Thermostable Phytase from a Bacillus sp.: Heterologous Production, Mutation, Characterization and Assay Development
Sammanfattning: Phytase is an important enzyme in the food/feed industry. It catalyzes the hydrolysis of phytate, an anti-nutrient compound present in cereals and grains, to release orthophosphate and myo-inositol-6-phosphate with lower degrees of phosphorylation. Phytic acid is a strong chelator capable of complexing with a variety of metal ions under neutral and alkaline conditions, as well as with proteins and starch under acidic conditions. Treatment with phytase increases not only the bioavailability of inorganic phosphorus but also the digestibility of proteins. Moreover, it improves absorption of minerals from food/feed. The action of phytase also contributes towards reducing the pollution in surface- and ground water caused by the phytate and phosphorus run-off from manure in intensive livestock regions. All the commercially available phytases are histidine acid phytases with optimum activities at low pH and with low thermostabilities. Alkaline phytases (also called -propeller phytases) are active at neutral or slightly alkaline conditions, calcium-dependent, and are quite thermostable so as to withstand the high temperatures during the pelleting of animal feeds. They can hence exhibit activities in the small intestine of animals as well as during storage of feeds. Alkaline phytases have several other potential applications. This thesis presents the studies on a phytase from Bacillus sp. MD2 isolated in Vietnam. The focus has been directed to (1) developing a new kinetic method to determine the phytase activity, (2) cloning, expression and production of the recombinant phytase from Bacillus sp. MD2, (3) the metal dependence of the catalytic properties and stability of the recombinant phytase, and (4) site-directed mutagenesis of the recombinant phytase to improve certain properties of the enzyme relevant for food/feed applications. A kinetic assay for phytases has been developed based on the turbidity reduction of phytate-protein complexes used as substrates. This method offered a reliable way to measure the enzyme activity of both histidine acid phytases and ß-propeller phytases. The method was found to be simpler, faster, and to measure the activity under conditions closer to those existing in the gastrointestinal tract of animals, thus making it more suitable for evaluating phytases for feed and food applications in comparison with the traditional method based on the release of phosphate from sodium phytate. The phytase gene from Bacillus sp. MD2 was cloned and expressed in Escherichia coli. Cultivation of the recombinant bacteria in a minimum medium using a fed-batch strategy combined with the control of the inorganic phosphate concentration resulted in a high level of production of the recombinant phytase. Lactose could be used as an alternative inducer to isopropyl--D-thiogalactoside, thus reducing the production cost. A significant amount of the expressed phytase (90% of the total active enzyme) leaked out into the medium, thereby facilitating the subsequent downstream processing. A close investigation of the effect of divalent metal ions on the activity and stability of the recombinant phytase was performed. Calcium played a critical role in stabilizing the enzyme and in activating the substrate (phytate) to fulfil the activity of the enzyme. Other metal ions (Ba2+, Mn2+, Mg2+ and Sr2+) could replace Ca2+ in the active site of the enzyme and recover more than 90% of the enzyme activity with the calcium complex substrate. On the other hand, the presence of Ca2+ on the phytate was crucial for an optimal expression of the phytase activity. The relationship between structure and function of the phytase was probed by site-directed mutagenesis. Single site mutations, S283R and E229V, on the catalytic surface increased the specific activity of the enzyme by 13 and 19%, respectively. Mutation of the catalytically important residue E227 to Ser shifted the optimum pH of the enzyme to the acidic side and simultaneously improved its acid stability. After 3 h of incubation at pH 2.6, the mutant phytase (E227S) retained over 80% of its initial activity while the wild-type phytase displayed only 40% of its original activity. Moreover, the E227S mutant phytase, unlike its wild-type, showed a higher relative activity towards calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate, thus suggesting a broader substrate specificity. Alkaline phytases have a lower specific activity than their acid counterparts. An increased knowledge of the enzymes for designing mutations to improve their specific activity and catalytic activity at low pH are thus necessary for rendering industrial applications possible.
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