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Phytase Enzyme Separation and Purification Technique



Phytase is a general term for enzymes that hydrolyze phytic acid and its salts to generate inositol and phosphoric acid. As a new type of feed additive,  phytase enzyme has great potential in fields such as animal nutrition and environmental protection. Phosphorus in plant tissues is mainly present in the form of sodium phytate, which is difficult to be absorbed by monogastric animals. In addition, inositol hexaphosphate molecules can chelate metal ions, which acts as an anti-nutrient factor and inhibits nutrient absorption. The incomplete utilization of phosphorus leads to eutrophication of water bodies through animal excretion. The fundamental solution to this cycling of phosphorus lies in solving the utilization problem of phosphorus, so the addition of microbe phytase in animal feed is gradually being promoted and applied.


Phytase enzyme pretreatment and concentration


For extracellular enzymes, the fermented liquid is centrifuged and filtered, and the supernatant is concentrated by ammonium sulfate fractionation precipitation or ultrafiltration. For intracellular enzymes, the cells need to be broken and separated. Ethanol and acetone precipitation and ammonium sulfate precipitation concentration are used. Many researchers have successfully isolated phytase industrial enzymes by ammonium sulfate precipitation with yields of 16.8%, 50%, and 78%, respectively. Salt precipitation, organic solvent precipitation, ultrafiltration and other methods have been successfully used in the preliminary isolation and purification of phytase from plants, bacteria, and fungi.


Phytase enzyme column chromatography purification


Chromatography has the advantages of fewer separation steps, strong specificity, and low activity loss, and it is the main technology for the separation and purification of phytase enzyme. Researchers purified phytase enzyme PhyA1 from Aspergillus oryzae A-1 by fiber filtration concentration and strong anion exchange column chromatography purification, with a relative molecular weight of about 45×103 and a purification ratio of 33.4. Studies of phytase enzyme separation and purification have shown that ion exchange chromatography with gelatin as the matrix is the most important step in the separation and purification of phytase enzyme, with a yield of 70.5% and a purification ratio of 1.9. Gel filtration chromatography as the final purification step can ultimately increase enzyme purification by four times with a yield of 57.7%.


Phytase enzyme immobilization


Immobilized phytase enzyme can improve enzyme stability and activity, and solve the problems of reduced enzyme activity and instability in feed additives. Researchers immobilized phytase on chitosan supports, used glutaraldehyde as the crosslinking agent, and studied the immobilization by covalent binding method. The study found that the optimal conditions for immobilization of industrial enzyme were 4% glutaraldehyde concentration, 65U/g enzyme loading, and 5h crosslinking time, with enzyme activity reaching 1665U/ml. Phytase enzyme immobilized in urea compounds and nanoclay showed increased stability under lower acidic conditions, as well as enhanced heat stability and resistance to protein hydrolysis. The high temperature during feed pelletization process reduces the thermal stability of phytase, and how to maintain enzyme stability in enzyme immobilization is still a topic worth further research.



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