Molecular Study of Beta Propeller Phytases

This dissertation, "Molecular Study of Beta Propeller Phytases" by Chi-wai, Cheng, 鄭智威, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled MOLECULAR STUDY OF BETA PROPELLER PHYTASES Submitted by CHENG CHI WAI for the degree of Master of Philosophy at The University of Hong Kong in August 2005 Phytate, a dominant organic phosphorus compound, is very stable in soils. Although a substantial amount of phytate is carried from terrestrial to aquatic systems, it is a minor component of organic phosphorus in coastal sediments. The ephemeral nature of phytate implies rapid hydrolysis under aquatic conditions. Among the four classes of known phytases that have been identified in terrestrial organisms, only beta propeller phytase-like sequences have been identified in the aquatic environment. A novel beta propeller phytase gene (phyS), cloned from Shewanella oneidensis MR-1, was found to encode a protein with two beta-propeller phytase domains. The characterization of recombinant full-length PhyS demonstrated that Domain II was the catalytic domain responsible for phytate hydrolysis. The full-length PhyS displayed a K of 83 M with -1 -1 a kcat of 175.9 min and Domain II displayed a K of 474 M with a kcat of 10.6 min . These results confirm that the phyS gene encodes a functional beta propeller phytase, which is expressed in S. oneidensis under P-deficient conditions. The presence of multiple sequences with a high similarity to phyS in aquatic environmental samples and the widespread occurrence of the Shewanella species in nature suggest that the beta propeller phytase family is the major class of phytases in the aquatic environment, and that it may play an important role in the recycling of phosphorus. Unlike Bacillus phytases, which do not carry disulfide bonding, many other beta propeller phytase-like sequences contain cysteine residues that may form disulfide bonds. By molecular modeling based on the comparison between the cysteine-containing beta propeller phytase-like sequences and Bacillus phytases, two amino acid residues of B. subtilis 168 phytase (168PhyA), Ser-161 and Leu-212, were mutated to cysteine residues by site-directed mutagenesis. The double cysteine mutant was secreted from B. subtilis, and the expression level of the mutant phytase was about 3.5-fold higher than that of the wild type. The disulfide bond had no effect on enzyme kinetics. The mutant phytase had a lower thermostability, which retained only 20% of its initial activity after 10 minutes of incubation above 70℃, while the wild type phytase retained 80% of its initial activity in the same condition. In the natural environment, beta propeller phytases can be subdivided into two groups: one with and the other without cysteine residues. Although the mutant phytase exhibited less stability against thermal inactivation, its higher expression yield suggests that cysteine-containing beta propeller phytase-like proteins may have higher stability towards protease susceptibility and hence, longer half-life in nature. DOI: 10.5353/th_b3638780 Subjects: Phytases - Genetics