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Localization of phytase in Selenomonas ruminantium and Mitsuokella multiacidus by transmission electron microscopy
C G D'Silva1, H D Bae, L J Yanke
1Agriculture and Agri-Food Canada Research Centre, Lethbridge, AB, Canada.
Abstract:
The localization of phytase (myo-inositol-hexaphosphate phosphohydrolase) in the ruminal bacteria, Selenomonas ruminantium JY35 and Mitsuokella multiacidus 46/5(2), was determined with transmission electron microscopy. Phosphate produced from the enzymatic dephosphorylation of the calcium salt of phytic acid is precipitated as calcium phosphate. The calcium is then replaced with lead to produce electron-dense lead phosphate. This deposition of lead phosphate localized phytase in S. ruminantium JY35 and M. multiacidus 46/5(2) to the outer membrane, and confirmed intracellular expression of the enzyme in Escherichia coli pSrP.2, the recombinant clone which possesses the gene (phyA) encoding phytase (phyA) in S. ruminantium.
Insights
Phytase, an enzyme crucial for breaking down phytic acid, was localized to the outer membrane of ruminal bacteria. This finding was confirmed using transmission electron microscopy in Selenomonas ruminantium and Mitsuokella multiacidus.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Phytase (myo-inositol-hexaphosphate phosphohydrolase) plays a key role in nutrient bioavailability.
- Understanding phytase localization in ruminal bacteria is essential for optimizing digestion and nutrient absorption in ruminants.
Purpose of the Study:
- To determine the precise cellular localization of phytase in the ruminal bacteria Selenomonas ruminantium and Mitsuokella multiacidus.
- To confirm the intracellular expression of the phytase gene (phyA) in a recombinant Escherichia coli clone.
Main Methods:
- Transmission electron microscopy was employed to visualize enzyme localization.
- A lead-based precipitation method was used to detect phosphate released by phytase activity.
- Recombinant Escherichia coli expressing the S. ruminantium phytase gene (phyA) were utilized for confirmation.
Main Results:
- Phytase was localized to the outer membrane of Selenomonas ruminantium JY35 and Mitsuokella multiacidus 46/5(2).
- The enzymatic activity resulted in the precipitation of electron-dense lead phosphate at the enzyme's location.
- Intracellular expression of phytase was confirmed in the recombinant Escherichia coli clone pSrP.2.
Conclusions:
- The outer membrane of S. ruminantium and M. multiacidus is the primary site of phytase activity.
- These findings provide crucial insights into the role of ruminal bacteria in phytic acid degradation.
- The study validates the use of electron microscopy for enzyme localization and confirms the functionality of the cloned phytase gene.