Freund's bacillus 379-1 of arctic origin and phytase produced by it and use thereof

Abstract

The present application relates to a freilandia 379-1 of arctic origin and a phytase produced by the same and application thereof, and belongs to the technical field of biological enzymes. Vreelandella The Latin name of the strain is Freilandia sp. 379-1; the strain was preserved in the China General Microbiological Culture Collection Center on December 22, 2025, the address is No. 1, Xibaheyi, Chaoyang District, Beijing, the preservation number is CGMCC NO. 37180. The phytase BPP651 produced by the strain has a nucleotide sequence as shown in SEQ ID NO. 2. The strain has phytase activity phytase BPP651, has strong phosphorus release activity on calcium phytate, has strong alkali resistance and excellent salt tolerance. These characteristics have great application prospects in the fields of degrading phytic acid, preparing feed additives for aquatic animal breeding, and treating high-salt phosphorus-containing wastewater.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme technology, specifically relating to an Arctic-derived Fritillaria 379-1 and the phytase it produces and its applications. Background Technology

[0002] Phytic acid is an anti-nutritional factor in animal feed, adversely affecting the digestion, absorption, and utilization of nutrients. Phytase can catalyze the hydrolysis of phytic acid to release inorganic phosphorus. As an animal feed additive, it can improve the utilization of phosphorus in feed by monogastric animals, reduce the cost of inorganic phosphorus additives, and reduce the environmental impact of phosphorus emissions from feces.

[0003] Phytases mainly include acidic phytases and neutral phytases. Acidic phytases have an optimal pH below 5.5, while neutral phytases have an optimal pH above 6.0. Currently, most commercially available phytases are derived from the acid phosphatase family, primarily adapted to the highly acidic gastric environment (pH 1.5-2.5) of terrestrial monogastric animals. The digestive tract of fish exhibits distinct compartmentalization: the stomach is acidic (pH 1.2-5.2), while the intestine is neutral to slightly alkaline. Therefore, acidic phytases may not be fully adapted to the digestive tract environment of all fish. Furthermore, the salt tolerance range of current acidic phytases is typically below 3.0%, and their activity is inhibited in seawater (approximately 3.0% NaCl) or high-salt environments. Therefore, developing multi-resistance phytases from extreme marine environments holds significant application potential in feed additives for marine aquaculture animals and even in the biological treatment of high-salt, phosphorus-containing wastewater. Summary of the Invention

[0004] To address the limitations of existing alkaline phytase resources and the lack of salt tolerance in commercially available acidic phytases, this invention aims to provide a strain of *Frederick* 379-1 derived from Arctic deep-sea sediments, capable of producing alkaline phytase. The *Frederick* 379-1 strain screened in this invention can grow and reproduce in seawater, exhibits phytic acid degradation activity, and its genome contains a β-propeller phytase gene. Recombinant phytase BPP651 was obtained through heterologous expression using the *E. coli* BL21(DE3) expression system. Recombinant phytase BPP651 possesses multiple excellent enzymatic properties: its specific activity reaches 246.2 U / mg (using calcium phytate as a substrate); the optimal temperature for recombinant phytase BPP651 is 40 °C; within a low temperature range of 0–15 °C, the enzyme retains more than 47.8% of its maximum activity, demonstrating good low-temperature adaptability; it has a wide pH tolerance range (7.0–12.0), with an optimal pH of 8.0; the optimal NaCl concentration is 3.0%, and it retains 83.8% of its maximum activity even in a high-salt environment of 23% NaCl. The phytase BPP651 provided by this invention exhibits alkali and salt tolerance, making it of significant application value in the fields of marine aquaculture animal feed additives and biological treatment of high-salt, phosphorus-containing wastewater.

[0005] The specific technical solution of this invention is as follows: One of the objectives of this invention is to provide a Vreelandella sp. 379-1 strain derived from Arctic deep-sea sediments. This strain was deposited on December 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.37180.

[0006] The 16S rRNA gene of *Vreelandella* sp. 379-1 is as follows:

[0007] A second objective of this invention is to provide phytase BPP651 produced by *Vreelandella* sp. 379-1, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] Nucleotide sequence of the phytase BPP651 gene (SEQ ID NO.2):

[0009] The amino acid sequence of the phytase BPP651 is as follows (SEQ ID NO.3): .

[0010] A third objective of this invention is to provide a recombinant bacterium containing the gene for phytase BPP651. The recombinant bacterium is prepared by introducing the gene for phytase BPP651 produced by *Vreelandella* sp. 379-1 into a pET-28a(+) plasmid vector, and then transforming the recombinant expression vector into *E. coli* BL21(DE3) host cells to obtain recombinant expression of phytase BPP651.

[0011] The fourth objective of this invention is to provide the application of the phytase BPP651 and / or the encoding gene of the phytase BPP651 and / or the strain in the degradation of phytic acid, the preparation of feed additives for aquaculture animals, and the treatment of high-salt phosphorus wastewater.

[0012] The fifth objective of this invention is to provide a product that degrades phytic acid, comprising live bacteria of Vreelandella sp. 379-1 and / or phytase BPP651.

[0013] The beneficial effects of this invention compared to the prior art are as follows: This invention provides the first strain of bacteria capable of degrading phytate from the Arctic marine environment, named *Vreelandella* sp. 379-1. This strain possesses phytase activity. The strain contains a phytase-encoding gene with a nucleotide sequence of 2136 bp, encoding 711 amino acid residues, named BPP651. Sequence analysis shows that BPP651 comprises a signal peptide and a catalytic domain, belonging to the β-propeller phytase family.

[0014] The phytase BPP651 described in this invention exhibits strong phosphorus-solubilizing activity against calcium phytate. Its optimal enzyme activity temperature is 40°C, optimal pH is 8.0, and optimal salinity is 3.0%. Phytase BPP651 retains 92.0% of its maximum activity even under strongly alkaline conditions at pH 12.0, demonstrating high alkali resistance; and retains 83.8% of its maximum activity in NaCl solutions with concentrations up to 23%, exhibiting excellent salt resistance. These properties suggest significant application prospects in the degradation of phytic acid, the preparation of feed additives for aquaculture animals, and the treatment of high-salt, phosphorus-containing wastewater. Attached Figure Description

[0015] Figure 1 The graph shows the phytate degradation activity of Vreelandella sp. 379-1; A shows the growth of the strain on R2A solid medium (containing bromocresol green) and the phytate degradation clear zone; B shows the functional verification results of the recombinant phytase BPP651 expressing strain E. coli BL21 (DE3) on modified Sperber solid medium. Figure 2 Phylogenetic tree diagram of the 16S rRNA gene of Vreelandella sp. 379-1; Figure 3 SDS-PAGE electrophoresis image of purified phytase BPP651. M: Protein molecular weight standard; 1: Purified phytase BPP651; 2: BCA protein quantitative sample; Figure 4Figure 1 shows the effect of temperature on the specific activity of phytase BPP651. Figure 5 The graph shows the effect of pH on the specific activity of phytase BPP651. Figure 6 The graph shows the effect of NaCl concentration on the specific activity of phytase BPP651. Detailed Implementation

[0016] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0017] Example 1. Obtaining Vreelandella sp. 379-1 strain and screening for phytase activity; The marine bacterium *Vreelandella* sp. 379-1 was isolated from sediments collected during the 92nd Chinese oceanographic expedition in 2025 in the Laptev Sea (77°N, 158°E, depth 787 m) of the Arctic Ocean. It was cultured in 30% strength R2A solid medium (Qingdao Haibo Biotechnology) prepared using natural seawater with a salinity of approximately 3%. The culture conditions were 15 °C for 7 days.

[0018] The strain was inoculated into a calcium phytate selection medium and incubated at 15 °C. The size of the clear zone was used to determine the strain's ability to degrade phytate. The calcium phytate selection medium consisted of: 30 g glucose, 5 g NH4NO3, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·4H2O, 3 g calcium phytate, 20 g agar, 1000 mL natural seawater, and a pH of 7.0–7.2. Figure 1 As shown in Figure A, after 10 days of cultivation, a clear zone appeared around the Vreelandella sp.379-1 colony on the calcium phytate selection medium, indicating that it has significant degradation activity for phytic acid.

[0019] Example 2. Classification, identification, nomenclature, and preservation of Vreelandella sp. 379-1 strain; 16S rRNA gene molecular identification: Using the strain's genomic DNA as a template, the 16S rRNA gene sequence was amplified by PCR using universal primers 27F and 1492R. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0020] The specific steps are as follows: Add 20–30 μL of ddH2O to a sterile 2 mL centrifuge tube. Pick a purified bacterial single colony, mix well, and heat in boiling water for 10 min to obtain a DNA template. Place on ice for later use. Using bacterial genomic DNA as a template, amplify the 16S rRNA gene fragment using the universal primers 27F (5'-AGAGTTGATCMTGGCTCAG-3' SEQ ID NO.4) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3' SEQ ID NO.5). PCR amplification system: 1 μL TransStart Taq DNA polymerase (2.5 U, TransGold, Beijing, China), 2 μL DNA, 1 μL primer (0.2 μmol / L final concentration), 5 μL buffer, 4 μL dNTPs, and add ddH2O to a final volume of 50 μL. PCR reaction conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 90 s, 30 cycles; final extension at 72 ℃ for 5 min. 2 μL of the PCR product was analyzed by 1% agarose gel electrophoresis and then sequenced.

[0021] Sequencing results were submitted to EzTaxon for comparison to determine the taxonomic position of the strain. The 16S rRNA gene sequence of strain 379-1 is most closely related to Vreelandella titanicae BH1 (99.97%), and it was preliminarily identified as Vreelandella sp., named Vreelandella sp. 379-1. A phylogenetic tree was constructed using MEGA 12.0 software with neighbor-joining, further demonstrating that strain Vreelandella sp. 379-1 and Vreelandella titanicae BH1 cluster on the same evolutionary branch, indicating the closest phylogenetic relationship. Figure 2 ).

[0022] Vreelandella sp. 379-1 has the accession number CGMCC No. 37180; the depositary institution is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; and the deposit date is December 22, 2025.

[0023] Example 3. Whole genome sequencing of Vreelandella sp. 379-1 and identification of the phytase BPP651 encoding gene; The whole genome of *Vreelandella* sp. 379-1 was sequenced by Beijing Novogene Technology Co., Ltd. Bioinformatics analysis and functional annotation of the *Vreelandella* sp. 379-1 genome were performed, identifying the coding gene sequence and amino acid sequence of phytase BPP651. The nucleotide sequence encoding BPP651 is shown in SEQ ID No. 2, totaling 2136 bp; the amino acid sequence is shown in SEQ ID No. 3, totaling 711 amino acids. Amino acid sequence analysis showed that the enzyme contains an N-terminal signal peptide and a catalytic domain. The catalytic domain consists of an N-terminal 5-leaf β-propeller domain and a C-terminal 7-leaf β-propeller domain, belonging to the β-propeller phytase family.

[0024] Example 4. Heterologous expression of phytase BPP651 from Vreelandella sp. 379-1; Phytase BPP651 gene synthesis: The signal peptide nucleotide sequence of BPP651 was removed to obtain the mature protein coding sequence (699 amino acids). After codon optimization, the gene synthesis was completed by Sangon Biotech (Shanghai) Co., Ltd.

[0025] Preparation of recombinant phytase BPP651 expression strain E. coli BL21(DE3): (1) Synthesis and cloning of target genes Based on the BPP651 phytase gene sequence, the full-length coding sequence was synthesized artificially after codon optimization. Nde I and Xho I restriction enzyme sites were introduced at the 5′ and 3′ ends of the sequence, respectively, via PCR to amplify the target fragment. The purified PCR product and the pET-28a(+) expression vector were double-digested with Nde I and Xho I, respectively. After recovering the linearized vector and the target fragment, they were ligated using T4 DNA ligase to construct the recombinant expression plasmid pET-28a-BPP651. The ligation product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing 50 μg / mL kanamycin, incubated overnight at 37 ℃. Single colonies were picked for colony PCR verification and Sanger sequencing. After confirming the sequence was correct, the recombinant plasmid was extracted for later use.

[0026] (2) Transformation and screening of expression strains Take 1 μL of the sequencing recombinant plasmid pET28a-BPP651 (approximately 50 ng / μL) and add it to 50 μL of E. coli BL21(DE3) competent cells. Mix gently and incubate on ice for 30 min. Heat shock at 42 °C for 45 s, then immediately incubate on ice for 2 min. Add 500 μL of SOC medium and incubate at 37 °C with shaking at 180 rpm for 45–60 min. Spread 100–200 μL of the bacterial culture onto LB agar plates containing 50 μg / mL kanamycin and incubate upside down at 37 °C for 12–16 h.

[0027] (3) Identification of positive clones Single colonies growing on the plate were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37 °C with shaking at 200 rpm. A portion of the bacterial culture was used for colony PCR verification. The target gene fragment was amplified using primers with the T7 promoter and T7 terminator. The amplified products were detected by 1% agarose gel electrophoresis; clones showing the expected band size were considered positive clones. The verified positive clone was the recombinant phytase BPP651 expressing strain E. coli BL21(DE3) / pET28a-BPP651.

[0028] (4) Preservation of strains A single colony of the validated positive clone was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C with shaking at 200 rpm until OD500 was reached. 600 Approximately 0.6–0.8. Mix 500 μL of bacterial culture with 500 μL of sterile 40% glycerol and store at -80°C for long-term storage.

[0029] Induction of expression: Phytase BPP651 expressing strain E. coli BL21(DE3) was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 ℃ and 180 rpm until OD200 was reached. 600 The pH was approximately 0.6–0.8. IPTG was added to a final concentration of 0.0005 mol / L, and the mixture was induced and cultured at 16 ℃ and 110 rpm for 16 h. The LB liquid medium consisted of 10 g peptone, 5 g yeast extract, 10 g NaCl, 1000 mL distilled water, and pH 7.0.

[0030] Functional validation: The induced recombinant bacterial culture was inoculated onto modified Sperber solid medium containing calcium phytate and BCIP (5-bromo-4-chloro-3-indolyl phosphate) and incubated statically at 16 °C for 48 h. The results showed that the colonies were distinctly dark blue with clear hydrolysis zones, confirming that the recombinant strain expressed active phytase (…). Figure 1(B) The modified Sperber solid medium consists of: 30g glucose, 5g NH4NO3, 0.5g MgSO4·7H2O, 0.5g KCl, 0.03g FeSO4·7H2O, 0.03g MnSO4·4H2O, 3g calcium phytate, 20g agar, 1000mL natural seawater, and a pH of 7.0–7.2.

[0031] Isolation and purification of recombinant protein: The induced bacterial culture was centrifuged at 12,000 rpm for 10 min at 4 ℃ to collect bacterial cells. The bacterial cells were resuspended in pre-cooled lysis buffer (0.02 mol / L Tris-HCl, 3% NaCl, pH 7.5) and sonicated in an ice-water bath (200 W, 3 s on, 5 s off, for a total of 10 min). The lysate was centrifuged at 12,000 rpm for 30 min at 4 ℃, and the supernatant was collected to obtain the crude enzyme solution.

[0032] The Ni-NTA affinity chromatography column was equilibrated with lysis buffer (0.02 mol / L Tris-HCl, 3% NaCl, pH 7.5). After adding the crude enzyme solution, the column was washed sequentially with lysis buffer and elution buffer containing 0.02 mol / L imidazole to remove impurities. The target protein was then eluted with elution buffer containing 0.25 mol / L imidazole, and the eluent was collected. The eluent was dialyzed against 0.02 mol / L Tris-HCl buffer (pH 7.5) to obtain purified BPP651 protein.

[0033] Protein purity assay: The purified protein was subjected to 12% SDS-PAGE electrophoresis. The target protein showed a single band with a molecular weight of approximately 75 kDa. Figure 3 This is consistent with the theoretical molecular weight of 75 kDa of the mature protein after the signal peptide is removed.

[0034] Example 5: Determination of recombinant phytase BPP651 activity; The enzyme activity assay method was based on the national standard method (GB / T 18634-2009), which used the molybdenum blue method to determine the release of inorganic phosphorus, with slight modifications.

[0035] Preparation of colorimetric reagent: (1) 100 g / L ammonium molybdate solution: Weigh 10 g of ammonium molybdate [(NH4)6Mo7O] 24Dissolve 4H2O] in water in a 50 mL beaker, and heat slightly if necessary. Then transfer to a 100 mL volumetric flask and make up to 100 mL to obtain an ammonium molybdate solution with a concentration of 100 g / L. (2) Sulfuric acid solution: Slowly add 50 mL of concentrated sulfuric acid to 50 mL of distilled water and cool for later use. (3) Molybdenum blue color reagent: Mix 2 mL of 100 g / L ammonium molybdate solution with 8 mL of sulfuric acid solution. Before use, add 10% ascorbic acid solution at a volume ratio of 1:10, mix well and use.

[0036] Standard assay system and procedure: The standard assay system (1.0 mL) consists of: 750 µL 0.1 mol / L Tris-HCl buffer (pH 8.0, containing 0.5 mol / L NaCl), 150 µL 0.01 mol / L calcium phytate solution, and 50 µL appropriately diluted enzyme solution. After reacting the system in a 30 ℃ water bath for 30 min, immediately add 1.0 mL of 10% (w / v) trichloroacetic acid (TCA) to terminate the reaction and mix well. Centrifuge at 12000 rpm for 5 min, take 1.0 mL of the supernatant, add 2.0 mL of molybdenum blue reagent, let stand at room temperature for 10 min, and measure the absorbance (OD) at 660 nm. 660 Each sample was tested in triplicate, and the average absorbance was recorded. An inorganic phosphorus standard curve was plotted using potassium dihydrogen phosphate (KH₂PO₄) as the standard, and the amount of inorganic phosphorus released in the reaction was calculated using a regression equation. The blank control was identical to the test samples in all aspects except that an equal volume of deionized water replaced 50 µL of the enzyme solution; the reagent composition, sample addition order, isothermal reaction conditions, TCA termination, centrifugation, and molybdenum blue colorimetric assay were all performed simultaneously. After the assay, the average OD₀ of the samples was recorded. 660 Value minus the mean OD of the blank control 660 The net absorbance is obtained by substituting it into the regression equation of the inorganic phosphorus standard curve, which allows for accurate calculation of the actual amount of inorganic phosphorus released by the enzyme-catalyzed reaction.

[0037] Enzyme activity calculation: One unit of phytase activity (U) is defined as the amount of enzyme required to release 1 μmol of inorganic phosphorus per minute from a 1.5 mmol / L calcium phytate substrate under certain conditions. In a sample, phytase activity is represented by X, with units of enzyme activity per milliliter (U / mL). The calculation formula is: In the formula, X: phytase activity (U / mL); y: amount of inorganic phosphorus (μmol) calculated by linear regression equation based on the absorbance of the actual sample solution; t: enzymatic reaction time (min); v: volume of enzyme solution added to the reaction system (mL); n: dilution factor of the sample.

[0038] Results: Under optimal conditions (40 °C, pH 8.0, 0.5 mol / L NaCl), with a reaction system containing 150 µL of 0.01 mol / L calcium phytate solution, the enzyme activity of BPP651 was 984.8 U / mL.

[0039] Protein content determination: The protein content in the purified BPP651 enzyme solution was determined using the Thermo BCA protein quantification kit. Bovine serum albumin (BSA) standards were prepared by serial dilution using protein preservation buffer (0.02 mol / L Tris-HCl, 3% NaCl, pH 7.5). Working solutions were prepared by mixing BCA reagent A and BCA reagent B (v / v = 50:1). Serially diluted BSA standards and appropriately diluted BPP651 enzyme solution were added to 200 μL of the working solution, and the mixture was reacted at 37 °C for 30 min. The reaction was then carried out at OD0.05. 562 The absorbance was measured at the specified wavelength. Each sample was tested in triplicate, and the average absorbance was taken. The protein content in the enzyme solution was calculated by regressing the linear equation of the standard curve.

[0040] In the sample, the protein content is expressed as P, with units of milligrams per milliliter (mg / mL), calculated using the following formula: In the formula, P: protein content in the sample (mg / mL); y: protein content calculated from the standard curve (mg / mL); n: dilution factor of the sample.

[0041] The results showed that the protein content in the purified BPP651 enzyme solution was 4 mg / mL.

[0042] Specific activity calculation: Specific activity refers to the number of enzyme activity units per unit weight of protein, expressed in U / mg. Formula for specific activity calculation: Specific activity (U / mg) = Enzyme activity (U / mL) / Protein content (mg / mL).

[0043] Calculation results: Under optimal reaction conditions (40 ℃, pH 8.0, 3% NaCl, 0.01 mol / L calcium phytate solution), the enzyme activity was measured to be 984.8 U / mL, and the protein content was 4 mg / mL. The specific activity of the heterologously expressed phytase BPP651 obtained in this invention was calculated to be 246.2 U / mg.

[0044] Example 6: Determination of the enzymatic properties of recombinant phytase BPP651; (1) Effect of reaction temperature on enzyme activity: In a reaction system of 3% NaCl and 150 µL of 0.01 mol / L calcium phytate solution (pH 8.0), enzyme activity was measured at 0, 15, 30, 40, 50, and 60 °C. The results showed that the optimal reaction temperature was 40 °C, with an enzyme activity of 976 U / mL and a specific activity of 244 U / mg. Figure 4 ).

[0045] (2) Effect of reaction pH on enzyme activity: In a reaction system containing 3% NaCl and 0.01 mol / L calcium phytate solution at 40 ℃, enzyme activity was measured at pH 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0. The results showed that the optimal pH was 8.0, with an enzyme activity of 974.4 U / mL and a specific activity of 243.6 U / mg. Figure 5 ).

[0046] (3) Effect of NaCl concentration on enzyme activity: Enzyme activity was measured at NaCl concentrations of 0%, 3%, 6%, 12%, 18%, and 23% in a reaction system containing 0.01 mol / L calcium phytate solution at 40 ℃. The results showed that the optimal NaCl concentration was 3.0%, with an enzyme activity of 984.8 U / mL and a specific activity of 246.2 U / mg. Figure 6 ).

Claims

1. A type of Frilanella bacteria derived from Arctic deep-sea sediments ( Vreelandella sp. 379-1, characterized in that, This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 22, 2025, with accession number CGMCC NO.37180.

2. The application of the *Frederick* 379-1 as described in claim 1 in the degradation of phytic acid and the preparation of feed additives for aquaculture animals.

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