An N-hydroxyamino-N'-acetyl indigo carmine and its fermentation method and application

Abstract

This application relates to the field of microbial fermentation technology, and specifically discloses an N-hydroxyamino-N'-acetylguanine blue, its fermentation method and application. The chemical name of the N-hydroxyamino-N'-acetylguanine blue is N-(5'-hydroxyamino-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3'] bipyridylmethylene)-acetamide, and the molecular formula is C 12 H 10 O6N4; compared with N-acetylguanine blue, the absorption of N-hydroxyamino-N'-acetylguanine blue is deeper in color at UV611, and at the same time, the formation of hydrogen bonds with amino groups on the fiber can enhance the dyeing property of the dye on the fiber and improve the wash fastness.

Description

Technical Field

[0001] The present application relates to the technical field of microbial fermentation, and in particular to an N-hydroxyamino-N'-acetyl indigoidine, its fermentation method and application. Background Art

[0002] Indigoidine is a natural blue pigment synthesized by microorganisms with antioxidant and antibacterial activities. It was first discovered from the plant pathogen Erwinia sp., and its molecular formula is C 10 H8N4O4. Its properties are similar to those of the chemically synthesized blue pigment indigo, and it can be used as a substitute for chemically synthesized blue dyes and pigments in industries such as textiles and food. The indigoidine pigment is catalyzed and polymerized from two molecules of glutamine in the microorganism by the non-ribosomal peptide synthetase indigoidine synthetase after being activated by 4'-phosphopantetheinyl transferase.

[0003] Previously, the applicant of the present application publicly applied for a patent with the application number CN2024107899337, which disclosed a method for constructing and applying a metabolic engineering bacterium for biosynthesizing N-acetyl indigoidine using glutamate as a substrate. In this application, the indigoidine synthetase encoding gene, 4'-phosphopantetheinyl transferase encoding gene, glutamine synthetase encoding gene, and N-acetylglutamate synthetase encoding gene of the engineering bacterium HG-N-Idg06 utilize glutamate to synthesize N-acetyl indigoidine. However, the almost insoluble property of N-acetyl indigoidine in water affects its dyeing effect, and additives need to be added to increase the dye uptake rate. Therefore, there is still room for improvement. Summary of the Invention

[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide an N-hydroxyamino-N'-acetyl indigoidine, its fermentation method and application.

[0005] To achieve the above purpose, the technical solution adopted by the present application is as follows:

[0006] The present application provides an N-hydroxyamino-N'-acetyl indigoidine, and the chemical name of the N-hydroxyamino-N'-acetyl indigoidine is N-(5'-hydroxyamino-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3'] bipyridine methylene)-acetamide, and its molecular formula is C 12 H 10 O6N4;

[0007] The chemical structure of the N-hydroxyamino-N'-acetyl indigoidine is shown in formula (I);

[0008]

[0009] Formula (I).

[0010] The present application provides a new natural blue pigment (N-hydroxyamino-N'-acetyl guanine blue). Through mass spectrometry, nuclear magnetic resonance, etc., its molecular structure is deduced as shown in formula (I), and the maximum absorption wavelength is 611 nm.

[0011] Compared with N-acetyl guanine blue, N-hydroxyamino-N'-acetyl guanine blue has a deeper absorption at UV611. At the same time, it can form hydrogen bonds with amino groups on fibers, which can enhance the dyeing property of the dye on fibers and improve the color fastness to washing. Therefore, the N-hydroxyamino-N'-acetyl guanine blue of the present application can improve the dyeing effect and color fastness to washing.

[0012] The present application also provides a method for fermenting the above-mentioned N-hydroxyamino-N'-acetyl guanine blue, including the following steps:

[0013] S1. Inoculate the activated recombinant strain seed fermentation broth into the basic fermentation medium for the first-stage fermentation culture, with the dissolved oxygen DO being 20-25%, and ferment to obtain the first-stage fermentation broth;

[0014] S2. Adjust the pH of the first-stage fermentation broth obtained in step S1, and then add a synthase inducer and a hydroxylase cofactor (Fe 2+ ) for the second-stage fermentation culture, with the dissolved oxygen DO being 40-45%, and ferment to obtain a fermentation product containing N-hydroxyamino-N'-acetyl guanine blue.

[0015] By adjusting the fermentation method, the present application can induce the generation of hydroxy-L-glutamine by using a synthase inducer, a hydroxylase cofactor and fermentation conditions (increasing dissolved oxygen), enabling the recombinant strain to switch from producing N-acetyl guanine blue to producing N-hydroxyamino-N'-acetyl guanine blue. Finally, the proportion of the produced N-hydroxyamino-N'-acetyl guanine blue can reach up to 90%, and its molecular structure is deduced by mass spectrometry and nuclear magnetic resonance.

[0016] The method of the present application includes the first-stage fermentation culture for cell proliferation of the bacteria and the second-stage fermentation culture for inducing the accumulation of hydroxy-L-glutamine. The first-stage fermentation is mainly to promote the rapid proliferation of strain cells, and the second-stage fermentation is mainly to induce the generation of hydroxy-L-glutamine and L-glutamine intermediates, inhibit the generation of N-acetyl guanine blue and promote the generation of N-hydroxyamino-N'-acetyl guanine blue.

[0017] As a preferred embodiment of the method for fermenting the above-mentioned N-hydroxyamino-N'-acetyl guanine blue in the present application, the basic fermentation medium includes the following components in mass concentration:

[0018] Yeast powder 5 - 10 g / L, 10% liquid glucose 20 - 40 g / L, ammonium sulfate 10 - 20 g / L, potassium dihydrogen phosphate 0.2 - 1 g / L, magnesium sulfate heptahydrate 0.2 - 0.4 g / L, manganese sulfate monohydrate 0.01 - 0.02 g / L, biotin 2 - 4 μg / L, VB1 0.2 - 0.4 mg / L, sodium glutamate 1 - 2 g / L, the solvent is water, pH 7.0.

[0019] The basic fermentation medium used in this application with the above formula can better ferment the recombinant strain, enabling the recombinant strain to produce N-acetylguanamine and then generate N-hydroxyamino-N'-acetylguanamine.

[0020] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanamine described in this application, the recombinant strain is obtained by introducing expression vector I and expression vector II into a strain;

[0021] The expression vector I is obtained by inserting the glutamine synthetase encoding gene EcglnA and the N-acetylglutamate synthetase encoding gene EcargA into two multiple cloning sites of a plasmid to obtain expression vector I;

[0022] The expression vector II is obtained by inserting the guanamine synthetase encoding gene bpsA and the 4'-phosphopantetheine transferase encoding gene entD into two multiple cloning sites of a plasmid to obtain expression vector II.

[0023] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanamine described in this application, the strain includes Escherichia coli.

[0024] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanamine described in this application, the plasmid includes the pRSFDuet-1 plasmid.

[0025] Preferably, the recombinant strain is obtained by introducing the expression vectors pRSFDuet-EcglnA-EcargA and pCDFDuet-bpsA-entD into Escherichia coli, and is HG-N-Idg06, which is derived from the patent with the patent number CN2024107899337 (the patent name is a construction method and application of a metabolic engineering bacterium for biosynthesizing N-acetylguanamine using glutamate as a substrate);

[0026] The expression vector pRSFDuet-EcglnA-EcargA is obtained by inserting the glutamine synthetase encoding gene EcglnA and the N-acetylglutamate synthetase encoding gene EcargA into two multiple cloning sites of the pRSFDuet-1 plasmid to obtain the expression vector pRSFDuet-EcglnA-EcargA;

[0027] The expression vector pCDFDuet-bpsA-entD is obtained by inserting the gene bpsA encoding indigoidine synthase and the gene entD encoding 4'-phosphopantetheinyl transferase into two multiple cloning sites of the plasmid pCDFDuet-1, resulting in the expression vector pCDFDuet-bpsA-entD.

[0028] Among them, the chassis strain of HG-N-Idg06 contains P450 monooxygenase. In the second fermentation stage, by adding an inducer, hydroxylase cofactor, and increasing dissolved oxygen, the expression of P450 monooxygenase in the chassis strain is co-induced to oxidize L-glutamine to hydroxy-L-glutamine, thereby inhibiting the engineering bacterium HG-N-Idg06 from synthesizing N-acetyl indigoidine using one molecule of glutamine and one molecule of N-acetylglutamine, and instead using one molecule of N-acetylglutamine and one molecule of hydroxy-L-glutamine to generate N-hydroxyamino-N'-acetyl indigoidine.

[0029] In this application, by adjusting the fermentation method, the recombinant strain HG-N-Idg06 is inhibited from generating N-acetyl indigoidine, and instead the generation of N-hydroxyamino-N'-acetyl indigoidine is promoted. Compared with N-acetyl indigoidine, the absorption of N-hydroxyamino-N'-acetyl indigoidine is at UV611 and the color is deeper. At the same time, it can form hydrogen bonds with amino groups on the fiber, which can enhance the dyeing property of the dye on the fiber and improve the color fastness to washing.

[0030] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetyl indigoidine described in this application, in the step S1, the conditions for the first-stage fermentation culture include: the temperature is 35-40 °C; the fermentation culture time is 0-24 h; the ventilation rate is 0.8-1.0 vvm.

[0031] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetyl indigoidine described in this application, in the step S2, the conditions for the second-stage fermentation culture include: the temperature is 20-30 °C; the fermentation culture time is 24-72 h; the ventilation rate is 1.8-2.0 vvm.

[0032] This application adopts the conditions of the first-stage fermentation culture and the second-stage fermentation culture. By controlling the temperature, fermentation culture time, ventilation rate, and rotation speed within the above ranges during the fermentation process, the generation of N-hydroxyamino-N'-acetyl indigoidine can be induced. The ventilation ratio is used to maintain dissolved oxygen. When the dissolved oxygen is higher than 40%, the oxygen-rich state appears, and hydroxy-L-glutamine is generated under the action of cofactors.

[0033] Preferably, the specific steps of the activation include:

[0034] Inoculate the glycerol bacterial solution containing the recombinant strain into a test tube filled with liquid medium, shake and culture it, and then transfer it to a conical flask filled with basic fermentation medium according to an inoculation volume of 0.5% - 2% of the volume of the fermentation medium, and add kanamycin with a final concentration of 50 μg / mL for culture. The culture conditions are: the culture temperature is 32 - 37°C, the rotation speed is 170 - 220 rpm, and the time is 8 - 10 h, until the OD 600 > 3 of the bacterial solution to obtain the activated bacterial solution.

[0035] The liquid medium includes LB liquid medium.

[0036] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine blue described in the present application,

[0037] In the step S2, the pH of the fermentation broth in the first stage is 6.0 - 6.5.

[0038] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine blue described in the present application, in the step S2, the fermentation broth in the first stage is subjected to a second-stage fermentation culture, and when the glucose concentration in the fermentation broth is lower than 2 g / L, a feeding medium is added dropwise;

[0039] The feeding medium includes components with the following concentrations:

[0040] Glucose 400 - 600 g / L, magnesium sulfate heptahydrate 2 - 4 g / L, ammonium chloride 40 - 80 g / L, and the solvent is water.

[0041] When the glucose concentration in the fermentation broth is lower than 2 g / L, a feeding medium is added dropwise, and finally the fermentation ends when the proportion of N-hydroxyamino-N'-acetylguanine blue is the largest to obtain the fermentation product.

[0042] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine blue described in the present application, in the step S2, the synthase inducer includes IPTG inducer; the hydroxylase cofactor includes ferrous sulfate heptahydrate.

[0043] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine blue described in the present application, the mass concentration of the IPTG inducer is 0.5 - 1.5 mM;

[0044] The mass concentration of the ferrous sulfate heptahydrate is 1.0 - 3.5 mM.

[0045] In this application, ferrous sulfate heptahydrate is used to induce and activate the activity of hydroxylation reaction enzymes, thereby promoting hydroxylation induction. Ferrous sulfate heptahydrate is dissolved in the feeding medium and added together with the feeding medium. The feeding medium after adding ferrous sulfate heptahydrate also contains 0.25 - 0.97 g / L of ferrous sulfate heptahydrate.

[0046] This application also provides a fermentation product, which includes by - product 1 and by - product 2;

[0047] The molecular formula of the by - product 1 is C 12 H 10 O6N4, and the chemical name of the by - product 1 is N-(5'-amino - 1'-hydroxy - 2,6,2',6'-tetraoxo - 1,6,1',6'-tetrahydro - 2H,2'H - [3,3']bipyridine - methylene)-acetamide;

[0048] The molecular formula of the by - product 2 is C 12 H 10 O6N4, and the chemical name of the by - product 2 is N-(5'-amino - 1 - hydroxy - 2,6,2',6'-tetraoxo - 1,6,1',6'-tetrahydro - 2H,2'H - [3,3']bipyridine - methylene)-acetamide;

[0049] The chemical structures of the by - product 1 and the by - product 2 are shown in formula (II);

[0050]

[0051] Formula Ⅱ.

[0052] This application also provides the application of the above N - hydroxyamino - N'-acetyl indigo carmine in fabric dyeing.

[0053] In this application, by adjusting the fermentation method, IPTG inducer is used to promote the engineering bacteria to generate intermediate metabolites to form N - hydroxyamino - N'-acetyl indigo carmine. The proportion of the finally produced N - hydroxyamino - N'-acetyl indigo carmine can reach up to 90%. Its molecular structure is inferred by mass spectrometry. At the same time, two new products with similar structures are obtained by this fermentation method. Compared with N - acetyl indigo carmine, N - hydroxyamino - N'-acetyl indigo carmine has a deeper absorption at UV611, and can form hydrogen bonds with amino groups on the fiber, which can enhance the dyeing property of the dye on the fiber and improve the color fastness to washing.

[0054] Compared with the prior art, this application has the following beneficial effects:

[0055] The present application provides an N-hydroxyamino-N'-acetyl indigo and its fermentation method and application. The fermentation method of the present application includes a first-stage fermentation culture for bacterial cell proliferation and a second-stage fermentation culture for inducing the accumulation of hydroxy-L-glutamine. The first-stage fermentation mainly promotes the rapid proliferation of strain cells, and the second-stage fermentation mainly induces the formation of hydroxy-L-glutamine and L-glutamine intermediates, inhibits the formation of N-acetyl indigo, and promotes the formation of N-hydroxyamino-N'-acetyl indigo. Compared with the production of N-acetyl indigo using the genetically engineered bacterium HG-N-Idg06 in Patent CN2024107899337, the present application induces the formation of the intermediate metabolite hydroxy-L-glutamine by changing the fermentation method conditions and combines it with N-acetyl-L-glutamine to form N-hydroxyamino-N'-acetyl indigo. Compared with N-acetyl indigo, N-hydroxyamino-N'-acetyl indigo has a deeper UV611 absorption color, and at the same time, it can form hydrogen bonds with amino groups on fibers, which can enhance the dyeing property of the dye on fibers and improve the wash fastness. Description of the Drawings

[0056] Figure 1 It is the fermentation time course curve of N-hydroxyamino-N'-acetyl indigo in a 5L fermenter in Example 1;

[0057] Figure 2 It is the high-performance liquid chromatography analysis chart of the 72h fermentation broth of N-hydroxyamino-N'-acetyl indigo in Example 1;

[0058] Figure 3 It is the high-performance liquid chromatography analysis chart of the pure product obtained by separating and purifying N-hydroxyamino-N'-acetyl indigo in Example 1, and the full wavelength absorption spectra of N-hydroxyamino-N'-acetyl indigo (A) and N-acetyl indigo (B);

[0059] Figure 4 It is the negative ion mode mass spectrum of N-hydroxyamino-N'-acetyl indigo;

[0060] Figure 5 It is the 1H-NMR chart of N-hydroxyamino-N'-acetyl indigo;

[0061] Figure 6 It is the 13C-NMR chart of N-hydroxyamino-N'-acetyl indigo;

[0062] Figure 7 It is the HSQC chart of N-hydroxyamino-N'-acetyl indigo;

[0063] Figure 8 It is the HMBC chart of N-hydroxyamino-N'-acetyl indigo;

[0064] Figure 91H-NMR spectrum of N-(5'-amino-1'-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridinylidene)-acetamide;

[0065] Figure 10 13C-NMR spectrum of N-(5'-amino-1'-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridinylidene)-acetamide;

[0066] Figure 11 1H-NMR spectrum of N-(5'-amino-1-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridinylidene)-acetamide;

[0067] Figure 12 13C-NMR spectrum of N-(5'-amino-1-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridinylidene)-acetamide;

[0068] Figure 13 High performance liquid chromatography analysis chart of the 72h fermentation broth of the 5L fermenter in Comparative Example 1;

[0069] Figure 14 Biosynthesis route of N-hydroxyamino-N'-acetylguanine blue. Detailed implementation mode

[0070] To better illustrate the purpose, technical solution and advantages of the present application, the present application will be further described below in conjunction with the drawings and specific embodiments.

[0071] In the following examples and comparative examples, the experimental methods used are all conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified, and the component raw materials used in each parallel experiment are the same kind.

[0072] The recombinant strains used in the following examples and comparative examples are: genetically engineered bacterium HG-N-Idg06 derived from patent CN2024107899337.

[0073] The following examples and comparative examples use a 5L fermenter for fermentation.

[0074] Example 1, an N-hydroxyamino-N'-acetylguanine blue and its fermentation method

[0075] This example provides an N-hydroxyamino-N'-acetylguanine blue and its fermentation method. The fermentation method includes the following steps:

[0076] (1) Inoculate the glycerol solution of the genetically engineered bacterium HG-N-Idg06 into a test tube containing LB liquid medium, shake and culture it, and then transfer it to a conical flask containing fermentation medium at an inoculation volume of 1% of the fermentation medium volume, and add kanamycin with a final concentration of 50 μg / mL for culture. The culture conditions are: temperature 35°C, rotation speed 200 rpm, time 8 h, until the OD 600 of the bacterial solution = 3.5, and the activated bacterial solution is obtained;

[0077] (2) Prepare 2 L of LB medium, then perform steam sterilization. After sterilization, cool it down to 35°C. Inoculate the activated bacterial solution obtained in step (1) into 2 L of LB medium at an inoculation amount of 0.1%, and culture it at 35°C for 10 h until the OD600nm of the bacterial solution = 5, and the activated recombinant strain seed fermentation broth is obtained;

[0078] (3) Add 1.2 L of basic fermentation medium (8 g / L of Shengqi yeast powder P801, 30 g / L of 10% liquid glucose, 15 g / L of ammonium sulfate, 0.5 g / L of potassium dihydrogen phosphate, 0.3 g / L of magnesium sulfate heptahydrate, 0.015 g / L of manganese sulfate monohydrate, 3 μg / L of biotin, 0.3 mg / L of VB1, 2 g / L of sodium glutamate) into a 5 L fermenter. Add the recombinant strain seed fermentation broth obtained in step (2) into the 5 L fermenter for the first-stage fermentation culture. Control the pH to 7.0, control the temperature to 35°C, aeration rate: 0.9 vvm, tank pressure 0.05 Mpa, stir and correlate the dissolved oxygen DO, and control the dissolved oxygen DO at 22%. Culture for 0 - 24 h to obtain the first-stage fermentation broth;

[0079] (4) Adjust the fermentation conditions of the first-stage fermentation broth for the second-stage fermentation culture. When the OD600nm at 24 h = 120, cool down to 25°C and add IPTG with a concentration of 0.7 mmol / L for the induction expression of the synthase, and add 2.5 mM / L of magnesium sulfate heptahydrate to promote the formation of hydroxylation reaction. Adjust the aeration ratio to 1.9 vvm, tank pressure 0.08 Mpa, maintain the dissolved oxygen DO at 42%, control the pH of the fermentation broth to 6.3, and culture for 24 - 72 h. When the glucose concentration in the fermentation broth is lower than 2 g / L, start to feed and supplement the medium (including the following components by mass concentration: 500 g / L of glucose, 3 g / L of magnesium sulfate heptahydrate, 60 g / L of ammonium chloride, 0.7 g / L of ferrous sulfate heptahydrate, and the solvent is water), and control the glucose concentration within the range of 0 - 5 g / L to obtain the second-stage fermentation broth;

[0080] (5) Take samples every 4 h to measure the biomass OD 600 and the yield of N-hydroxyamino-N'-acetylguanine blue. The change trend during the fermentation process is as Figure 1 shown.

[0081] When fermentation reaches 72 h, the production of N-hydroxyamino-N'-acetylguanamine stops increasing and reaches the highest proportion. The fermentation is ended to obtain the fermentation product.

[0082] The maximum biomass OD during fermentation 600 is 118.5 ( Figure 1 ), and the production of N-hydroxyamino-N'-acetylguanamine in the final discharged fermentation broth is 15.08 g / L ( Figure 1 ), with a proportion of 90.0% ( Figure 2 ).

[0083] Example 2, an N-hydroxyamino-N'-acetylguanine blue and its fermentation method

[0084] (1) Inoculate the glycerol bacterial solution of the genetically engineered bacterium HG-N-Idg06 into a test tube containing LB liquid medium, shake and culture it, and then transfer it to a conical flask containing fermentation medium at an inoculation volume of 1% of the fermentation medium volume, and add kanamycin with a final concentration of 50 μg / mL for culture. The culture conditions are: temperature 32°C, rotation speed 170 rpm, time 8 h, until the OD600 of the bacterial solution = 3.1, to obtain the activated bacterial solution;

[0085] (2) Prepare 2 L of LB medium, then perform steam sterilization. After the sterilization is completed, cool it down to 32°C. Inoculate the activated bacterial solution obtained in step (1) into 2 L of LB medium at an inoculation amount of 0.1%, and culture it at 32°C for 10 h until the OD600nm of the bacterial solution = 4, to obtain the activated recombinant strain seed fermentation broth;

[0086] (3) Add 1.2 L of basic fermentation medium (5 g / L of Shengqi yeast powder P801, 20 g / L of 10% liquid glucose, 10 g / L of ammonium sulfate, 0.2 g / L of potassium dihydrogen phosphate, 0.2 g / L of magnesium sulfate heptahydrate, 0.01 g / L of manganese sulfate monohydrate, 2 μg / L of biotin, 0.2 mg / L of VB1, 1 g / L of sodium glutamate) to a 5 L fermenter, add the recombinant strain seed fermentation broth obtained in step (2) to the 5 L fermenter for the first-stage fermentation culture, control the pH to 6.8, control the temperature to 32°C, aeration rate: 0.8 vvm, tank pressure 0.05 Mpa, stir and correlate the dissolved oxygen DO, control the dissolved oxygen DO at 20%, and culture for 0 - 24 h to obtain the first-stage fermentation broth;

[0087] (4) Adjust the fermentation conditions of the first-stage fermentation broth for the second-stage fermentation culture. When the OD600nm at 24 h is 120, cool down to 20 °C and add IPTG at a concentration of 0.5 mmol / L for the induced expression of the synthase. 1 mM / L of ferrous sulfate heptahydrate promotes the formation of hydroxylation reaction. Adjust the aeration ratio to 1.8 vvm, the tank pressure to 0.08 Mpa, maintain the dissolved oxygen DO at 40%, control the pH of the fermentation broth at 6.0, and culture for 24 - 72 h. When the glucose concentration in the fermentation broth is lower than 2 g / L, start to feed and supplement the culture medium (including the following components by mass concentration: glucose 400 g / L, magnesium sulfate heptahydrate 2 g / L, ammonium chloride 40 g / L, ferrous sulfate heptahydrate 0.5 g / L, and the solvent is water), and control the glucose concentration within the range of 0 - 5 g / L to obtain the second-stage fermentation broth;

[0088] (5) Sample every 4 h to measure the biomass OD600 and the yield of N-hydroxyamino-N'-acetylguanamine blue. The changing trends during the fermentation process are as Figure 1 shown.

[0089] (6) When the fermentation reaches 72 h, the yield of N-hydroxyamino-N'-acetylguanamine blue stops increasing and reaches the highest proportion. End the fermentation to obtain the fermentation product.

[0090] The maximum biomass OD600 during the fermentation process is 110.5, and the yield of N-hydroxyamino-N'-acetylguanamine blue in the final discharged fermentation broth is 12.52 g / L, with a proportion of 83.5%.

[0091] Example 3, an N-hydroxyamino-N'-acetylguanine blue and its fermentation method

[0092] (1) Inoculate the glycerol bacterial solution of the genetically engineered bacterium HG-N-Idg06 into a test tube containing LB liquid medium, shake and culture, and then transfer it to a conical flask containing fermentation medium according to an inoculation volume of 1% of the fermentation medium volume, and add kanamycin at a final concentration of 50 μg / mL for culture. The culture conditions are: temperature 37 °C, rotation speed 220 rpm, time 8 h, until the OD600 of the bacterial solution = 4.0 to obtain the activated bacterial solution;

[0093] (2) Prepare 2 L of LB medium, then perform steam sterilization. After the sterilization is completed, cool down to 37 °C. Inoculate the activated bacterial solution obtained in step (1) into 2 L of LB medium at an inoculation amount of 0.1%, and culture at 37 °C for 10 h until the OD600nm of the bacterial solution = 6 to obtain the activated recombinant strain seed fermentation broth;

[0094] (3) Add 1.2 L of the basic fermentation medium (10 g / L of Saccharomyces cerevisiae powder P801, 40 g / L of 10% liquid glucose, 20 g / L of ammonium sulfate, 1 g / L of potassium dihydrogen phosphate, 0.4 g / L of magnesium sulfate heptahydrate, 0.02 g / L of manganese sulfate monohydrate, 4 μg / L of biotin, 0.4 mg / L of VB1, 2 g / L of sodium glutamate) to a 5 L fermenter. Add the recombinant strain seed fermentation broth obtained in step (2) to the 5 L fermenter for the first-stage fermentation culture. Control the pH at 7.2, the temperature at 37 °C, the aeration rate at 1.0 vvm, the tank pressure at 0.05 Mpa, and stir to correlate with the dissolved oxygen DO. Control the dissolved oxygen DO at 25% and culture for 0 - 24 h to obtain the first-stage fermentation broth;

[0095] (4) Adjust the fermentation conditions of the first-stage fermentation broth for the second-stage fermentation culture. When the OD600nm at 24 h = 120, lower the temperature to 30 °C and add IPTG at a concentration of 1.0 mmol / L for the induced expression of the synthase. Promote the formation of hydroxylation reaction with 3.5 mM / L of ferrous sulfate heptahydrate. Adjust the aeration ratio to 2.0 vvm, the tank pressure to 0.08 Mpa, maintain the dissolved oxygen DO at 45%, control the pH of the fermentation broth at 6.5, and culture for 24 - 72 h. When the glucose concentration in the fermentation broth is lower than 2 g / L, start to feed the feeding medium (including the following components by mass concentration: 600 g / L of glucose, 4 g / L of magnesium sulfate heptahydrate, 80 g / L of ammonium chloride, 0.97 g / L of ferrous sulfate heptahydrate, and the solvent is water), and control the glucose concentration within the range of 0 - 5 g / L to obtain the second-stage fermentation broth;

[0096] (5) Take samples every 4 h to measure the biomass OD600 and the yield of N-hydroxyamino-N'-acetylguanamine blue. The change trend during the fermentation process is as Figure 1 shown.

[0097] (6) When fermenting to 72 h, the yield of N-hydroxyamino-N'-acetylguanamine blue stops increasing and reaches the highest proportion. End the fermentation to obtain the fermentation product.

[0098] The maximum biomass OD600 during the fermentation process is 116.2. The yield of N-hydroxyamino-N'-acetylguanamine blue in the final discharged fermentation broth is 13.87 g / L, and the proportion is 85.4%.

[0099] From the above results, it can be seen that the yield of N-hydroxyamino-N'-acetylguanamine blue in the final discharged fermentation broth of Example 1 is 15.08 g / L, and the proportion is 90.0%; the yield of N-hydroxyamino-N'-acetylguanamine blue in the final discharged fermentation broth of Example 2 is 12.52 g / L, and the proportion is 83.5%; the yield of N-hydroxyamino-N'-acetylguanamine blue in the final discharged fermentation broth of Example 3 is 13.87 g / L, and the proportion is 85.4%.

[0100] Examples using the above fermentation method all obtained N-hydroxyamino-N'-acetylguanine blue, and the yield and proportion were relatively high under suitable fermentation conditions, proving that the second-stage fermentation culture of this application adopted the above fermentation conditions, and added a synthase inducer and a cofactor (Fe 2+ ), which could induce the expression of the product synthase, inhibit the synthesis of N-acetylguanine blue, and promote the formation of N-hydroxyamino-N'-acetylguanine blue. Based on the existing genetically engineered bacterium HG-N-Idg06, this application adjusted the fermentation method to obtain a new substance. The structure has not been reported in the literature. It shows a dark blue color in solution, and the maximum light absorption value is 610 nm ( Figure 3 A in), and it is inferred to be a new type of blue pigment.

[0101] Example 4, extraction, purification and structure identification of N-hydroxyamino-N'-acetylguanine blue product

[0102] The fermentation product of N-hydroxyamino-N'-acetylguanine blue in Example 1 was extracted, purified, and its structure was identified. The final N-hydroxyamino-N'-acetylguanine blue fermentation broth obtained in Example 1 was centrifuged at 12,000 g for 20 min. The obtained supernatant was the N-hydroxyamino-N'-acetylguanine blue and the fermentation broth solution. Four times the volume fraction of absolute ethanol was added to the supernatant, and the impurities in the fermentation broth such as water-soluble proteins, salts, and sugars were removed by the water extraction and alcohol precipitation method. The obtained solution was centrifuged, the bottom precipitate was discarded, and the supernatant was collected. This was the crude extract of N-hydroxyamino-N'-acetylguanine blue. Then it was washed with ethanol 1-2 times and freeze-dried to obtain the pure product of N-hydroxyamino-N'-acetylguanine blue ( Figure 3 ).

[0103] The above samples were sent to the Analysis and Testing Center of Nanjing Normal University for LC-MS detection and NMR detection.

[0104] Figure 4 is the LC-MS detection chart; Figure 5 is the 1H-NMR chart of N-hydroxyamino-N'-acetylguanine blue; Figure 6 is the 13C-NMR chart of N-hydroxyamino-N'-acetylguanine blue; Figure 7 is the HSQC chart of N-hydroxyamino-N'-acetylguanine blue; Figure 8 is the HMBC chart of N-hydroxyamino-N'-acetylguanine blue.

[0105] The relative molecular mass of acetylguanine blue is 290.07, and the relative molecular mass of the hydroxyl group is 17. The LC-MS detection results ( Figure 4 ) show that the relative molecular mass of the sample sent for testing is 306.07, which is consistent with the theoretical value of N-hydroxyamino-N'-acetylguanine blue.

[0106] The 1H NMR chemical shift assignments of N-hydroxyamino-N'-acetylguanine blue are as follows: 1H NMR (400 MHz, DMSO-d6) δ 10.57 (brs, 1H), 8.98 (s, 1H), 8.73 (s, 1H), 8.12 (s, 1H), 2.00 (s, 3H).

[0107] The 13C NMR chemical shift assignments of N-hydroxyamino-N'-acetylguanine blue are as follows: 13C NMR (101 MHz, DMSO-d6) δ 168.45, 165.03, 161.19, 159.61, 140.41, 132.50, 130.45, 125.05, 121.66, 116.5, 115.70, 23.94.

[0108] By assigning the two-dimensional HSQC spectrum of the product and excluding the solvent-related signals, three sets of direct carbon-hydrogen correlations were obtained, namely (δ 8.98 (s, 1H), δ 130.45), (δ 8.12 (s, 1H), δ 116.5), and (δ 2.00 (s, 3H), 23.94), corresponding to the imine proton, the carbon-hydrogen in the double bond, and the direct carbon-hydrogen of the methyl group in the molecule, respectively. Combining with the information of the long-range correlation between a methyl group and a carbonyl carbon in the HMBC spectrum, it is suggested that there is an acetyl group in the molecule. Analyzing the carbon spectrum data, the molecule has a structural feature similar to N-acetyl-guanine blue. Based on the mass-to-charge ratio given in ESI-MS, the calculated molecular weight is 306. Combining with the structural features of N-acetylguanine blue, it is suggested that there is a substituted hydroxyl group corresponding to an isolated broad peak δ 10.57 (brs, 1H) in the 1H NMR spectrum, and this hydroxyl group is connected to the amino group outside N-acetylguanine blue.

[0109] Finally, the structure of the compound was determined as shown in formula (I), so it was named N-hydroxyamino-N'-acetylguanine blue. This structure has not been reported in the literature and is a completely new substance. This new substance appears dark blue in solution with a maximum light absorption value of 611 nm, and is inferred to be a new type of blue pigment.

[0110] The chemical structure of the said N-hydroxyamino-N'-acetylguanine blue is shown in formula (I);

[0111]

[0112] Formula (I).

[0113] Example 5, extraction and purification of the fermentation product in Example 1

[0114] During the extraction and purification of the fermentation product in Example 1, two other by-products were purified and structurally characterized. The insoluble matter in the fermentation broth was removed by centrifugation, and the macroporous resin D101 was used for gradient elution with ethanol aqueous solution, column chromatography (developing agent: ethyl acetate: methanol: water = 15:5:3) purification and freeze-drying to obtain the product.

[0115] The above samples were sent to the Analysis and Testing Center of Nanjing Normal University for LC-MS detection and NMR detection.

[0116]

[0117] By-product 1;

[0118] By-product 1 has structural characteristics similar to those of the product. Analysis of the proton signals at δ 7.65 (s, 1H) and 7.63 (s, 1H) in the hydrogen spectrum indicates the presence of two vinylic hydrogen protons in the molecule, corresponding to two pairs of double bonds on the parent nucleus. The isolated singlet at δ 5.89 (brs, 1H) may be the proton signal of an active hydrogen. The signal at δ 2.15 (s, 3H) indicates the presence of a methyl group in the molecule. The amino proton signal at δ 6.01 (s, 2H) in the hydrogen spectrum, combined with the mass-to-charge ratio given by ESI-MS, calculates that the molecular weight is the same as that of the product. It is speculated that the difference lies in the substitution position of the hydroxyl group. By comparing the hydrogen spectrum of the parent nucleus N-acetylguanamine, it is speculated that the substituted hydroxyl group is connected to the NH at the 1 position. The structure of by-product 1 is determined to be N-(5'-amino-1'-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridine methylene)-acetamide.

[0119]

[0120] By-product 2;

[0121] The test results of by-product 1 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H), 9.00(s, 1H), 7.73 (d, 1H), 7.56 (d, 1H), 5.46 (brs, 1H), 5.36 (s, 1H), 2.08 (s,3H). See Figure 11 .

[0122] The test results of by-product 2 are as follows: 13C NMR (101 MHz, DMSO-d6) δ 170.85, 167.62,165.49, 165.60, 162.56, 137.68, 136.77, 130.16, 128.65, 125.38, 122.61,25.68. SeeFigure 12 。

[0123] Byproduct 2 also has the structural characteristics of the product and is extremely similar to the spectral data of the product. By analyzing the spectral data of the parent nucleus hydroxyguanamine blue and calculating the molecular weight obtained by ESI-MS, there is also a hydroxyl substituent in byproduct 2, which is different from byproduct 1 and is connected to the NH at the 1 position. Thus, the structure of byproduct 2 is determined to be N-(5'-amino-1-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3'] bipyridine methylene)-acetamide.

[0124] Example 6, application of N-hydroxyamino-N'-acetylguanine blue in dyeing

[0125] Dye the cotton fabric with the N-hydroxyamino-N'-acetylguanamine blue prepared in Examples 1 to 3 above and byproducts 1 and 2 in Example 5 according to the following process: 2% (o.w.f) of guanamine blue, reduce with 2 times of sodium dithionite, adjust to pH 5 with 0.5 mL / L of citric acid, dye at 25 °C for 10 min, and the bath ratio is 1:30.

[0126] After the dyeing is completed, take out the dyed fabric sample, wash it, dry it, and solve the K / S value of the dye by using the Lambert-Beer law. Test the various properties of the dye according to the following standards, and record the test results in Table 1 below. The color fastness to water is tested according to GB / T 5713-2013;

[0127] Table 1

[0128]

[0129] Comparative Example 1

[0130] Compared with Example 1, the difference is only that ferrous sulfate heptahydrate is not added, and the other conditions are the same.

[0131] When the fermentation reaches 72 h, the fermentation ends. The maximum biomass OD600 during the fermentation process is 118.2. N-Hydroxyamino-N'-acetylguanamine blue is not produced in the final fermentation broth, while the yield of N-acetylguanamine is 9.85 g / L and the purity is 90.5%.

[0132] Compared with Example 1, the strain grows normally under the conditions of this Comparative Example 1. Since the cofactor (Fe 2+ ) is not added, the P450 monooxygenase cannot be induced to oxidize L-glutamine to hydroxy-L-glutamine, and finally N-hydroxyamino-N'-acetylguanamine blue cannot be produced. Without changing other conditions, the engineered bacterium HG-N-Idg06 can still produce N-acetylguanamine ( Figure 13 ).

[0133] Comparative Example 2

[0134] Compared with Example 1, the difference is only that ferrous sulfate heptahydrate is replaced with ferrous chloride heptahydrate, and the other conditions are the same.

[0135] When the fermentation reached 72 h, the fermentation ended. The maximum biomass OD600 during the fermentation was 121.2, and the yield of N-hydroxyamino-N'-acetylguanamine in the final fermented broth was 14.69 g / L, with a proportion of 88.7%.

[0136] Compared with Example 1, the strain grew normally under the conditions of this Comparative Example 2. Ferrous sulfate heptahydrate was replaced with ferrous chloride heptahydrate to provide cofactor (Fe 2+ ), which induced P450 monooxygenase to oxidize L-glutamine to hydroxy-L-glutamine. Finally, the yield of N-hydroxyamino-N'-acetylguanamine was only 14.69 g / L.

[0137] Comparative Example 3

[0138] Compared with Example 1, the difference is that the dissolved oxygen DO in the second-stage fermentation is 30%, and the other conditions are the same.

[0139] When the fermentation reached 72 h, the fermentation ended. The maximum biomass OD600 during the fermentation was 122.2, and the yield of N-hydroxyamino-N'-acetylguanamine in the final fermented broth was 8.56 g / L, with a proportion of 50%.

[0140] Compared with Example 1, the strain grew normally under the conditions of this Comparative Example 3. Since the dissolved oxygen content was 30% which was less than 40%, it failed to promote the large-scale oxidation of P450 monooxygenase to convert L-glutamine into hydroxy-L-glutamine. Finally, the yield of N-hydroxyamino-N'-acetylguanamine was only 8.56 g / L, and N-acetylguanamine was generated.

[0141] Comparative Example 4

[0142] Compared with Example 1, the difference is that the dissolved oxygen DO in the second-stage fermentation is 60%, and the other conditions are the same.

[0143] When the fermentation reached 72 h, the fermentation ended. The maximum biomass OD600 during the second-stage fermentation was 105.2, and the yield of N-hydroxyamino-N'-acetylguanamine in the final fermented broth was 4.48 g / L, with a proportion of 90.2%.

[0144] Compared with Example 1, the strain did not grow normally under the second-stage fermentation conditions of this Comparative Example 4. Since the dissolved oxygen content was 60% which was much higher than 45%, an oxygen-rich state occurred and a large amount of peroxides were synthesized to inhibit the growth of the strain. Finally, the yield of N-hydroxyamino-N'-acetylguanamine was only 4.48 g / L.

[0145] Comparing Example 1 and Comparative Example 4, Example 1 involves a first stage of cell proliferation and a second stage where an inducer, hydroxylase cofactor (Fe 2+ ions), and increased dissolved oxygen are used together to induce the expression of P450 monooxygenase in the chassis strain to oxidize L-glutamine to hydroxy-L-glutamine, thereby inhibiting the production of cyan blue by the engineered bacterium HG-N-Idg06 and instead using one molecule of L-glutamine and one molecule of hydroxy-L-glutamine to produce N-hydroxyamino-N'-acetyl cyan blue. It is possible to achieve the conversion of the engineered bacterium HG-N-Idg06 from not producing N-acetyl cyan blue to producing N-hydroxyamino-N'-acetyl cyan blue, with the yield of N-hydroxyamino-N'-acetyl cyan blue being 15.08 g / L ( Figure 1 ), and the proportion being 90.0% ( Figure 2 ).

[0146] Table 2

[0147]

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A N-hydroxyamino-N'-acetylindigotin, characterized in that, The chemical structure of the N-hydroxyamino-N'-acetylguanine blue is shown in formula (I); Formula (I).

2. A method for fermenting N-hydroxyamino-N'-acetyl indigo blue as described in claim 1, characterized in that, It includes the following steps: S1. Inoculate the activated recombinant strain seed fermentation broth into the basic fermentation medium for the first-stage fermentation culture, with the dissolved oxygen DO being 20-25%, and ferment to obtain the first-stage fermentation broth; S2. Adjust the pH of the first-stage fermentation broth obtained in step S1, then add a synthase inducer and a hydroxylase cofactor for the second-stage fermentation culture, with the dissolved oxygen DO being 40-45%, and ferment to obtain a fermentation product containing N-hydroxyamino-N'-acetylguanine blue; The recombinant strain is obtained by introducing expression vector I and expression vector II into a strain; The expression vector I is obtained by inserting the glutamine synthetase-encoding gene EcglnA and the N-acetylglutamate synthetase-encoding gene EcargA into two multiple cloning sites of a plasmid; The expression vector II is obtained by inserting the guanine blue synthase-encoding gene bpsA and the 4'-phosphopantetheine transferase-encoding gene entD into two multiple cloning sites of a plasmid; The strain is Escherichia coli; the plasmid is pRSFDuet-1 plasmid; In step S1, the conditions for the first-stage fermentation culture include: the temperature is 35-40 °C; the fermentation culture time is 0-24 h; the ventilation rate is 0.8-1.0 vvm; In step S2, the conditions for the second-stage fermentation culture include: the temperature is 20-30 °C; the fermentation culture time is 24-72 h; the ventilation rate is 1.8-2.0 vvm; In step S2, the synthase inducer is IPTG inducer; the hydroxylase cofactor is ferrous sulfate heptahydrate.

3. The method according to claim 2, characterized in that, The basic fermentation medium includes the following components by mass concentration: Yeast powder 5-10 g / L, 10% liquid glucose 20-40 g / L, ammonium sulfate 10-20 g / L, potassium dihydrogen phosphate 0.2-1 g / L, magnesium sulfate heptahydrate 0.2-0.4 g / L, manganese sulfate monohydrate 0.01-0.02 g / L, biotin 2-4 μg / L, VB1 0.2-0.4 mg / L, sodium glutamate 1-2 g / L, the solvent is water, pH 7.

0.

4. The method according to claim 2, wherein In step S2, the pH of the first-stage fermentation broth is 6.0-6.

5.

5. The method according to claim 2, wherein In step S2, when the first-stage fermentation broth is subjected to the second-stage fermentation culture, a feeding medium is added by flow when the glucose concentration in the fermentation broth is lower than 2 g / L; The feeding medium includes the following components by concentration: Glucose 400-600 g / L, magnesium sulfate heptahydrate 2-4 g / L, ammonium chloride 40-80 g / L, the solvent is water.

6. The method according to claim 2, wherein The mass concentration of the IPTG inducer is 0.5-1.5 mM; The mass concentration of the ferrous sulfate heptahydrate is 1.0-3.5 mM.

7. The application of the N-hydroxyamino-N'-acetylguanine blue according to claim 1 in fabric dyeing.

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