CN111607575B - A kind of transaminase PHTA, preparation method and application - Google Patents

Abstract

The invention relates to transaminase PHTA, the amino acid sequence of which is SEQ ID NO.1. The optimum temperature of the transaminase PHTA of the invention is 35 ℃, the optimum pH is 8.0, and the transaminase PHTA still maintains more than 50 percent of activity within the pH range of 6 to 8. Under the optimal temperature and the optimal pH, the degradation rate of the transaminase PHTA of the invention to HFB1 is 100 percent; the transaminase PHTA of the invention has excellent property, can be applied to agriculture, feed, food and other industries, and reduces the harm of fumonisins FB1 to the health of animals and human beings.

Description

A kind of transaminase PHTA, preparation method and application

technical field

The invention belongs to the field of agricultural biotechnology, in particular to a transaminase PHTA, a preparation method and an application.

Background technique

Fumonisins are a class of structurally related metabolites mainly produced by Fusarium and Fusarium proliferators. Twenty-eight fumonisin analogs have been identified so far, which are divided into four series A, B, C and P. Among them, fumonisin B1 (FB1) is the most common and most toxic fumonisin. It causes leukomalacia in horses and pulmonary edema in pigs, and also affects the liver and immune system in poultry. In addition, FB1 is hepatotoxic to most mammals.

Mitigation strategies to eliminate fumonisin contamination in food and feed are divided into physical, chemical and biological principles. Physical methods, including dry and wet milling, soaking, heating, and the use of adsorbents. However, these strategies have limitations, either because the instrumentation is expensive or because of the loss of certain nutrients in the feed. Ammoniation and alkalization are the most common chemical detoxification methods, but their application is limited due to their potential toxicity and negative impact on the taste and nutritional quality of raw materials. Biological detoxification technology can reduce the toxicity of mycotoxins without affecting the quality of food and feed, and is considered as a promising detoxification strategy. Compared with microbial preparations, enzyme preparations have higher stability in storage. Therefore, the development of enzyme preparations that degrade mycotoxins can well curb the production of toxins in contaminated food crops and restore economic losses.

Through searching, no patent publications related to the patent application of the present invention have been found.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a transaminase PHTA, a preparation method and an application. The transaminase PHTA can degrade the amino group in the fumonisin to relieve its toxicity.

The technical scheme adopted by the present invention to solve its technical problems is:

A transaminase PHTA, the amino acid sequence of the transaminase PHTA is SEQ ID NO.1.

The preparation method of the above-mentioned transaminase PHTA, the steps are as follows:

(1) transforming the host cell with the recombinant vector comprising the gene encoding the transaminase PHTA to obtain a recombinant strain;

(2) Cultivate the recombinant strain to induce the expression of transaminase PHTA;

(3) Separation and purification of the obtained transaminase PHTA.

Moreover, the host cell in the step (1) is a large intestinal cell, a Saccharomyces cerevisiae cell or a S. polymorpha cell.

Use of the transaminase PHTA as described above in the degradation of fumonisins.

A transaminase PHTA gene encoding the above-mentioned transaminase PHTA.

Also, the nucleotide sequence of the gene is SEQ ID NO.2.

A recombinant vector comprising the transaminase PHTA gene as described above.

The recombinant vector pET28a(+)-PHTA containing the transaminase PHTA gene as described above.

A recombinant strain comprising the transaminase PHTA gene as described above.

Moreover, the recombinant strain is Escherichia coli.

The advantages and positive effects obtained by the present invention are:

1. The optimum temperature of the transaminase PHTA of the present invention is 35°C, the optimum pH is 8.0, and it still maintains 50% in the range of pH 6-8

above activity. Under the optimum temperature and optimum pH, the degradation rate of HFB1 by the transaminase PHTA of the present invention is 100%;

The transaminase PHTA of the invention has excellent properties, and the enzyme can be applied to industries such as agriculture, feed and food, so as to reduce the harm of the fumonisin FB1 to the health of animals and humans.

2. The transaminase PHTA of the present invention can degrade and catalyze the hydrolysis of the amino group of fumonisin FB1 (HFB1) and eliminate the activity of fumonisins. Since amino groups are one of the key functional groups that make fumonisins toxic, removal of amino groups is a key point to detoxify fumonisins. The transaminase PHTA of the invention has the activity of degrading and hydrolyzing the fumonisin HFB1, and can be applied to industries such as agriculture, feed and food to reduce the harm of the fumonisins to the health of animals and humans.

Description of drawings

Fig. 1 is the SDS-PAGE purification diagram of transaminase PHTA in the present invention; wherein, M: protein marker; 1: transaminase PHTA;

Fig. 2 is a schematic diagram showing the degradation effect of transaminase PHTA in the present invention;

Fig. 3 is the optimum temperature diagram showing transaminase PHTA in the present invention;

Figure 4 is a graph showing the optimum pH of the transaminase PHTA in the present invention.

Detailed ways

The embodiments of the present invention will be described in detail below. It should be noted that the embodiments are descriptive, not restrictive, and cannot limit the protection scope of the present invention.

The raw materials used in the present invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional methods in the art unless otherwise specified.

A transaminase PHTA, the amino acid sequence of the transaminase PHTA is SEQ ID NO.1.

The preparation method of the above-mentioned transaminase PHTA, the steps are as follows:

(1) transforming the host cell with the recombinant vector comprising the gene encoding the transaminase PHTA to obtain a recombinant strain;

(2) Cultivate the recombinant strain to induce the expression of transaminase PHTA;

(3) Separation and purification of the obtained transaminase PHTA.

Preferably, in the step (1), the host cell is a large intestine cell, a Saccharomyces cerevisiae cell or a Saccharomyces cerevisiae cell, preferably Escherichia coli BL21 (DE3).

Use of the transaminase PHTA as described above in the degradation of fumonisins.

A transaminase PHTA gene encoding the above-mentioned transaminase PHTA.

Preferably, the nucleotide sequence of the gene is SEQ ID NO.2.

A recombinant vector comprising the transaminase PHTA gene as described above.

The recombinant vector pET28a(+)-PHTA containing the transaminase PHTA gene as described above.

A recombinant strain comprising the transaminase PHTA gene as described above.

Preferably, the recombinant strain is Escherichia coli.

Specifically, described transaminase PHTA, its amino acid sequence is as shown in SEQ ID NO.1:

SEQ ID NO. 1:

MTRQRAKDAELRERAYRVVPGGVYGHLSTALLPSGYPQFFRRGKGAHLWDVDDNMYIDYLCAYGPNLFGYGFEPIERAAVRQQNLGDTLTGPTEALVELAEAFVGMVTHADWAMFCKNGTDANTIALMISRAHTGRATVLVAEGAYHGAAPWSTPRPAGVTAGDRANIVTYRYNDPESLAAAYRAHRHDLAAIFATPFRHEVFADQEDLNVTYARLARELCDQAGALLVVDDVRAGFRIARDCSWSPSGVQPDLSAWGKCFANGYPISAVLGSDKARKAAAEIFVTGSFWMSATPMAAAIEGLKQIRETDYLERLVESGLALRHGLQRQAAAHGFTLRQTGPAQMPQILFEEDPDFRVGYAWAEACVERGVYFSPYHNMFLSTAHTDGVIRRTLEVTDVAFETVKRQRGSLRTPAQLVPYAQEMAARLAT

Among them, the enzyme gene encodes 430 amino acids and has no signal peptide. Therefore, the theoretical molecular weight of the mature transaminase PHTA is 48.21kDa.

The present invention provides encoding the above-mentioned transaminase PHTA. The genome sequence of the gene is shown in SEQ ID NO.2:

SEQ ID NO. 2:

ATGACTAGACAGAGAGCTAAGGACGCTGAGTTGAGAGAGAGAGCCTACAGAGTTGTTCCAGGTGGTGTTTACGGTCACTTGTCCACTGCTTTGTTGCCATCTGGTTACCCACAGTTCTTCAGACGTGGTAAGGGTGCTCATTTGTGGGATGTTGACGACAACATGTACATCGACTACTTGTGTGCCTACGGTCCAAACTTGTTCGGTTACGGTTTCGAGCCAATTGAGAGAGCTGCTGTCAGACAACAAAACTTGGGTGACACTTTGACTGGTCCAACCGAGGCTTTGGTTGAATTGGCTGAGGCTTTCGTTGGTATGGTTACTCATGCTGACTGGGCCATGTTCTGCAAGAACGGTACTGACGCTAACACTATCGCCTTGATGATTTCCAGAGCACACACTGGTAGAGCCACTGTTTTGGTTGCTGAAGGTGCTTATCATGGTGCTGCACCTTGGTCTACTCCAAGACCTGCTGGTGTTACTGCTGGTGATAGAGCTAACATCGTCACCTACAGATACAACGACCCAGAATCTTTGGCTGCTGCTTACAGAGCCCATAGACATGACTTGGCTGCTATCTTCGCTACCCCATTCAGACACGAAGTTTTCGCTGATCAAGAGGACCTGAACGTTACCTACGCTAGATTGGCCAGAGAATTGTGTGATCAGGCTGGTGCCTTGTTGGTTGTTGATGATGTTAGAGCCGGTTTCAGAATCGCCAGAGACTGTTCTTGGTCCCCATCTGGTGTTCAACCAGATTTGTCTGCTTGGGGTAAGTGTTTCGCTAACGGTTACCCAATCTCCGCTGTTTTGGGTTCTGACAAGGCTAGAAAAGCTGCCGCCGAGATTTTCGTTACTGGTTCTTTTTGGATGTCCGCCACTCCAATGGCTGCCGCTATTGAAGGTTTGAAGCAGATCAGAGAGACTGACTACTTGGAGAGATTGGTCGAATCCGGTTTGGCTTTGAGACACGGTCTGCAAAGACAAGCTGCTGCTCACG GTTTTACCTTGAGACAAACTGGTCCAGCTCAGATGCCACAGATTTTGTTCGAAGAGGACCCAGACTTCAGAGTTGGTTATGCTTGGGCTGAAGCCTGTGTTGAAAGAGGTGTTTACTTCTCCCCATACCACAACATGTTCTTGTCCACCGCTCACACTGACGGTGTTATCAGAAGAACTTTGGAGGTTACCGACGTCGCCTTCGAGACTGTTAAGAGACAAAGAGGTTCCCTGATAGACCCCAGCTCAATTGGTTCCATAGCTAGACT

The present invention isolates and clones the transaminase PHTA by the method of PCR, and the analysis result of the complete DNA sequence shows that the full length of the open reading frame sequence (ORF) of the transaminase PHTA gene is 1290 bp.

The present invention also provides a recombinant vector comprising the above-mentioned transaminase PHTA, and the preferred recombinant vector is named pET28a-PHTA. The transaminase PHTA gene of the present invention is inserted into the expression vector between suitable restriction enzyme sites, so that its nucleotide sequence is operably linked to the expression control sequence. As a most preferred embodiment of the present invention, it is preferable to insert the detoxification enzyme gene of the present invention between the EcoR I and Xhol I restriction sites on plasmid pET28a, so that the nucleotide sequence is located in the T7 promoter downstream and regulated by it, the recombinant colon expression plasmid pET28a-PHTA was obtained.

More specifically, relevant preparation and detection are as follows:

Test materials and reagents:

1. Strain and carrier: The present invention obtains a new transaminase PHTA. The E. coli expression vector pET28a(+) and strain BL21(DE3) were preserved in the laboratory.

2. Enzymes and other biochemical reagents: Endonuclease was purchased from TaKaRa Company, and ligase was purchased from Invitrogen Company. Purchased from Sigma company, other reagents are domestic (all can be purchased from common biochemical reagent companies).

3. Culture medium:

E. coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).

Note: The following unspecified molecular biology experimental methods are carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) by J. Sambrook, or according to the kit and product instructions. .

1. Cloning of transaminase PHTA

The gene fragment of transaminase PHTA was obtained by artificial chemical synthesis, and an endonuclease site EcoR I was introduced at the 5' end and an endonuclease site Xhol I was introduced at the 3' end.

2. Preparation of recombinant transaminase PHTA

The expression vector pET28a is double-enzyme cut (EcoR I+XholI), and the gene encoding transaminase PHTA is double-enzyme cut (EcoR I+XholI), and the gene fragment encoding mature transaminase is cut out and the expression vector pET28a is connected to obtain a gene containing transaminase. The recombinant plasmid pET28a-PHTA of PHTA was transformed into E. coli BL21 (DE3) to obtain a recombinant E. coli strain BL21/PHTA.

Take the BL21 (DE3) strain containing the plasmid, inoculate it in 100 mL of LB medium, shake at 37 °C and 220 rpm for about 2 hours, add 1 mM IPTG, and place it at 25 °C and 220 rpm for induction. After about 20 hours, measure the intracellular and extracellular levels. Transaminase activity. The activity of transaminase was detected in the cells. After purification by nickel column, the results of SDS-PAGE showed that the recombinant transaminase was expressed. As shown in Figure 1, lane 1 is the result after purification.

3. Determination of the properties of recombinant transaminases

The enzyme activity of transaminase was detected by high performance liquid chromatography, and the specific method was as follows:

(1) HFB1 standard stock solution: prepared into a standard solution with a concentration of 100 μg/mL, and stored at -20°C.

(2) Sample preparation: Take 850 μL of the purified transaminase enzyme solution, add 100 μL of HFB1 standard stock solution and 50 μL of pyruvate solution to make the final concentration of HFB1 10 μg/mL, 37 ° C, 220 rpm, and culture for 20 min in the dark.

(3) Sample derivatization: Take 100 μL of the sample to be tested, add 400 μL 50% acetonitrile water, 500 μL OPA derivatization solution, mix for 30 s, inject the sample within 2 minutes of derivatization, and filter the membrane to be tested. The enzymatic activity of the transaminase PHTA was determined by comparison with the peak profile of the HFB1 standard.

1. Determination of the degradation ability of fumonisin-degrading enzymes

100 μL of HFB1 solution was added to 900 μL of a mixture of transaminase PHTA enzyme solution and pyruvate (citric acid-disodium hydrogen phosphate buffer, pH=7.0) to make the final concentration of HFB1 10 μg/mL. The reaction solution was placed under the conditions of 37° C. and pH=7, and the reaction was carried out for 12 h, and the solution without adding the purified transaminase PHTA was used as a control. After the reaction is completed, boil in boiling water for 10 min to inactivate the enzyme. After cooling to room temperature, it was passed through the membrane and detected by high performance liquid chromatography.

The results are shown in Fig. 2, wherein Fig. 2a shows the mixed solution of buffer solution and HFB1, and Fig. 2b shows the reaction solution of enzyme solution and HFB1. It can be seen that HFB1 will have the highest peak at 11.646min, and HFB1 is not detected in the solution added with purified recombinant transaminase PHTA. Therefore, it can be concluded that the transaminase PHTA has the ability to completely degrade HFB1, and can completely degrade 10 μg/mL HFB1 within 12 h.

2. Determination of the optimum temperature for fumonisin-degrading enzymes

Using HFB1 as the substrate, take 100 μL of the substrate and add 850 μL of enzyme solution and 50 μL of pyruvate solution, the final concentration is 10 μg/mL, in citric acid-disodium hydrogen phosphate buffer (pH 7.0) buffer system and at different temperatures , react for 1h, then boil in boiling water for 10min to inactivate the enzyme. After cooling to room temperature, it was passed through the membrane and detected by high performance liquid chromatography.

As shown in Fig. 3, the optimum temperature of transaminase PHTA was 35°C. When the temperature is higher than 35°C, the activity of the enzyme shows a continuous decline, and when the temperature is 70°C, the relative activity is only no higher than 10%.

The optimum temperature of transaminase PHTA at 35°C is very close to the mammalian body temperature of 37°C. When transaminase PHTA is used in animal feed, it will play a greater role in animals.

3. Determination of the optimum pH of transaminase PHTA

The purified recombinant transaminase PHTA was subjected to enzymatic reaction under different pH buffers to determine its optimum pH. The buffer gradient of the selected buffer solution was 100mM citric acid-disodium hydrogen phosphate (pH3.0–8.0), 100 mM Tris-HCl (pH 7.0-9.0), 100 mM Glycine-NaOH (pH 9.0-12.0). The transaminase PHTA was reacted with the substrate HFB1 (final concentration of 1 μg/mL) in the above different buffers at 37 °C for 1 h, boiled in boiling water for 10 min, and detected by high performance liquid chromatography.

As a result, as shown in Fig. 4, the optimum pH of the transaminase PHTA was 8.0. Between pH=3-8, the relative enzyme activity of transaminase PHTA increased continuously, from 20% to 100%, but after pH was greater than 8, it showed a great gradient decline. When pH=10, the relative enzyme activity less than 20%. Transaminase PHTA has a high acid-base energy capacity, and whether it is added to feed storage or enters the stomach of animals, it will maintain a certain vitality to achieve the purpose of degrading toxins.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, therefore , the scope of the present invention is not limited to the contents disclosed in the embodiments.

sequence listing

<110> Tianjin University of Science and Technology

<120> A kind of transaminase PHTA, preparation method and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 430

<212> PRT

<213> Amino acid sequence of transaminase PHTA (Unknown)

<400> 1

Met Thr Arg Gln Arg Ala Lys Asp Ala Glu Leu Arg Glu Arg Ala Tyr

1 5 10 15

Arg Val Val Pro Gly Gly Val Tyr Gly His Leu Ser Thr Ala Leu Leu

20 25 30

Pro Ser Gly Tyr Pro Gln Phe Phe Arg Arg Gly Lys Gly Ala His Leu

35 40 45

Trp Asp Val Asp Asp Asn Met Tyr Ile Asp Tyr Leu Cys Ala Tyr Gly

50 55 60

Pro Asn Leu Phe Gly Tyr Gly Phe Glu Pro Ile Glu Arg Ala Ala Val

65 70 75 80

Arg Gln Gln Asn Leu Gly Asp Thr Leu Thr Gly Pro Thr Glu Ala Leu

85 90 95

Val Glu Leu Ala Glu Ala Phe Val Gly Met Val Thr His Ala Asp Trp

100 105 110

Ala Met Phe Cys Lys Asn Gly Thr Asp Ala Asn Thr Ile Ala Leu Met

115 120 125

Ile Ser Arg Ala His Thr Gly Arg Ala Thr Val Leu Val Ala Glu Gly

130 135 140

Ala Tyr His Gly Ala Ala Pro Trp Ser Thr Pro Arg Pro Ala Gly Val

145 150 155 160

Thr Ala Gly Asp Arg Ala Asn Ile Val Thr Tyr Arg Tyr Asn Asp Pro

165 170 175

Glu Ser Leu Ala Ala Ala Tyr Arg Ala His Arg His Asp Leu Ala Ala

180 185 190

Ile Phe Ala Thr Pro Phe Arg His Glu Val Phe Ala Asp Gln Glu Asp

195 200 205

Leu Asn Val Thr Tyr Ala Arg Leu Ala Arg Glu Leu Cys Asp Gln Ala

210 215 220

Gly Ala Leu Leu Val Val Asp Asp Val Arg Ala Gly Phe Arg Ile Ala

225 230 235 240

Arg Asp Cys Ser Trp Ser Pro Ser Gly Val Gln Pro Asp Leu Ser Ala

245 250 255

Trp Gly Lys Cys Phe Ala Asn Gly Tyr Pro Ile Ser Ala Val Leu Gly

260 265 270

Ser Asp Lys Ala Arg Lys Ala Ala Ala Glu Ile Phe Val Thr Gly Ser

275 280 285

Phe Trp Met Ser Ala Thr Pro Met Ala Ala Ala Ile Glu Gly Leu Lys

290 295 300

Gln Ile Arg Glu Thr Asp Tyr Leu Glu Arg Leu Val Glu Ser Gly Leu

305 310 315 320

Ala Leu Arg His Gly Leu Gln Arg Gln Ala Ala Ala His Gly Phe Thr

325 330 335

Leu Arg Gln Thr Gly Pro Ala Gln Met Pro Gln Ile Leu Phe Glu Glu

340 345 350

Asp Pro Asp Phe Arg Val Gly Tyr Ala Trp Ala Glu Ala Cys Val Glu

355 360 365

Arg Gly Val Tyr Phe Ser Pro Tyr His Asn Met Phe Leu Ser Thr Ala

370 375 380

His Thr Asp Gly Val Ile Arg Arg Thr Leu Glu Val Thr Asp Val Ala

385 390 395 400

Phe Glu Thr Val Lys Arg Gln Arg Gly Ser Leu Arg Thr Pro Ala Gln

405 410 415

Leu Val Pro Tyr Ala Gln Glu Met Ala Ala Arg Leu Ala Thr

420 425 430

<210> 2

<211> 1290

<212> DNA/RNA

<213> Transaminase PHTA gene (Unknown)

<400> 2

atgactagac agagagctaa ggacgctgag ttgagagaga gagcctacag agttgttcca 60

ggtggtgttt acggtcactt gtccactgct ttgttgccat ctggttaccc acagttcttc 120

agacgtggta agggtgctca tttgtgggat gttgacgaca acatgtacat cgactacttg 180

tgtgcctacg gtccaaactt gttcggttac ggtttcgagc caattgagag agctgctgtc 240

agacaacaaa acttgggtga cactttgact ggtccaaccg aggctttggt tgaattggct 300

gaggctttcg ttggtatggt tactcatgct gactgggcca tgttctgcaa gaacggtact 360

gacgctaaca ctatcgcctt gatgatttcc agagcacaca ctggtagagc cactgttttg 420

gttgctgaag gtgcttatca tggtgctgca ccttggtcta ctccaagacc tgctggtgtt 480

actgctggtg atagagctaa catcgtcacc tacagataca acgacccaga atctttggct 540

gctgcttaca gagcccatag acatgacttg gctgctatct tcgctacccc attcagacac 600

gaagttttcg ctgatcaaga ggacctgaac gttacctacg ctagattggc cagagaattg 660

tgtgatcagg ctggtgcctt gttggttgtt gatgatgtta gagccggttt cagaatcgcc 720

agagactgtt cttggtcccc atctggtgtt caaccagatt tgtctgcttg gggtaagtgt 780

ttcgctaacg gttacccaat ctccgctgtt ttgggttctg acaaggctag aaaagctgcc 840

gccgagattt tcgttactgg ttctttttgg atgtccgcca ctccaatggc tgccgctatt 900

gaaggtttga agcagatcag agagactgac tacttggaga gattggtcga atccggtttg 960

gctttgagac acggtctgca aagacaagct gctgctcacg gttttacctt gagacaaact 1020

ggtccagctc agatgccaca gattttgttc gaagaggacc cagacttcag agttggttat 1080

gcttgggctg aagcctgtgt tgaaagaggt gtttacttct ccccatacca caacatgttc 1140

ttgtccaccg ctcacactga cggtgttatc agaagaactt tggaggttac cgacgtcgcc 1200

ttcgagactg ttaagagaca aagaggttcc ctgagaaccc cagctcaatt ggttccatac 1260

gctcaagaaa tggctgctag actggctact 1290

Claims (1)

1. The application of transaminase PHTA in degrading fumonisin B1 is characterized in that the amino acid sequence of the transaminase PHTA is SEQ ID NO.1, and the application is a non-disease treatment purpose.

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