Marine-derived salt-tolerant neutral phytase PHYBB1 as well as coding gene and application of marine-derived salt-tolerant neutral phytase PHYBB1

Abstract

The invention relates to marine-derived salt-tolerant neutral phytase PHYBB1 as well as a coding gene and application thereof, and belongs to the technical field of biological enzymes. According to the invention, firstly, a bacteroides SM25 is provided, the strain is preserved in the China Center for Type Culture Collection on June 23, 2025, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M20251440. The invention further provides a coding gene of the salt-tolerant neutral phytase PHYBB1, the nucleotide sequence of the coding gene is as shown in SEQ ID No. 1, and the amino acid sequence of the coding gene is as shown in SEQ ID No. 2. According to the present invention, the provided Rosevirgica sp. SM25 is the new species of Bacteroides family, the strain can produce the salt-tolerant neutral phytase PHYBB1, and the phytase has high phosphate solubilizing activity on sodium phytate and corn-soybean meal mixture phytate phosphorus, has good salt tolerance, and is suitable for being developed into the mariculture animal feed additive, such that the salt-tolerant neutral phytase PHYBB1 can be used to produce the salt-tolerant neutral phytase PHYBB1, and the salt-tolerant neutral phytase PHYBB1 can be used to produce the salt-tolerant neutral phytase PHYBB1, such that the salt-tolerant neutral phytase PHYBB1 can be used to produce the mariculture animal feed additive,

Description

TECHNICAL FIELD

[0001] The present application relates to a salt-tolerant neutral phytase PHYBB1 of marine origin, a coding gene thereof and an application thereof, and belongs to the technical field of biological enzymes. BACKGROUND

[0002] Phytic acid is an anti-nutritional factor in animal feed, which has an adverse effect on the digestion, absorption and utilization of nutrients. For many years, animal nutritionists have been working hard to find an economic and effective way to solve the influence of this anti-nutritional factor. Phytase can hydrolyze phytic acid to release inorganic phosphorus, and therefore, phytase as an animal feed additive plays an important role in improving the utilization of phosphorus in feed by monogastric animals, reducing the cost of inorganic phosphorus addition and the impact of monogastric animal excretion on the environment.

[0003] Phytases generally include acid phytase and neutral phytase. Among them, the optimum pH of acid phytase is lower than 5.5, and the optimum pH of neutral phytase is above 6.0. At present, the development and application technology of acid phytase is very mature, and acid phytase as a feed additive has been widely used in terrestrial animal feed. Since the pH of the digestive tract of most economic fish is neutral, neutral phytase is very suitable for use as a feed additive for fish in aquaculture. China's aquaculture industry is developed, and the market for neutral phytase as an aquaculture feed additive is large, but there is still a lack of neutral phytase products on the market at present, so it is necessary to accelerate the development and application technology research and development of neutral phytase.

[0004] Aquaculture includes freshwater aquaculture and marine aquaculture. Due to the large difference in salinity between freshwater and seawater, the salt tolerance of neutral phytase required in freshwater aquaculture feed and marine aquaculture feed is different. Freshwater aquaculture feed is applied to freshwater environment, and the neutral phytase used does not need to have salt tolerance. But seawater contains about 3% salinity, and the neutral phytase applied to marine aquaculture feed needs to have salt tolerance to ensure high activity in seawater environment.

[0005] Most of the existing neutral phytases are derived from terrestrial organisms and do not have salt tolerance, and are only suitable for development as freshwater aquaculture feed additives, but not suitable for development as marine aquaculture feed additives. Therefore, it is necessary to develop salt-tolerant neutral phytase of marine origin for marine aquaculture feed additives. SUMMARY

[0006] In view of the deficiencies of the prior art, the application provides a salt-tolerant neutral phytase PHYBB1 of marine origin, a coding gene and application thereof.The neutral phytase PHYBB1 has the highest activity in a 0.5M NaCl solution equivalent to the salinity of seawater, and still has 84.41% activity in a 1M NaCl solution, and has good salt tolerance.The salt-tolerant neutral phytase PHYBB1 has a phosphorus release effect on phytic acid in animal feed, and can be used as a feed additive for seawater aquaculture, and can improve the utilization rate of phosphorus in feed by seawater aquaculture animals.

[0007] The application also provides a Bacteroides (Roseivirga spongicola) SM25 for producing the salt-tolerant neutral phytase PHYBB1.

[0008] The technical scheme of the application is as follows:

[0009] A Bacteroides (Roseivirga spongicola) SM25, which is preserved in the China Center for Type Culture Collection (CCTCC) on June 23, 2025, and the address is Wuhan University, Wuchang District, Wuhan City, Hubei Province, China, and the preservation number is CCTCC NO: M20251440.

[0010] A coding gene of the salt-tolerant neutral phytase PHYBB1, and the nucleotide sequence is shown in SEQ ID No. 1.

[0011] The salt-tolerant neutral phytase PHYBB1 expressed by the coding gene, and the amino acid sequence is shown in SEQ ID No. 2.

[0012] According to the application, preferably, the salt-tolerant neutral phytase PHYBB1 and the coding gene thereof are both derived from the Bacteroides (Roseivirga spongicola) SM25.

[0013] A recombinant plasmid vector, which comprises the coding gene of the salt-tolerant neutral phytase PHYBB1.

[0014] According to the application, preferably, the carrier plasmid of the recombinant plasmid vector is a pET-22b(+) plasmid.

[0015] A recombinant cell, which comprises the coding gene of the salt-tolerant neutral phytase PHYBB1 or the recombinant plasmid vector.

[0016] According to the application, preferably, the host cell of the recombinant cell is an Escherichia coli; and more preferably, the host cell is an Escherichia coli BL21(DE3).

[0017] The salt-tolerant neutral phytase PHYBB1 and / or the coding gene of the salt-tolerant neutral phytase PHYBB1 are used for degrading phytic acid and preparing feed for aquaculture animals.

[0018] Technical features of the present application:

[0019] The present application carries out whole genome sequencing on Roseivirga spongicola SM25 isolated from the sea, and finds the nucleotide sequence of the coding gene of enzyme PHYBB1 from the strain genome through gene function annotation. Then the amino acid sequence of enzyme PHYBB1 is determined by searching and analyzing the protein database of the whole genome annotation of Roseivirga spongicola SM25. The nucleotide sequence of enzyme PHYBB1 is 1047 bp, which encodes 348 amino acid residues.

[0020] Sequence analysis shows that enzyme PHYBB1 contains signal peptide and catalytic domain two parts, and belongs to the beta-propeller phytase subclass of neutral phytase BPPhys subclass. Then primers are designed according to the gene sequence, and the gene of enzyme PHYBB1 is cloned from the genomic DNA of Roseivirga spongicola SM25 by PCR technology and heterologously expressed in Escherichia coli, and the recombinant expression strain of enzyme PHYBB1 is obtained. The recombinant expression strain is cultured and separated and purified, and enzyme PHYBB1 is obtained. The properties of the purified enzyme PHYBB1 are determined, and the results show that the enzyme has strong phosphorus release activity on sodium phytate. And its optimum enzyme activity temperature is 40℃, and the optimum pH is 6.5, which fully shows that enzyme PHYBB1 is a neutral phytase.

[0021] At the same time, the neutral phytase PHYBB1 has the highest activity in 0.5M NaCl solution equivalent to seawater salinity, and still has 84.41% activity in 1M NaCl solution, indicating that it has good salt tolerance, and it is a salt-tolerant neutral phytase PHYBB1. And the 2-hour phytate phosphorus release rate of corn-soybean meal mixture is 36%. Therefore, the salt-tolerant neutral phytase PHYBB1 is suitable for use as a feed additive for marine aquaculture animals.

[0022] Advantages:

[0023] 1、The Roseivirga spongicola SM25 provided by the present application is a new strain of Bacteroidaceae, which can produce salt-tolerant neutral phytase PHYBB1, and has good application potential in the fields of degrading phytic acid and preparing feed for aquatic animals.

[0024] 2, The optimal enzyme activity temperature of the salt-tolerant neutral phytase PHYBB1 provided by the application is 40 DEG C, the optimal pH is 6.5, and the salt-tolerant neutral phytase PHYBB1 has very high phosphorus releasing activity on sodium phytate and corn-soybean meal mixture phytate. And the activity is the highest in 0.5M NaCl solution corresponding to seawater salinity, and still has 84.41% activity in 1M NaCl solution, has very good salt tolerance, and is suitable for development as a feed additive for marine animal culture. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is SDS-PAGE electrophoretogram of the purified salt-tolerant neutral phytase PHYBB1 expressed by heterologous expression.

[0026] In the figure, M: protein molecular weight marker (marker); 1: supernatant after ultrasonic crushing of recombinant E. coli BL21 bacteria; 2: component of supernatant after ultrasonic crushing flowing through nickel column; 3: Lysis buffer eluted nickel column component; 4: Wash buffer eluted component; 5: nickel column purified neutral phytase PHYBB1.

[0027] Figure 2 It is the optimal enzyme activity temperature of the salt-tolerant neutral phytase PHYBB1.

[0028] Figure 3 It is the thermal stability of the salt-tolerant neutral phytase PHYBB1.

[0029] Figure 4 It is the optimal pH of the salt-tolerant neutral phytase PHYBB1.

[0030] Figure 5 It is the pH stability of the salt-tolerant neutral phytase PHYBB1.

[0031] Figure 6 It is the influence of calcium ion concentration on the activity of the salt-tolerant neutral phytase PHYBB1.

[0032] Figure 7 It is the influence of NaCl concentration on the activity of the salt-tolerant neutral phytase PHYBB1.

[0033] Figure 8 It is the influence of metal ions on the activity of the salt-tolerant neutral phytase PHYBB1.

[0034] Figure 9 It is the enzyme kinetics parameter analysis of the salt-tolerant neutral phytase PHYBB1. DETAILED DESCRIPTION

[0035] The technical solutions of the application will be further described below in combination with examples, but the scope of protection of the application is not limited thereto.

[0036] Roseivirga spongicola SM25 in the example, which was preserved in China Center for Type Culture Collection on June 23, 2025, address: Wuhan University, Wuchang District, Wuhan City, Hubei Province, preservation number: CCTCC NO: M20251440.

[0037] Strains and vectors: Escherichia coli DH5α was purchased from Novagen Biotech Co., Ltd., Escherichia coli BL21 (DE3) was purchased from GenScript Biotech Co., Ltd., and vector pET-22b (+) was purchased from GenScript Biotech Co., Ltd.

[0038] Enzymes and kits: PCR enzyme was purchased from Beijing Zongshijin Biotechnology Co., Ltd., restriction endonuclease was purchased from Thermo Fisher Scientific, ligase was purchased from Yixing Biotech Co., Ltd., DNA extraction kit was purchased from Bioteke, plasmid extraction kit and gel purification and recovery kit were purchased from Omega, and BCA protein quantitative analysis kit was purchased from Thermo.

[0039] Culture medium formula:

[0040] 2216E liquid medium: 0.5wt% peptone, 0.1wt% yeast powder, prepared with artificial seawater, pH 8.0.

[0041] LB liquid medium: 1wt% peptone, 0.5wt% yeast powder, 1wt% NaCl, prepared with distilled water, pH 8.0.

[0042] LB solid medium: 1wt% peptone, 0.5wt% yeast powder, 1wt% NaCl, 1.5wt% agar, prepared with distilled water, pH 8.0.

[0043] Example 1: Acquisition of salt-tolerant neutral phytase PHYBB1 encoding gene and construction of its recombinant plasmid vector

[0044] 1. Extraction of Roseivirga spongicola SM25 genomic DNA and whole genome sequencing.

[0045] Roseivirga spongicola SM25 genomic DNA was extracted, and the specific method referred to the steps of extracting genomic DNA in the Bioteke genomic extraction kit instruction manual.

[0046] 2. Identification of salt-tolerant neutral phytase PHYBB1 amino acid sequence and encoding gene nucleotide sequence

[0047] The genomic DNA of the Bacteroides SM25 extracted in the above step was subjected to whole genome sequencing by Beijing LiHua Huada Gene Technology Co., Ltd.

[0048] After obtaining the whole genome by sequencing, the amino acid sequence of the enzyme PHYBB1 was determined by searching the protein database of the whole genome of the Bacteroides SM25, and the coding gene sequence was further obtained, and it was determined that the enzyme PHYBB1 was an extracellular protease secreted by the Bacteroides SM25.

[0049] The nucleotide sequence encoding the enzyme PHYBB1 is shown in SEQ ID No. 1, which is 1047 bp; the amino acid sequence is shown in SEQ ID No. 2, which is 348 amino acid residues. Analysis of the amino acid sequence shows that the enzyme contains a signal peptide and a catalytic domain, and belongs to the neutral phytase BPPhys subclass (the beta-propeller phytase subclass).

[0050] 3. Construction of enzyme PHYBB1 recombinant plasmid vector

[0051] 3.1. Design and synthesis of primers

[0052] According to the Bacteroides R. spongicola gene sequence information obtained by genome sequencing, the following two primers were designed:

[0053] F: 5'-CTTTAAGAAGGAGATATACATATGGTACCCGCAGTTGC-3';

[0054] R: 5'-GGTGGTGGTGGTGGTGCTCGAGTTTCTTGAGTAATGAGTCC-3'.

[0055] The primers were artificially synthesized by Qingdao Qianke Biological Co., Ltd. according to the sequence information.

[0056] 3.2. Gene sequence amplification and product recovery by PCR

[0057] (1) PCR amplification was carried out with F and R as primers and genomic DNA as template; the PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 10 min.

[0058] PCR amplification system (50 μL) is: 29 μL of sterile distilled water, 5 x Fast Pfu buffer 10 μL, dNTP mixture 5 μL, primer F (10 μM) 2 μL, primer R (10 μM) 2 μL, genomic DNA 1 μL, Fast Pfu DNA polymerase 1 μL, supplemented with ddH2O to 50 μL.

[0059] (2) The PCR amplification product was subjected to 1% agarose gel electrophoresis, and a DNA fragment of about 1000 bp was obtained. Then the Omega DNA recovery kit was used according to the instructions to recover the amplified DNA fragment, and the coding gene PHYBB1 of the enzyme PHYBB1 was obtained.

[0060] (3) The coding gene PHYBB1 and the vector pET-22b(+) were subjected to double enzyme digestion reaction with Nde I and Xho I respectively,

[0061] The enzyme digestion reaction system of the coding gene PHYBB1 (20 μL) is: 2 μL of enzyme digestion buffer, 6 μL of gene PHYBB1, 1 μL of Nde I endonuclease, 1 μL of Xho I endonuclease, supplemented with ddH2O to 20 μL.

[0062] The enzyme digestion reaction system of the vector pET-22b(+) (20 μL) is: 2 μL of enzyme digestion buffer, 6 μL of vector pET-22b(+), 1 μL of Nde I endonuclease, 1 μL of Xho I endonuclease, supplemented with ddH2O to 20 μL.

[0063] (4) The enzyme digestion product was subjected to 1 wt% agarose gel electrophoresis, and then the Omega DNA recovery kit was used according to the instructions to recover the amplified DNA fragment.

[0064] 3.3, Construction of recombinant plasmid vector

[0065] (1) DNA fragment and cloning vector ligation

[0066] The recovered DNA fragment was ligated with the double-digested pET-22b vector, and the ligation reaction system was: vector pET-22b(+) 1 μL, DNA fragment 1.5 μL, 2 x Hieff Clone 2.5 μL.

[0067] Cover tightly, flick the centrifuge tube with your fingers, mix the sample, and centrifuge for 2 sec on the centrifuge, concentrate the sample at the bottom of the tube, connect at 50°C for 15 min.

[0068] (2) The expression vector was transformed into E. coli DH5a, and the ligated recombinant pET22b vector was transformed into E. coli DH5a competent cells by heat shock transformation according to the heat shock transformation method in the Guide to Molecular Cloning Experiments. The transformants were sent to Qingdao Qikai Biological Co., Ltd. for sequencing verification, and the recombinant plasmid vector pET-22b-PHYBB1 for heterologous expression of PHYBB1 was obtained.

[0069] Example 2: Heterologous expression and separation and purification of salt-tolerant neutral phytase PHYBB1

[0070] 1. Heterologous expression of enzyme PHYBB1 in E. coli BL21 (DE3)

[0071] The constructed recombinant plasmid vector pET-22b-PHYBB1 was transformed into E. coli BL21 (DE3) competent cells by heat shock transformation according to the heat shock transformation method in the Guide to Molecular Cloning Experiments, and was plated on LB solid medium containing 100 μg / mL ampicillin for overnight culture, and the recombinant strain was selected. The selected recombinant strain was inoculated into LB liquid medium containing 100 μg / mL ampicillin, and was cultured at 37°C, 180 rpm until OD 600 = 0.6-0.8, and then was transferred to a shaker at 18°C and 110 rpm, and IPTG was added to a final concentration of 0.5 mM, and was induced for 12 h to obtain the recombinant strain pET-22b-PHYBB1.

[0072] 2. Separation and purification of enzyme PHYBB1

[0073] The bacteria in 1 L of the recombinant strain fermentation broth were collected by centrifugation at 4°C and 6000 rpm for 5 min. The bacteria were resuspended with pre-cooled Lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0), and subjected to pressure disruption at 4°C and 1000 bar for three times until the bacterial solution was clear. The disrupted bacterial solution was centrifuged at 11000 rpm for 1 h, and the supernatant was collected to obtain a crude enzyme solution. The Ni-NT column was pre-equilibrated with Lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0), and the crude enzyme solution was added until it flowed through the affinity column. The affinity column was washed with Lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0) to remove unbound impurities. Then, the column was washed with Wash buffer (50 mM Tris-HCl, 100 mM NaCl, 15 mM imidazole, pH 8.0) to remove non-specifically bound proteins. Finally, the target protein was eluted from the affinity column with Elution buffer (50 mM Tris-HCl, 100 mM NaCl, 250 mM imidazole, pH 8.0), and the eluted product was collected. The collected sample was concentrated to 2.5 mL using an ultrafiltration tube. The desalting column was washed with 3-5 column volumes of molecular sieve buffer (10 mM Tris-HCl, 100 mM NaCl, pH 8.0), and the concentrated 2.5 mL enzyme solution was added until it flowed through the desalting column. Then, 3.5 mL of molecular sieve buffer (10 mM Tris-HCl, 100 mM NaCl, pH 8.0) was added for elution, and the eluted product was collected to obtain the final purified enzyme PHYBB1.

[0074] The purified enzyme PHYBB1 was subjected to SDS-PAGE electrophoresis to detect the purity, and the electrophoresis result is shown in Figure 1

[0075] As shown in Figure 1 , the molecular weight of the enzyme PHYBB1 is about 40 kDa, which is consistent with the theoretical value, indicating that the enzyme PHYBB1 is successfully expressed heterologously.

[0076] Example 3: Activity determination of salt-tolerant neutral phytase PHYBB1

[0077] 1. Definition of enzyme activity unit

[0078] Under the optimal conditions of the enzyme, 1 μmol of inorganic phosphorus released per minute from 5.0 mmol / L sodium phytate is defined as one phytase activity unit, which is represented by U.

[0079] 2. Enzyme activity determination

[0080] ​According to the volume ratio of ammonia water to water = 1:3, prepare the ammonia water solution.

[0081] According to the volume ratio of nitric acid to water = 1:2, prepare the nitric acid solution.

[0082] Take 10g of ammonium molybdate [(NH4)6Mo7O 24 4H2O] in a 50mL beaker, dissolve with water, and heat if necessary, then transfer to a 100mL volumetric flask, add 1mL of ammonia water solution, and dilute to 100mL with water to obtain a 100g / L ammonium molybdate solution.

[0083] Take 0.235g of ammonium metavanadate (NH4VO3) in a 50mL beaker, add 2mL of nitric acid solution and a small amount of water, and stir with a magnet stirring bar under light-proof conditions, then transfer to a 100mL brown volumetric flask, dilute to 100mL with water to obtain a 2.35g / L ammonium metavanadate solution, which is effective for one week under light-proof conditions.

[0084] According to the volume ratio of nitric acid solution: ammonium molybdate solution: ammonium metavanadate solution = 2:1:1, prepare the stop and color developing solution.

[0085] According to the national standard method (GB / T 18634-2009), the phytase enzyme activity is determined, and the specific method is as follows:

[0086] Take 100μL of PHYBB1 enzyme solution diluted with Tris-HAC (0.1M, pH=6.5, containing 4mM Ca 2+ ), add 200μL of 7.5mM sodium phytate substrate prepared with Tris-HAC (0.1mM, pH=6.5, containing 4mM Ca 2+ ), and react for 30min at 40℃, then add 200μL of stop and color developing solution. The control group first adds the stop and color developing solution, then adds the enzyme solution, and the other steps are the same. After standing for 10min, the absorbance value is measured at 415nm wavelength. Each sample is made in triplicate, and the average absorbance value is taken. Potassium dihydrogen phosphate is used as the standard, and the amount of inorganic phosphorus in the sample is calculated by the standard curve using the regression straight line equation.

[0087] In the sample, the activity of enzyme PHYBB1 is represented by X, with the unit of enzyme activity unit per gram (U / g) or enzyme activity unit per milliliter (U / mL), calculated according to formula (1).

[0088]

[0089] In the formula: X represents the activity of enzyme PHYBB1 in the sample, with the unit of enzyme activity unit per gram (U / g) or enzyme activity unit per milliliter (U / mL);

[0090] y - the amount of inorganic phosphorus calculated from the absorbance value of the actual sample solution by the linear regression equation, in units of micromole (pmol);

[0091] t - enzymatic reaction time, in units of minutes (min);

[0092] n - the dilution multiple of the sample;

[0093] m - sample mass, in units of grams (g) or milliliters (mL).

[0094] The determination result is that the enzyme activity of the enzyme PHYBB1 is 22.45 U / mL, and the enzyme PHYBB1 can effectively degrade sodium phytate and has strong phosphorus release activity on sodium phytate, which fully shows that the enzyme PHYBB1 is a neutral phytase.

[0095] 3. Protein content determination method

[0096] The protein content in the neutral phytase PHYBB1 enzyme solution is determined by using the BCA protein quantitative analysis kit of Thermo Company, and the specific method is as follows:

[0097] Gradient dilution bovine serum albumin (BSA) is used to prepare a standard sample, BCA reagent A and BCA reagent B are mixed (the ratio of reagent A to reagent B = 50:1) to prepare a working solution. 200 μL of the working solution is added to gradient dilution bovine serum albumin (BSA) standard sample and 20 μL of neutral phytase PHYBB1 enzyme solution diluted with Tris-HAC (0.1M, pH = 6.5, containing 4mM Ca 2+ ) respectively, and the absorbance value is determined at 595 nm after reaction at 37°C for 30 min. Each sample is made in triplicate, and the average value of the absorbance values is calculated by the regression straight line equation to calculate the protein content in the enzyme solution.

[0098] In the sample, the protein content is represented by X, in units of milligrams per milliliter (mg / mL), and calculated according to formula (2):

[0099] X = y x n (2)

[0100] In the formula, X is the protein content in the sample, in units of milligrams per milliliter (mg / mL);

[0101] y - the amount of inorganic phosphorus calculated from the absorbance value of the actual sample solution by the linear regression equation, in units of micromole (pmol);

[0102] n - the dilution multiple of the sample.

[0103] The determination result is that the protein content in the neutral phytase PHYBB1 enzyme solution is 7.77 mg / mL.

[0104] 4. Specific activity calculation

[0105] Specific Activity refers to the number of enzyme activity units in unit weight of protein, generally expressed as U / mg protein.

[0106] Specific Activity Formula: Specific Activity (U / mg) = Enzyme Activity (U / mL) / Protein Content (mg / mL).

[0107] Calculation Result: The specific activity of the heterologously expressed neutral phytase PHYBB1 of the application is 2.89 U / mg.

[0108] Example 4: Property determination of salt-tolerant neutral phytase PHYBB1

[0109] 1. Optimum temperature of neutral phytase PHYBB1 and thermal stability of enzyme:

[0110] Determination of optimum temperature: According to the method described in Example 3, the enzyme activity of neutral phytase PHYBB1 under different temperature conditions (10, 20, 30, 40, 50, 60, 70, 80℃) was determined in a pH = 6.5 buffer system (containing 4mM Ca 2+ ), and the temperature corresponding to the highest enzyme activity was the optimum enzyme activity temperature of the enzyme. The enzyme activity at this point was taken as 100%, and the enzyme activity at other temperatures was compared with it to obtain the relative activity. The results are shown in Figure 2 .

[0111] As can be seen from Figure 2 , the optimum temperature of neutral phytase PHYBB1 is 40℃.

[0112] Determination of thermal stability: The neutral phytase PHYBB1 enzyme solution was incubated at 40℃, 60℃ and 100℃ for 5, 10, 15, 20, 25 and 30 min, respectively; then the activity of neutral phytase PHYBB1 was determined according to the method described in Example 3 in a pH = 6.5 buffer system (containing 4mM Ca 2+ ) at 40℃. The activity measured without incubation treatment was taken as 100%, and the residual enzyme activity under the condition was expressed as the percentage of the initial enzyme activity based on the enzyme activity measured after incubation. The results are shown in Figure 3 .

[0113] As can be seen from Figure 3 , after 30 min of treatment at 40℃, the residual enzyme activity of neutral phytase PHYBB1 was 89%; after 30 min of treatment at 60℃, the residual enzyme activity of neutral phytase PHYBB1 was 50%, and even after 10 min of treatment at 100℃, the residual enzyme activity of neutral phytase PHYBB1 could still maintain 30%, indicating that neutral phytase PHYBB1 has good thermal stability.

[0114] 2. Optimal pH and pH stability of neutral phytase PHYBB1

[0115] Determination of optimal pH: Following the method described in Example 3, at 40°C, buffer solutions of different pH values ​​(containing 4 mM Ca) were prepared. 2+ The activity of neutral phytase PHYBB1 was determined in [the experiment]. The pH at which the enzyme activity was highest is the optimal pH for the enzyme. The enzyme activity at this pH was taken as 100%, and the relative activity at other pH values ​​was calculated by comparing this value. The results are shown below. Figure 4 As shown.

[0116] The buffer solutions with different pH values ​​were: 0.1M HAc-NaAc, pH 4.5–6.0; 0.1M Tris-HAc, pH 6.0–7.0; and 0.1M Tris-HCl, pH 7.0–9.0.

[0117] Depend on Figure 4 It is known that the optimal pH for neutral phytase PHYBB1 is 6.5.

[0118] pH stability was determined: Neutral phytase PHYBB1 enzyme solution was treated for 30 min at 37℃ in Tris-HCl buffer at different pH values ​​(4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.0), and then further treated in a 40℃, pH 6.5 buffer system (containing 4 mM Ca). 2+ Phytase activity was determined in [the following text is incomplete and requires further context to translate accurately]. The activity measured using the untreated enzyme solution was taken as 100%. The residual enzyme activity under those conditions was expressed as the percentage of the enzyme activity measured after treatment relative to the initial enzyme activity. The results are as follows: Figure 5 As shown.

[0119] Depend on Figure 5 It can be seen that the neutral phytase PHYBB1 is relatively stable at pH 6.0-8.0, with the remaining enzyme activity all above 80%, indicating that this enzyme has good pH stability under alkaline conditions.

[0120] 3. Optimal calcium ion concentration for neutral phytase PHYBB1:

[0121] Determination of optimal calcium ion concentration: Following the method described in Example 3, at 40°C, pH = 6.5, and different calcium concentrations... 2+ The activity of neutral phytase PHYBB1 was measured under concentrations of (1, 2, 3, 4, 5, 6 mM). The highest enzyme activity corresponds to the highest Ca2+ concentration. 2+ The concentration is the optimal Ca concentration for this enzyme. 2+ Concentration, with the enzyme activity at that location taken as 100%, other Ca 2+ The relative activity was determined by comparing the enzyme activity at a certain concentration with that at another concentration, and the results are as follows:Figure 6 As shown.

[0122] Depend on Figure 6 It is known that the optimal calcium ion concentration for neutral phytase PHYBB1 is 4 mM.

[0123] 4. Optimal NaCl concentration for neutral phytase PHYBB1

[0124] Determination of the optimal NaCl concentration: Following the method described in Example 3, in a buffer system (containing 4 mM Ca) at 40°C and pH = 6.5. 2+ The activity of neutral phytase PHYBB1 was measured under different NaCl concentrations (0, 0.5, 1.0, 1.5, and 2.0 M). The NaCl concentration corresponding to the highest enzyme activity is the optimal NaCl concentration for this enzyme. The enzyme activity at this concentration is taken as 100%, and the relative activity at other NaCl concentrations is calculated by comparing it to this optimal concentration. The results are shown below. Figure 7 As shown.

[0125] Depend on Figure 7 It is known that the optimal NaCl concentration for neutral phytase PHYBB1 is 0.5M, and it still retains 84% ​​activity in 1M NaCl, demonstrating excellent salt tolerance. This indicates that PHYBB1 is not only a neutral phytase, but also a salt-tolerant neutral phytase, making it suitable as a feed additive for marine aquaculture animals.

[0126] 5. Effects of metal ions on the activity of salt-tolerant neutral phytase PHYBB1

[0127] Different concentrations of different metal ions and chemical reagents were added to the enzyme-catalyzed reaction system to study their effects on enzyme activity. The final concentration of each substance was 1 mM.

[0128] Specifically, following the method described in Example 3, using a solution containing 4 mM Ca 2+ Phytate (5 mM) was used as a substrate in 4 mM Ca 2 + The activity of salt-tolerant neutral phytase PHYBB1 was determined under the conditions of 40℃ and pH=6.5. The activity measured using the enzyme solution without added metal ions was taken as 100%, and the relative activity was obtained by comparing it with the enzyme activity after treatment with other metal ions. The results are as follows: Figure 8 As shown.

[0129] Depend on Figure 8 It can be seen that, except for Li+ which has a weak activating effect on the activity of salt-tolerant neutral phytase PHYBB1, most ions have an inhibitory effect on the activity of salt-tolerant neutral phytase PHYBB1, such as Zn. 2+ Fe 2+ Fe 3+ Cu 2+The activity of salt-tolerant neutral phytase PHYBB1 was almost completely inhibited, and SDS also almost completely inhibited its activity.

[0130] 6. Kinetic parameter analysis of salt-tolerant neutral phytase PHYBB1

[0131] According to the method described in Example 3, 4 mM Ca 2+ The activity of salt-tolerant neutral phytase PHYBB1 was almost completely inhibited, and SDS also almost completely inhibited its activity. 2+ ) at 40°C and pH = 6.5 buffer system (containing 4 mM Ca Figure 9 ) and further calculated the corresponding reaction rate, the Km value and Vmax were obtained by Lineweaver-Burk method, the results are shown in

[0132] As can be seen from Figure 9 , the Km of salt-tolerant neutral phytase PHYBB1 is 0.85 mM, and the Vmax is 3.74 μmol / min / mg. And the kcat is calculated to be 2468.99 s -1 .

[0133] Example 4: Determination of the phosphorus release rate of salt-tolerant neutral phytase PHYBB1 on corn-soybean meal mixture phytate phosphorus

[0134] The ability of salt-tolerant neutral phytase PHYBB1 to hydrolyze phytate phosphorus in corn-soybean meal mixture was determined in pH = 6.5 buffer system (containing 4 mM Ca 2+ ) at 20°C and 40°C, respectively.

[0135] The specific method is as follows: 9 mL of Tris-HAc (0.1 M, pH = 6.5, containing 4 mM Ca 2+ ) was added to 1 g of corn-soybean meal mixture (corn to soybean meal mass ratio = 7:3) to obtain a mixed solution; then the mixed solution was preheated at 20°C and 40°C for 30 min, respectively, 1 mL of salt-tolerant neutral phytase PHYBB1 enzyme solution diluted with Tris-HAc (0.1 M, pH = 6.5, containing 4 mM Ca 2+ ) was added (the amount of enzyme added was according to 1500 U / kg of corn-soybean meal mixture), and the reaction was carried out in a shaker at 20°C and 40°C, respectively, for 2 h, then 30% trichloroacetic acid was added to terminate the reaction, and the content of inorganic phosphorus released in the reaction solution was determined. The total phosphorus content in corn-soybean meal mixture was determined according to the method in the group standard (T / CBFIA02002-2020), and the phosphorus release rate of salt-tolerant neutral phytase PHYBB1 hydrolyzing corn-soybean meal mixture at 20°C and 40°C for 2 h was further calculated.

[0136] The results of the determination show that the phosphorus release rates of the salt-tolerant neutral phytase PHYBB1 in hydrolyzing corn-soybean meal mixture for 2 hours at 20 DEG C and 40 DEG C are 32% and 36% respectively, which further proves that the enzyme PHYBB1 provided by the application is a salt-tolerant neutral phytase, has high phosphorus release activity on sodium phytate and corn-soybean meal mixture phytate phosphorus, and is suitable for development as an aquatic animal feed additive.

Claims

1. A strain of Bacteroides (Roseivirga spongicola) SM25, characterized in that, This strain was deposited on June 23, 2025, at the China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCCNO: M20251440.

2. A gene encoding a salt-tolerant neutral phytase PHYBB1, characterized in that, The nucleotide sequence is shown in SEQ ID No.

1.

3. The salt-tolerant neutral phytase PHYBB1 encoding the gene expressed according to claim 2, characterized in that, The amino acid sequence is shown in SEQ ID No.

2.

4. A recombinant plasmid vector, characterized in that, The recombinant plasmid vector contains the encoding gene of the salt-tolerant neutral phytase PHYBB1 as described in claim 2.

5. The recombinant plasmid vector as described in claim 4, characterized in that, The recombinant plasmid vector is pET-22b(+) plasmid.

6. A recombinant cell, characterized in that, The recombinant cell contains the encoding gene of the salt-tolerant neutral phytase PHYBB1 as described in claim 2 or the recombinant plasmid vector as described in claim 4.

7. The recombinant cell as described in claim 6, characterized in that, The host cell used for the recombinant cells was Escherichia coli BL21(DE3).

8. The application of the encoding gene of salt-tolerant neutral phytase PHYBB1 as described in claim 3 and / or salt-tolerant neutral phytase PHYBB1 as described in claim 2 in the degradation of phytic acid and the preparation of feed for aquaculture animals.

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