed for phytase activity as described above.

Results

Effect of CIBENZA® EP150 on Phytase in the Absence of Feed

To determine if CIBENZA® EP150 could interfere with phytase activity under acidic conditions such as those present in the upper GIT, the two enzymes were mixed in phytase assay buffer and then phytase activity was determined. CIBENZA® EP150 had no effect on the activity of phytase under normal phytase assay conditions (Figure 1.).

To determine if prolonged incubation of the fungal phytase with CIBENZA® EP150 would lead to reduced phytase activity, experiments were performed in which enzymes were incubated together under acidic conditions for up to one hour at 37°C.

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The results demonstrated that CIBENZA® EP150 had little or no effect on the activity of the fungal phytase over time (Figure 2.). In separate experiments, it was shown that CIBENZA® EP150 did not affect phytase activity from a bacterial source when incubated for up to one hour under acidic conditions at 37°C (Figure 3.).

Three independent 50 g samples were taken from each feed sample and tested using the ISO method 30024. As anticipated, phytase activity was completely recovered from the feed when no CIBENZA® EP150 was present (Figure 4.). In the presence of CIBENZA EP150, there was no apparent loss in added phytase activity indicating that CIBENZA® EP150 had only a minimal effect on phytase activity in the presence of feed.



®NOVUS and CIBENZA EP150 are trademarks of Novus International, Inc., and are registered in the United States and other countries.

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