Method for directionally preparing indigo blue or indirubin by biocatalytically converting indole glycoside fermentation liquor

Abstract

The invention discloses a method for directionally preparing indigo blue or indirubin by biocatalytically converting indoleglycoside fermentation liquor, which is characterized in that beta-glucosidase Asbg1 and flavin-dependent monooxygenase MaFMO are combined, and the indoleglycoside fermentation liquor is used as a substrate to directionally prepare the indigo blue or indirubin. A glucose dehydrogenase (GDH)-mediated NADPH circulating system is introduced as a process supply cofactor, and then process conditions are optimized and regulated, so that a process for directionally preparing the indigo blue or indirubin by taking the indole glycoside fermentation liquor as a substrate is established, and the directional preparation of the indigo blue or indirubin is realized. The process provides a method for realizing green and efficient directional preparation of indigo blue or indirubin.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for directional preparation of indole glycoside fermentation broth into indigo or indirubin through biocatalytic conversion. Background Art

[0002] Indigo and its isomer, indirubin, are important natural pigments with a long history of use in industries such as food, medicine, dyeing, and cosmetics. Global annual demand for indigo is approximately 80,000 tons. Its distinctive deep blue hue is often used for denim dyeing, food preparation, and biochemical indicator preparation. Indirubin is the core active ingredient in the traditional Chinese medicine Indigo Naturalis. As the active ingredient in Danggui Longhui Wan (Danggui Longhui Wan), it exhibits significant anti-cancer activity and is clinically used in the treatment of chronic myeloid leukemia.

[0003] Currently, the industrial production of indigo and indirubin relies primarily on three methods: plant extraction, chemical synthesis, and microbial conversion. Plant extraction typically involves extracting the precursor indole glycosides from plants such as Amaranthus serrata, followed by enzymatic hydrolysis and oxidation to produce the target products. However, due to the scarcity of plant resources, the complex extraction process, and the low yield and purity, it is difficult to meet the needs of large-scale production. Chemical synthesis, with its advantages of high efficiency, high product purity, and simple process, is currently the main method for producing indigo and indirubin. However, the synthesis requires the use of toxic catalysts, generates heavy metal-containing wastewater, and produces toxic byproducts, posing serious environmental and safety risks. Since the first report of microbial enzyme-based indigo synthesis in 1928, this method has attracted attention for its environmentally friendly nature. Indigo-producing plants are rich in indole glycosides, the precursors required for the synthesis of indigo and indirubin. By removing the glucose at the 3-position using β-glucosidase, indole glycosides undergo spontaneous oxidative condensation in an alkaline and aerobic environment to produce indigo or indirubin. However, there are still many bottlenecks in the actual application process. For example, the indole glycoside products obtained by heterologous biosynthesis pathways, due to the complexity and diversity of their chemical composition, can strongly inhibit the activity of enzyme preparations such as β-glucosidase and monooxygenase, thereby directly weakening the rate and efficiency of the enzymatic reaction, affecting the yield and purity of the final product, and thus posing a severe challenge to the economy and feasibility of the entire production process. In addition, the complex and diverse components of indole glycoside products can also cause pH fluctuations in the reaction system. The presence of potential competitive substrates or inhibitors leads to a decrease in the selectivity of the target product, which seriously hinders the effective production of the target product. Summary of the Invention

[0004] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0005] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0006] One of the objectives of the present invention is to provide a method for the targeted production of indigo or indirubin by biocatalytic conversion of indole glycoside fermentation broth. After pretreatment of the indole glycoside fermentation broth, β-glucosidase is used in combination with flavin-dependent monooxygenase, assisted by glucose dehydrogenase that can generate NADPH to provide a cofactor, and finally, the process reaction conditions are regulated to achieve the targeted production of indigo or indirubin.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for directional preparation of indole glycoside fermentation broth by biocatalytic conversion, comprising:

[0008] β-glucosidase and flavin-dependent monooxygenase are added to the indole glycoside fermentation broth, based on 200 mg / L indole glycoside, with a reaction system pH of 7.0-10.0, a potassium dihydrogen phosphate-disodium hydrogen phosphate buffer concentration of 50-200 mM, a temperature of 35-55° C., a reaction shaking speed of 50-250 r / min, and a reaction time of 30-300 min;

[0009] Wherein, the β-glucosidase is directly used in combination with the flavin-dependent monooxygenase, or the recombinant bacteria expressing the β-glucosidase and the flavin-dependent monooxygenase are used in combination;

[0010] The β-glucosidase is derived from bacteria or fungi and can be obtained by microbial culture, fermentation, separation and purification, or by gene cloning, expression, separation and purification;

[0011] The flavin-dependent monooxygenase includes one from: Hydrogenovibrio sp. JE_KL2, Pseudomonadotabacterium and Methylophagasp. strain SK1.

[0012] As a preferred embodiment of the method for the directed production of indigo by biocatalytic conversion of indole glycoside fermentation broth of the present invention, the β-glucosidase is Asbg1 derived from the surface soil of a winter wheat field, the nucleotide sequence encoding the Asbg1 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2;

[0013] The flavin-dependent monooxygenase is derived from MaFMO of Methylophagasp. strain SK1. The nucleotide sequence encoding the MaFMO is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0014] As a preferred embodiment of the method for the directional preparation of indigo by biocatalytic conversion of indole glycoside fermentation broth of the present invention, the amount of β-glucosidase Asbg1 added to the reaction system is 0.02-0.14 mg / mL, based on 200 mg / L indole glycoside; and the amount of flavin-dependent monooxygenase MaFMO added is 0.17-1.00 mg / mL.

[0015] As a preferred embodiment of the method for the directional preparation of indigo by biocatalytic conversion of indole glycoside fermentation broth of the present invention, the amount of β-glucosidase Asbg1 added to the reaction system is 0.09 mg / mL, based on 200 mg / L indole glycoside; and the amount of flavin-dependent monooxygenase MaFMO added is 0.67 mg / mL.

[0016] As a preferred embodiment of the method for the directional production of indole glycosides by biocatalytic conversion of indole glycoside fermentation broth of the present invention, the method further comprises: adding glucose dehydrogenase to the reaction system; supplementing 100 to 600 μM NADP in the initial reaction system based on 200 mg / L indole glycosides; + After the reaction was carried out for 10 to 60 minutes, 0.124 to 0.744 U / mL glucose dehydrogenase was added;

[0017] The glucose dehydrogenase comprises one of the following sources: Bacilus subtilis and Burkholderia cenocepacia.

[0018] As a preferred embodiment of the method for the directed production of indigo by biocatalytic conversion of indole glycoside fermentation broth of the present invention, the glucose dehydrogenase is derived from the glucose dehydrogenase GDH of Bacilus subtilis, the nucleotide sequence encoding the GDH is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0019] As a preferred embodiment of the method for preparing indigo by biocatalytic conversion of indole glycoside fermentation broth, the reaction system is supplemented with 200 μM NADP based on 200 mg / L indole glycoside. + After the reaction was carried out for 30 minutes, 0.620 U / mL glucose dehydrogenase GDH was added, the reaction pH was 7.5, the concentration of potassium dihydrogen phosphate-disodium hydrogen phosphate buffer was 100 mM, the temperature was 40°C, and a static reaction mode was adopted.

[0020] Another object of the present invention is to provide a method for preparing indirubin by biocatalytic conversion of indole glycoside fermentation broth, comprising:

[0021] β-glucosidase, flavin-dependent monooxygenase and glucose dehydrogenase were added to the indole glycoside fermentation broth, and the concentration of potassium dihydrogen phosphate-disodium hydrogen phosphate buffer was 50-200 mM based on 200 mg / L indole glycoside. The reaction pH was 7.0-10.0, the amount of cysteine ​​added was 3-7 mM, the reaction shaking speed was 50-250 r / min, the temperature was 35-55 ° C, and NADP + The addition amount is 100-600 μM, and the reaction time is 30-300 min;

[0022] Wherein, the β-glucosidase, flavin-dependent monooxygenase and glucose dehydrogenase are directly used in combination, or a recombinant bacterium expressing β-glucosidase is used in combination with a recombinant bacterium expressing flavin-dependent monooxygenase and glucose dehydrogenase;

[0023] The β-glucosidase is derived from bacteria or fungi and can be obtained by microbial culture, fermentation, separation and purification, or by gene cloning, expression, separation and purification;

[0024] The flavin-dependent monooxygenase comprises one of the following sources: Hydrogenovibrio sp. JE_KL2, Pseudomonadotabacterium and Methylophagasp. strain SK1;

[0025] The glucose dehydrogenase comprises one of the following sources: Bacilus subtilis and Burkholderia cenocepacia.

[0026] As a preferred embodiment of the method for the directed production of indirubin by biocatalytic conversion of indole glycoside fermentation broth of the present invention, the β-glucosidase is Asbg1 derived from the surface soil of a winter wheat field, the nucleotide sequence encoding the Asbg1 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2;

[0027] The flavin-dependent monooxygenase is derived from MaFMO of Methylophagasp. strain SK1, the nucleotide sequence encoding the MaFMO is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4;

[0028] The glucose dehydrogenase is derived from the glucose dehydrogenase GDH of Bacilus subtilis. The nucleotide sequence encoding the GDH is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0029] Based on 200 mg / L indole glycoside, in the reaction system, the addition amount of β-glucosidase Asbg1 is 0.02-0.14 mg / mL; the addition amount of flavin-dependent monooxygenase MaFMO is 0.33-1.33 mg / mL; and the addition amount of glucose dehydrogenase GDH is 0.124-0.620 U / mL.

[0030] As a preferred embodiment of the method for the directional preparation of indirubin by biocatalytic conversion of indole glycoside fermentation broth of the present invention, the method comprises the following steps: using indole glycoside fermentation broth as a substrate, based on 200 mg / L indole glycoside, adding 0.11 mg / mL of β-glucosidase Asbg1; adding 1.00 mg / mL of flavin-dependent monooxygenase MaFMO; and performing the reaction with glucose dehydrogenase GDH. The concentration of potassium dihydrogen phosphate-disodium hydrogen phosphate buffer is 100 mM, the reaction pH is 9.0, the amount of cysteine ​​added is 4 mM, the reaction is allowed to stand at a temperature of 50°C, the amount of glucose dehydrogenase GDH added is 0.372 U / mL, and the amount of NADP is 0. + The amount added was 100 μM).

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a method for the targeted production of indigo or indirubin by biocatalytic conversion of indole glycoside fermentation broth, creating a new pathway and technology for the targeted production of indigo or indirubin. The present invention first pretreats the indole glycoside fermentation broth, then combines β-glucosidase Asbg1 with flavin-dependent monooxygenase MaFMO, and adds glucose dehydrogenase GDH to provide the cofactor NADPH. This establishes a process for the targeted production of indigo or indirubin using the indole glycoside fermentation broth as a substrate, providing a rational approach for the green and sustainable production of indigo or indirubin. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0034] Figure 1 Schematic diagram of the process flow;

[0035] Figure 2 Schematic diagram of process optimization for the biocatalytic conversion of indole glycoside fermentation broth to produce indigo (A. reaction pH; B. buffer concentration; C. reaction temperature; D. oscillation speed);

[0036] Figure 3 The effect of GDH and co-substrate on the biocatalytic conversion of indole glycoside fermentation broth to produce indigo (A. GDH addition amount; B. GDH addition time; C. NADP + Added amount; D. Glucose added amount);

[0037] Figure 4 The reaction process analysis diagram for the biocatalytic conversion of indole glycoside fermentation broth to produce indigo;

[0038] Figure 5 Schematic diagram of process optimization for the biocatalytic conversion of indole glycoside fermentation broth to produce indirubin (A. buffer concentration; B. reaction pH; C. cysteine ​​addition amount; D. oscillation speed; E. reaction temperature);

[0039] Figure 6 The effect of GDH and co-substrate on the biocatalytic conversion of indole glycoside fermentation broth to produce indirubin (A. GDH addition amount; B. NADP + Added amount; C. Glucose added amount);

[0040] Figure 7 This is an analysis diagram of the reaction process for the targeted preparation of indirubin from the biocatalytic conversion of indole glycoside fermentation broth. DETAILED DESCRIPTION

[0041] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0042] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0043] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0044] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0045] In the embodiment, the culture medium used is:

[0046] (1) Luria-Bertani (LB) medium: Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, dissolve in distilled water, and dilute to 1 L (adjust pH to 7.0–7.5 with sodium hydroxide). Sterilize at 121°C for 20 min. When preparing LB solid medium, add 20 g / L agar powder before autoclaving.

[0047] (2) Terrific Broth (TB) medium: Solution A: Weigh 12 g of tryptone, 24 g of yeast extract, and 5 g of inulin, dissolve in distilled water, and dilute to 900 mL. Sterilize at 121°C for 20 min. Solution B: Weigh 16.43 g of K2HPO4 and 2.31 g of KH2PO4, dilute to 100 mL with water. Prepare before use, sterilize with a 0.22 μm filter in a clean bench, and add to Solution A.

[0048] The relevant buffer solutions used in the examples were purchased from Quanshijin (Beijing) Biotechnology Co., Ltd.

[0049] The indole glycosides used in the examples were purchased from Nanjing Yuanzhi Biological Co., Ltd.

[0050] The test method used in the embodiment of the present invention is:

[0051] Molar conversion rate = (yield of indigo or indirubin / 262.29) / (concentration of indole glycoside fermentation broth / 295.29) × 100%.

[0052] Example 1 Cloning and expression of β-glucosidase Asbg1, flavin-dependent monooxygenase MaFMO, and glucose dehydrogenase GDH

[0053] (1) Cloning of the β-glucosidase gene Asbg1

[0054] β-glucosidase Asbg1 (GenBank accession No. AF494423.2) was codon-optimized according to the preferred codons of Escherichia coli and synthesized by Sangon Biotech (Shanghai) Co., Ltd. in the pET-28a vector to generate the recombinant plasmid pET-28a-Asbg1. The nucleotide sequence encoding Asbg1 is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0055] (2) Cloning of the flavin-dependent monooxygenase gene MaFMO

[0056] The flavin-dependent monooxygenase MaFMO (GenBank accession No. KF660588) was codon-optimized according to the preferred codons of Escherichia coli and synthesized by Sangon Biotech (Shanghai) Co., Ltd. in the pETDuet-1 vector, generating the recombinant plasmid pETDuet-1-MaFMO. The nucleotide sequence encoding MaFMO is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.

[0057] (3) Cloning of the glucose dehydrogenase gene GDH

[0058] Glucose dehydrogenase GDH (GenBank accession No. EC1.1.1.47) was codon-optimized according to the preferred codons of Escherichia coli and synthesized in pETDuet-1 by Sangon Biotech (Shanghai) Co., Ltd. to obtain the recombinant plasmid pETDuet-1-GDH. The nucleotide sequence encoding GDH is shown in SEQ ID NO. 5, and the amino acid sequence is shown in SEQ ID NO. 6.

[0059] (4) Expression and purification of β-glucosidase Asbg1

[0060] The recombinant Escherichia coli BL21 (DE3) containing the plasmid pET-28a-Asbg1 was activated in a 5 mL LB tube (final concentration of kanamycin was 50 mg / L), cultured at 37°C for 12 h, and then inoculated into 50 mL TB liquid medium. The culture was shaken at 37°C and 180 rpm until the OD 600 =1.4. Without the addition of inducers, the culture was continued at 25°C for 22 hours. The culture medium was centrifuged at 8000 rpm for 10 minutes, and the cells were harvested. The cells were then resuspended in 1× PBS (pH 7.4) and sonicated at an appropriate power (3 seconds, 4 seconds intervals, for a total of 99 times). The supernatant was then centrifuged at 12000 rpm for 10 minutes to obtain the crude recombinant Asbg1 enzyme solution.

[0061] The crude recombinant Asbg1 enzyme solution was filtered through a 0.22 μm membrane and purified using a Ni-NTA prepacked gravity column. The column was equilibrated with 2 to 5 column volumes of 25 mm NaH2PO4-Na2HPO4 buffer, followed by 2 column volumes of crude enzyme solution for binding. Finally, the recombinant protein was eluted with buffer containing varying concentrations of imidazole (20, 50, 100, 200, and 400 mM). The 50 mM and 100 mM imidazole eluates were collected. After 48 hours of dialysis, pure recombinant Asbg1 enzyme solution was obtained.

[0062] (5) Expression of flavin-dependent monooxygenase MaFMO

[0063] The recombinant Escherichia coli BL21 (DE3) containing the plasmid pETDuet-1-MaFMO was activated in 5 mL LB tubes (final concentration of ampicillin was 100 mg / L), cultured at 37°C for 12 h, and then inoculated into 300 mL LB liquid medium. The culture was shaken at 37°C and 180 rpm until the OD reached 0. 600 =0.6-0.8, add 0.1 mM IPTG, and continue culturing at 18°C ​​for 20 hours. Centrifuge the culture medium at 8000 rpm for 10 minutes, then collect the cells. Resuspend the cells in 1× PBS (pH 7.4) and sonicate at an appropriate power (3 seconds, 4 seconds intervals, 99 times). Centrifuge at 12000 rpm for 10 minutes. The resulting supernatant is the crude recombinant MaFMO enzyme solution.

[0064] (6) Expression of glucose dehydrogenase GDH

[0065] The recombinant Escherichia coli BL21 (DE3) containing the plasmid pETDuet-1-GDH was activated in a 5 mL LB tube (the final concentration of ampicillin was 100 mg / L), cultured at 37°C for 12 h, and then inoculated into 300 mL LB liquid medium. The culture was shaken at 37°C and 180 rpm until the OD 600 =0.6-0.8, add 0.6 mM IPTG, and continue culturing at 28°C for 22 hours. Centrifuge the culture medium at 8000 rpm for 10 minutes, then collect the cells. Resuspend the cells in 1× PBS (pH 7.4) and sonicate at an appropriate power (3 seconds, 4 seconds intervals, 99 times). Centrifuge at 12000 rpm for 10 minutes. The resulting supernatant is the crude recombinant GDH enzyme solution.

[0066] Example 2 Pretreatment of indole glycoside fermentation broth

[0067] (1) Preparation of indole glycoside fermentation broth

[0068] Escherichia coli, containing genes for tryptophanase, flavin-dependent monooxygenase, glucosyltransferase, glucose dehydrogenase, cellobiose phospholyase, and glucose-1-phosphate uridyltransferase, is used as the host bacteria. Heterologous indole glycoside synthesis is achieved in TB medium supplemented with 1% inulin as the carbon source, using tryptophan as the substrate and supplemented with 0.1 mM IPTG, cellobiose, and a small amount of glucose for 48-72 hours.

[0069] (2) Pretreatment of indole glycoside fermentation broth

[0070] After the heterologous synthesis reaction of indole glycosides is completed, the fermentation broth is centrifuged at 12,000 rpm for 20 minutes to separate the bacterial cells. The resulting supernatant is the indole glycoside fermentation broth. Equal proportions of ultrapure water are added to the indole glycoside fermentation broth, and after oscillation and mixing, the broth is centrifuged at 12,000 rpm for 15 minutes to remove the water-insoluble precipitate. The supernatant is adjusted to pH 7.0 using a glycine-NaOH buffer with a pH of 9.0. After oscillation and mixing, the broth is centrifuged at 12,000 rpm for 15 minutes to remove the precipitate. The fermentation broth is then boiled using a microwave method over medium-high heat until the volume is half of the initial volume. The broth is then centrifuged again at 12,000 rpm for 15 minutes to remove the precipitate. This operation is repeated to concentrate the fermentation broth. Finally, the pH of the concentrated fermentation broth is again adjusted to 7.0 using a glycine-NaOH buffer to obtain the indole glycoside concentrate.

[0071] Example 3: Directed production of indole glycosides or indirubin from fermentation broth using biocatalytic conversion

[0072] The total reaction volume (100 μL) contained 200 mg / L indole glycoside concentrate, 0.02 mg / mL Asbg1 enzyme, and 1.50 mg / mL MaFMO enzyme. The Eppendorf tube was incubated at 45°C for 1 hour. After completion, 1 mL of DMF was added to terminate the reaction. The sample was sonicated until the colored precipitate was completely dissolved. After centrifugation at 12,000 rpm for 10 minutes, the product was filtered through a 0.22 μm membrane and assayed for indigo or indirubin content using HPLC.

[0073] The yield of indigo or indirubin was determined using an HPLC 1200 high-performance liquid chromatograph (Agilent, USA) with an XDB-C18 column (4.6 × 250 mm; id, 5 μm) at a flow rate of 0.8 mL / min and a column temperature of 30°C. The HPLC analysis lasted 10 minutes, with the mobile phase consisting of water and acetonitrile in a ratio of 30%:70%, and the wavelength was 290 nm.

[0074] Example 4 Optimization of conditions for the directional production of indole glycosides from fermentation broth using biocatalytic conversion

[0075] (1) Optimization of enzyme addition

[0076] Based on Example 3, the addition amounts of Asbg1 enzyme and MaFMO enzyme were adjusted to find the optimal enzyme addition ratio for the directional production of indigo. As shown in Table 1, the addition amounts of Asbg1 enzyme and MaFMO enzyme and the corresponding indigo yield results are recorded.

[0077] Table 1

[0078]

[0079] (2) Optimization of process conditions

[0080] Based on Example 3, the process conditions, including reaction pH (6.0-9.0), glycine-NaOH buffer concentration (50-200 mM), reaction temperature (40-55 ° C) and shaking speed (50-300 rpm), were optimized. Figure 2 As shown, Figure 2 a is the reaction pH optimization result, which shows that at a pH of 7.5, the indigo yield reaches the maximum value, and the presence of the by-product indirubin is not detected, but the yield is low. Figure 2 b is the optimization result of the glycine-NaOH buffer concentration that needs to be supplemented in the system. The results show that when the additional concentration of Gly-NaOH buffer is 100 mM, the indigo production is significantly improved, with an increase of 51.6%. Figure 2 c is the reaction temperature optimization result, and the results show that the optimal reaction temperature is 40℃. Figure 2 d is the effect of oscillation reaction rate on the reaction. The results show that the introduction of oscillation reaction significantly inhibits the yield of indigo, that is, static reaction is the optimal reaction condition.

[0081] (3) Effects of GDH and cosubstrate glucose

[0082] On the basis of Example 3, the addition amount of glucose dehydrogenase (0.124-0.744 U / mL) and the addition time (10-60 min), as well as NADP + The experimental results are shown in Figure 2. Figure 3 As shown, Figure 3 a is the optimization result of the addition amount of glucose dehydrogenase. The results show that the optimal addition amount of glucose dehydrogenase is 0.620 U / mL. Figure 3 b is the optimization result of the addition timing of glucose dehydrogenase. The results show that adding GDH after 30 minutes of reaction can promote the increase of indigo production. Figure 3 c is NADP + The results of the optimization of the addition amount showed that the addition of 200 μM NADP + The GDH-mediated NADPH cycle can be initiated. Figure 3 d is the optimization result of the glucose addition amount. The results show that since the glucose in the indole glycoside hydrolysate can serve as one of the co-substrates required by GDH, there is no need to supplement glucose.

[0083] (4) Reaction process analysis

[0084] On the basis of Example 3, the reaction process was analyzed, that is, the reaction was terminated after 30 to 5 hours. Figure 4 As shown, the results indicate that the initial concentration of indole glycoside in the reaction system was 471.28 mg / L, while no indole glycoside was detected in the system at this time. By 150 minutes into the reaction, the indole glycoside was almost completely consumed, approaching zero, while the indole yield reached 204.57 mg / L. At this point, the molar conversion reached 99.1%. Thereafter, even with extended reaction times, the indole yield remained almost constant, and the residual indole glycoside content remained constant, indicating that the reaction had reached equilibrium.

[0085] Example 5 Optimization of conditions for the directional production of indirubin from biocatalytic conversion of indole glycoside fermentation broth

[0086] (1) Optimization of enzyme addition

[0087] Based on Example 3, the addition amounts of Asbg1 enzyme and MaFMO enzyme were adjusted to find the optimal process enzyme addition ratio for the directional preparation of indirubin. As shown in Table 2, the addition amounts of Asbg1 enzyme and MaFMO enzyme and the corresponding indirubin yield results were recorded.

[0088] Table 2

[0089]

[0090]

[0091] (2) Optimization of process conditions

[0092] Based on Example 3, the process conditions were optimized, including the concentration of glycine-NaOH buffer (50-200 mM), reaction pH (6.0-9.0), cysteine ​​addition (3-8 mM), shaking speed (50-300 rpm), and reaction temperature (40°C-55°C). Figure 5 As shown, Figure 5 a is the optimization result of the glycine-NaOH buffer concentration, indicating that the optimal glycine-NaOH buffer concentration is 100 mM. Compared with the control group without added buffer, the yield of the target product at this concentration increased by 86.2%. Figure 5 b shows the pH optimization results of the reaction system, which show that the optimal pH is 9. As the reaction environment shifts further toward alkalinity, the yield of indirubin decreases, and no product is generated at pH 10. Figure 5 Figure c shows the optimized cysteine ​​concentration added to the reaction system. The results show that 4 mM is the optimal cysteine ​​concentration. When the amount of cysteine ​​added exceeds this optimal value, the indirubin yield decreases. Figure 5 d is the effect of the oscillating reaction rate on the reaction. The results show that the oscillating reaction has a negative impact on the formation of the product, that is, with the increase of the rotation speed, the output of indirubin shows a decreasing trend, so the oscillating reaction should be selected. Figure 5 e is the temperature optimization result of the reaction system, and the results show that 50℃ is the optimal temperature.

[0093] (3) Effects of GDH and cosubstrate glucose

[0094] Based on Example 3, the addition amount of glucose dehydrogenase (0.124-0.620 U / mL) and NADP + The experimental results are shown in Figure 2. Figure 6 As shown, Figure 6 a is the optimization result of the addition amount of glucose dehydrogenase. The results show that the optimal addition amount of glucose dehydrogenase is 0.620 U / mL. Figure 6 b is NADP + The results of the optimization of the addition amount showed that only 100 μM NADP was needed. + A reaction system can be successfully constructed that can effectively replace the direct use of NADPH and significantly promote the increase in indigo carmine production. Figure 6 c is the optimization result of the glucose addition amount, which shows that there is no need to add additional glucose as a co-substrate for the GDH reaction.

[0095] (4) Reaction process analysis

[0096] Based on Example 3, the reaction process was analyzed, that is, the reaction was terminated after 30 to 300 minutes. Figure 7 As shown in the figure, the results show that at the initial stage of the reaction, the indole glycoside concentration was set at 360.68 mg / L, at which point no indirubin was detected in the system. At 150 minutes into the reaction, the indole glycoside concentration dropped significantly to 89.54 mg / L. At this point, the indirubin yield reached 62.12 mg / L, corresponding to a molar conversion of 51.6%, indicating that the reaction reached equilibrium.

[0097] The present invention provides a method for the targeted production of indigo or indirubin by biocatalytic conversion of indole glycoside fermentation broth, creating a new process and technology for the preparation of indigo or indirubin. The present invention uses β-glucosidase in combination with flavin-dependent monooxygenase to biocatalytically convert pretreated indole glycoside fermentation broth as a substrate to produce indigo or indirubin, and is assisted by the NADPH circulation system mediated by glucose dehydrogenase to provide a cofactor ( Figure 1), and further adjust the process conditions to achieve the directional preparation of indigo or indirubin.

[0098] For the directional preparation of indigo, the optimal conditions are a reaction pH of 7.5, a potassium dihydrogen phosphate-sodium dihydrogen phosphate buffer concentration of 100mM, a temperature of 40°C, a static reaction, the addition of 0.09mg / mL Asbg1 and 0.67mg / mL MaFMO. The reaction system also needs to be supplemented with 200μM NADP. + No additional glucose was added, and after 30 minutes of reaction, 0.620 U / mL of glucose dehydrogenase (GDH) was added. After 150 minutes of reaction, the maximum indigo yield was 204.57 mg / L, with a molar conversion rate of 99.1%.

[0099] For the directional preparation of indirubin, the optimal conditions were: potassium dihydrogen phosphate-sodium dihydrogen phosphate buffer with a concentration of 100 mM, a reaction pH of 9.0, 4 mM cysteine, a static reaction temperature of 50°C, 0.372 U / mL of glucose dehydrogenase GDH, and NADP + The addition amount of 100 μM was used, and no additional glucose was added. 0.11 mg / mL Asbg1 and 1.00 mg / mL MaFMO were added. After 150 minutes of reaction, the maximum indirubin yield was 62.12 mg / L, with a molar conversion of 51.6%.

[0100] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for producing indigo by biocatalytic conversion of indole glycoside fermentation broth, characterized by: include, β-glucosidase and flavin-dependent monooxygenase are added to the indole glycoside fermentation broth, based on 200 mg / L indole glycoside, with a reaction system pH of 7.0-10.0, a potassium dihydrogen phosphate-disodium hydrogen phosphate buffer concentration of 50-200 mM, a temperature of 35-55° C., a reaction shaking speed of 50-250 r / min, and a reaction time of 30-300 min; Wherein, the β-glucosidase is directly used in combination with the flavin-dependent monooxygenase, or the recombinant bacteria expressing the β-glucosidase and the flavin-dependent monooxygenase are used in combination; The β-glucosidase is derived from bacteria or fungi and can be obtained by microbial culture, fermentation, separation and purification, or by gene cloning, expression, separation and purification; The flavin-dependent monooxygenase includes one from: Hydrogenovibrio sp. JE_KL2, Pseudomonadotabacterium and Methylophagasp. strain SK1.

2. The method for producing indigo by biocatalytic conversion of indole glycoside fermentation broth according to claim 1, characterized in that: The β-glucosidase is Asbg1 from the surface soil of winter wheat fields. The nucleotide sequence encoding the Asbg1 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; The flavin-dependent monooxygenase is derived from MaFMO of Methylophagasp. strain SK1. The nucleotide sequence encoding the MaFMO is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

3. The method for producing indigo by biocatalytic conversion of indole glycoside fermentation broth according to claim 1, wherein: Based on 200 mg / L indole glycoside, the added amount of β-glucosidase Asbg1 in the reaction system is 0.02-0.14 mg / mL; the added amount of flavin-dependent monooxygenase MaFMO is 0.17-1.00 mg / mL.

4. The method for producing indigo by biocatalytic conversion of indole glycoside fermentation broth according to claim 1, wherein: Based on 200 mg / L indole glycoside, the added amount of β-glucosidase Asbg1 in the reaction system was 0.09 mg / mL; the added amount of flavin-dependent monooxygenase MaFMO was 0.67 mg / mL.

5. The method for producing indigo by biocatalytic conversion of indole glycoside fermentation broth according to claim 1, characterized in that: The method further includes adding glucose dehydrogenase to the reaction system; based on 200 mg / L indole glycoside, the initial reaction system is supplemented with 100-600 μM NADP + After the reaction was carried out for 10 to 60 minutes, 0.124 to 0.744 U / mL glucose dehydrogenase was added; The glucose dehydrogenase includes one of the following sources: Bacilus subtilis and Burkholderiacenocepacia.

6. The method for producing indigo by biocatalytic conversion of indole glycoside fermentation broth according to claim 1, wherein: The glucose dehydrogenase is derived from the glucose dehydrogenase GDH of Bacilus subtilis. The nucleotide sequence encoding the GDH is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.

6.

7. The method for producing indigo by biocatalytic conversion of indole glycoside fermentation broth according to claim 1, wherein: Based on 200 mg / L indole glycoside, 200 μM NADP was added to the reaction system. + After the reaction was carried out for 30 minutes, 0.620 U / mL glucose dehydrogenase GDH was added, the reaction pH was 7.5, the concentration of potassium dihydrogen phosphate-disodium hydrogen phosphate buffer was 100 mM, the temperature was 40°C, and a static reaction mode was adopted.

8. A method for producing indirubin by biocatalytic conversion of indole glycoside fermentation broth, characterized by: include, β-glucosidase, flavin-dependent monooxygenase and glucose dehydrogenase were added to the indole glycoside fermentation broth, and the concentration of potassium dihydrogen phosphate-disodium hydrogen phosphate buffer was 50-200 mM based on 200 mg / L indole glycoside. The reaction pH was 7.0-10.0, the amount of cysteine ​​added was 3-7 mM, the reaction shaking speed was 50-250 r / min, the temperature was 35-55 ° C, and NADP + The addition amount is 100-600 μM, and the reaction time is 30-300 min; Wherein, the β-glucosidase, flavin-dependent monooxygenase and glucose dehydrogenase are directly used in combination, or a recombinant bacterium expressing β-glucosidase is used in combination with a recombinant bacterium expressing flavin-dependent monooxygenase and glucose dehydrogenase; The β-glucosidase is derived from bacteria or fungi and can be obtained by microbial culture, fermentation, separation and purification, or by gene cloning, expression, separation and purification; The flavin-dependent monooxygenase comprises one of the following sources: Hydrogenovibrio sp. JE_KL2, Pseudomonadotabacterium and Methylophagasp. strain SK1; The glucose dehydrogenase includes one of the following sources: Bacilus subtilis and Burkholderiacenocepacia.

9. The method for producing indirubin by biocatalytic conversion of indole glycoside fermentation broth according to claim 1, wherein: The β-glucosidase is Asbg1 from the surface soil of winter wheat fields. The nucleotide sequence encoding the Asbg1 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; The flavin-dependent monooxygenase is derived from MaFMO of Methylophagasp. strain SK1, the nucleotide sequence encoding the MaFMO is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; The glucose dehydrogenase is derived from the glucose dehydrogenase GDH of Bacilus subtilis. The nucleotide sequence encoding the GDH is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.

6. Based on 200 mg / L indole glycoside, in the reaction system, the addition amount of β-glucosidase Asbg1 is 0.02-0.14 mg / mL; the addition amount of flavin-dependent monooxygenase MaFMO is 0.33-1.33 mg / mL; and the addition amount of glucose dehydrogenase GDH is 0.124-0.620 U / mL.

10. The method for producing indirubin by biocatalytic conversion of indole glycoside fermentation broth according to claim 1, characterized in that: The indole glycoside fermentation broth was used as the substrate, and the addition amount of β-glucosidase Asbg1 was 0.11 mg / mL based on 200 mg / L indole glycoside; the addition amount of flavin-dependent monooxygenase MaFMO was 1.00 mg / mL and glucose dehydrogenase GDH was used for the reaction. The concentration of potassium dihydrogen phosphate-disodium hydrogen phosphate buffer was 100 mM, the reaction pH was 9.0, the addition amount of cysteine ​​was 4 mM, the reaction was allowed to stand at 50°C, the addition amount of glucose dehydrogenase GDH was 0.372 U / mL, and NADP + The amount added was 100 μM.

Images