Mutant strain of high-yield tryptophan side chain oxidase and application of mutant strain

Abstract

The invention relates to the technical field of microorganisms, and provides a mutant strain of high-yield tryptophan side chain oxidase and application of the mutant strain. The bacterial strain is Pseudomonas sp. WDR-032, and the preservation number of the bacterial strain is CCTCC (China Center for Type Culture Collection) NO: M 2025204. The mutant strain pseudomonas sp. WDR-032 disclosed by the invention is used for highly expressing the tryptophan side chain oxidase, and compared with initial pseudomonas (ATCC 29574), the tryptophan side chain oxidase has the advantage that the enzyme activity of the tryptophan side chain oxidase is improved by 2 to 3 times.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a mutagenized strain with high yield of tryptophan side-chain oxidase and its application. Background Art

[0002] Tryptophan side-chain oxidase (TSO) is a hydroxylase that can degrade tryptophan and is found in Pseudomonas (ATCC29574). It can oxidize the side chain of tryptophan to β-ketotryptophan in vitro. Tryptophan side-chain oxidase can oxidize the α-carbon at the 3rd position of the indole ring of tryptophan, and the products generated under different pH reaction conditions are also different, and 3-indole glycol aldehyde, N-acetyl-α,β-dehydrotryptamide, etc. can be generated. In addition, it has been found that tumor tissues are metabolically active in tryptophan, and the content of tryptophan in the tumor microenvironment is low. Low concentrations of tryptophan have an inhibitory effect on the proliferation of tumor cells. Therefore, the preparation of tryptophan side-chain oxidase is of great significance.

[0003] Currently, the sequence information of tryptophan side-chain oxidase is unknown, and it is impossible to obtain an expression strain through genetic engineering transformation. It can only be obtained from the lysate supernatant of Pseudomonas. According to reports, tryptophan side-chain oxidase in the supernatant of Pseudomonas is a multi-enzyme complex, and its action mode is relatively complex. Currently, the method for obtaining TSO enzyme from the supernatant of Pseudomonas is relatively fixed. Generally, tryptophan analogs are immobilized on a carrier, and TSO enzyme is separated by affinity chromatography. The yield of TSO enzyme obtained by this method is relatively low and depends on the expression abundance of the TSO enzyme in the Pseudomonas background. Therefore, a Pseudomonas strain with high yield of tryptophan side-chain oxidase is needed.

[0004] Based on the problem of low background expression of TSO enzyme derived from Pseudomonas as described above, the present application proposes a mutagenized strain of Pseudomonas with high yield of tryptophan side-chain oxidase and its application. Summary of the Invention

[0005] Based on the problem of low background expression of TSO enzyme derived from Pseudomonas as described above, the present invention proposes a mutagenized strain with high yield of tryptophan side-chain oxidase and its application.

[0006] The technical solution of the present invention is realized as follows: On the one hand, the present invention provides a mutagenized strain with high yield of tryptophan side-chain oxidase, and the strain is Pseudomonas sp. WDR-032, and the preservation number is CCTCC NO: M2025204.

[0007] Among them, the mutagenesis and screening method of Pseudomonas sp. WDR-032 includes the following steps:

[0008] S1: Streak inoculate the cryopreserved Pseudomonas (ATCC 29574) on an LB solid medium (containing 100 μg / mL ampicillin antibiotic), and culture it in the dark at 37 °C for 16 h in an inverted position. Pick a single colony of Pseudomonas and transfer it to 20 mL of LB liquid medium (containing 100 μg / mL ampicillin antibiotic), and culture it at 37 °C and 220 rpm until the logarithmic growth phase to obtain a bacterial solution in the logarithmic growth phase.

[0009] The composition of the LB liquid medium is as follows: 10 g / L tryptone, 100 μg / mL ampicillin antibiotic, 5 g / L yeast extract, 5 g / L sodium chloride, and the pH value is 7.0.

[0010] 2 wt% agar is added to the LB liquid medium to prepare the LB solid medium.

[0011] S2: Gradient dilute the bacterial solution prepared in step S1 with the mutagenesis protectant 3% chlorogenic acid (CAS No.: 327-97-9, purchased from sigmaaldrich) and 0.01 mol / L PBS buffer (pH = 7.3), namely the original solution, 10 -1 dilution, 10 -2 dilution, 10 -3 dilution, 10 -4 dilution. Take 5 mL of the 10 -4 dilution and place it in a petri dish, add magnetic beads, stir and shake, and irradiate it under an ultraviolet lamp for 100 s, 110 s, and 120 s respectively. The power of the ultraviolet lamp is 30 W, and the irradiation distances are 20 cm, 25 cm, and 30 cm respectively. Then dilute and spread on the plate, and culture it in the dark at 37 °C for 18 h.

[0012] S3: Pick 500 single colony strains, inoculate them into the screening medium at an inoculation amount of 1%, culture them at 37 °C and 220 rpm for 5 h, then take 200 μL of the bacterial solution, centrifuge it at 8000 rpm for 3 min, take 100 μL of the supernatant and measure the change of tryptophan in a 96-well plate. Since the change amount of tryptophan is positively correlated with the enzyme activity, the enzyme activity of tryptophan side chain oxidase can be obtained by detecting the change of tryptophan.

[0013] In a second aspect, the present invention provides an application of a mutagenized strain with high yield of tryptophan side chain oxidase in the production of tryptophan side chain oxidase.

[0014] In a third aspect, the present invention provides a method for producing tryptophan side chain oxidase, and the method is to use Pseudomonas sp. WDR-032 to produce tryptophan side chain oxidase.

[0015] The mutagenized strain with high yield of tryptophan side chain oxidase and its application of the present invention have the following beneficial effects compared with the prior art:

[0016] (1) The sequence information of tryptophan side chain oxidase is unknown, and the expression strain cannot be genetically engineered. It can only be obtained from the lysate supernatant of Pseudomonas. The expression level of tryptophan side chain oxidase in Pseudomonas itself is low, which poses a great obstacle to scientific research. In the present invention, Pseudomonas sp. WDR-032 with high yield of tryptophan side chain oxidase was obtained by ultraviolet-induced mutation of Pseudomonas. This strain can highly express tryptophan side chain oxidase, and the enzyme activity can reach 15000-18000 IU / mL.

[0017] (2) Ultraviolet light belongs to a physical mutagen. As the most commonly used mutagenesis method, the research on its mutagenesis mechanism and action law is also the most in-depth. Ultraviolet light can penetrate bacterial cells and directly act on DNA, causing the formation of pyrimidine dimers in the DNA molecule. The appearance of dimers will weaken the hydrogen bond between the double bonds and cause the double-stranded structure to twist and deform, hindering the normal base pairing, which may cause mutations or death. UV mutagenesis has the characteristics of low cost, simple operation, high mutation rate and high safety, and is widely used in breeding and screening. At present, UV-induced mutation will cause certain damage to cells, and the cell viability will be greatly reduced, affecting the efficiency of cell screening. The extract of Ailanthus altissima, chlorogenic acid, can scavenge free radicals in cells, increase the cell viability by 2-3 times, and greatly improve the screening efficiency.

[0018] (3) The tryptophan side chain oxidase expressed by Pseudomonas can degrade exogenous tryptophan to provide substances required for its own growth. Tryptophan is added to the culture medium as a carbon source for cultivation. According to the consumption ratio of tryptophan per unit time, the expression level of tryptophan side chain oxidase in Pseudomonas can be indicated. Using this as a screening method can efficiently screen out strains with high expression of tryptophan side chain oxidase. Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 It is a comparison chart of the number of mutagenized Pseudomonas colonies before and after the addition of chlorogenic acid. Detailed Embodiments

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] I. Mutation method and screening method of Pseudomonas with high-yield tryptophan side-chain oxidase

[0023] The mutation method and screening method of Pseudomonas with high-yield tryptophan side-chain oxidase include the following steps:

[0024] S1: Streak-inoculate the cryopreserved Pseudomonas (ATCC 29574) on an LB solid medium (containing 100 μg / mL ampicillin antibiotic), and culture it in the dark at 37 °C for 16 h in an inverted position. Pick a single colony of Pseudomonas into 20 mL of LB liquid medium (containing 100 μg / mL ampicillin antibiotic), and culture it at 37 °C and 220 rpm until the logarithmic growth phase to obtain a bacterial solution in the logarithmic growth phase.

[0025] The composition of the LB liquid medium is: 10 g / L of tryptone, 100 μg / mL ampicillin antibiotic, 5 g / L of yeast extract, 5 g / L of sodium chloride, and the pH value is 7.0.

[0026] The LB solid medium is added with 2 wt% agar on the basis of the LB liquid medium.

[0027] S2: Gradient-dilute the bacterial solution prepared in step S1 with a mutagenesis protective agent, 3% chlorogenic acid (CAS No.: 327-97-9, purchased from sigmaaldrich) and 0.01 mol / L PBS buffer (pH = 7.3), namely the stock solution, 10 -1 dilution, 10 -2 dilution, 10 -3 dilution, 10 -4 dilution. Take 5 mL of the 10 -4 dilution into a petri dish, add magnetic beads, stir and oscillate, and irradiate under an ultraviolet lamp for 100 s, 110 s, and 120 s respectively. The power of the ultraviolet lamp is 30 W, and the irradiation distances are 20 cm, 25 cm, and 30 cm respectively. Then dilute and spread on a plate, and culture in the dark at 37 °C for 18 h.

[0028] Taking the example of irradiating for 100 s and the irradiation distance of 20 cm, the experimental group adds chlorogenic acid, and the control group (without chlorogenic acid, only PBS buffer). After ultraviolet lamp irradiation, the bacterial solution is diluted and spread on a plate, and cultured in the dark at 37 °C for 18 h. Detect the number of colonies, and the results are shown in Figure 1 .

[0029] Figure 1 As shown in Figure 1 , after adding chlorogenic acid to the experimental group, the number of Pseudomonas colonies obtained after mutagenesis was 1,200 - 1,400, and the number of Pseudomonas colonies obtained after mutagenesis in the control group (without chlorogenic acid, only PBS buffer) was 500 - 600, and the number of colonies increased by 2 - 3 times (see Figure 1 ).

[0030] The reason is as follows: During ultraviolet mutagenesis, cells will be strongly affected by external factors (such as DNA damage caused by ultraviolet radiation, attack of reactive oxygen free radicals, etc.), resulting in damage to the molecular structures of cell membranes, DNA, and proteins, and even leading to cell death in severe cases. As a strong antioxidant, chlorogenic acid can capture and neutralize ROS, thereby effectively reducing the intracellular oxidative stress level, protecting the cell structure, and reducing the risk of cell killing. Chlorogenic acid can also protect the enzyme proteins and membrane-bound proteins inside the cells, maintain normal metabolic activities and energy supply, provide favorable conditions for cell survival, and then enhance the cell repair ability and stress resistance ability, thus significantly increasing the cell survival rate, which is of great significance for subsequent mutant screening and improving the experimental success rate.

[0031] S3: Pick 500 monoclonal strains and inoculate them into the screening medium at an inoculation amount of 1%, and screen out the strains with high enzyme activity by high-throughput method.

[0032] The formula of the screening medium is: 2 g / L tryptophan, 10 g / L sodium chloride, 1 wt% potassium nitrate, 0.1 wt% magnesium sulfate, and 50 mM calcium carbonate, pH 7.3.

[0033] Specifically, take the same number of Pseudomonas in S2 and inoculate them into the screening medium at an inoculation amount of 1%, culture at 37°C and 220 rpm for 5 h, then take 200 μL of the bacterial liquid, centrifuge at 8,000 rpm for 3 min, and take 100 μL of the supernatant into a 96-well plate to measure the change of tryptophan. The change amount of tryptophan is positively correlated with the enzyme activity.

[0034] The determination of the enzyme activity of tryptophan side-chain oxidase in the invention is based on GB / T 15400-2018 and is appropriately adjusted. The adjusted determination method is:

[0035] 1. Preparation of tryptophan standard curve: Prepare a 500 μg / mL tryptophan stock solution using a buffer solution of 50 mmol / L citrate, 100 mmol / L sodium chloride, and pH = 5.5. Accurately pipette 0 μL, 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL of the tryptophan stock solution into 1.5 mL centrifuge tubes, and make up the volume of the solution to 100 μL with the buffer solution. At this time, the corresponding concentrations of tryptophan are 0 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL respectively. Add 100 μL of 10% potassium hydroxide solution to each centrifuge tube, mix well, then add 500 μL of 1 wt% p-dimethylaminobenzaldehyde solution, mix well, and let it react at room temperature for 5 min. Add 20 μL of 0.2 wt% sodium nitrite solution to each tube, mix well, and let it stand at room temperature for 5 min. Measure the absorbance values of the standards using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 590 nm. Draw a working curve based on the measured values.

[0036] 2. Groups are as follows: Take 100 μL of the culture medium supernatant, measure it according to the measurement method in step 1, substitute the measured value into the standard curve to convert it into tryptophan concentration, and compare the changes before and after the reaction to obtain the enzyme activity.

[0037] The applicant carried out multiple rounds of mutagenesis screening according to the above method, and finally obtained a mutagenized strain with the highest enzyme activity. The enzyme activity reached 18120 IU / mL, which was more than 3 times higher than that of the non-mutagenized original Pseudomonas (enzyme activity was 4500 IU / mL).

[0038] The applicant named the Pseudomonas with the highest enzyme activity as Pseudomonas sp. WDR-032. The applicant deposited it with the China Center for Type Culture Collection, Wuhan University, Wuhan, China on February 13, 2025, and the deposit number is CCTCC NO: M2025204.

[0039] II. Optimization of the screening medium formula

[0040] The screening medium formula is: 2 - 5 g / L tryptophan, 10 - 12 g / L sodium chloride, 1 - 2 wt% potassium nitrate, 0.1 - 0.5 wt% magnesium sulfate, and 50 - 100 mM calcium carbonate, pH 7.2 - 7.4.

[0041] Using Pseudomonas sp. WDR-032 as the experimental object, after adjusting it to the same OD, it was inoculated into the screening medium shown in Table 1 respectively, cultured at 37 °C and 220 rpm for 5 h. 100 μL was taken and the OD value was measured with an ultraviolet spectrophotometer. The OD value is proportional to the number of bacteria. At the same time, 200 μL of the bacterial liquid was taken, centrifuged at 8000 rpm for 3 min, and 100 μL of the supernatant was taken to measure the change of tryptophan in a 96-well plate. Since the change amount of tryptophan is positively correlated with the enzyme activity, the enzyme activity of tryptophan side-chain oxidase can be obtained by measuring the change amount of tryptophan.

[0042] Table 1 Formulation of the screening medium

[0043] Example 1 Example 2 Example 3 Example 4 Tryptophan (g / L) 2 3 5 4 Sodium chloride (g / L) 10 11 12 10 Potassium nitrate (wt%) 1 2 1.5 1.5 Magnesium sulfate (wt%) 0.1 0.3 0.5 0.2 Calcium carbonate (mM) 50 70 100 80 pH 7.3 7.2 7.4 7.3 Enzyme activity (IU / mL) 18120 18100 18150 18090

[0044] In the present invention, tryptophan is added as a carbon source to the medium for culture. According to the consumption ratio of tryptophan per unit time, the expression level of tryptophan side-chain oxidase in Pseudomonas is indicated. Using this as a screening method, strains with high expression of tryptophan side-chain oxidase can be efficiently screened, and the enzyme activity can reach more than 18000 IU / mL.

[0045] III. Method for producing tryptophan side-chain oxidase using Pseudomonas sp. WDR-032

[0046] The method for producing tryptophan side-chain oxidase using Pseudomonas sp. WDR-032 includes the following steps

[0047] S1, DCC catalyzes the coupling of tryptophan and amino agarose

[0048] First, weigh 150 mg of tryptophan and dissolve it in 30 mL of N,N-dimethylformamide (DMF), then add 630 mg of dicyclohexylcarbodiimide (DCC) and 20 mg of triethylamine, and stir magnetically at room temperature for 2 h. Observe that the color of the solution changes from colorless and transparent to light yellow oily substance, which indicates the activation of tryptophan.

[0049] Measure 10 mL of amino agarose microspheres, wash away the microsphere storage solution with 50 mL of deionized water, drain the water, and balance the microspheres with 50 mL of N,N-dimethylformamide; since the reaction catalyzed by DCC needs to be carried out under anhydrous conditions, water is likely to generate insoluble intermediate urea, so this process must strictly ensure anhydrous.

[0050] Take 5 mL of the balanced amino agarose microspheres in a beaker, then add 10 mL of the activated tryptophan, and stir magnetically at 4 °C for 12 h to complete the coupling of tryptophan and amino agarose microspheres.

[0051] S2. Add the conjugated microspheres into a gravity column, collect the outflowing liquid, wash the microspheres with N,N-dimethylformamide, collect the washing solution, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect until the absorbance value under the A325nm absorption peak is 0. Add 20% ethanol and store at 4°C to complete the preparation of the tryptophan side-chain oxidase affinity chromatography adsorbent.

[0052] S3. Purify tryptophan side-chain oxidase with the affinity chromatography adsorbent

[0053] S31. Add 5 mL of the adsorbent from step S2 into a 30 mL gravity column, wash it with 50 mL of deionized water, and then balance the adsorbent with 50 mL of the equilibration solution; the equilibration solution formula is: 50 mmol / L citrate, 100 mmol / L sodium chloride, pH = 5.5.

[0054] S32. Take 20 mL of the crude enzyme solution from Example 1 (original enzyme activity 18120 IU / mL) and add it to the adsorbent equilibrated in step S31. React on a 4°C mixer for 12 h; after the reaction, first elute the adsorbent with 50 mmol / L citrate, 20 mmol / L sodium chloride, pH = 5.5 until the Bradford reagent does not change color; then elute with 200 mmol / L citrate, 20 mmol / L sodium chloride, pH = 5.5 until the Bradford reagent does not change color; finally, elute with 500 mmol / L citrate, 20 mmol / L sodium chloride, pH = 5.5 until the Bradford reagent does not change color; use the method in the first part to detect the enzyme activity of the collected eluate components with different salt concentrations.

[0055] After purification, the enzyme activity can be increased by more than 10 times, reaching 194000 IU / mL.

[0056] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A mutagenized strain with high tryptophan side-chain oxidase activity, characterized by: The strain is Pseudomonas sp. WDR-032, and the deposit number is CCTCC NO: M 2025204.

2. Use of a mutagenized strain with high tryptophan side-chain oxidase productivity according to claim 1 in the production of tryptophan side-chain oxidase.

3. A method for producing tryptophan side-chain oxidase, characterized in that: The method is to use Pseudomonas sp. WDR-032 according to claim 1 to produce tryptophan side-chain oxidase.

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