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Mutant strain capable of highly producing phytase and application of mutant strain

A technology of mutant bacteria and phytase, applied in the field of mutant strains with high production of phytase, can solve the problems of phosphorus pollution, low utilization rate of phytate digestion, etc.

Active Publication Date: 2021-05-11
QINGDAO VLAND BIOTECH GRP +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Animals take in inorganic phosphorus with food, and 80% will be excreted with feces. This is because monogastric animals have low digestion and utilization of phytate, and most of the inorganic phosphorus in food exists in the form of phytate , which has caused very serious phosphorus pollution to the global environment

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] The construction of embodiment 1 phytase recombinant expression strain

[0015] The applicant obtained a heat-resistant phytase by using protein engineering technology, and then optimized the codon of the phytase gene according to the codon preference of Trichoderma, and synthesized the phytase by General Biosystems (Anhui) Co., Ltd. coding nucleotide sequence.

[0016] Using the synthesized nucleotide sequence as a template, primers were designed to amplify the phytase gene fragment. The reaction conditions were: denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 s, renaturation at 56°C for 30 s, extension at 72°C for 70 s, and after 30 cycles, incubation at 72°C for 10 min.

[0017] The phytase gene fragment obtained above and the expression vector pVL002 were double-digested with restriction endonucleases XbaI and MluI respectively, and the target fragment was recovered from the gel, and the double-digested fragment of the phytase gene obtained by ...

Embodiment 2

[0034] Example 2 UV mutagenesis and screening

[0035] The mutations caused by ultraviolet mutagenesis are very random, and the effects of mutations are also random and difficult to predict. Therefore, in order to obtain effective positive mutations, technicians usually need to carry out multiple rounds of ultraviolet mutagenesis, the workload of screening is relatively large, and there is a possibility that effective positive mutations cannot be obtained. However, because ultraviolet mutagenesis requires simple equipment, low cost, and a large number of mutants can be obtained in a short period of time, it is still a commonly used method of mutagenesis selection.

[0036] The applicant used Trichoderma reesei UE-5 as the starting strain, and carried out genetic modification on it through ultraviolet mutagenesis to further increase its phytase production.

[0037] 1. Determine the fatality rate:

[0038] Trichoderma reesei UE-5 was inoculated on a PDA plate and cultured at 3...

Embodiment 3

[0046] Embodiment 3 The heat resistance test of phytase

[0047] Treat the fermentation supernatants of the starting bacterium Trichoderma reesei UE-5 and the mutant bacterium Trichoderma reesei UEM-5 respectively at 90°C for 3 minutes, and then quickly cool them with ice-water mixture, then measure the fermentation supernatants respectively The residual enzyme activity of phytase in the Taking the initial enzyme activity before heat treatment as 100%, the enzyme activity residual rate was calculated, and the results are shown in Table 1.

[0048] Enzyme activity residual rate (%) = (initial enzyme activity - enzyme activity after heat treatment) / initial enzyme activity × 100%

[0049] Table 1 Residual rate of phytase activity in Trichoderma reesei fermentation supernatant after heat treatment

[0050] Trichoderma reesei 90°C for 3 minutes UE-5 82.5% UEM-5 84%

[0051] It can be seen from the data in Table 1 that after treatment at 90°C for 3 m...

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Abstract

The invention belongs to the technical field of microbial engineering modification, and provides a trichoderma reesei mutant strain capable of highly producing phytase and application of the trichoderma reesei mutant strain. The mutant strain is obtained through ultraviolet mutagenesis screening, the preservation number is CCTCC NO: M2019893, the temperature-resistant phytase can be efficiently expressed, and the wide application of the phytase in the field of feed is favorably promoted.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering transformation, and in particular relates to a high-yield phytase mutant strain and application thereof. Background technique [0002] Phytic acid, namely myo-inositol hexakisphosphate, referred to as PA. Phytic acid widely exists in various tissues of animals and plants, and the content of phosphorus is very high. 60%-90% of the total phosphorus content of plants exists in the form of phytate phosphorus in the phytate complex. Animals take in inorganic phosphorus with food, and 80% will be excreted with feces. This is because monogastric animals have low digestion and utilization of phytate, and most of the inorganic phosphorus in food exists in the form of phytate , which has caused very serious phosphorus pollution to the global environment. It is well known that phytic acid has a very strong ability to chelate metal ions. Under neutral or alkaline conditions, it can chelate s...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N9/16C12N13/00C12R1/885
CPCC12N9/16C12Y301/03008C12N13/00
Inventor 李瑞宋清清许丽红刘士成黄亦钧
Owner QINGDAO VLAND BIOTECH GRP
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