A method for preparing L-alanine at high temperature

Abstract

The present invention relates to the field of enzyme catalysis technology, in particular to a method for preparing L-alanine at high temperature, in the presence of an aspartate β-decarboxylase mutant, L-aspartate can be catalyzed to generate L-alanine at 45°C-60°C. Wherein, the amino acid sequence of the aspartate β-decarboxylase mutant is shown in SEQ ID NO: 3. The present invention uses an aspartate β-decarboxylase mutant with better heat resistance and higher enzyme activity to prepare L-alanine, which can effectively reduce wastewater discharge, effectively reduce the cost of industrial production of L-alanine, and improve reaction efficiency and the optical purity of the product.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme catalysis, and specifically relates to a method for preparing L-alanine at high temperature. Background Art

[0002] L-Alanine, or L-α-aminopropionic acid, is a small non-essential amino acid in the human body. Alanine is one of the most widely used amino acids in protein construction and is involved in the metabolism of tryptophan and vitamin pyridoxine. It participates in sugar and acid metabolism, can improve immunity, and provide energy for muscle tissue, brain and central nervous system, strengthen the immune system, and shows cholesterol-lowering effects in animals. L-Alanine is an amino acid with important value and has been used in many fields such as food, pharmaceutical and chemical industries. L-Alanine can be used as a raw material for the synthesis of amino acid surfactant chelating agent MGDA, and is also an important intermediate for the synthesis of vitamin B6, sweetener alitame and aminopropanol (used in the production of levofloxacin hydrochloride).

[0003] In chemical synthesis, propionic acid chlorination is a common method for synthesizing L-alanine. However, this method has the disadvantages of poor product quality, long synthesis route, low yield, high cost, and serious environmental pollution, and has been basically eliminated. For decades, the commercial production of L-alanine has been carried out by aspartate β-decarboxylase (ASD) using the substrate L-aspartate. Chibata catalyzed the production of L-alanine with 40% L-aspartate as the catalytic substrate, and the yield of L-alanine exceeded 90% within 72 hours (I.Chibata, T.Kakimoto and J.Kato, Appl.Microbiol., 1965, 13, 638–645). Fusee fixed the whole cells of P.dacunhae with polyurethane prepolymer, and continuously converted L-aspartate into L-alanine within 31 days by providing 23% L-aspartate with 0.1mM α-ketoglutarate and 0.1mM 5-pyridoxal phosphate (PLP) every day. Due to its high activity towards L-aspartic acid, ASD has been widely used in industry to produce L-alanine (MC Fusee and JE Weber, Appl. Environ. Microbiol., 1984, 48, 694-698).

[0004] With the development of gene editing and metabolic engineering, the production of L-alanine is shifting towards an economical fermentation method. Zhou et al. knocked out alanine racemase from wild-type Escherichia coli and knocked in L-alanine dehydrogenase genes, and finally achieved a yield of 67.2 g / L at the shake flask level (Li Zhou, Can Deng, Wen-Jing Cui, Zhong-Mei Liu, Zhe-Min Zhou. Efficient L-alanine production by a thermo-regulated switch in Escherichia coli. Applied Biochemistry and Biotechnology, 2016, 178: 324-337). Zhang used an engineered Escherichia coli W (Escherichia coli W) to produce 113.8 g / L L-alanine in 48 hours using glucose as a carbon source (X. Zhang, K. Jantama, JC Moore, KT Shanmugam and LO Ingram, Appl. Microbiol. Biotechnol., 2007, 77, 355–366). Jojima et al. used engineered bacteria of Corynebacterium glutamicum to produce 98 g / L L-alanine in 32 hours under anaerobic conditions (T.Jojima, M.Fujii, E.Mori, M.Inui and H.Yukawa, Appl.Microbiol.Biotechnol., 2010, 87, 159–165). Zhou et al. reduced the inhibition of L-alanine on cell growth by regulating the temperature of engineered Escherichia coli and obtained 120.8 g / L L-alanine in 40 hours (L.Zhou, C.Deng, W.Cui, Z.Liu and Z.Zhou, Appl.Biochem.Biotechnol., 2016, 178, 324–337).

[0005] However, compared with enzyme catalysis, the fermentation method for producing L-alanine has obvious disadvantages such as poor optical purity, many by-products, and difficulty in purification. Especially under anaerobic conditions, many intracellular soluble substances and by-products are released due to cell lysis after long-term cultivation. Aminopropanol has high requirements for impurities due to the hydrogenation reaction involved, and the fermentation method indicators are far from meeting the qualified requirements. The cost of further purification even exceeds the production cost of L-alanine, making the fermentation route narrow in application and only able to meet some chemical needs, such as MGDA synthesis. With the increasing demand for L-alanine as a drug precursor, it is becoming increasingly necessary to produce L-alanine with high optical purity.

[0006] Aspartate β-decarboxylase (EC 4.1.1.12) is an enzyme that catalyzes the following chemical reaction, L-aspartate = L-alanine + CO2. Therefore, this enzyme has one substrate, L-aspartate, and two products, L-alanine and CO2. This reaction is the basis of the industrial synthesis of L-alanine. This enzyme belongs to the family of lyases, in particular carboxyl lyases, which cleave carbon-carbon bonds. The systematic name of this enzyme class is L-aspartate 4-carboxyl lyase (L-alanine forming). Other commonly used names include desulfurase, aminomalonate decarboxylase, aspartate β-decarboxylase, aspartate ω-decarboxylase, aspartate ω-decarboxylase, aspartate β-decarboxylase, L-aspartate β-decarboxylase, cysteine ​​sulfenic acid desulfurase, L-cysteine ​​sulfenic acid desulfurase and L-aspartate 4-carboxyl lyase. This enzyme is involved in alanine and aspartate metabolism and cysteine ​​metabolism. It uses a cofactor, pyridoxal phosphate (PLP). The gene Asd encoding L-aspartate-β-decarboxylase has been found to be widely present in microorganisms such as Mycobacterium pseudomonas, Pseudomonas, Alcaligenes faecalis, Acetobacter, and Clostridium perfringens.

[0007] CN1155716C obtains a strain of Pseudomonas having L-aspartate-β-decarboxylase activity by mutagenesis, and the catalytic conversion rate of the substrate L-aspartate is close to 100%, but it takes 5 days for the catalytic reaction, which is too long.

[0008] CN109536429A heterologously expresses L-aspartate β-decarboxylase from Acinetobacter sp.ACNIH2, and produces L-alanine by whole-cell catalysis, and performs whole-cell reaction (oscillation reaction) at 37°C and 200r / min. However, there are three main problems: (1) The OD600 of the bacterial cell in the reaction system is 300, and the cost of the bacterial cell alone far exceeds the value of the product. (2) The substrate concentration is too low, only 800mM sodium aspartate. (3) The reaction temperature is too low, only 37°C, which is not conducive to the generation of high-concentration products from high-concentration substrates.

[0009] CN105018405A discloses an L-aspartate-β-decarboxylase gene derived from Comamonas testosteroni CICC11023s. After 9 hours of reaction, the yield of L-alanine is only 112.7 g / L, the production rate is 12.5 g / (L·h), and the conversion rate is only 93.9%, which cannot meet the cost requirements of industrial-scale production.

[0010] CN114107270A discloses a mutant E88R with improved enzyme activity and stability under alkaline conditions. After incubation in a water bath at 45 and 55°C for 30 minutes, the residual enzyme activity of the mutant enzyme E88R at 45°C and 50°C was 85% and 59%, respectively. However, due to the large loss of enzyme activity, no higher temperature was tested.

[0011] Therefore, finding a method for preparing L-alanine at high temperature has become one of the problems that need to be solved urgently in this field. Summary of the invention

[0012] The object of the present invention is to provide a method for preparing L-alanine at high temperature.

[0013] To achieve the above object, the present invention provides the following technical solutions:

[0014] A method for preparing L-alanine at high temperature comprises: in the presence of an aspartate beta-decarboxylase mutant, catalyzing L-aspartate to generate L-alanine at 45-60°C.

[0015] The amino acid sequence of the aspartate β-decarboxylase mutant is shown in SEQ ID NO:3.

[0016] The pH of the catalytic reaction is 6.5 to 7.5.

[0017] The concentration of the crude enzyme solution of the aspartate β-decarboxylase mutant is 25 g / L.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The aspartate β-decarboxylase mutant used in the method of the present invention exhibits better thermal stability than the wild-type aspartate β-decarboxylase, and has a more sustained activity at 45°C-60°C.

[0020] (2) The method for preparing L-alanine at high temperature of the present invention: ① Accelerate the kinetic reaction and effectively shorten the reaction cycle;

[0021] ② The enzyme extraction process is simplified, and the host cell protein denatures, condenses and precipitates at high temperature, making it easy to separate from the target protein; ③ The cooling system of the reaction is not demanding, thereby reducing energy consumption and costs; ④ The protein with improved stability can be transported and stored at room temperature, and the shelf life is effectively extended; ⑤ Under the conditions of high-temperature catalytic reaction, the growth of miscellaneous bacteria is avoided, thereby reducing the contamination of the product by bacterial metabolites; ⑥ High temperature helps to increase the solubility of the substrate and improves the production efficiency per unit volume.

[0022] (3) The aspartate β-decarboxylase mutant of the present invention is used for the preparation of L-alanine from L-aspartate at high temperature. The reaction temperature is higher and the substrate solubility is higher, so the substrate concentration can be effectively increased, the kinetic reaction can be effectively accelerated, and the production efficiency can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of thin layer chromatography of substrate and product standards are shown in Figure 2. The upper colored band is the product L-alanine, and the lower colored band is the substrate aspartic acid.

[0024] Figure 2 The results of the 4-hour reaction in Example 5 are shown in the figure. The left side shows the mutant reaction, and the right side shows the reaction before mutation.

[0025] Figure 3 The results of the 6-hour reaction in Example 5 are shown in the figure. The left side shows the mutant reaction, and the right side shows the reaction before mutation.

[0026] Figure 4 The results of the 4-hour reaction in Example 6 are shown on the left side using hydrochloric acid and on the right side using formic acid.

[0027] Figure 5 The results are shown in Example 7 after 2 hours of reaction. DETAILED DESCRIPTION

[0028] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The instruments and reagents used in the present embodiment are all commercially available products unless otherwise specified.

[0029] Example 1 Obtaining the wild-type aspartate β-decarboxylase gene sequence

[0030] The secondary structure and codon preference of the aspartate β-decarboxylase gene from Myxococcus sp. were adjusted by total gene synthesis to achieve high expression in E. coli. Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for design, and the annealing temperature (Tm) difference was controlled within 3°C, the primer length was controlled within 60 bases, and the obtained primers were dissolved in double distilled water and added to the following reaction system, so that the final concentration of each primer was 30nM and the final concentration of the first and last primers was 0.6μM.

[0031] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μl

[0032] Place the prepared PCR reaction system in the Biori XP cycler gene amplification instrument and perform amplification according to the following program: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0033] The DNA fragment obtained by PCR was gel-cleaved and purified, and cloned into the NdeI / XhoI site of pET30a by homologous recombination. A single clone was selected for sequencing. The sequenced DNA sequence was SEQ ID NO: 2, named ASDIwt, and its corresponding amino acid sequence was SEQ ID NO: 1.

[0034] Example 2 Obtaining the gene sequence of aspartate β-decarboxylase mutant

[0035] Aspartate beta-decarboxylase mutant, which is derived from the wild-type aspartate beta-decarboxylase of Example 1, can catalyze L-aspartate to produce L-alanine at high temperature. The aspartate beta-decarboxylase mutant shows stronger thermal stability than the wild-type aspartate beta-decarboxylase. Aspartate beta-decarboxylase mutant and polynucleotide encoding such mutant can be prepared using methods commonly used by those skilled in the art. The mutant can be obtained by in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR and directed evolution methods, etc., encoding the enzyme.

[0036] Through the method of total gene synthesis, the secondary structure and codon preference of the gene were adjusted to achieve high expression in E. coli. Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for design, and the difference in annealing temperature (Tm) was controlled within 3°C, the primer length was controlled within 60 bases, and the obtained primers were dissolved in double distilled water and added to the following reaction system, so that the final concentration of each primer was 30nM, and the final concentration of the first and last primers was 0.6μM.

[0037] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μl

[0038] Place the prepared PCR reaction system in the Biori XP cycler gene amplification instrument and perform amplification according to the following program: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0039] The DNA fragment obtained by PCR was purified by gel excision and cloned into the NdeI / XhoI site of pET30a by homologous recombination. A single clone was selected for sequencing. The sequenced DNA sequence was SEQ ID NO: 4, named ASDI, and its corresponding amino acid sequence was SEQ ID NO: 3.

[0040] Example 3 Shake flask expression test

[0041] A single colony of E. coli containing the expression vector was picked and inoculated into 10 ml of autoclaved medium: 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 0.493 g / L magnesium sulfate heptahydrate, 0.027 g / L ferric chloride hexahydrate, 5 g / L glycerol, 0.8 g / L glucose, and kanamycin was added to 50 mg / L. Incubate overnight at 30°C, 250 rpm. The next day, take a 1L Erlenmeyer flask and inoculate it into 100ml of autoclaved culture medium at an inoculation ratio of 1:100: 10g / L tryptone, 5g / L yeast extract, 3.55g / L disodium hydrogen phosphate, 3.4g / L potassium dihydrogen phosphate, 2.68g / L ammonium chloride, 0.71g / L sodium sulfate, 0.493g / L magnesium sulfate heptahydrate, 0.027g / L ferric chloride hexahydrate, 5g / L glycerol, 0.3g / L glucose, and add kanamycin to 50mg / L. Cultivate at 30℃ until the bacterial OD is 5-6, and immediately place the Erlenmeyer flask in a 25℃ shaker at 250rpm for 1 hour. Add IPTG to a final concentration of 0.1mM and continue to culture at 25℃ and 250rpm for 16 hours. After the culture is completed, the culture solution is centrifuged at 4℃ and 12000g for 20 minutes to collect the wet bacteria. Then the bacterial precipitate was washed twice with distilled water, and the bacterial cells were collected and stored at -70°C. At the same time, a small amount of bacterial cells was taken for SDS-PAGE detection.

[0042] Example 4 Fed-batch fermentation

[0043] Fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai Guoqiang) with a reactor capacity of 15 L and a working volume of 8 L. The culture medium used was 24 g / L yeast extract, 12 g / L peptone, 0.4% glucose, 2.31 g / L dihydrogen phosphate and 12.54 g / L potassium dihydrogen phosphate, pH 7.0. The primary inoculation strain was used to prepare 200 ml of culture, which was inoculated at OD 2.0. During the entire fermentation process, the temperature was maintained at 37°C, and the dissolved oxygen concentration during the fermentation process was automatically controlled at 30% by the stirring rate (rpm) and the ventilation supply cascade control, while the pH value of the culture medium was maintained at 7.0 by 50% (v / v) orthophosphoric acid and 30% (v / v) ammonia water. During the fermentation process, when a significant rise in dissolved oxygen occurred, feeding was started. The feed solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When OD600 was about 50.0 (wet weight was about 100 g / L), the temperature was controlled at 28°C and expression was induced with 0.15 mM IPTG.

[0044] Example 5 Comparison of reaction rates

[0045] 50mM pH 6.5 TrisHCl, final concentration 250gL L aspartic acid (adjusted to 7.5 with ammonia water before use), adjusted to pH 7.5, added 2mM MgCl2, 0.2mM PLP, set the temperature to 56 degrees, and finally added 10g / L of the two crude enzyme solutions. The results of the thin layer chromatography of the substrate and product standards are shown in Figure 1 Take 4-hour and 6-hour samples and spot the plate. The results are shown in Figure 2 , Figure 3 It can be seen that no substrate remained 6 hours after the mutation, while before the mutation, due to the rapid inactivation of the protein under high temperature conditions, only half of the product could be reacted and no longer increased, indicating that the enzyme activity had been completely lost at this time.

[0046] Example 6

[0047] 50mM pH 6.5 TrisHCl, final concentration 300gL L aspartic acid (adjust to 6.5 with ammonia water before use), adjust to pH 7.5, add 2mM MgCl2, 0.2mM PLP, set the temperature to 56 degrees, and finally add 5g / L of two crude enzyme solutions. In the middle, use hydrochloric acid and formic acid to control the pH to 6.5, take a 4-hour sample plate, such as Figure 4 As shown, it is shown that the effect of adjusting the pH with formic acid is very poor and is not conducive to the reaction.

[0048] Example 7 Rapid Response Test

[0049] 50mM pH 7.5 TrisHCl, final concentration 250gL L aspartic acid (adjust pH 6.5 with ammonia water before use), add 2mM MgCl2, 0.2mM PLP, set the temperature to 48℃, and finally add 25g / L crude enzyme solution. After 2 hours, the plate shows that the reaction is complete. The results are shown in Figure 5 .

[0050] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing L-alanine at high temperature, characterized in that: In the presence of an aspartate β-decarboxylase mutant, L-aspartate is catalyzed to produce L-alanine at 45°C-60°C, and the pH of the catalytic reaction is 6.5-7.5; the amino acid sequence of the aspartate β-decarboxylase mutant is shown in SEQ ID NO:

3.

2. The method for preparing L-alanine at high temperature according to claim 1, characterized in that: The crude enzyme solution concentration of the aspartate β-decarboxylase mutant was 25 g / L.

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