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Acid phytase appa, its mutant and its preparation

A phytase and mutant technology, applied in the field of phytase APPA, can solve the problems of reducing the effective utilization of nutrients, waste of phosphorus sources, water and soil pollution, etc.

Active Publication Date: 2012-02-15
FUJIAN FUDA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, phosphorus in the form of phytate phosphorus is difficult to be utilized due to the lack of enzymes capable of decomposing phytic acid in monogastric animals, and its utilization rate is only 0-40%.
Phytate phosphorus cannot be effectively used by animals, which causes many problems in the feeding process. First, it causes waste of phosphorus sources. On the one hand, the phosphorus sources in the feed cannot be effectively used; , and additional inorganic phosphorus must be added to the feed, which increases the cost of feed
In actual production, when inorganic phosphorus is added, it often causes animal poisoning due to residual elements such as fluorine and heavy metals in inorganic phosphorus
Second, the formation of high-phosphorus feces will pollute the environment. About 85% of the phytate phosphorus in the feed will be directly excreted by animals. A large amount of phosphorus in the feces will seriously pollute the water and soil
Third, phytate phosphorus is also an anti-nutritional factor, which will combine with various metal ions Zn during the digestion and absorption process in the gastrointestinal tract of animals. 2+ , Ca 2+ 、Cu 2+ , Fe 2+ etc. and protein sequestration into corresponding insoluble complexes, thus reducing the effective utilization of these nutrients by animals
[0006] Although there are various types and properties of phytase, there is no really ideal wild-type phytase that can be used in production practice.

Method used

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  • Acid phytase appa, its mutant and its preparation
  • Acid phytase appa, its mutant and its preparation
  • Acid phytase appa, its mutant and its preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 The cloning and acquisition of the Yersia phytase-encoding gene appa

[0034] Dissolve the genomic DNA of Yersinia frederiksenii from the Academy of Military Medical Sciences with 30 μL sterile water and set aside. The degenerate primers were designed and synthesized according to the conserved sequence of neutral phytase:

[0035] FI: 5'-GTKSTKAWWKTGAGYCGCCA-3' (SEQ ID NO: 17)

[0036] R I: 5'-TWKGCMAKRTTRGTATCATG-3' (SEQ ID NO: 18).

[0037] Among them, K is G or T or U, S is C or G, W is A or T, Y is C or T or U, M is A or C, N is A, C, G or T or U, R is A or G.

[0038]Degenerate PCR amplification was performed using Yersia genomic DNA as a template. The PCR reaction parameters were as follows: denaturation at 94°C for 3 minutes and cooling to 4°C; denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 1 min, and incubation at 72°C for 10 min after 32 cycles. The conserved fragment of phytase was obtained, and the fragment...

Embodiment 2

[0051] Example 2 Obtaining of mutant phytase coding gene appa-S22T

[0052] Apply the Quick-Change Gene Mutation Kit (Stratagene Company) to design primers

[0053] S22T (+): 5'-GTGTTCGTTCACCGACCAAACAAACACAGCT-3' (SEQ ID NO: 13) and S22T (-): 5'-AGCTGTGTTTGTTTGGTCGGTGAACGAACAC-3' (SEQ ID NO: 14), with Figure 6a The sequence shown is a template, and the 51st position of the amino acid sequence is single-point mutated by PCR to obtain a gene containing the coding sequence of the phytase appa-S22T mutant. The gene uses ATG as the starting codon and consists of 1326 bases Base pair composition, encoding 441 amino acids and a stop codon taa (SEQ ID NO: 6). Among them, the first 1-87 bases encode a signal peptide with a length of 29 amino acids (see Figure 6b ), wherein the phytase appa-S22T mutant coding sequence is shown in SEQ ID NO: 4, and the encoded amino acid sequence is shown in SEQ ID NO: 3, wherein the 22nd amino acid is threonine (Thu22).

Embodiment 3

[0054] Embodiment 3 The preparation of recombinant phytase

[0055] Expression primers were designed according to the sequence obtained in Example 1 (SEQ ID NO: 5):

[0056] appaF,5'-GTT GGATCC GCAACCTGGTGGTTTACACTTTG-3', (SEQ ID NO: 15)

[0057] appaR, 5′-GAC GCGGCCGC TTAAATATGGCAGGCTGGTTCTATC-3' (SEQ ID NO: 16), wherein, restriction sites BamHI and NotI are respectively introduced into the ends of the primers. Using the Yersiniafrederiksenii genomic DNA as a template, carry out PCR amplification.

[0058] The amplified product was digested with EcoR I and Not I, and the resulting digested fragment was connected to the BamHI-Not I site of the vector pET-22b to obtain the recombinant plasmid pET-appa containing the gene encoding the phytase of the present invention. Escherichia coli E. coli BL21 (DE3) was transformed with the obtained recombinant plasmid pET-appa to obtain a recombinant strain.

[0059] Expression primers were designed according to the coding sequence (...

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Abstract

The present invention relates to the field of genetic engineering, specifically, the present invention relates to a kind of phytase APPA derived from bacteria Yersiniafrederiksenii and its gene, the single amino acid site mutant APPA-S22T of the phytase and its coding gene, and comprising the Recombinant vectors and applications of genes. The invention provides a new phytase APPA, the amino acid sequence of which is shown in SEQ ID NO.1, and the invention provides the gene appa encoding the above-mentioned phytase. The invention obtains a phytase whose optimal pH value is 2.5, and its ability to adapt to the environment of the gastrointestinal tract of feeding animals is better than the currently used phytase. The present invention also obtains the single amino acid site mutant APPA-S22T of this phytase, its optimal pH value becomes 4.5, and its specific activity, pH stability and thermal stability are all improved, these properties make it in Feed has wider application and higher application value.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a phytase APPA, its single amino acid site mutant, genes encoding the enzyme and the mutant, and recombinant vectors and applications comprising these genes. Background technique [0002] Phosphorus is an essential mineral that all animals need for growth. It is very important to provide adequate phosphorus in animal diets. Long-term phosphorus deficiency can lead to rickets in growing animals and osteoporosis in adult animals, thereby affecting the growth performance of animals. At present, inorganic phosphorus such as calcium hydrogen phosphate is added to animal feed to meet the animal's demand for phosphorus. However, animal diets that are mainly plant-based feeds are rich in phosphorus, but only because they mainly exist in the form of phytate phosphorus that animals cannot use. [0003] Phytate phosphorus is the basic storage form of phosphorus a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/63C12N1/21C12R1/19
Inventor 叶秀云周建武张洋靳伟刚罗鋆琳李仁宽赖庆安徐黄兆
Owner FUJIAN FUDA BIOTECH
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