Citrobacter freundii and application thereof

Abstract

The invention discloses citrobacter freundii and application thereof, the citrobacter freundii is excellent in performance and capable of producing hyaluronidase at high yield, and the obtained hyaluronidase is high in enzyme activity and excellent in performance and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a novel Citrobacter freundii, and also to the use of this bacterium to obtain hyaluronidase through fermentation. Background Technology

[0002] Hyaluronidase is a type of glycosidase that can degrade hyaluronic acid and some glycosaminoglycans. Based on differences in origin, catalytic mechanism, and substrate specificity, hyaluronidases are classified into three categories: The first category consists of microbial hyaluronidases (EC 4.2.2.1), which cleave the β-1,4 glycosidic bond of hyaluronic acid via a β-racemic reaction, producing unsaturated oligosaccharides with an N-acetylglucosamine reducing end; the second category, represented by hyaluronidase (EC 3.2.1.36), mainly originates from the salivary glands of leeches and belongs to the endo-β-glucuronidase family, capable of hydrolyzing the β-1,3 glycosidic bond of hyaluronic acid, producing saturated oligosaccharides with a D-glucuronic acid reducing end; the third category consists of endo-β-N-acetylglucosamineases (EC 3.2.1.35), mainly derived from the testes of mammals and animal venom, capable of hydrolyzing the β-1,4 glycosidic bond of hyaluronic acid, producing saturated oligosaccharides with an N-acetylglucosamine reducing end. Compared to animal-derived enzymes, microbial enzymes are characterized by high yield, high activity, diverse properties, and ease of heterologous recombination expression. Therefore, screening microorganisms that can produce hyaluronidase with high enzyme activity and novel hyaluronidases with excellent properties is of great research significance. Summary of the Invention

[0003] In response to the problems existing in the prior art, the present invention screened and obtained a high-performance strain of Citrobacter freundii. This strain can ferment and produce hyaluronidase, and the obtained hyaluronidase has high enzyme activity and excellent performance.

[0004] This invention provides a high-performance strain of Citrobacter freundii (Citrobacter freundii). Citrobacter freundii The strain, named ZQF03, was obtained through environmental screening by adjusting the ionic strength of the hyaluronic acid hydrolysate. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 1.61949.

[0005] The present invention also provides a microbial agent comprising the above-mentioned Citrobacter freundii ( Citrobacter freundii ZQF03. This bacterial agent may contain only Citrobacter freundii ( Citrobacter freundii ZQF03 is a microorganism, and may also contain other microorganisms and / or excipients.

[0006] Furthermore, the bacterial agent can be either a solid formulation or a liquid formulation.

[0007] The present invention also provides the above-mentioned Citrobacter freundii ( Citrobacter freundii The use of ZQF03 or bacterial agent in the preparation of hyaluronidase. This *Citrobacter freundii* CGMCC No. 1.61949 can be used to ferment and produce hyaluronidase.

[0008] This invention also provides a method for preparing hyaluronidase, the method comprising using the above-mentioned Citrobacter freundii ( Citrobacter freundii The steps for producing hyaluronidase by fermentation of ZQF03.

[0009] Furthermore, the preparation of the hyaluronidase specifically includes the following steps: preparing the above-mentioned Citrobacter freundii (… Citrobacter freundii ZQF03 was used for seed culture and fermentation culture. The resulting fermentation broth was centrifuged and the supernatant was collected to prepare an enzyme solution containing hyaluronidase.

[0010] Furthermore, a plate culture step is included before seed culture.

[0011] Furthermore, the culture medium for the plate culture consists of: 5-15 g / L peptone, 5-10 g / L yeast extract, 5-10 g / L sodium chloride, 10-20 g / L agar powder, and water as the remainder.

[0012] Specifically, all culturable Citrobacter freundii ( Citrobacter freundii All plate culture media are applicable, and no further limitations are made here.

[0013] Furthermore, the temperature for plate culture is 25℃ to 42℃, for example, 25℃, 26℃, 27℃, 28℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, or 42℃.

[0014] Furthermore, the plate culture time is 18 to 24 hours, for example, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

[0015] Furthermore, the temperature for seed cultivation is 25℃ to 42℃, preferably 28℃ to 40℃, for example, 25℃, 26℃, 27℃, 28℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, and 42℃.

[0016] Furthermore, the seed culture time is 4h to 20h, preferably 7h to 15h, for example, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, or 20h.

[0017] Furthermore, the seed culture medium consists of: 5-15 g / L peptone, 5-10 g / L yeast extract, 5-10 g / L sodium chloride, and water to balance.

[0018] Specifically, all culturable Citrobacter freundii ( Citrobacter freundii All seed culture media are applicable, and no further limitations are made here.

[0019] Furthermore, the fermentation culture temperature is 25℃~42℃, preferably 28℃-40℃, for example, it can be 25℃, 26℃, 27℃, 28℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃.

[0020] Furthermore, the fermentation culture time is 12 h to 36 h, preferably 15 h to 25 h, for example, it can be 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, or 36 h.

[0021] Furthermore, the fermentation medium consists of: 5-15 g / L peptone, 10-20 g / L yeast extract, 1-5 g / L K2HPO4·3H2O, 0.5-2 g / L MgSO4·7H2O, 0.5-2 g / L hyaluronic acid, and water as the remainder.

[0022] Specifically, all fermentation media capable of culturing Citrobacter freundii are applicable, without further limitations.

[0023] Furthermore, the molecular weight of the hyaluronic acid in the fermentation medium is 200 kDa - 1500 kDa, for example, it can be 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, or 1500 kDa.

[0024] Furthermore, the inoculum amount during fermentation is 1wt% to 10wt%, preferably 4-10wt%, for example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0025] Preferably, both seed culture and fermentation culture are carried out under stirring.

[0026] Furthermore, the method includes a step of purifying the enzyme solution containing hyaluronidase to obtain hyaluronidase. The purification method includes, but is not limited to, one or more combinations of operations such as salting out with ammonium sulfate, ultrafiltration, and chromatography.

[0027] The present invention also provides a hyaluronidase, which is produced by Citrobacter freundii (Citrobacter freundii) Citrobacter freundii ZQF03 is obtained by fermentation, preferably prepared according to the above method.

[0028] Furthermore, the suitable temperature for the hyaluronidase is 25-42℃, for example, it can be 25℃, 26℃, 27℃, 28℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, preferably 38℃.

[0029] Furthermore, the suitable pH of the hyaluronidase is 4.5-5.5, for example, it can be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5, preferably 4.5.

[0030] The present invention also provides the application of the above-mentioned hyaluronidase in the preparation of hyaluronic acid or its salts of different molecular weights.

[0031] This invention has independently screened and obtained a strain of Citrobacter freundii. This bacterium has excellent performance, can produce high levels of hyaluronidase, and the hyaluronidase obtained has high enzyme activity and excellent performance.

[0032] Preservation Information The Citrobacter freundii described in this invention ( Citrobacter freundii ZQF03 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No.1.61949, deposited on October 21, 2025, at Institute of Microbiology, Chinese Academy of Sciences, No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0033] Figure 1 A plate screening diagram showing 14 single colonies; Figure 2 The colony morphology of Citrobacter freundii ZQF03 strain on solid culture medium; Figure 3 The graph shows the results of the pH measurement for hyaluronidase. Figure 4 This is a graph showing the results of the optimal temperature determination for hyaluronidase. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present invention, and are not intended to limit the present invention.

[0035] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The invention is further described below with reference to specific embodiments, but is not intended to limit the scope of the invention.

[0036] Example 1: Strain Screening and Identification Process The enzymatic digestion system was set to 2L, and the hyaluronidase enzyme solution of recombinant *Sinomenis serratus* (obtained by the method described in CN114350691B) was diluted with pure water to a final volume of 1.0 × 10⁻⁶. 5 Add sodium acetate powder to the enzymatic hydrolysis system until the conductivity reaches 688 μS / cm, and adjust the pH to 5.5 with acetic acid. Add 200 g of high molecular weight HA (1000-1300 kDa), control the water bath temperature at 38℃, and carry out enzymatic hydrolysis for 24 h at high stirring speed.

[0037] Take an appropriate amount of the above-mentioned enzymatic hydrolysate and dilute it with water in a gradient to 10. -5 10 -6 10 -7 10 -8 Five concentrations were prepared and plated onto LB agar plates, incubated overnight at 37°C. Well-grown single colonies were picked and streaked onto selection medium. Each single colony was plated twice, one for selection and the other as a backup. Incubation was carried out at 37°C for 8-12 hours. The plates were then covered with a suitable amount of 2 mol / L glacial acetic acid, incubated for 10 minutes, and single colonies with a clear transparent zone were selected. Figure 1 As shown, 14 single colonies were screened, of which 2... # and 3 # The presence of a clear zone in the strain indicates that these two single colonies are capable of effectively secreting hyaluronidase to degrade hyaluronic acid.

[0038] The components of the screening medium were: 10 g / L peptone, 10 g / L yeast extract, 2 g / L K2HPO4·3H2O, 1 g / L MgSO4·7H2O, 1 g / L hyaluronic acid (molecular weight 1000 kDa - 1300 kDa), 20 g / L agar powder, and 10 g / L BSA.

[0039] 2 on the tablet # 3 # Colonies were inoculated separately into LB liquid medium and incubated overnight at 37°C. The OD values ​​of the two bacterial cultures were then adjusted using sterile purified water. 600 Adjust to the same value, centrifuge, collect the supernatant, and perform enzyme activity detection. The results showed that 2 # The enzyme activity was 2201 U / mL, 3 # The enzyme activity was 9527 U / mL, 3 # The enzyme activity of the strain is much higher than 2 # strain, therefore stored for 3 # strains.

[0040] 3 # Solid culture medium plates of the strain ( Figure 2 Observations and identification were conducted, and the results showed that the colonies of this strain on hyaluronic acid solid medium plates were raised, milky white in color, with a smooth and moist surface, and easy to pick up. Under an optical microscope, the cells appeared as short rods. This strain is a Gram-negative rod-shaped bacterium, without capsules, spores, or flagella.

[0041] 3 # Single colonies of the strain were inoculated into 5 mL of LB liquid medium and incubated overnight at 37°C. Genomes were extracted using the Tiangen kit.

[0042] Gene amplification was performed using 16S rDNA primers, and the amplified products were sequenced. The universal primers used for gene amplification are: Primer F: 5'-AGAGTTTGATCCTGGCTCAG-3' Primer R: 5'-GGTTACCTTGTTACGACTT-3' The reaction system used for gene amplification is as follows, with a total volume of 50 μL.

[0043] PCR process: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 51℃ annealing for 30 s, 72℃ extension for 90 s, 34 cycles, 72℃ extension for 15 min, 4℃ incubation.

[0044] Sequencing results showed that the 16S rDNA sequence of this strain was 1447 bp in length. Sequence BLAST alignment in the NCBI database revealed that the 16S rDNA sequence of this strain shared over 99% homology with relevant sequences of *Citrobacter freundii*, ultimately classifying it as *Citrobacter freundii*. This strain was named ZQF03 and deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 1.61949.

[0045] Example 2: Production of hyaluronidase by fermentation of Citrobacter freundii ZQF03 (1) Take Citrobacter freundii ZQF03 and culture it on a plate to obtain activated single colonies; (2) Inoculate a single colony into a sterilized seed culture medium and incubate at 38°C and 220 rpm for 8 hours to obtain a seed culture. (3) Inoculate the seed liquid into the sterilized fermentation medium at an inoculation rate of 5wt%, and culture it at 38℃ and 220 rpm for 16h to obtain a bacterial solution containing hyaluronidase.

[0046] The culture medium for plate culture consists of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder, and water as the remainder.

[0047] The seed culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and water to balance.

[0048] The fermentation medium consists of: 10 g / L peptone, 10 g / L yeast extract, 2 g / L K2HPO4·3H2O, 1 g / L MgSO4·7H2O, 1 g / L hyaluronic acid (molecular weight 1000 kDa - 1300 kDa), and water as the balance.

[0049] (4) Post-treatment of the above-mentioned bacterial solution containing hyaluronidase is performed as follows: (i) Centrifuge the bacterial solution containing hyaluronidase obtained in step (3) to remove the bacterial cells. The centrifugation conditions are: 4°C, 9000 rpm for 5 min. (ii) Take the supernatant of the centrifuged liquid, which is the crude hyaluronic acid enzyme solution.

[0050] Example 3: Production of hyaluronidase by fermentation of Citrobacter freundii ZQF03 (1) Take Citrobacter freundii ZQF03 for plate culture to obtain plate culture; (2) Inoculate the plate culture into the sterilized seed culture medium and incubate at 30℃ and 220 rpm for 12 h to obtain the seed liquid; (3) The seed liquid was inoculated into the sterilized fermentation medium at an inoculation rate of 8wt%, and cultured at 30℃ and 220 rpm for 20h to obtain a bacterial solution containing hyaluronidase.

[0051] The culture medium for plate culture consists of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder, and water as the remainder.

[0052] The seed culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and water to balance.

[0053] The fermentation medium consists of: 10 g / L peptone, 10 g / L yeast extract, 2 g / L K2HPO4·3H2O, 1 g / L MgSO4·7H2O, 1 g / L hyaluronic acid (molecular weight 1000 kDa - 1300 kDa), and water as the balance.

[0054] (4) Post-treatment of the above-mentioned bacterial solution containing hyaluronidase is performed as follows: (i) Take the bacterial solution containing hyaluronidase obtained in step (3) and centrifuge to remove the bacterial cells. The centrifugation conditions are: 4℃, 9000 rpm for 5 min. (ii) Take the supernatant of the centrifuged liquid, which is the crude hyaluronic acid enzyme solution.

[0055] Example 4: Production of hyaluronidase by fermentation of Citrobacter freundii ZQF03 (1) Take Citrobacter freundii ZQF03 for plate culture to obtain plate culture; (2) Inoculate the plate culture into the sterilized seed culture medium and incubate at 35℃ and 220 rpm for 10 h to obtain the seed liquid; (3) Inoculate the seed liquid into the sterilized fermentation medium at an inoculation rate of 2wt%, and culture it at 30℃ and 220 rpm for 18h to obtain a bacterial solution containing hyaluronidase.

[0056] The culture medium for plate culture consists of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder, and water as the remainder.

[0057] The seed culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and water to balance.

[0058] The fermentation medium consists of: 10 g / L peptone, 10 g / L yeast extract, 2 g / L K2HPO4·3H2O, 1 g / L MgSO4·7H2O, 1 g / L hyaluronic acid (molecular weight 1000 kDa - 1300 kDa), and water as the balance.

[0059] (4) Post-treatment of the above-mentioned bacterial solution containing hyaluronidase is performed as follows: (i) Take the bacterial solution containing hyaluronidase obtained in step (3) and centrifuge to remove the bacterial cells. The centrifugation conditions are: 4℃, 9000 rpm for 5 min. (ii) Take the supernatant of the centrifuged liquid, which is the crude hyaluronic acid enzyme solution.

[0060] Comparative Example 1: Production of hyaluronidase by fermentation of Citrobacter freundii (CGMCC NO.1.10604) The crude hyaluronic acid enzyme solution was produced by fermentation according to the method in Example 2, except that the strain used was Citrobacter freundii CGMCC NO.1.10604, which came from the China General Microbiological Culture Collection Center.

[0061] Comparative Example 2: Production of hyaluronidase by fermentation of Citrobacter freundii (CGMCC NO.1.10629) The crude hyaluronic acid enzyme solution was produced by fermentation according to the method in Example 2, except that the strain used was Citrobacter freundii CGMCC NO. 1.10629, which came from the China General Microbiological Culture Collection Center.

[0062] Example 5: Determination of enzyme activity in crude hyaluronic acid enzyme solution 1. Preparation of glucose standard curve: Add 0 μL, 50 μL, 75 μL, 100 μL, 150 μL, 175 μL, and 200 μL of glucose standard solution (2 mg / mL) to 2 mL DNS solution (Solepro), respectively. Add water to make up to 3 mL, boil in a boiling water bath for 10 min, cool to room temperature, and then add water to make up to 10 mL. Measure the absorbance at 540 nm. Prepare a standard curve with absorbance as the abscissa and glucose mass concentration as the ordinate.

[0063] 2. Preparation of hyaluronic acid solution: Add 14.705g of sodium citrate to 1L of water and stir until completely dissolved to obtain a 50mM sodium citrate solution. Add 10.52g of citric acid monohydrate to 1L of water and stir until completely dissolved to obtain a 50mM citric acid solution. Slowly add the 50mM citric acid solution to the 50mM sodium citrate solution while adjusting the pH until the pH reaches 5.5, thus obtaining a citric acid buffer solution with a concentration of 50 mmol / L and a pH of 5.5.

[0064] Add 200 mg of hyaluronic acid with a molecular weight of 1000 kDa-1300 kDa to 100 ml of citrate buffer and stir until completely dissolved to obtain a hyaluronic acid solution with pH 5.5.

[0065] 3. Mix 800 μL of hyaluronic acid solution with 100 μL of crude hyaluronic acid enzyme solution (Examples 2-4 and Comparative Examples 1-2), add citrate buffer to a final volume of 1 mL, and react in a 38°C water bath for 15 min. Immediately boil for 2 min to terminate the reaction, and cool to room temperature. Add 1 mL of the reaction solution to 2 mL of DNS solution (Solepro), boil in a water bath for 10 min, cool to room temperature, and then add water to a final volume of 10 mL. Measure the absorbance at 540 nm to obtain the sample absorbance value.

[0066] Hyaluronidase solution (Examples 2-4 and Comparative Examples 1-2) was boiled for 5 min to inactivate the enzyme. After cooling to room temperature, 800 μL of hyaluronic acid solution was mixed with 100 μL of the inactivated crude hyaluronic acid enzyme solution (Examples 2-4 and Comparative Examples 1-2), and citrate buffer was added to bring the volume to 1 mL. The mixture was then reacted in a 38°C water bath for 15 min. The reaction was immediately stopped by boiling for 2 min, and then cooled to room temperature. 1 mL of the reaction solution was added to 2 mL of DNS solution (Solepro), boiled in a water bath for 10 min, cooled to room temperature, and then water was added to bring the volume to 10 mL. The absorbance was measured at 540 nm to obtain the absorbance value for the blank control.

[0067] Subtract the absorbance of the blank control from the absorbance of the sample, and substitute the difference into the standard curve from step 1 to obtain the difference in reducing sugar concentration (μg / mL) for the glucose reducing equivalent. Multiply the reducing sugar difference by 4 to obtain the hourly conversion value, divide it by the enzyme dosage (100μL) multiplied by 1000, and obtain the enzyme activity per milliliter. The formula for calculating enzyme activity (U / mL) is: Enzyme activity (U / mL) = (difference in reducing sugar concentration of samples × 4 × 1000) / 100.

[0068] The enzyme activities of hyaluronidase in each example and comparative example are shown in Table 1 below: Table 1 As can be seen from Table 1, the hyaluronidase produced by Citrobacter freundii of the present invention has high enzyme activity, and the yield of hyaluronidase produced using the strain of the present invention is high.

[0069] Example 6: Determination of the optimal pH for enzymes 1. Preparation of citric acid and hyaluronic acid solutions at different pH values: Add 14.705g of sodium citrate to 1L of water and stir until completely dissolved to obtain a 50mM sodium citrate solution. Add 10.52g of citric acid monohydrate to 1L of water and stir until completely dissolved to obtain a 50mM citric acid solution.

[0070] Slowly add 50 mM citric acid solution to 50 mM sodium citrate solution, adjusting the pH as you add, until the pH values ​​are 4.5, 5.0, 5.5, 6.0, and 6.5, respectively, to obtain citric acid buffer solutions with a concentration of 50 mmol / L and pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5.

[0071] 200 mg of hyaluronic acid with a molecular weight of 1000 kDa-1300 kDa was added to 100 ml of citrate buffer solution with a concentration of 50 mmol / L and pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5, respectively. The solution was stirred until completely dissolved to obtain hyaluronic acid solutions with pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5.

[0072] 2. Mix 800 μL of hyaluronic acid solutions with different pH values ​​(4.5, 5.0, 5.5, 6.0, 6.5) with 100 μL of crude hyaluronic acid enzyme solution (Example 4), and then add citric acid solutions with pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5 to a final volume of 1 mL. React at 38°C for 15 min, then boil for 2 min to immediately terminate the reaction, and cool to room temperature. Add 1 mL of the reaction solution to 2 mL of DNS solution (Solepro), boil in a water bath for 10 min, cool to room temperature, and then add water to a final volume of 10 mL. Measure the absorbance at 540 nm to obtain the sample absorbance value.

[0073] Hyaluronidase solution (Example 4) was boiled for 5 min to inactivate it, and then cooled to room temperature. 800 μL of hyaluronic acid solutions with different pH values ​​(4.5, 5.0, 5.5, 6.0, 6.5) were mixed with 100 μL of the inactivated crude hyaluronic acid enzyme solution (Example 4), and then brought to a final volume of 1 mL with citrate buffer solutions at pH 4.5, 5.0, 5.5, 6.0, and 6.5, respectively. The mixture was then reacted in a 38°C water bath for 15 min. The reaction was immediately stopped by boiling for 2 min, and then cooled to room temperature. 1 mL of the reaction solution was added to 2 mL of DNS solution (Solepro), boiled in a water bath for 10 min, cooled to room temperature, and then water was added to a final volume of 10 mL. The absorbance was measured at 540 nm to obtain the absorbance value for the blank control.

[0074] Subtract the absorbance of the blank control from the absorbance of the sample, and substitute the difference into the standard curve of Example 5 to obtain the difference in reducing sugar concentration (μg / mL) of glucose reducing equivalent. Calculate the enzyme activity according to the following formula.

[0075] Enzyme activity (U / mL) = (Difference in reducing sugar concentration of samples × 4 × 1000) / 100 Using the hyaluronidase solution (Example 4) at a reaction pH of 4.5 as 100% enzyme activity, the relative enzyme activities at reaction pH conditions of 5.0, 5.5, 6.0, and 6.5 were calculated. The results are as follows: Figure 3 As shown in the figure, hyaluronidase exhibits good enzyme activity in the pH range of 4.5-5.5, with the optimal pH being 4.5.

[0076] Example 7 Determination of the optimal temperature for enzymes 800 μL of pH 5.5 hyaluronic acid solution was mixed with 100 μL of crude hyaluronic acid enzyme solution from Example 4, and the volume was brought up to 1 mL with pH 5.5 citric acid solution. The mixture was reacted in water baths at 25°C, 30°C, 35°C, 38°C, and 45°C for 15 min each, and then the reaction was stopped immediately by boiling for 2 min. The mixture was then cooled to room temperature. 1 mL of the reaction solution was added to 2 mL of DNS solution (Solepro), boiled in a water bath for 10 min, cooled to room temperature, and then water was added to bring the volume up to 10 mL. The absorbance was measured at 540 nm to obtain the sample absorbance value.

[0077] Hyaluronidase solution (Example 4) was boiled for 5 min to inactivate it, cooled to room temperature, and 800 μL of pH 5.5 hyaluronic acid solution was mixed with 100 μL of the inactivated crude hyaluronic acid enzyme solution (Example 4). The mixture was then brought to a final volume of 1 mL with pH 5.5 citrate buffer. The mixture was reacted in water baths at 25°C, 30°C, 35°C, 38°C, and 45°C for 15 min each. The reaction was immediately stopped by boiling for 2 min, and then cooled to room temperature. 1 mL of the reaction solution was added to 2 mL of DNS solution (Solepro), boiled in a water bath for 10 min, cooled to room temperature, and then water was added to a final volume of 10 mL. The absorbance was measured at 540 nm to obtain the absorbance value for the blank control.

[0078] Subtract the absorbance of the blank control from the absorbance of the sample, and substitute the difference into the standard curve of Example 5 to obtain the difference in reducing sugar concentration (μg / mL) of glucose reducing equivalent. Calculate the enzyme activity according to the following formula.

[0079] Enzyme activity (U / mL) = (Difference in reducing sugar concentration of samples × 4 × 1000) / 100 Using the enzyme activity measured at a reaction temperature of 38°C with hyaluronidase solution (Example 4) as 100%, the relative enzyme activity at reaction temperatures of 25°C, 30°C, 35°C, 38°C, and 45°C was calculated.

[0080] The results are as follows Figure 4As shown in the figure, hyaluronidase has good enzyme activity at 25-38℃, with the optimal temperature being 38℃.

Claims

1. A type of Citrobacter freundii ( Citrobacter freundii ZQF03, characterized by: The accession number is CGMCCNo.1.61949.

2. A microbial agent, characterized in that: Contains the Citrobacter freundii as described in claim 1 ( Citrobacter freundii ZQF03.

3. The Citrobacter freundii as described in claim 1 ( Citrobacter freundii The application of ZQF03 or the bacterial agent according to claim 2 in the preparation of hyaluronidase.

4. A method for preparing hyaluronidase, characterized in that: Including the use of Citrobacter freundii as described in claim 1 ( Citrobacter freundii The steps for producing hyaluronidase by fermentation of the microbial agent as described in ZQF03 or claim 2.

5. The preparation method according to claim 4, characterized in that: include: The Citrobacter freundii as described in claim 1 ( Citrobacter freundii ZQF03 was used for seed culture and fermentation culture. The resulting fermentation broth was centrifuged and the supernatant was collected to prepare an enzyme solution containing hyaluronidase.

6. The preparation method according to claim 5, characterized in that: The seed culture temperature is 25-42℃, and preferably, the culture time is 4-20h.

7. The preparation method according to claim 4 or 5, characterized in that: The fermentation culture temperature is 25-42℃, preferably, the culture time is 12-36h, and more preferably, the inoculum amount is 1-10wt.

8. The preparation method according to claim 5, characterized in that: in Seed culture includes a plate culture step; Preferably, after centrifugation and obtaining the supernatant, a purification step is also included; Preferably, the purification method includes one or more of the following: salting out with ammonium sulfate, ultrafiltration, and chromatography.

9. Hyaluronidase prepared according to any one of claims 4-8.

10. The use of the hyaluronidase prepared by any of the preparation methods of claims 4-8 and / or the hyaluronidase of claim 9 in the preparation of hyaluronic acid or its salts of different molecular weights.

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