A high conversion rate β-alanine bioenzyme synthesis method and complete set of equipment

Abstract

The present invention relates to a high-conversion β-alanine bioenzyme synthesis method, which comprises the following steps: after acrylic acid and ammonia solution are uniformly mixed in a mixer (one), the obtained mixed solution (one) is introduced into a mixer (two), acrylic acid ammonia oxidase is added, and after uniform mixing, the obtained mixed solution (two) is introduced into a pipeline reactor for conversion reaction to obtain a conversion solution containing β-alanine. The preparation method of the present invention realizes the continuous production of the β-alanine bioenzyme method, significantly reduces side reactions, and significantly improves the reaction rate and the effective conversion rate of acrylic acid.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a beta-alanine bioenzyme synthesis method with high conversion rate and a complete set of equipment thereof. Background Art

[0002] β-Alanine is the only β-type non-protein amino acid that exists in nature and is widely used in medicine, feed, food and other fields.

[0003] CN 108383742 A discloses a method for producing β-alanine by chemical method, wherein an ammonia water mixture and acrylic acid are respectively introduced into a pipeline reactor for continuous reaction, and then the reaction product is subjected to deamination, dehydration, alcohol precipitation and crystallization treatment to obtain β-alanine. The effective conversion rate of acrylic acid in this method can reach 96%. However, the reaction needs to be carried out under high temperature (100-150°C) and high pressure (1.0MPa) conditions, and there are many side reactions (such as acrylic acid self-polymerization) and large energy consumption.

[0004] CN1285730C discloses a method for synthesizing β-alanine by biological method. In this method, microorganisms capable of producing acrylic acid ammoxidase are used to culture the corresponding aminase in a seed culture medium containing acrylic acid and a fermentation culture medium containing acrylic acid, respectively. The aminase generated by biological transformation is then added to an ammonia solution containing 1% to 50% (g / ml) acrylic acid to synthesize β-alanine under the action of the enzyme. The product purity can reach 98%, but its acrylic acid conversion rate is only about 60%.

[0005] CN109385415A discloses a method for converting acrylic acid into β-alanine using an aspartase variant, wherein the conversion rate of acrylic acid is above 84%.

[0006] CN110923272A discloses an aspartic acid mutant capable of bioconverting acrylic acid into β-alanine, the mutant has four mutation points at the same time, and the conversion rate of acrylic acid is as high as 99.8%. However, the above conversion rate is not the effective conversion rate of acrylic acid into β-alanine.

[0007] Compared with chemical synthesis, the reaction conditions of the bio-enzyme synthesis method of β-alanine are relatively mild, and it is more suitable for the conversion of acrylic acid into β-alanine by adding ammonia. However, the bio-enzyme synthesis method has defects such as low conversion rate, and it is urgently needed to study and explore bio-enzyme synthesis methods with higher conversion rates. Summary of the invention

[0008] The object of the present invention is to provide a high conversion rate β-alanine bioenzymatic synthesis method, comprising the following steps:

[0009] (1) After uniformly mixing acrylic acid and an aqueous ammonia solution in a mixer (1), introducing the obtained mixed solution (1) into a mixer (2), adding acrylic acid ammonia oxidase, and uniformly mixing to obtain a mixed solution (2);

[0010] (2) The obtained mixed solution (II) is introduced into a pipeline reactor for conversion reaction to obtain a conversion solution containing β-alanine.

[0011] In the preferred technical solution of the present invention, the acrylic acid ammonia oxidase is an acrylic acid ammonia oxidase solution, and the acrylic acid ammonia oxidase solution is preferably selected from any one or a combination of fermentation broth or concentrated solution obtained by fermenting a microbial strain that can express acrylic acid ammonia oxidase in a culture medium.

[0012] In the preferred technical solution of the present invention, the concentration is selected from any one of vacuum concentration, reduced pressure concentration, membrane concentration, normal pressure concentration, ultrafiltration concentration, centrifugal concentration or a combination thereof.

[0013] According to a preferred technical solution of the present invention, the weight-to-volume ratio (w / v / v) of acrylic acid: aqueous ammonia solution: acrylic acid ammonia oxidase solution is 1:1.5-2.0:0.6-1.5, preferably 1:1.6:0.7.

[0014] According to a preferred technical solution of the present invention, the enzyme activity of the acrylic acid ammoxidase solution is 50-2000 U / mL, preferably 500-1500 U / ml, and more preferably 800-1200 U / mL.

[0015] According to a preferred technical solution of the present invention, the microbial strain is a wild-type strain, preferably any one of wild-type Escherichia coli and wild-type Sarcina luteus.

[0016] According to a preferred technical solution of the present invention, the microbial strain is a genetically engineered strain, preferably any one of a genetically engineered strain of Escherichia coli and a genetically engineered strain of Sarcina luteus.

[0017] According to a preferred technical solution of the present invention, the concentration of the aqueous ammonia solution is 15%-45%, preferably 20%-40%, and more preferably 20%-30%.

[0018] According to a preferred technical solution of the present invention, the mixed liquid (I) stays in the mixer (I) for 5 to 60 seconds, preferably 10 to 50 seconds, and more preferably 20 to 45 seconds.

[0019] According to a preferred technical solution of the present invention, the mixing temperature of the mixed liquid (I) in the mixer (I) is 45 to 95°C, preferably 50 to 70°C, and more preferably 55 to 65°C.

[0020] According to a preferred technical solution of the present invention, the mixed liquid (II) stays in the mixer (II) for 5 to 40 seconds, preferably 8 to 35 seconds, and more preferably 10 to 30 seconds.

[0021] In the technical solution of the present invention, the mixer (1) and the mixer (2) are any one of a static mixer and a dynamic mixer or a combination thereof, preferably a static mixer.

[0022] According to a preferred technical solution of the present invention, the reaction temperature of the mixed solution (II) in the pipeline reactor is 30-65°C, preferably 35-60°C, and more preferably 40-55°C.

[0023] According to a preferred technical solution of the present invention, the reaction pressure of the mixed solution (II) in the pipeline reactor is 0 to 1.0 MPa, preferably 0 to 0.8 MPa, and more preferably 0 to 0.5 MPa.

[0024] According to a preferred technical solution of the present invention, the reaction time of the mixed solution (II) in the pipeline reactor is 5 to 180 min, preferably 30 to 150 min, and more preferably 30 to 120 min.

[0025] According to a preferred technical solution of the present invention, a mixer is further provided in the pipeline reactor, and the mixer is preferably a static mixer, and more preferably an SK type static mixer.

[0026] According to a preferred technical solution of the present invention, the obtained conversion liquid containing β-alanine is purified to obtain β-alanine, and the preferred purification process includes ultrafiltration, deamination, crystallization, and separation steps.

[0027] According to a preferred technical solution of the present invention, the prepared β-alanine is subjected to a recrystallization purification process, and the recrystallization preferably includes the steps of dissolving, decolorizing, crystallizing, separating, and drying.

[0028] In a preferred technical solution of the present invention, the separation is selected from any one of filtration, centrifugation, membrane treatment or a combination thereof.

[0029] In a preferred technical solution of the present invention, the collected separated solid is washed and then dried, and the solvent used for the washing is preferably water.

[0030] In a preferred technical solution of the present invention, the drying is selected from any one of vacuum drying, reduced pressure drying, normal pressure drying, spray drying, and boiling drying, or a combination thereof.

[0031] According to a preferred technical solution of the present invention, the prepared β-alanine is subjected to crystallization purification treatment for no less than 1 time, preferably 2-4 times.

[0032] According to a preferred technical solution of the present invention, the ultrafiltration treatment is to filter the obtained β-alanine conversion liquid in a closed pre-coat drum filter and then perform ultrafiltration treatment, preferably ultrafiltration membrane treatment.

[0033] According to a preferred technical solution of the present invention, the ultrafiltration concentrate collected from the ultrafiltration treatment of the β-alanine conversion liquid is used to prepare organic fertilizer.

[0034] According to a preferred technical solution of the present invention, the deamination treatment is flash deamination.

[0035] According to a preferred technical solution of the present invention, the gas phase collected during the deammoniation treatment of the obtained ultrafiltration clear liquid is condensed and recovered, and then fed back to the mixing step of liquid ammonia and water for recycling.

[0036] According to a preferred technical solution of the present invention, the mother liquor collected in the separation process is returned to any one of the ultrafiltration and decolorization steps for recycling.

[0037] In a preferred technical solution of the present invention, the decolorization treatment is any one of activated carbon and adsorbent treatment.

[0038] According to the preferred technical solution of the present invention, the activated carbon used for decolorization is then incinerated or used as a raw material for preparing organic fertilizer for comprehensive utilization.

[0039] Another object of the present invention is to provide a complete set of equipment for the bio-enzyme synthesis of β-alanine, wherein the complete set of equipment comprises a mixer (one), a mixer (two), and a pipeline reactor.

[0040] In the technical solution of the present invention, the mixer (1) and the mixer (2) are any one of a static mixer and a dynamic mixer or a combination thereof, wherein a static mixer is preferred, a static pipeline mixer is more preferred, and a static pipeline SK type packing mixer is most preferred.

[0041] According to a preferred technical solution of the present invention, the pipeline reactor is composed of sub-reactors connected in series, preferably at least two sub-reactors connected in series, and more preferably at least three sub-reactors connected in series.

[0042] According to a preferred technical solution of the present invention, a mixing device is further provided in the pipeline reactor, preferably a static mixing device, more preferably an SK type static mixing device.

[0043] According to a preferred technical solution of the present invention, the complete set of equipment also includes an ultrafiltration device, a deamination device, a crystallization device, a dissolution device, a decolorization device, a crystallization device, a separation device, and a drying device, which are preferably connected in series.

[0044] Another object of the present invention is to provide a method for recycling waste generated by a β-alanine bioenzyme synthesis method, comprising the following steps:

[0045] (1) After uniformly mixing acrylic acid and an aqueous ammonia solution in a mixer (1), introducing the obtained mixed solution (1) into a mixer (2), adding acrylic acid ammonia oxidase, and uniformly mixing to obtain a mixed solution (2);

[0046] (2) introducing the obtained mixed solution (II) into a pipeline reactor for conversion reaction to obtain a conversion solution containing β-alanine;

[0047] (3) Purifying the obtained conversion solution containing β-alanine to obtain β-alanine. Preferably, the purification process includes ultrafiltration, deamination, crystallization, and separation steps.

[0048] According to a preferred technical solution of the present invention, the obtained conversion liquid containing β-alanine is subjected to ultrafiltration treatment, and the ultrafiltration concentrate collected during the deammoniation treatment of the obtained ultrafiltration clear liquid is used to prepare organic fertilizer.

[0049] According to a preferred technical solution of the present invention, the gas phase collected during the deammoniation treatment of the obtained ultrafiltration clear liquid is condensed and recovered, and then fed back to the mixing step of liquid ammonia and water for recycling.

[0050] In the preferred technical solution of the present invention, the crystallization mother liquor collected during the crystallization treatment is returned to any one of the ultrafiltration and dissolution steps for recycling.

[0051] According to the preferred technical solution of the present invention, the activated carbon used for decolorization is then incinerated or used as a raw material for preparing organic fertilizer for comprehensive utilization.

[0052] Another object of the present invention is to provide a complete set of recycling equipment for the bio-enzyme synthesis of β-alanine, the complete set of equipment comprising a batching kettle, a mixer (one), a mixer (two), a pipeline reactor, an ultrafiltration device, a deamination device, a crystallization device, a separation device, a dissolving device, a decolorization device, a crystallization device, a separation device, and a drying device, preferably connected in series in sequence.

[0053] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0054] Unless otherwise stated, the present invention is tested according to the following method:

[0055] 1. Enzyme activity detection method

[0056] The present invention defines the amount of acrylic acid ammoxidase required for catalyzing the conversion of 1 mmol acrylic acid to generate 1 mmol β-alanine per minute at 45° C. as its enzyme activity unit (1U).

[0057] Preparation of acrylic acid substrate solution: Weigh 288.24 g of acrylic acid, add it into a 1 L beaker, add ammonia water and deionized water, adjust the pH to 9.5 ± 0.05, and make the volume 1 L.

[0058] Method for detecting the enzyme activity of unit volume of acrylic acid ammonia oxidase solution: add 0.2 ml of acrylic acid ammonia oxidase solution to 2 ml of acrylic acid substrate solution in a 45°C water bath, shake well, react for 30 minutes, and use high pressure liquid chromatography to detect the enzyme activity and calculate the amount of β-alanine generated.

[0059] High-pressure liquid chromatography detection conditions: instrument model: Agilent 1200Serie; chromatographic column: Nucleosil 100C184.6×250mm 5μm; column temperature: 25℃; UV detection wavelength: 210nm; acquisition time: 10min; injection volume: 10μL; flow rate: 1.00mL / min. Mobile phase composition: phase A: phase B = 95:10, wherein the preparation of phase A: weigh 1.44g KH2PO4 and dissolve it in 1L double distilled water, and adjust the pH to 2.6 with phosphoric acid.

[0060] According to the above-mentioned high pressure liquid chromatography detection conditions, a β-alanine concentration standard curve was prepared, and the β-alanine concentration in the conversion solution before and after the reaction was detected.

[0061] Calculation of enzyme activity per unit volume of acrylic acid ammonia oxidase solution: Enzyme activity (U / ml) = β-alanine concentration in the conversion product (mmol / L) * (acrylic acid substrate solution volume + acrylic acid ammonia oxidase solution volume) / (reaction time * acrylic acid ammonia oxidase solution volume)

[0062] 2. Calculation method of effective conversion rate of acrylic acid

[0063] Effective conversion rate of acrylic acid = molar concentration of β-alanine in the conversion solution / initial molar concentration of acrylic acid in the mixed solution (II).

[0064] 3. Detection method of β-alanine purity

[0065] Weigh the required amount of β-alanine standard and prepare it into standard solutions with concentrations of 0, 0.4, 0.8, 1.2, 1.6, and 1.0 g / L, respectively. According to the "method for detecting the enzyme activity of acrylic acid ammonia oxidase solution" of the present invention, draw a β-alanine standard curve that correlates concentration and peak area.

[0066] The β-alanine prepared in the present invention is prepared into a 1 g / L sample solution. According to the "method for detecting the enzyme activity of acrylic acid ammonia oxidase solution" of the present invention, the obtained liquid phase detection peak area is substituted into the β-alanine standard curve to calculate the concentration of β-alanine in the sample solution.

[0067] β-alanine purity = β-alanine concentration of sample solution / (1 g / L)×100%.

[0068] Compared with the prior art, the present invention has the following beneficial technical effects:

[0069] 1. The preparation method of the present invention realizes the continuous production of β-alanine by bioenzymatic method, significantly reduces the side reaction, and significantly improves the reaction rate and the effective conversion rate of acrylic acid.

[0070] 2. The complete set of equipment of the present invention significantly improves the utilization rate of reaction raw materials and reaction equipment, significantly reduces side reactions and energy consumption, and significantly improves the reaction rate and effective conversion rate of acrylic acid.

[0071] 3. The preparation method of the present invention has the advantages of mild reaction, continuous production, better cost, and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 β-Alanine bioenzyme synthesis process flow chart DETAILED DESCRIPTION

[0073] The present invention will be further described below in conjunction with specific embodiments so that those skilled in the art can understand the present invention, but the present invention is not limited thereby.

[0074] Reference Example 1 Preparation of acrylic acid ammonia oxidase solution

[0075] The enzyme solution of this reference example was prepared by referring to the method of Example 1 of CN109385415A:

[0076] Example 1. Preparation and performance evaluation of aspartase mutants

[0077] 1. Cloning

[0078] A plasmid containing a gene encoding the aspartase and its variants is constructed using methods known in the art, and the resulting recombinant plasmid is transformed into a suitable host cell.

[0079] In this embodiment, patent CN201710659654.9 discloses an enzyme that can catalyze the addition of acrylic acid to amino groups and its expression strain, and transforms the aspartase encoding gene from Bacillus subtilis to obtain multiple mutants. The present invention selects the 373rd mutant and transforms the aspartase encoding gene from Bacillus subtilis (see sequence 1). The mutant gene combination encoding the variant is as follows: A59T / C60G / A267T / C560T / G561T / T772A / A774T / G963T / A971T /

[0080] A972T / A976T / A977G / T978C / A1166T / A1167C. In this embodiment, the vector plasmid used is specifically pET21a. Using the gene fragment containing the gene encoding aspartase and its variants as a template, TATGGCTAGCATGACTGGTatgaataccgatgttcgtattg and GCTAGTTATTGCTCAGCGGttttcttccagcaattcccg are used as primers to PCR amplify the corresponding nucleotide gene fragment. Afterwards, the gene fragment obtained by the PCR amplification and pET21a are mixed in a molar ratio of 1:1, and a plasmid containing the gene encoding the aspartase variant is constructed by the Gibson assembly method (see: Ji Zhicheng et al., Construction of plant expression vector using the Gibson Assembly method, Journal of South China Agricultural University, 2014, 35 (5): 112-116). Using Escherichia coli BL21 (DE3) as a host cell, a recombinant plasmid containing the aspartase encoding gene is introduced into the host cell to obtain an expression strain capable of expressing the aspartase variant (ie, the acrylic acid aminoase of the present invention).

[0081] Sequence 1:

[0082]

[0083]

[0084] 2. Expression

[0085] Use autoinduction medium to culture host cells containing the recombinant plasmid constructed in step 1. The composition of the autoinduction medium is as follows: peptone 10g / L, yeast powder 5g / L, disodium hydrogen phosphate 3.55g / L, potassium dihydrogen phosphate 3.4g / L, ammonium chloride 2.68g / L, sodium sulfate 0.71g / L, magnesium sulfate heptahydrate 0.493g / L, ferric chloride hexahydrate 0.27g / L, 100% glycerol 20mL / L, glucose 0.5g / L, lactose 2g / L, ampicillin sodium 50mg / L. The host cells containing the recombinant plasmid were inoculated in the autoinduction medium and fermented in batch fermentation. Shake and culture for 20 hours at 30°C and 200rpm.

[0086] 3. Cell Collection

[0087] The following three methods can be used: ① Centrifugation: centrifuge the cell culture medium at 4000g for 10 minutes to collect cells; ② Hollow fiber membrane filtration: filter the cell culture medium with a 0.22 micron hollow fiber membrane to collect cells; ③ Ceramic membrane filtration: filter with a 50KDa ceramic membrane to collect cells. Centrifugation is preferred to collect cells under laboratory conditions (this example uses this method).

[0088] The collected cells were diluted with water to obtain an acrylic acid ammonia oxidase solution, and the enzyme activity in the acrylic acid ammonia oxidase solution was measured according to the enzyme activity measurement method of the present invention, and the enzyme activity was 800 U / ml.

[0089] Embodiment 1-11 Preparation of β-alanine

[0090] The preparation method of β-alanine comprises the following steps:

[0091] 1. According to the ratio of acrylic acid: ammonia water: acrylic acid ammonia oxidase solution described in Table 1, take the required amount of acrylic acid (1000 kg) and 21% ammonia water, pump them into the mixer (I) and mix them for 45 seconds, then pump the obtained mixed solution (I) into the mixer (II), add the acrylic acid ammonia oxidase solution prepared in Reference Example 1, mix evenly for 30 seconds, and then enter the pipeline reactor at a flow rate of 191 L / h to react (reaction temperature see Table 1) for 120 minutes to obtain β-alanine conversion solution.

[0092] 2. The obtained β-alanine conversion liquid is filtered through a ceramic membrane and the clear liquid is collected. The obtained clear liquid is subjected to flash deamination treatment, and the obtained deaminated liquid is subjected to decolorization treatment. The decolorization conditions are: 0.5% (w / v) activated carbon, maintained at 55°C for 60 minutes. The obtained decolorized clear liquid is evaporated and concentrated to a β-alanine content of 800g / L at 90°C and -0.9MPa. The obtained slurry (I) is cooled to below 40°C and centrifuged to obtain centrifuged wet crystals (I). The centrifuged wet crystals (I) are redissolved in water to a β-alanine content of 450g / L and decolorized. The decolorization conditions are: 0.5% (w / v) activated carbon, maintained at 55°C for 60 minutes. The obtained decolorized clear liquid is evaporated and concentrated at 90°C and -0.9MPa to a β-alanine content of 800g / L. The obtained crystal slurry (II) is cooled to below 40°C and centrifuged. At the end of centrifugation, 5% of the volume of the centrifuged crystal slurry is added with water to rinse the crystals, which are then dried to obtain pure β-alanine.

[0093] The effective conversion rate of acrylic acid and the purity of β-alanine in Examples 1-11 were tested according to the method of the present invention. The results are shown in Table 1. In Table 1, the units of acrylic acid, ammonia water and acrylic acid are kg, L and L respectively.

[0094] Table 1

[0095]

[0096] Comparative Example 1

[0097] The preparation method of β-alanine comprises the following steps:

[0098] (1) According to the ratio of acrylic acid: ammonia water: acrylic acid ammonia oxidase solution (w:v:v) of 1:1.6:0.7, the required amount of acrylic acid (1000 kg) and 21% ammonia water were pumped into a mixer (I) and mixed for 45 seconds. The obtained mixed solution (I) was pumped into a mixer (II), and the acrylic acid ammonia oxidase solution prepared in Reference Example 1 was added. After uniform mixing for 30 seconds, the mixture was put into a tank reactor and continued to react at 45°C. The feeding was terminated and the reaction was carried out for 120 minutes to obtain a β-alanine conversion solution.

[0099] (2) The obtained β-alanine conversion liquid is filtered through a ceramic membrane and the clear liquid is collected. The obtained clear liquid is subjected to flash deamination treatment, and the obtained deaminated liquid is subjected to decolorization treatment. The decolorization conditions are: 0.5% (w / v) activated carbon, maintained at 55°C for 60 minutes. The obtained decolorized clear liquid is evaporated and concentrated to a β-alanine content of 800g / L at 90°C and -0.9MPa. The obtained crystal slurry (I) is cooled to below 40°C and centrifuged to obtain centrifuged wet crystals (I). The centrifuged wet crystals (I) are redissolved in water to a β-alanine content of 450g / L and decolorized. The decolorization conditions are: 0.5% (w / v) activated carbon, maintained at 55°C for 60 minutes. The obtained decolorized clear liquid is evaporated and concentrated at 90°C and -0.9MPa to a β-alanine content of 800g / L. The obtained crystal slurry (II) is cooled to below 40°C and centrifuged. At the end of centrifugation, 5% of the volume of the centrifuged crystal slurry is added with water to rinse the crystals, and then pure β-alanine is obtained.

[0100] Comparative Example 2

[0101] The preparation method of β-alanine comprises the following steps:

[0102] (1) According to the ratio of acrylic acid: ammonia water: acrylic acid ammonia oxidase solution (w:v:v) of 1:1.6:0.7, the required amount of acrylic acid (1000 kg) and 21% ammonia water were pumped into a mixer (1), and the acrylic acid ammonia oxidase solution prepared in Reference Example 1 was added. After uniform mixing for 30 seconds, the mixture was introduced into a pipeline reactor at a flow rate of 191 L / h and reacted (reaction temperature 45°C) for 120 minutes to obtain a β-alanine conversion solution.

[0103] (2) The obtained β-alanine conversion liquid is filtered through a ceramic membrane and the clear liquid is collected. The obtained clear liquid is subjected to flash deamination treatment, and the obtained deaminated liquid is subjected to decolorization treatment. The decolorization conditions are: 0.5% (w / v) activated carbon, maintained at 55°C for 60 minutes. The obtained decolorized clear liquid is evaporated and concentrated to a β-alanine content of 800g / L at 90°C and -0.9MPa. The obtained crystal slurry (I) is cooled to below 40°C and centrifuged to obtain centrifuged wet crystals (I). The centrifuged wet crystals (I) are redissolved in water to a β-alanine content of 450g / L and decolorized. The decolorization conditions are: 0.5% (w / v) activated carbon, maintained at 55°C for 60 minutes. The obtained decolorized clear liquid is evaporated and concentrated at 90°C and -0.9MPa to a β-alanine content of 800g / L. The obtained crystal slurry (II) is cooled to below 40°C and centrifuged. At the end of centrifugation, 5% of the volume of the centrifuged crystal slurry is added with water to rinse the crystals, and then β-alanine is obtained.

[0104] The effective conversion rate of acrylic acid and the purity of β-alanine in Comparative Example 1-2 were determined according to the method of the present invention. The results are shown in Table 2.

[0105] Table 2

[0106] serial number Effective conversion rate of acrylic acid (%) Purity of β-alanine (%) Comparative Example 1 86.1 85.2 Comparative Example 2 80.5 77.4

[0107] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A method for synthesizing β-alanine with high conversion rate by bioenzyme, comprising the following steps: (1) After uniformly mixing acrylic acid and an aqueous ammonia solution in a mixer (1), introducing the obtained mixed solution (1) into a mixer (2), adding acrylic acid ammonia oxidase, and uniformly mixing to obtain a mixed solution (2); The acrylic acid ammonia oxidase is an acrylic acid ammonia oxidase solution, and the enzyme activity of the acrylic acid ammonia oxidase solution is 50-2000U / mL; The concentration of the ammonia solution is 15%-45%; The weight-to-volume ratio (w / v / v) of the acrylic acid: ammonia solution: acrylic acid ammonia oxidase solution is 1:1.5-2.0:0.6-1.5; (2) introducing the obtained mixed solution (II) into a pipeline reactor for conversion reaction to obtain a conversion solution containing β-alanine; The reaction temperature of the mixed solution (II) in the pipeline reactor is 35-60°C.

2. The method according to claim 1, characterized in that The acrylic acid ammonia oxidase solution is selected from any one or a combination of fermentation broth or concentrated solution obtained by fermenting microorganisms containing a strain capable of expressing acrylic acid ammonia oxidase in a culture medium.

3. The method according to claim 2, characterized in that The concentration is selected from any one of vacuum concentration, reduced pressure concentration, membrane concentration, normal pressure concentration, ultrafiltration concentration, centrifugal concentration or a combination thereof.

4. The method according to claim 1, characterized in that The weight-to-volume ratio (w / v / v) of the acrylic acid:ammonia solution:acrylic acid ammonia oxidase solution is 1:1.6:0.

7.

5. The method according to claim 1, characterized in that The enzyme activity of the acrylic acid ammonia oxidase solution is 500-1500 U / ml.

6. The method according to claim 2, characterized in that The microbial strain is a wild-type strain.

7. The method according to claim 6, characterized in that The microbial strain is any one of wild-type Escherichia coli and wild-type Sarcina luteus.

8. The method according to claim 2, characterized in that The microbial strain is a genetically engineered strain.

9. The method according to claim 8, characterized in that The microbial strain is any one of a genetically engineered strain of Escherichia coli and a genetically engineered strain of Sarcina luteus.

10. The method according to claim 1, characterized in that The concentration of the ammonia solution is 20%-40%.

11. The method according to claim 1, characterized in that The mixed liquid (1) stays in the mixer (1) for 5 to 60 seconds.

12. The method according to claim 1, characterized in that The mixing temperature of the mixed liquid (1) in the mixer (1) is 45-95°C.

13. The method according to claim 1, characterized in that The mixed solution (2) stays in the mixer (2) for 5 to 40 seconds.

14. The method according to claim 1, wherein: The mixer (1) and the mixer (2) are any one of a static mixer and a dynamic mixer or a combination thereof.

15. The method according to claim 1, wherein: The reaction temperature of the mixed solution (II) in the pipeline reactor is 40-55°C.

16. The method according to claim 1, wherein: The reaction pressure of the mixed liquid (II) in the pipeline reactor is 0-1.0 MPa.

17. The method according to claim 1, wherein: The reaction time of the mixed solution (II) in the pipeline reactor is 5 to 180 minutes.

18. The method of claim 1, wherein: A mixer is also arranged in the pipeline reactor, and the mixer is a static mixer.

19. The method according to claim 1, wherein: The obtained conversion liquid containing β-alanine is purified to obtain β-alanine, and the purification process includes ultrafiltration, deamination, crystallization and separation steps.

20. The method of claim 19, wherein: The prepared β-alanine is subjected to a recrystallization purification process, wherein the recrystallization comprises the steps of dissolving, decolorizing, crystallizing, separating and drying.

21. The method according to claim 19 or 20, characterized in that The separation is selected from any one of filtration, centrifugation, membrane treatment or a combination thereof.

22. The method according to claim 21, characterized in that The collected separated solid was washed and then dried.

23. The method according to claim 20 or 22, characterized in that The drying is selected from any one of vacuum drying, reduced pressure drying, normal pressure drying, spray drying, boiling drying or a combination thereof.

24. The method of claim 19, wherein: The number of crystallization purification treatments for the obtained β-alanine is 2-4 times.

25. The method of claim 19, wherein: The ultrafiltration treatment is to filter the prepared beta-alanine conversion liquid in a closed pre-coating drum filter and then perform ultrafiltration treatment.

26. The method of claim 19, wherein: The ultrafiltration concentrate collected during the ultrafiltration treatment of the β-alanine conversion solution is used to prepare organic fertilizer.

27. The method of claim 19, wherein: The deamination treatment is flash deamination.

28. The method of claim 19, wherein: The gas phase collected during the deammoniation treatment of the obtained ultrafiltration clear liquid is condensed and recovered, and then fed back to the mixing step of liquid ammonia and water for recycling.

29. The method of claim 20, wherein: The mother liquor collected during the separation process is returned to any one of the ultrafiltration and decolorization steps for recycling.

30. The method of claim 20, wherein: The decolorization treatment is any one of activated carbon and adsorbent treatment.

31. The method of claim 30, wherein: The activated carbon used for decolorization can be incinerated or used as a raw material for preparing organic fertilizer for comprehensive utilization.

32. A complete set of equipment for implementing the method described in any one of claims 1 to 31, wherein the complete set of equipment comprises a mixer (one), a mixer (two), and a pipeline reactor connected in series in sequence.

33. The device according to claim 32, characterized in that The mixer (1) and the mixer (2) are static mixers, dynamic mixers or a combination thereof.

34. The device according to claim 33, characterized in that The mixer (1) and the mixer (2) are static pipeline SK type filler mixers.

35. The device according to claim 32, characterized in that The pipeline reactor is composed of sub-reactors connected in series.

36. The device according to claim 32, characterized in that The pipeline reactor is also provided with a mixing device.

37. The device according to claim 32, characterized in that The complete set of equipment also includes an ultrafiltration device, a deammoniation device, a first crystallization device, a dissolving device, a decolorization device, a second crystallization device, a separation device, and a drying device.

38. A method for recycling waste generated by a β-alanine bioenzyme synthesis method, comprising the following steps: (1) After uniformly mixing acrylic acid and an aqueous ammonia solution in a mixer (1), introducing the obtained mixed solution (1) into a mixer (2), adding acrylic acid ammonia oxidase, and uniformly mixing to obtain a mixed solution (2); The acrylic acid ammonia oxidase is an acrylic acid ammonia oxidase solution, and the enzyme activity of the acrylic acid ammonia oxidase solution is 50-2000U / mL; The concentration of the ammonia solution is 15%-45%; The weight-to-volume ratio (w / v / v) of acrylic acid: ammonia solution: acrylic acid ammonia oxidase solution is 1:1.5-2.0:0.6-1.5; (2) introducing the obtained mixed solution (II) into a pipeline reactor for conversion reaction to obtain a conversion solution containing β-alanine; The reaction temperature of the mixed solution (II) in the pipeline reactor is 30-65°C; (3) Purifying the obtained conversion solution containing β-alanine to obtain β-alanine.

39. The method of claim 38, wherein: The purification process includes ultrafiltration, deamination, crystallization and separation steps.

40. The method of claim 39, wherein: The obtained conversion liquid containing β-alanine is subjected to ultrafiltration treatment, and the ultrafiltration concentrate collected during the deammoniation treatment of the obtained ultrafiltration clear liquid is used to prepare organic fertilizer.

41. The method of claim 39, wherein: The gas phase collected during the deammoniation treatment of the obtained ultrafiltration clear liquid is condensed and recovered, and then fed back to the mixing step of liquid ammonia and water for recycling.

42. The method of claim 39, wherein: The crystallization mother liquor collected during the crystallization process is returned to any step of ultrafiltration or dissolution for recycling.

43. The method of claim 39, wherein: The mother liquor collected during the separation process is returned to any one of the ultrafiltration and decolorization steps for recycling.

44. The method of claim 43, wherein: The activated carbon used for decolorization can be incinerated or used as a raw material for preparing organic fertilizer for comprehensive utilization.

45. A complete set of recycling equipment for implementing the method described in any one of claims 38 to 44, wherein the complete set of equipment includes a batching kettle, a mixer (one), a mixer (two), a pipeline reactor, an ultrafiltration device, a deamination device, a first crystallization device, a separation device, a dissolving device, a decolorization device, a second crystallization device, a separation device, and a drying device.

46. ​​The recycling equipment according to the method described in claim 45, wherein the batching kettle, mixer (1), mixer (2), pipeline reactor, ultrafiltration device, deamination device, first crystallization device, separation device, dissolution device, decolorization device, second crystallization device, separation device, and drying device are connected in series in sequence.

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