A mutant strain of vibrio fluvialis producing alginate lyase and application thereof
Patent Information
- Application Number
- CN202310494025.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-26
- Publication Date
- 2026-09-29
- Estimated Expiration
- 2043-04-26
AI Technical Summary
[0003]除褐藻外，少数细菌如假单胞菌属(Pseudomonas)、固氮菌属(Azotobacter)、弧菌属(Vibrio)也会产生褐藻胶裂解酶；由于现有微生物的产酶量少、酶活低，限制了其进一步开发应用，因此寻找新的高效产酶菌、提高菌株发酵酶活力是迫切需要解决的问题
[0022]有益效果：本发明提供了一株高产褐藻胶裂解酶的弗氏弧菌C3-2，弗氏弧菌C3-2所产生褐藻胶裂解酶的酶活力较野生菌株提升了40％以上，且褐藻胶裂解酶对碱性环境的耐受性更强，同时遗传特性稳定，具有良好的工业发展前景。
Smart Images
Figure CN116606769B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial mutagenesis breeding technology, specifically to a mutant strain of Vibrio freundii that produces alginate lyase and its applications. Background Technology
[0002] Alginate is a high-molecular-weight polysaccharide found in the cell walls of brown algae. It is primarily composed of β-D-mannuronate acid (M) and its C5 epimer α-L-guluronic acid (G), polymerized through random arrangement of 1,4-glycosidic bonds. Currently, alginate has become a sustainable resource with production second only to cellulose, possessing significant application value. Alginate lyases are key enzymes in the preparation of alginate oligosaccharides. Alginate lyases neutralize the 1,4-glycosidic bonds between monomers through β-elimination reactions. Most microbial alginate lyases possess the ability to simultaneously degrade both uronic acid fragments. The unsaturated bonds generated during degradation may improve certain physicochemical properties of alginate oligosaccharides, thereby enhancing their bioactivity.
[0003] Besides brown algae, a few bacteria, such as Pseudomonas, Azotobacter, and Vibrio, also produce alginate lyase. However, the low enzyme production and activity of existing microorganisms limit their further development and application. Therefore, finding new, highly efficient enzyme-producing bacteria and improving the fermentation enzyme activity of strains are urgent problems that need to be solved.
[0004] Mutagenesis is one of the effective methods to improve enzyme production levels in microbial fermentation and has become an important technique in microbial mutagenesis breeding. Atmospheric and ambient temperature plasma (ARTP) mutagenesis breeding refers to the generation of highly active plasma jets under ambient temperature and pressure, causing diverse damage to cells. These jets are rich in high-energy chemically active particles that can damage the genetic material of microorganisms, alter cell membrane structure and permeability, and change protein structure, thereby inducing gene mutations and leading to changes in microbial traits and metabolism. Diethyl sulfate (DES) is a chemical mutagen that can induce changes in the base pairs of microorganisms, causing gene mutations. Both ARTP and DES mutagenesis breeding are efficient and rapid microbial genome mutagenesis breeding techniques, which are simple to operate, low in cost, and highly efficient. Summary of the Invention
[0005] To address the aforementioned issues, this invention provides a Vibrio furnissii mutant strain that produces alginate lyase. The applicant used Vibrio furnissii C1 as the starting strain and, after four ARTP mutagenesis and two DES mutagenesis, screened for a mutant strain with significantly increased alginate lyase production. The enzyme activity was 41% higher than the wild-type strain, and the genetic characteristics were stable. This strain was named Vibrio furnissii C3-2.
[0006] The technical solution of this invention is as follows:
[0007] A strain of Vibrio furnissii C3-2 was deposited on March 27, 2023, at the China Center for Type Culture Collection (CCTCC) with accession number M2023411, located at Wuhan University, Wuhan, China.
[0008] The Vibrio freundii C3-2 strain was obtained by screening Vibrio freundii C1 as the starting strain through ARTP mutagenesis and DES mutagenesis.
[0009] Preferably, the method for culturing Vibrio flexneri C3-2 includes the following steps:
[0010] After streaking and purifying Vibrio freundii C3-2, single colonies were picked and inoculated into seed culture medium and incubated at 28±2℃ and 150~200r / min to obtain Vibrio freundii C3-2 bacterial suspension.
[0011] Preferably, the seed culture medium comprises: 5.0g peptone, 1.0g yeast extract, 2.0g sodium alginate, 0.01g ferric phosphate, 1L seawater buffer solution, pH 7.4-7.6; wherein the seawater buffer solution comprises: 30g sea salt, 1L deionized water.
[0012] The Vibrio freundii C3-2 strain can be used to produce alginate lyase, and the yield of alginate lyase is significantly higher than that of the original strain Vibrio freundii C1.
[0013] Preferably, the method for producing alginate lyase from Vibrio freundii C3-2 includes the following steps:
[0014] The above-mentioned Vibrio freundii C3-2 bacterial suspension was inoculated into the fermentation medium at an inoculation amount of 1-5% of the fermentation medium volume. The fermentation was carried out at a constant temperature of 28±2℃ and 150-200r / min to obtain the fermentation broth. The supernatant was obtained by centrifugation, and the supernatant was the crude alginate lyase solution.
[0015] Preferably, the fermentation medium comprises: 1.5g potassium dihydrogen phosphate (KH2PO4), 6.0g peptone, 4.0g sodium alginate, 0.01g ferrous sulfate (Fe2SO4), 0.01g magnesium sulfate heptahydrate (MgSO4·7H2O), 1L seawater buffer, pH 7.0; wherein the seawater buffer comprises: 30g sea salt and 1L deionized water.
[0016] The optimal reaction temperature for the alginate lyase produced by Vibrio freundii C3-2 in this invention is 20°C, and it can exhibit more than 60% enzyme activity in the range of 15–40°C. The optimal reaction pH is 8.0, and it can exhibit more than 60% enzyme activity in substrate environments with a pH of 7.0–9.0. Furthermore, it can still exhibit about 80% enzyme activity in an alkaline environment with a pH of 9.0. This indicates that the alginate lyase produced by Vibrio freundii C3-2 has a stronger tolerance to alkaline environments.
[0017] The methods for ARTP mutagenesis and DES mutagenesis specifically include the following steps:
[0018] S1, Preparation of bacterial suspension: First, Vibrio freundii C1 was streaked and purified on a solid plate. Then, a single colony with good growth was picked and inoculated into a seed culture medium. After incubation to the logarithmic growth phase, the bacterial cells were collected by centrifugation, washed twice with an equal volume of seawater buffer, and then resuspended with an equal volume of seawater buffer and diluted to OD. 600 A bacterial suspension with a concentration of approximately 0.4;
[0019] S2, ARTP mutagenesis treatment: Take 10 μL of the bacterial suspension described in S1 and spread it evenly on a sterile small iron plate provided with the ARTP mutagenesis instrument; set the ARTP mutagenesis instrument power to 120W, airflow rate to 10 SLM (Standard Liter per Minute), cooling water circulation system to 20℃, and mutagenesis time to 20s; after mutagenesis, quickly remove the small iron plate with sterile pointed forceps and place it into a 1.5mL centrifuge tube containing 990μL of sterile seawater buffer. Vortex and mix for 1 min. After mixing, dilute with seawater buffer 10 μL in a laminar flow hood. 3 Take 100 μL and spread it evenly on a solid plate. Spread 3 solid plates for each time gradient as parallels. After the bacterial culture on the surface of the plate dries, seal it with sealing film and invert it in a constant temperature biochemical incubator. Incubate at 28℃ for 12 h and then carry out subsequent screening. The strain with the highest enzyme activity selected is used as the starting strain for the next round of ARTP mutagenesis and iterative mutagenesis is carried out.
[0020] S3, DES mutagenesis treatment: The screened ARTP mutagenesis strains were streaked and purified. After single colonies grew, they were inoculated into 20 mL of seed culture medium and cultured in a constant temperature shaker at 28℃ and 200 r / min until OD500. 600The value was approximately 1.0; 1 mL of seed culture was mixed with 1 mL of DES solution to form the reaction solution, and mutagenesis was induced for 20 min in a constant temperature shaker at 30℃ and 200 r / min. Then, 2 mL of sodium thiosulfate solution was quickly added as a terminator, and the reaction was terminated by vortexing. The reaction solution was diluted 10% with seawater buffer. 3 Take 100 μL and spread it evenly on a solid plate. Spread 3 solid plates for each time gradient as parallels. After the bacterial solution on the surface of the plate is dried, seal it with sealing film and invert it in a constant temperature biochemical incubator. After incubation at 28℃ for 12 h, screen to obtain strains with relatively high enzyme activity. Use this strain as the starting strain for the next round of DES mutagenesis and carry out iterative mutagenesis.
[0021] After four ARTP mutagenesis and two DES mutagenesis, the desired mutant strain Vibrio furnissii C3-2 was screened using a combination of plate screening and DNS enzyme activity assay. The enzyme activity of alginate lyase produced by Vibrio furnissii C3-2 was increased by about 41% compared with that of the wild strain Vibrio furnissii C1.
[0022] Beneficial effects: This invention provides a high-yield alginate lyase-producing Vibrio freundii C3-2 strain. The alginate lyase produced by Vibrio freundii C3-2 has an enzyme activity that is more than 40% higher than that of the wild strain, and the alginate lyase has stronger tolerance to alkaline environments. At the same time, it has stable genetic characteristics and has good prospects for industrial development. Attached Figure Description
[0023] Figure 1 The results of enzyme activity assays for the mutant strains obtained from the first round of ARTP mutagenesis and the starting strain Vibrio freundii C1;
[0024] Figure 2 The results of enzyme activity assays for mutant strains obtained from the second round of ARTP mutagenesis and starting strains 1-6 are shown.
[0025] Figure 3 The results of enzyme activity assays for the mutant strain obtained from the third round of ARTP mutagenesis and the starting strain 2-2 are shown.
[0026] Figure 4 The results of enzyme activity assays for the mutant strains obtained from the fourth round of ARTP mutagenesis and the starting strain 3-3 are shown.
[0027] Figure 5 The results of enzyme activity assays for the mutant strains obtained from the first round of DES mutagenesis and the starting strain C2 are shown.
[0028] Figure 6 The results of enzyme activity assays for the mutant strain obtained from the second round of DES mutagenesis and the starting strain C2-22 are shown.
[0029] Figure 7Line graphs showing the growth of the strain before and after the mutation;
[0030] Figure 8 Line graphs showing enzyme production of the strain before and after mutation;
[0031] Figure 9 Bar chart showing the enzyme activity of crude enzyme solution before and after mutation;
[0032] Figure 10 Line graphs showing enzyme activity at different reaction temperatures;
[0033] Figure 11 Line graphs showing enzyme activity at different reaction pH levels. Detailed Implementation
[0034] The following description is based on specific embodiments:
[0035] Example:
[0036] The wild strain used in this embodiment was Vibrio furnissii C1, which was obtained through previous screening in our laboratory. After 16S rDNA identification and comparison with data in the Gene Bank, it showed 99% similarity to many Vibrio sp. strains. The results showed that it was most closely related to Vibrio furnissii, and it was named Vibrio furnissii C1.
[0037] The experimental reagents used in this embodiment are as follows:
[0038] Seawater buffer (3% sea salt): 30g sea salt, 1L deionized water (ddH2O);
[0039] Seed culture medium: 5.0g peptone, 1.0g yeast extract, 2.0g sodium alginate, 0.01g ferric phosphate, 1L seawater buffer, pH 7.4-7.6;
[0040] Solid seed culture medium: 5.0g peptone, 1.0g yeast extract, 2.0g sodium alginate, 0.01g ferric phosphate, 1L seawater buffer, 15g agar powder, pH 7.4-7.6;
[0041] Screening medium: 5.0 g peptone, 1.0 g yeast extract, 5.0 g sodium alginate, 1 L seawater buffer, 15 g agar powder, pH 7.4–7.6;
[0042] Fermentation medium: potassium dihydrogen phosphate (KH2PO4) 1.5g, peptone 6.0g, sodium alginate 4.0g, ferrous sulfate (Fe2SO4) 0.01g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.01g, seawater buffer 1L, pH 7.0;
[0043] Mutagen: 1 mL diethyl sulfite, 99 mL ddH2O;
[0044] Terminating agent: 25g sodium thiosulfate, 75mL ddH2O.
[0045] I. Preparation of bacterial suspension:
[0046] First, Vibrio freundii C1 was streaked and purified on solid seed culture medium. Then, healthy single colonies were picked and inoculated onto the seed culture medium and cultured until the logarithmic growth phase. The cells were collected by centrifugation, washed twice with an equal volume of seawater buffer, and then resuspended with an equal volume of seawater buffer and diluted to OD200. 600 The bacterial suspension was 0.4.
[0047] II. ARTP mutagenesis treatment:
[0048] Take 10 μL of bacterial suspension and spread it evenly on a sterile small iron plate provided with the ARTP mutagenesis instrument. Set the ARTP mutagenesis instrument power to 120W, airflow rate to 10 SLM (Standard Liter per Minute), and cooling water circulation system to 20℃. Mutagenesis times were 0 s, 5 s, 10 s, 20 s, 30 s, and 45 s. After mutagenesis, quickly remove the small iron plate with sterile pointed forceps and transfer it to a 1.5 mL centrifuge tube containing 990 μL of sterile seawater buffer. Vortex and mix for 1 min. After mixing, dilute with seawater buffer 10 μL in a clean bench. 3 Take 100 μL and spread it evenly on a solid plate. Spread 3 solid plates for each time gradient as parallels. After the bacterial solution on the surface of the plate dries, seal it with sealing film and invert it in a constant temperature biochemical incubator. Incubate at 28℃ for 12 h and then carry out subsequent screening. The strain with the highest enzyme activity is selected as the starting strain for the next round of ARTP mutagenesis. Iterative mutagenesis is carried out. High-yielding strains of alginate lyase are screened by plate screening and DNS enzyme activity assay.
[0049] Determination of ARTP mutagenesis time: The lethality of the strain increased significantly with increasing mutagenesis time. The lethality reached approximately 67% after 5 seconds of mutagenesis, approximately 90% after 20 seconds, and as high as 99.9% after 40 seconds. Studies indicate that a positive mutation rate is relatively high when the mutagenesis lethality is around 90%, therefore 20 seconds is the optimal mutagenesis time.
[0050] ARTP mutagenesis iteration effect: An increasing number of iterations may have a cumulative effect on mutant strains. The optimal mutant strain obtained in the previous round of screening is used as the starting strain for the next round of ARTP mutagenesis, and iterative mutagenesis is performed in conjunction with the optimal mutagenesis time. Statistical analysis is performed on the enzyme activities of the mutant strains obtained from each subsequent mutagenesis rescreening and the starting strain. The screening process is as follows: Figures 1-4 As shown.
[0051] The enzyme activity assay results of the mutant strain obtained from the first round of ARTP mutagenesis and the starting strain Vibrio freundii C1 are as follows: Figure 1 As shown, the triangles represent the starting strains for each round of mutagenesis, and the circles represent the mutants with the highest enzyme activity screened after each round of mutagenesis. The mutants with the highest enzyme activity obtained in the first round of ARTP mutagenesis are 1-6, and the second round of ARTP mutagenesis is carried out using mutants 1-6 as the starting strains.
[0052] The enzyme activity assay results of the mutant strains obtained from the second round of ARTP mutagenesis and the starting strains 1-6 are as follows: Figure 2 As shown, the mutant strain with the highest enzyme activity obtained from the second round of ARTP mutagenesis was 2-2, and the third round of ARTP mutagenesis was carried out using mutant strain 2-2 as the starting strain.
[0053] The enzyme activity assay results of the mutant strain obtained from the third round of ARTP mutagenesis and the starting strain 2-2 are as follows: Figure 3 As shown, the mutant strain with the highest enzyme activity obtained from the third round of ARTP mutagenesis was 3-3, and the fourth round of ARTP mutagenesis was carried out using mutant strain 3-3 as the starting strain.
[0054] The enzyme activity assay results of the mutant strain obtained from the fourth round of ARTP mutagenesis and the starting strain 3-3 are as follows: Figure 4 As shown, the mutant strain with the highest enzyme activity obtained from the fourth round of ARTP mutagenesis was 4-20; Figure 4 It can be seen that after four rounds of iterative mutagenesis, the strain exhibited a "fatigue effect," and ARTP mutagenesis no longer resulted in a significant increase in enzyme activity. The fourth round of ARTP mutagenesis screened out the mutant strain 4-20 with the highest activity. Preliminary tests showed that the enzyme activity was about 15% higher than that of the starting strain Vibrio flexneri C1. It was named Vibrio flexneri C2, and Vibrio flexneri C2 was preserved and used as the starting strain for DES mutagenesis.
[0055] III. DES-induced mutagenesis treatment:
[0056] Preparation of bacterial suspension: The selected mutant strain Vibrio freundii C2 was streaked and purified. After single colonies grew, they were inoculated into 20 mL of seed culture medium and cultured in a constant temperature shaker at 28 °C and 200 r / min until OD200. 600 It is around 1.0;
[0057] DES mutagenesis: 1 mL of seed culture was mixed with 1 mL of 1% DES solution to prepare the reaction solution. Mutagenesis was induced at 30℃ and 200 rpm for 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, respectively. Then, 2 mL of 25% sodium thiosulfate was rapidly added as a terminator, and the mixture was vortexed to terminate the reaction. The reaction solution was diluted 10 μL with seawater buffer. 3Take 100 μL and spread it evenly on a solid plate. Spread 3 solid plates for each time gradient as parallels. After the bacterial solution on the surface of the plate is dried, seal it with sealing film and invert it in a constant temperature biochemical incubator. After incubation at 30℃ for 12 h, the strain with relatively high enzyme activity is screened to obtain the strain. This strain is used as the starting strain for the next round of DES mutagenesis. High-yielding strains of alginate lyase are screened by plate screening method and DNS enzyme activity assay.
[0058] Determination of DES mutagenesis time: The lethality rate was calculated by counting colonies at different mutagenesis times. The lethality rate was 87% when mutagenesis lasted 15 minutes, 96% when mutagenesis lasted 20 minutes, and 100% when mutagenesis lasted more than 25 minutes. The 20-minute mutagenesis time with a lethality rate of 96% was selected as the optimal mutagenesis time for DES mutagenesis.
[0059] DES mutagenesis iteration effect: The increase in iteration rounds may have a cumulative effect on the fermentation activity of mutant strains. The best mutant strain obtained in the previous round of screening is used as the starting strain for the next round of DES mutagenesis, and iterative mutagenesis is performed in conjunction with the optimal mutagenesis time. The enzyme activities of the mutant strains obtained from each subsequent mutagenesis rescreening are statistically compared with those of the starting strain. The screening process is as follows: Figures 5-6 As shown.
[0060] The enzyme activity assay results of the mutant strain obtained from the first round of DES mutagenesis and the starting strain Vibrio freundii C2 are as follows: Figure 5 As shown, the triangles represent the starting strains for each round of mutagenesis, and the circles represent the mutants with the highest enzyme activity screened after each round of mutagenesis. The mutant with the highest enzyme activity obtained in the first round of DES mutagenesis is C2-22. The second round of DES mutagenesis was carried out using mutant C2-22 as the starting strain.
[0061] The enzyme activity assay results of the mutant strain obtained from the second round of DES mutagenesis and the starting strain C2-22 are as follows: Figure 6 As shown, the mutant strain with the highest enzyme activity obtained from the second round of DES mutagenesis was C3-2. After two rounds of iterative mutagenesis, the strain exhibited a "fatigue effect," and DES mutagenesis no longer resulted in a significant increase in enzyme activity. The second round of DES mutagenesis screened out the C3-2 strain with the highest activity. Preliminary tests showed that the enzyme activity was about 20% higher than that of the starting strain, Vibrio furnissii C2, and it was named "Vibrio furnissii C3-2".
[0062] Vibrio furnissii C3-2 was deposited at the China Center for Type Culture Collection (CCTCC) on March 27, 2023, with accession number CCTCC No: M2023411, located at Wuhan University, Wuhan, China.
[0063] The operation steps of the plate screening method are as follows:
[0064] The strain was inoculated onto a screening medium plate and incubated at 28°C for 24 hours until a single colony grew. After treatment with CaCl2 solution and standing for 30 minutes, color development was observed. A certain range of sodium alginate degradation was formed around the colony, and a white hydrolysis zone could be observed with the naked eye. By observing the size of the hydrolysis zone, positive mutant strains with increased enzyme activity could be preliminarily screened.
[0065] The operation steps of the DNS enzyme activity assay are as follows:
[0066] The fermentation broth of the strain was centrifuged at 12000 r / min for 10 min at 4℃, and the supernatant was the crude enzyme solution. 2 mL of 0.5% (w / v, g / mL) sodium alginate substrate solution and 100 μL of the crude enzyme solution were added to a 25 mL colorimetric tube to construct the enzyme reaction system. The control group did not add enzyme solution. After incubating in a 30℃ water bath for 30 min, 2 mL of DNS reagent was added. The control group was supplemented with enzyme solution. The reaction was terminated by boiling in water for 5–10 min, cooled to room temperature with ice water, and then diluted to 25 mL with pure water. OD was measured. 550 The absorbance A at the control group was measured, and the OD was measured at the control group. 550 The absorbance B at a certain point is used to calculate enzyme activity as follows:
[0067] Enzyme activity (U / mL) = (AB) × 1000 × n / K / t, where:
[0068] AB — Absorbance of the reducing sugar produced;
[0069] n—the dilution factor of the enzyme solution;
[0070] t — enzyme reaction time;
[0071] K – Slope of the standard curve.
[0072] Experimental example:
[0073] (1) Enzyme production capacity assay
[0074] Mutations in bacterial strains can affect their growth status and enzyme production time. Slow growth and excessively long enzyme production times increase costs in industrial production or practical applications. Therefore, the growth rate and enzyme production time of a strain directly determine its practical value in industrial production. This experiment measured the growth rate and enzyme production time of the mutated strain. The specific operational steps are as follows:
[0075] The high-yielding mutant strain *Vibrio freundii* C3-2 obtained through screening was streaked and purified from the starting strain *Vibrio freundii* C1 and the ARTP mutant strain *Vibrio freundii* C2. Then, single colonies with good growth were picked and inoculated into seed culture medium and cultured at 28℃ and 200 rpm. 1 mL of the seed culture, cultured to the logarithmic growth phase, was inoculated into an Erlenmeyer flask containing 100 mL of sterile fermentation medium and fermented at 28℃ and 200 rpm. Samples were taken every two hours to determine the growth rate and enzyme activity of the strain. The growth trend is as follows: Figure 7 As shown, the enzyme production trend is as follows Figure 8 As shown.
[0076] Depend on Figure 7 It can be seen that the mutant strains *Vibrio flexneri* C2 and C3-2 reached the logarithmic growth phase at around 4 hours, with a greater growth rate than the original strain *Vibrio flexneri* C1. Furthermore, the growth rate of *Vibrio flexneri* C3-2 was significantly greater than that of *Vibrio flexneri* C1 during the 12–24 h period. Figure 8 It can be seen that after 20 hours of fermentation, the enzyme production of Vibrio flexneri C1 showed a decreasing trend, while the enzyme production capacity of the mutant strain continued to increase. Moreover, the enzyme production capacity of Vibrio flexneri C3-2 was higher than that of Vibrio flexneri C1.
[0077] (2) Determination of genetic stability of mutant strains
[0078] Since the stability of the newly screened strain is unknown, it is necessary to perform multiple subcultures to test its genetic stability. The specific operation steps are as follows: the high-yield mutant strain Vibrio freundii C3-2 obtained by screening was subcultured five times consecutively with the starting strain Vibrio freundii C1 and the ARTP mutant strain Vibrio freundii C2. The enzyme activity of their fermentation broth was tested under the same conditions, and the results are shown in Table 1.
[0079] Table 1. Results of fermentation enzyme activity experiments of mutant strains
[0080] 0 100 100 100 1 99.8±1.02 103.2±1.37 102.77±2.31 2 101.3±1.44 100.3±2.01 100.6±0.71 3 97.8±1.75 99.86±0.34 98.37±0.95 4 103.16±2.14 98.36±1.52 99.33±1.45 5 98.5±0.89 99.55±1.41 98.67±0.49
[0081] As shown in Table 1, the enzyme activity of each generation of fermentation broth was retained by more than 95% compared with the first generation during five consecutive passages. Therefore, the mutant strain Vibrio freundii C3-2 obtained by screening has good genetic stability and high industrial value.
[0082] (3) Enzyme activity assay
[0083] Under the premise of stable genetic inheritance of mutant strains, three strains with the same growth state—Vibrio freundii C1, the ARTP mutant Vibrio freundii C2, and Vibrio freundii C3-2—were selected for re-testing enzyme activity. First, streaking was performed for purification. Then, single colonies with good growth were picked and inoculated into seed culture medium and cultured at 28℃ and 200 rpm. 1 mL of the seed culture, cultured to the logarithmic growth phase, was inoculated into an Erlenmeyer flask containing 100 mL of sterile fermentation medium and fermented at 28℃ and 200 rpm for 24 hours. The fermentation broth was obtained, and the enzyme activity of the three strains was tested according to the DNS enzyme activity assay method described above. The results are as follows: Figure 9 As shown.
[0084] Among them, the enzyme activity of Vibrio flexneri C2 was 203 U / mL as determined by the DNS enzyme activity assay, which is 19.4% higher than that of the wild-type Vibrio flexneri C1 (170 U / mL). A genetically stable high-yield alginate lyase mutant strain, Vibrio flexneri C3-2, was obtained by two DES mutagenesis of Vibrio flexneri C2. The enzyme activity of Vibrio flexneri C3-2 reached 240 U / mL, which is 41.2% higher than that of the wild-type Vibrio flexneri C1.
[0085] (4) Enzymatic property determination
[0086] During mutation, the deletion or translocation of bases can lead to mutations in key amino acids that determine the enzymatic properties of a strain, thus altering the enzyme's enzymatic properties. Studying the stability of mutant strains' enzymes can prevent enzyme loss and waste caused by temperature and pH changes during enzymatic reactions, which is of great significance in industrial production and practical applications. To investigate the enzymatic properties of the mutant strain, this experiment streaked the selected mutant strain *Vibrio freundii* C3-2 with the starting strain *Vibrio freundii* C1 and the ARTP mutant strain *Vibrio freundii* C2 for purification. Then, single colonies with good growth were inoculated into seed culture medium and incubated at 28℃ and 200 rpm for 12 h until the logarithmic growth phase OD... 600 The concentration was approximately 1.0. 1 mL of seed culture was inoculated into an Erlenmeyer flask containing 100 mL of sterile fermentation medium. Fermentation was carried out at 28°C and 200 rpm for 24 hours on a shaker, followed by centrifugation. The supernatant was then used as the crude enzyme solution. The enzyme activities of each enzyme were measured at different reaction temperatures and pH values. The specific procedures are as follows:
[0087] ① Optimal temperature detection of enzyme: Using 0.5% (w / v, g / mL) sodium alginate solution prepared with phosphate buffer as substrate, 2 mL of sodium alginate substrate solution and 100 μL of crude enzyme solution were added to a 25 mL colorimetric tube. The control group did not add enzyme solution to construct the enzyme reaction system. The reaction was carried out at 20℃, 30℃, 40℃, 50℃ and 60℃ for 30 min respectively, and 2 mL of DNS reagent was added to each reaction. The control group was supplemented with enzyme solution. The reaction was terminated by boiling in water for 5 min, cooled to room temperature with ice water, and then diluted to 25 mL with pure water. The relative enzyme activity at different reaction temperatures was determined with the maximum enzyme activity as 100%.
[0088] The results are as follows Figure 10 As shown, the optimal reaction temperature for mutant strains Vibrio flexneri C2 and C3-2, as well as wild-type strain Vibrio flexneri C1, is 20℃. Furthermore, the enzymes can exhibit more than 60% activity in reactions between 15 and 40℃, but are almost inactivated at 60℃. This may be related to the fact that the experimental strains are Vibrio flexneri derived from low-temperature marine environments.
[0089] ② Optimal pH detection of the enzyme: Sodium alginate substrate solutions were prepared using phosphate buffer solutions of different pH values, resulting in substrate pH values of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. 2 mL of sodium alginate substrate solution and 100 μL of crude enzyme solution were added to 25 mL colorimetric tubes, respectively. The control group received no enzyme solution. The enzyme reaction system was constructed and reacted at 20 °C for 30 min. The relative enzyme activity at different reaction pH values was measured, with maximum enzyme activity defined as 100%.
[0090] The results are as follows Figure 11 As shown, the optimal reaction pH of the alginate lyase produced by the mutant strains remained unchanged before and after the mutation. Both strains exhibited maximum activity in a substrate environment of pH 8.0, and could maintain more than 60% enzyme activity in substrate environments of pH 7.0–9.0. In addition, the alginate lyase produced by the mutant Vibrio flexneri C2 and Vibrio flexneri C3-2 could still maintain about 80% enzyme activity in an alkaline environment of pH 9.0, indicating that the alginate lyase produced by the mutant strains was more tolerant to alkaline environments.
[0091] In summary, this invention provides a high-yield alginate lyase-producing Vibrio freundii C3-2 strain. The alginate lyase produced by Vibrio freundii C3-2 has an enzyme activity that is more than 40% higher than that of the wild strain, and the alginate lyase is more tolerant to alkaline environments. At the same time, it has stable genetic characteristics and has good prospects for industrial development.
Claims
1. A strain of Vibrio flexneri ( Vibrio furnissii C3-2, this strain was deposited at the China Center for Type Culture Collection on March 27, 2023, with accession number CCTCC No: M2023411, address: Wuhan University, Wuhan, China.
2. The method for culturing Vibrio flexneri C3-2 according to claim 1, characterized in that, The process includes the following steps: After purifying Vibrio freundii C3-2 by streaking, a single colony is picked and inoculated into a seed culture medium and cultured at a constant temperature of 28±2℃ and 150~200r / min to obtain Vibrio freundii C3-2 bacterial suspension.
3. The cultivation method as described in claim 2, characterized in that, The seed culture medium consists of: 5.0g peptone, 1.0g yeast extract, 2.0g sodium alginate, 0.01g ferric phosphate, 1L seawater buffer solution, pH 7.4-7.6; wherein the seawater buffer solution consists of: 30g sea salt, 1L deionized water.
4. The use of Vibrio freundii C3-2 as described in claim 1 in the production of alginate lyase.
5. A method for producing alginate lyase, characterized in that, The Vibrio freundii C3-2 strain described in claim 1 is used as the fermentation strain.
6. The method for producing alginate lyase as described in claim 5, characterized in that, The process includes the following steps: inoculating Vibrio freundii C3-2 bacterial suspension into the fermentation medium at an inoculation volume of 1-5% of the fermentation medium volume, fermenting at a constant temperature of 28±2℃ and 150-200 r / min to obtain the fermentation broth, centrifuging to collect the supernatant, which is the crude alginate lyase solution.
7. The method for producing alginate lyase as described in claim 6, characterized in that, The fermentation medium consists of: 1.5g potassium dihydrogen phosphate, 6.0g peptone, 4.0g sodium alginate, 0.01g ferrous sulfate, 0.01g magnesium sulfate heptahydrate, 1L seawater buffer, pH 7.0; wherein the seawater buffer consists of: 30g sea salt and 1L deionized water.
8. The method for producing alginate lyase as described in claim 5, characterized in that, The reaction temperature of the alginate lyase produced by Vibrio freundii C3-2 is 15–40℃, and the reaction pH is 7.0–9.
0.
9. The method for producing alginate lyase as described in claim 5, characterized in that, The reaction temperature for the alginate lyase produced by Vibrio freundii C3-2 is 20℃, and the reaction pH is 8.0.