CN110643590B

CN110643590B - Fungus-derived beta-propeller type recombinant phytase r-AoPhytase as well as expression strain and application thereof

Info

Publication number: CN110643590B
Application number: CN201911074264.0A
Authority: CN
Inventors: 王永中; 侯贤娟; 沈振; 孔小卫; 盛康亮; 汪静敏
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2019-11-06
Filing date: 2019-11-06
Publication date: 2022-03-22
Anticipated expiration: 2039-11-06
Also published as: CN110643590A

Abstract

The invention discloses a fungal-source beta-propeller type recombinant phytase r-AoPhytase, an expression strain and application thereof, wherein the original source of the beta-propeller type recombinant phytase r-AoPhytase is nematophagous fungi Arthrobotryy oligosopropa, and the amino acid sequence of the beta-propeller type recombinant phytase r-AoPhytase is one of the following amino acid sequences: (1) SEQ ID No: 1; (2) SEQ ID No:1 amino acid sequence which is substituted, deleted or added with one or more amino acid residues and encodes the same functional protein; (3) and SEQ ID No:1 a mutated amino acid sequence having more than 80% homology. The recombinant phytase r-AoPhytase has high specific enzyme activity, is close to neutral optimum pH and high optimum temperature, has the capability of promoting the release of inorganic phosphorus and soluble minerals in different feed samples, and is suitable for feed processing and production.

Description

Fungus-derived beta-propeller type recombinant phytase r-AoPhytase as well as expression strain and application thereof

Technical Field

The invention belongs to the field of molecular biology and genetic engineering, and particularly relates to a beta-propeller type recombinant phytase r-AoPhytase derived from fungi as well as an expression strain and application thereof.

Background

Phytase preparations have a wide range of applications in animal and human nutrition. It hydrolyzes phytate (inositol-1, 2, 3, 4, 5, 6-hexaphosphate) to produce lower inositol phosphate derivatives and inorganic phosphoric acid (Mullaney, Daly,&ullah, 2000). Phytate is considered the major storage form of phosphate and inositol in plants, and phytic acid derivatives account for 60-80% of the phosphorus in plant-derived feeds. Phytic acid is also a polyanionic chelating agent which can be used in combination with several major nutritionally valuable divalent cations, such as Ca²⁺，Mg²⁺，Zn²⁺，Cu²⁺，Fe²⁺And Mn²⁺Formation of a Complex (Chen)&Headquaters, 2000). Phytases are widely used in commercial poultry, pig and fish feeds to increase the availability of phosphorus, minerals, amino acids, etc. The phytate molecules and the nutrients bound thereto are not absorbed by the digestive tract, but are enzymatically degraded by phytase, facilitating their absorption. The phytase added into the feed can also obviously improve the utilization rate of mineral elements of calcium and magnesium and trace elements of zinc, copper, iron, manganese and the like (Lonnerdal, 2000). Besides, the addition of the phytase in the feed can improve the digestion and absorption of protein and amino acid by animals, because the phytase can release the protein complexed with the phytic acid while hydrolyzing the phytic acid to release phosphorus, thereby facilitating the action of various proteases secreted by the digestive tract; meanwhile, endogenous protease, amylase, lipase and the like combined with phytic acid can be released, so that the digestion utilization rate is improved. Phytase may also beThe phytase is used in food processing, and the phytase added in food can improve the utilization rate of phosphorus and other mineral elements, and can also improve the utilization rate of nutrients such as protein, starch and fat (Haefner et al, 2005).

Phytase is widely found in animals and plants, such as rice, wheat, corn (Ullah & Gibson,1988), soybean, etc., and contains phytase in small intestine of mammals and red blood cells of vertebrates, but its content is very low and its activity is very low. The phytase activity is higher in rumen of ruminant such as cattle. Phytase is also present in microorganisms (bacteria, fungi and yeasts), such as Aspergillus in moulds. The most studied at present are the production of phytase by microorganisms, protein isolation and purification and the results of analysis of physicochemical properties, which indicate that they are the best sources for the industrialization of phytase (Lei, Porres, Mullaney, & Brinch-Pedersen, 2007).

BPP-type phytases are a newly discovered class of alkaline phosphatase enzymes, and are the most abundant and structurally diverse phytases in nature. To date, all known BPP-type phytases are derived from bacteria, in particular bacillus (Singh & satylararayana, 2015). For example, the phytases phyC and TS-Phy isolated from Bacillus subtilis and Bacillus amyloliquefaciens, respectively, belong to the BPPs (Ha et al, 2000). The prior BPP type phytase from bacteria has lower specific enzyme activity, and the optimum pH and the optimum temperature are not suitable for the application of feed processing technology.

Aiming at the technical requirement, the invention develops the BPP phytase derived from fungi. We firstly take the nematophagous fungus Arthrobotry oligosopropra as a starting material, develop the beta-propeller phytase Aophytase from the fungus and a recombinant preparation method thereof, and find that the BPP type recombinant phytase from the fungus has higher specific enzyme activity, is close to neutral optimum pH and higher optimum temperature, has the capability of promoting the release of inorganic phosphorus and soluble minerals in different feed samples, and is suitable for application in feed processing and production.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a fungal source beta-propeller type recombinant phytase r-AoPhytase, an expression strain and application thereof. The recombinant phytase r-AoPhytase has high specific enzyme activity, is close to neutral optimum pH and high optimum temperature, has the capability of promoting the release of inorganic phosphorus and soluble minerals in different feed samples, and is suitable for application in feed processing and production.

The original source of the fungus-derived beta-propeller type recombinant phytase r-AoPhytase is nematophagous fungus Arthrobotryy oligosopropa, which consists of 782aa amino acid, has the molecular weight of 84.2KDa and the isoelectric point of 5.58, and the amino acid sequence of the beta-propeller type recombinant phytase r-AoPhytase is one of the following amino acid sequences:

(1) SEQ ID No: 1;

(2) SEQ ID No:1 amino acid sequence which is substituted, deleted or added with one or more amino acid residues and encodes the same functional protein;

(3) and SEQ ID No:1 a mutated amino acid sequence having more than 80% homology.

The coding gene for coding the beta-propeller type recombinant phytase is one of the following nucleotide sequences:

(1) SEQ ID No: 2 or SEQ ID No: 3;

(2) SEQ ID No: 2 or SEQ ID No:3 nucleotide sequences with one or more nucleotide residues substituted, deleted or added and encoding the same functional proteins;

(3) encoding SEQ ID No:1 a polynucleotide sequence of a protein sequence;

(4) and SEQ ID No: 2 or SEQ ID No: 3a mutant nucleotide sequence having more than 80% homology.

The invention relates to a recombinant expression strain of a fungal source beta-propeller type recombinant phytase r-AoPhytase, which is classified and named as: pichia pastoris GS115(5 'PAOX 1:: pPIC. alpha. ZA-Aophytase) (Pichia pastoris GS115 (5' PAOX1:: pPIC. alpha. ZA-Aophytase)), deposited Unit: china Center for Type Culture Collection (CCTCC), address: wuhan university, preservation date: year 2019, month 10, day 8, accession number: CCTCC NO: m2019775.

Firstly, chemically synthesizing the r-AoPhytase gene, carrying out double enzyme digestion by using EcoRI and SalI, connecting the gene with an expression plasmid vector, and transforming a connecting product into a competent cell to obtain an expression vector; then linearizing the expression vector by Sac I enzyme, electrically transforming the linearized plasmid into an expression host cell, and carrying out colony PCR identification by using primers P1 and P2 to obtain a recombinant engineering strain capable of recombinantly expressing r-AoPhytase.

The nucleotide sequence of the primer P1 is shown as SEQ ID No: 4, the nucleotide sequence of the primer P2 is shown as SEQ ID No: 5, respectively.

The expression plasmid vector is selected from pPICZ alpha A, pPICZ alpha B, pPICZ alpha C, pPIC6 alpha A, pPIC6 alpha B, pPIC6 alpha C, pPICZA, pPICZB, pPICZC, pPIC9K, pPIC3.5K and the like.

The expression host is selected from Pichia pastoris SMD1168(H), SMD1165, SMD1163, GS115, X-33, MP-36, MC100-3 or KM71 (H).

The expression vector is preferably pPICZ alpha A-Aophytase, and the expression host cell is preferably Pichia pastoris GS 115.

The method for preparing the beta-propeller type recombinant phytase r-AoPhytase by using the recombinant expression strain comprises the following steps:

step 1: inoculating the recombinant expression strain into 10mL-1L BMGY medium at a ratio of 1:1000, culturing at 30 ℃ for about 48h by using a shaker at 220rpm, and measuring OD600 to be 2-8;

step 2: centrifuging the culture at 1500rpm for 10min, discarding supernatant, resuspending the precipitate in 100mL-5L BMMY medium, measuring OD600 as 0.4-4.0, and inducing at 30 deg.C for 72h with 220rpm shaker;

and step 3: centrifuging the culture solution at 4 deg.C and 8000rpm for 10min, collecting supernatant, and preparing into crude enzyme solution;

and 4, step 4: adding 5mL of Ni NTA Beads 6FF into the crude enzyme solution, incubating at 4 ℃ for 2h, adding the incubated product into an empty column tube, and collecting the flow-through solution.

And 5: washing the filler by using 20mM imidazole buffer solution, and eluting impurities; then, the target protein is eluted by using 40-500mmol/L imidazole buffer solution in a grading way to obtain purified r-AoPhytase protein.

The preparation method of the fungal beta-propeller type recombinant phytase r-AoPhytase is described by taking an expression plasmid vector pPICZ alpha A and an expression host Pichia pastoris GS115 as an example, and specifically comprises the following steps:

step 1: according to the amino acid sequence SEQ ID NO.1 and the nucleotide sequence SEQ ID NO.2(Genbank accession NO.22896793) of the AoPhytase, carrying out codon optimization aiming at a Pichia pastoris expression system to obtain a gene sequence (SEQ ID NO.3 in a sequence table) of the AoPhytase or a mutant sequence which has homology of more than 80% with the nucleotide sequence SEQ ID NO.2 or SEQ ID NO. 3;

step 2: carrying out double enzyme digestion on the nucleotide fragments by using EcoRI and SalI, connecting the nucleotide fragments with a vector pPICZ alpha A subjected to the same double enzyme digestion at 16 ℃ overnight, and transforming a connecting product into a competent DH5 alpha cell to obtain an expression vector pPICZ alpha A-Aophytase;

and step 3: adopting Sac I enzyme to linearize expression vector pPICZ alpha A-Aophytase;

and 4, step 4: electrically converting the linearized plasmid pPICZ alpha A-Aophytase into pichia pastoris GS 115;

and 5: carrying out colony PCR identification on a transformation plate YPDSZ by using primers P1 and P2 to obtain a positive transformant, wherein the nucleotide sequence of the primer P1 is shown as SEQ ID NO.4, and the nucleotide sequence of the primer P1 is shown as SEQ ID NO. 5;

step 6: carrying out induction expression of the recombinant protein in pichia pastoris: inoculating positive clones into 1mL BMGY medium, and culturing at 30 ℃ until OD600 is 2-6 (about 48 h); centrifuging the culture at 1500rpm for 10min, removing the supernatant, adding 2mL BMMY culture medium to resuspend the thallus precipitate, culturing at 30 ℃ for 72h, and performing induced expression;

and 7: 1mL of the induced microbial suspension was collected, centrifuged, and the supernatant was transferred to an ultrafiltration tube and concentrated to 40. mu.l for SDS-PAGE analysis.

Further, the amplification culture and separation purification process of the recombinant expression of the fungal beta-propeller type phytase r-AoPhytase comprises the following steps:

1. inoculating the recombinant pichia pastoris engineering bacteria into 10mL-1L BMGY medium, preferably 50mL BMGY medium at a ratio of 1:1000, culturing for about 48h at 30 ℃ in a shaking table at 220rpm, and measuring OD600 to be 2-8, preferably OD600 to be 4.6;

2. centrifuging the culture at 1500rpm for 10min, discarding supernatant, resuspending the precipitate with 100mL-5L BMMY medium, preferably 200mL BMMY medium, measuring OD600 ═ 0.4-4.0, preferably OD600 ═ 1.1, and inducing at 220rpm with shaking at 30 deg.C for 72 h;

3. centrifuging the culture solution at 4 deg.C and 8000rpm for 10min, collecting supernatant, and preparing into crude enzyme solution;

4. adding 5mL of Ni NTA Beads 6FF into the crude enzyme solution, incubating at 4 ℃ for 2h, adding the incubated product into an empty column tube, and collecting the flow-through solution.

5. Washing the packing material with 20mM Imidazole buffer (10mM Na2HPO4, 2mM KH2PO4, 0.8% NaCl, 0.02% KCl, 5% Glycerol, 20mM Imidazole, pH6.0) to elute impurities;

6. the target protein is fractionated with 40-500mmol/L imidazole buffer, preferably with 100mM or 250mM imidazole eluent (containing 10mM Na2HPO4, 2mM KH2PO4, 0.8% NaCl, 0.02% KCl, 5% Glycerol, pH6.0) to obtain purified r-AoPhytase protein.

7. And (3) determining the purity of the recombinant protein of the purified r-AoPhytase protein prepared by 100mM or 250mM imidazole eluate or the purified r-AoPhytase protein samples eluted by 100mM or 250mM imidazole eluate by combining the eluate and the eluate, and determining the protein concentration by using a BCA method.

The application of the beta-propeller type recombinant phytase r-AoPhytase is that the beta-propeller type recombinant phytase r-AoPhytase is used as a phytase preparation in the processing and production processes of feeds, can remarkably promote the release of inorganic phosphorus in soybean meal, and remarkably promote the soluble release of mineral ions such as Ca, Fe, Zn, Mg and the like in durum wheat flour and black-foot millet flour.

The optimal pH value of the beta-propeller type recombinant phytase r-AoPhytase is 7.5, and the optimal temperature is 50 ℃; ca²⁺，Na⁺，K⁺Has effect in enhancing enzyme activity; li⁺，Mg²⁺Slight or almost no inhibition of the enzyme activity; fe³⁺，Ni²⁺，Cu²⁺，Zn²⁺，Mn²⁺SDS and EDTA have strong inhibitory effect on enzyme activity to different degrees, wherein EDTA and Zn²⁺、Mn²⁺The inhibition effect on the activity of the compound is most remarkable.

The kinetic parameters of the beta-propeller type recombinant phytase r-AoPhytase are as follows: vmax is 40-200 mu mol min^-1mg^-1Preferably Vmax 72.46. mu. mol min^-1mg^-1(ii) a Km ═ 0.1 to 2.5mM, preferably Km ═ 1.015 mM; kcat 2-50s^-1Preferably Kcat ═ 13.57s^-1。

The specific enzyme activity of the beta-propeller type recombinant phytase r-AoPhytase is far higher than that of BPP type phytase derived from other bacteria, such as bacillus, purple bacillus, terribacterium, serratia and the like, and is 50U-100U/mg, preferably 74.71U/mg.

Compared with the prior art, the invention has the beneficial effects that:

1. the specific enzyme activity of the fungal beta-propeller type recombinant phytase r-AoPhytase provided by the invention is 74.71U/mg, which is far higher than that of BPP type phytase from bacterial sources, such as bacillus, purple bacillus, terribacterium, serratia and the like.

2. The codon preference of pichia pastoris is overcome by a codon optimization method, so that the recombinant protein obtains higher secretion expression; the recombinant protein is secreted in the culture solution, so that the cost of downstream separation and purification is reduced.

3. The optimum pH of the fungus beta propeller type recombinant phytase r-AoPhytase and the phytase preparation thereof is about 7.5 and is closer to neutral; the optimum temperature is higher and is 50 ℃; can be effectively applied to the industrial processing and production process of the feed, and effectively promotes the release of inorganic phosphorus and soluble mineral substances in feed samples.

Drawings

FIG. 1 shows pPICZ α A-phytase plasmid and the linearization of the digestion. The constructed plasmid pPICZ alpha A-phytase is digested by SacI, and then the linearization effect is detected by agarose gel electrophoresis. M: DL5000 DNA Marker; 1: pPICZ alpha A-phytase plasmid; 2: the position of the linearized plasmid pPICZ alpha A-phytase on the gel is more than 5000bp, which is consistent with the size of 5767bp of a target fragment.

FIG. 2 is the integration of AoPhytase in the Pichia genome and its induced expression. (A) A schematic diagram of the integration of linearized pPICZalphaA-phytase in the yeast genome; (B) PCR identification of positive transformants on a bleomycin screening plate; (C) after the positive transformant induces the expression, SDS-PAGE analysis of the recombinant protein in the fermentation liquor is carried out.

FIG. 3 is a Ni NTA agarose affinity chromatography purified r-AoPhytase and its SDS-PAGE and Western Blotting analysis. (A) Obtaining r-AoPhytase components from imidazole eluents with different concentrations through a Ni NTA agrose affinity chromatography column; (B) SDS-PAGE analysis of the purified r-AoPhytase; (C) western Blotting analysis of purified r-AoPhytase.

FIG. 4 is a phosphorus calibration curve. 1ml of AMES developing solution was added to 1ml of the phosphorus standard measuring solution, and after a water bath at 50 ℃ for 20 minutes, the mixture was cooled to room temperature, and the OD value was measured at 700 nm. And drawing an OD-phosphorus concentration standard curve by taking the OD value as a vertical coordinate and the phosphorus concentration as a horizontal coordinate. Linear fitting and regression gave the linear equation y 6.2417x +0.0723, R2 0.9971.

FIG. 5 is a graph showing the effect of pH, temperature, surfactant, chelating agent and various metal ions on the activity of recombinant AoPhytase enzyme. (A) The pH value is active on the recombinant AoPhytase enzyme; (B) the temperature is used for activating the recombinant AoPhytase enzyme; (C) the influence of the surfactant, the chelating agent and different metal ions on the activity of the recombinant AoPhytase.

FIG. 6 shows kinetic parameters of recombinant AoPhytase enzyme. (A) When the enzyme content is constant, the release amount of inorganic phosphorus is gradually increased along with the increase of the phytic acid concentration of a substrate; (B) the reciprocal of the reaction velocity V vs substrate concentration [ S]The reciprocal of (a) was plotted against (B) to obtain a fitted curve of y 0.014+ 0.0138. From this calculation, the Vmax of Aophytase was 72.46. mu. mol min^- ¹mg^-1，Km＝1.015mM Kcat＝13.57s^-1。

FIG. 7 is the effect of recombinant AoPhytase on the release of inorganic phosphorus or minerals in feed samples. (A) With the increase of the amount of the recombinant phytase, the release amount of inorganic phosphorus in the soybean meal is correspondingly increased; (B) the amount of total minerals in 1g durum wheat flour or black currant powder; (C) the addition of the recombinant phytase promotes the obvious release of minerals in the durum wheat flour; (D) the addition of the recombinant phytase promotes the significant release of minerals in the castanea henryi powder.

Detailed Description

The technical solution of the present invention is further explained by the following examples, but the scope of the present invention is not limited in any way by the examples. The methods carried out in the examples are, unless otherwise specified, conventional.

Example 1: AoPhytase codon optimization and recombinant expression thereof in pichia pastoris

According to the amino acid sequence SEQ ID NO.1 and the nucleotide sequence SEQ ID NO.2(Genbank accession NO.22896793) of AoPhytase, codon optimization is carried out on a Pichia pastoris expression system to obtain the AoPhytase gene sequence (SEQ ID NO.3 in a sequence table). Then, the gene fragment optimized by the double enzyme digestion of EcoRI and SalI is connected with a vector pPICZ alpha A subjected to the double enzyme digestion at 16 ℃ overnight, the connection product is transformed into a competent DH5 alpha cell, and a positive transformant is screened on an LB solid medium plate containing Zeocin resistance. As shown in FIG. 1, an expression vector pPICZ α A-Aophytase was obtained.

Mu.g of plasmid pPICZ alpha A-Aophytase was taken, 2. mu.l of restriction enzyme Fast digest Sac I enzyme and 10 XFastdigest Buffer 50. mu.l were added, ddH2O to 500. mu.l were supplemented, and the reaction was carried out overnight at 37 ℃ to linearize it. After the completion of the enzyme digestion reaction, 500. mu.l of isopropanol was added to the reaction system and vigorously shaken, centrifuged at 12000rpm for 10min, the supernatant was discarded, and the precipitate was added with 1mL of 75% ethanol, and mixed by inversion. After centrifugation at 12000rpm for 10min, the supernatant was discarded, and the pellet was reconstituted with 20. mu.l of sterile water to dissolve the linearized plasmid. The linearization effect was checked by agarose gel electrophoresis (FIG. 1).

The linearized plasmid pPICZ alpha A-Aophytase is transformed into Pichia pastoris GS115, and Aophytase gene and its screening gene ble expression cassette are integrated into 5 'promoter region (5' PAOX1) of AOX1 gene on Pichia pastoris GS115 genome by using single recombination (single cross) mechanism (FIG. 2A). And adding 10 mu L of linearized plasmid into 100 mu L of yeast competent cell GS115, uniformly mixing, and transferring to a 0.2cm ice-precooled electric transformation cup. After ice bath for 5min, setting the voltage to be 1.5 kV; a capacitance of 25 μ F; and a resistor of 200 omega is used for electric shock conversion. Immediately transferring into 500. mu.L of 1M sorbitol solution, standing at room temperature for 20min, adding 500. mu.L of YPD medium, and incubating at 30 deg.C and 220rpm with shaking for 1 h. 100. mu.L of the above solution was spread on YPDSZ plates and cultured in an inverted state at 30 ℃ for about 2 to 3 days. Identified by colony PCR using primer 5' AOX

(5'-GACTGGTTCCAATTGACAAGC-3') and 3' AOX (5'-GCAAATGGCATTCTGACATCC-3') 20 clones were picked from YPDSZ plates and PCR verified for positive clones. The PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 20s, and extension at 72 ℃ for 1min for 30 cycles; finally, extension is carried out for 10min at 72 ℃. As shown in FIG. 2B, 20 transformant clones were selected on the bleomycin screening plate for PCR verification, and all the transformants were positive.

And finally, carrying out induction expression of the recombinant protein in pichia pastoris. 10 positive clones were selected, inoculated into 1mLBMGY medium, and cultured at 30 ℃ to OD₆₀₀About 2 to about 6 (about 48 h). The culture is centrifuged at 1500rpm for 10min, the supernatant is discarded, 2mL of BMMY culture medium is added to resuspend the thallus precipitate, and the thallus precipitate is cultured at 30 ℃ for 72h for induction expression. After induction, 1mL of the induced microbial suspension was collected, centrifuged at 12000rpm for 5min, and the supernatant was collected in an ultrafiltration tube and concentrated to 40. mu.l. The concentrated supernatant was taken out into a 1.5mL centrifuge tube, and a Loading Buffer was added to the tube, followed by a 10min boiling water bath. The prepared samples were finally subjected to SDS-PAGE analysis. As shown in FIG. 2C, 10 positive transformants were randomly selected and induced to express with methanol for 72 hours, and the fermentation supernatant was concentrated and analyzed by SDS-PAGE to find a band at 66.2-116KD, indicating that the engineering bacteria can efficiently induce and express the recombinant phytase with methanol and secrete it into the culture medium.

Example 2: purification of recombinant AoPhytase

1. Preparation of crude enzyme solution: the recombinant pichia pastoris engineering bacteria are inoculated into 50mL BMGY medium at the ratio of 1:1000, cultured at 220rpm of 30 ℃ for about 48h, and the OD600 is measured to be 4.6. The culture was then centrifuged at 1500rpm for 10min, the supernatant discarded, and the pellet resuspended in 200mL BMMY media and measured to OD600 ═ 1.1, 220rpm, and induced at 30 ℃ for 72 h. Centrifuging the above culture solution at 4 deg.C and 8000rpm for 10min to obtain supernatant as crude enzyme solution.

2. And (3) separating and purifying phytase: 5mL of Ni NTA Beads 6FF was added to the supernatant and incubated at 4 ℃ for 2 h. Adding the incubated product into an empty column tube, and collecting effluent liquid. The packing was washed with 20mM Imidazole buffer (10mM Na2HPO4, 2mM KH2PO4, 0.8% NaCl, 0.02% KCl, 5% Glycerol, 20mM Imidazole, pH6.0) and the wash was collected. Then eluting with 100 and 250mM imidazole buffer solution, and collecting the eluent. Finally, the eluate was dialyzed at a ratio of 1:100 to an imidazole-free buffer (10mM Na2HPO4, 2mM KH2PO4, 0.8% NaCl, 0.02% KCl, 5% Glycerol, pH 6.0). As shown in FIG. 3A, electrophoretically pure phytase protein was obtained at elution with 100-250mM imidazole eluate. FIGS. 3B-C show the results of SDS-PAGE and Western Blotting analysis, respectively, indicating that recombinant Aophytase has achieved SDS-PAGE purity and molecular weight of 95-116 kD.

Example 3: analysis of enzyme Activity of recombinant AoPhytase

1. Drawing a phosphorus determination standard curve: 0, 2, 4, 6, 8 and 10ml of phosphorus standard stock solutions are measured in a 100ml volumetric flask, diluted to the scale and prepared into standard curve measuring solutions with phosphorus contents of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mmol/L. Taking six 15ml centrifuge tubes, adding the standard determination solution with the concentration of 1ml, adding 1ml AMES color development solution, heating in water bath at 50 deg.C for 20min, cooling to room temperature, determining OD value at 700nm, and drawing OD-phosphorus concentration standard curve, as shown in FIG. 4.

2. And (3) enzyme activity determination: after diluting the pure enzyme solution at a concentration of 0.088mg/ml 10-fold, 100. mu.l of the diluted enzyme solution was mixed with 900. mu.l of a substrate solution (2mM sodium phytate dissolved in 100mM Tris-HCl buffer, pH 7.0), and 1ml of 10% trichloroacetic acid was added to the control group, followed by incubation at 37 ℃ for 30 minutes. The reaction was stopped by adding 1ml of 10% trichloroacetic acid to the reaction sample, and 950. mu.l of the substrate solution was added to the control sample. After cooling, 2ml of a color developing solution was added, and the absorbance was measured at A700. The experiment was repeated three times. Phytase activity units (U) are defined as: the enzyme amount required for releasing 1 mu mol of inorganic phosphorus from sodium phytate per minute at 37 ℃ is one enzyme activity unit. The enzyme activity was calculated as follows:

enzyme activity ═ OD-OD₀)×N]V (K × T); the specific enzyme activity is calculated according to the following formula: specific enzyme activity (U)/amount of enzyme added to the test system (mg).

Note: the unit of enzyme activity (U) is mu mol P/min;

OD: measuring the absorbance of the sample at 700 nm; OD₀: absorbance at 700nm of the control sample;

n, dilution times of the samples; k, standard curve slope; t, enzymatic reaction time.

The specific enzyme activity of the recombinant phytase was determined to be 74.71U/mg, calculated according to the above formula (Table 1). Table 1 also lists the specific enzyme activities of the BPP-type recombinant phytases of different bacterial origins reported by other groups. By comparison, we found that the specific activity of the recombinant AoPhytase derived from fungi is much higher than that of BPP-type phytase derived from other bacteria, such as Bacillus, purple bacillus, terribacterium, Serratia, etc.

TABLE 1 specific enzyme Activity of recombinant AoPhytase and its comparison with specific Activity of different recombinant phytases (BPP-type)

Example 4: influence of pH value, temperature, metal ions, surfactant and complexing agent on activity of recombinant AoPhytase

1. And (3) optimum pH determination: the following pH buffers were used: glycine-HCl (100mM, ph2.0-3.5), sodium acetate-acetic acid (100mM, ph3.5-6.5), Tris-HCl (100mM, ph6.5-9.0), and glycine-NaOH (100mM, ph9.0-10), 2mM sodium phytate solution was prepared as a substrate solution. Taking 4 parts of substrate solution with different pH values, taking a mixed solution of 10% trichloroacetic acid added in one part and enzyme solution added in the other part as a reference, carrying out enzyme activity determination according to the enzyme activity determination method, repeating the steps for three times, determining the change of the light absorption value of the substrate solution at A700, and plotting the pH value and the relative activity of the enzyme to obtain the optimal pH value. As shown in FIG. 5A, significant phytase activity was shown between pH5-8, with an optimum pH of 7.5, which is consistent with the BPP-type phytase being a basic phytase.

2. Optimum temperature measurement: sodium phytate was dissolved in 100mM Tris-HCl buffer pH 7.0 to prepare 2mM sodium phytate as a substrate solution. The phytase activity was calculated as described above for the enzyme activity measurement by measuring the change in absorbance at 700nm by setting the temperature change at intervals of 10 ℃ from 20 to 70 ℃. The temperature was plotted against the relative activity of the enzyme to find the optimum temperature. As shown in FIG. 5B, the activity of the recombinant phytase was changed within the range of 20-80 ℃ and the optimum temperature was found to be 50 ℃.

3. The influence of different metal ions, surfactants and complexing agents on the enzyme activity: 2mM sodium phytate substrate solutions were prepared using 100mM Tris-HCl (pH 7.0) buffer, to which 1mM Ca was added respectively²⁺，Na⁺，K⁺，Li⁺，Mg²⁺，Fe³⁺，Ni²⁺，Cu²⁺，Zn²⁺，Mn²⁺SDS and EDTA. Then 100. mu.l of the 10-fold diluted enzyme solution was mixed with 900. mu.l of the substrate solution to determine the phytase activity, and the effect of metal ions, surfactants and chemical complexing agents on the activity of the purified enzyme was analyzed. Blank Tris-HCl buffer was used as a control. The mixed sample was incubated at 37 ℃ for 30 minutes. The reaction was stopped by adding 1ml of 10% trichloroacetic acid to the test sample, and 950. mu.l of the substrate solution to the control sample. After cooling, 2mL of a developing solution was added, and the absorbance was measured at A700. Each set of experiments was repeated three times. FIG. 5C shows the effect of surfactant SDS, chelating agent EDTA and different metal ions on the activity of recombinant AoPhytase enzyme. We have found Ca²⁺，Na⁺，K⁺With a slight enhancement of the enzyme activity, Li⁺，Mg²⁺Has slight or almost no effect on the enzyme activity, Fe³⁺，Ni²⁺，Cu²⁺，Zn²⁺，Mn²⁺SDS and EDTA have strong inhibitory effects on enzyme activity to various degrees. Wherein EDTA, Zn²⁺，Mn²⁺The inhibition effect on the activity of the compound is most obvious,the inhibition rates respectively reach 47.7%, 68.2% and 97.7%.

Example 5: determination of kinetic parameters of the group AoPytase

The specific enzyme activity and kinetic parameters of the recombinant phytase are determined under optimal reaction conditions (optimal pH and optimal temperature, pH7.5, 50 ℃). Sodium phytate reaction solutions (100mM Tris-HCl, pH7.5) were prepared at different concentrations, such as 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0mM, and 100. mu.l of the enzyme solution (0.5U) was mixed with 900. mu.l of the substrate solution. After incubation at 50 ℃ for 10 minutes, the reaction was stopped by adding 1ml of 10% trichloroacetic acid. In the control group, 100. mu.l of the enzyme solution was added, 1ml of 10% trichloroacetic acid was further added, and 950. mu.l of the substrate solution was finally added after incubation at 50 ℃ for 10 minutes. Finally, after cooling the sample, 2mL of a color developing solution was added, absorbance was measured at a700 to quantify the inorganic phosphorus released by the reaction, and the experiment was repeated three times. Kinetic constants were calculated using the Lineweaver-Burk plot. As shown in FIG. 6A, at a constant enzyme content, the amount of inorganic phosphorus released gradually increased with the increase in the phytic acid concentration of the substrate. As shown in FIG. 6B, the substrate concentration [ S ] is plotted on the ordinate as the reciprocal of the reaction rate V]The reciprocal of (d) is plotted on the abscissa, and L-B is plotted to obtain a fitted curve of y ═ 0.014+ 0.0138. When x is equal to 0, 1/Vmax is equal to 0.0138, and Vmax is equal to 72.46 mu mol min^-1mg^-1When y is 0, 1/Km is-0.9857, and Km is 1.015 mM; according to the theoretical molecular weight of 84.195kD, the Kcat is calculated to be 13.57s^-1。

Example 6: recombinant AoPytase promotes hydrolysis of phytate phosphorus in soybean meal

The amount of phosphorus released from soybean meal (0.5U, 1U, 1.5U, 2U, 2.5U) was measured at different enzyme levels. 1g of soy flour was added to a 50ml centrifuge tube followed by 9ml of buffer (100mM Tris-HCl, pH7.5) for a total of 15 tubes. The centrifuge tubes were incubated at 37 ℃ for 60 minutes with shaking (150rpm) and then 1ml of enzyme preparation was added to the soy flour solution to a final enzyme amount of 0.5-2.5U/g soy flour, respectively. Using 10ml of 15% trichloroacetic acid as a control, 3 tubes were repeated for each sample. After the reaction, 10ml of 15% trichloroacetic acid was added to the sample tube to be tested to terminate the reaction, and 1ml of the enzyme preparation was added to the control tube. After cooling to normal temperature, the reaction solution was centrifuged at 5000rpm for 5 min. 2ml of the supernatant was collected from each tube, and 2ml of a developing solution was added thereto, and absorbance was measured at A700. As shown in fig. 7A, the recombinant phytase significantly promoted the release of inorganic phosphorus in soy flour. As the amount of enzyme increases, the amount of phosphorus released increases accordingly.

Example 7: recombinant AoPyrase promotes release of minerals from durum wheat flour and red sorrel

1. And (3) total mineral content determination: durum wheat flour and Panicum virgatum flour, each 1g, were weighed separately and dissolved in 5mlddH2O, and the mixture was incubated at 50 ℃ for 2 h. 6ml of an acid mixture (nitric acid: sulfuric acid: perchloric acid in a volume ratio of 10: 1: 4) was added to each tube, the mixture was dried at 200 ℃ in a fume hood, the dried sample was treated at 450 ℃ in a muffle furnace to obtain a white powder, the powder was dissolved in 1ml of 35% concentrated hydrochloric acid, and the volume was adjusted to 10ml with ddH 2O. The sample was filtered and treated with 100-fold dilution. Three replicates per sample were tested. Figure 7B shows the total mineral content in durum wheat flour and finger millet flour.

2. Determination of mineral Release solubility: durum wheat flour and Panicum virgatum powder each 1g were weighed separately, resuspended in 6ml buffer (140mM NaCl, 5mM KCl), phytase (20U) was added thereto, the volume of the solution was made up to 10ml with ddH2O, incubated at 50 ℃ for 2h, and centrifuged at 5000rpm for 20 min. The supernatant was diluted 50 times and subjected to ICP-MS measurement. Samples without phytase were used as controls and each sample was replicated three times. As shown in fig. 7C, the recombinant phytase significantly promoted the release of Ca, Fe, Zn and Mg ions in durum flour, with an increase in the release rates of 13.2%, 4.3%, 10.5% and 20.5%, respectively, compared to the control. As shown in fig. 7D, the recombinant phytase also significantly promoted the release of Ca, Fe, Zn and Mg ions from the castanea mollissima powder, and the release rates were increased by 32.1%, 1.8%, 4.3% and 1.6%, respectively, compared to the control.

Reference documents:

Chen,J.,&Headquarters,E.(2000).Phytase in animal nutrition and waste management:a BASF reference manual 1996|Clc.ASA Technical Bulletin Vol.AN29.

Ha,N.C.,Oh,B.C.,Shin,S.,Kim,H.J.,Oh,T.K.,Kim,Y.O.,...Oh,B.H.(2000).Crystal structures of a novel,thermostable phytase in partially and fully calcium-loaded states.Nat Struct Biol,7(2),147-153.doi:10.1038/72421

Haefner,S.,Knietsch,A.,Scholten,E.,Braun,J.,Lohscheidt,M.,&Zelder,O.(2005).Biotechnological production and applications of phytases.Appl Microbiol Biotechnol,68(5),

588-597.doi:10.1007/s00253-005-0005-y Huang,H.,Shao,N.,Wang,Y.,Luo,H.,Yang,P.,Zhou,Z.,...Yao,B.(2009).A novel beta-propeller phytase from Pedobacter nyackensis MJ11 CGMCC 2503with potential as an aquatic feed additive.Appl Microbiol Biotechnol,83(2),249-259.doi:10.1007/s00253-008-1835-1Lei,X.G.,Porres,J.M.,Mullaney,E.J.,&Brinch-Pedersen,H.(2007).Phytase:Source,Structure and Application.505-529.doi:10.1007/1-4020-5377-0_29

Lonnerdal,B.(2000).Dietary factors influencing zinc absorption.J Nutr,130(5S Suppl),1378s-1383s.

Mullaney,E.J.,Daly,C.B.,&Ullah,A.H.(2000).Advances in phytase research.Adv Appl Microbiol,47,157-199.

Reddy,C.S.,Achary,V.M.,Manna,M.,Singh,J.,Kaul,T.,&Reddy,M.K.(2015).Isolation and molecular characterization of thermostable phytase from Bacillus subtilis(BSPhyARRMK33).Appl Biochem Biotechnol,175(6),3058-3067.doi:10.1007/s12010-015-1487-4

Singh,B.,&Satyanarayana,T.(2015).Fungal phytases:characteristics and amelioration of nutritional quality and growth of non-ruminants.J Anim Physiol Anim Nutr(Berl),99(4),646-660.doi:10.1111/jpn.12236

Ullah,A.H.,&Gibson,D.M.(1988).Purification and characterization of acid phosphatase fromcotyledons of germinating soybean seeds.Archives of Biochemistry&Biophysics,260(2),503-513.Viader-Salvadó,J.M.,A Gallegos-López,J.,Gerardo

J.,Castillo-Galván,M.,Rojo,A.,&Guerrero-Olazaran,M.(2010).Design of Thermostable Beta-Propeller Phytases withActivity over a Broad Range of pHs and Their Overproduction by Pichia pastoris(Vol.76).Yao,M.-Z.,Lu,W.-L.,Chen,T.-G.,Wang,W.,Fu,Y.-J.,Yang,B.-S.,&Liang,A.-H.(2013).Effect of metals ions on thermostable alkaline phytase from Bacillus subtilis YCJS isolated from soybean rhizosphere soil.Annals of Microbiology,64(3),1123-1131.doi:10.1007/s13213-013-0751-5

Zhang,R.,Yang,P.,Huang,H.,Shi,P.,Yuan,T.,&Yao,B.(2011).Two types of phytases(histidine acid phytase and beta-propeller phytase)in Serratia sp.TN49 from the gut of Batocera horsfieldi(coleoptera)larvae.Curr Microbiol,63(5),408-415.doi:10.1007/s00284-011-9995-0

Zhang,R.,Yang,P.,Huang,H.,Yuan,T.,Shi,P.,Meng,K.,&Yao,B.(2011).Molecular and biochemical characterization of a new alkaline beta-propeller phytase from the insect symbiotic bacterium Janthinobacterium sp.TN115.Appl Microbiol Biotechnol,92(2),317-325.doi:10.1007/s00253-011-3309-0

sequence listing

SEQ ID NO.1：

MKSSHNLSGLILLCLMGYIHPTSAADKFSITLPITARTSSVESDSAAVYYPSKSKYSPIFIGNDGSAETGGFHVYELYGKRSDALVKELGAYKTGRSKLVEVVYGEDRDFVVTLSMSDGMFRVFEVDGKNGVRGLKAEKLVRGDFSAMCTWKSKVGEYVYVLGKRWGYRFLVREKKGGRGVEVVQTQEFGIPIEPNSCTVSPEGKVFLAGDSGKVFSFLAVDETAAPKIVEVGELGGGDEVKGLKIYHGKKDTYLLVGLEDGIEVFDIKKLGSSLGKIQFDDEELEVGDFAVHQTSAKGYEDGSIVFAGEDGEGKFFGVSSLTPLFKALGKGKLNTKYDPRDCTDNHARPKKCANLSDCNGYGYCPKDSRDKKATCDCFPGLTGKTCNKITCPSNCTSPSHGTCTGPNICTCIPPFTGENCATLAVPARYETEESGGADGDDPAIWIHPTDKTKSRIITTVKSEVGSGLGVYDLKGKRTGGVSGGEPNNVDVLYGVEFAGRKVDLAVAACRADDTICIYEITPTGDLVTIPGGVQPLPPAVKELEKKFKVYGSCVYHSPKTGAYHIFVNSKSSLYLQFQLSATTDGKLNTTLVRHFYAGNRGQVEGCVVDDENSSLFLGEEPYGIWSYDAEPDQPAVGTLVDNTVVDGGKLHADVEGVTLVYGKTKKEGYIIVSCQGYSEYNIYQRYPPHEFVMSFSIPDNKEKGVDRVTNTDGITAVGANLGKEWPYGMVVVHDDVNEAAGGGVRADATFKIVGLGDILGNKAVKELGLLKGVDENWDPRK

SEQ ID NO.2：

SEQ ID NO.3：

gaattcGCTGACAAGTTCTCCATCACTTTGCCAATTACTGCCAGAACCTCTTCCGTTGAATCTGACTCTGCTGCCGTTTACTACCCATCCAAGTCTAAGTACTCCCCAATCTTCATCGGTAACGACGGTTCTGCTGAAACTGGTGGTTTTCACGTCTACGAGTTGTACGGTAAGAGATCCGACGCCTTGGTCAAAGAATTGGGTGCTTACAAGACCGGTAGATCCAAGTTGGTTGAGGTTGTTTACGGTGAGGACAGAGACTTCGTTGTCACTTTGTCTATGTCCGACGGTATGTTCAGAGTGTTCGAGGTCGATGGTAAGAACGGTGTCAGAGGTTTGAAGGCCGAGAAGTTGGTCAGAGGTGATTTCTCCGCTATGTGTACCTGGAAGTCTAAGGTTGGTGAGTACGTCTACGTCCTGGGTAAAAGATGGGGTTACAGATTCTTGGTCCGTGAGAAGAAAGGTGGTAGAGGTGTTGAGGTCGTTCAGACTCAAGAGTTCGGTATTCCAATCGAGCCAAACTCCTGTACTGTTTCCCCAGAAGGTAAGGTTTTCTTGGCTGGTGACTCCGGTAAGGTGTTTTCTTTTTTGGCCGTTGACGAGACTGCTGCCCCAAAGATAGTTGAAGTTGGTGAACTTGGTGGTGGTGACGAGGTTAAGGGTTTGAAGATCTACCACGGTAAGAAGGACACCTACTTGTTGGTTGGTTTGGAGGACGGTATCGAGGTGTTCGACATTAAGAAGTTGGGATCCTCCTTGGGTAAGATCCAATTCGACGACGAAGAGTTGGAAGTCGGTGATTTCGCTGTTCATCAGACTTCCGCTAAGGGTTACGAAGATGGTTCCATCGTTTTCGCTGGTGAAGATGGTGAGGGAAAGTTCTTCGGTGTTTCCTCCTTGACTCCTCTGTTCAAGGCTCTTGGTAAGGGTAAGCTGAACACCAAGTACGACCCAAGAGACTGTACTGACAACCACGCTAGACCAAAGAAGTGTGCTAACTTGTCCGACTGCAACGGTTACGGTTACTGTCCAAAGGACTCCAGAGACAAGAAGGCTACTTGCGACTGTTTTCCAGGTTTGACCGGTAAGACCTGCAACAAGATTACCTGTCCATCCAACTGCACTTCCCCATCTCATGGTACTTGTACCGGTCCAAACATCTGCACTTGTATCCCACCATTCACTGGTGAGAACTGTGCTACTTTGGCTGTTCCAGCTAGATACGAGACTGAAGAATCTGGTGGTGCTGATGGTGATGACCCAGCTATTTGGATTCACCCAACTGACAAGACCAAGTCCAGAATCATCACCACCGTTAAGTCCGAAGTTGGTTCCGGTTTGGGTGTCTACGATCTGAAGGGTAAGAGAACCGGTGGTGTTTCAGGTGGTGAACCTAACAACGTTGACGTCTTGTACGGTGTTGAGTTCGCCGGTAGAAAGGTTGACTTGGCTGTTGCTGCTTGTAGAGCTGACGACACCATCTGTATCTACGAGATCACTCCAACCGGTGACTTGGTTACTATTCCAGGTGGTGTTCAACCATTGCCACCAGCTGTCAAAGAGCTGGAAAAGAAGTTCAAGGTCTACGGTTCCTGCGTCTACCACTCTCCAAAGACTGGTGCTTACCACATCTTCGTCAACTCCAAGTCCTCCTTGTACTTGCAGTTCCAGTTGTCCGCTACTACTGACGGAAAGTTGAACACTACCCTGGTCAGACACTTCTACGCTGGTAACAGAGGTCAAGTTGAGGGTTGTGTTGTTGACGACGAAAACTCTTCCCTGTTCTTGGGTGAAGAACCATACGGTATTTGGTCCTACGATGCTGAACCAGACCAACCTGCTGTTGGTACTTTGGTTGACAACACTGTCGTTGACGGTGGTAAGTTGCACGCTGATGTTGAGGGTGTTACCTTGGTTTACGGAAAGACCAAGAAAGAGGGTTACATCATCGTTTCTTGCCAGGGTTACTCCGAGTACAACATCTACCAAAGATACCCACCACACGAGTTCGTCATGTCCTTCTCTATCCCTGACAACAAAGAGAAGGGCGTTGACAGAGTTACCAACACTGACGGTATTACTGCCGTTGGTGCCAACTTGGGTAAAGAATGGCCATACGGAATGGTTGTTGTCCACGACGATGTTAACGAAGCTGCTGGTGGTGGCGTTAGAGCTGATGCTACTTTTAAGATTGTCGGTCTGGGTGACATCTTGGGTAACAAGGCTGTGAAAGAGTTGGGTTTGCTGAAGGGTGTTGATGAGAACTGGGACCCAAGAAAGgtcgacgaattc:EcoRI；gtcgac:SacI

SEQ ID NO.4：

5'-GACTGGTTCCAATTGACAAGC-3'

SEQ ID NO.5：

5'-GCAAATGGCATTCTGACATCC-3'

SEQUENCE LISTING

<110> university of Anhui

<120> fungal-source beta-propeller-type recombinant phytase r-AoPhytase, and preparation method and application thereof

<130>

<160> 5

<170> PatentIn version 3.1

<210> 1

<211> 782

<212> PRT

<213> Arthrobotrys oligospora

<400> 1

Met Lys Ser Ser His Asn Leu Ser Gly Leu Ile Leu Leu Cys Leu Met

1 5 10 15

Gly Tyr Ile His Pro Thr Ser Ala Ala Asp Lys Phe Ser Ile Thr Leu

20 25 30

Pro Ile Thr Ala Arg Thr Ser Ser Val Glu Ser Asp Ser Ala Ala Val

35 40 45

Tyr Tyr Pro Ser Lys Ser Lys Tyr Ser Pro Ile Phe Ile Gly Asn Asp

50 55 60

Gly Ser Ala Glu Thr Gly Gly Phe His Val Tyr Glu Leu Tyr Gly Lys

65 70 75 80

Arg Ser Asp Ala Leu Val Lys Glu Leu Gly Ala Tyr Lys Thr Gly Arg

85 90 95

Ser Lys Leu Val Glu Val Val Tyr Gly Glu Asp Arg Asp Phe Val Val

100 105 110

Thr Leu Ser Met Ser Asp Gly Met Phe Arg Val Phe Glu Val Asp Gly

115 120 125

Lys Asn Gly Val Arg Gly Leu Lys Ala Glu Lys Leu Val Arg Gly Asp

130 135 140

Phe Ser Ala Met Cys Thr Trp Lys Ser Lys Val Gly Glu Tyr Val Tyr

145 150 155 160

Val Leu Gly Lys Arg Trp Gly Tyr Arg Phe Leu Val Arg Glu Lys Lys

165 170 175

Gly Gly Arg Gly Val Glu Val Val Gln Thr Gln Glu Phe Gly Ile Pro

180 185 190

Ile Glu Pro Asn Ser Cys Thr Val Ser Pro Glu Gly Lys Val Phe Leu

195 200 205

Ala Gly Asp Ser Gly Lys Val Phe Ser Phe Leu Ala Val Asp Glu Thr

210 215 220

Ala Ala Pro Lys Ile Val Glu Val Gly Glu Leu Gly Gly Gly Asp Glu

225 230 235 240

Val Lys Gly Leu Lys Ile Tyr His Gly Lys Lys Asp Thr Tyr Leu Leu

245 250 255

Val Gly Leu Glu Asp Gly Ile Glu Val Phe Asp Ile Lys Lys Leu Gly

260 265 270

Ser Ser Leu Gly Lys Ile Gln Phe Asp Asp Glu Glu Leu Glu Val Gly

275 280 285

Asp Phe Ala Val His Gln Thr Ser Ala Lys Gly Tyr Glu Asp Gly Ser

290 295 300

Ile Val Phe Ala Gly Glu Asp Gly Glu Gly Lys Phe Phe Gly Val Ser

305 310 315 320

Ser Leu Thr Pro Leu Phe Lys Ala Leu Gly Lys Gly Lys Leu Asn Thr

325 330 335

Lys Tyr Asp Pro Arg Asp Cys Thr Asp Asn His Ala Arg Pro Lys Lys

340 345 350

Cys Ala Asn Leu Ser Asp Cys Asn Gly Tyr Gly Tyr Cys Pro Lys Asp

355 360 365

Ser Arg Asp Lys Lys Ala Thr Cys Asp Cys Phe Pro Gly Leu Thr Gly

370 375 380

Lys Thr Cys Asn Lys Ile Thr Cys Pro Ser Asn Cys Thr Ser Pro Ser

385 390 395 400

His Gly Thr Cys Thr Gly Pro Asn Ile Cys Thr Cys Ile Pro Pro Phe

405 410 415

Thr Gly Glu Asn Cys Ala Thr Leu Ala Val Pro Ala Arg Tyr Glu Thr

420 425 430

Glu Glu Ser Gly Gly Ala Asp Gly Asp Asp Pro Ala Ile Trp Ile His

435 440 445

Pro Thr Asp Lys Thr Lys Ser Arg Ile Ile Thr Thr Val Lys Ser Glu

450 455 460

Val Gly Ser Gly Leu Gly Val Tyr Asp Leu Lys Gly Lys Arg Thr Gly

465 470 475 480

Gly Val Ser Gly Gly Glu Pro Asn Asn Val Asp Val Leu Tyr Gly Val

485 490 495

Glu Phe Ala Gly Arg Lys Val Asp Leu Ala Val Ala Ala Cys Arg Ala

500 505 510

Asp Asp Thr Ile Cys Ile Tyr Glu Ile Thr Pro Thr Gly Asp Leu Val

515 520 525

Thr Ile Pro Gly Gly Val Gln Pro Leu Pro Pro Ala Val Lys Glu Leu

530 535 540

Glu Lys Lys Phe Lys Val Tyr Gly Ser Cys Val Tyr His Ser Pro Lys

545 550 555 560

Thr Gly Ala Tyr His Ile Phe Val Asn Ser Lys Ser Ser Leu Tyr Leu

565 570 575

Gln Phe Gln Leu Ser Ala Thr Thr Asp Gly Lys Leu Asn Thr Thr Leu

580 585 590

Val Arg His Phe Tyr Ala Gly Asn Arg Gly Gln Val Glu Gly Cys Val

595 600 605

Val Asp Asp Glu Asn Ser Ser Leu Phe Leu Gly Glu Glu Pro Tyr Gly

610 615 620

Ile Trp Ser Tyr Asp Ala Glu Pro Asp Gln Pro Ala Val Gly Thr Leu

625 630 635 640

Val Asp Asn Thr Val Val Asp Gly Gly Lys Leu His Ala Asp Val Glu

645 650 655

Gly Val Thr Leu Val Tyr Gly Lys Thr Lys Lys Glu Gly Tyr Ile Ile

660 665 670

Val Ser Cys Gln Gly Tyr Ser Glu Tyr Asn Ile Tyr Gln Arg Tyr Pro

675 680 685

Pro His Glu Phe Val Met Ser Phe Ser Ile Pro Asp Asn Lys Glu Lys

690 695 700

Gly Val Asp Arg Val Thr Asn Thr Asp Gly Ile Thr Ala Val Gly Ala

705 710 715 720

Asn Leu Gly Lys Glu Trp Pro Tyr Gly Met Val Val Val His Asp Asp

725 730 735

Val Asn Glu Ala Ala Gly Gly Gly Val Arg Ala Asp Ala Thr Phe Lys

740 745 750

Ile Val Gly Leu Gly Asp Ile Leu Gly Asn Lys Ala Val Lys Glu Leu

755 760 765

Gly Leu Leu Lys Gly Val Asp Glu Asn Trp Asp Pro Arg Lys

770 775 780

<210> 2

<211> 2349

<212> DNA

<213> Arthrobotrys oligospora

<400> 2

atgaagtcct cacataacct ctcggggtta atcctccttt gtttgatggg gtacatccat 60

cccaccagcg ctgccgataa attctcaatc acactcccaa tcacagctcg gacttcatcc 120

gtcgaatccg atagcgcagc agtctactac ccttctaaat ccaagtactc accaatattc 180

atcggtaacg acgggtctgc cgaaaccggc ggattccatg tttatgaact atacggcaaa 240

cgaagtgatg ccttggtgaa agagcttgga gcgtacaaaa ctgggaggag taagttggtg 300

gaggtggttt atggtgaaga tagggacttt gtggttacgc tgtccatgtc ggatgggatg 360

tttagggtct ttgaggttga tggaaagaat ggagtgaggg gtcttaaggc ggaaaagctc 420

gttaggggtg atttttcggc gatgtgtacg tggaagagta aggttggaga gtacgtgtat 480

gttttgggga agagatgggg gtataggttc ttggttaggg agaagaaggg cgggagggga 540

gtcgaggttg tccaaacaca agaatttgga attcctattg aaccgaattc gtgtactgtt 600

tcacctgaag ggaaggtatt cttggcgggt gatagtggga aggttttctc cttcttagca 660

gttgacgaaa ctgctgcgcc aaagatcgta gaagttgggg agttgggcgg tggtgatgag 720

gttaagggcc tcaagatata tcatggaaag aaagatactt atcttctcgt tggattggaa 780

gatggaatcg aagtctttga tatcaagaaa ctgggaagtt cattaggaaa gatccagttc 840

gacgatgaag agctcgaggt tggagatttt gcagtacacc agacaagtgc gaagggatat 900

gaggatggga gtattgtttt tgcaggggag gatggggaag ggaagttctt tggagtcagt 960

tctttgactc ctttgtttaa agcattaggg aagggaaagc tgaacacgaa gtacgaccca 1020

cgagactgta ccgacaacca tgcgaggccg aagaagtgcg ccaatctttc tgattgcaat 1080

ggatacggat actgcccgaa agattctcga gataagaagg ccacctgcga ttgtttcccc 1140

gggcttacag gaaagacatg taataaaatt acctgcccat cgaactgtac atccccatcc 1200

catggaactt gtactggtcc aaacatctgt acttgcatcc cacccttcac cggcgaaaac 1260

tgtgcaaccc tagctgttcc agctagatac gaaacagaag agagtggcgg agcagatgga 1320

gatgatcccg ccatatggat tcatccaacc gacaaaacaa agtcgaggat tattacaaca 1380

gtaaagagtg aggttggaag tggacttggg gtatacgatt tgaagggcaa gagaacggga 1440

ggtgttagcg gcggtgaacc gaataatgtg gatgttttgt atggagttga atttgcaggg 1500

aggaaggttg atttggcagt ggcggcttgt agagctgatg atactatctg tatatacgaa 1560

atcaccccca ccggcgattt agtcaccatc ccaggcggcg ttcaacctct cccgccagcc 1620

gtcaaagaac tagagaagaa attcaaagtt tacggctcct gcgtctacca ttctcccaaa 1680

acaggagcat atcatatttt cgtcaactcc aaatcctcgc tgtatcttca attccagctt 1740

tctgcgacta cagacggaaa attgaatact actttggtgc gacactttta tgcaggaaat 1800

agaggacagg tagaaggctg tgtggtggat gacgagaact ctagcctttt ccttggtgaa 1860

gagccgtatg gaatttggag ttatgatgcc gaacctgatc aaccggcggt tggaacacta 1920

gtcgataaca cagtcgttga cggtggaaaa cttcatgccg atgtcgaggg agtcacacta 1980

gtctatggaa agactaaaaa agaaggatac atcatcgttt cctgtcaagg atacagtgaa 2040

tacaatattt accaacgata tccaccccat gagtttgtca tgagctttag cattccggat 2100

aataaagaaa agggcgtgga tagagtcacg aatacagatg ggattactgc tgttggagcg 2160

aacttgggga aggaatggcc ctatggaatg gtggttgtac atgatgatgt taatgaagct 2220

gcgggtggag gggttagagc cgacgccaca tttaagatcg taggattggg ggatattttg 2280

ggcaataagg cagtgaagga actgggtctt ttgaaagggg tggatgagaa ctgggatcct 2340

agaaagtag 2349

<210> 3

<211> 2286

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

gaattcgctg acaagttctc catcactttg ccaattactg ccagaacctc ttccgttgaa 60

tctgactctg ctgccgttta ctacccatcc aagtctaagt actccccaat cttcatcggt 120

aacgacggtt ctgctgaaac tggtggtttt cacgtctacg agttgtacgg taagagatcc 180

gacgccttgg tcaaagaatt gggtgcttac aagaccggta gatccaagtt ggttgaggtt 240

gtttacggtg aggacagaga cttcgttgtc actttgtcta tgtccgacgg tatgttcaga 300

gtgttcgagg tcgatggtaa gaacggtgtc agaggtttga aggccgagaa gttggtcaga 360

ggtgatttct ccgctatgtg tacctggaag tctaaggttg gtgagtacgt ctacgtcctg 420

ggtaaaagat ggggttacag attcttggtc cgtgagaaga aaggtggtag aggtgttgag 480

gtcgttcaga ctcaagagtt cggtattcca atcgagccaa actcctgtac tgtttcccca 540

gaaggtaagg ttttcttggc tggtgactcc ggtaaggtgt tttctttttt ggccgttgac 600

gagactgctg ccccaaagat agttgaagtt ggtgaacttg gtggtggtga cgaggttaag 660

ggtttgaaga tctaccacgg taagaaggac acctacttgt tggttggttt ggaggacggt 720

atcgaggtgt tcgacattaa gaagttggga tcctccttgg gtaagatcca attcgacgac 780

gaagagttgg aagtcggtga tttcgctgtt catcagactt ccgctaaggg ttacgaagat 840

ggttccatcg ttttcgctgg tgaagatggt gagggaaagt tcttcggtgt ttcctccttg 900

actcctctgt tcaaggctct tggtaagggt aagctgaaca ccaagtacga cccaagagac 960

tgtactgaca accacgctag accaaagaag tgtgctaact tgtccgactg caacggttac 1020

ggttactgtc caaaggactc cagagacaag aaggctactt gcgactgttt tccaggtttg 1080

accggtaaga cctgcaacaa gattacctgt ccatccaact gcacttcccc atctcatggt 1140

acttgtaccg gtccaaacat ctgcacttgt atcccaccat tcactggtga gaactgtgct 1200

actttggctg ttccagctag atacgagact gaagaatctg gtggtgctga tggtgatgac 1260

ccagctattt ggattcaccc aactgacaag accaagtcca gaatcatcac caccgttaag 1320

tccgaagttg gttccggttt gggtgtctac gatctgaagg gtaagagaac cggtggtgtt 1380

tcaggtggtg aacctaacaa cgttgacgtc ttgtacggtg ttgagttcgc cggtagaaag 1440

gttgacttgg ctgttgctgc ttgtagagct gacgacacca tctgtatcta cgagatcact 1500

ccaaccggtg acttggttac tattccaggt ggtgttcaac cattgccacc agctgtcaaa 1560

gagctggaaa agaagttcaa ggtctacggt tcctgcgtct accactctcc aaagactggt 1620

gcttaccaca tcttcgtcaa ctccaagtcc tccttgtact tgcagttcca gttgtccgct 1680

actactgacg gaaagttgaa cactaccctg gtcagacact tctacgctgg taacagaggt 1740

caagttgagg gttgtgttgt tgacgacgaa aactcttccc tgttcttggg tgaagaacca 1800

tacggtattt ggtcctacga tgctgaacca gaccaacctg ctgttggtac tttggttgac 1860

aacactgtcg ttgacggtgg taagttgcac gctgatgttg agggtgttac cttggtttac 1920

ggaaagacca agaaagaggg ttacatcatc gtttcttgcc agggttactc cgagtacaac 1980

atctaccaaa gatacccacc acacgagttc gtcatgtcct tctctatccc tgacaacaaa 2040

gagaagggcg ttgacagagt taccaacact gacggtatta ctgccgttgg tgccaacttg 2100

ggtaaagaat ggccatacgg aatggttgtt gtccacgacg atgttaacga agctgctggt 2160

ggtggcgtta gagctgatgc tacttttaag attgtcggtc tgggtgacat cttgggtaac 2220

aaggctgtga aagagttggg tttgctgaag ggtgttgatg agaactggga cccaagaaag 2280

gtcgac 2286

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

gactggttcc aattgacaag c 21

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

gcaaatggca ttctgacatc c 21

Claims

1. A method for preparing beta-propeller type recombinant phytase r-AoPhytase is characterized by comprising the following steps:

step 1: the recombinant expression strain is inoculated into 10mL-1L BMGY medium at the ratio of 1:1000v/v, cultured for 48h at 30 ℃ by a shaking table at 220rpm, and the OD is measured₆₀₀2-8, obtaining a culture;

step 2: the culture was centrifuged at 1500rpm for 10min, the supernatant discarded, and the pellet resuspended using 100mL-5L BMMY medium, and OD measured₆₀₀Inducing with shaking table at 220rpm for 72 hr at 30 deg.C under 0.4-4.0 to obtain culture solution;

and step 3: centrifuging the culture solution at 4 ℃ and 8000rpm for 10min, collecting supernatant, and preparing to obtain crude enzyme solution;

and 4, step 4: adding 5mL of Ni NTA Beads 6FF into the crude enzyme solution, incubating at 4 ℃ for 2h, adding the incubated product into an empty column tube, and collecting flow-through solution;

and 5: washing the filler by using 20mM imidazole buffer solution, and eluting impurities; then, the target protein is eluted by 40-500mmol/L imidazole buffer solution in a grading way to obtain purified r-AoPhytase protein;

the preservation number of the recombinant expression strain is CCTCC NO: m2019775.

2. The method for preparing a recombinant phytase r-AoPhytase according to claim 1, wherein the recombinant expression strain is prepared by:

firstly, chemically synthesizing an r-AoPhytase gene, carrying out double enzyme digestion by using EcoRI and SalI, connecting the gene with an expression plasmid vector pPICZ alpha A, and transforming a connecting product into a competent DH5 alpha cell to obtain an expression vector pPICZ alpha A-AoPhytase; then linearizing the expression vector by Sac I enzyme, and electrically transforming the linearized plasmid into expression host cell Pichia pastoris GS115 to obtain the recombinant engineering strain capable of recombinantly expressing r-AoPhytase, wherein the nucleotide sequence of the r-AoPhytase gene is shown in SEQ ID NO: 3.

3. The method for preparing a recombinant phytase r-AoPhytase according to claim 1 or 2, wherein the amino acid sequence of the phytase r-AoPhytase is as shown in SEQ ID NO. 1.

4. The application of beta-propeller type recombinant phytase r-AoPhytase in preparing phytase preparations for feed processing and production is disclosed, wherein the amino acid sequence of the recombinant phytase r-AoPhytase is shown as SEQ ID NO. 1.