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Phytase mutant

A technology of mutants and phytase, applied in the direction of enzymes, hydrolytic enzymes, plant gene improvement, etc., can solve the problems of low enzyme activity level and inability to meet the requirements of aquaculture, and achieve broad prospects, improved tolerance, Effect of heat resistance improvement

Active Publication Date: 2020-06-02
QINGDAO VLAND BIOTECH GRP +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme activity level of existing phytase products under neutral pH conditions is generally low, which cannot meet the requirements of aquaculture.

Method used

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Embodiment 1

[0040] Example 1 Screening of Phytase Mutants Tolerant to Neutral pH Environment

[0041] 1.1 Mutant screening

[0042]In order to improve the tolerance of Escherichia coli-derived phytase PHY-M (the amino acid sequence is SEQ ID NO: 1, and its coding nucleotide sequence is SEQ ID NO: 2) under neutral pH conditions, the applicant first entrusted Shanghai Jierui Bioengineering Co., Ltd. optimized and synthesized the above sequence according to the codon preference of Pichia pastoris, and added EcoRI and NotI restriction sites at the 5' and 3' ends of the synthetic sequence, respectively.

[0043] The protein structure analysis of the PHY-M gene synthesized above shows that the protein has two structural domains: 134 amino acid residues at the N-terminus and 152 amino acid residues at the C-terminus together form domain 1, and the remaining 124 amino acid residues in the middle form the structure Domain 2, the conserved sequence and active center are all located in domain 1, an...

Embodiment 2

[0081] Example 2 Screening of high temperature resistant phytase mutants

[0082] 2.1 Mutant screening

[0083] In order to improve the heat resistance of the phytase mutant PHY3 (its amino acid sequence is SEQ ID NO: 5, and its coding nucleotide sequence is SEQ ID NO: 6), the applicant further carried out a large number of mutations on the enzyme through directed evolution technology filter.

[0084]Using the PHY3 gene as a template, using the primers PHY-M-F1 and PHY-M-R1 described in Example 1, PCR amplification was performed with the GeneMorphII Random Mutation PCR Kit (Stratagene), and the PCR product was recovered from the gel, EcoRI, Not I After enzyme digestion, it was ligated with the pET21a vector after the same enzyme digestion, 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 with a toothpick. In a 96-well plate, add 150 ul of LB+Amp medium containing 0.1m...

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Abstract

The invention relates to the technical field of biology, and in particular relates to a phytase mutant, a DNA molecule for encoding the phytase mutant, a vector and a host cell. The optimal pH value of the phytase mutant is 5.5, under neutral pH, the tolerance is significantly improved, under the pH of 6.0, the relative enzyme activity of the mutant reaches 72.80%-79.25%, under the pH of 6.5, therelative enzyme activity of the mutant can still reach 32.99%-40.95%, so that unexpected technical effect is achieved, and wide applications of phytase in aquatic feed is facilitated.

Description

technical field [0001] The invention relates to the technical field of protein engineering transformation, in particular to a phytase mutant and application thereof. Background technique [0002] Phytic acid, also known as phytic acid or cyclohexyl phosphate, widely exists in plant seeds and is the main storage form of phosphorus in plant feed, but phytate phosphorus cannot be directly absorbed by animals and must be digested In the channel, it is first hydrolyzed into inorganic phosphate. Aquatic animals, especially fish, lack endogenous phytase in their digestive system and cannot utilize phytate phosphorus in feed. Most of it is excreted with feces, causing serious water environment pollution and waste of phosphorus. However, aquatic animals have a higher demand for phosphorus in feed than livestock and poultry, especially carps, which are stomachless fish and have no gastric acid secretion. Usually, calcium hydrogen phosphate or calcium dihydrogen phosphate is added to ...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/81C12N15/80C12N1/15C12N1/19C12R1/84C12R1/885C12R1/66
CPCC12N9/16C12Y301/02006C12Y301/03008C12N15/81C12N15/80Y02E50/10
Inventor 吴秀秀李玉强刘扬黄亦钧
Owner QINGDAO VLAND BIOTECH GRP
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