Phytase

Phytic acid (phytate) has a strong complexing ability and usually combines with mineral elements such as Ca²⁺, Mg²⁺, Zn²⁺ and K⁺ to form insoluble salts. Therefore, phytic acid is an anti-nutritional factor that greatly reduces the nutritional effectiveness of trace minerals. This property of phytic acid causes imbalances in elements such as calcium, magnesium, zinc, and potassium in humans and animals. Phytase is a generic term for a class of enzymes that catalyze the hydrolysis of phytic acid and its salts to inositol and phosphate. Adding phytase to animal feed to release phosphorus from phytic acid can not only improve the utilization of phosphorus in food and feed, but also degrade phytic acid protein complexes, reduce the chelation of phytate to trace elements, and improve the utilization of plant protein by animals the nutritional value of feed.

Sources

Phytase is widely present in animal and plant tissues and microbial cells. In plants, phytase is mainly present in the seed. In animals, phytase is mainly found in the red blood cells and plasma of vertebrates, as well as in the small intestine of mammals. Microorganisms are the most convenient and economical source of phytase. The phytase-producing microorganisms mainly include bacteria, fungi, and yeasts. Among them, the amount of phytase secreted by mold is relatively high, especially Aspergillus has a strong ability to produce phytase, which is considered to be the main producing enzyme.

Classification

Phytase is a class of phosphatases that hydrolyze phytic acid. According to their action on substrates, they can be divided into two types, namely 3-phytase (EC 3.1.3.8) and 6-phytase (EC 3.1. 3.26). 3-Phytase degrades phytate to phosphoinositide and orthophosphoric acid starting from the 3rd carbon site. 6-Phytase degrades phytate to phosphoinositide and orthophosphoric acid starting from the 6th carbon site. Plants and E. coli phytase belong to 6-phytase, while fungi and most bacterial phytase belong to 3-phytase. Phytase can be divided into three categories according to structural differences and hydrolysis methods, namely histidine acid phosphatase (HAPs), β-propeller phytases (BPP) and purple acid phosphatase (PAP).

Catalytic Mechanism

Phytase catalyzes the breakdown of phytate (phytic acid) into inositol and phosphoric acid. The phytase cleaves the phosphate groups on the phytic acid one by one to form intermediates IP5, IP4, IP3, and IP, and the final products are inositol and phosphoric acid. The catalytic mechanisms of phytase from different sources are different. When 3-phytase acts on phytic acid, it first hydrolyzes the ester bond from the 3rd carbon site of phytic acid to release inorganic phosphorus, and then releases phosphorus from other carbon sites in turn, finally esterifying the entire phytic acid. This enzyme requires the participation of divalent magnesium ions (Mg²⁺) in the catalytic process. 6-Phytase first catalyzes phytic acid at the 6th carbon site to release inorganic phosphorus. The complete decomposition of 1g phytic acid theoretically releases 281.6mg of inorganic phosphorus. Phytase can only decompose phytic acid into inositol phosphate, which cannot be completely broken down into inositol and phosphoric acid. To completely break down inositol phosphates, acid phosphatase is needed. Acid phosphatase can completely decompose monophosphate and diphosphate into inositol and phosphoric acid. The catalytic mechanism of most microbial-derived phytase is as follows:

Phytic acid→D-myo-Inositol-1,2,4,5,6-pentaphosphate→D-myo-Inositol-1,2,3,4,5-pentaphosphate→D-myo-Inositol-1,2,5,6-tetraphosphate→D-myo-Inositol-1,2,5-triphosphate or D-myo-Inositol-1,2,6-triphosphate→D-myo-Inositol-1,2-diphosphate→Myo-inositol-2-phosphate

Figure 1. The hydrolysis of phytate by phytase into inositol, phosphate and other divalent elements. (Yao, M.; et al. 2012)

Properties

The molecular weight of phytase varies greatly from source to source, and the difference is mainly due to the glycosylation of phytase. The optimum pH of phytase is generally between 2 to 6. Plant-derived phytase has an optimum pH of 4.0-7.5, most of which is 5.0-6.0, which is not suitable for action in the stomach of single-stomach livestock. The optimum pH for bacterially derived phytase is generally neutral or alkaline. The optimum pH of the fungal phytase is 2.5-7.0. The optimum temperature of phytase is in the range of 40-60°C, and the optimum temperature of phytase from different sources is quite different. Most divalent cations (Ca²⁺, Fe²⁺, Zn²⁺, Mg²⁺, Cu²⁺, etc.) inhibit the activity of the enzyme due to strong complexation with the substrate (phytic acid). Compounds such as oxalic acid and citric acid also reduce the enzyme activity of phytase by reacting with the side chain of key amino acids in the active center of phytase. The presence of common substrate competitive inhibitors can also adversely affect the action of the enzyme.

Applications

Treating food with food-grade phytase can decompose phytate, reduce the chelation of phytic acid to trace elements, and improve the nutritional value of food. Phytase can enzymatically modify soy protein in soybean processing, thereby improving its nutritional value and commercial value. The addition of phytase to the bread production process can remove phytic acid from the dough. Impregnation is one of the production processes for corn syrup to soften the kernels and break the cell walls. Microbial phytase can accelerate this process, improves the isolation of embryos, obtains high yields of starch and gluten, and improves the quality of corn syrup.

Adding phytase to animal feed to release phosphorus from phytic acid can not only improve the utilization of phosphorus in food and feed, but also degrade phytic acid protein complexes, reduce the chelation of phytate to trace elements, and improve the utilization of plant protein by animals the nutritional value of feed. It also reduces the amount of organic phosphorus in animal waste and reduces pollution to nature.

Reference

1. Yao, M.; et al. Phytases: crystal structures, protein engineering and potential biotechnological applications. Journal of Applied Microbiology, 2012, 112(1):1.