CN113698790A

CN113698790A - Method for extracting indigo from indigo pigment engineering bacterium fermentation liquor

Info

Publication number: CN113698790A
Application number: CN202110875041.5A
Authority: CN
Inventors: 程雷; 王成涛; 袁栋栋; 王玥; 潘紫荆
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2021-07-30
Filing date: 2021-07-30
Publication date: 2021-11-26

Abstract

The invention discloses a method for extracting indigo from the fermentation broth of indigo pigment engineering bacteria, comprising the following steps: (a) breaking the cells of the indigo pigment engineering bacteria in the fermentation broth, and removing impurities in the fermentation broth; (b) ) reducing the indigo in the fermentation broth to form a leuco body to obtain a leuco body solution; after removing the insoluble impurities in the leuco body solution, the leuco body is oxidized to obtain an indigo precipitate. In this application, through the comparative analysis of different oxidation methods, the optimal reaction conditions for purifying indigo are proposed. Using the indigo purification method in this application, the final bacterial cell removal rate in the fermentation broth reaches 96.3%, and the removal rate of impurity proteins reaches 96.7%. , the indigo extraction and refining yield reached 89.8%.

Description

Method for extracting indigo from indigo pigment engineering bacterium fermentation liquor

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a method for extracting indigo from an indigo engineering bacterium fermentation broth.

Background

The indigo pigment is one of the oldest dyes known in the world, is bright and durable in color and has a long history in China; the "cyan" from blue to blue "in the" Xun & Chou "study also confirmed the long history of use of indigo, which is indigo, from clothes unearthed from Mawang heaps. The indigo is easy to color and has unique color tone, and has wide application prospect in the industries of food, medicine, printing and dyeing, cosmetics and the like. However, due to the limitation of the current extraction process, the indigo fermented by microorganisms contains a large amount of impurities, so that the application of the indigo in the fields of food, cosmetics and medicines is limited. At present, more researches are carried out, but the related reports on how to improve the separation and extraction yield are very few. The purity of indigo is affected due to the large amount of bacteria and protein impurities in the fermented indigo pigment, and the application of the indigo pigment in the fields of food, cosmetics, medicines and the like is limited. Therefore, the research on the downstream processing technology of the indigo pigment has certain practical significance. Lays a foundation for the application of the indigo in the fields of food, cosmetics and medicines.

The impurities in the fermentation liquor are removed by using a centrifugal method, the centrifugation is a main means for separating thalli and products in the current industrial production, the method is simple and convenient, most impurities in the fermentation liquor can be removed by the centrifugation, but the indigo and the thalli cannot be separated, because the indigo has extremely low solubility in water, and the thalli and the impure proteins are insoluble in water, at the moment, the method for removing the thalli and the impure proteins is required to further remove the impurities. Extraction is one of the main means for extracting pigment, but indigo is a substance which is slightly soluble in water, ethanol, glycerol and propylene glycol, insoluble in oil and fat, and some organic reagents have toxicity, so the indigo cannot be obtained by dissolution and extraction.

Disclosure of Invention

In order to solve the problem that indigo is difficult to extract from the fermentation broth of the engineering bacteria of indigo blue, the invention provides a method for extracting indigo from the fermentation broth of the engineering bacteria of indigo blue, which is realized by adopting the following technical scheme:

a method for extracting indigo from indigo blue engineering bacteria fermentation liquor at least comprises the following steps:

(a) and (3) breaking thalli of the indigo pigment engineering bacteria in the fermentation liquor, and removing the impure proteins in the fermentation liquor.

(b) And reducing the indigo in the fermentation liquor to form a leuco body to obtain a leuco body solution. Removing insoluble impurities in the leuco body solution, and oxidizing the leuco body to obtain indigo precipitate.

Optionally, the indigo pigment engineering bacterium is integrated with a fragment I on the genome of a host; the fragment I contains a styrene monooxygenase gene styA and a styrene monooxygenase gene styB;

optionally, the host is selected from any one of e.coli;

alternatively, the e.coli is e.coli DH5 α.

Optionally, the integration is a nucleotide sequence that replaces the host integration site with fragment I; the integration site is selected from any one of trpR, trpB and hisD.

Optionally, the coding sequence of the styrene monooxygenase gene styA is shown as SEQ ID No. 1;

the coding sequence of the styrene monooxygenase gene styB is shown as SEQ ID NO. 2;

optionally, the fragment I further comprises an inducible promoter, an operon;

arranging an inducible promoter, an operon, a styrene monooxygenase gene styA and a styrene monooxygenase gene styB in sequence;

optionally, the inducible promoter is inducible promoter cat;

the operon is the lactose operon lac.

Optionally, the method for disrupting bacterial cells in step (a) comprises the following steps: adding lysozyme into the fermentation liquor for reaction to break the cell wall of the indigo blue engineering bacteria.

Optionally, the conditions for adding lysozyme to the fermentation liquor for reaction comprise: the enzyme adding amount of the lysozyme added into the fermentation liquor is 0.5 to 4 percent.

Optionally, the amount of lysozyme added to the fermentation broth is 2% -4%.

Optionally, the lysozyme is added to the fermentation broth in an amount of 2%.

Optionally, the stirring speed in the reaction process is 300-900 r/min.

Optionally, the stirring speed in the reaction process is 500-700 r/min.

Optionally, the stirring speed in the reaction process is 600 r/min.

Optionally, the pH value is 6-8, the temperature is 35-45 ℃ and the reaction time is 0.5-6h in the reaction process.

Optionally, the reaction process is carried out at pH 7 and temperature 40 deg.C for 3 h.

Optionally, the method for removing the hetero-protein in the fermentation broth in the step (a) is as follows: adding protease into the fermentation liquor to react and decompose the hybrid protein.

Optionally, the conditions for adding protease to the fermentation broth for reaction include: the enzyme adding amount of the protease added into the fermentation liquor is 1.5 to 5 percent.

Optionally, the protease is added to the fermentation broth in an amount of 4% to 5%.

Alternatively, the protease is added to the fermentation broth in an amount of 4%.

Optionally, the stirring speed in the reaction process is 300-700 r/min.

Optionally, the stirring speed is 500-.

Optionally, the stirring speed is 500r/min during the reaction.

Optionally, the pH value is 1.5-3.5, the temperature is 45-55 ℃ and the reaction time is 0.5-2.5h in the reaction process.

Alternatively, the pH value is 2.5, the temperature is 50 ℃ and the reaction time is 1.5h in the reaction process.

Optionally, in the step (b), the fermentation liquid from which the impure proteins are removed is centrifuged, and the precipitate obtained by centrifugation is added to a reducing agent solution to reduce indigo to form leuco bodies, so as to obtain a leuco body solution.

Optionally, centrifuging the fermentation liquor without the hybrid protein for 20min by a small high-speed centrifuge 9000r/min, discarding the supernatant, drying to obtain crude indigo, weighing a proper amount of crude indigo, and adding a reducing agent solution into the crude indigo produced by fermentation by a full-bath reduction method under the water bath condition to reduce the indigo to form a leuco body.

Optionally, the precipitate obtained by centrifugation is reacted with a reducing agent solution for 20-40min under the condition of water bath at 50-70 ℃.

Optionally, the precipitate obtained by centrifugation is reacted with a reducing agent solution under the condition of water bath at 60 ℃ for 30 min.

Optionally, the reducing agent solution contains a base and a reducing agent.

Optionally, the reducing agent comprises at least one of thiourea dioxide or sodium dithionite.

Optionally, the base is sodium hydroxide.

Optionally, the precipitate: the mass ratio of the reducing agent is 1: 0.5-0.7.

Optionally, during the indigo reduction, the indigo product: sodium hydroxide: thiourea dioxide 1.00:0.61: 0.66.

Optionally, in the step (b), removing insoluble impurities in the leuco body solution by centrifugation, and then adding an oxidizing agent into the leuco body solution to react to obtain indigo precipitate.

Optionally, after the indigo is fully reduced, centrifuging for 5min by using a small high-speed centrifuge 11000r/min, and pouring the supernatant into a clean beaker to obtain a leuco body solution.

Optionally, the oxidizing agent is a hydrogen peroxide solution, and the volume ratio of the hydrogen peroxide solution to the leuco body solution is 1: 1-12.

Optionally, the volume ratio of the hydrogen peroxide solution to the leuco solution is 1: 4-6.

Optionally, the volume ratio of the hydrogen peroxide solution to the leuco solution is 1: 6.

Optionally, the leuco body solution is reacted with an oxidizing agent at 30-70 deg.C and pH 6-11 for 0.5-2.5 h.

Optionally, the leuco body solution is reacted with an oxidizing agent at 35-50 deg.C and pH 9-10.5 for 1.5-2 h.

Alternatively, the leuco solution was reacted with the oxidant at 40 ℃ for 1.5h at pH 10.

The beneficial effects that this application can produce include:

according to the method, the thallus is firstly broken by using lysozyme, the foreign protein in the fermentation liquor is removed by using protease, and then the indigo is reduced and separated from the thallus by using the characteristic that the indigo is easily reduced into salt dissolved in water, so that the purer indigo is obtained by further extracting and refining. Finally, the indigo is oxidized from the salt dissolved in water by using an oxidizing agent for extracting and refining pure indigo particles.

According to the method, through comparative analysis of different oxidation modes, the optimal reaction conditions for purifying the indigo are provided, the final thallus removal rate in the fermentation liquor by adopting the indigo purification method in the application reaches 96.3%, the removal rate of the foreign proteins in the fermentation liquor reaches 96.7%, and the indigo extraction and refining yield reaches 89.8%.

Drawings

FIG. 1 is a diagram showing the construction of the integration recombinant plasmids pHS-AVC-01(A), pHS-AVC-02(B), and pHS-AVC-03 (C);

FIG. 2 shows the integrated recombinant plasmid pHS-AVC-01 and its restriction enzyme-digestion-verified electrophoresis;

FIG. 3 shows the integrated recombinant plasmid pHS-AVC-02 and its restriction enzyme-verified electrophoresis;

FIG. 4 shows an integrated recombinant plasmid pHS-AVC-03 and its restriction enzyme-digestion-verified electrophoresis;

FIG. 5 is a PCR electrophoretogram of colonies integrating recombinant plasmids pHS-AVC-01(A), pHS-AVC-02(B), and pHS-AVC-03 (C);

FIG. 6 is a comparison map of integrated sequences and sequencing of pHS-AVC-01(A), pHS-AVC-02(B), pHS-AVC-03 (C); in the figure, left arm is homologous left arm, right arm is homologous right arm, Promoter is Promoter, and repeat region is repetitive region;

FIG. 7 is an electrophoretogram of a pHS-AVC-01 targeting fragment;

FIG. 8 is an electrophoretogram of pHS-AVC-02, pHS-AVC-03 targeting fragments;

FIG. 9 is a PCR identification chart of colony of engineering bacterium E.coliAB-01;

FIG. 10 is a PCR identification chart of the colonies of engineering bacteria E.coliAB-02 and E.coliAB-03;

FIG. 11 shows the effect of different amounts of lysozyme on the degree of bacterial removal;

FIG. 12 shows the effect of lysozyme on the degree of removal of bacteria at different stirring rates;

FIG. 13 shows the cell contents at each stage in example 1;

FIG. 14 is a graph of the effect of different protease dosages on protein removal in indigo products;

FIG. 15 is a graph of the effect of different agitation rates on protein removal in indigo product;

FIG. 16 shows the protein content at each stage in example 1;

FIG. 17 is a graph of the effect of different reducing agents on the reduction of indigo product; wherein (a) is the content of residual impurities in the indigo product, (b) is the reduction potential value, and (c) is the absorbance of unreduced substrate;

FIG. 18 shows the effect of different oxidants on the refining yield of indigo;

FIG. 19 shows the effect of hydrogen peroxide dosage on refining yield of indigo;

FIG. 20 is a graph showing the effect of oxidation time on the yield of purified indigo from oxidant extraction;

FIG. 21 is a graph showing the effect of temperature on the yield of purified indigo from oxidant extraction;

FIG. 22 is a graph showing the effect of pH on the yield of purified indigo from oxidant extraction.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

The raw materials in the examples of the present application were all purchased commercially, unless otherwise specified.

In the embodiment, the strain using the indigo pigment engineering bacteria is derived from a wild type indigo synthesis strain P.putida B3, a genetic engineering technical means is utilized, an indigo pigment synthesis key enzyme styrene monooxygenase gene styAB is heterologously expressed in an escherichia coli system, and fermentation is carried out by utilizing a fermentation tank to produce indigo. The construction method of the indigo pigment engineering bacteria adopted in the embodiment comprises the following steps:

(1) materials and reagents

The indigo blue standard is purchased from Sigma-Aldrich company, U.S. A, and tryptophan, indole, kanamycin, chloramphenicol, sodium hydroxide, N-dimethylformamide, dodecahydrate and disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulfate, yeast extract, tryptone, soybean peptone, kanamycin, etc. are purchased from pinellia ternata Biotech Limited; PrimeSTAR HS (Premix), 10000DL DNA Marker, available from Bao bioengineering (Dalian) Inc.; a centrifugal column type ultra-pure total RNA rapid extraction Kit, a FastKing RT Kit (With gDNase) reverse transcription Kit, a SuperRealPreMix Plus (SYBR Green) Kit, a high-purity plasmid miniprep Kit, a multifunctional DNA purification recovery Kit and the like are purchased from Beijing Tiangen Biochemical technology Co., Ltd.

(2) Apparatus and device

C1000 Touch PCR instrument, PowerPac Basic electrophoresis apparatus (equipped with horizontal electrophoresis tank), CFX96 Touch fluorescent quantitative PCR detection system, U.S. BIO-RAD company; 3K15 high speed centrifuge, SIGMA, usa; SPX-150B Biochemical incubator, Shanghai Boxun medical Bioinstrumentation, Inc.; BioSpectrum gel imager, UVP, USA; small high speed centrifuges, SIGMA, usa; vortex oscillator, IKA corporation, germany; electronic balance (0.01g), shanghai pu chun measuring instruments ltd; continuous wavelength multifunctional microplate reader spectra max 13, Molecular Devices, usa; constant temperature shaking table, Shanghai Bo news medical bio instruments GmbH; 10 in 1L mini-fermentor, Shanghai Bailun Bio Inc.

(3) The strains and plasmids used and the strains and plasmids constructed

The strains and plasmids used and the strains and plasmids constructed therefrom are shown in Table 1

TABLE 1 strains and plasmids

It should be noted that:

in the embodiment of the present application, the wild type strain Pseudomonas (Pseudomonas putida) B3 stored in the experiment is used as the indigo pigment synthesis related gene (styrene monooxygenase gene) styAB, but the implementation of the scheme provided by the present application can not be realized by using the strain, but the Pseudomonas carrying the indigo pigment synthesis related genes styA and styB can be used as the source of the indigo pigment synthesis related genes styA and styB for the present application, for example, the strain with the collection number of cgmm ccno.12202 disclosed in the patent application with the publication number of CN105802986A can be used in the present application; alternatively, the nucleotide sequence of the gene styAB involved in the synthesis of indigo pigment disclosed in the present application may be used in the present application.

Coli DH5 α, pHS-AVC-LW, pKD46, pCP20 are commercially available.

The recombinant vectors pHS-AVC-01, pHS-AVC-02, pHS-AVC-03, and recombinant strains E.coli AB-01 and E.coli AB-02 disclosed in the present application can be obtained by repeating the methods disclosed in the present application.

(4) Culture medium

The culture medium was prepared as follows:

LB liquid Medium (g/L): 5.0 yeast extract, 10.0 tryptone, 10.0 NaCI, pH 7.0, and high temperature sterilization at 121.0 deg.C for 15.0 min.

LB solid Medium (g/L): 5.0 yeast extract, 10.0 tryptone, 10.0 NaCI, 15.0 agar, pH 7.0, and high temperature sterilization at 121.0 deg.C for 15.0 min.

Tryptophan fermentation Medium (g/L): na (Na)₂HPO₄·12H₂O 17.0，KH₂PO₄ 3.0，NH₄Cl 1.0，NaCl 0.5，MgSO₄0.1, 3.0 yeast extract powder, 1.6 substrate tryptophan and 3.0121.0 ℃ high-temperature sterilization of lactose for 15.0 min.

Indole fermentation medium (g/L): na (Na)₂HPO₄·12H₂O 17.0，KH₂PO₄ 3.0，NH₄Cl 1.0，NaCl 0.5，MgSO₄0.1, 3.0 yeast extract powder, 3.0 soy peptone and 1.6 indole substrate, and sterilizing at 121.0 ℃ for 15.0 min.

Preparing antibiotics:

kanamycin (Kan): a stock solution was prepared at a concentration of 10.0mg/mL, kanamycin at a concentration of 50.0. mu.g/mL was used, and the stock solution was stored at 4 ℃ until use.

Chloramphenicol (Chl): a stock solution was prepared at a concentration of 10.0mg/mL, kanamycin at a concentration of 100.0. mu.g/mL was used, and the stock solution was stored at 4 ℃ until use.

Ampicillin (Amp): a stock solution was prepared at a concentration of 10.0mg/mL, kanamycin at a concentration of 50.0. mu.g/mL was used, and the stock solution was stored at 4 ℃ until use.

The inoculum amount in the present application refers to the ratio of the volume of the seed liquid to the volume of the fermentation liquid (i.e., the total volume of the seed liquid and the culture medium), for example, an inoculum amount of 1.2% refers to a volume of the seed liquid of 1.2 and a volume of the fermentation liquid (i.e., the total volume of the seed liquid and the culture medium) of 100.

In this application, styAB means that the styrene monooxygenase gene styA and the styrene monooxygenase gene styB are linked together in the order of the styrene monooxygenase gene styA and the styrene monooxygenase gene styB along the direction of expression.

1 construction of recombinant vector

1.1 primer design

Some primers of the present application are shown in table 2.

TABLE 2 primer List

1.2 PCR amplification of homology arms, indigo Synthesis related genes styA and styB

E.coli DH5 alpha trpR is used as an integration site to amplify a left arm and a right arm, wherein the size of the left arm is 358bp (the sequence is shown as SEQ ID NO. 3), and the size of the right arm is 357bp (the sequence is shown as SEQ ID NO. 4); e.coli DH5 alpha trpB is used as an integration site to amplify a left arm and a right arm, wherein the size of the left arm is 353bp (the sequence is shown as SEQ ID NO. 5), and the size of the right arm is 351bp (the sequence is shown as SEQ ID NO. 6); e.coli DH5 alpha hisD is used as an integration site to amplify a left arm and a right arm, wherein the size of the left arm is 346bp (the sequence is shown as SEQ ID NO. 7), and the size of the right arm is 369bp (the sequence is shown as SEQ ID NO. 8).

Extracting wild type indigo synthetic strain Pseudomonas (Pseudomonas putida) B3 as a whole genome template, and performing Primer design by using Primer Premier 5.0 according to a styrene monooxygenase gene sequence (GenBank access No. DQ177365.1) in GeneBank to amplify styrene monooxygenase gene styA and styrene monooxygenase gene styB, wherein the sequences are respectively shown as SEQ ID No.1 and SEQ ID No. 2.

1.3 construction of styAB Gene-integrating recombinant plasmid

According to the principle of constructing a homologous recombination vector by a one-step method, gene fragments on two sides of three integration sites trpR, trpB and hisD genes of escherichia coli are respectively used as homologous fragments, a promoter and a resistance gene are connected with a homologous arm and a target gene fragment, and finally, plasmid pHS-AVC-LW is used as a template to construct integrated recombinant plasmids pHS-AVC-01, pHS-AVC-02 and pHS-AVC-03 through homologous recombination.

1.4 transformation of styAB Gene and recombinant plasmid ligation products into E.coli competence

Preparing E.coli DH5 alpha electrotransformation competence, respectively electrotransforming 3 integrated recombinant plasmids constructed in 1.3, screening three resistance corresponding to Chl culture medium, Kan culture medium and Kan culture medium to obtain positive monoclonal, and sequencing to verify the insertion sequence.

2 targeting fragment acquisition

2.1 plasmid extraction

1.4 the genome DNA extraction of the positive clones without errors is verified by adopting a bacterial genome DNA extraction kit (centrifugal column type) of the company Baitach, and the specific operation is shown in the kit specification.

2.2 PCR amplification of the targeting fragment

The DNA concentrations of three sets of integrated recombinant plasmids pHS-AVC-01, pHS-AVC-02 and pHS-AVC-03 extracted in 2.1 were measured, and the extracted concentrations were about 200 ng/. mu.L, and the PCR reaction system using pHS-AVC-01 as a template is shown in Table 3:

TABLE 3 PCR reaction System for targeting Gene fragments

The PCR reaction with pHS-AVC-01 as a template was carried out as follows:

the PCR reaction system using pHS-AVC-02 as a template is shown in Table 4:

TABLE 4 PCR reaction System for targeting Gene fragments

The PCR reaction of pHS-AVC-02 was carried out as follows:

the PCR reaction system for pHS-AVC-03 is shown in Table 5:

TABLE 5 PCR reaction System for targeting Gene fragments

The PCR reaction of pHS-AVC-03 was carried out as follows:

2.3 Gene fragment gel electrophoresis and product recovery

The agarose gel electrophoresis was performed in standard fashion.

The gel electrophoresis product is recovered by adopting a centrifugal column type multifunctional DNA purification recovery kit of Beijing Baitaike company, and the specific operation is shown in the kit specification. 3 construction of recombinant strains

3.1 inducible expression of Red Gene

Transformation of pKD46 plasmid into E.coli DH 5. alpha. competence, recovery of culture at 30 ℃ for 1h, ampicillin (Amp)⁺) The plates were incubated overnight at 30 ℃. Picking single clone to LB test containing AmpIncubate overnight in tubes.

Inoculating to LB medium with Amp at a ratio of 1:100, adding L-arabinose to a final concentration of 1mM, culturing at 30 deg.C, OD_600nmNot more than 0.6, and preparing electrotransformation competence.

3.2 electric transfer targeting fragment

And (3) uniformly mixing the targeting DNA fragment obtained in the step (2.2) with electrotransfer competent cells (100 mu L of competent cells + about 200ng of targeting DNA fragment, transferring the targeting DNA fragment into a precooled electric rotating cup, taking three monoclonals from the electrotransfer competent plate prepared in the step (3.1) respectively for electric shock transformation, adding an LB culture medium after electric shock, putting the mixture at the temperature of 30 ℃ for recovery for 2h, coating the mixture on a corresponding double-resistance (Chl + Amp or Kan + Amp) plate, and culturing the mixture at the temperature of 30 ℃ overnight.

3.3 identification of engineering bacteria

From the double-resistant plates cultured in step 3.2, 10. mu.L of ddH was picked up₂And O, preparing bacterial suspension, taking 1 mu L of bacterial suspension as a template, and taking F-0151/R-0151, F-0152/R-0152 and F-0153/R-0153 as primers to carry out PCR, wherein the system is shown in Table 6:

TABLE 6 identification of strains PCR reaction System

The PCR conditions were the same as those described in example 2. Electrophoresis was performed on a 1% agarose gel for 30min, and the results were detected and recorded by a gel imager.

The remaining 9. mu.L of bacterial suspension was placed in the corresponding resistance (Kan)⁺Or Chl⁺) LB medium was cultured at 42 ℃ to remove plasmid pKD46, and then LB plate was streaked (Kan)⁺Or Chl⁺) Then, the resulting single colony is subjected to Amp⁺And (3) sensitive detection, namely, the strain sensitive to Amp is the strain with the plasmid pKD46 removed. After extracting strain DNA, preserving the strain by glycerol, and then sending the strain to a company for sequencing.

3.4 knock-out of Kan + or Chl + resistance Gene

3.3 electroporation of pCP20 plasmid in the error-free recombinant Strain⁺Plates were incubated overnight at 30 ℃.

Picking single clone into test tube added with Amp, culturing to OD at 30 DEG C₆₀₀About 0.4-0.6, heat shock at 37 deg.C for 1h (heat-activated protein expression), after overnight culture at 30 deg.C, PCR verified Kan⁺Or Chl⁺Whether or not to eliminate (or to add kanamycin or chloramphenicol directly into the test tube for verification).

The correct bacteria are streaked, separated and monocloned, cultured at 42 ℃, and then Amp is carried out⁺The pCP20 plasmid was verified to be eliminated in a fire tube on a plate.

4 analysis of target Gene expression

4.1 Total RNA extraction of engineering bacteria

The extraction of total RNA in the thalli adopts a centrifugal column type ultra-pure total RNA rapid extraction kit of Beijing Baitaike company, and the specific operation steps are shown in the kit specification.

4.2 reverse transcription of Total RNA into cDNA

The ReverTra Ace qPCR RT Master Mix reverse transcription kit from TOYOBO (Shanghai) corporation, Toyobo, was selected for cDNA synthesis, and the specific operation method is described in the specification.

4.3RT-PCR analysis

According to the sequence information of the styA and styB genes, primer Design and specificity evaluation are carried out by Beacon Design software. Primer sequences are shown in Table 7.

TABLE 7RT-PCR primers

Gene	Primer name	Numbering in sequence listing	Sequence (5'-3')
				styA	F-A	SEQ ID NO.20	GGCGAGCTGATTGAGATTC
styA	R-A	SEQ ID NO.21	TTTTGCCGTTATTGAGGGT
				styB	F-B	SEQ ID NO.22	AAAAGATGTGGTGGTGGAT
styB	R-B	SEQ ID NO.23	TGCTGAAGAATGCCGATAA
				16s	F-16s	SEQ ID NO.24	CCACCTGGACTGATACT
16s	R-16s	SEQ ID NO.25	GCACCTGTCTCAATGTT

The test adopts SYBR Green Realtime PCR Master Mix kit from TOYOBO (Shanghai) of Toyobo, and fluorescence quantification is carried out by SYBR Green method, and the reaction system is shown in Table 8.

TABLE 8 RT-PCR reaction System

The RT-qPCR reaction was performed as follows:

calculating relative expression amount: real-time fluorescence quantification with 16s gene as internal reference, E.coli-styAB-01 strain at different fermentation times under 1.2mg/mL tryptophan concentration or 0.01 wt% indole as control and 2^-ΔΔCtThe method performs relative quantitative calculation. The numerical values represent the fold change in the expression level of the objective gene in the experimental group relative to the control group. Calculating the delta Ct value by the formula:

△△Ct＝(Ct_{target gene}-Ct_{Internal reference gene}) Experimental group- (Ct)_{Target gene}-Ct_{Internal reference gene}) Control group

Analysis of Experimental results

All experiments were repeated 3 times, significance analysis and mapping were performed on the data using OriginPro 2018 software, with significance for p < 0.01 < 0.05, very significant for p < 0.01, and insignificant for p > 0.05.

1 construction of recombinant plasmid map

The plasmid construction map of the integrative engineering bacteria is shown in figure 1 by analyzing and designing according to SnapGene software, wherein A is pHS-AVC-01, B is pHS-AVC-02, C is pHS-AVC-03, and subsequent experiments are carried out according to the map.

Construction and enzyme digestion verification of 2 styrene monooxygenase gene styAB integrated recombinant plasmid

The enzyme digestion verification principle is as follows: the pHS-AVC-LW skeleton vector is provided with 1 EcoRV enzyme cutting site, and the tyB gene is provided with 1 EcoRV enzyme cutting site, so that if the recombinant plasmid is successfully constructed, the recombinant plasmid contains two EcoRV enzyme cutting sites, and two bands are obtained after enzyme cutting and electrophoresis by using EcoRV enzyme.

The gene segments at both sides of the trpR gene of the escherichia coli are used as homologous segments, a PCR amplification promoter and a resistance gene are connected with homologous arms and a target gene segment, and the integrated recombinant plasmid pHS-AVC-01 of trpR left arm-Chl-lac promoter-sty AB-trpR right arm is constructed. The total plasmid size is 6692bp, the gel electrophoresis and enzyme digestion electrophoresis (FIG. 2) of the recombinant plasmid show that lane 1 is the gene band constructed by plasmid pHS-AVC-01, and lane 2 shows two bands of 4170bp and 2522bp by enzyme digestion verification of EcoRV, which proves that the recombinant plasmid can be cut and the bands have the same size, indicating that the recombinant plasmid pHS-AVC-01 is constructed.

The gene segments at both sides of the trpB gene of the escherichia coli are used as homologous segments, a PCR amplification promoter and a resistance gene are connected with a homologous arm and a target gene segment, and the integrated recombinant plasmid pHS-AVC-02 of trpB left arm-Kan-lac promoter-sty AB-trpB right arm is constructed. The total plasmid size is 6742bp, the gel electrophoresis diagram and the enzyme digestion electrophoresis diagram (FIG. 3) of the recombinant plasmid show that the lane 1 is the gene band constructed by the plasmid pHS-AVC-02, and the lane 2 shows two bands of 4255bp and 2487bp by enzyme digestion verification of EcoRV, which proves that the recombinant plasmid can be cut and is consistent with the expected band size, and shows that the recombinant plasmid pHS-AVC-02 is constructed.

The gene segments at both sides of the escherichia coli hisD gene are used as homologous segments, a PCR amplification promoter and a resistance gene are connected with a homologous arm and a target gene segment, and the integrated recombinant plasmid pHS-AVC-03 of hisD left arm-Kan-lac promoter-sty AB-hisD right arm is constructed. The total plasmid size is 6763bp, the gel electrophoresis and the enzyme digestion electrophoresis (FIG. 4) of the recombinant plasmid show that the lane 1 is the gene band constructed by the plasmid pHS-AVC-03, and the lane 2 shows two bands of 4258bp and 2505bp by the enzyme digestion verification of EcoRV, which proves that the recombinant plasmid can be cut and is consistent with the expected band size, indicating that the recombinant plasmid pHS-AVC-03 is constructed.

3 colony PCR verification

PCR verification is respectively carried out on three recombinant plasmids pHS-AVC-01, HS-C-0151-2 and HS-C-KAN for pHS-AVC-02 and HS-C-KAN for HS-C-0151-3 for pHS-AVC-03 by using identification primers HS-C-0151-1 and HS-AVC-0063-7, and the three recombinant plasmids all have bright bands as can be seen from figure 5, so that the success of plasmid construction is proved; wherein A is pHS-AVC-01, B is pHS-AVC-02, and C is pHS-AVC-03.

4 sequencing verification of plasmid integration sequence

The three recombinant plasmids were sequenced and verified, and it can be seen from FIG. 6 that the left-arm-styAB-right-arm fragments of the three plasmids were all integrated correctly, and the plasmid integrated sequence was verified to be correct again, and the plasmid was successfully constructed, wherein A is pHS-AVC-01, B is pHS-AVC-02, and C is pHS-AVC-03.

5 PCR amplification of the targeting fragment

The primers F-0151/R-0151, F-0152/R-0152 and F-0153/R-0153 are used for carrying out PCR amplification on the three plasmids respectively, and the base numbers of the pHS-AVC-01, pHS-AVC-02 and pHS-AVC-03 target fragments are 3683bp, 3740bp and 3761bp in sequence. FIG. 7 is an electrophoretogram of pHS-AVC-01 targeting fragment, which has been subjected to 6 sets of parallel experiments, wherein lanes 1-6 all have bright bands between 2000-4000bp, which proves the success of obtaining pHS-AVC-01 targeting fragment; in FIG. 8, lanes 1-6 are the electrophoresis of the targeting fragment pHS-AVC-02, lanes 7-12 are the electrophoresis of the targeting fragment pHS-AVC-03, all of which are 6 parallel groups, and it can be seen that there is a bright band between 2000-4000bp, which is the target targeting fragment, and the target fragment is recovered by gel cutting for further experiments.

6 identification of engineering bacteria

And (4) plating and culturing the strains after the electrotransfer is finished, and selecting a single bacterial colony to identify the engineering bacteria. In FIG. 9, lane 1 is a blank control group, lanes 2-4 are different single colonies on the same plate after the pHS-AVC-01 targeting fragment is electroporated, the colony template is identified by using the primer F-0151F/R-0151, lane 2 has an obvious band at 4000bp of 2000-plus,

lanes

3 and 4 have no required fragment, and the single colony in lane 2 is proved to be a strain with successful construction, and subsequent experiments can be continued, so that the E.coliAB-01 construction is successful.

In FIG. 10,

lanes

1 and 5 are blank control groups, lanes 2 to 4 are different single colonies on the same plate after the pHS-AVC-02 targeting fragment is electrically transferred and coated, the colony template is identified by using the primer F-0152/R-0152, lane 2 has an obvious band between 2000 and 4000bp,

lanes

3 and 4 have no obvious band, it is proved that the colony in lane 2 is the required strain,

lanes

3 and 4 are false positive strains, therefore, the strain in lane 6 is further cultured, and the E.coliAB-02 strain is constructed; lanes 6-7 are different single colonies on the same plate after the pHS-AVC-03 targeted fragment is electrically transferred and coated, the colony template is identified by using the primer F-0153/R-0153, no obvious band exists at 4000bp of 2000-plus, although the colony grows out on the resistant plate, the gene fragment is not integrated completely, the gene fragment is false positive, and the integration efficiency of the integration site is not high, so the E.coliAB-03 is not constructed successfully.

In summary, the E.coliAB-01 and E.coliAB-02 strains are successfully constructed, and subsequent experiments can be carried out.

Reagents used in the following examples: protease (CAS:9001-92-7, 250U/mg), lysozyme (CAS:12650-88-3, 10000U/mg), Total protein kit, thiourea dioxide, sodium dithionite, sodium hydroxide, sodium chloride, hydrochloric acid, trichloroacetic acid, N-N-dimethylformamide, hydrogen peroxide, kanamycin, and the like were purchased from pinellia Biotech Ltd.

The apparatus used in the examples: electronic balance (0.01g), shanghai pu chun measuring instruments ltd; continuous wavelength multifunctional microplate reader spectra max 13, Molecular Devices, usa; 3K15 high speed centrifuge, SIGMA, usa; constant temperature shaking table, Shanghai Bo news medical bio instruments GmbH; an electric heating constant temperature water bath, Shanghai Bocheng industries, Ltd; ORP oxidation-reduction potentiometers, Shanghai Sanxin Meter factories; magnetic stirrers, Van Dall laboratory instruments Inc. of Changzhou; 10 in 1L mini-fermentor, Shanghai Bailun Bio Inc.

Preparation of indigo fermentation broths used in the examples: preparing a seed solution: 200 mu L of E.coliAB-01 indigo engineering bacteria liquid preserved at the temperature of 20 ℃ is taken from a clean bench, inoculated into 20mL LB liquid culture medium containing 50.0 mu g/mL kanamycin, put into a shaking table at the temperature of 37 ℃, activated at 200r/min for 12h, and then transferred twice for standby.

Preparing fermentation liquor: inoculating the seed solution into 500mL of fermentation medium with the inoculation amount of 1.2%, setting the temperature of the fermentation tank at 31 ℃, the rotation speed at 200.0r/min, the aeration ratio at 0.5vvm, the pH value at 7.0, correcting the dissolved oxygen by 100%, and culturing for 36h to obtain the indigo fermentation liquid.

Wherein:

Fermentation medium (g/L): na (Na)₂HPO₄·12H₂O 17.0，KH₂PO₄ 3.0，NH₄Cl 1.0，NaCl 0.5，MgSO₄0.1, 3.0 yeast extract powder, 3.0 soybean peptone and 1.6121.0 ℃ substrate tryptophan, and sterilizing for 15.0 min.

5% trichloroacetic acid solution: 50g of trichloroacetic acid is weighed by a beaker, 600mL of distilled water is added for dissolution, and then the solution is transferred into a volumetric flask, and the volume is adjusted to 1L by distilled water for standby.

Lysozyme solution: a lysozyme solution was prepared at a concentration of 10mg/mL and stored at 4 ℃ until use.

Protease solution: a protease solution was prepared at a concentration of 10mg/mL, and the solution was dissolved completely by slow stirring and stored at 4 ℃.

Preparing antibiotics: a stock solution was prepared at a concentration of 10.0mg/mL, and stored at 4 ℃ using kanamycin at a concentration of 50.0. mu.g/mL.

The calculation method of the protein removal rate: total protein content: suspending the fermentation liquid with physiological saline, adding lysozyme lysate for repeated freeze thawing to break cell wall (lysate formula: 50mmol Tris-HCL (pH 8.5-9.0), 2mmol EDTA,100mmol NaOH, 0.5% TritonX-100, 0.5mg/mL lysozyme). Placing in 43 deg.C water bath for 30min, freezing in-80 deg.C refrigerator for 20min, rapidly placing in 37 deg.C water bath for 10min, and repeating the operation for 4 times. After wall breaking, protein content was determined according to the instructions of the total protein kit.

Protein content in the sample: equal volume of the treated sample solution was taken and the above procedure was repeated to determine the protein content according to the total protein kit instructions.

Protein removal rate ═ (total protein content-protein content in sample)/total protein content × 100%

The calculation method of the thallus removal rate comprises the following steps: collecting solution containing thallus (fermentation broth, lysozyme-treated leuco solution, and indigo-oxidized leuco solution)Color body solution) at 9000r for 20min, discarding supernatant, and collecting precipitate. 5mL of distilled water was added to the precipitate, mixed well, and centrifuged at 90000r for 20min to remove the residual medium in the precipitate. Washing for 3 times, collecting the final precipitate, adding 20mL 5% trichloroacetic acid solution into the precipitate, mixing, water bathing at 80 deg.C for 30min, immediately cooling in ice water. Centrifuging at 8000r at 4 deg.C for 10min, collecting supernatant, and diluting with 5% trichloroacetic acid (OD)_260nmBetween 0.2 and 0.8), determining the OD₂₆₀ _nmAbsorbance values and concentration were calculated.

Removal rate of cell (C)_{NA, fermentation broth}－C_{NA, sample})/C_{NA, fermentation broth}×100％

In the formula: c_{NA, fermentation broth}: the concentration (mg/mL) of total bacteria in the fermentation broth;

C_{NA, sample}: the concentration of the bacterial cells in the sample (mg/mL).

The method for calculating the extraction and refining yield of the indigo comprises the following steps: oxidizing leuco body with oxidant to form indigo particle, eluting indigo particle insoluble in water and partially dissolved in organic reagent with water for three times, filtering and centrifuging to obtain pure indigo. Dissolving the obtained pure indigo with N-N-dimethylformamide solution, measuring the absorbance by using an enzyme-labeling instrument, calculating the concentration, and comparing with the initial indigo concentration to calculate the indigo extraction and refining yield.

Extracting and refining indigo blue with yield ═ C_Pure/C_Initial×100％

In the formula: c_Initial: initial concentration of indigo product;

C_pure: extracting pure indigo blue.

Example 1

The method for extracting the indigo from the indigo engineering bacterium fermentation liquor comprises the following steps:

removing thalli in the fermentation liquor: 50mL of fermentation liquor is taken and added with lysozyme for reaction. The enzymolysis reaction conditions are as follows: when the temperature is 40 ℃ and the pH value is 7, the enzyme adding amount is 2 mu g/mL, the stirring speed is 600r/min, and the reaction time is 2 h. Detection shows that the final thallus removing rate in the fermentation liquor reaches 96.3%.

Then adding protease to remove the foreign protein in the fermentation liquor. The enzymolysis reaction conditions are as follows: the enzyme dosage is 4%, the stirring speed is 500r/min and the reaction time is 0.5h when the temperature is 50 ℃ and the pH value is 2.5. Through detection, the removal rate of the foreign protein in the fermentation liquor reaches 96.7%.

Centrifuging the liquid from which the thalli and proteins in the thalli are removed for 20min at 9000r/min by using a small high-speed centrifuge, discarding the supernatant, drying to obtain a crude indigo, weighing a proper amount of the crude indigo, preparing a solution (the crude indigo is 1.00:0.61:0.66) by adding distilled water, sodium hydroxide and thiourea dioxide by using a full-bath reduction method under the water bath condition, and reducing the prepared solution in a water bath kettle at 60 ℃ for 30 min. After the indigo is fully reduced, centrifuging for 5min by using a small high-speed centrifuge 11000r/min, and pouring the supernatant into a clean beaker to obtain a leuco body solution.

Adding 30 wt% of hydrogen peroxide solution into the leuco body solution, wherein the volume ratio of the hydrogen peroxide solution to the leuco body solution is 1:6, and carrying out oxidation reaction for 1.5h under the conditions that the pH value is 10 and the temperature is 40 ℃ to obtain the indigo particles. The detection shows that the extraction and refining yield of the indigo reaches 89.8 percent.

Example 2

In the process of using lysozyme to remove the thallus of the fermentation liquor, the enzyme adding amount is respectively 0.5%, 1%, 1.5%, 2.5%, 3%, 3.5% and 4%, and the rest steps are the same as the example 1. The results of comparing the cell removal rate with the enzymatic method in example 1 are shown in FIG. 11: in the 2h before the reaction, the thallus removal rate is increased along with the extension of the reaction time, and in the 2h before the reaction, the thallus removal rate is increased along with the increase of the lysozyme dosage, but after the reaction for 2h, the lysozyme dosage is increased from 2 percent to 4 percent, and the influence on the thallus removal rate is very small. In order to reduce the cost of the enzymolysis process, 2 percent of the using amount of lysozyme is selected as the optimal condition, and the thallus removal rate is 80.7 percent.

Example 3

In the process of using lysozyme to remove the thalli of the fermentation liquor, the stirring speed is respectively 300, 400, 500, 700, 800 and 900r/min, and the rest steps are the same as the steps in the example 1. The results of comparing the cell removal rate with the enzymatic method in example 1 are shown in FIG. 12: the cell removal rate increased with increasing stirring rate within 2h before the reaction, and then increased slowly, and a maximum of 83.3% was obtained at a stirring rate of 600 r/min. Along with the prolonging of time, the lysozyme is mechanically damaged by high-speed stirring, so that the enzyme activity is reduced, and the thallus removal rate is reduced. Therefore, stirring at 600r/min was used as the optimum condition.

Example 4

The cell contents of the fermentation liquid, the fermentation liquid after lysozyme reaction and the indigo blue crude product reconstituted solution in example 1 were measured, and the results are shown in fig. 13: after the 16.7% of thalli are removed by lysozyme enzymolysis, the number of the thalli remained in the crude indigo is reduced to 10.3% after the crude indigo is prepared by a crude indigo preparation process.

Example 5

In the process of removing the heteroprotein in the fermentation liquor by using the protease, the enzyme addition amounts are respectively 1.5%, 2%, 2.5%, 3%, 3.5%, 4.5% and 5%, and the rest steps are the same as those in example 1. As a result of comparing the protein removal rate with that of example 1, as shown in FIG. 14, it can be seen from FIG. 14 that the protein removal rate gradually increased with the increase of the protease amount, the protein removal rate was relatively high in the range of 4% to 5% of the enzyme amount, and the reaction time became stable after exceeding 0.5 h. When the enzyme adding amount is lower, the mixed protein is in an excessive state compared with the enzyme, in this case, the reaction rate can be improved by adding the enzyme into the fermentation liquor, the enzyme and the mixed protein in the fermentation liquor gradually reach a saturated state along with the continuous increase of the enzyme amount, the enzyme amount is continuously increased, and the increase of the reaction rate is not influenced any more. Therefore, the optimum condition was selected to be 4% of the enzyme addition amount, and the protein removal rate was 69%.

Example 6

In the process of using protease to remove the foreign proteins in the fermentation liquor, the stirring speed is respectively 300, 400, 600 and 700r/min, and the rest steps are the same as the example 1. The results of comparing the cell removal rate with the enzymatic method in example 1 are shown in FIG. 15: the protein removal rate increased first and then leveled off with increasing stirring rate and reached a maximum at 500 r/min. This indicates that the combination of the protease and the hetero-protein is affected at a low stirring speed, so that the reaction is incomplete, and the protease is damaged at an excessively high stirring speed, so that the activity is reduced. Therefore, the optimum stirring rate was 500r/min, and the protein removal rate was 77.8%.

Example 7

The contents of protein in the fermentation liquid, the fermentation liquid after lysozyme reaction and the indigo crude product reconstituted solution in example 1 were measured, and the results are shown in fig. 16: after protease enzymolysis is carried out to remove the residual 22.2 percent of the impure protein, and after the crude product preparation process is carried out, the quantity of the residual impure protein in the crude indigo product is reduced to 14.7 percent.

Example 8

The reducing agent thiourea dioxide used in example 1 was replaced with sodium dithionite and the rest of the procedure was the same as in example 1. And comparing the reduction potential value, the residual impurity content and the absorbance of unreduced substrates in the reduction process of the leuco body solution obtained by the two reducing agents.

As can be seen from fig. 17, when thiourea dioxide is used as the reducing agent, the residual impurity content is lower than that when sodium dithionite is used as the reducing agent, the reduction potential value is higher, that is, the reduction capability is stronger, and the absorbance of the unreduced substrate is lower, which indicates that the amount of the reduced indigo is larger. When thiourea dioxide is used as a reducing agent, the residual thallus content is 6.4 percent, and the residual impurity protein content is 5.8 percent; when sodium hydrosulfite is used as a reducing agent, the residual thallus content is 7.4 percent, and the residual impurity protein content is 7.1 percent.

In conclusion, the effect of thiourea dioxide as a reducing agent is better than that of sodium hydrosulfite, so that the leuco body solution prepared by using thiourea dioxide as a reducing agent is continuously used for screening and optimizing the oxidizing agent.

Comparative example 3

The oxidizing agent hydrogen peroxide solution used in example 1 was replaced with air or water, respectively, and the rest of the procedure was the same as in example 1. As can be seen from FIG. 18, the highest extraction and purification yield of hydrogen peroxide as an oxidizing agent was 65.3%, and hydrogen peroxide was used as a catalyst.

Example 9

The ratio of the hydrogen peroxide solution to the leuco solution in example 1 was replaced with 1: 12. 1: 10. 1: 8. 1: 4. 1: 2. 1:1, the rest of the procedure was the same as in example 1. And measuring the content of residual thallus, the content of residual hybrid protein and the extraction and refining rate of the indigo in the obtained indigo. As shown in FIG. 19, the impurity content gradually decreased with the increase of the amount of hydrogen peroxide, the purification yield of indigo blue gradually increased, the effect was the best when the ratio of the amount of hydrogen peroxide to the leuco solution was 1:6, and the trend gradually decreased when the ratio of hydrogen peroxide to leuco solution exceeded 1:6, indicating that too little hydrogen peroxide reacted with leuco incompletely, but too much hydrogen peroxide did not increase the oxidation ability any more, so 1:6 was selected as the optimum ratio. At this time, the residual cell content was 5.4%, the residual hetero-protein content was 4.8%, and the indigo extraction and purification yield was 78.3%, indicating that there was still a loss of nearly 22% of indigo at this time.

Example 10

The reaction time of the hydrogen peroxide solution and the leuco solution in example 1 was respectively replaced by 0.5, 1.0, 2.0 and 2.5h, and the rest of the procedure was the same as in example 1. And measuring the content of residual thallus, the content of residual hybrid protein and the extraction and refining rate of the indigo in the obtained indigo. As can be seen from fig. 20, the impurity content gradually decreased with the increase of the oxidation time, the purification yield of indigo gradually increased, and became slow after reaching 1.5h, which indicates that 1.5h is the optimum oxidation time, the reaction could not be completed with a shorter time, and the reaction rate slowly increased even after exceeding 1.5h, so 1.5h was selected as the optimum reaction time. At this time, the residual cell content was 4.9%, the residual hetero-protein content was 4.7%, and the indigo extraction purification yield was 83.6%.

Example 11

The reaction temperatures of the hydrogen peroxide solution and the leuco solution in example 1 were changed to 30, 50, 60, and 70 ℃ respectively, and the rest of the procedure was the same as in example 1. And measuring the content of residual thallus, the content of residual hybrid protein and the extraction and refining rate of the indigo in the obtained indigo. It can be seen from fig. 21 that the impurity content decreases and increases with the increase of temperature, the refining yield of indigo extraction increases and decreases, the impurity content is the least at 40 ℃, the oxidation effect is the most obvious, and the extraction yield is the highest, because the reaction is slow at too low temperature, and too high temperature can destroy the leuco body structure, and can not sufficiently oxidize the leuco body solution into indigo particles. So that the optimal oxidation temperature of 40 ℃ is finally selected. At this time, the residual cell content was 4.5%, the residual hetero-protein content was 4.3%, and the indigo extraction purification yield was 85.9%.

Example 12

The pH of the reaction of the hydrogen peroxide solution with the leuco solution in example 1 was changed to 6, 7, 8, 9, 11, respectively, and the rest of the procedure was the same as in example 1. And measuring the content of residual thallus, the content of residual hybrid protein and the extraction and refining rate of the indigo in the obtained indigo. As can be seen from fig. 22, the impurity content reached the lowest level at pH10, at which time the residual cell content was only 3.7%, the residual protein content was only 3.3%, and the indigo extraction yield reached 89.8%. The weak alkaline environment is more suitable for indigo extraction, impurities are gradually reduced along with the increase of the extraction rate, hydrogen peroxide and leuco bodies are incompletely reacted due to the over-strong alkalinity, and partial indigo cannot be oxidized out, so that the pH10 is selected as the optimal condition finally. At this time, the residual thallus content is 3.7%, the residual hybrid protein content is 3.3%, and the indigo extraction and refining yield can reach 89.8%.

In summary, the invention firstly removes impurities by an enzymatic hydrolysis method, and establishes the optimal removal process parameters: the final thallus removal rate in the fermentation liquor is increased from 53.1 percent to 96.3 percent, and when the temperature is 40 ℃ and the pH value is 7, the removal effect is best when the enzyme adding amount is 2 percent and the stirring speed is 600 r/min; the removal rate of the foreign protein in the fermentation liquor is increased from 66.2 percent to 96.7 percent, and the removal effect is best when the enzyme adding amount is 4 percent and the stirring speed is 500r/min at the temperature of 50 ℃ and the pH value of 2.5. Then reducing the indigo into sodium salt dissolved in water by using a reducing agent, centrifuging to remove residual impurities, and finding by comparing the reducing agent that thiourea dioxide has stronger reducibility and better effect than sodium hydrosulfite. Finally, extracting and refining the indigo from the leuco body solution by using an oxidant, taking thiourea dioxide as a reducing agent and hydrogen peroxide as an oxidant, wherein the extracting and refining yield of the indigo is improved from 65.3 percent to 89.8 percent, and the optimal oxidation conditions are as follows: oxidizing agent: leuco solution was 1:6 at pH10, oxidation time 1.5h, temperature 40 ℃. The process solves partial problems of extraction and refining of indigo engineering bacteria products, and can realize industrial development of producing indigo by microbial fermentation.

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

Sequence listing

<110> Beijing university of Industrial and commercial

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<400> 11

cgcatcgagc gagcacgtac 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cggcattctt cagcaagcgt 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tcaggccgca atagtgggtg 20

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aacgtgctgg cttatgacgc ttact 25

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cggtgattct ggaaaaagtg cgtga 25

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tatgttttag acaacggcca gggtg 25

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gccaatggga atggtcgggt 20

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gaagtcatcg ccctgctgcc a 21

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

agcggttgag cgtttgctga gtaag 25

<210> 20

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggcgagctga ttgagattc 19

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ttttgccgtt attgagggt 19

<210> 22

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

aaaagatgtg gtggtggat 19

<210> 23

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tgctgaagaa tgccgataa 19

<210> 24

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ccacctggac tgatact 17

<210> 25

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gcacctgtct caatgtt 17

Claims

1. A method for extracting indigo from indigo blue engineering bacteria fermentation liquor is characterized by at least comprising the following steps:

(a) breaking thallus of indigo blue engineering bacteria in the fermentation liquor, and removing hybrid protein in the fermentation liquor;

(b) reducing indigo in the fermentation liquor to form a leuco body to obtain a leuco body solution; removing insoluble impurities in the leuco body solution, and oxidizing the leuco body to obtain indigo precipitate.

2. The method for extracting indigo from fermentation broth of indigo pigment engineering bacterium according to claim 1, wherein the indigo pigment engineering bacterium is a host with fragment I integrated into its genome; the fragment I contains a styrene monooxygenase gene styA and a styrene monooxygenase gene styB;

preferably, the host is selected from any one of e.coli;

preferably, the e.coli is e.coli DH5 α;

preferably, the integration is a nucleotide sequence that replaces the host integration site with fragment I; the integration site is selected from any one of trpR, trpB and hisD.

3. The method for extracting indigo from fermentation broth of indigo engineering bacteria as claimed in claim 2, wherein the coding sequence of styrene monooxygenase gene styA is shown in SEQ ID No. 1;

preferably, the fragment I further comprises an inducible promoter, an operon;

preferably, the inducible promoter is an inducible promoter cat;

the operon is the lactose operon lac.

4. The method for extracting indigo from fermentation broth of indigo blue engineering bacteria according to claim 1, wherein the method for disrupting bacterial cells in step (a) comprises the following steps: adding lysozyme into the fermentation liquor for reaction to break the cell wall of the indigo blue engineering bacteria;

preferably, the conditions for adding lysozyme to the fermentation broth for reaction include: adding lysozyme into the fermentation liquor with the enzyme adding amount of 0.5-4 mug/mL;

preferably, the enzyme adding amount of the lysozyme added into the fermentation liquor is 2-4 mug/mL;

preferably, the stirring speed is 300-900r/min in the reaction process;

preferably, the stirring speed in the reaction process is 500-700 r/min;

preferably, the pH value is 6-8, the temperature is 35-45 ℃ and the reaction time is 0.5-6h in the reaction process.

5. The method for extracting indigo from fermentation broth of indigo blue engineering bacteria according to claim 1, wherein the method for removing hetero-protein in the fermentation broth in step (a) comprises: adding protease into the fermentation liquor to react to decompose the hybrid protein;

preferably, the conditions for adding protease to the fermentation broth for reaction include: adding protease into the fermentation liquor with the enzyme adding amount of 1.5-5 mug/mL;

preferably, the protease is added into the fermentation liquor in an amount of 4-5 mug/mL;

preferably, the stirring speed in the reaction process is 300-700 r/min;

preferably, the stirring speed is 500-600r/min in the reaction process;

preferably, the pH value is 1.5-3.5, the temperature is 45-55 ℃ and the reaction time is 0.5-2.5h in the reaction process.

6. The method for extracting indigo from fermentation broth of indigo engineering bacteria according to claim 1, wherein the fermentation broth from which impurity proteins are removed is centrifuged in step (b), and the precipitate obtained by centrifugation is added with a reducing agent solution to reduce indigo to form leuco bodies, thereby obtaining a leuco body solution;

preferably, the precipitate obtained by centrifugation is reacted with a reducing agent solution for 20-40min under the condition of water bath at 50-70 ℃.

7. The method for extracting indigo from fermentation broth of indigo blue engineering bacteria as claimed in claim 6, wherein the reducing agent solution contains alkali and 2mg/mL reducing agent;

preferably, the reducing agent comprises at least one of thiourea dioxide or sodium dithionite;

preferably, the alkali is sodium hydroxide;

preferably, the precipitate: the mass ratio of the reducing agent is 1: 0.5-0.7.

8. The method for extracting indigo from fermentation broth of indigo engineering bacteria according to claim 1, wherein the step (b) comprises removing insoluble impurities from the leuco body solution by centrifugation, and adding oxidant into the leuco body solution to react to obtain indigo precipitate.

9. The method for extracting indigo from indigo blue engineering bacteria fermentation liquor according to claim 8, wherein the oxidant is hydrogen peroxide solution, and the volume ratio of the hydrogen peroxide solution to the leuco body solution is 1: 1-12;

preferably, the volume ratio of the hydrogen peroxide solution to the leuco solution is 1: 4-6.

10. The method for extracting indigo from indigo blue engineering bacteria fermentation broth as claimed in claim 8, wherein the leuco body solution reacts with oxidant at 30-70 deg.C and pH 6-11 for 0.5-2.5 h;

preferably, the leuco body solution is reacted with the oxidizing agent at 35-50 deg.C and pH 9-10.5 for 1.5-2 h.