CN105925629A - Method for synthesizing butanediamine through microbial transformation - Google Patents

Abstract

The invention relates to a method for synthesizing butanediamine through microbial transformation. According to the method, an ornithine decarboxylase gene is expressed by virtue of a microorganism recombinant strain and a substrate, namely ornithine, is transformed into butanediamine, so that the butanediamine is obtained. The invention can solve the problem of the extracellular transport of the butanediamine by constructing an escherichia coli recombinant strain for the secretory expression of ornithine decarboxylase into periplasmic cavity of escherichia coli and by adding a certain concentration of the substrate, namely the ornithine, to a fermentation broth so that the ornithine is converted into the butanediamine by virtue of the ornithine decarboxylase in the periplasmic cavity; therefore, the method is low in production cost, economic and environment-friendly and is simple and convenient to operate; and the yield of the butanediamine is improved.

Description

A kind of method that converts synthetic butanediamine by microorganism

technical field

The invention belongs to the technical field of genetically engineered bacteria, in particular to a method for synthesizing butanediamine through microbial conversion. The method performs biotransformation of ornithine in fermentation broth by overexpressing ornithine decarboxylase, thereby effectively increasing the production of butanediamine output.

Background technique

1,4-Butanediamine (butanediamine for short), also known as putrescine, is a kind of nitrogen-containing, positively charged small molecule aliphatic compound, and is a biological amine (including putrescine, Amine, spermidine and cadaverine, etc.) in the simplest one. In the cell, butanediamine is produced by the decarboxylation of ornithine under the action of ornithine decarboxylase. Like the decarboxylation product of lysine, cadaverine, it is a component in the odor of corpse decay, and is a toxic base. present in spoilage. Butylenediamine is widely used. Appropriate addition of butanediamine in agriculture can slow down the rotting time of fruits and vegetables, thereby prolonging the storage time; in medicine, butanediamine is used as a judgment indicator for some diseases; in industry, butanediamine can be synthesized by polymerization with adipic acid A new high-quality polyamide, nylon 46, has important industrial uses.

At present, the methods for synthesizing butanediamine include chemical synthesis and biosynthesis. The chemical synthesis method has harsh conditions, pollutes the environment, and destroys the ecology. The biosynthesis method and microbial transformation method can directly produce the products needed by human beings on a large scale.

The production of butanediamine by biosynthesis is to directly synthesize butanediamine through the fermentation of sugar by microorganisms. In order to increase the production of butanediamine, according to the regulation of butanediamine synthesis in Escherichia coli. Modification of the metabolic pathway for the synthesis of butanediamine. Patent publication CN 103403147A reports a method for synthesizing butanediamine by using a recombinant strain of Corynebacterium glutamicum to ferment sugars: by blocking the biosynthesis pathway from ornithine to arginine of Corynebacterium glutamicum, increasing the Intracellular glutamate levels, enhancement of the biosynthetic pathway from glutamate to ornithine and introduction of extracellular ornithine deshuttle to produce butanediamine. Patent Publication WO06/005603 also reports the improvement of butanediamine production by removing degradation or using putrescine's spermidine and acetylputrescine synthesis pathways. However, due to the intracellular synthesis of butanediamine cannot be effectively secreted outside the cell, resulting in accumulation in the cell and inhibiting the activity of lysine decarboxylase, affecting the production of butanediamine.

The microbial conversion method is to use microbial cells to express ornithine decarboxylase to convert the input substrate ornithine into butanediamine. The patent application of the present invention secretes and expresses ornithine decarboxylase into the periplasmic cavity of Escherichia coli by constructing a recombinant strain of E. coli, and then puts a certain concentration of substrate ornithine into the fermentation broth, and the ornithine decarboxylase in the Converting ornithine into butanediamine avoids the problem of extracellular transport of butanediamine. Therefore, there is an essential difference from the above-mentioned patent publications.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a method for the synthesis of butanediamine through microbial transformation, the method constructs microbial recombinant strains that can secrete and express ornithine decarboxylase gene, ferment the recombinant bacterial strain and induce The ornithine decarboxylase gene is secreted and expressed in the periplasmic cavity, and the ornithine put into the fermentation broth is converted into butanediamine, which solves the problem of the extracellular transport of butanediamine.

The technical solution adopted by the present invention to solve the problems of the technologies described above is:

The invention discloses a method for synthesizing butanediamine through microbial conversion. The method utilizes microbial recombinant strains to express ornithine decarboxylase gene, and converts substrate ornithine into butanediamine to obtain butanediamine.

And, the steps are as follows:

Constructing a microbial recombinant strain capable of expressing the ornithine decarboxylase gene, fermenting the microbial recombinant strain to induce the expression of the ornithine decarboxylase gene to obtain a fermentation broth, and then adding ornithine to the fermentation broth expressing the ornithine decarboxylase, Ornithine decarboxylase converts the substrate ornithine into butanediamine to obtain butanediamine.

Moreover, the concentration of the ornithine is 5-40g/L.

Moreover, the ornithine concentration is 10-30 g/L.

Moreover, the ornithine concentration is 15-25g/L.

Moreover, the microbial recombinant strains are recombinant strains of Escherichia coli, recombinant strains of Corynebacterium glutamicum, recombinant strains of Bacillus aspartate and recombinant strains of lactic acid bacteria;

Alternatively, the ornithine decarboxylase gene is derived from the speC gene of Escherichia coli.

And, the construction method of described microbial recombinant bacterial strain is as follows:

The ornithine decarboxylase gene is connected to the secretion expression carrier of the microorganism, and the microorganism is transformed, and the recombinant strain of the microorganism capable of secreting and expressing the ornithine decarboxylase gene is obtained by screening.

Moreover, the ornithine decarboxylase gene is derived from microorganisms or higher plants.

Moreover, the microorganism is Escherichia coli, lactic acid bacteria (Lactobacillus), coccidioides immits (Coccidioidesimmits) or Morganella morganii (Morganellamorganii); or, the higher plant is datura, artichoke, ice leaf sunflower, Orchid or aspen.

And, the steps are as follows:

⑴Construction of butanediamine-producing engineering strain PUT-C1

Use the kit to extract the genomic DNA of Escherichia coli as a template for amplifying the ornithine decarboxylase gene speC; design primers according to the speC sequence of the E. coli ornithine decarboxylase gene speC provided by NCBI, and amplify speC respectively; then the speC fragment is passed through After EcoRI and Nco I digestion, clone into pET22b+ between the same restriction sites; transform E. coli E.coliBL21(DE3), and obtain butanediamine-producing E. coli recombinant strain PUT-C1 after screening;

The primer sequences designed to construct the recombinant strain PUT-C1 are as follows (SEQUENCE1, SEQUENCE2):

⑵Determination of the enzyme activity of ornithine decarboxylase SpeC

① Preparation of crude enzyme solution

1) Take a loop of recombinant Escherichia coli PUT-C1 from the plate and inoculate it in 50mL liquid LB with corresponding resistance, and culture it overnight at 37°C and 200r/min on a shaker for 12h;

2) transfer 1% of the inoculum into a new LB liquid medium, and culture on a shaker at 37°C and 200rpm;

3) When OD ₆₀₀ =1.0, add a final concentration of 1mM IPTG, and then induce at 37°C and 200r/min for 6h;

4) Take 30mL culture solution in a 50mL centrifuge tube and centrifuge at 6000r/min for 8min;

5) Discard the supernatant, add 10 mL of sterile phosphate buffer solution, vortex to mix, add PBS to 20 mL, centrifuge at 6000 r/min for 8 min; repeat the steps once;

6) Add 10mL PBS, sonicate the cell suspension for 30min, sonicate for 2s, pause for 3s, power 40%;

7) Centrifuge at 8000r/min at 4°C for 15min, remove the supernatant of cell debris and obtain the crude enzyme solution;

② SDS-PAGE analysis of ornithine decarboxylase SpeC

Soak the gel to be used in the buffer solution, and use a micro-sampler to add samples to the gel holes according to the number; connect the electrophoresis instrument, turn on the power, adjust the current to 20-30mA, and maintain a constant current until the sample migrates to When the separation gel is 1cm lower, turn off the power; after the electrophoresis is completed, remove the electrophoresis gel, and carefully cut off a small piece at the corner of the gel as a mark, pour the Coomassie brilliant blue staining solution for staining for 1 hour; pour out the staining solution , rinse the gel in water for several times; add decolorization solution, and start to replace the decolorization solution every 2 hours until the background of the gel is clear;

PUT-C1 induced expression and SDS-PAGE analysis;

③Enzyme activity assay

1) Take 800 μL of phosphate buffer solution with different pH in a 5 mL sterile stoppered graduated tube, and preheat it in a 37°C water bath for 5 minutes; 3 in parallel;

2) Add 100 μL of crude enzyme solution, add 100 μL of L-arginine mother solution, and start timing for 30 minutes; the control group uses sterile water instead of L-arginine;

3) Place the stoppered graduated tube on ice, and quickly add 500 μL of 2M NaOH to terminate the reaction;

4) Derivatization, the production amount of butanediamine is detected on the column;

④ Detection results of ornithine decarboxylase activity

According to the HPLC detection butanediamine conversion result, the activity of ornithine decarboxylase SpeC is detected;

(3) Shake flask fermentation of recombinant strain PUT-C1 and determination of butylamine production

① Shake bottle fermentation

Pick a ring of monoclonal recombinant strains on the plate and inoculate them into 5mL LB medium for 10-12h, and insert 1% of the inoculum into a 250mL Erlenmeyer flask containing 50mL LB medium, at 37°C, 200r/h Min culture to OD ₆₀₀ = 1.0, add final concentration of 1mM IPTG, then induce at 37°C, 200r/min for 6h; add substrate ornithine, the concentration of ornithine is 0g/L, 10g/L respectively , 20g/L, 30g/L and 40g/L, three in parallel for each group; culture at 37°C, 200r/min for 12h; centrifuge the fermentation broth at 12000r/min for 5min, take the supernatant and store it at 4°C;

② HPLC detection of butanediamine

In the experiment, the national standard method was used to detect the content of butanediamine. By derivatizing the water sample before the column, the water sample was derivatized with benzoyl chloride and then extracted with ether.

1) Derivatization and extraction of samples

Take 1 mL of the supernatant and add it to a 5 mL graduated test tube with a stopper, add 500 μL of 2mol/L NaOH solution and 10 μL of benzoyl chloride, shake in a water bath at 37°C for 20 min, vortex for 30 s every 5 min, add 0.5 g of NaCl, 1 mL of diethyl ether and shake for 30 s to separate Take the upper layer of ether into another centrifuge tube. After the ether is completely volatilized, add 500 μL of methanol to dissolve, and pass through the membrane as a sample for HPLC detection; the derivation and extraction steps of the standard solution are the same as the sample processing steps;

2) HPLC determination

a Chromatographic conditions

A) Chromatographic column: C18 chromatographic column, 5μm, 4.6×250mm, or equivalent;

B) column temperature: room temperature;

C) Mobile phase: A (acetonitrile), B (0.02mol/L ammonium acetate), gradient elution conditions are as follows:

D) Detection wavelength: 254nm.

E) Injection volume: 10 μL;

F) flow rate: 1mL/min;

b Liquid chromatography analysis and determination

Start the machine, preheat it, rinse the chromatographic column with the mobile phase, and start sampling after the baseline is stable for 30 minutes;

The supernatant of the fermentation broth was diluted and derivatized for HPLC analysis, and the content of butanediamine was calculated by the peak area.

Advantage and positive effect of the present invention:

The method of the present invention secretes and expresses ornithine decarboxylase into the periplasmic cavity of Escherichia coli by constructing a recombinant bacterial strain of E. The conversion of ornithine into butanediamine solves the problem of extracellular transport of butanediamine, reduces production costs, is economical and environmentally friendly, and is simple and convenient to operate, increasing the output of butanediamine.

Description of drawings

Fig. 1 is E.coli genome DNA figure among the present invention;

Fig. 2 is the PCR product figure of speC among the present invention;

Fig. 3 is the pET22b-speC double enzyme digestion result figure among the present invention;

Fig. 4 is the SDS-PAGE analysis chart of the induced expression product in the present invention;

Fig. 5 is the standard product figure of butanediamine in the present invention;

Fig. 6 is sample chromatogram among the present invention;

Fig. 7 is the standard curve figure of butanediamine content in the present invention;

Fig. 8 is the output figure of the butanediamine of different charging amounts among the present invention.

detailed description

Below in conjunction with the examples, the present invention is further described, the following examples are illustrative, not limiting, and the protection scope of the present invention cannot be limited by the following examples.

The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

Technical means used in the present invention:

The gene sequence of the present invention relates to the ornithine decarboxylase gene, and its sequence is a known sequence. Plasmid pET22b+, molecular manipulation techniques, microbial culture techniques, detection of butanediamine, etc. are well known to those skilled in the art.

Example 1

The invention discloses a method for synthesizing butanediamine through microbial transformation, the method utilizes microbial recombinant strains to express ornithine decarboxylase gene, and converts substrate ornithine into butanediamine.

The steps can be as follows:

Constructing a microbial recombinant strain capable of expressing the ornithine decarboxylase gene, fermenting the microbial recombinant strain to induce the expression of the ornithine decarboxylase gene to obtain a fermentation broth, and then adding ornithine to the fermentation broth expressing the ornithine decarboxylase, Ornithine decarboxylase converts the substrate ornithine into butanediamine;

Wherein, the concentration of ornithine may be 5-40g/L, preferably 10-30g/L, and more preferably 15-25g/L.

Further, the microbial recombinant strains are recombinant strains of Escherichia coli, recombinant strains of Corynebacterium glutamicum, recombinant strains of Bacillus aspartate and recombinant strains of lactic acid bacteria;

Alternatively, the ornithine decarboxylase gene is derived from the speC gene of Escherichia coli.

Further, the construction method of the microbial recombinant strain is as follows:

The ornithine decarboxylase gene is connected to the secretion expression carrier of the microorganism, and the microorganism is transformed, and the recombinant strain of the microorganism capable of secreting and expressing the ornithine decarboxylase gene is obtained by screening.

Further, the ornithine decarboxylase gene can be derived from microorganisms such as Escherichia coli, lactic acid bacteria (Lactobacillus), coccidioides immits (Coccidioidesimmits), Morganella morganii (Morganellamorganii) and the like; it can also be derived from Datura Stramonium , artichoke (Cynarascolymus), ice leaf sunflower (Mesembryanthemum crystallinum), orchid (Cymbidium), poplar (poplar) and other higher plants; more preferably the speC gene derived from Escherichia coli.

Example 2

A method for synthesizing butanediamine through microbial conversion, the specific steps are as follows:

(1) Construction of butanediamine-producing engineering strain PUT-C1

The ornithine decarboxylase gene was connected to the expression plasmid pET22b+ to construct the plasmid pET22b-speC, which was transformed into E.coliBL21(DE3) to construct the engineering strain E.coli BL21(DE3)/pET22b-speC.

Specifically, use the kit to extract the genomic DNA of Escherichia coli as a template for amplifying the ornithine decarboxylase gene speC, as shown in Figure 1; design primers according to the sequence of the Escherichia coli ornithine decarboxylase gene speC provided by NCBI, and amplify the speC, as shown in Figure 2; then the speC fragment was digested by EcoRI and Nco I and cloned into pET22b+ between the same restriction sites to obtain the plasmid pET22b-speC, as shown in Figure 3; transformed into Escherichia coli E.coli BL21(DE3), After screening, the butanediamine-producing Escherichia coli recombinant strain PUT-C1 was obtained.

The primer sequences designed to construct the recombinant strain PUT-C1 are shown in Table 1:

Table 1: Primers required for the construction of recombinant bacteria PUT-C1

(2) Determination of the enzyme activity of ornithine decarboxylase SpeC

The method steps for measuring the activity of ornithine decarboxylase SpeC are as follows:

1. Preparation of crude enzyme solution

1) Take a loop of the recombinant strain from the plate and inoculate it into 50 mL of liquid LB with corresponding resistance, and culture it overnight at 37° C. on a shaker at 200 r/min for 12 hours.

2) Transfer 1% of the inoculum into new LB liquid medium, and culture on a shaker at 37° C. and 200 rpm.

3) When OD ₆₀₀ =1.0, add a final concentration of 1 mM IPTG, and then induce at 37° C. and 200 r/min for 6 h.

4) Take 30mL culture solution in a 50mL centrifuge tube and centrifuge at 6000r/min for 8min.

5) Discard the supernatant, add 10 mL of sterile phosphate buffered saline (PBS), vortex to mix, add PBS to 20 mL, and centrifuge at 6000 r/min for 8 min. repeat step once

6) Add 10 mL of PBS, sonicate the cell suspension for 30 min, 2 s, 3 s intermittent, 40% power.

7) Centrifuge at 8000r/min for 15min at low temperature at 4°C, remove the supernatant of cell debris and obtain the crude enzyme solution.

2. SDS-PAGE analysis of ornithine decarboxylase SpeC

Soak the gel to be used in the buffer solution, and add samples to the gel holes according to the number with a micro-sampler. Connect the electrophoresis instrument, turn on the power, adjust the current to 20-30mA, keep a constant current, and turn off the power when the sample migrates to the bottom 1cm of the separation gel. After the electrophoresis is completed, remove the electrophoresis gel, carefully cut off a small piece at the corner of the gel as a marker, and pour into Coomassie brilliant blue staining solution for staining, about 1h. Pour off the staining solution and rinse the gel several times in water. Add the destaining solution and start to replace the destaining solution every 2 hours until the background of the gel is clear.

PUT-C1 was induced to express and analyzed by SDS-PAGE. The electrophoresis results are shown in Figure 4. The molecular weight of ornithine decarboxylase is about 80kD.

3. Determination of enzyme activity

1) Take 800 μL of phosphate buffer solution with different pH in a 5 mL sterile stoppered graduated tube, and preheat it in a 37°C water bath for 5 minutes. 3 in parallel.

2) Add 100 μL of crude enzyme solution, add 100 μL of L-arginine mother solution, and start timing for 30 minutes. In the control group, sterile water was used instead of L-arginine.

3) Place the stoppered graduated tube on ice, and quickly add 500 μL of 2M NaOH to stop the reaction.

4) Derivatization, put on the column to detect the generation amount of butanediamine.

4. Detection results of ornithine decarboxylase activity

According to the results of HPLC detection of the conversion of butanediamine, the activity of ornithine decarboxylase SpeC was 17.48 U/mL.

(3) Determination of shake flask fermentation and butylamine output of recombinant strain PUT-C1

1. Shake flask fermentation

Pick a ring of monoclonal recombinant strains on the plate and inoculate them into 5mL LB medium for 10-12h, and insert 1% of the inoculum into a 250mL Erlenmeyer flask containing 50mL LB medium, at 37°C, 200r/h Min culture to OD ₆₀₀ =1.0, add final concentration of 1mM IPTG, and then induce at 37°C, 200r/min for 6h. Put in the substrate ornithine, wherein the concentrations of ornithine are 0g/L, 10g/L, 20g/L, 30g/L and 40g/L, respectively, and each group has three parallels. Cultivate at 37°C, 200r/min for 12h. The fermentation broth was centrifuged at 12000r/min for 5min, and the supernatant was taken and stored at 4°C.

2. HPLC detection of butanediamine

In the experiment, the national standard method was used to detect the content of butanediamine. By derivatizing the water sample before the column, the water sample was derivatized with benzoyl chloride and then extracted with ether. UV detection. This method has high sensitivity, butanediamine has a linear relationship in the range of 2-40mg/L.

1) Derivatization and extraction of samples

Take 1 mL of the supernatant and add it to a 5 mL graduated test tube with a stopper, add 500 μL of 2mol/L NaOH solution and 10 μL of benzoyl chloride, shake in a water bath at 37°C for 20 min, vortex for 30 s every 5 min, add 0.5 g of NaCl, 1 mL of diethyl ether and shake for 30 s to separate layer. Take the ether in the upper layer into another centrifuge tube, wait for the ether to volatilize completely, add 500 μL of methanol to dissolve, pass through the membrane and use it as a sample for HPLC detection. The derivation and extraction steps of the standard solution are the same as the sample processing steps.

2) HPLC determination

a Chromatographic conditions

A) Chromatographic column: C18 chromatographic column, 5μm, 4.6×250mm, or those with equivalent performance.

B) Column temperature: room temperature.

C) mobile phase: A (acetonitrile), B (0.02mol/L ammonium acetate), gradient elution conditions are shown in Table 2.

D) Detection wavelength: 254nm.

E) Injection volume: 10 μL.

F) Flow rate: 1mL/min

b Liquid chromatography analysis and determination

Turn on the machine, preheat it, rinse the column with mobile phase, and start sampling after the baseline is stable for 30 minutes

Table 2 gradient elution conditions

After diluting the supernatant of the fermentation broth by a certain number of times, it was derivatized for HPLC analysis. The liquid chromatograms of the standard and sample were shown in Figure 5 and Figure 6, respectively, and the butanediamine derivative peaked at about 6.8 minutes. According to the standard curve in Figure 7, the content of butanediamine was calculated by the peak area.

The standard curve and its linear regression equation of peak area (y)-butanediamine hydrochloride sample concentration (x) were generated by external standard method. Can draw from Fig. 4, adopt external standard method to generate the standard curve and its linear regression equation of peak area (y)-butanediamine hydrochloride sample concentration (x).

y=16.342x+5.34 formula (1)

Wherein, y is the peak area, x is the butanediamine hydrochloride concentration, and the equation linear regression correlation coefficient (Rs) is 0.999.

The content of butanediamine in different samples was determined according to the standard curve. Each sample was repeated three times, and the average value of butanediamine production was obtained. The result is shown in Figure 8. As can be seen from Figure 8, the output of butanediamine in the fermentation broth without ornithine input is 0; when the concentration of input ornithine is 10g/L, the output of butanediamine is 500±17mg/L; When the concentration of ornithine is 20g/L, the output of butanediamine is 1690±64mg/L; when the concentration of input ornithine is 30g/L, the output of butanediamine is 1240±27mg/L; When the concentration is 40g/L, the output of butanediamine is 696±8mg/L. It shows that when the substrate concentration is 20g/L, the accumulation of butanediamine in the fermentation broth is the largest, and the expression of ornithine decarboxylase to convert ornithine into butanediamine can effectively increase the production of butanediamine.

Claims (10)

1. the method passing through microorganism Synthesis butanediamine, it is characterised in that: described method utilizes microorganism recombinant bacterial strain Express ornithine decarboxylase gene, substrate ornithine is changed into butanediamine, obtains butanediamine.

Method by microorganism Synthesis butanediamine the most according to claim 1, it is characterised in that: step is as follows:

Structure can express the microorganism recombinant bacterial strain of ornithine decarboxylase gene, and ferment this microorganism recombinant bacterial strain induction ornithine The expression of decarboxylase gene, obtains fermentation liquid, then adds ornithine in the fermentation liquid expressing ODC Ornithine decarboxylase, and ornithine takes off Substrate ornithine is changed into butanediamine by carboxylic acid, obtains butanediamine.

Method by microorganism Synthesis butanediamine the most according to claim 2, it is characterised in that: described ornithine Concentration be 5-40g/L.

Method by microorganism Synthesis butanediamine the most according to claim 2, it is characterised in that: described ornithine Concentration is 10-30g/L.

Method by microorganism Synthesis butanediamine the most according to claim 2, it is characterised in that: described ornithine Concentration is 15-25g/L.

Method by microorganism Synthesis butanediamine the most according to claim 1 and 2, it is characterised in that: described micro- Biological recombination bacterial strain is E. coli recombinant stain, Corynebacterium glutamicum recombinant bacterial strain, millet straw bacillus cereus recombinant bacterial strain and lactic acid Bacterium recombinant bacterial strain；

Or, described ornithine decarboxylase gene derives from colibacillary speC gene.

Method by microorganism Synthesis butanediamine the most according to claim 1 and 2, it is characterised in that: described micro- The construction method of biological recombination bacterial strain is as follows:

Ornithine decarboxylase gene is connected on the secretion expression carrier of microorganism, and microbial, screening obtains secreting Express the microorganism recombinant bacterial strain of ornithine decarboxylase gene.

Method by microorganism Synthesis butanediamine the most according to claim 1 and 2, it is characterised in that: described bird Propylhomoserin decarboxylase gene derives from microorganism or higher plant.

Method by microorganism Synthesis butanediamine the most according to claim 5, it is characterised in that: described microorganism For escherichia coli, lactic acid bacteria (Lactobacillus), Blastomyces coccidioides (Coccidioidesimmits) or morganella morganii (Morganellamorganii)；Or, described higher plant is Flos Daturae, Carlina acaulis, ice plant, Cymbidium ensifolium (L.) Sw. or Cortex Populi dividianae.

Method by microorganism Synthesis butanediamine the most according to claim 1 and 2, it is characterised in that: step As follows:

(1) produce the structure of butanediamine engineered strain PUT-C1

Test kit is utilized to extract colibacillary genomic DNA, as the template of amplification ornithine decarboxylase gene speC；Root The escherichia coli ornithine decarboxylase gene speC primers provided according to NCBI, amplifies speC respectively；Then speC Between fragment is by EcoRI and Nco I enzyme action rear clone to the identical restriction enzyme site of pET22b+；Convert E. coli BL21 (DE3), through screening, it is thus achieved that produce butanediamine Escherichia coli recombinant strain PUT-C1；

Building the primer sequence designed by recombinant bacterium PUT-C1 is SEQUENCE1, SEQUENCE2；

(2) the enzyme activity determination of ODC Ornithine decarboxylase SpeC

1. the preparation of crude enzyme liquid

1) from flat board, take ring Escherichia coli recombinant strain PUT-C1 be inoculated in the 50mL liquid LB of corresponding resistant, 37 DEG C, 200r/min shaker overnight cultivates 12h；

2) in the LB fluid medium transferring new by 1% inoculum concentration, 37 DEG C, cultivate on shaking table under conditions of 200rpm；

3) OD is worked as₆₀₀When=1.0, add final concentration 1mM IPTG, then at 37 DEG C, under conditions of 200r/min, induce 6h；

4) taking 30mL culture fluid in 50mL centrifuge tube, 6000r/min is centrifuged 8min；

5) abandoning supernatant, add 10mL sterile phosphate buffer solution, vortex oscillation mixes, and adds PBS to 20mL, and 6000 R/min is centrifuged 8min；Repeat step once；

6) 10mL PBS, cell suspension supersound process 30min, ultrasonic 2s, intermittently 3s, power 40% are added；

7) under 4 DEG C of low temperature, 8000r/min is centrifuged 15min, removes cell debris supernatant and is crude enzyme liquid；

2. the SDS-PAGE of ODC Ornithine decarboxylase SpeC analyzes

By stand-by soak in buffer, it is loaded in gel pore by the microsyringe number of pressing；Connect electrophresis apparatus, open Power supply, regulation electric current, to 20-30mA, keeps constant current, time at sample migration to separation gel lower end 1cm, closes power supply； After electrophoresis, take off running gel, and carefully cut a fritter as labelling at gel one jiao, pour Coomassie brilliant blue dye into Color liquid carries out the 1h that dyes；Pour out dyeing liquor, gel is rinsed for several times in water；Add destaining solution, start to change every 2h Destaining solution once, until gel clear background；

PUT-C1 carries out abduction delivering and SDS-PAGE analyzes；

3. enzyme activity determination

1) phosphate buffer taking 800 μ L differences pH is filled in graded tube in the aseptic tool of 5mL, as 37 DEG C of water-baths preheating 5min； Parallel 3；

2) add 100 μ L crude enzyme liquids, add L-arginine mother solution 100 μ L, start timing 30min；Matched group sterilized water Substitute L-arginine；

3) tool plug graded tube is placed on ice, is rapidly added 500 μ L 2MNaOH and terminates reaction；

4) derivative, the growing amount of upper prop detection butanediamine；

4. ornithine decarboxylase activity testing result

Butanediamine conversion results, the activity of detection ODC Ornithine decarboxylase SpeC is detected according to HPLC；

(3) the shake flask fermentation of recombinant bacterial strain PUT-C1 and the mensuration of butylamine yield

1. shake flask fermentation

On flat board, picking one ring monoclonal recombinant bacterial strain is inoculated into containing cultivating 10-12h in 5mL LB culture medium, with 1% Inoculum concentration is linked in the 250mL triangular flask equipped with 50mL LB culture medium, 37 DEG C, and 200r/min cultivates to OD₆₀₀=1.0, Add final concentration 1mM IPTG, then at 37 DEG C, under conditions of 200r/min, induce 6h；Put into substrate ornithine, wherein The concentration of ornithine is respectively 0g/L, 10g/L, 20g/L, 30g/L and 40g/L, often organizes parallel three；37 DEG C, 200r/min Cultivate 12h；Fermentation liquid 12000r/min is centrifuged 5min, takes supernatant 4 DEG C preservation；

2. HPLC detects butanediamine

Experiment uses the content of National Standard Method detection butanediamine, and by water sample is carried out column front derivation, water sample is through Benzenecarbonyl chloride. derivatization Extracting with ether afterwards, extract uses high performance liquid chromatography ultraviolet detection after solvent switch is dissolved in methanol；

1) the derivative and extraction of sample

Take 1mL supernatant to add in 5mL tool plug scale test tube, add 500 μ L 2mol/LNaOH solution, 10 μ L benzene first Acyl chlorides, 37 DEG C of water-bath shaking 20min, every 5min vortex vibration 30s, add 0.5g NaCl, 1mL ether vibration 30s Stratification；Upper strata ether is taken to another centrifuge tube, treats ether volatilization completely, add 500 μ L methanol and dissolve, after crossing film As HPLC detection sample；Derivative and the same sample handling procedure of extraction step of standard solution；

2) HPLC measures

A chromatographic condition

A) chromatographic column: C18 chromatographic column, 5 μm, 4.6 × 250mm, or the suitable person of performance；

B) column temperature: room temperature；

C) flowing phase: A (acetonitrile), B (0.02mol/L ammonium acetate), condition of gradient elution is as follows:

D) detection wavelength: 254nm.

E) sample size: 10 μ L；

F) flow velocity: 1mL/min；

B liquid-phase chromatographic analysis measures

Start, preheating, use flowing to rinse chromatographic column mutually, after baseline stability 30min, start sample introduction；

Derive after fermented liquid supernatant is diluted and carry out HPLC analysis, gone out the content of butanediamine by calculated by peak area.