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Date of Award
Campus Access Dissertation
Doctor of Philosophy in Chemistry (PhD)
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Department of Chemistry
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Acid phytases from fungal sources are used as animal feed supplements to alleviate the environmental contamination and nutritional issues associated with the high concentration of phytic acid in seeds (5-8% of the dry cell weight). Acid phytases function optimally at acidic pH values, however there is need for phytases that operate over a broader pH range. Additionally, there are concerns about potential allergic responses to the incorporation of fungal proteins into the diets of animals for human consumption. A variety of phytases with a range of catalytic and thermal stability properties is needed to be effective in different animals. Our lab has isolated an alkaline phytase from Lilium longiflorum and expressed it in Pichia pastoris. This dissertation describes the: (1) enhancement of the intracellular expression yield of alkaline phytase by employing a constitutive promoter, (2) establishment of extracellular expression of alkaline phytase by employing different secretion signals, (3) delineation of structure-activity relationships of mutant alkaline phytase, (4) development of colorimetric assays to rapidly screen for alkaline phytase activity in a large number of clones, and (5) development of NMR methods for the structural investigation of inositol pyrophosphates.
The intracellular yield of alkaline phytase was investigated using two different promoters: the tightly-regulated promoter of the alcohol oxidase 1 gene (PAOX1) and the constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP). Employing PGAP resulted in a four-fold increase in intracellular expression (100-120 mg/L) over the use of PAOXI. Extracellular expression constructs with various mutant forms of the rLlAlp2 gene downstream of either the alpha-mating factor secretion signal (α-MF) or the chicken lysozyme (CL) secretion signal were generated and extracellular expression levels investigated. The data suggest that with α-MF-driven secretion, deletion of both N- and C-termini native signal peptides (m2-rLlALP2) enhanced extracellular expression eight- to ten-fold with the constitutive promoter PGAP (80-100 mg/L) compared to PAOXI (~10 mg/L).
To delineate structure-activity relationships, the catalytic activity of m2-rLlALP2 was compared to the wt-phytase. Catalytic activity over a broad pH range is important to the feed industry as this enables the enzyme to efficiently digest phytic acid during passage through the GI tracts of different animals. Thermal stability is also important because the enzyme is exposed to approximately 80 °C for 10 min during the feed pelleting process. At pH 9.0 and 6.0, m2-rLlALP2 retained 90% and 40% activity, respectively, compared to 50% and 10% retained by wt-phytase. At 60 °C and 55 °C, m2-rLlALP2 retained 35% and 70% activity, respectively, compared to 15% and 50% retained by wt-phytase. Thus, the improved activity over a broad pH range and temperature profile suggest that the deletion of amino acids at the N- and C-terminal domains resulted in beneficial catalytic properties.
Mutagenesis can be used to change the catalytic properties of enzymes. Mutagenesis experiments generate hundreds of clones so a rapid assay to screen for phytase activity in clones is critical. The current standard assay involves many steps and is time-consuming (requires 24 h). Colorimetric assays were developed using the substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and para-nitrophenyl phosphate (p-NPP). These assays yield colored products visible to the eye and can be completed in less than one hour after cell collection.
Although inositol phosphates have been investigated for over a hundred years, inositol pyrophosphates have been discovered only recently. The low abundance and facile hydrolysis of the unstable pyrophosphate bond make structural analysis of these highly polar compounds challenging. NMR spectroscopy provides unambiguous structural information when sufficient quantities (1-5 mg) of compounds are available. Pyrophosphorylation resulted in significant (δ 8.0-12.0) upfield shifts of both the Pα and Pβ resonances in 31P-NMR. However, 1H-NMR of 4-InsP7 and 1,5-InsP8 revealed small (δ 0.01-0.20) upfield shifts in the 1H resonances geminal, vicinal, and two carbons removed from the pyrophosphate moiety. Thus, 31P-NMR provides clear evidence for pyrophosphorylation. The two-dimensional NMR methods Total Correlation Spectroscopy (TOCSY) and Heteronuclear Multiple Quantum Coherence (HMQC) were modified to enable conclusive structural assignment of pyrophosphorylated compounds. The results show that the pyrophosphate moiety may be used as a handle to identify all 1H resonances on an individual inositol polyphosphate in a complex spectrum.
Teymorian, Sasha, "Extracellular expression of alkaline phytase in Pichia pastoris and Development of Nuclear Magnetic Resonance spectroscopy methods for structural investigation of inositol polyphosphates", Campus Access Dissertation, Michigan Technological University, 2015.