CN115948376A - Thermostable aspartate beta-decarboxylase - Google Patents

Abstract

The invention relates to the technical field of enzyme catalysis, in particular to thermostable aspartate beta-decarboxylase, the amino acid sequence of which is shown as SEQ ID NO:3, has higher thermal stability compared with the wild-type aspartate beta-decarboxylase. The aspartate beta-decarboxylase has more lasting activity at 45-60 ℃, and can effectively shorten the reaction period; and can be transported and stored at room temperature, effectively prolonging shelf life. In addition, under the condition of high-temperature catalytic reaction, the growth chance of mixed bacteria is avoided, so that the pollution of the metabolic products of bacteria to products is reduced; is helpful to increase the solubility of the substrate and improve the production efficiency of unit volume.

Description

Thermostable aspartate beta-decarboxylase

Technical Field

The invention relates to the technical field of enzyme catalysis, in particular to thermostable aspartate beta-decarboxylase.

Background

L-alanine, L-alpha-aminopropionic acid, is a small, non-essential amino acid in humans, and alanine, one of the most widely used amino acids in protein construction, is involved in the metabolism of tryptophan and the vitamin pyridoxine. It is involved in sugar and acid metabolism, enhances immunity, supplies energy to muscle tissue, brain and central nervous system, enhances immune system, and shows cholesterol lowering effect in animals. L-alanine is an important amino acid and has been used in many fields such as food, pharmaceutical and chemical industries. The L-alanine can be used as a raw material for synthesizing an amino acid surfactant chelating agent MGDA, and is also an important intermediate for synthesizing vitamin B6, sweetener alitame and aminopropanol (used for producing levofloxacin hydrochloride).

In chemical synthesis, propionic acid chlorination is a common method for synthesizing L-alanine. However, the method has the defects of poor product quality, long synthesis route, low yield, high cost, serious environmental pollution and the like, and is basically eliminated at present. For decades, the commercial production of L-alanine has been carried out by aspartate beta-decarboxylase (ASD) using the substrate L-aspartate. Chibata catalyzes the production of L-alanine by using 40 percent of L-aspartic acid as a catalytic substrate, and the yield of the L-alanine in 72 hours exceeds 90 percent (I.Chibata, T.Kakimoto and J.Kato, appl.Microbiol.,1965,13,638-645). Fusee immobilizes p.dachunhae whole cells with a polyurethane prepolymer, and converts L-aspartic acid to L-alanine continuously over 31 days by providing 23% L-aspartic acid per day with 0.1mM alpha ketoglutaric acid and 0.1mM pyridoxal 5-phosphate (PLP). ASD is industrially well-established for the production of L-alanine due to its high activity on L-aspartic acid (m.c. fusee and j.e. weber, appl.environ.microbiol.,1984,48,694-698).

With the development of gene editing and metabolic engineering, the production of L-alanine is shifting to an economical fermentation process. Zhou et al knocked wild-type E.coli out for alanine racemase and knocked in the L-alanine dehydrogenase gene, resulting in a yield of 67.2g/L at shake flask level (Li Zhou, can Deng, wen-lacing Cui, zhong-Mei Liu, zhe-Min Zhou. Effective L-alkane production by a thermal-regulated switch in Escherichia coli. Applied Biochemistry and Biotechnology,2016, 178-324-337. Zhang utilizes an engineered e.coli W (e.coli W) that produces 113.8g/L L-alanine (x.zhang, k.jantama, j.c.moore, k.t.shanmugamand l.o.ingram, appl.microbiol.biotechnol.,2007,77,355-366) within 48 hours using glucose as a carbon source. Jojima et al produced 98g/L L-alanine (T. Jojima, M. Fujii, E. Mori, M. Inuiand H. Yukawa, appl. Microbiol. Biotechnol.,2010,87,159-165) using C.glutamicum engineered bacteria under anaerobic conditions for 32 hours. Zhou et al, engineered E.coli by temperature regulation, reduced the inhibition of cell growth by L-alanine, yielded 120.8g/L L-alanine (L.Zhou, C.Deng, W.cui, Z.Liu and Z.Zhou, appl.biochem.Biotechnol.,2016,178, 324-337) within 40 hours.

However, compared with enzyme catalysis, the fermentation method for producing L-alanine has the obvious defects of poor optical purity, more byproducts, difficult purification and the like. Especially under anaerobic conditions, many intracellular solutes and by-products are released by cell lysis after prolonged culture. The aminopropanol has high impurity requirement in hydrogenation reaction, and the fermentation method index can not reach the qualified requirement, so that the cost of further purification even exceeds the production cost of L-alanine, so that the fermentation route has narrow application range, and can only meet partial chemical requirements, such as MGDA synthesis. With the increasing demand for L-alanine as a prodrug, it becomes increasingly necessary to produce L-alanine with high optical purity.

Aspartate beta-decarboxylase (EC 4.1.1.12) is an enzyme that catalyzes the chemical reaction L-aspartate = L-alanine + CO ₂ . Thus, this enzyme has one substrate, L-aspartic acid, and two products, L-alanine and CO ₂ . This reaction is the basis for the industrial synthesis of L-alanine. This enzyme belongs to the family of lyases, in particular carboxy lyases, which cleave carbon-carbon bonds. The system name of this enzyme class is L-aspartate 4-carboxy lyase (L-alanine formation). Other names commonly used include desulfonatase, aminomalonate decarboxylase, aspartate beta-decarboxylase, aspartate omega-decarboxylase, aspartate beta-decarboxylase, L-aspartate beta-decarboxylase, cysteine sulfinate desulfatase, L-cysteine sulfinate desulfatase, and L-aspartate 4-carboxy lyase. The enzyme is involved in alanine and aspartate metabolism and cysteine metabolism. It uses pyridoxal phosphate (PLP), a cofactor. The gene Asd encoding L-aspartate-beta-decarboxylase was found to be widely present in Mycobacterium pseudotuberculosis, pseudomonas spAnd acetobacter, clostridium perfringens and the like.

CN1155716C obtains a pseudomonad with L-aspartic acid-beta-decarboxylase activity by mutagenesis, the catalytic conversion rate of the substrate L-aspartic acid is close to 100 percent, but 5 days are needed for catalytic reaction, and the time is too long.

CN109536429A heterologously expresses L-aspartate beta-decarboxylase from Acinetobacter sp. However, there are three major problems: (1) The OD600 of the thalli in the reaction system is 300, and the cost of the thalli is far more than the value of the product. (2) substrate concentration was too low, only 800mM sodium aspartate. (3) The reaction temperature is too low, namely 37 ℃, which is not beneficial to the generation of high-concentration products from high-concentration substrates.

CN105018405A discloses an L-aspartic acid-beta-decarboxylase gene from Comamonas testosteroni CICC 11023s, the yield of L-alanine is only 112.7g/L, the production rate is 12.5 g/(L.h), the conversion rate is only 93.9 percent after 9 hours of reaction, and the cost requirement of industrial scale production cannot be met.

CN114107270A discloses a mutant E88R with improved enzyme activity and stability under alkaline conditions, and after incubation in a water bath kettle at 45 and 55 ℃ for 30min, the residual enzyme activity of the mutant E88R at 45 ℃ and 50 ℃ is 85% and 59% respectively. However, higher temperatures were not tested due to greater loss of enzyme activity.

Therefore, the search for a more thermostable aspartate beta-decarboxylase has become one of the problems to be solved in the art.

Disclosure of Invention

The invention aims to provide a thermostable aspartate beta-decarboxylase.

In order to achieve the purpose, the invention provides the following technical scheme:

a thermostable aspartate β -decarboxylase having the amino acid sequence set forth in SEQ ID NO:3, respectively.

The thermostable aspartate beta-decarboxylase of the present invention has higher thermostability than wild aspartate beta-decarboxylase, and the amino acid sequence of the wild aspartate beta-decarboxylase is shown as SEQ ID NO:1 is shown.

The invention also provides a polynucleotide which can encode the aspartate beta-decarboxylase.

Further, the polynucleotide sequence is shown in SEQ ID NO:4, respectively.

Compared with the prior art, the invention has the beneficial effects that:

(1) Compared with the wild aspartic acid beta-decarboxylase, the thermostable aspartic acid beta-decarboxylase of the invention has better thermostability and longer activity at 45-60 ℃.

(2) The thermostable aspartate beta-decarboxylase of the present invention has a number of advantages: (1) the kinetic reaction is accelerated, and the reaction period can be effectively shortened; (2) the enzyme extraction process is simplified, and the host cell protein is denatured, coagulated and precipitated at high temperature and is easy to separate from the target protein; (3) the requirement on a cooling system of the reaction is not high, so that the energy consumption and the cost are reduced; (4) the protein with improved stability can be transported and stored at room temperature, and the shelf life is effectively prolonged; (5) under the condition of high-temperature catalytic reaction, the growth chance of mixed bacteria is avoided, so that the pollution of metabolic products of the bacteria to products is reduced; (6) the high temperature helps to increase the solubility of the substrate and increases the production efficiency per unit volume.

(3) The aspartate beta-decarboxylase is used for preparing L-alanine by catalyzing L-aspartate at high temperature, and has higher reaction temperature and higher substrate solubility, so that the substrate concentration can be effectively increased, the kinetic reaction can be effectively accelerated, and the production efficiency can be improved.

(4) The thermostable aspartate beta-decarboxylase of the invention has wide application prospect in the fields of chemical industry, food, pharmacy, energy development and the like.

Drawings

FIG. 1 shows the thin layer chromatography results of the substrate and product standards. The upper color development band is the product L-alanine, and the lower color development band is the substrate aspartic acid.

FIG. 2 shows the results of 6 hours of reaction in example 5, with mutant reaction on the left and pre-mutation reaction on the right.

FIG. 3 shows the results of the 20-hour reaction in example 5, with the left side showing the mutant reaction and the right side showing the pre-mutation reaction.

FIG. 4 shows the results of 6 hours of reaction in example 6, with mutant reaction on the left and pre-mutation reaction on the right.

FIG. 5 shows the results of example 7 after 2 hours of reaction.

FIG. 6 shows the results of the reaction test in example 8 with different enzyme amounts.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention. The apparatus and reagents used in this example were all commercially available products unless otherwise specified.

Example 1 acquisition of wild-type aspartic acid beta-decarboxylase Gene sequence

The secondary structure and codon preference of Streptomyces sp-derived aspartate beta-decarboxylase gene are adjusted by a whole-gene synthesis method to achieve high expression in Escherichia coli. The design was carried out using Primer Premier (http:// Primer3.Ut. Ee /) and OPTIMIZER (http:// genes. Uv. Es/OPTIMIZER /), and the difference of annealing temperatures (Tm) was kept within 3 ℃ and the length of primers was kept within 60base, and the obtained primers were dissolved in double distilled water and added to the reaction system such that the final concentration of each Primer was 30nM and the final concentration of the head and tail primers was 0.6. Mu.M.

2mM dNTP mix(2mM each dNTP)	5μl
		10×Pfu buffer	5μl
Pfu DNA polymerase(10U/μl)	0.5μl
		ddH 2 O	The total volume of the reaction system was adjusted to 50. Mu.l

The prepared PCR reaction system is placed in a Bori XP cycler gene amplification instrument and amplified according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 120s at 72 ℃ and 35x.

The DNA fragment obtained by PCR was purified by gel cutting and cloned into the NdeI/XhoI site of pET30a by homologous recombination. Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO:2, designated ASDHwt, the corresponding amino acid sequence of which is SEQ ID NO:1.

EXAMPLE 2 acquisition of aspartate beta-decarboxylase mutant Gene sequences

The mutant aspartate beta-decarboxylase, which is derived from the wild-type aspartate beta-decarboxylase of example 1, catalyzes the formation of L-alanine from L-aspartate at high temperature. The mutant aspartate beta-decarboxylase shows stronger thermal stability compared with wild aspartate beta-decarboxylase. Aspartate beta-decarboxylase mutants and polynucleotides encoding such mutants can be prepared using methods commonly used by those skilled in the art. Mutants can be obtained by in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR and directed evolution methods etc. encoding the enzyme.

The secondary structure and codon preference of the gene are adjusted by a whole-gene synthesis method so as to realize high expression in escherichia coli. Using Primer Premier (http:// Primer3.Ut. Ee /) and OPTIMIZER

(http:// genes. Urv. Es/OPTIMIZER /) was designed, and the difference of annealing temperature (Tm) was controlled to be within 3 ℃ and the length of the primer was controlled to be within 60base, 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 head and tail primers was 0.6. Mu.M.

2mM dNTP mix(2mM each dNTP)	5μl
		10×Pfu buffer	5μl
Pfu DNA polymerase(10U/μl)	0.5μl
		ddH 2 O	The total volume of the reaction system was adjusted to 50. Mu.l

The prepared PCR reaction system is placed in a Bori XP cycler gene amplification instrument and amplified according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 120s at 72 ℃ and 35x.

The DNA fragment obtained by PCR was purified by gel cutting and cloned into the NdeI/XhoI site of pET30a by homologous recombination. Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO:4, designated as ASDH and the corresponding amino acid sequence is SEQ ID NO:3.

example 3 Shake flask expression assay

Coli single colonies containing the expression vector were picked and inoculated into 10ml of autoclaved medium: 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, 0.8g/L glucose, and kanamycin to 50mg/L. The culture was carried out at 30 ℃ and 250rpm overnight. Taking a 1L triangular flask the next day, and carrying out the following steps: 100 into 100ml of autoclaved medium: 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 kanamycin to 50mg/L. After culturing at 30 ℃ until the cell OD 5-6 was reached, the flask was immediately placed in a shaker at 25 ℃ and cultured at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 hours at 250 rpm. After completion of the culture, the culture was centrifuged at 12000g at 4 ℃ for 20 minutes to collect wet cells. Then the bacterial pellet is washed twice with distilled water, and the bacterial is collected and preserved at-70 ℃. Meanwhile, a small amount of thalli is taken for SDS-PAGE detection.

Example 4 fed-batch fermentation

The fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai Seisaku) with a reactor capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium hydrogen phosphate as the medium, pH 7.0. Primary inoculum preparation 200ml culture, OD2.0 inoculation. Throughout the fermentation, the temperature was maintained at 37 ℃, the dissolved oxygen concentration during fermentation was automatically controlled at 30% by the agitation rate (rpm) and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% (v/v) orthophosphoric acid and 30% (v/v) aqueous ammonia. During the fermentation, when a large amount of dissolved oxygen rises, feeding is started. The feed solution contained 9%w/v peptone, 9%w/v yeast extract, 14% w/v glycerol. When OD600 was about 50.0 (wet weight was about 100 g/L), the temperature was controlled at 28 ℃ and expression was induced with 0.15mM IPTG.

Example 5 comparison of reaction rates

50mM pH 7.5TrisHCl, final concentration 250gL L aspartic acid (initially adjusted to 7.5 with ammonia), adjusted to pH7.5 post addition of 2mM MgCl ₂ 0.2mM PLP, temperature set to 60 ℃ and finally 5g/L of the two crude enzyme solutions were added. The results of thin layer chromatography of the substrate and product standards are shown in FIG. 1. The results of the 6-hour and 20-hour sample spotting plates are shown in FIGS. 2 and 3. It was found that no substrate remained 20 hours after the mutation, whereas the protein was rapidly inactivated at high temperature before the mutation, and there was almost no reaction.

EXAMPLE 6 determination of optimum pH

50mM pH 7.5TrisHCl, final concentration 250gL L L aspartic acid (used after pH adjustment with ammonia), pH 5.0, 5.5, 6.0, 6.5, 7, 7.5, 8 respectively, and 2mM MgCl added ₂ 0.2mM PLP, the temperature was set to 50 ℃ and 25g/L of the crude enzyme solution was added. The 6 hour sample point plate was checked for substrate residue and product formation and the results are shown in FIG. 4, with all reactions completed except for pH5, 8 and only a small amount of substrate remained at pH 8.

Example 7 fast response test

50mM pH 7.5TrisHCl, final concentration 250gL L L aspartic acid (used after pH6.5 adjustment with ammonia), 2mM MgCl ₂ 0.2mM PLP, the temperature was set to 45 ℃ and 25g/L of the crude enzyme solution was added. The time point was 2 hours, indicating that the reaction was complete, and the results are shown in FIG. 5.

Example 8 reaction testing of different enzyme amounts

a.50mM pH 7.5TrisHCl, final concentration 150g/L L aspartic acid (used after pH6.5 adjustment with ammonia), 2mM MgCl was added ₂ 0.2mM PLP, set the temperature to 55 ℃, and finally 1g/L of wet cells were added. Plate was spotted for 2 hours.

b.50mM pH 7.5TrisHCl, final concentration 150g/L L aspartic acid (used after pH6.5 adjustment with ammonia), 2mM MgCl was added ₂ 0.2mM PLP, the temperature was set to 55 ℃ and 5g/L of the crude enzyme solution was added. Plate was spotted for 2 hours.

c.50mM pH 7.5TrisHCl, final concentration 150g/L L aspartic acid (used after pH6.5 adjustment with ammonia), 2mM MgCl was added ₂ 0.2mM PLP, the temperature was set to 55 ℃ and 10g/L of the crude enzyme solution was finally added. Plate was dosed for 2 hours.

d.50mM pH 7.5TrisHCl, final concentration 150g/L L aspartic acid (pH 6.5 adjusted with ammonia and then adjustedWith) 2mM MgCl was added ₂ 0.2mM PLP, the temperature was set to 55 ℃ and 25g/L of the crude enzyme solution was finally added. Plate was spotted for 2 hours.

e.50mM pH 7.5TrisHCl, final concentration 150g/L L aspartic acid (used after pH6.5 adjustment with ammonia), 2mM MgCl was added ₂ 0.2mM PLP, the temperature was set to 55 ℃ and 50g/L of the crude enzyme solution was finally added. Plate was spotted for 2 hours.

As shown in FIG. 6, the reaction was completed in 2 hours except for 1g/L of wet bacteria, indicating that the reaction time can be controlled by adjusting the amount of enzyme used.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (4)

1. A thermostable aspartate β -decarboxylase which is characterized by: the amino acid sequence is shown as SEQ ID NO:3, respectively.

2. The thermostable aspartate beta-decarboxylase according to claim 1, characterized in that: has higher thermal stability than the wild aspartate beta-decarboxylase, and the amino acid sequence of the wild aspartate beta-decarboxylase is shown as SEQ ID NO:1 is shown.

3. A polynucleotide encoding the aspartate beta-decarboxylase of any one of claims 1 or 2.

4. The polynucleotide of claim 3, wherein: the sequence is shown as SEQ ID NO:4, respectively.

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