Nitrilase and its application in the biological preparation of β-alanine

Abstract

The present invention discloses a nitrilase and its application in the biological preparation of β-alanine. In the first stage, the nitrilase NIT II obtained by site-directed mutagenesis is reacted with a substrate acrylonitrile to obtain acrylic acid, and the tolerance of the enzyme to high-concentration substrates is improved by preparing immobilized cells. In the second stage, aspartase is reacted with acrylic acid to obtain β-alanine. Compared with the method using fumaric acid and acrylic acid as raw materials, the acrylonitrile used in the present invention is cheaper, has a lower molecular weight of acrylonitrile, consumes less ammonia water, and has higher atom economy.

Description

Technical Field

[0001] The invention relates to the fields of biological fermentation and genetic engineering, in particular to nitrilase and its application in the biological preparation of beta-alanine. Background Art

[0002] β-alanine is the only β-type amino acid that exists in nature. It is also a non-protein amino acid that exists in some bacteria, fungi and plants. β-alanine and its derivatives are widely used in the fields of medicine, chemical industry, food, etc. In medicine, β-alanine is used to synthesize pantothenic acid, calcium pantothenate, carnosine, sodium pamidronate and balsalazide; in chemical industry, it is used in electroplating and as a water flocculant; in food, it can make pickled products quickly taste good, thereby shortening the pickling time, effectively enhancing the role of seasoning and preventing oxidation. It can also be used to synthesize sweeteners, improve the taste of alcoholic beverages and organic acids, and prevent beer and sparkling wine from aging.

[0003] The production of β-alanine at home and abroad is mainly based on chemical synthesis, that is, it is synthesized using acrylic acid or acrylonitrile as substrates. Under high temperature and high pressure conditions, acrylic acid, acrylate or acrylate reacts with ammonia to produce β-alanine, but the reaction process will cause reversible binding of acrylic acid and β-alanine, which affects the yield of β-alanine. Similarly, under high temperature and high pressure conditions, acrylonitrile and ammonia water can react in one step to obtain β-alanine, and the reaction route can also be adjusted to first synthesize β-aminopropionitrile, and then hydrolyze it under certain conditions to obtain β-alanine. Although the raw materials of this method are easy to obtain and the cost is low, a large amount of inorganic salts are generated during the hydrolysis process, which makes it difficult to purify the product and affects the quality of subsequent products.

[0004] The biological method mainly synthesizes β-alanine through aspartase and L-aspartate-α-decarboxylase, catalyzing fumaric acid to first synthesize L-aspartic acid, and then decarboxylating to generate β-alanine. This method converts fumaric acid into β-alanine with a molecular weight that changes from large to small, poor atom economy, low catalytic substrate concentration, and high catalytic cost. The Chinese patent "Biosynthesis of β-alanine" (publication number CN 110923272 A, publication date March 27, 2020) discloses a technology for synthesizing β-alanine using acrylic acid as a raw material through aspartase. Although this method uses cheaper acrylic acid with a molecular weight that increases from small to large, and the atom economy is significantly improved compared to the method using fumaric acid as a raw material, this method is at the laboratory level, the reaction system is 100 ml, the substrate concentration is only 60 g / L, the reaction temperature is as high as 48-51 ° C, the energy consumption is high, and it is not suitable for industrial production. In addition, a large amount of ammonia water needs to be added to the reaction system, and the conversion liquid is also accompanied by about 60 g / L of ammonium acetate, which affects the post-product separation and the quality indicators of the product ammonium salt are easily exceeded. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a nitrilase which uses acrylonitrile as a substrate to catalyze the production of acrylic acid with a high conversion rate under a more suitable reaction environment; another purpose of the present invention is to provide a gene encoding the nitrilase, a recombinant vector and a host cell; a further purpose of the present invention is to provide a method for preparing the nitrilase; a further purpose of the present invention is to use the nitrilase in the biological preparation of β-alanine, and to provide a specific method for the biological preparation of β-alanine.

[0006] Technical solution: In order to achieve the above-mentioned invention object, the present invention provides a nitrilase NIT II, ​​comprising an amino acid sequence as shown in SEQ ID No: 1, and a gene encoding the same as shown in SEQ ID No: 2. The sequence is derived from a wild-type nitrilase NIT I (GenBank: ASY91980.1) of Achromobacter denitrificans, and the nucleotide sequence is shown in SEQ ID No: 4, and the corresponding amino acid sequence is shown in SEQ ID NO: 3.

[0007] The nitrilase NIT II provided by the invention is obtained by site-directed mutagenesis on the basis of NIT I. The engineered strain cultured after the mutation has a higher conversion rate and a lower optimum temperature for enzyme activity, is suitable for industrial production, and has extremely high application value.

[0008] The aforementioned SEQ ID NO: 2 nucleotide sequence can be constructed into a recombinant expression vector through existing bioengineering technology, for example, using any of pET, pGEX, and pMAL vectors, containing necessary resistance genes, and transfected into competent host cells after recombination, including but not limited to any of Escherichia coli, Bacillus subtilis, and Pichia pastoris, to obtain an improved strain, and produce the enzyme protein expressing the target sequence through culture and fermentation.

[0009] For site-directed mutagenesis, a wild-type nitrilase comprising an amino acid sequence as shown in SEQ ID No: 3 is subjected to site-directed mutagenesis, and the specific mutation sites are: F at position 54 is mutated to Y, M at position 113 is mutated to L, F at position 168 is mutated to L, F at position 192 is mutated to N, A at position 193 is mutated to E, L at position 201 is mutated to T, and Q at position 248 is mutated to A.

[0010] As a further optimization of the present invention, the wild nitrilase is subjected to enzyme immobilization treatment, which can effectively improve the service life of the enzyme in industrial production. As a preferred embodiment, after fermentation and culture of the mutant strain, the bacterial cells are centrifuged and crushed, and the supernatant is taken as a crude enzyme liquid, and the crude enzyme liquid is subjected to immobilization treatment, which is to add diatomaceous earth and polyethyleneimine to the crude enzyme liquid and mix them evenly, and then add glutaraldehyde for cross-linking to obtain immobilized nitrilase.

[0011] More preferably, the crude enzyme solution concentration of the reaction system is 50-100 g / L, and 3-6 g / L of diatomaceous earth, 0.1-0.15 wt% of polyethyleneimine and 0.1-0.25 wt% of glutaraldehyde are added accordingly. The enzyme solution concentration used in the present invention is very low, and its catalytic efficiency can be effectively guaranteed by immobilization treatment. On this basis, there is no need to replace the reaction system, and aspartase can be added to enter the next biocatalytic process, thereby greatly improving production efficiency.

[0012] The nitrilase provided by the present invention is different from the prior art. It uses acrylonitrile as a substrate for biocatalysis to obtain acrylic acid, and then uses acrylic acid catalytic enzyme to further catalyze acrylic acid to obtain β-alanine. The catalytic reaction route is as follows:

[0013]

[0014] Compared with the method of preparing β-alanine using fumaric acid and acrylic acid as raw materials, the acrylonitrile used in the present invention is cheaper, has a lower molecular weight, has higher atom economy, and its pH is not acidic, so there is no need to add a large amount of ammonia water for neutralization. In view of the efficient conversion characteristics of the aforementioned mutant nitrilase, the optimal pH temperature of the catalytic reaction in the two stages is not much different, so they can be carried out in the same reaction system.

[0015] Specifically, acrylonitrile is used as a reaction substrate, 0.1-10 g / L of the aforementioned nitrilase or immobilized nitrilase is added, and the first stage reaction is carried out under an environment of 30-37°C and pH 7.0-8.0. After the reaction is complete, 1-20 g / L of mutant aspartase is added to the reaction system, and the temperature is adjusted to 37-45°C and pH 8.5-9.0 for the second stage reaction to obtain β-alanine. It is worth noting that the strain selected by the present invention has a lower optimum temperature and the loss of production resources is much less than that of the prior art. Its efficient conversion rate can be catalyzed for acrylonitrile with a substrate concentration of not less than 200 g / L. Under suitable reaction conditions, the conversion rate of the nitrilase after catalysis for 3 hours is not less than 99.8%. Preferably, when no acrylonitrile is detected in the reaction system, the catalytic reaction is deemed to be finished, and then a small amount of ammonia water is used to adjust the pH to 8.5-9.0. Since the enzyme has a magnesium ion binding site at the catalytic midpoint, a small amount of magnesium acetate is added to improve the enzyme activity, so that the overall conversion rate of acrylonitrile after two catalysis in one reaction system is not less than 98%. The general addition amount is 0.05-5g / L.

[0016] In order to improve the overall conversion rate, in addition to providing a nitrilase with a higher conversion rate, the catalytic efficiency of the acrylic acid catalytic enzyme should be further optimized. The present invention provides a preferred acrylic acid catalytic enzyme, which is a mutant aspartase ASP II, which is derived from a wild-type thermophilic urea bacillus (Ureibacillus thermophilus). The original gene ASP I of the bacterium does not have the ability to catalyze acrylic acid to β-alanine. The mutant aspartase is a site-directed mutation of the amino acid sequence of GenBank number QBK25944.1, wherein the A at position 54 is mutated to S, the T at position 69 is mutated to E, the L at position 128 is mutated to K, the T at position 187 is mutated to I, the N at position 321 is mutated to I, the K at position 324 is mutated to L, and the T at position 460 is mutated to I. The mutant aspartase mentioned above is similarly prepared into a recombinant expression vector, transferred into competent host cells, cultured amplified and then inoculated for fermentation, and the suspension is obtained after centrifugal crushing to obtain a crude improved aspartate enzyme solution. After 8 hours of reaction in the second stage, the conversion rate can reach no less than 99.5%.

[0017] The present invention utilizes site-directed mutagenesis, and the nitrilase NIT II obtained based on the above method exhibits excellent activity to the substrate acrylonitrile, but free cells are difficult to tolerate high concentrations of acrylonitrile. After being prepared into immobilized cells, it can tolerate high concentrations of acrylonitrile substrates, greatly reducing the inhibitory effect of acrylonitrile on the enzyme; since acrylonitrile residues will inhibit the activity of aspartase, resulting in a significant decrease in the reaction rate in the second stage, the high conversion rate characteristics of nitrilase can shorten the time for the complete reaction in the first stage and improve production efficiency. There is no need to consider the recycling problem when immobilizing nitrilase, and aspartase can be directly added after the conversion reaction of acrylic acid is completed for the next reaction. The improved ASP II provided by the present invention exhibits high activity to acrylic acid, can tolerate high concentrations of acrylic acid, has high conversion efficiency, requires less enzyme, and has low enzyme cost. Compared with the current synthesis method using fumaric acid and acrylic acid as substrates, it consumes less ammonia water, has lower cost, and has higher atom economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is Example 5, the effect of acrylonitrile concentration on aspartase. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the percentages are all mass percentages.

[0020] Example 1

[0021] 1. Nitrilase NIT II. Construction of expression strain

[0022] The nitrilase NIT II provided in this example is derived from the wild-type nitrilase of Achromobacter denitrificans (GenBank: ASY91980.1, SEQ ID No: 3), and the nucleotide sequence is shown in SEQ ID No: 4.

[0023] SEQ ID No:3 was subjected to site-directed mutagenesis, wherein the F at position 54 was mutated to Y, the M at position 113 was mutated to L, the F at position 168 was mutated to L, the F at position 192 was mutated to N, the A at position 193 was mutated to E, the L at position 201 was mutated to T, and the Q at position 248 was mutated to A, and the amino acid sequence NIT II after mutation was obtained, as shown in SEQ ID No:1. After the codons of the nucleotide sequence SEQ ID No:2 were optimized, Shanghai Jierui Bioengineering Co., Ltd. was commissioned to synthesize the nucleic acid sequence SEQ ID No.2 connected to the vector pET-26b(+), and introduced into Escherichia coli BL21 star(DE3) competent cells were transformed, ice-bathed for 30 minutes, heat-shocked in a 45°C water bath for 90 seconds, and ice-bathed for 2 minutes before evenly spreading the bacterial solution on a plate containing LB solid medium with kanamycin resistance. After culturing at 37°C for 18 hours, single clones were picked and transferred to 4 mL of LB liquid medium with kanamycin resistance, and cultured at 37°C and 220 rpm for 14 hours. The mutant nitrilase expression strain BL21 star(DE3) / pET-26b(+)-NIT II was obtained.

[0024] 2. Aspartase ASP II. Construction of expression strain

[0025] Wild-type aspartase ASP II (GenBank: QBK25944.1, SEQ ID No: 7) is derived from Ureibacillus thermophilus. SEQ ID No: 7 was subjected to site-directed mutagenesis, wherein the A at position 54 was mutated to S, the T at position 69 was mutated to E, the L at position 128 was mutated to K, the T at position 187 was mutated to I, the N at position 321 was mutated to I, the K at position 324 was mutated to L, and the T at position 460 was mutated to I. The amino acid sequence of ASP II after mutation is shown in SEQ ID No: 5. According to the nucleotide sequence of ASP II, the nucleotide sequence shown in SEQ ID No: 6 was obtained after codon optimization. It was commissioned to be synthesized by Shanghai Jierui Bioengineering Co., Ltd. and introduced into Escherichia coli BL21 star (DE3) competent cells for transformation. After ice bathing for 30 minutes, heat shock was performed in a 45°C water bath for 90 seconds. After ice bathing for 2 minutes, the bacterial liquid was evenly spread on a plate containing LB solid medium with kanamycin resistance. After culturing at 37°C for 18 hours, a single clone colony was picked and added to 4 mL of LB liquid medium with kanamycin resistance, and cultured at 37°C and 220rpm for 14 hours. The mutant aspartase expression strain BL21 star (DE3) / pET-26b (+) -ASP II was obtained.

[0026] 3. Preparation of Nitrilase and Aspartase

[0027] The above strains were inoculated into 4 mL of LB liquid culture medium containing 50 mg / L kanamycin, and cultured at 37°C and 180 rpm for 12 hours. The bacterial liquid was inoculated into 600 mL of TB liquid culture medium, and cultured at 37°C and 180 rpm until OD600 reached about 2. IPTG with a final concentration of 0.3 mM was added for induction, and the induction was carried out at 25°C for 16 hours. Then, the bacterial cells containing carbonyl reductase were collected by centrifugation at 5000 rpm for 20 minutes, and wet bacterial cells containing nitrilase and aspartase were obtained respectively. The aspartase bacterial cells were directly used for the enzyme catalysis reaction in the second stage, and the nitrilase was used for further preparation of immobilized cells.

[0028] 4. Preparation of immobilized nitrilase

[0029] a. Prepare 100 g / L bacterial suspension with tap water from the wet bacteria obtained by centrifugation, add 6 g / L (m / v) diatomaceous earth and stir for 10 min;

[0030] b. Add 0.15wt% polyethyleneimine, stir at a temperature of 20-25°C for 1h;

[0031] c. Add 0.25wt% glutaraldehyde, stir and cross-link at a temperature of 20-25°C for 1h, then wash repeatedly with tap water several times until the filtrate is colorless and transparent, and drain the liquid to obtain immobilized nitrilase, one of which is used for the first stage of enzyme catalysis reaction.

[0032] Example 2 Preparation of β-alanine by biocatalytic system

[0033] 1. Preparation of acrylic acid from acrylonitrile

[0034] In 1L of the catalyst system, 200g of acrylonitrile and 750g of water were added. The pH was generally around 7.2 and no further adjustment was required. 4g of the immobilized nitrilase obtained in Example 1 was added. The mixture was mechanically stirred at 37°C. After reacting for 3h, the conversion rate reached 99.98%.

[0035] 2. Preparation of β-alanine from acrylic acid

[0036] The conversion solution was heated to 42° C., 0.1 g of magnesium acetate was added, the pH was adjusted to 9.0 with ammonia water, 8 g of aspartase obtained in Example 1 was added, and after stirring for 8 hours, the acrylic acid concentration was 329.5 g / L and the conversion rate was 99.8%.

[0037] Example 3 Preparation of β-alanine by 300L biocatalytic system

[0038] 1. Preparation of acrylic acid from acrylonitrile

[0039] In a 300L catalytic system, 60kg acrylonitrile and 225kg water were added. Without adjusting the pH, 1.2kg immobilized nitrilase was added. After stirring at 37°C and reacting for 3h, the acrylic acid concentration was 270g / L and the conversion rate was 99.95%.

[0040] 2. Preparation of β-alanine from acrylic acid

[0041] The above conversion solution was heated to 42°C, 30g of magnesium acetate was added, the pH was adjusted to 9.0 with ammonia water, 24kg of aspartase was added, and after stirring for 8h, the β-alanine concentration reached 325g / L and the conversion rate was 99.6%.

[0042] 3. Post-treatment of β-alanine conversion solution

[0043] The conversion liquid is passed through a ceramic membrane to remove the bacteria and immobilized cells, concentrated at 55-65°C to a β-alanine concentration of about 500 g / L, and 2.5 times the volume of ethanol is added to crystallize at 0-4°C overnight. β-alanine crystals are obtained by filtration and drying. The yield of one crystallization is 89%, the purity is greater than 99.99%, and the content is greater than 99.0%.

[0044] Example 4

[0045] In order to verify the effect of immobilized nitrilase on substrate tolerance, this example prepared acrylonitrile conversion systems containing 50 / 100 / 150 / 200 / 250 g / L, respectively, added 1% (m / V) of free nitrilase cells and immobilized cells obtained in Example 1, respectively, reacted at 37° C. for 12 h and 24 h to detect the residual acrylonitrile concentration. The results are shown in Table 1.

[0046] Table 1 Transformation results of free cells and immobilized cells at different acrylonitrile concentrations

[0047]

[0048] As shown in Table 1, when the acrylonitrile concentration is 100 g / L, it has an inhibitory effect on free cells. When the acrylonitrile concentration is greater than 150 g / L, the reaction is almost difficult to proceed. However, the immobilized cells can still react normally in 250 g / L acrylonitrile conversion solution.

[0049] Example 5 Effect of acrylonitrile concentration on aspartase

[0050] Preparation of ammonium acrylate solution: Take 300g acrylic acid, add 200g water, add 0.1g magnesium acetate, adjust the pH to 9.0 with ammonia water, and dilute to 1L with water for later use.

[0051] Take 100 mL of the above ammonium acrylate solution, add 2% aspartase obtained in Example 1, add different concentrations of acrylonitrile, so that the acrylonitrile concentration in the conversion solution is 0.25 / 0.5 / 1 / 1.5 / 2% (m / V), react at 42°C for 8h and 16h, and detect the amount of β-alanine produced. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that when the residual acrylonitrile in the conversion liquid is greater than 0.25%, acrylonitrile inhibits aspartase and the reaction rate is significantly reduced. When the residual acrylonitrile in the conversion liquid is greater than 1.5%, the inhibitory effect is more obvious. Therefore, when using aspartase to convert acrylic acid to β-alanine, it is necessary to ensure that the residual acrylonitrile is less than 0.25%, and try to wait until the acrylonitrile is consumed before adding aspartase.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nitrilase, the amino acid sequence of the nitrilase is shown in SEQ ID No:

1.

2. The expression gene of the nitrilase according to claim 1, comprising the nucleotide sequence shown in SEQ ID No:

2.

3. A recombinant expression vector comprising the nucleotide sequence shown in SEQ ID NO:

2. A host cell comprising the recombinant expression vector according to claim 3.

5. A method for preparing nitrilase, characterized in that: The wild-type nitrilase comprising the amino acid sequence shown in SEQ ID No: 3 is subjected to site-directed mutagenesis, wherein the F at position 54 is mutated to Y, the M at position 113 is mutated to L, the F at position 168 is mutated to L, the F at position 192 is mutated to N, the A at position 193 is mutated to E, the L at position 201 is mutated to T, and the Q at position 248 is mutated to A, to obtain the nucleotide sequence of the mutant SEQ ID No: 2, and the recombinant expression vector is prepared and transfected into a competent host cell, and then fermented, cultured, and centrifuged to obtain the mutant.

6. The method for preparing the nitrilase according to claim 5, characterized in that: Diatomaceous earth and polyethyleneimine are added to the crude enzyme solution obtained after centrifugal separation and mixed evenly, and then glutaraldehyde is added for cross-linking to obtain immobilized nitrilase.

7. The method for preparing the nitrilase according to claim 6, characterized in that: For 50-100 g / L crude enzyme solution, 3-6 g / L diatomaceous earth, 0.1-0.15 wt% polyethyleneimine and 0.1-0.25 wt% glutaraldehyde are added accordingly.

8. The use of the nitrilase in the biological preparation of β-alanine according to claim 1, characterized in that: In a reaction system, acrylic acid is obtained by catalysis with acrylonitrile as substrate, and β-alanine is obtained by catalysis of acrylic acid catalyzer.

9. The use according to claim 8, characterized in that: The substrate concentration of acrylonitrile is not less than 200 g / L, and the conversion rate after catalysis by the nitrilase for 3 hours is not less than 99.8%.

10. A biological preparation method of β-alanine, characterized in that: Acrylonitrile is used as a reaction substrate, the nitrilase according to claim 5 or the immobilized nitrilase according to claim 6 is added, and a first-stage reaction is carried out at 30-37° C. and pH 7.0-8.

0. After the reaction is complete, acrylic acid catalyzed enzyme is added to the reaction system to carry out a second-stage reaction to obtain β-alanine.

11. The biological preparation method of β-alanine according to claim 10, characterized in that: The acrylic acid catalytic enzyme is a mutant aspartic acid enzyme derived from thermophilic urea bacillus ( Ureibacillus thermophilus); the mutant aspartase is obtained by subjecting the amino acid sequence of GenBank No. QBK25944.1 to site-directed mutagenesis, wherein the A at position 54 is mutated to S, the T at position 69 is mutated to E, the L at position 128 is mutated to K, the T at position 187 is mutated to I, the N at position 321 is mutated to I, the K at position 324 is mutated to L, and the T at position 460 is mutated to I.

Images