N-hydroxyamino-N '-acetyl ornamental blue as well as fermentation method and application thereof

Abstract

The invention relates to the technical field of microbial fermentation, and particularly discloses N-hydroxyamino-N '-acetyl blue as well as a fermentation method and application thereof. The chemical name of the N-hydroxyamino-N '-acetyl blue is N-(5'-hydroxyamino-2, 6, 2 ', 6'-tetraoxo-1, 6, 1 ', 6'-tetrahydro-2H, 2 'H-[3, 3'] bipyridyl methylene)-acetamide, and the molecular formula of the N-hydroxyamino-N '-acetyl blue is C12H10O6N4. Compared with N-acetyl blue, the absorption of N-hydroxyamino-N '-acetyl blue is darker in UV611 color, and meanwhile, the N-hydroxyamino-N'-acetyl blue and amino on the fiber form a hydrogen bond, so that the dyeing property of the dye on the fiber can be enhanced, and the color fastness to washing can be improved.

Description

Technical Field

[0001] This application relates to the technical field of microbial fermentation, and particularly relates 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 H 8 N 4 O 4 . 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. Indigoidine pigment is formed by the catalytic polymerization of two molecules of glutamine in microorganisms by the non-ribosomal peptide synthetase indigoidine synthetase after activation by 4'-phosphopantetheinyl transferase.

[0003] Previously, the applicant of this 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 this 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 in this application is as follows: This 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 the molecular formula is C 12 H 10 O 6 N 4 ; The chemical structure of the N-hydroxyamino-N'-acetyl indigoidine is shown in formula (I); Formula (I).

[0006] The present application provides a new natural blue pigment (N-hydroxyamino-N'-acetylguanine 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.

[0007] Compared with N-acetylguanine blue, N-hydroxyamino-N'-acetylguanine blue has a deeper UV611 absorption color. 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. Therefore, the N-hydroxyamino-N'-acetylguanine blue of the present application can improve the dyeing effect and color fastness to washing.

[0008] The present application also provides a method for fermenting the above-mentioned N-hydroxyamino-N'-acetylguanine blue, which 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, 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'-acetylguanine blue.

[0009] By adjusting the fermentation method, the present application can induce the formation 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-acetylguanine blue to producing N-hydroxyamino-N'-acetylguanine blue. Finally, the proportion of the produced N-hydroxyamino-N'-acetylguanine blue can reach up to 90%, and its molecular structure is deduced by mass spectrometry and nuclear magnetic resonance.

[0010] The method of the present application includes the first-stage fermentation culture for cell proliferation and the 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-acetylguanine blue and promotes the formation of N-hydroxyamino-N'-acetylguanine blue.

[0011] As a preferred embodiment of the method for fermenting the above-mentioned N-hydroxyamino-N'-acetylguanine blue of the present application, the basic fermentation medium includes the following components in 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, 1 - 2 g / L of sodium glutamate, the solvent is water, pH 7.0.

[0012] The basic fermentation medium with the above formula is adopted in this application, which can better ferment the recombinant strain, enabling the recombinant strain to produce N-acetylguanamine and then generate N-hydroxyamino-N'-acetylguanamine.

[0013] 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; 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; 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.

[0014] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanamine described in this application, the strain includes Escherichia coli.

[0015] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanamine described in this application, the plasmid includes the pRSFDuet-1 plasmid.

[0016] Preferably, the recombinant strain is obtained by introducing expression vectors pRSFDuet-EcglnA-EcargA and pCDFDuet-bpsA-entD into Escherichia coli, and it is HG-N-Idg06, which is derived from the patent with the patent number CN2024107899337 (the patent title is A Construction Method and Application of a Metabolic Engineering Bacterium for Biosynthesizing N-acetylguanamine Using Glutamate as a Substrate); 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; The expression vector pCDFDuet-bpsA-entD is obtained by inserting the guanamine synthetase encoding gene bpsA and the 4'-phosphopantetheine transferase encoding gene entD into two multiple cloning sites of the pCDFDuet-1 plasmid.

[0017] 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 using one molecule of glutamine and one molecule of N-acetylglutamine to synthesize N-acetylguanine blue and instead using one molecule of N-acetylglutamine and one molecule of hydroxy-L-glutamine to generate N-hydroxyamino-N'-acetylguanine blue.

[0018] This application inhibits the production of N-acetylguanine blue by the recombinant strain HG-N-Idg06 by adjusting the fermentation method and promotes the production of N-hydroxyamino-N'-acetylguanine blue instead. Compared with N-acetylguanine blue, the absorption of N-hydroxyamino-N'-acetylguanine blue is at UV611 and the color is deeper. At the same time, forming a hydrogen bond with the amino group on the fiber can enhance the dyeing property of the dye on the fiber and improve the wash fastness.

[0019] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine blue 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 volume is 0.8-1.0 vvm.

[0020] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine blue 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 volume is 1.8-2.0 vvm.

[0021] 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 volume, and rotation speed within the above ranges during the fermentation process, the production of N-hydroxyamino-N'-acetylguanine blue 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 the cofactor.

[0022] Preferably, the specific steps of the activation include: Inoculate the glycerol bacterial solution containing the recombinant strain into a test tube containing a liquid medium, shake and culture, and then transfer it to a conical flask containing a 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 of the bacterial solution 600 >3, to obtain the activated bacterial solution.

[0023] The liquid medium includes LB liquid medium.

[0024] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine described in the present application, In the step S2, the pH of the fermentation broth in the first stage is 6.0 - 6.5.

[0025] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine 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 a feeding medium is added when the glucose concentration in the fermentation broth is lower than 2 g / L; The feeding medium comprises components with the following concentrations: Glucose 400 - 600 g / L, magnesium sulfate heptahydrate 2 - 4 g / L, ammonium chloride 40 - 80 g / L, and the solvent is water.

[0026] When the glucose concentration in the fermentation broth is lower than 2 g / L, the feeding medium is added, and finally the fermentation ends when the proportion of N-hydroxyamino-N'-acetylguanine is the largest, obtaining a fermentation product.

[0027] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine described in the present application, in the step S2, the synthase inducer comprises IPTG inducer; the hydroxylase cofactor comprises ferrous sulfate heptahydrate.

[0028] As a preferred embodiment of the method for fermenting N-hydroxyamino-N'-acetylguanine described in the present application, 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.

[0029] The present application uses ferrous sulfate heptahydrate 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 further comprises ferrous sulfate heptahydrate 0.25 - 0.97 g / L.

[0030] The present application also provides a fermentation product, and the fermentation product comprises by-product 1 and by-product 2; The molecular formula of the by-product 1 is C 12 H 10 O 6 N 4 , 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; The molecular formula of the by-product 2 is C 12 H10 O 6 N 4 , the chemical name of 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; The chemical structures of the said by-product 1 and by-product 2 are shown in formula (II); Formula II.

[0031] This application also provides the application of the above N-hydroxyamino-N'-acetyl indigo carmine in fabric dyeing.

[0032] This application adjusts the fermentation method to use IPTG inducer 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% at most. 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, the absorption of N-hydroxyamino-N'-acetyl indigo carmine is at UV611 with a deeper color. At the same time, it can form hydrogen bonds with amino groups on the fiber, which can enhance the dyeing property of the dye to the fiber and improve the color fastness to washing.

[0033] Compared with the prior art, this application has the following beneficial effects: This application provides an N-hydroxyamino-N'-acetyl indigo carmine and its fermentation method and application. The fermentation method of this application includes the first-stage fermentation culture for bacterial body proliferation 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. The second-stage fermentation is mainly to induce the generation of hydroxy-L-glutamine and L-glutamine intermediates, inhibit the generation of N-acetyl indigo carmine, and promote the generation of N-hydroxyamino-N'-acetyl indigo carmine. Compared with using the genetically engineered bacterium HG-N-Idg06 in Patent CN2024107899337 to generate N-acetyl indigo carmine, this application induces the generation of intermediate metabolite hydroxy-L-glutamine by changing the fermentation method conditions and combines N-acetyl-L-glutamine to form N-hydroxyamino-N'-acetyl indigo carmine. Compared with N-acetyl indigo carmine, the absorption of N-hydroxyamino-N'-acetyl indigo carmine is at UV611 with a deeper color. At the same time, it can form hydrogen bonds with amino groups on the fiber, which can enhance the dyeing property of the dye to the fiber and improve the color fastness to washing. Description of the Drawings

[0034] Figure 1 It is the fermentation time course curve of N-hydroxyamino-N'-acetyl indigo carmine in a 5L fermenter in Example 1; Figure 2High performance liquid chromatography analysis chart of the 72-hour fermentation broth of N-hydroxyamino-N'-acetylguanine in Example 1; Figure 3 High performance liquid chromatography analysis chart of the pure product obtained by separation and purification of N-hydroxyamino-N'-acetylguanine in Example 1, and full wavelength absorption spectra of N-hydroxyamino-N'-acetylguanine (A) and N-acetylguanine (B); Figure 4 Negative ion mode mass spectrum of N-hydroxyamino-N'-acetylguanine; Figure 5 1H-NMR chart of N-hydroxyamino-N'-acetylguanine; Figure 6 13C-NMR chart of N-hydroxyamino-N'-acetylguanine; Figure 7 HSQC chart of N-hydroxyamino-N'-acetylguanine; Figure 8 HMBC chart of N-hydroxyamino-N'-acetylguanine; Figure 9 1H-NMR chart of N-(5'-amino-1'-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridine methylene)-acetamide; Figure 10 13C-NMR chart of N-(5'-amino-1'-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridine methylene)-acetamide; Figure 11 1H-NMR chart of N-(5'-amino-1-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridine methylene)-acetamide; Figure 12 13C-NMR chart of N-(5'-amino-1-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridine methylene)-acetamide; Figure 13 High performance liquid chromatography analysis chart of the 72-hour fermentation broth of the 5L fermenter in Comparative Example 1; Figure 14 Biosynthetic route of N-hydroxyamino-N'-acetylguanine. Detailed implementation manners

[0035] 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 accompanying drawings and specific embodiments.

[0036] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified, and the component raw materials used in each parallel experiment are of the same kind.

[0037] The recombinant strains used in the following examples and comparative examples are: the genetically engineered bacterium HG-N-Idg06 derived from Patent CN2024107899337.

[0038] The following examples and comparative examples use a 5L fermenter for fermentation.

[0039] Example 1: An N-hydroxyamino-N'-acetylguanine blue and its fermentation method This example provides an N-hydroxyamino-N'-acetyl indigo carmine and its fermentation method. The fermentation method includes the following steps: (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 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 = 3.5 to obtain the activated bacterial solution; (2) Prepare 2L of LB medium, then perform steam sterilization. After the sterilization is completed, cool it down to 35°C. Inoculate the activated bacterial solution obtained in step (1) into 2L of LB medium at an inoculation amount of 0.1%, and culture at 35°C for 10 h until the OD600nm of the bacterial solution = 5 to obtain the activated recombinant strain seed fermentation broth; (3) Add 1.2L 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 5L fermenter. Add the recombinant strain seed fermentation broth obtained in step (2) into the 5L fermenter for the first-stage fermentation culture. Control the pH to 7.0, control the temperature at 35°C, aeration volume: 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; (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 25 °C and add IPTG at a concentration of 0.7 mmol / L for the induction expression of the synthase. 2.5 mM / L ferrous sulfate heptahydrate promotes the formation of hydroxylation reaction. Adjust the aeration ratio to 1.9 vvm, the tank pressure to 0.08 Mpa, maintain the dissolved oxygen DO at 42%, control the pH of the fermentation broth at 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 culture medium (including the following components by mass concentration: glucose 500 g / L, magnesium sulfate heptahydrate 3 g / L, ammonium chloride 60 g / L, ferrous sulfate heptahydrate 0.7 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; (5) Take samples every 4 h to measure the biomass OD 600 and the yield of N-hydroxyamino-N'-acetylguanamine blue. The change trend during the fermentation process is as Figure 1 shown.

[0040] (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, and obtain the fermentation product.

[0041] The maximum biomass OD during the fermentation process 600 is 118.5 ( Figure 1 ), and the yield of N-hydroxyamino-N'-acetylguanamine blue in the final discharged fermentation broth is 15.08 g / L ( Figure 1 ), and the proportion is 90.0% ( Figure 2 ).

[0042] Example 2: An N-hydroxyamino-N'-acetylguanine blue and its fermentation method (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 32 °C, rotation speed 170 rpm, time 8 h, until the OD600 of the bacterial solution is 3.1 to obtain the activated bacterial solution; (2) Prepare 2 L of LB medium, then perform steam sterilization. After the sterilization is completed, cool 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 at 32 °C for 10 h until the OD600nm of the bacterial solution is 4 to obtain the activated recombinant strain seed fermentation broth; (3) Add 1.2 L of basic fermentation medium (5 g / L of Saccharomyces cerevisiae 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) 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 at 6.8, the temperature at 32 °C, the aeration rate at 0.8 vvm, the tank pressure at 0.05 Mpa, and stir to correlate the dissolved oxygen (DO). Control the DO at 20% and culture for 0 - 24 h to obtain the first-stage fermentation broth. (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 20 °C and add IPTG with a concentration of 0.5 mmol / L for the induction expression of synthase. Add 1 mM / L of ferrous sulfate heptahydrate to promote the formation of hydroxylation reaction. Adjust the aeration ratio to 1.8 vvm and the tank pressure to 0.08 Mpa. Maintain the DO at 40% and control the pH of the fermentation broth at 6.0. 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: 400 g / L of glucose, 2 g / L of magnesium sulfate heptahydrate, 40 g / L of ammonium chloride, 0.5 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. (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.

[0043] (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.

[0044] 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%.

[0045] Example 3: An N-hydroxyamino-N'-acetylguanine blue and its fermentation method (1) Inoculate the glycerol bacterial solution of the genetically engineered bacterium HG-N-Idg06 into a test tube containing LB liquid medium and shake culture. 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 with 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. (2) Prepare 2 L of LB medium, then perform steam sterilization. After the sterilization is completed, cool it 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 it at 37°C for 10 h until the OD600nm of the bacterial solution reaches 6 to obtain the activated recombinant strain seed fermentation broth. (3) Add 1.2 L of basic fermentation medium (10 g / L of Shengqi yeast 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) 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 at 7.2, the temperature at 37°C, the ventilation 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. (4) Adjust the fermentation conditions of the first-stage fermentation broth for the second-stage fermentation culture. When the OD600nm at 24 h reaches 120, cool down to 30°C and add IPTG with a concentration of 1.0 mmol / L for the induction expression of synthase. Add 3.5 mM / L of ferrous sulfate heptahydrate to promote the formation of hydroxylation reaction. Adjust the ventilation 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. (5) Take samples every 4 h to measure the biomass OD600 and the yield of N-hydroxyamino-N'-acetylguanamine. The change trend during the fermentation process is as Figure 1 shown.

[0046] (6) When fermenting to 72 h, the yield of N-hydroxyamino-N'-acetylguanamine stops increasing and reaches the highest proportion. End the fermentation to obtain the fermentation product.

[0047] The maximum biomass OD600 during the fermentation process is 116.2, and the yield of N-hydroxyamino-N'-acetylguanamine in the final discharged fermentation broth is 13.87 g / L, with a proportion of 85.4%.

[0048] As can be seen from the above results, the yield of N-hydroxyamino-N'-acetylguanine blue in the final fermentation broth of Example 1 was 15.08 g / L, accounting for 90.0%. The yield of N-hydroxyamino-N'-acetylguanine blue in the final fermentation broth of Example 2 was 12.52 g / L, accounting for 83.5%. The yield of N-hydroxyamino-N'-acetylguanine blue in the final fermentation broth of Example 3 was 13.87 g / L, accounting for 85.4%.

[0049] All the examples using the above fermentation method obtained N-hydroxyamino-N'-acetylguanine blue, and the yield and proportion were relatively high under suitable fermentation conditions, which proved 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 presents a dark blue color in solution, and the maximum light absorption value is 610 nm ( Figure 3 A), and it is inferred to be a new type of blue pigment.

[0050] Example 4: Extraction, purification and structure identification of N-hydroxyamino-N'-acetylguanine blue product The fermentation product of N-hydroxyamino-N'-acetylguanine blue in Example 1 was extracted, purified and structurally identified. The final N-hydroxyamino-N'-acetylguanine blue fermentation broth of Example 1 was centrifuged at 12000 g for 20 min. The obtained supernatant was the solution of N-hydroxyamino-N'-acetylguanine blue and the fermentation broth. Four times the volume fraction of absolute ethanol was added to the supernatant to remove impurities such as water-soluble proteins, salts and sugars in the fermentation broth by the method of water extraction and alcohol precipitation. 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 ).

[0051] The above samples were sent to the Analysis and Testing Center of Nanjing Normal University for LC-MS detection and NMR detection.

[0052] 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.

[0053] The relative molecular mass of acetylguanine blue is 290.07, and the relative molecular mass of hydroxyl group is 17. The LC-MS detection result ( Figure 4 ) shows that the relative molecular mass of the submitted sample is 306.07, which is consistent with the theoretical value of N-hydroxyamino-N'-acetylguanine blue.

[0054] The 1H NMR chemical shift assignment of N-hydroxyamino-N'-acetylguanine blue is 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). The 13C NMR chemical shift assignment of N-hydroxyamino-N'-acetylguanine blue is 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. By assigning the two-dimensional HSQC spectrum of the product and excluding the solvent-related signals, three sets of direct carbon-hydrogen correlation information 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), which correspond to the imine proton, the carbon-hydrogen correlation in the double bond, and the direct carbon-hydrogen correlation 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 structural characteristics similar to N-acetylguanine blue. Based on the mass-to-charge ratio given in ESI-MS, the calculated molecular weight is 306. Combining with the structural characteristics 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.

[0055] 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, and the maximum light absorption value is 611 nm, suggesting that it is a new type of blue pigment.

[0056] The chemical structure of the said N-hydroxyamino-N'-acetylguanine blue is shown in formula (I); Formula (I).

[0057] Example 5: Extraction and purification of the fermentation product in Example 1 During the process of extracting and purifying the fermentation product in Example 1 of this experiment, it was found that two other by-products were purified and structurally identified. The insoluble substances in the fermentation broth were removed by centrifugation, and the macroporous resin D101 was used for gradient elution with ethanol aqueous solution, column chromatography (developer: ethyl acetate: methanol: water = 15:5:3) purification and freeze-drying to obtain them.

[0058] The above samples were sent to the Analysis and Testing Center of Nanjing Normal University for LC-MS detection and NMR detection.

[0059] By-product 1; 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 it is due to the different positions of the hydroxyl substitution. By comparing the hydrogen spectrum of the parent nucleus N-acetylguanine blue, 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.

[0060] By-product 2; The detection result of by-product 1 is: 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 。

[0061] The detection result of by-product 2 is: 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. See Figure 12 。

[0062] By-product 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 by-product 2, which is different from by-product 1 and is connected to the NH at the 1-position, thus determining the structure of by-product 2 as N-(5'-amino-1-hydroxy-2,6,2',6'-tetraoxo-1,6,1',6'-tetrahydro-2H,2'H-[3,3']bipyridine methylene)-acetamide.

[0063] Example 6: Application of N-hydroxyamino-N'-acetylguanine blue in dyeing Dye the cotton fabric with the N-hydroxyamino-N'-acetylguanamine blue prepared in the above Examples 1 to 3 and by-products 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.

[0064] After the dyeing is completed, take out the dyed fabric sample, wash it, dry it, and use the Lambert-Beer law to solve the K / S value of the dye. 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; Table 1 Comparative Example 1 Compared with Example 1, the difference is only that ferrous sulfate heptahydrate is not added, and the other conditions are the same.

[0065] 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 generated in the final fermentation broth, while the yield of N-acetylguanamine is 9.85 g / L and the purity is 90.5%.

[0066] Compared with Example 1, under the conditions of this Comparative Example 1, the strain grows normally. Due to the absence of the cofactor (Fe 2+ ), the P450 monooxygenase cannot be induced to oxidize L-glutamine to hydroxy-L-glutamine, and finally N-hydroxyamino-N'-acetylguanamine blue cannot be generated. Without changing other conditions, the engineered bacterium HG-N-Idg06 can still generate N-acetylguanamine ( Figure 13 ).

[0067] Comparative Example 2 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.

[0068] When fermentation reached 72 h, the fermentation ended. The maximum biomass OD600 during the fermentation process was 121.2. The yield of N-hydroxyamino-N'-acetylguanamine in the final fermented broth was 14.69 g / L, accounting for 88.7%.

[0069] Compared with Example 1, under the conditions of this Comparative Example 2, the strain grew normally. Ferrous sulfate heptahydrate was replaced with ferrous chloride heptahydrate to provide cofactor (Fe 2+ ), which induced the P450 monooxygenase to oxidize L-glutamine to hydroxy-L-glutamine. Finally, the yield of N-hydroxyamino-N'-acetylguanamine was only 14.69 g / L.

[0070] Comparative Example 3 Compared with Example 1, the difference lies in that the dissolved oxygen DO in the second-stage fermentation was 30%, and the other conditions were the same.

[0071] When fermentation reached 72 h, the fermentation ended. The maximum biomass OD600 during the fermentation process was 122.2. The yield of N-hydroxyamino-N'-acetylguanamine in the final fermented broth was 8.56 g / L, accounting for 50%.

[0072] Compared with Example 1, under the conditions of this Comparative Example 3, the strain grew normally. Since the dissolved oxygen content was 30% which was less than 40%, it failed to promote the P450 monooxygenase to oxidize a large amount of L-glutamine to hydroxy-L-glutamine. Finally, the yield of N-hydroxyamino-N'-acetylguanamine was only 8.56 g / L, and N-acetylguanamine was produced.

[0073] Comparative Example 4 Compared with Example 1, the difference lies in that the dissolved oxygen DO in the second-stage fermentation was 60%, and the other conditions were the same.

[0074] When fermentation reached 72 h, the fermentation ended. The maximum biomass OD600 during the second-stage fermentation process was 105.2. The yield of N-hydroxyamino-N'-acetylguanamine in the final fermented broth was 4.48 g / L, accounting for 90.2%.

[0075] Compared with Example 1, under the second-stage fermentation conditions of this Comparative Example 4, the strain grew abnormally. 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.

[0076] Comparing Example 1 and Comparative Example 4, Example 1 adopted a process including a first stage for cell proliferation and a second stage using an inducer and a hydroxylase cofactor (Fe 2+ions) and increased dissolved oxygen jointly induced the expression of P450 monooxidase in the chassis strain to oxidize L-glutamine to hydroxy-L-glutamine, thereby inhibiting the production of indigo in the engineered bacteria HG-N-Idg06 and using one molecule of L-glutamine and one molecule of hydroxy-L-glutamine to produce N-hydroxyamino-N'-acetyl indigo indigo. It can achieve the goal of converting the engineered bacteria HG-N-Idg06 from producing N-acetyl indigo to producing N-hydroxyamino-N'-acetyl indigo indigo, and the yield of N-hydroxyamino-N'-acetyl indigo indigo is 15.08g / L ( Figure 1 ), accounting for 90.0% ( Figure 2 ).

[0077] Table 2 Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. An N-hydroxyamino-N'-acetyl indole, characterized in that: The chemical structure of the N-hydroxyamino-N'-acetyl indole is shown in formula (I); Formula (I).

2. A method for fermenting the N-hydroxyamino-N'-acetyl indigo plant as claimed in claim 1, characterized in that: The following steps are involved: S1. Inoculate the activated recombinant strain seed fermentation liquid into the basic fermentation medium for the first stage fermentation culture, the dissolved oxygen DO is 20-25%, and the first stage fermentation liquid is obtained by fermentation; S2. Adjust the pH of the first-stage fermentation broth obtained in step S1, then add a synthase inducer and a hydroxylase cofactor to carry out a second-stage fermentation culture, with the dissolved oxygen DO being 40-45%, and fermenting to obtain a fermentation product containing N-hydroxyamino-N'-acetyl indigo.

3. The method according to claim 2, characterized in that The basic fermentation medium includes the following components in mass concentrations: 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, VB10.2~0.4 mg / L, sodium glutamate 1~2 g / L, solvent is water, pH 7.

0.

4. The method according to claim 2, characterized in that The recombinant strain is constructed by introducing expression vector I and expression vector II into the strain; The expression vector I is a plasmid in which the glutamine synthetase encoding gene EcglnA and the N-acetylglutamate synthetase encoding gene EcargA are inserted into two multiple cloning sites to obtain the expression vector I; The expression vector II is a plasmid in which the cyanobacterium synthase encoding gene bpsA and the 4'-phosphopantetheinyl transferase encoding gene entD are inserted into two multiple cloning sites to obtain the expression vector II.

5. The method according to claim 4, characterized in that The strains include Escherichia coli.

6. The method according to claim 4, characterized in that The plasmids include the pRSFDuet-1 plasmid.

7. The method according to claim 2, characterized in that In step S1, the conditions of the first stage fermentation culture include: temperature of 35-40°C; fermentation culture time of 0-24h; ventilation volume of 0.8-1.0vvm.

8. The method according to claim 2, characterized in that In step S2, the conditions for the second stage fermentation culture include: temperature of 20-30°C; fermentation culture time of 24-72h; and ventilation volume of 1.8-2.0vvm.

9. The method according to claim 2, characterized in that In step S2, the pH of the fermentation liquid in the first stage is 6.0-6.

5.

10. The method according to claim 2, characterized in that In the step S2, the first stage fermentation broth is subjected to the second stage fermentation culture, and when the glucose concentration of the fermentation broth is lower than 2 g / L, feed medium is added; The feed medium includes the following components at the following concentrations: Glucose 400~600 g / L, magnesium sulfate heptahydrate 2~4g / L, ammonium chloride 40~80 g / L, the solvent is water.

11. The method according to claim 2, characterized in that In the step S2, the synthetase inducer includes IPTG inducer; and the hydroxylase cofactor includes ferrous sulfate heptahydrate.

12. The method according to claim 11, characterized in that 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.

13. A fermentation product, characterized in that The fermentation products include byproduct 1 and byproduct 2; The chemical structures of the byproduct 1 and byproduct 2 are shown in Formula II; Formula II.

14. Use of N-hydroxyamino-N'-acetyl indigo as claimed in claim 1 in fabric dyeing.

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