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Phytase mutant and preparation method thereof

A technology of mutants and phytase, applied in the field of phytase mutants and its preparation, can solve problems such as strategies that have not been adopted for transformation, and achieve the effects of improving catalytic efficiency, improving catalytic efficiency, and enhancing pH tolerance

Pending Publication Date: 2022-07-12
NANJING YIXIAN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The content of the invention described in the above-mentioned patent is that a single-point mutation of phytase is performed to improve the heat resistance and enzymatic activity of the mutant, and a strategy of modifying the amino acid of its catalytic amino acid part is not adopted. Design and construction mutant

Method used

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  • Phytase mutant and preparation method thereof
  • Phytase mutant and preparation method thereof
  • Phytase mutant and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0030] Wild phytase PhyAn is derived from Aspergillus neoniger , belonging to the HAP family, the conserved amino acid sequence of the phytase catalytic region of this family is RHGX 1 RX 2 P, where X 1 and X 2 It is a variable amino acid residue, that is, phytases of the HAP family with the same conserved catalytic site will produce differences in these two amino acids, and the catalytic region sequence of phytase PhyAn is RHGARYP.

[0031] The invention aims at improving the catalytic efficiency and pH stability of the wild phytase PhyAn, and rationally designs its amino acid sequence to construct a mutant.

[0032] The catalytic efficiency is related to the binding affinity of the enzyme to the substrate, and then is closely related to the steric hindrance of the amino acid side chain in the catalytic region; secondly, the pH tolerance of the enzyme is inseparable from the charge of the amino acid, and the enzyme plays a key role in the activity of the charged part. Ami...

Embodiment 2

[0037] Example 2: Expression of phytase PhyAn and its mutant A63G+Y65H in Pichia pastoris GS115.

[0038] (1) Phytase PhyAn and its mutant A63G+Y65H were transformed into Pichia GS115;

[0039] The expression vectors of phytase PhyAn and its mutant A63G+Y65H were extracted with Plasmid DNA Kit and linearized with Sac1 restriction endonuclease. The purified products will be transformed . Transfer 10 μL of the enzyme digestion product to be transformed into 100 μL of Pichia GS115 competent cells, and transfer the above mixed samples to a 2 mm electroporation cup and incubate on ice for 15 min at a working voltage of 2 kV and pulse time. The electric shock was performed under the condition of about 0.5ms. Immediately after the end, 1 mL of pre-cooled 1 M sorbitol solution was added. After mixing, it was transferred to a new 1.5 mL centrifuge tube and placed in a 30 °C incubator for 1 incubation. h, spread in YPD medium after centrifugation, and cultured at 30 °C for 3-5 days. ...

Embodiment 3

[0046] Analysis of Enzymatic Properties of Recombinant Phytase PhyAn and Its Mutant A63G+Y65H

[0047] The activity detection of phytase was carried out according to the molybdenum vanadium method given in the national standard of the People's Republic of China "GB / T 18634-2009". The specific determination method is as follows: dilute the phytase enzyme solution with 0.25 mol / L acetic acid buffer at pH 5.5 to an appropriate multiple, take 0.2 mL of the diluted enzyme solution into a 25 mL test tube, add 1.8 mL of acetic acid buffer, and vortex to mix After homogenization, the test tube was placed in a 37 °C water bath to preheat for 5 min; then 4 mL of 7.5 mmol / L sodium phytate solution was added to the test tube, and after mixing, the mixed sample was placed in a 37 °C water bath for 30 min. Add 4 mL of stop solution to the test tube, mix by vortex and let stand for 10 min at room temperature, and measure its absorbance at 415 nm wavelength.

[0048] The enzymatic activity o...

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Abstract

The invention relates to a phytase mutant and a preparation method thereof, the amino acid sequence of the phytase mutant is as shown in SEQ ID No.2, the phytase mutant is mutated on the basis of the amino acid sequence of wild phytase PhyAn as shown in SEQ ID NO.1, the mutation site and sequence of the mutant are A63G + Y65H, and the amino acid sequence of the phytase mutant is as shown in SEQ ID NO.1. In other words, alanine at the 63 site and tyrosine at the 65 site are substituted by glycine and histidine at the same time. Compared with wild phytase PhyAn, the mutant disclosed by the invention has the advantages that the catalytic efficiency of the mutant is improved, which means that the substitution of the glycine residue at the 63 site reduces the steric hindrance of the phytase, so that the combination of the enzyme and the substrate is facilitated, and the catalytic efficiency of the phytase is improved; and the pH tolerance of the mutant is improved at the same time, which means that the histidine at the 65 site changes the quantity of charged amino acids in a catalytic region, thereby being beneficial to enhancing the pH tolerance of the mutant.

Description

technical field [0001] The invention relates to the fields of genetic engineering and enzyme engineering, in particular to a phytase mutant and a preparation method thereof. Background technique [0002] Phytic acid (phytic acid) is widely found in plants in nature, with six phosphate groups that act as a reservoir of phosphoric acid. Phytic acid combines with calcium, magnesium, iron, zinc and other mineral elements and nutrients such as protein to easily form a protein-phytic acid-mineral element complex, which greatly reduces the absorption and utilization of nutrients and some plant foods and some plant proteins. Nutritional potency of minerals in isolates. In addition, pigs, fish and other animals lack phytase and cannot hydrolyze phytic acid well, and phosphorus is a mineral element necessary for their growth, which makes the bioavailability of phosphorus in phytic acid very low and its growth Requires a high intake of inorganic phosphorus. Therefore, adding phytase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/81C12N1/19A23K20/189C12R1/84
CPCC12N9/16C12Y301/03026C12N15/815A23K20/189Y02P60/87
Inventor 牟海津梁青平刘哲民周思嘉
Owner NANJING YIXIAN BIOTECHNOLOGY CO LTD
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