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Fixedpoint mutation modified phytase

A site-directed mutation and phytase technology, applied in the field of phytase, can solve the problems of no use value, waste of cost, loss of enzyme activity, etc., and achieve the effect of promoting large-scale industrialization and improving heat resistance

Inactive Publication Date: 2008-03-19
广东中大南海海洋生物技术工程中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there is a short high temperature process in the phytase granulation process, generally at 75°C-93°C, most commercial phytases cannot withstand high temperature, and a large amount of enzyme activity will be lost during the high temperature granulation process, resulting in a lot of wasted cost
The optimum temperature of the high-temperature phytase isolated from mesophilic microorganisms is 70°C to 80°C. Although it has good temperature resistance, its enzyme activity at 37°C is extremely low and has no use value; and the source Phytases such as Aspergillus niger have high enzyme activity at 37°C, but they cannot withstand the high temperature during granulation

Method used

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  • Fixedpoint mutation modified phytase
  • Fixedpoint mutation modified phytase
  • Fixedpoint mutation modified phytase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: PCR amplification and sequencing of Aspergillus niger phytase gene

[0027] 1. The present invention uses the phytase gene (PhyA) sequence of Aspergillus niger origin as a reference, designs and synthesizes two oligonucleotide primers downstream of the signal peptide, extracts Aspergillus niger total DNA by the method of phenol extraction, and passes PCR The target gene PhyA was amplified by the method, and the nucleotide sequence of the PCR product was determined.

[0028] The two PCR primers are as follows:

[0029] F1: TCTCTCGAGAAAAGTCCAAGTCCTGCGATACGGT

[0030] R1: AAGAATGCGGCCGCTTATCAACTAAAGCACTCTCCCC

[0031] The PCR reaction system is as follows:

[0032] Template

2ul

F1

1ul

R1

1ul

2×GC buffer I

5ul

high fidelity enzyme

0.5ul

DNTP

8uI

wxya 2 o

32.5uI

[0033] PCR program setting:

[0034] Pre-denaturation at 95°C for 5 minutes

[0035] 94°C, 30s; 65°C, 30s;...

Embodiment 2

[0038] Embodiment 2: the connection of phytase gene PHYA and cloning vector pPlCZαA

[0039] PhyA and the cloning vector pPlCZαA (as shown in Figure 1) were subjected to double digestion with restriction endonuclease Xho I / Not I respectively, and the digestion conditions were as follows:

[0040] PCR fragment digestion system (20ul)

[0041] Digest at 37°C for 10 hours, recover two target fragments after electrophoresis, and connect them with T4DNA ligase. The connection system is as follows:

[0042] PCR fragment

[0043] Ligated overnight at 16°C, the electrophoresis results showed a fragment of about 5kb, and the electrophoresis results after double digestion with Xho l / Not l showed two bands of 3.6kb and 1.4kb, indicating that the ligation was successful, and the pPhyA vector (PhyA is the code name of the phytase gene transformed by site-directed mutation), that is, pPICZαA-PHYA as shown in FIG. 3 .

Embodiment 3

[0044] Example 3: Site-directed mutagenesis

[0045] After repeated experiments and exploration, we determined to carry out site-directed mutagenesis on the amino acids at the following positions:

[0046] For the 23rd amino acid mutation, design the following primers:

[0047] F2: 5'TTGTACTCGCCATTCTTTTC3'

[0048] mutation site

[0049] R2: 5'GCCCCATAGATGAGAAGTCG3'

[0050] For the 53rd amino acid mutation, the following primers were designed:

[0051] F3: 5'TTGCGCCATGGAGCGCGGTA3'

[0052] mutation site

[0053] R3: 5'TAGCACCTGTACCAAGGTGA3'

[0054] For the 136th amino acid mutation, the following primers were designed:

[0055] F4: 5' TAC GGTCGGGTTATTGCTTC3'

[0056] mutation site

[0057] R4: 5'GCCTGAGGCGCGAATAAACG3'

[0058] For the 137th amino acid mutation, the following primers were designed:

[0059] F5: 5' GGT CGGGTTATGCTTCGGG3'

[0060] mutation site

[0061] R 5 : 5'GTAGCCTGAGGCGCGAATAA3'

[0062] For the 195th amino acid mutation, th...

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Abstract

The present invention relates to phytase which is obtained through the gene engineering reconstruction, and provides the phytase which is reconstructed through the rite-directed mutagenesis, the phytase is produced by manufacturing a plurality of amino acids in the phytase which is derived from aspergillus niger and has the serial number of the amino acid of EQ ID No.1 to be replaced, and the replacement of the amino acid comprises a 23rd replacement, a 53rd replacement, a 136th replacement, a 137th replacement, and a 195th replacement. The present invention discloses the optimal and reconstructive phytase and the corresponding DNA sequence, and also discloses the production method and the application of the phytase. The phytase of the present invention is better in the stand up quality of the temperature, and the pH range which is most suitable for the enzyme activity is also improved; further, a mutant gene is highly and effectively expressed through a bioreactor, to lead the phytase after the reconstruction and the rite-directed mutagenesis to be highly and effectively expressed, thereby finally achieving the requirement of industrialized production.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a phytase obtained through genetic engineering transformation. Background technique [0002] Phytate, Phytic acid, IP6, also known as phytate, has a complex structure. The phytic acid molecule contains 6 phosphate groups and is rich in phosphorus. Phytic acid is an important storage form of phosphorus in feed. It is precisely because the 6 phosphate groups on the phytic acid molecule have a very strong negative charge, which can combine with many cations (calcium, magnesium, phosphorus, copper, zinc, manganese) to form a chelate, which is not easy to hydrolyze , Phosphorus and cations on phytic acid molecules are not easily utilized by animals, so they are called anti-nutritional factors. Generally, the utilization rate of phosphorus in the feed for animals is only 10%-30%, and additional inorganic phosphate is needed, and the commonly used ones are calcium hydrogen phosphate an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C07K14/37C12N15/55C12N15/63A23K1/165A23K20/189
Inventor 徐安龙荆琛峰梁东
Owner 广东中大南海海洋生物技术工程中心有限公司
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