Author
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Phillippy, Brian |
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Mullaney, Edward |
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Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 5/9/1997 Publication Date: N/A Citation: N/A Interpretive Summary: Phytase is an enzyme that breaks down phytic acid, which is a compound found in seeds that pigs and chickens cannot digest. The fungal phytase used as a feed supplement was cloned in bacteria to permit its improvement through genetic engineering. An enzyme with increased heat stability would withstand the commercial feed manufacturing process and help reduce groundwater pollution by phytic acid in animal waste. Technical Abstract: The gene (phyA) for the Aspergillus niger phytase with optima at pH 5.5 and 2.2 was expressed in Escherichia coli under the control of the lacUV5 promoter. A 56 kDa fusion protein comprised of phytase linked to an S-Tag leader peptide accumulated in inclusion bodies at 30 dec C. The yield of unglycosylated recombinant phytase purified from 50 ml cultures by anion exchange chromatography of solubilized inclusion body protein was 10 mg. The refolded enzyme had an activity of 1.5 umol mg to the -1 min to the -1 at 37 deg C, but most of the protein was in the form of inactive aggregates. Recombinant phytase displayed a single pH optimum at pH 5.1 and was not active above 55 deg C. As with A. Niger phytase, the initial breakdown product observed was inositol 1,2,4,5,6-pent hosphate. Km values for the hydrolysis of phytic acid and p-nitrophenylphosphate were 96 uM and 2.0 mM, respectively, at pH 4.5. |
