CN113637650A - Alanine dehydrogenase SaAD and coding gene and application thereof - Google Patents

Abstract

The invention discloses alanine dehydrogenase SaAD and a coding gene and application thereof. The protein provided by the invention is derived from a helicobacter sp (NM), is an alanine dehydrogenase, is named as SaAD protein, and is a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table. The invention also protects the application of the SaAD protein as alanine dehydrogenase. The invention has important application prospect in the fields of relevant medicines, food industry, health care and the like.

Description

Alanine dehydrogenase SaAD and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to alanine dehydrogenase SaAD and a coding gene and application thereof.

Background

Alanine Dehydrogenase (EC 1.4.1.1) belongs to amino acid Dehydrogenase, and can reversibly catalyze Alanine oxidative deamination to generate pyruvic acid and reduce NAD⁺Generating NADH. The reaction product pyruvic acid is used in medicine industry to synthesize antiphlogistic, analgesic, antituberculotic, antiviral, anthelmintic, etc. and as food additive for food industry. Besides, pyruvic acid is widely applied to the fields of biotechnology diagnostic reagents, weight loss and health care and the like.

Alanine dehydrogenases are widely distributed and reported to exist in plants, microorganisms and algae, but more alanine dehydrogenases derived from microorganisms are reported. To date, alanine dehydrogenases have been found in microorganisms such as Helicobacter pylori (Helicobacter aurantii), Bacillus subtilis (Bacillus subtilis), Thermus thermophilus (Thermus thermophilus), candida xanthus (myococcus xanthophylls), Rhodobacter capsulatus (Rhodobacter capsulatus), Streptomyces fradiae (Streptomyces fragilis), Enterobacter aerogenes (Enterobacter), bifidobacterium farfarinosus (bifidobacterium wadrachium), Streptomyces clavuligerus (Streptomyces clavuligerus), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Rhizobium japonicum (Rhizobium japonicum), and chlorella flava (archaeoglobulus). However, alanine dehydrogenase derived from a microorganism belonging to the genus Salinispirum (Salinispiillum sp.) has not been reported.

Disclosure of Invention

The invention aims to provide alanine dehydrogenase SaAD and a coding gene and application thereof.

The protein provided by the invention is derived from a helicobacter sp (NM), is an alanine dehydrogenase and is named as SaAD protein, and the amino acid sequence is shown as a sequence 1 in a sequence table. Due to the particularity of the amino acid sequence, any peptide protein fragment containing the amino acid sequence shown in the sequence 1 in the sequence table or a mutant thereof is within the protection scope of the invention as long as the peptide protein fragment has over 99 percent of homology with the amino acid sequence and has the function of alanine dehydrogenase. In particular, the alteration comprises a substitution and/or deletion and/or addition and/or substitution of one or several amino acid residues in the amino acid sequence.

The coding gene of the SaAD protein also belongs to the protection scope of the invention.

Preferably, the base sequence of the gene is shown as a sequence 2 in the sequence table, and the gene sequence is derived from Salinispirillum sp.NM and consists of 1122 bases. Due to the specificity of the nucleotide sequence, any variant of the polynucleotide shown in sequence 2 in the sequence table is within the scope of the present invention as long as it has more than 90% homology with the polynucleotide. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Variants of the polynucleotide include substitution variants, deletion variants and insertion variants.

The recombinant expression vector, the expression cassette or the recombinant microorganism containing the gene all belong to the protection scope of the invention.

The invention also protects the application of the SaAD protein as alanine dehydrogenase. When the SaAD protein is used as alanine dehydrogenase, the temperature is 20-60 deg.C, and the pH is 6-10. When the SaAD protein is used as the alanine dehydrogenase, the temperature used is 50 ℃ and the pH used is 8.

The alanine dehydrogenase SaAD provided by the invention has high enzyme activity, wide reaction temperature and wide reaction pH.

Salinispira sp.NM has been deposited at 26.4.2021 at China center for type culture Collection (address: university of Wuhan, China; zip code: 430072) with the collection number of CCTCC NO: M2021460.

The invention has important application prospect in the fields of relevant medicines, food industry and the like.

Drawings

FIG. 1 is a photograph of strain NM.

FIG. 2 is a phylogenetic tree of strain NM.

FIG. 3 is an electrophoretogram of a SaAD protein solution.

FIG. 4 shows the relative enzyme activity results when the optimum pH was measured.

FIG. 5 shows the relative enzyme activity results when the optimal reaction temperature was measured.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 isolation, identification and preservation of Salicospira NM

First, separate

Taking 50 mu L of the alkali lake sample, adding 450 mu L of the alkali lake filtrate, and uniformly pumping to obtain 10^-1Concentration samples, diluted sequentially to give 10^-1、10^-2、10^-3、10^-4、10^-5Concentration of the sample. Taking 20 μ L and 40 μ L of alkaline lake water sample stock solution and diluted sample, respectively spreading on YMAH, TSAH, LBH, 2216EH and other solid culture medium, culturing in 35 deg.C constant temperature incubator for 3-4 days, and observing growth condition. Selecting colonies with different colors and morphologies, purifying and culturing by a three-region scribing method, and repeating the scribing and purifying process for 3-4 times to obtain pure colonies.

II, identification

The purified strain was streaked in LBH solid medium at three regions, cultured at 35 ℃ for 2 days, and the morphology, size, flagella presence and absence of cells were observed by a transmission electron microscope. Single colonies on LBH solid plates were picked for gram staining according to standard methods and the bacteria were observed for gram staining using an inverted fluorescence microscope. Inoculating the strain in a semi-solid culture medium by adopting a puncture method, then putting the strain in an incubator at 37 ℃ for 2d, and observing the motility and the aerobic condition of the strain.

LBH liquid culture medium with NaCl of different concentrations (0%, 1.0%, 3.0%, 5.0% and 10.0% (w/v)) is prepared, inoculated and placed in a 35 ℃ shaking table for shake cultureCulturing for 7 days, sampling every 24h, and measuring OD with UV-2450 spectrophotometer₆₀₀Detecting the growth condition of the bacteria to determine the optimal salinity and salinity growth range of the strain; for the determination of the pH value of the strain, liquid LBH culture media with different pH values (7.0, 8.0, 9.0, 10.0, 11.0, 11.5 and 12.0) are prepared. After inoculation, placing the mixture in a shaking table at 37 ℃ for shaking culture for 7d, sampling every 24h, and taking a non-inoculated culture medium with the same culture condition as a blank control to determine the optimal growth pH range; the temperature gradients were set at 4 deg.C, 10 deg.C, 25 deg.C, 28 deg.C, 30 deg.C, 37 deg.C, 40 deg.C, 45 deg.C and 50 deg.C, respectively, to determine the optimal growth temperature range of the strain.

The Biolog GEN III identification plate is used for detecting the utilization condition of the strain on different carbon sources.

The strains are identified by using API 20NE and API 32GN kits, such as nitrate reduction test, nitrite reduction test, enzyme activity characteristic and carbon source utilization test and the like. NM strain Salinispiillum sp.and reference strain Salinispiillum marinum GCWy1^TThe results are shown in Table 1.

TABLE 1

Extracting the genome DNA of the strain by using a genome extraction kit, and amplifying the 16SrDNA sequence of the strain by using a PCR (polymerase chain reaction) technology. The PCR system adopted in the experiment is as follows: 4. mu.L of template DNA, 1. mu.L of primer 27F, 1. mu.L of primer 1492R, 5. mu.L of 10 XTaq Buffer, 4. mu.L of dNTP, 0.8. mu.L of Taq DNA polymerase, and 34.2. mu.L of sterilized ultrapure water. And verifying the PCR amplification product by using agarose gel electrophoresis, and if the size of the band accords with the expected experimental result, sending the PCR amplification product to commercial sequencing. And if the sequencing result is correct, performing gel cutting recovery on the PCR amplification product, cloning a target strip by using a pClone007 Simple Vector Kit cloning Kit, connecting the cloned target gene with a plasmid pMD18-T, finally transferring the recombinant plasmid into Escherichia coli E.coli DH5 alpha, and strictly performing the transformation process according to the operation instruction of a competent cell E.coli DH5 alpha. And uploading the measured 16S rDNA sequence result to https:// www.ezbiocloud.net/website for sequence comparison analysis, determining the species of the isolated bacteria, and downloading the 16S rDNA sequence of the bacteria with higher affinity with the strain. Phylogenetic trees of the strains were constructed using the MEGA 7.0 software by selecting appropriate outliers and based on the 16S rDNA gene sequences of the isolated strains and their related strains. Phylogenetic analysis of bacteria was performed using the nearest neighbor ligation (NJ). Based on 1000 replicates, the topology of the phylogenetic tree was determined using Bootstrap analysis. See fig. 2.

The above identification results show that the strain NM belongs to the family Saccharospirilludae (Saccharospirilluae), the genus Salinispirillum (Salinispirillum).

III, preservation

Salinispira sp.NM has been deposited at 26.4.2021 at China center for type culture Collection (address: university of Wuhan, China; zip code: 430072) with the collection number of CCTCC NO: M2021460.

Example 2 preparation of alanine dehydrogenase (SaAD protein)

Through a large amount of sequence analysis, comparison and functional verification, a new protein is found from the helicobacter salina NM and named as SaAD protein, as shown in a sequence 1 of a sequence table. The gene of SaAD protein coded in the helicobacter pylori NM is named as SaAD gene, and the coding frame is shown as a sequence 2 in a sequence table.

Construction of recombinant plasmid

1. Taking the genome DNA of the salt spirillum NM as a template, adopting a primer pair consisting of AD-F and AD-R to carry out PCR amplification, and recovering a PCR amplification product.

AD-F：5’-CCCAAGCTTTCAGACCGGC-3’；

AD-R：5’-CGGGATCCATGGACTTCG-3’。

2. And (3) connecting the PCR amplification product obtained in the step (1) with a pET-28a vector to obtain a recombinant plasmid pET-28 a-SaAD.

pET-28a Vector (pET-28a Vector): novagen, catalog No. 69864-3.

Through sequencing, the recombinant plasmid pET-28a-SaAD has a DNA molecule shown in a sequence 2 in a sequence table.

Secondly, preparing recombinant bacteria

The recombinant plasmid pET-28a-SaAD was introduced into Escherichia coli BL21(DE3) to obtain recombinant bacterium A.

The pET-28a vector was introduced into Escherichia coli BL21(DE3) to obtain recombinant strain B.

Third, expression of proteins

1. The recombinant strain was inoculated into a liquid LB medium containing 50. mu.g/mL kanamycin and subjected to shaking culture at 37 ℃ and 150rpm for 12 hours to obtain a seed solution.

2. 1 volume portion of the seed liquid was inoculated into 99 volume portions of liquid LB medium containing 50. mu.g/mL kanamycin, and cultured at 37 ℃ with shaking at 200rpm until OD₆₀₀The value was about 0.6, at which time IPTG inducer was added so that the concentration in the system became 0.5mmol/L, followed by shaking culture at 200rpm at 25 ℃ for 6 hours (induction expression), followed by centrifugation at 8000 Xg for 10min at 4 ℃ to collect the cell pellet.

3. And (3) taking the precipitate obtained in the step (2), washing with a Tris-HCl buffer solution (0.05M, pH 7.5), suspending in the Tris-HCl buffer solution (0.05M, pH 7.5), carrying out ultrasonication (ultrasonication parameters: power of 250W, 3s per ultrasonication, 3s stopping, total time of 30min), centrifuging at 4 ℃ and 10000 Xg for 10min, and collecting a supernatant.

The recombinant bacterium A is subjected to the supernatant obtained in the step, and named as crude enzyme liquid A.

And (3) carrying out the steps on the recombinant bacterium B to obtain supernatant, and naming the supernatant as crude enzyme solution B.

Fourth, purifying the protein

And (4) filtering the crude enzyme liquid A obtained in the step three by using a microfiltration membrane with the pore diameter of 0.22 mu m, and collecting the filtrate. Taking the filtrate, and adopting Superdex^TM20010/60 separating and purifying with gel chromatographic column. Mixing Superdex^TM20010/60 the gel chromatography column was connected to a fast protein liquid phase system with PBS buffer (50mM, pH 8.0) as the mobile phase at a flow rate of 0.05 mL/min. Collecting the solution after passing through the column with the retention volume of 18-20mL corresponding to the elution peak, namely the SaAD eggWhite solution. The electrophoretogram of the SaAD protein solution is shown in figure 3, with only one protein band, and corresponds to the predicted molecular weight (about 39.4 kDa).

Example 3 enzymatic Properties of alanine dehydrogenase (SaAD protein)

PBS buffer (50mM, pH 8.0): 1.44g of sodium dihydrogen phosphate, 0.24g of potassium dihydrogen phosphate, 0.20g of potassium chloride and 8.00g of sodium chloride were weighed, dissolved in 800mL of ultrapure water, adjusted to pH 8.0 with HCl, and the volume was adjusted to 1L.

Alanine reaction (10 mM): 1.1g L-alanine was weighed, dissolved in PBS buffer and made to volume of 1L.

NAD⁺Solution (80 μ M): 0.0531g of NAD are weighed out⁺Dissolving with PBS buffer solution, and diluting to 1L.

The reaction principle is as follows: the alanine dehydrogenase can catalyze the oxidative deamination reaction of L-alanine, the product is pyruvic acid, and NAD is simultaneously removed⁺Reduced to NADH, which has a maximum absorbance at 340 nm.

First, influence of pH on alanine dehydrogenase Activity

The SaAD protein solution prepared in example 2 was diluted to 2 volumes with a buffer solution, and the diluted solution was used as a test solution.

The detection method comprises the following steps: adding 200 μ L of test solution and 700 μ L, NAD of alanine reaction solution⁺The solution (100. mu.L) was reacted at room temperature for 2min, and then OD was measured_340nmThe value is obtained.

The following buffers were used respectively: a phosphate buffer at pH 6.0, a phosphate buffer at pH 7.0, a phosphate buffer at pH 8.0, a carbonate buffer at pH 9.0, a carbonate buffer at pH 10.0. The formulation of the phosphate buffer is shown in Table 2. The formulation of the carbonate buffer is shown in Table 3.

TABLE 2

pH	0.2M aqueous disodium hydrogen phosphate solution (mL)	0.2M sodium dihydrogen phosphate aqueous solution (mL)
			6.0	61.5	438.5
7.0	305	195
			8.0	473.5	26.5

TABLE 3

pH	0.05M aqueous sodium carbonate solution (mL)	0.05M aqueous sodium bicarbonate solution (mL)
			8.0	50	450
9.0	150	350
			10.0	300	200

The optimum pH of the SaAD protein is 8. OD when buffer solution corresponding to optimum pH is used_340nmThe value was taken as 100%, and the relative value when each buffer was used was calculated as the relative enzyme activity. The results are shown in FIG. 4.

Second, influence of temperature on the Activity of alanine dehydrogenase

The SaAD protein solution prepared in example 2 was diluted to 2 volumes with PBS buffer and used as a test solution.

The detection method comprises the following steps: adding 200 μ L of test solution and 700 μ L, NAD of alanine reaction solution⁺The solution (100 μ L) was reacted at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, and 60 deg.C for 2min, and the OD was measured_340nmThe value is obtained.

The optimum reaction temperature is 50 ℃. OD at optimum reaction temperature_340nmThe value was taken as 100%, and the relative values at various reaction temperatures were calculated as relative enzyme activities. The results are shown in FIG. 5.

Third, determination of enzyme activity

Enzyme activity (1U) is defined as: reduction of 1. mu. mol NAD per minute⁺The amount of enzyme required.

The detection method comprises the following steps: adding 200 μ L of test solution and 700 μ L, NAD of alanine reaction solution⁺The solution (100. mu.L) was reacted at room temperature for 2min, and then OD was measured_340nmThe value is obtained.

The crude enzyme solution A prepared in example 2 was used as a test solution, and the enzyme activity was 320U/mL.

The crude enzyme solution B prepared in example 2 was used as a test solution, and the enzyme activity was 0U/mL.

The SaAD protein solution prepared in example 2 was used as a test solution, and the enzyme activity per unit volume of the test solution was measured. The protein concentration in the SaAD protein solution prepared in example 2 was examined. And dividing the enzyme activity of the test solution in unit volume by the protein content of the test solution in unit volume to obtain the specific activity of the protein, wherein the value is 646U/mg.

Example 4 use of alanine dehydrogenase (SaAD protein) for the preparation of 3-fluoro-pyruvate

1. Procedure of experiment

(1) The SaAD protein solution prepared in example 2 was diluted to 80U/mL with PBS buffer (100mM, pH 8.0) as a test solution;

(2) the total volume of the reaction was 50mL, containing 50mM of 3-fluoro-L-alanine, 2mM of NAD⁺80U/mL SaAD protein solution in PBS buffer (100mM, pH 8.0), a reaction temperature of 50 ℃, a stirring rate of 120rpm, and a reaction time of 10 hours.

(3) The reaction mixture was analyzed in an SCR-101H (300 mm. times.7.9 mm) column at 20 ℃ and a flow rate of 0.8mL/min, with 5. mu.L of an aqueous perchloric acid solution (0.01M, pH 3.8) as a mobile phase, and UV detection was maintained at 200 nm.

2. Use of alanine dehydrogenase (SaAD protein) for preparing 3-fluoro-pyruvate

After the reaction is finished, HPLC analysis shows that the conversion rate of the 3-fluoro-L-alanine is 97.4%, which indicates that the enzyme has larger application potential in the aspect of preparation of pyruvic acid.

Sequence listing

<110> China university of Petroleum (east China)

<120> alanine dehydrogenase SaAD and coding gene and application thereof

<130> 2021.6.8

<141> 2021-06-16

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 373

<212> PRT

<213> Salinum sp (Salinispirilum sp.)

<400> 1

Met Tyr Val Gly Val Pro Lys Glu Ile Lys Asn His Glu Tyr Arg Val

1 5 10 15

Gly Leu Ile Pro Ala Ala Val Arg Glu Leu Thr Val Ala Gly His Gln

20 25 30

Val Phe Val Glu Asn Asp Ala Gly Ala Ala Ile Gly Tyr Ser Asn Glu

35 40 45

Asp Tyr Glu Ala Val Gly Ala Thr Ile Val Gln Gln Ala Ser Asp Val

50 55 60

Phe Glu Arg Ala Glu Met Ile Val Lys Val Lys Glu Pro Gln Ala Glu

65 70 75 80

Glu Arg Ala Leu Leu Arg Pro His His Val Leu Phe Thr Tyr Leu His

85 90 95

Leu Ala Pro Asp Ala Pro Gln Leu Lys Gly Leu Met Asp Ser Gly Ala

100 105 110

Thr Cys Ile Ala Tyr Glu Thr Val Thr Asp Val His Gly Arg Leu Pro

115 120 125

Leu Leu Ala Pro Met Ser Glu Val Ala Gly Arg Met Ala Val Gln Ala

130 135 140

Gly Ala His Cys Leu Glu Lys Ser Met Gly Gly Ser Gly Met Leu Leu

145 150 155 160

Gly Gly Val Pro Gly Val Ala Pro Ala Lys Val Thr Ile Val Gly Gly

165 170 175

Gly Val Val Gly Gln Asn Ala Leu Ala Ile Ala Val Gly Met Gly Ala

180 185 190

Gln Val Thr Val Leu Asp Arg Ser Met Asp Val Leu Arg Arg Leu Asp

195 200 205

Gln Val Tyr Ser Asn Arg Ile Thr Thr Leu Phe Ser Thr Arg Glu Ala

210 215 220

Leu Glu Ser Ser Val Val His Ser Asp Leu Val Ile Gly Ala Val Leu

225 230 235 240

Ile Pro Gly Ala Ala Ala Pro Lys Leu Ile Thr Arg Glu Met Ile Lys

245 250 255

Thr Met Ala Ala Gly Ser Val Val Val Asp Val Ala Ile Asp Gln Gly

260 265 270

Gly Cys Met Glu Thr Ser Lys Pro Thr Thr His Ser Glu Pro Thr Tyr

275 280 285

Val Val Asp Gly Val Val His Tyr Cys Val Ala Asn Met Pro Gly Gly

290 295 300

Val Ala Arg Thr Ala Thr Gln Ala Leu Asn Asn Ala Thr Leu Pro Phe

305 310 315 320

Val Leu Gln Leu Ala Asn Lys Gly Ala Gln Gln Ala Leu Leu Asp Asn

325 330 335

Thr His Leu Arg Asn Gly Leu Asn Val Tyr Arg Gly Glu Val Thr Tyr

340 345 350

Ala Glu Val Ala Glu Ala Arg Gly Met Pro Phe Arg Glu Ala Leu Asp

355 360 365

Ala Leu Gln Lys Gly

370

<210> 2

<211> 1122

<212> DNA

<213> Salinum sp (Salinispirilum sp.)

<400> 2

atgtatgtcg gtgtcccgaa agaaatcaag aatcacgaat accgtgttgg gctgatccca 60

gcggcagtgc gcgagttgac ggtggcaggt catcaggtct ttgtcgagaa cgacgccggt 120

gcggcgatcg gctacagcaa tgaggactat gaggcggttg gggcgactat tgtgcagcag 180

gcgtcggatg tgttcgagcg tgccgagatg atcgttaaag taaaggagcc gcaagccgaa 240

gagcgtgccc tattgcgtcc gcatcatgtg ctgtttacct acttgcatct ggcacccgat 300

gcgccgcagc taaaaggcct gatggacagt ggggcgacct gcatagctta tgaaactgtc 360

accgatgtgc atgggcgctt gcctttgttg gcgcccatgt cggaagtggc ggggcgtatg 420

gccgtgcaag cgggcgcaca ctgtttggaa aaatccatgg gtggcagtgg aatgctattg 480

ggcggtgtac cgggcgtggc acccgccaag gtgactattg tcggtggtgg tgtggtgggg 540

caaaatgcgc tcgccatcgc agtcggcatg ggagcacagg tgactgtgtt ggaccgcagt 600

atggatgtat tgcggcgttt agatcaggtt tacagcaacc gcattaccac cctgttctca 660

acccgtgaag ccttggaaag cagtgtggtg cattccgacc tggtgatcgg tgccgtactc 720

attcctggtg ccgcagcccc taaattgatc acccgtgaaa tgatcaaaac catggcggca 780

ggcagtgtcg ttgtggatgt cgccatcgac caagggggtt gcatggaaac ttcgaagccg 840

accacgcaca gcgagccgac ttacgttgta gacggggtgg tgcactactg tgtcgccaat 900

atgcccggtg gtgtggcgcg cacggccaca caggcgctga ataacgccac cttgcccttt 960

gtgttacaac tggccaataa aggcgcgcag caagcgctgt tggacaatac gcacctgcgc 1020

aatgggctga atgtgtaccg tggtgaagta acctatgcgg aagttgcgga agctcgaggt 1080

atgccgtttc gtgaggcttt agatgcatta caaaaaggat aa 1122

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

cggatccatg tatgtcggtg t 21

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ccggaagctt ttatcctttt tgt 23

Claims (5)

1. The protein coded by the alanine dehydrogenase gene is characterized in that an amino acid sequence is shown as a sequence 1 in a sequence table.

2. A gene encoding the protein of claim 1.

3. The gene as claimed in claim 2, characterized in that the nucleotide sequence is contained in the nucleotide sequence as set forth in sequence 2 of the sequence listing.

4. A recombinant expression vector, expression cassette or recombinant microorganism comprising the gene of claim 2 or 3.

5. Use of the protein of claim 1 as an alanine dehydrogenase.

Images