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Production of Phytase as Animal Feed Additive
Date: 2009-6-13 Click: 4203


Background   

Phosphorus is an essential element for the growth and development of all animals, playing key roles in skeletal structure and in vital metabolic pathways. As such, all animal diets must contain adequate amounts of this element.

 

The principle storage form of phosphorus in feedstuffs of plant origin is the phytic acid (inositol hexaphosphate). Phytic acid and the salts and esters of phytic acid are known as phytases. Phytates are contained in all kinds of nuts, cereals, beans, seeds, spores and pollens in an amount of 1-3% and up to 80% of the phosphorus in grains and seeds.

 

Phytate phosphorous is readily available to ruminants, as certain microorganisms present in the rumen produce a kind of phosphor hydrolyses enzyme, phytase, which catalyze the conversion of phytate to inositol and available inorganic phosphate. In contrast, only 0-40% of phytate phosphorous is available to mono gastric animals such as pigs and poultry as lake phytase-producing microorganisms in their gut.

1.      Due to this inability of mono gastric animals to utilize phytate, it is therefore necessary to supplement the diets with expensive sources of available inorganic phosphate such as dicalcium phosphate or meat and bone meal.

2.      Phytaste phosphorus passes through the intestinal tract and ends up in the feces causing environmental problems in areas of intensive livestock production.

3.      In addition, phytate is considered to be anti-nutritional factors. Phytate forms complexes with essential minerals such as calcium, zinc, magnesium, iron, and may also react with proteins, thereby decreasing the bio-availability of protein and nutritionally important factors.

 

Phytase (myo-inositol hexaphosphate phosphohydrolase, EC3.1.3.8) are enzymes which catalyze the hydrolysis of phytate into myo-inositol and inorganic phosphate. The addition of microbial phytase to the feedstuff of mono gastric animals was described as long as 25 years ago. Addition of this phytase to feed substantially improved phosphorus utilization reduced excretion of phosphorus in the feces and counteracted the ant-nutritional properties of phytate.

1.      Increase in phosphorous availability by 60%

2.      Reducing or even eliminating the need for supplemental dietary inorganic phosphorous.

3.      Decrease in the excretion of phosphorous in the feces by over 30%

4.      Additionally, such a treatment can also counteraction the anty-nutritional properties associated with unhydrolyzed phytate and increase in both weight gain and feed intake of animals.

 

Although abroad range of microorganisms can produce phytase, the expression levels of phytase in tem are too low for economic considerations. Due to the high cost of phytase production, application of phytase has not been widespread. For economic considerations, inorganic phosphorous supplement of mono gastric animal feedstuffs is still the method of choice in many cases. Obviously, any commercially viable procedure for the production of a large quantity of phytase would be of tremendous value both from the feed-conversion and environment point of view.

Towards the ultimate goal of improving the production of phytase and reducing its production cost, here we have developed recombinant yeast, which can greatly improve phytase production levels.

 

Advantages in Phytase Production by Recombinant Yeast

Yeast Pichia Pastoris have been successfully employed as host for the industrial production of extra cellular phytase. The advantages of the technique include:

1.      The expression level of phytase in recombinant Pichia Pastoris is very high, and the amount of expressed phytase reaches to 5gram/liter medium (equal to 15000IU/mL. One unit of phytase was defined at the amount of enzyme which was able to liberate I mol of inorganic phosphate from substrate per minute) in industrial fermentation.

2.      Large scale, high-density fermentation technology for production of phytase has well established, and fermentation method is simple and easy.

3.      Large scale downstream processing for phytase production has well established, and processing methods is very simple and easy.

4.      The fermentation medium, a defined mixture of glucose, salts and trace elements that is easy to get, is inexpensive and free of toxins.

5.      The microorganism selected for the purpose of producing phytase is Pichia pastoris due to its acceptance as safe for human use through experience with the microorganism used as a source of single-cell protein (SCP) from 1970s.

6.      The overall production cost of phytase products is very low. The production cost of 1 liter of liquid phytase products (5000IU/mL) or 1 kg of solid phytase products (5000IU/g) is about 1 US dollar, which can be added in 10,000kg of feedstuff.

7.      The equipment of producing phytase by recombinant yeast is simple. The equipment in general fermentation factory can meet the needs for phytase production.

 

Production of Phytase

Strain Inoculum Seed Flask Preparation Seed Fermentor Preparation Fermentation of Producing Phytase Phytase Secreted Into the Growth Medium Harvesting Supernatant Without Cells Liquid Phytase Products Mixing With Stuffing Drying Solid Phytase Products

 

Construction of Recombinant Prchia Pastoris Producing Phytase

High-level expression of phytase with high specific activity is an effective way to improve phytase fermentation potency and reduce its production cost. The gene appA encoding Escherchia coli phytase AppA with high specific activity was modified and artificially synthesized according to the bias in codon choice of the high expression gene in pichia pastoris without changing the amino acid sequence of the AppA. The modified gene, appA-m, was inserted in the pichia pastor is expression vector pPIC9, and then introduced into the Pichia Pastoris by electroporation. The Pichia Pastoris recombinants for phytase over expression were screened by enzyme activity analysis and SDS-PAGE. The result of southern blotting analysis of the recombinant yeast indicated that only one copy of the appA-m gene was integrated into the genome of Pichia Pastoris. The result of Northern analysis of the recombinant yeast showed that the modified gene was effectively transcribed. SDS-PAGE analysis of the phytase expressed in Pichia Pastoris revealed that the phytase was overexpressed and secreted into the medium supernatant. There are three phytase with apparent molecular in approximately 50kD, 52kD and 54kD respectively in the media, which are larger in the size than the native phytase from E.coli. The results of N-terminal sequencing and deglycosylation of the expressed phytase in Pichia Pastoris proved that the expressed phytase were glycosylated protein with different glycosylation degree. The expressed phytase Pichia Pastoris shared similar pH and temperature optima to those of the natural from E.coli and had highly resistant to pepsin digestion. In 30000-L fermentor, after induced by 0.5% methanol, the expression level of phytase protein was 5mg/mL, and the phytase activity (fermentation potency) exceeded 15000IU/mL, which was the highest among those of all kinds of recombinant strains reported now.

 

Cooperation Intention

One-off techniques transfer(US1,000,000)

1.      To provide the strain for production of phytase

2.      To provide all of the technology of manufacture of the phytase product, including the techniques of storage and growth of the strain, the techniques of fermentation to produce phytase, the techniques of phytase processing, the techniques of quality testing of phytase product and so on.

3.      To provide technical documentation about the use standard of phytase product in different animals.

4.      To assist the end-user with designation or remake for the fermentation factory.

5.      To provide technical directions and training.

 

Cooperation Demands

1.      There have available fermentation factories or have intention to build new fermentation factories.

2.      There have available raw materials for phytase production, such as glucose, ammonium hydroxide, and methanol and so on.

3.      There are in good condition of power and water supplies.

4.      Phytases produced by the technology will be restricted from either production or sale in china.

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