CN113801863B - A kind of engineering strain of apocynum tannase whole cell catalyst and its preparation method and application - Google Patents

Abstract

The invention relates to the technical field of bioengineering, in particular to an engineering strain of apocynum tannase whole-cell catalyst and its preparation method and application. The whole-cell catalyst engineering strain of apocynum tanninase comprises the gene of apocynum tanninase shown in SEQ ID NO:1, and can express the apocynum tanninase shown in SEQ ID NO:2. The invention clones and obtains a new tannase gene from the herb plant Apocynum apocynum in vivo, constructs a shuttle expression vector, and obtains a whole-cell catalyst engineering bacterial strain anchoring the apocynum tannase on the cell wall surface through induction. The whole-cell catalyst engineering strain has high heat resistance, can catalyze tannin substrates such as epicatechin gallate, remove the bitter taste of apocynum tea, obviously improve the flavor of apocynum tea, and has broad application prospects.

Description

A kind of Apocynum tannase whole cell catalyst engineering strain and its preparation method and application

technical field

The invention relates to the technical field of bioengineering, in particular to an engineering strain of apocynum tannase whole-cell catalyst and its preparation method and application.

Background technique

The wild plant "Apocynum venetum" (scientific name: A. venetum L.) has the functions of clearing blood vessel wall deposits, softening blood vessels, lowering blood pressure and clearing fat, and stabilizing blood pressure all year round. Since ancient times, apocynum has played a very special role in health in the lives of Chinese people. According to ancient pharmacopoeia records such as "Compendium of Materia Medica", "Compendium of Materia Medica" and other ancient pharmacopoeias, Apocynum tea has the functions of calming palpitations, stopping dizziness, eliminating phlegm and relieving cough, and strengthening the heart and diuresis. After brewing with boiling water, the color is green and light, and the taste is bitter and astringent. Sour, can inhibit the increase of blood pressure, and lower the blood pressure of hypertensive patients, but the content of tannins in Apocynum tea is too much, which will easily lead to the aggravation of bitterness and astringency of tea and affect the flavor.

Tannin is a phenolic compound ubiquitous in plants. It is a secondary metabolite formed by plants in the process of survival and adaptation to resist microorganisms such as viruses and fungi or environmental stress. They are widely accumulated in tea, fruits, In various plant foods such as vegetables, it is the main compound that determines the astringency. For a long time, the key genes that control the synthesis and hydrolysis pathways of plant tannins have not been clear, and have become a research hotspot of great concern in the plant community at home and abroad. At present, there is no report on tannase (Tannse, EC3.1.1.20, TA) and its gene and application in Apocynum apocynum.

Contents of the invention

In view of this, the present invention provides an engineering strain of apocynum tannase whole-cell catalyst and its preparation method and application. The whole-cell catalyst engineering strain can catalyze the hydrolysis of tannin substrates such as epicatechin gallic acid, methyl gallate, gallic acid acyl glucose, propyl gallate, etc., and improve the flavor of apocynum tea.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides an apocynum tannase gene, which is the nucleotide sequence shown in SEQ ID NO:1.

The present invention also provides an apocynum tannase gene, which is a nucleotide sequence with more than 80% homology with the nucleotide sequence shown in SEQ ID NO:1.

The present invention also provides an apocynum tannase, which has the amino acid sequence shown in SEQ ID NO:2.

The present invention also provides a recombinant vector, which comprises the above-mentioned apocynum tannase gene and a shuttle vector.

In the present invention, the shuttle vector is pYD1.

The present invention also provides an engineering strain of the whole-cell catalyst of apocynum tannase, which comprises the above-mentioned recombinant vector and the basic strain.

In the present invention, the basic strain is Saccharomyces cerevisiae.

The present invention also provides the preparation method of this apocynum tannase whole-cell catalyst engineering strain, comprising the following steps:

Step 1, synthesizing the nucleotide sequence shown in SEQ ID NO: 1;

Step 2, inserting the nucleotide sequence shown in SEQ ID NO: 1 into the shuttle vector to obtain the recombinant vector;

Step 3, transfer the recombinant vector into the basic strain to obtain the recombinant strain;

Step 4. Induce the expression of the recombinant strain to obtain the whole-cell catalytic engineering strain of apocynum tannase anchored on the cell wall surface as shown in SEQ ID NO:2.

The invention also provides the application of the apocynum tannase whole-cell catalyst engineering strain in preparing apocynum tea.

The invention also provides an apocynum tea, the raw material of which comprises apocynum leaves and an engineering strain of apocynum tannase whole-cell catalyst.

The invention provides an engineering strain of apocynum tannase whole-cell catalyst and its preparation method and application. The apocynum tannase whole-cell catalyst engineering strain comprises the apocynum tannase gene shown in SEQ ID NO:1, and can express the apocynum tannase shown in SEQ ID NO:2. Beneficial effects of the present invention:

The recombinant apocynum tannase whole-cell catalyst engineering strain of the invention can improve the flavor of apocynum tea. The whole-cell catalyst engineering strain of apocynum tannase acts on apocynum tea soup, which can reduce the content of ester catechins and eliminate part of the bitter taste; increase the solubility of iron, calcium, magnesium and zinc plasma, as well as the solid content and Extract yield.

The optimal reaction temperature of the whole-cell catalyst of Apocynum tanninase is 80°C, and the stability of the enzyme activity is higher when the temperature is below 80°C, showing a relative remaining enzyme activity of more than 85%. By anchoring the tannase derived from the Apocynum plant on the surface of the cell wall of Saccharomyces cerevisiae, the whole-cell catalyst strain formed has high catalytic activity towards epicatechin gallate, and its heat resistance is significantly higher than that of other reported plants or Tannase of fungal origin.

In addition, the tannase derived from the apocynum plant is anchored on the surface of the cell wall of Saccharomyces cerevisiae, and after the enzymatic reaction, 60-70% of the whole-cell catalyst strain can be recovered by low-speed centrifugation at 4000 rpm, realizing the solidification and recycling of the enzyme .

Description of drawings

Fig. 1 is a kind of apocynum tannase whole cell catalyst engineering strain substrate activity analysis of embodiment 2;

Figure 2 and Figure 3 are the enzyme activity stability analysis of a kind of Apocynum tannase whole cell catalyst engineering strain in Example 3.

Detailed ways

The invention discloses an engineering strain of apocynum tannase whole-cell catalyst and its preparation method and application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

The method for preparing Apocynum tanninase of the present invention comprises the following steps:

Step 1, synthesizing the nucleotide sequence shown in SEQ ID NO: 1 in the sequence table;

Step 2. According to the nucleotide sequence of step 1, construct the pYD1-avta shuttle vector and transform Escherichia coli, after verification, obtain a large number of recombinant pYD1-avta vectors through Escherichia coli amplification and plasmid extraction;

Step 3, transforming the pYD1-avta vector obtained in step 2 into the Saccharomyces cerevisiae EBY100 strain, and inducing expression to obtain the Saccharomyces cerevisiae whole-cell catalytic engineering of the amino acid sequence anchored on the cell wall surface as shown in SEQ ID NO: 2 strain.

Step 4. Induce and express pYD1-avta/S.cerevisiae EBY100 whole-cell catalyst engineering strain, respectively use epicatechin gallate, methyl gallate, gallate acyl glucose, propyl gallate, etc. as substrates, and carry out Its substrate characteristics and enzymatic properties detection.

The concrete method of described step 1 comprises the following steps:

1) total RNA is extracted from the Apocynum leaf sample, and the first strand of cDNA (complementary deoxyribonucleic acid) is synthesized with reverse transcriptase;

2) using cDNA as a template, using upstream primer 1 and downstream primer 2 to perform PCR (polymerase chain reaction) amplification;

Upstream primer 1: 5'G GAATTC ATGGGTTGCCCTTGTGGAAAC3'

Downstream primer 2: 5'C AAGCTT TTGATTGCAAGAGCAGCTTGAG3'

The PCR reaction system is: 2 μL cDNA template, 25 μL 2×PrimerSTAR Max DNA Polymerase, 1 μL upstream primer 1, 1 μL downstream primer 2, supplemented with ddH ₂ O (double distilled water) until the total reaction system is 50 μL; the reaction conditions for PCR are: ①94°C, 5min; ②94°C, 15s; ③55°C, 15s; ④72°C, 30s; ⑤Repeat steps ②-④25 cycles; ⑥72°C, 10min;

3) The PCR product is sequenced after purification and recovery, and the base sequence shown in SEQ ID NO: 1 in the sequence table is obtained.

In the second step, the vector backbone of the recombinant expression vector is the pYD1 shuttle expression vector; the shuttle strain is Escherichia coli Top10; the whole-cell catalyst engineering strain is Saccharomyces cerevisiae (EBY100).

The concrete method of described step 2 comprises the following steps:

1) Carry out double digestion of the obtained nucleotide sequence and the pYD1 shuttle expression vector, and the double digestion products are purified and recovered, and then ligated with T4 DNA ligase to obtain the recombinant vector ligation product;

2) Mix the ligation product of the recombinant expression vector with Escherichia coli competent cells (E.coli DH5α), place it in an ice bath for 20 minutes, put it in a water bath at 42°C for 90 seconds, take it out and put it in an ice bath for 5 minutes again; then add it to LB ( Luria-Bertani medium) liquid medium, and at 37°C, under the condition of 150rpm, shake culture for 1h; take the bacterial liquid in LB solid medium containing 50mg/L kanamycin, spread evenly, and cultivate overnight at 37°C , to obtain clonal colonies;

3) Pick a monoclonal colony and inoculate it in LB culture medium containing kanamycin, shake overnight at 37°C and 250rpm, take the bacterial solution for preliminary identification by colony PCR, and extract the plasmid (pYD1-avta ) After further enzyme digestion (EcoRI/HindIII) identification is correct, send the sample for sequencing, and obtain the recombinant engineering strain with correct sequencing;

4) After the recombinant engineering strains with correct sequencing were selected and multiplied, a large number of pYD1-avta recombinant vectors were extracted.

The concrete method of described step 3 comprises the following steps:

1) After mixing about 2 μg of the pYD1-avta recombinant vector with 50 μL of Saccharomyces cerevisiae EBY100 competent cells, conduct electric shock transformation under the conditions of 1.5kV, 25μF, and 200Ω;

2) The above transformation solution was inoculated into 10 mL of SD-CAA medium and cultured overnight at 30°C and 250 rpm until the OD600 value of the bacterial solution concentration reached 2-5. Centrifuge at room temperature at 5,000rpm for 5min to collect the bacterial cells, transfer them to 20-40mL SG-CAA induction medium, induce culture at 20°C-25°C, 250rpm for 48h, and then take the induced bacterial cells for colony PCR sequencing, immunization Fluorescence and western-blotting verification. The whole cell catalyst engineering strain of Saccharomyces cerevisiae whose cell wall surface anchored amino acid sequence as shown in SEQ ID NO: 2 was obtained.

The concrete method of described step 4 comprises the following steps:

Take 1 μg pYD1-avta/S.cerevisiae EBY100 whole-cell catalyst engineering strain, add vitamin C solution with a final concentration of 0.2mM, epicatechin gallate, gallic acid acylglucose, methyl gallate, and a final concentration of 300μM, respectively. Substrate solution such as propyl gallate and phosphate buffer solution with pH 7.4, until the total reaction system is 50 μL; after 10 minutes of reaction at 35°C, 50 μL of methanol reaction termination solution was added; after centrifugation at 12000g at 4°C for 15 minutes, the upper The clear liquid was analyzed by liquid chromatography to detect the types and contents of substrates and corresponding products in the solution after the reaction.

Using the specific tannin with the highest catalytic activity as the substrate, the optimal reaction temperature of the recombinant yeast whole cells was determined in the temperature range of 10-90 °C under the optimal pH value; similarly, the recombinant yeast whole cells were treated at different temperatures After 1 hour of treatment under the same conditions, the relative remaining enzyme activity and temperature stability after treatment at different temperatures were measured; using buffer solutions with different pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0) The relative enzyme activity of recombinant yeast whole cells under different pH conditions; after the recombinant yeast whole cells were placed at 4°C for 1 hour under different pH conditions, the remaining relative enzyme activity and pH stability were measured.

All reagents and instruments used in the present invention can be purchased from the market.

Below in conjunction with embodiment, further set forth the present invention:

Example 1

The apocynum tannase gene is the nucleotide sequence shown in SEQ ID NO: 1 in the sequence table; the apocynum tannase gene is the amino acid sequence shown in SEQ ID NO: 2 in the sequence table.

1. Synthesis of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing

(1) Extraction of total RNA from Apocynum leaf sample

TotalRNA Kit II(50)的说明书进行并略做修改。对于所用到的塑料器皿均用DEPC(焦碳酸二乙酯)水处理；玻璃器皿在180℃烘烤6h；其他所有的试剂也均用DEPC处理水配制，121℃高温灭菌20min。The total RNA of Apocynum leaf samples was extracted according to the RNA extraction kit of Promega Company

The instructions for the TotalRNA Kit II (50) were carried out with minor modifications. The plastic utensils used were treated with DEPC (diethylpyrocarbonate) water; the glass utensils were baked at 180°C for 6h; all other reagents were also prepared with DEPC-treated water, and sterilized at 121°C for 20min.

①Take about 100 mg of Apocynum leaf sample, wash it with DEPC treated water, put it into a mortar pre-cooled with liquid nitrogen, and immediately add liquid nitrogen to grind it into powder;

Reagent提取液继续研磨匀浆，并将匀浆转入经DEPC水处理过的1.5mL离心管中，加入0.2mL的氯仿，剧烈摇动离心管15s，静置5min；② Immediately add 1mL of liquid nitrogen before the liquid nitrogen evaporates

Continue grinding and homogenizing the Reagent extract, transfer the homogenate into a 1.5mL centrifuge tube treated with DEPC water, add 0.2mL of chloroform, shake the centrifuge tube vigorously for 15 seconds, and let stand for 5 minutes;

③Centrifuge at 12000×g for 15 min at 4°C, the solution is divided into three layers, the lower layer is a phenol-chloroform liquid phase, the middle layer is a white film, and the upper layer is an aqueous phase. Transfer the upper aqueous phase into another DEPC-treated 1.5mL centrifuge tube, add 1/3 volume of absolute ethanol and vortex quickly for 15s;

④ Take 700 μL of the mixed solution of the upper aqueous phase in step ③ and add it to the collection column, centrifuge at 12000×g for 30-60 s at room temperature, and discard the effluent;

⑤ Add 700 μL RNA Wash Buffer, centrifuge at 12000×g for 1 min at room temperature, discard the effluent, and repeat this washing step once;

⑥Put a 1.5mL centrifuge tube treated with DEPC water on the collection column, add 30-50μL of 70°C preheated DEPC water, incubate at room temperature for 5 minutes, and centrifuge at room temperature ≥13000×g for 2 minutes. The obtained RNA solution is tested and placed in Store in -70°C refrigerator for later use. The results of denatured agarose gel electrophoresis of total RNA are shown in Figure 1. Clear characteristic band patterns of 28S, 18S and 5.8S can be seen, indicating that the integrity of the RNA is good and no significant degradation has occurred.

(2) cDNA synthesis of Apocynum leaf sample by reverse transcription

The first strand of cDNA was synthesized according to the instructions of Reverse Transcritase M-MLV, a reverse transcription kit from TAKARA Company. All reagents used were prepared with DEPC-treated water, and all experimental operations were carried out in a clean bench.

First, add: total 1 μL RNA (100 μg/mL), 1 μL Oligo(dT)12-18 (50 μM), 5 μL RNase free ddH ₂ O to a 0.2 mL centrifuge tube treated with DEPC water, mix well and centrifuge briefly, After incubating at 70°C for 5 minutes, place it on ice for more than 10 minutes, centrifuge briefly for a few seconds so that all the mixed solution is collected at the bottom of the centrifuge tube, and add 2 μL 5×M-MLV Buffer, 0.5 μL dNTP Mix (10Mm), 0.25 μL RNase Inhibitor (40U/μL), 0.25μL RTaseM-MLV (RNase H-) (200U/μL). After mixing, incubate at 42°C for 60 minutes, briefly centrifuge, place at 70°C for 15 minutes, and then cool on ice. The obtained cDNA solution is used for PCR amplification.

(3) PCR reaction amplification apocynum tannase gene

Using the above cDNA as a template, primers were designed according to the unigene sequence of the transcriptome sequencing of Apocynum leaf samples, and EcoRI and HindIII restriction sites were introduced into the primers to amplify the cDNA sequence of Apocynum tannase gene.

Upstream primer 1: 5'G GAATTC ATGGGTTGCCCTTGTGGAAAC 3'

Downstream primer 2: 5'C AAGCTT TTGATTGCAAGAGCAGCTTGAG3'

PCR reaction system: 2 μL cDNA template, 25 μL 2×PrimerSTAR Max DNA Polymerase, 1 μL primer 1, 1 μL primer 2, supplemented with ddH ₂ O to a total reaction system of 50 μL. Reaction program: ①94°C, 5min; ②94°C, 15s; ③55°C, 15s; ④72°C, 30s; ⑤Repeat steps ②-④25 cycles; ⑥72°C, 10min;

Take 5 μL of PCR amplification product and add 6× loading buffer for agarose gel electrophoresis detection. The PCR products were sent to a sequencing company for sequencing.

The total length of the Apocynum tanninase gene cDNA obtained in this embodiment is 1026bp, the start codon is ATG, the stop codon is TGA, the sequence has no introns, has a complete open reading frame, and encodes 341 amino acids; The nucleotide sequence is as follows:

ATGGGGAAACGTCAGAAGTTTCCTGGTGTCAATGAAGAACTGCAGAAGATAATTGATGGAAATATGGATGAAGCGGGGGCAAGGAGGCGTGCTCGTGAGGCATTTAAGGATATTCAGCTTTCAATTGATCATGTCTTGTTCAAGATGCCATGCAAGGGTTTGAAGATGAAGGAGTCATATGAAGTGAACTCTAAAGGATTGGAAATTTTCAC GAAAAGTTGGCTTCCGGAGGCCAGTTTCTCCAAAAGCAGTGGTTTTTTTTGTCATGGATATGGAGACACTTGCACCTTTTTCTTTGAAGGAATTGCTCGGAAGTTGGCAACTGCTGGTTATGGAGTATTTGCTATGGATTATCCGGGATTTGGTCTTTCTGACGGTCTTCATGCCTATATTCCAAACTTTGATGCACTGGTAGATGCTGTGATTGAGC ATTATTCAAAAAGTAAAAGACAATCCAGATTTCAGTTCTCTGCCAAGTTTCCTGTTCGGAGAATCTATGGGTGGAGCAATAGCTCTGAAGGTGCACCTGAAACAACCTGACTCATGGACTGGAGCTATTCTTGTTGCCCCTATGTGTAAAATTGCAGATGACATGGTTCCACCATGGGTGGTGACACAGTTTCTTATTGGTGTGGCAAAAGTTCTTCCAAGACATAAGT TAGTTCCACAAAGGATTTAGCTGACTTGGCATTCAGAGATGAGAAGAAGAAAAAATTGGCAAAATACAATGTAATTGCTTATAAGCATAAACCACGTTTACGAACAGCTGTGGAGATGCTGAACACCCACCAGGAGATAGAGCAAACATTGGAAAAAGTATCTTTGCCATTGTTGATCCTTCATGGGAAGGCTGATGTAATTACTGATCCATCTGTAAGTAAGG CCTTATATGAGAAAGCAAGCAGTACAGACAAAGAAACTTATTCTCTATGATGATGCTTATCATTCTCTTGAGGGTGAGCCAGATGAGATGATTCTCAAAGTGTTTGGAGACATAATTTCTTGGTTAGATGCTCATAGTTGA

The specific amino acid sequence is as follows:

MGKRQKFPGVNEELQKIIDGNMDEAGARRRAREAFKDIQLSIDHVLFKMPCKGLKMKESYEVNSKGLEIFTKSWLPEASSPKAVVFFCHGYGDTCTFFFEGIARKLATAGYGVFAMDYPGFGLSDGLHAYIPNFDAVDAVIEHYSKVKDNDFSSLPSFLFGESMGGAIALKVHLKQPDSWTGA ILVAPMCKIADDMVPPWVVTQFLIGVAKVLPRHKLVPQKDLADLAFRDEKKKKLAKYNVIAYKHKPRLRTAVEMLNTTQEIEQTLEKVSLPLLILHGKADVITDPSVSKALYEKASSTDKKLILYDDAYHSLLEGEPDEMILKVFGDIISWLDAHS

2. Construction of pYD1-avta shuttle carrier and whole-cell catalyst engineering bacteria

After purification and recovery of the PCR amplification product, it was subjected to double enzyme digestion with the pYD1 shuttle vector. After the double enzyme digestion product was purified and recovered, it was ligated with T4 DNA ligase overnight at 16°C to obtain the recombinant expression vector ligation product. The ligation system is: 2 μL of product recovered from double digestion of Apocynum tannase gene, 6 μL of product recovered from double digestion of pET-30a, 1 μL of 10×T4 DNA ligase buffer, and 1 μL of T4 DNA ligase.

Mix the constructed shuttle expression vector ligation product with 50 μL of Escherichia coli competent cells (E.coli DH5α), ice-bath for 30 minutes, then water-bath at 42°C for 90 seconds, take it out quickly, and ice-bath again for 5 minutes; then add 600 μL LB liquid culture medium, 37°C, 150rpm shaking culture for 1h; take 200μL of bacterial liquid on LB solid medium (containing kanamycin 50mg/L) and spread evenly, and culture overnight at 37°C to obtain clone colonies.

Pick a monoclonal colony and inoculate it in 2 mL of LB (containing kanamycin) culture solution at 37°C and 250 rpm for overnight shaking, take 1 μL of the bacterial solution for preliminary identification by colony PCR, extract the plasmid (pYD1-avta) from the positive bacterial solution, and further After enzyme digestion (EcoRI/HindIII) was identified correctly, the sample was sent for sequencing. The engineered strains with correct sequencing were used for induction expression experiments or added glycerol to a final concentration of 10%, and stored at -70°C.

A large number of pYD1-avta plasmid vectors were extracted, and about 2 μg of pYD-avta recombinant vector was mixed with 50 μL of Saccharomyces cerevisiae EBY100 competent cells, and electric shock transformation was carried out under the conditions of 1.5kV, 25μF, 200Ω; then the above transformation solution was inoculated into Cultivate overnight in 10mL SD-CAA medium at 30°C and 250rpm until the OD600 value of the bacterial solution reaches 2-5. Centrifuge at room temperature at 5,000rpm for 5min to collect the bacterial cells, transfer them to 20-40mL SG-CAA induction medium, induce culture at 20°C-25°C, 250rpm for 48h, and then take the induced bacterial cells for colony PCR sequencing, immunization Fluorescence and western-blotting verification. The whole-cell tannin catalytic engineering strain of Saccharomyces cerevisiae whose cell wall surface-anchored amino acid sequence is shown in SEQ ID NO: 2 is obtained.

Embodiment 2 A kind of Apocynum tannase whole cell catalyst engineering strain substrate activity analysis

Take 1 μg pYD1-avta/S.cerevisiae EBY100 whole-cell catalyst engineering strain, add vitamin C solution with a final concentration of 0.2mM, epicatechin gallate, gallic acid acylglucose, and methyl gallate with a final concentration of 300μM , propyl gallate and other substrate solutions, and pH 7.4 phosphate buffer solution, until the total reaction system is 50 μL; 35 ° C, after 10 minutes of reaction, add 50 μ L of methanol reaction termination solution; The supernatant was analyzed by liquid chromatography to detect the types and contents of substrates and corresponding products in the solution after the reaction. The experimental results are shown in Figure 1. The whole-cell catalyst engineered bacteria have catalytic activity on tannin substrates such as epicatechin gallate, acyl gallate, methyl gallate, and propyl gallate. The corresponding enzyme kinetic parameters (Kcat/Km(S ^-1 M ^-1 )) are: epicatechin gallate 77.86±1.12; galloacylglucose 3.21±0.24; Propyl acid 10.05±1.23. Among them, the catalytic effect of the whole-cell catalyst engineering bacteria on catechin gallate-like tannin substrates is significantly higher than that of other tannin-like substrates.

Embodiment 3 A kind of Apocynum tanninase whole cell catalyst engineering strain enzyme activity stability analysis

Apocynum tannase whole cell catalyst engineering strain is placed in the buffer solution of different pH (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0), according to the method in embodiment 2 4 ℃ The relative enzyme activity and pH stability of the recombinant yeast whole cells were measured under different pH conditions after standing for 1 hour. As can be seen from Figure 2, the most suitable reaction pH value of this apocynum tannase whole-cell catalyst engineering strain is 6.0, and the enzyme activity stability between pH 2～7 is relatively high, showing a relative residual of more than 80%. Enzyme activity.

Under optimum pH value condition, in the temperature scope of 20～90 ℃, measure the optimum reaction temperature of Apocynum tannase whole cell catalyst engineering bacterial strain; Its optimum reaction temperature 80 ℃ as shown in Figure 3, at 80 ℃ The stability of the enzyme activity is higher when the enzyme activity is lower than 85% relative remaining enzyme activity.

Embodiment 4 A kind of Apocynum tanninase whole cell catalyst engineering strain improves the flavor of Apocynum tea flavor

Add 0.1% (g/ml) of Apocynum tanninase whole cell catalyst engineering bacterial strain to 10% (g/ml) of Apocynum tea soup, after 20 minutes, the ester type catechin content in the tea soup tends to be stable Among them, the reduction of epicatechin gallate content is the most significant, compared with the control group without adding tannase whole-cell catalyst engineering strain, the content is reduced by 72.18%, and the tea soup is bright in color, pure in fragrance, mellow in taste, slightly bitter taste. In this embodiment, the addition amount of the Apocynum tannase whole-cell catalyst engineered strain is in the range of 0-0.1%, and the enzymolysis time is 15-20 minutes, which can achieve the best effect of tea soup with light taste and pure aroma.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Institute of Hemp, Chinese Academy of Agricultural Sciences

<120> An engineering strain of apocynum tannase whole-cell catalyst and its preparation method and application

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gtgattgagc attattcaaa agtaaaagac aatccagatt tcagttctct gccaagtttc 480

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Met Gly Lys Arg Gln Lys Phe Pro Gly Val Asn Glu Glu Leu Gln Lys

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Ile Ile Asp Gly Asn Met Asp Glu Ala Gly Ala Arg Arg Arg Ala Arg

20 25 30

Glu Ala Phe Lys Asp Ile Gln Leu Ser Ile Asp His Val Leu Phe Lys

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Met Pro Cys Lys Gly Leu Lys Met Lys Glu Ser Tyr Glu Val Asn Ser

50 55 60

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

65 70 75 80

Pro Lys Ala Val Val Phe Phe Cys His Gly Tyr Gly Asp Thr Cys Thr

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Phe Phe Phe Glu Gly Ile Ala Arg Lys Leu Ala Thr Ala Gly Tyr Gly

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Val Phe Ala Met Asp Tyr Pro Gly Phe Gly Leu Ser Asp Gly Leu His

115 120 125

Ala Tyr Ile Pro Asn Phe Asp Ala Leu Val Asp Ala Val Ile Glu His

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Tyr Ser Lys Val Lys Asp Asn Pro Asp Phe Ser Ser Leu Pro Ser Phe

145 150 155 160

Leu Phe Gly Glu Ser Met Gly Gly Ala Ile Ala Leu Lys Val His Leu

165 170 175

Lys Gln Pro Asp Ser Trp Thr Gly Ala Ile Leu Val Ala Pro Met Cys

180 185 190

Lys Ile Ala Asp Asp Met Val Pro Pro Trp Val Val Thr Gln Phe Leu

195 200 205

Ile Gly Val Ala Lys Val Leu Pro Arg His Lys Leu Val Pro Gln Lys

210 215 220

Asp Leu Ala Asp Leu Ala Phe Arg Asp Glu Lys Lys Lys Lys Lys Leu Ala

225 230 235 240

Lys Tyr Asn Val Ile Ala Tyr Lys His Lys Pro Arg Leu Arg Thr Ala

245 250 255

Val Glu Met Leu Asn Thr Thr Gln Glu Ile Glu Gln Thr Leu Glu Lys

260 265 270

Val Ser Leu Pro Leu Leu Ile Leu His Gly Lys Ala Asp Val Ile Thr

275 280 285

Asp Pro Ser Val Ser Lys Ala Leu Tyr Glu Lys Ala Ser Ser Thr Asp

290 295 300

Lys Lys Leu Ile Leu Tyr Asp Asp Ala Tyr His Ser Leu Leu Glu Gly

305 310 315 320

Glu Pro Asp Glu Met Ile Leu Lys Val Phe Gly Asp Ile Ile Ser Trp

325 330 335

Leu Asp Ala His Ser

340

Claims (9)

1. An apocynum tanninase gene, characterized in that it is the nucleotide sequence shown in SEQ ID NO:1. 2. An Apocynum tanninase, characterized in that it is the amino acid sequence shown in SEQ ID NO:2. 3. A recombinant vector, characterized in that, the recombinant vector comprises the Apocynum tanninase gene and a shuttle carrier according to claim 1. 4. The recombinant vector according to claim 3, wherein the shuttle vector is pYD1. 5. An engineering bacterial strain of apocynum tannase whole-cell catalyst, characterized in that, said engineering bacterial strain comprises the recombinant carrier and basic bacterial strain described in claim 3 or 4. 6. Apocynum tannase whole cell catalyst engineering bacterial strain according to claim 5, is characterized in that, described basic bacterial strain is Saccharomyces cerevisiae. 7. the preparation method of the described apocynum tannase whole cell catalyst engineering strain of claim 5 or 6, is characterized in that, comprises the following steps: Step 1, synthesizing the nucleotide sequence shown in SEQ ID NO: 1; Step 2, inserting the nucleotide sequence shown in SEQ ID NO: 1 into the shuttle vector to obtain the recombinant vector; Step 3, transfer the recombinant vector into the basic strain to obtain the recombinant strain; Step 4. Induce the expression of the recombinant strain to obtain the whole-cell catalytic engineering strain of Apocynum tannase anchored on the cell wall surface as shown in SEQ ID NO:2. 8. the application of claim 5 or 6 described apocynum tannase whole cell catalyst engineering bacterial strains in the preparation of apocynum tea. 9. Apocynum tea, is characterized in that, its raw material comprises apocynum leaf and claim 5 or 6 described apocynum tannase whole-cell catalyst engineering bacterial strains.

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