Neutral phytase mutant and application thereof

A technology of phytase and mutants, applied in the field of genetic engineering, can solve problems such as unfavorable acid phytase action, achieve the effects of reducing breeding costs, reducing pollution, and improving enzyme activity levels

Active Publication Date: 2015-09-16
青岛玛斯特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the phytase currently on the market is mainly acidic phytase, which has the highest activity under the conditions of pH 2.5 and 5.5, while in the digestive tract of anergic aquatic animals, due to the absence of gastric acid secretion, the pH in the digestive tract is about neutral, not Conducive to the action of acid phytase

Method used

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  • Neutral phytase mutant and application thereof
  • Neutral phytase mutant and application thereof
  • Neutral phytase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 The acquisition of phytase gene

[0019] According to the gene sequence in the public gene database, the codon of the synthetic gene was optimized and the phytase gene PHYA (sequence is SEQ ID NO: 2) derived from Aspergillus niger was artificially synthesized, and the amino acid sequence encoded by it was SEQ ID NO: 2. ID NO: 1.

[0020] PCR primers and reaction conditions are as follows:

[0021] Primer 1 (F): GCGCGAATTCATGAGCTTCCGGTCCCTG

[0022] Primer 2 (R): TAAAGCGGCCGCTTAGGCGAAGCACTCGGC

[0023] The reaction conditions were: denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 seconds, renaturation at 56°C for 30 seconds, extension at 72°C for 1.5 minutes, and after 30 cycles, incubation at 72°C for 10 minutes. The results of agarose electrophoresis showed that the size of the PHYA gene was 1395bp.

Embodiment 2

[0024] Example 2 Construction and Screening of Phytase Mutants

[0025] In order to improve the enzyme activity level of phytase PHYA under neutral conditions, the applicant screened a large number of mutations of the enzyme by directed evolution technology.

[0026] With the PHYA gene as a template, the primers 1 and 2 described in Example 1 were used for PCR amplification with the GeneMorph II random mutation PCR kit (Stratagene), and the PCR product was recovered by gel, and EcoRI and NotI were digested with the same enzyme. The final pET21a vector was ligated, transformed into Escherichia coli BL21(DE3), spread on LB+Amp plate, and cultured upside down at 37°C. After the transformants appeared, pick them one by one to a 96-well plate with a toothpick, and add 150ul to each well LB+Amp medium containing 0.1mM IPTG, cultivated at 37°C and 220rpm for about 6 hours, centrifuged to discard the supernatant, resuspended the bacteria with pH 5.0 buffer, and repeatedly freeze-thawe...

Embodiment 3

[0030] Example 3 Construction of recombinant expression vector

[0031] The gene fragments of phytase mutants PHYAT3 and PHYAT5 were amplified by PCR, and XbaI sites were introduced at both ends of the primers. The primer sequences are as follows:

[0032] Primer 3 (F): GC TCTAGA ATGAGCTTCCGGTCCCTG

[0033] Primer 4 (R): GC TCTAGA TTAGGCGAAGCACTCGGC

[0034] The PCR reaction conditions were as follows: denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 seconds, renaturation at 56°C for 30 seconds, extension at 72°C for 1.5 minutes, and after 30 cycles, incubation at 72°C for 10 minutes. The results of agarose gel electrophoresis showed that the PHYAT3 and PHYAT5 genes were fragments with a size of 1395 bp.

[0035] The phytase mutant PHYAT3 and PHYAT5 gene fragments obtained above and the expression vector pGAU were subjected to single-digestion with restriction endonuclease XbaI respectively, and the digestion conditions were as follows:

[0036] ...

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Abstract

The invention provides a neutral phytase mutant and an application thereof. Phytase PHYA is transformed through a random mutation method to obtain mutant protein with the amino acid sequence being SEQ ID NO: 3. The screened phytase mutant PHYAT3 optimal reaction pH is 6.5, more than 80% of enzyme activity can be maintained within pH 6.5-7.0, 20% of enzyme activity can be maintained when pH is 8.0, and accordingly, an unexpected technical effect is obtained. Compared with wild neutral phytase, the enzyme activity within the neutral range of the phytas mutant PHYAT3 is improved apparently, and the optimal reaction temperature is not changed apparently. The phytase mutant can be widely applied to aquatic feeds, and accordingly, adding of inorganic phosphate into the feeds is reduced, the breeding cost is reduced, and pollution of aquatic livestock fecal phosphorus to the environment can be reduced effectively.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a neutral phytase mutant and its application in aquatic feed. technical background [0002] Phosphorus is one of the important mineral elements necessary for fish. The requirement of phosphorus for fish is higher than that of other mineral elements, but excessive phosphorus in feed is considered to be one of the important factors causing eutrophication of water body. Therefore, it is an important way to reduce the pollution of water environment by improving the utilization rate of phosphorus in raw materials by fish and properly adjusting the phosphorus content in feed to make it closer to the demand of fish to reduce the discharge of phosphorus from farmed fish. [0003] As an enzyme produced by microbial fermentation, phytase can decompose phytic acid and phytate, promote the decomposition of phytic acid and phytate in feed, and make endogenous enzymes an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/80C12N1/15A23K1/165C12R1/685
CPCC12N9/16C12Y301/03008
Inventor 徐晓东张霞吴秀秀王华明
Owner 青岛玛斯特生物技术有限公司
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