A strain of Acinetobacter garneri capable of hydrolyzing m-aminoacetanilide and its application

Abstract

A strain of Acinetobacter garneri that hydrolyzes m-aminoacetanilide and its application, relating to the field of microorganisms. Acinetobacter gerneri )CCZU‑DM3 is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee, with the deposit number of CGMCC No.25885. The strain can be used to hydrolyze m-aminoacetanilide, and the applicable culture conditions are: temperature 25°C to 37°C, pH 6.0 to 8.0. The strain of the present invention can catalyze the hydrolysis of m-aminoacetanilide to completely convert it into m-phenylenediamine, with high conversion efficiency, simple operation, low equipment requirements and low operating cost.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Acinetobacter garneri capable of hydrolyzing m-aminoacetanilide and application thereof. Background Art

[0002] Due to the vast territory, complex terrain and diverse climate of my country, there are many types of microorganisms in my country. People can discover new strains of microorganisms in different latitudes, terrains and climates. Discovering the species diversity and genetic diversity of microorganisms, discovering new metabolites with diverse functions and different enzymatic mechanisms are important foundations for discovering innovative drugs and developing special functional products.

[0003] Microbial metabolites often have unique, novel and complex chemical structures. They must contain biological enzymes with special functions. For example, reductase can catalyze the reduction of nitro compounds to corresponding amine compounds, and amidase can catalyze the hydration of nitrile compounds to corresponding amides and organic acid compounds.

[0004] Acinetobacter species are widely distributed in nature and have important clinical and ecological significance. Some strains of this genus, such as Acinetobacter baumannii A.baumannii It is a common cause of multidrug-resistant (MDR) infections worldwide, with its cumulative resistance and extraordinary ability to survive in hospital environments (Mohammad Al-Tamimi et al., 2022). Monika et al. (2019) isolated a new bacterial source of xanthine oxidase from soil, namely, Acinetobacter calcoaceticus with high oxidase activity. A. calcoaceticus RL2-M4, xanthine oxidase (EC 1.17.3.2) is a key enzyme in purine metabolism and has potential applications in the food and pharmaceutical industries. Mohammad Mahbub Kabir et al. (2018) Isolation of a novel Cr(VI)-reducing bacterium from tannery effluent and solid waste A. gerneri MKPF7 is used to repair and detoxify hexavalent chromium in polluted environments. However, there are few reports on other functions of this bacterium at home and abroad. A. gerneri CCZU-DM3 can catalyze the hydrolysis of m-aminoacetanilide into m-phenylenediamine, and has a high conversion efficiency of up to 100%. However, in the prior art, there is no report on the specific conversion of m-aminoacetanilide by microorganisms. Summary of the invention

[0005] Technical problem to be solved: In view of the problem that there is a lack of effective m-aminoacetanilide-specific conversion technology in the prior art, the present invention provides a strain of Acinetobacter garneri that hydrolyzes m-aminoacetanilide and its application, and the strain can efficiently and specifically convert m-aminoacetanilide.

[0006] Technical solution: A strain of Acinetobacter garneri that hydrolyzes m-aminoacetanilide ( Acinetobacter gerneri )CCZU-DM3, its deposit number is CGMCC No.25885.

[0007] A method for screening Acinetobacter garneri CCZU-DM3 that hydrolyzes m-aminoacetanilide as described above, comprising the following steps:

[0008] Step 1. Transfer the wastewater sample in the biochemical pool to an inorganic salt medium containing 100 mg / L of m-aminoacetanilide at a 1% inoculation volume. After shaking culture for 5 to 7 days, transfer it to a new inorganic salt medium containing a higher concentration of m-aminoacetanilide at a 1% inoculation volume. Continuously acclimate and transfer several times to obtain an active acclimatization solution.

[0009] Step 2. The obtained acclimation solution is streaked and cultured to obtain Acinetobacter garneri that hydrolyzes m-aminoacetanilide. The details are as follows: Take the acclimation solution and dilute it with sterile physiological saline (0.85% sodium chloride solution) to 10 -2 ~10 -6 times. Take 10 μL of the diluted acclimation solution and spread it on solid culture medium plates containing 100 mg / L m-aminoacetanilide inorganic salts, and place it in a biochemical incubator at 30-32 ℃ for three days. Select single colonies with obvious differences on the culture plate, and purify and culture them on LB solid culture medium by plate streaking separation. After five consecutive purifications, a single strain was obtained and stored at -80 ℃.

[0010] Preferably, the colony morphology of the Acinetobacter garneri CCZU-DM3 is round, light yellow, smooth on the surface, and the bacteria are small and short rod-shaped.

[0011] Preferably, the seed liquid preparation method of the Acinetobacter gerneri CCZU-DM3 is as follows: streak the activated Acinetobacter gerneri CCZU-DM3 on LB solid culture medium, culture at a constant temperature of 30-32°C, culture for 22-24 hours, pick up the single colony into LB liquid culture medium after seeing a complete single colony, and then culture at 30-32°C, 180 r / min shaking for 12-14 hours, centrifuge at 4000 r / min, 10 min, 4°C, discard the supernatant, rinse with physiological saline, repeat centrifugation 3 times, obtain a bacterial suspension, adjust OD 600nm=0.8, that is, seed liquid.

[0012] The application of the above-mentioned Acinetobacter garneri CCZU-DM3 that hydrolyzes m-aminoacetanilide in catalyzing the hydrolysis of m-aminoacetanilide.

[0013] The application of the above-mentioned Acinetobacter garneri that hydrolyzes m-aminoacetanilide in transforming m-aminoacetanilide.

[0014] Preferably, the conversion conditions are to use m-aminoacetanilide as the sole carbon source, and the transformation of Acinetobacter gellosa CCZU-DM3 is carried out at a temperature of 25 to 37°C, a pH of 6.0 to 8.0, and a rotation speed of 100 to 250 r / min. When the initial concentration of m-aminoacetanilide is 500 mg / L, the conversion rate of m-aminoacetanilide can reach 100% after 5 hours.

[0015] The strain CCZU-DM3 has strong adaptability to the environment and good temperature adaptability. The activity conversion rate is relatively high in the temperature range of 25 ℃ to 37 ℃. It is sensitive to pH and can convert m-aminoacetanilide well in the pH range of 6.0 to 8.0, but the over-acidic or over-alkaline environment will inhibit the effect of the strain. Its oxygen demand range is wide, and it can work at a speed of 0r / min to 250 r / min. The initial concentration of m-aminoacetanilide can be quickly converted in the range of 100 mg / L to 1000 mg / L.

[0016] Beneficial effects: (1) The present invention provides Acinetobacter sp. The highly efficient m-aminoacetanilide transformation bacterium CCZU-DM3 of the bacterial strain provides a type of m-aminoacetanilide transformation bacterium; it has a high transformation efficiency and can carry out m-aminoacetanilide transformation in a targeted manner;

[0017] (2) The screening process of the present invention is simple and controllable, has excellent conversion effect, is environmentally friendly, highly efficient and energy-saving, and can also avoid causing pollution.

[0018] Deposit information: The deposit date of the strain of the present invention is October 10, 2022, the deposit number is: CGMCC No. 25885, and the classification name is Acinetobacter gerneri ( Acinetobacter gerneri )CCZU-DM3, the name of the depository is the General Microbiology Center of China Culture Collection Administration, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Acinetobacter garneri in Example 1 of the present invention gerneri ) Phylogenetic tree of CCZU-DM3.

[0020] Figure 2 For Acinetobacter gerneri in Example 1 of the present invention ( Acinetobacter gerneri ) Growth status of CCZU-DM3 plate culture medium.

[0021] Figure 3 It is the GC-MS chart of the intermediate aminoacetanilide conversion product of Example 2 of the present invention.

[0022] Figure 4 LC-MS diagram of the intermediate aminoacetanilide conversion product of Example 2 of the present invention.

[0023] Figure 5 The aminoacetanilide conversion product of Example 2 of the present invention 1 H-NMR spectrum.

[0024] Figure 6 This is the effect of the initial concentration of intermediate aminoacetanilide on the ability of the strain to convert meta-aminoacetanilide in Example 3 of the present invention.

[0025] Figure 7 This is the effect of culture temperature on the ability of the strain to convert m-aminoacetanilide in Example 3 of the present invention.

[0026] Figure 8 The figure shows the effect of the shaking speed on the ability of the strain to convert m-aminoacetanilide in Example 3 of the present invention.

[0027] Fig. 9 This is the effect of pH value on the ability of the strain to convert m-aminoacetanilide in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. However, it should be understood that these embodiments are only for illustrative purposes and should not be construed as limitations on the implementation of the present invention.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0030] Example 1: Screening and identification of Acinetobacter CCZU-DM3

[0031] 1. Material preparation

[0032] (1) Water sample materials

[0033] Wastewater samples from the biochemical pool of Zhejiang Wanfeng Chemical Co., Ltd.

[0034] (2) Culture medium

[0035] Inorganic salt culture medium (g / L): NaCl 1, K2HPO4 1, (NH4)2SO4 1, MgSO4 0.2, FeSO4 1, MnCl2 1, ZnSO4 1.

[0036] Inorganic salt solid culture medium (g / L): NaCl 1, K2HPO4 1, (NH4)2SO4 1, MgSO4 0.2, FeSO4 1, MnCl21, ZnSO4 1, agar powder 15.

[0037] LB liquid medium (g / L): tryptone 10, yeast powder 5, NaCl 10.

[0038] LB solid medium (g / L): tryptone 10, yeast powder 5, NaCl 10, agar powder 15.

[0039] 2. Domestication and screening of strain CCZU-DM3

[0040] (1) Acclimation of CCZU-DM3

[0041] Add 100 mL of inorganic salt culture medium to a 250 mL conical flask and seal it with gauze. After sterilization, add 1 mL of wastewater sample to the culture medium, the wastewater sample accounts for 1% of the volume of the culture medium, add 10 mg of m-aminoacetanilide to the above culture medium, the concentration of m-aminoacetanilide is 100 mg / L, place it on a shaker at 180 r / min and 30-32 °C for 5-7 days, and then transfer the above culture medium to another fresh m-aminoacetanilide inorganic salt culture medium at a 1% inoculation amount, and the concentration of m-aminoacetanilide in the culture medium is 150 mg / L. Continuously acclimate and transfer several times (the content of m-aminoacetanilide increases by 50 mg / L each time), and measure the transformation effect. When there is obvious transformation for three consecutive generations, an active acclimatization solution is obtained. Use m-aminoacetanilide as the sole carbon source.

[0042] (2) Screening of CCZU-DM3

[0043] Prepare 1000 mL of 0.85% saline, pipette 9 mL into a test tube, sterilize and set aside. Add 10 mg of m-aminoacetanilide to 100 mL of sterilized inorganic salt solid medium, shake evenly and pour on a plate. After the medium solidifies, use a pipette to pipette 1 mL of active acclimation solution into the above test tube as 10 -1 Dilution, then draw 1 mL10 -1 Dilute the solution into another test tube, and dilute to 10 -2 , 10 -3 , 10 -4 , 10 -5, 10 -6 , take 10 μL10 -2 ~10 -6 The dilution was spread on an inorganic salt solid plate, the plate was inverted in a constant temperature incubator, the temperature was set to 30°C, and cultured for 2 to 5 days (3 days in this embodiment). After colonies grew, single colonies with good morphology, different colors, and different forms were selected, and streaked on fresh LB solid medium for separation and purification. After five consecutive purifications, a single strain was obtained, and the strain was stored at -80°C.

[0044] Add 10 mg of m-aminoacetanilide to 100 mL of sterilized inorganic salt medium, inoculate the isolated and purified pure strain, and culture it in a constant temperature shaking incubator at 180 r / min and 30-35 °C. Detect the concentration change of m-aminoacetanilide by high performance liquid chromatography. Select the strain that can effectively transform the m-aminoacetanilide in the medium, named CCZU-DM3, and store it at -80 °C.

[0045] The DNA identification was commissioned to Shanghai Shenggong Biotechnology Co., Ltd. The 16S ribosomal DNA gene sequence characteristics of the above strains were compared and analyzed with the database using analytical methods, such as Figure 1 , and found that the strain belonged to Acinetobacter ( Acinetobacter sp. ), which is speculated to be Acinetobacter gerneri The colony morphology of the above strains is: Figure 2 , round, light yellow, smooth surface, small, short rod-shaped.

[0046] Example 2: Application of strain CCZU-DM3 to catalyze hydrolysis of m-aminoacetanilide

[0047] Prepare 100 mL of LB liquid medium and take 3 mL into a test tube. Streak the activated Acinetobacter cerevisiae CCZU-DM3 on the LB solid medium, culture the LB solid medium at a constant temperature of 30-32 °C for 22-24 hours, and pick up a single colony with a sterile inoculation loop into the LB liquid medium after seeing a complete single colony. Tilt the test tube 45° and place it in a shaker. Culture at 30-32 °C and 180 r / min for 12-14 hours, centrifuge the bacterial solution (4000 r / min, 10 min, 4 °C), discard the supernatant, rinse with 0.85% saline, repeat centrifugation 3 times, and obtain a bacterial suspension. Adjust the OD 600nm =0.8, that is, seed liquid.

[0048] Take 1 mL of the prepared seed solution and add it to a sterilized inorganic salt medium containing 500 mg / L of m-aminoacetanilide. Incubate at 30-32 °C and 180 r / min with shaking. Stop the reaction after 6 h. According to conventional HPLC analysis, the content of the conversion product is about greater than 98%.

[0049] The cells were removed by centrifugation, and the supernatant was filtered through a 0.22 μm filter membrane, extracted with dichloromethane, and the upper aqueous phase was removed. A portion was subjected to GC-MS and LC-MS detection, and the remaining portion was rotary evaporated to obtain the corresponding transformation product, which was dissolved in deuterated DMSO and then analyzed. 1 H-NMR detection.

[0050] GC-MS showed that the conversion product was m-phenylenediamine, such as Figure 3 The retention time of the conversion product was consistent with that of the m-phenylenediamine standard. LC-MS analysis showed that Figure 4 The molecular weight of the transformed product was 108, which was the same as that of the standard. 1 The H-NMR data are also consistent with the standard, such as Figure 5 As shown. Thus, it was determined that Acinetobacter CCZU-DM3 could catalyze the hydrolysis of m-aminoacetanilide and convert it into m-phenylenediamine.

[0051] Example 3: Application of strain CCZU-DM3 in transforming m-aminoacetanilide

[0052] A certain amount of m-aminoacetanilide and the above-prepared seed solution were added to the sterilized inorganic salt medium to prepare a 100 mL transformation system. The initial concentration, temperature, rotation speed and other conditions of m-aminoacetanilide were artificially changed, and samples were taken at regular intervals to measure the residual concentration of m-aminoacetanilide in the medium.

[0053] Optimization of the initial concentration of m-aminoacetanilide. 1% seed solution was inoculated into an inorganic salt medium, and m-aminoacetanilide was added to make the initial concentrations of 100 mg / L, 200 mg / L, 500 mg / L, 700 mg / L, and 1000 mg / L, respectively. The medium was placed in a shaker and cultured at 30°C, natural pH, and 180 r / min. Samples were taken at regular intervals to measure the concentration changes of m-aminoacetanilide. The results showed that the initial concentration of m-aminoacetanilide could be rapidly converted in the range of 100 mg / L to 1000 mg / L, such as Figure 6 .

[0054] Optimization of culture temperature. 1% seed liquid was inoculated into an inorganic salt medium containing 500 mg / L m-aminoacetanilide, and the culture conditions were 20℃, 25℃, 30℃, 37℃, and 42℃, respectively. The medium was placed in a shaker, and cultured at natural pH and 180 r / min for 5 hours. The concentration change of m-aminoacetanilide was measured. The results showed that the optimal temperature for the conversion of m-aminoacetanilide by Acinetobacter CCZU-DM3 was 25~37℃. When the temperature was 30℃, the conversion rate of m-aminoacetanilide was the highest, reaching 93%. Figure 7 .

[0055] Optimization of shaking speed. 1% seed liquid was inoculated into an inorganic salt medium containing 500 mg / L m-aminoacetanilide, and the shaking speed was adjusted to 0 r / min, 50 r / min, 100 r / min, 150 r / min, 180 r / min, and 250 r / min, respectively. The medium was placed in a shaker and cultured at 30°C and natural pH for 5 hours. The concentration change of m-aminoacetanilide was measured. The results showed that the optimal speed for the conversion of m-aminoacetanilide by Acinetobacter CCZU-DM3 was 100-250 r / min. When the shaking speed was 100 r / min, the conversion rate of m-aminoacetanilide was the highest, reaching 98%. Figure 8 .

[0056] Optimization of pH conditions. 1% seed liquid was inoculated into an inorganic salt medium containing 500 mg / L m-aminoacetanilide, and the pH of the medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. The medium was placed in a shaker and cultured at 30°C and 180 r / min for 5 h, and then the concentration change of m-aminoacetanilide was measured. The results showed that the optimal pH for the conversion of m-aminoacetanilide by Acinetobacter CCZU-DM3 was 6.0-8.0. When the pH was 8.0, the conversion rate of m-aminoacetanilide was the highest, which could reach 100%. Fig. 9 .

[0057] By comparison, it was found that when the initial concentration of m-aminoacetanilide was 500 mg / L, CCZU-DM3 had the best conversion effect of m-aminoacetanilide under the conditions of temperature 30 ℃, pH 8.0 and rotation speed 100 r / min, and the conversion rate of m-aminoacetanilide could reach 100% after 5 h.

Claims

1. A strain of Acinetobacter garneri that hydrolyzes m-aminoacetanilide ( Acinetobacter gerneri )CCZU-DM3, its deposit number is CGMCC No.25885.

2. The strain of Acinetobacter garnerii CCZU-DM3 that hydrolyzes m-aminoacetanilide according to claim 1, characterized in that: The colony morphology of the Acinetobacter garneri CCZU-DM3 is round, light yellow, with a smooth surface, and the bacteria are small and short rod-shaped.

3. The strain of Acinetobacter garnerii CCZU-DM3 that hydrolyzes m-aminoacetanilide according to claim 1, characterized in that: The preparation method of the seed liquid of the Acinetobacter garneri CCZU-DM3 is as follows: streaking the activated Acinetobacter garneri CCZU-DM3 on an LB solid culture medium, culturing at a constant temperature of 30-32°C for 22-24 hours, picking up a single colony and transferring it to an LB liquid culture medium after a complete single colony is seen, culturing at 30-32°C with shaking at 180 r / min for 12-14 hours, centrifuging at 4000 r / min for 10 minutes at 4°C, discarding the supernatant, and then washing with physiological saline, repeatedly centrifuging 3 times to obtain a bacterial suspension, and adjusting the OD 600nm =0.8, that is, seed liquid.

4. Use of the m-aminoacetanilide hydrolyzing Acinetobacter garneri CCZU-DM3 according to claim 1 in catalyzing the hydrolysis of m-aminoacetanilide.

5. Use of the m-aminoacetanilide hydrolyzing Acinetobacter garneri according to claim 1 in transforming m-aminoacetanilide.

6. The use according to claim 5, characterized in that: The transformation conditions are as follows: m-aminoacetanilide is used as the sole carbon source, and Acinetobacter galneri CCZU-DM3 is transformed at a temperature of 25 to 37°C, a pH of 6.0 to 8.0, and a rotation speed of 100 to 250 r / min.

Images