CN114645039B

CN114645039B - Mutant salicylic acid decarboxylase, strain and application thereof in degradation of ginkgolic acid

Info

Publication number: CN114645039B
Application number: CN202210444588.4A
Authority: CN
Inventors: 戴玉杰; 宋园园; 胡彦营; 贺傲; 孙国娟; 李嘉欣; 孟璇; 景文杰; 贾士儒
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2022-04-26
Filing date: 2022-04-26
Publication date: 2024-02-23
Anticipated expiration: 2042-04-26
Also published as: CN114645039A

Abstract

The invention discloses a mutant salicylic acid decarboxylase for efficiently degrading ginkgolic acid, which is mutant enzyme Sdc-P191A and/or mutant enzyme Sdc-Y64; the amino acid sequence of the mutant enzyme Sdc-P191A is shown as SEQ ID NO.2, wherein the 191 th amino acid of the salicylic acid decarboxylase Sdc with the amino acid sequence of SEQ ID NO.1 is changed from proline to alanine; the 64 th amino acid of the salicylic acid decarboxylase Sdc with the amino acid sequence of SEQ ID NO.1 of the mutant enzyme Sdc-Y64T is changed from tyrosine to threonine, and the amino acid sequence of the mutant enzyme Sdc-Y64T is shown as SEQ ID NO. 3. According to the invention, partial amino acids are selected for site-directed mutagenesis, and two mutant salicylic acid decarboxylases are successfully constructed, so that two mutant enzymes which are compared with wild sdc and can efficiently degrade ginkgolic acid are obtained.

Description

Mutant salicylic acid decarboxylase, strain and application thereof in degradation of ginkgolic acid

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to application of mutant salicylic acid decarboxylase Sdc-P191A and Sdc-Y64T in degradation of ginkgolic acid.

Background

Semen Ginkgo is a Chinese medicine containing various active ingredients, and is commonly used for relieving cough, moistening lung, eliminating phlegm, treating leukorrhagia, reducing urination, etc., and also used for treating pulmonary tuberculosis, etc.; the health-care food is also a health-care food, can preserve health, is popular as a royal tribute, and is popular with people.

In modern researches, the gingko contains more linoleic acid, has certain improvement effect on hyperlipidemia and hypertension, can prevent cardiovascular diseases, and can poison the culture of cancer cells to a certain extent; the vitamin E in the gingko has the effects of strengthening the oxidation resistance of a human body and scavenging free radicals, protecting cell membranes and prolonging the survival time of red blood cells; the research proves that the beta-carotene in the gingko has extremely strong oxidation resistance to lipid, and can also play a role in preventing cancers, cardiovascular diseases and cataract; the presence of the ginkgo protein, however, gives the ginkgo the following characteristics: (1) antioxidant and antiaging; (2) increasing serum hemolysin in vivo; (3) Improving the function of resisting radiation injury, hematopoietic function and immunity function in vivo of human body; (4) Has stronger scavenging function to the in vitro active oxygen such as hydroxyl free radical, superoxide anion and the like; (5) has a certain protection effect on DNA damage. Although the ginkgo has strong medical and edible value, and researches are continuously carried out by researchers, the utilization rate of the ginkgo is still low, because the toxic component, ginkgolic acid, is not well treated, and thus, the development and the utilization of the ginkgo are severely limited. Therefore, the removal of the toxic substance ginkgolic acid from ginkgo becomes a big hot spot in the research today.

There are many reports on the extraction and measurement method of ginkgolic acid, decarboxylation and degradation of ginkgolic acid are one of important means, and biological research methods show that salicylic acid decarboxylase (Sdc) from candida has a decarboxylation and degradation function on ginkgolic acid, and can decarboxylate ginkgolic acid into a nontoxic product, but previous researches show that the wild type salicylic acid decarboxylase has low degradation performance on ginkgolic acid. Therefore, partial mutation sites are selected, and the salicylic acid decarboxylase is directionally modified by utilizing a site-directed mutagenesis technology, so that a novel strain capable of efficiently degrading ginkgolic acid is researched. The method has important significance for development and utilization of special gingko resources in China, safe production of gingko products, reduction of production cost and the like.

By searching, the following patent publications related to the present patent application are found:

1. the application of salicylic acid decarboxylase to degradation of ginkgolic acid (CN 104770624A) is disclosed in the sequence 1, the enzyme is salicylic acid decarboxylase (Sdc), and the cell disruption liquid containing pET21a (+) -Sdc/E.coli-BL21 (DE 3) of the Sdc can obviously reduce the content of ginkgolic acid.

By contrast, the above publication discloses degradation of ginkgolic acid by wild type Sdc, but the degradation capability is relatively low, and the invention selects partial loci to carry out mutation transformation on the wild type Sdc based on PCR technology, so as to obtain two mutant strains with high degradation capability of ginkgolic acid compared with the wild type Sdc. Thus, the present patent application is substantially different from the above patent publications.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an application of mutant salicylic acid decarboxylase Sdc-P191A and Sdc-Y64T in degradation of ginkgolic acid.

The technical scheme adopted for solving the technical problems is as follows:

a mutant salicylic acid decarboxylase for efficiently degrading ginkgolic acid, wherein the mutant salicylic acid decarboxylase is mutant enzyme Sdc-P191A and/or mutant enzyme Sdc-Y64;

the amino acid sequence of the mutant enzyme Sdc-P191A is shown as SEQ ID NO.2, and the 191 th amino acid of the salicylic acid decarboxylase Sdc with the amino acid sequence of SEQ ID NO.1 is changed from proline to alanine; the 64 th amino acid of the salicylic acid decarboxylase Sdc with the amino acid sequence of SEQ ID NO.1 of the mutant enzyme Sdc-Y64T is changed from tyrosine to threonine, and the amino acid sequence of the mutant enzyme Sdc-Y64T is shown as SEQ ID NO. 3.

Further, the optimum pH of the mutant enzyme Sdc-P191A is 5.5, the optimum temperature is 50 ℃, the mutant enzyme Sdc-P191A still has the activity of degrading ginkgolic acid after 4 hours, the temperature stability of the mutant enzyme Sdc-P191A is slightly improved compared with that of a wild type Sdc, and the degradation capacity of ginkgolic acid of the mutant enzyme Sdc-P191A is improved by 116.74 percent compared with that of the non-mutant wild type Sdc.

Further, the optimum action pH of the mutant enzyme Sdc-Y64T is 5.5, the optimum action temperature is 40 ℃, the optimum action temperature is reduced by 10 ℃ compared with the optimum action temperature of the wild type Sdc, and the strong ginkgolic acid degradation capacity can be maintained within 1-4h at 40 ℃; and the degradation capacity of ginkgolic acid is improved by 105.18% compared with that of the non-mutated wild type sdc.

The nucleotide sequence of the mutant type salicylic acid decarboxylase Sdc-P191A gene is shown as SEQ ID NO.4, and the nucleotide sequence of the mutant type salicylic acid decarboxylase Sdc-Y64T gene is shown as SEQ ID NO. 5.

A recombinant plasmid for expressing a mutant salicylic acid decarboxylase as described above in a host cell, said recombinant plasmid being a vector suitable for expression in e.

Further, the recombinant plasmid is a gene containing SEQ ID NO.4 and/or SEQ ID NO. 5.

A mutant engineering strain comprising a recombinant plasmid as described above.

Furthermore, the mutant engineering strain is a recombinant strain obtained by taking escherichia coli E.coli BL21 (DE 3) as a host strain and transforming the corresponding recombinant plasmid into the host strain.

The application of the mutant salicylic acid decarboxylase in efficiently degrading ginkgolic acid.

Further, the ginkgolic acid is a 2-hydroxy-6-alkyl benzoic acid compound, and concretely, ginkgolic acid R=C13:0, ginkgolic acid R=C15:0, ginkgolic acid R=C15:1, ginkgolic acid R=C17:1 and ginkgolic acid R=C17:2.

The beneficial effects obtained by the invention are as follows:

1. according to the invention, partial amino acids are selected for site-directed mutagenesis, and two mutant salicylic acid decarboxylases Sdc-P191A and Sdc-Y64T are successfully constructed, so that two mutant enzymes Sdc-P191A and Sdc-Y64T which are compared with wild type Sdc and can efficiently degrade ginkgolic acid are obtained.

The mutation enzyme Sdc-P191A is successfully constructed by changing the proline at 191 th position into alanine, the optimal action pH value of the mutation enzyme is 5.5, the optimal action temperature is 50 ℃, the mutation enzyme still has a certain activity of degrading ginkgolic acid after 4 hours, the temperature stability of the mutation enzyme Sdc-P191A is slightly improved compared with that of a wild type Sdc, and the degradation capacity of ginkgolic acid is improved by 116.74 percent compared with that of the non-mutated wild type Sdc. The mutant enzyme has improved degradation capability to ginkgolic acid.

The 64-bit tyrosine is changed into threonine, so that the mutant enzyme Sdc-Y64T is successfully constructed, the optimal action pH of the mutant enzyme is 5.5, the optimal action temperature is 40 ℃, the optimal action temperature is reduced by 10 ℃ compared with the optimal action temperature of the wild type Sdc, and the strong capacity of degrading ginkgolic acid can be maintained for a long time at 40 ℃. And the degradation capacity of ginkgolic acid is improved by 105.18% compared with that of the non-mutated wild type sdc. The degradation capability of the enzyme to ginkgolic acid is proved to be improved.

Drawings

FIG. 1 is a diagram of a site-directed mutagenesis route in accordance with the present invention;

FIG. 2 is a sequence alignment of the gene sdc and the mutant gene of the present invention;

FIG. 3 is a diagram showing SDS-PAGE results of mutant enzymes of the present invention; wherein M is a protein molecular weight standard, control is crude enzyme after induction of a control strain, P-Sdc is crude enzyme after induction of a wild strain, P-Y64T is crude enzyme after induction of a mutant strain Y64T, and P-P191A is crude enzyme after induction of a mutant strain P191A;

FIG. 4 is a graph showing the decarboxylation performance of wild-type and mutant Sdc versus ginkgolic acid in the present invention;

FIG. 5 is a graph showing decarboxylation performance of the Sdc and its mutant enzymes at various temperatures in the present invention;

FIG. 6 is a graph showing decarboxylation performance of the Sdc and its mutant enzymes at various pH values in the present invention;

FIG. 7 is a graph showing the effect of temperature on the stability of Sdc and its mutant enzymes in the present invention; wherein A:40 ℃; b:50 ℃;

FIG. 8 is a PCR identification chart of a mutant plasmid of the present invention;

FIG. 9 is a diagram showing the identification of a mutant plasmid of the present invention by double cleavage.

Detailed Description

The present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.

The raw materials used in the invention are conventional commercial products unless otherwise specified, the methods used in the invention are conventional methods in the art unless otherwise specified, and the mass of each substance used in the invention is conventional.

Preferably, the mutant enzyme Sdc-P191A has an optimum action pH of 5.5, an optimum action temperature of 50 ℃ and an activity of degrading ginkgolic acid after 4 hours, the temperature stability of the mutant enzyme Sdc-P191A is slightly improved compared with that of a wild type Sdc, and the degradation capacity of ginkgolic acid is improved by 116.74% compared with that of an unmutated wild type Sdc.

Preferably, the optimum action pH of the mutant enzyme Sdc-Y64T is 5.5, the optimum action temperature is 40 ℃, the optimum action temperature is reduced by 10 ℃ compared with the optimum action temperature of the wild type Sdc, and the strong ginkgolic acid degradation capacity can be maintained within 1-4h at 40 ℃; and the degradation capacity of ginkgolic acid is improved by 105.18% compared with that of the non-mutated wild type sdc.

Preferably, the recombinant plasmid is a gene containing SEQ ID NO.4 and/or SEQ ID NO. 5.

Preferably, the mutant engineering strain is a recombinant strain obtained by taking escherichia coli E.coli BL21 (DE 3) as a host strain and transforming a corresponding recombinant plasmid into the host strain.

Preferably, the ginkgolic acid is a 2-hydroxy-6-alkyl benzoic acid compound, and specifically, ginkgolic acid R=C13:0, ginkgolic acid R=C15:0, ginkgolic acid R=C15:1, ginkgolic acid R=C17:1) and ginkgolic acid R=C17:2.

Specifically, the preparation and detection of the correlation are as follows:

the invention extracts the sdc gene from candida, adopts an inverse PCR transformation method based on PCR, takes plasmids pET21a-sdc as templates, designs a pair of partial base reverse complementary primers, completes the mutation process through one PCR reaction, digests a small amount of residual templates by using Dpn I, then carries out recombination cyclization, and finally verifies the mutant plasmids through transformation and screening. Obtaining mutant strains with complete mutation. The mutation scheme is shown in FIG. 1.

Then pET21a was used as an expression vector for the mutant sdc gene, E.coil-BL21 (DE 3) as a host cell. Each pET21 a-pdc mutant plasmid was constructed, expressed in e.coll BL21 (DE 3), and protein expression and enzyme activity measurement were performed using the unmutated pET21 a-pdc expression protein as a control, and the activity of the site-directed mutant pdc enzyme and the change in enzymatic properties were studied.

Wherein, the ginkgolic acid refers to a 2-hydroxy-6-alkyl benzoic acid compound and comprises ginkgolic acid (R=C13:0), ginkgolic acid (R=C15:0), ginkgolic acid (R=C15:1) ginkgolic acid (R=C17:1) ginkgolic acid (R=C17:2) and the like.

The specific operation mode and the steps are as follows:

1. determining mutation sites: the present invention determines 32 mutation sites as shown in FIG. 4.

2. Construction of mutant plasmids: the invention constructs site-directed mutagenesis plasmid according to the site-directed mutagenesis route diagram of figure 1, which mainly comprises three steps.

(1) Inverse PCR amplification Using the plasmid pET21a (+) -pdc constructed in the laboratory as a template (referred to simply as pET21 a-pdc in the present invention), inverse PCR was performed using the mutation primers shown in Table 1 (only the construction primers of the strains finally determined in the present invention are shown in the table), and after the completion of the reaction, 1% agarose gel electrophoresis was performed on 2. Mu.L of the amplified product, and the results are shown in FIG. 8. And verifying the result to be correct, and carrying out the next step.

(2) Template plasmid removal

After the end of the PCR amplification reaction, in order to prevent a small amount of the remaining template plasmid from affecting the later screening, it is necessary to remove it by digestion with the restriction enzyme DpnI before proceeding to the next step, so as not to form a false positive transformant. The PCR amplification products were mixed uniformly by preparing a reaction system according to Table 2, and digested at 37℃for 10min.

(3) Recombination reactions

The PCR-mediated site-directed mutagenesis product is linear, and can be used for screening mutants after cyclization by T4 ligase, the reaction system is shown in Table 3, after the components are uniformly mixed, the mixture is placed in a metal bath at 22 ℃ for incubation for 1h, the solution on the pipe wall is collected at the bottom of the pipe in a transient centrifugation way, the T4 ligase is inactivated after incubation for 5min at 70 ℃, and the final product can be directly used for conversion or stored at-20 ℃ for standby.

In order to improve the success rate of mutation reaction, the use amount of template plasmid is reduced as much as possible on the premise of ensuring that the plasmid can be amplified correctly by inverse PCR. The DpnI digestion products were detected by 1% agarose electrophoresis, and the detection results were consistent with those of FIG. 8, with the correct band size. If there are few bands, it is indicated that the product is amplified specifically, it can be used directly in the subsequent recombination cyclization, and if there are more bands, it is necessary to purify the target band first, and the purification operation is performed by referring to the manual of the biological DNA gel recovery kit.

3. Colony validation and sequencing: transferring the recombinant connection product into E.coli DH5 alpha by using a chemical conversion method; 3-5 transformants were randomly picked from the transformed plates and inoculated into about 5mL of the plates containing Amp (100. Mu.g. Multidot.mL) ^-1 ) In the LB liquid medium of (2), shaking culture is carried out for 12-16h at 37 ℃ at 180rpm, the culture is collected for plasmid extraction, and PCR and enzyme digestion identification are respectively carried out on each extracted mutant plasmid so as to detect whether the mutant plasmid contains target gene fragments with corresponding sizes. Mutant plasmids with correct PCR and double restriction enzyme identification were sequenced.

4. Introduction into recipient cells: the plasmid successfully subjected to site-directed mutagenesis was transformed into E.coli BL21 (DE 3) by chemical transformation to construct a mutant pdc gene expression strain, named "p-mutation name", and simultaneously, pET21a empty plasmid and unmutated pET21 a-pdc plasmid were also transformed into E.coli BL21 (DE 3) as controls, named p-control and p-Sdc, respectively. And performing PCR verification and enzyme digestion verification, wherein the enzyme digestion verification result is shown in figure 9.

5. Protein induction expression: inoculating the mutant strain into Amp with concentration of 100 mug.mL ^-1 In the LB liquid medium of (2), shake culturing is carried out for 12 hours at 37 ℃ and 160rpm rotating speed to obtain seed liquid; inoculating 1% seed solution into new Amp with concentration of 100 μg.mL ^-1 LB liquid culture of (C)In the medium, the culture was carried out at 37℃and 160rpm for about 4 hours to reach an OD600 of about 0.6, IPTG was added to a final concentration of 0.1mM, and induction was carried out at 25℃and 120rpm for 18 hours.

6. Preparing crude enzyme solution: centrifuging the culture solution at 5000rpm and 4 ℃ for 0min, collecting thalli, then resuspending the thalli by using an ultrasonic crushing buffer solution, and carrying out ultrasonic crushing in an ice bath under the following ultrasonic conditions: the power is 40%, the operation is 3s, the interval is 7s, and the total ultrasonic time is 10min. Centrifuging the crushed cell disruption solution at 4 ℃ and 14000rpm for 15min, wherein the supernatant is crude enzyme solution.

7. Mutant enzyme SDS-PAGE analysis: SDS-PAGE analysis of the crude enzyme solution resulted in a band of approximately 40kDa for both the wild-type Sdc and each mutant Sdc, consistent with the theoretical molecular weight of 39.9kDa for the salicylic acid decarboxylase predicted by PredictProtein. Since the size of each mutant enzyme is uniform, only the non-mutant Sdc and the SDS-PAGE results of two of the mutant Sdc enzymes are shown in fig. 3.

8. Preparing a standard: 1.0mg of each of ginkgolic acid A and ginkgolic acid B which are standard substances is accurately weighed, dissolved by 10mL of methanol, and prepared according to the formula A: b is 1:2, the concentration is 0.1 mg.mL ^-1 Is prepared from gingko total acid standard solution.

9. Reaction of the substrate with the enzyme solution: taking 1mL of crude enzyme solution, uniformly mixing with 2mL of PBS buffer solution, and taking the total volume of a reaction system as 4mL, wherein the amount of ginkgolic acid is 0.1 mg.mL ^-1 The pH was adjusted to 5.5, oxygen was introduced for 5min, the reaction was carried out at 40℃for 4h, and 100. Mu.L of 12M HCl was added to terminate the reaction.

10. Sample preparation and detection: after the reaction, vacuum freeze drying to constant weight, adding methanol for dissolution, filtering with a 0.22 μm filter membrane, and detecting the content of ginkgolic acid by HPLC.

11. Measurement of enzyme Activity: as a result of the difference in the effect of the mutation on the enzyme activity, as shown in FIG. 4, the mutant enzymes Sdc-E8A, sdc-H169A, sdc-H224A and Sdc-D298A showed little degradation ability for ginkgolic acid, whereas the enzyme activities of the mutant strains Sdc-Y64T and Sdc-P191A were markedly increased, and the base sequence changes of the mutant strains Sdc-Y64T and Sdc-P191A with the wild-type Sdc were shown in FIG. 2.

12. The mutant enzymes Sdc-P191A and Sdc-Y64T having significantly increased enzyme activities were purified, and then the mutant enzymes were subjected to determination of optimum pH (fig. 6), optimum temperature (fig. 5) and temperature stability (fig. 7): the degradation capability of the Sdc-P191A and the Sdc-Y64T mutant enzyme on ginkgolic acid is respectively improved by 116.74 percent and 105.18 percent under the conditions of the optimal pH value of 5.5 and the optimal temperature of 40 ℃ compared with the non-mutant wild type Sdc.

TABLE 1

TABLE 2

TABLE 3 Table 3

Salicylic acid decarboxylase sdc in candida:

mutant enzyme Sdc-P191A:

mutant enzyme Sdc-Y64T:

nucleotide sequence of the mutase Sdc-P191A gene:

nucleotide sequence of mutant enzyme Sdc-Y64T gene:

although 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, and therefore the scope of the invention is not limited to the disclosure of the embodiments.

Sequence listing

<110> university of Tianjin science and technology

<120> mutant salicylic acid decarboxylase, strain and application thereof in degradation of ginkgolic acid

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<210> 1

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<213> salicylic acid decarboxylase sdc (Unknown) in Candida

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Glu Leu Ser Asn Lys His Gly Ile Gly Tyr Thr Ile Tyr Ser Ile Tyr

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Ser Pro Gly Pro Gln Gly Trp Thr Glu Arg Ala Glu Cys Glu Glu Tyr

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Ala Arg Glu Cys Asn Asp Tyr Ile Ser Gly Glu Ile Ala Asn His Lys

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Asp Arg Met Gly Ala Phe Ala Ala Leu Ser Met His Asp Pro Lys Gln

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Ala Ser Glu Glu Leu Thr Arg Cys Val Lys Glu Leu Gly Phe Leu Gly

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Ala Leu Val Asn Asp Val Gln His Ala Gly Pro Glu Gly Glu Thr His

130 135 140

Ile Phe Tyr Asp Gln Pro Glu Trp Asp Ile Phe Trp Gln Thr Cys Val

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Asp Leu Asp Val Pro Phe Tyr Leu His Pro Glu Pro Pro Phe Gly Ser

165 170 175

Tyr Leu Arg Asn Gln Tyr Glu Gly Arg Lys Tyr Leu Ile Gly Pro Pro

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Val Ser Phe Ala Asn Gly Val Ser Leu His Val Leu Gly Met Ile Val

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<213> mutant enzyme Sdc-P191A (Unknown)

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Asp Arg Met Gly Ala Phe Ala Ala Leu Ser Met His Asp Pro Lys Gln

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Ala Ser Glu Glu Leu Thr Arg Cys Val Lys Glu Leu Gly Phe Leu Gly

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Ala Leu Val Asn Asp Val Gln His Ala Gly Pro Glu Gly Glu Thr His

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Ile Phe Tyr Asp Gln Pro Glu Trp Asp Ile Phe Trp Gln Thr Cys Val

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Asp Leu Asp Val Pro Phe Tyr Leu His Pro Glu Pro Pro Phe Gly Ser

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Ala Ala Gln Thr Lys Glu Lys Ala Glu Leu Tyr Ile Ala Pro Asn Asn

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Glu Leu Ser Asn Lys His Gly Ile Gly Tyr Thr Ile Tyr Ser Ile Thr

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Ser Pro Gly Pro Gln Gly Trp Thr Glu Arg Ala Glu Cys Glu Glu Tyr

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Ala Arg Glu Cys Asn Asp Tyr Ile Ser Gly Glu Ile Ala Asn His Lys

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Asp Arg Met Gly Ala Phe Ala Ala Leu Ser Met His Asp Pro Lys Gln

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Ala Ser Glu Glu Leu Thr Arg Cys Val Lys Glu Leu Gly Phe Leu Gly

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Ala Leu Val Asn Asp Val Gln His Ala Gly Pro Glu Gly Glu Thr His

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Ile Phe Tyr Asp Gln Pro Glu Trp Asp Ile Phe Trp Gln Thr Cys Val

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Asp Leu Asp Val Pro Phe Tyr Leu His Pro Glu Pro Pro Phe Gly Ser

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Glu His Cys Ser Arg Pro Leu Ala Lys Ser Arg Gly Asp Val Phe Ala

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Glu Lys Pro Leu Leu His Tyr Phe Arg Asn Asn Ile Trp Leu Thr Thr

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Ser Gly Asn Phe Ser Thr Glu Thr Leu Lys Phe Cys Val Glu His Val

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Gly Ala Glu Arg Ile Leu Phe Ser Val Asp Ser Pro Tyr Glu His Ile

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Asp Val Gly Cys Gly Trp Tyr Asp Asp Asn Ala Lys Ala Ile Met Glu

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Ala Val Gly Gly Glu Lys Ala Tyr Lys Asp Ile Gly Arg Asp Asn Ala

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Lys Lys Leu Phe Lys Leu Gly Lys Phe Tyr Asp Ser Glu Ala

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<210> 4

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<212> DNA

<213> nucleotide sequence of mutant enzyme Sdc-P191A Gene (Unknown)

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atgcgcggaa aggtttctct cgaggaggcg ttcgagcttc ccaagttcgc tgcccagacc 60

aaggagaagg ccgagctcta catcgccccc aacaaccgcg accggtactt tgaggagatt 120

ctcaacccgt gcggcaaccg tctcgagctt tcgaacaagc acggtatcgg ctacaccatc 180

tactctatct actcgcctgg tccgcaggga tggaccgagc gcgccgagtg tgaggagtac 240

gcgcgcgagt gcaacgacta catctcgggc gagattgcca atcacaagga ccggatgggt 300

gcctttgccg ctctgtcgat gcacgacccc aagcaggcgt ccgaggagct tacccgctgc 360

gttaaagagc tcggtttcct cggcgcgctc gtcaacgacg tgcagcacgc cggacccgaa 420

ggcgagaccc acatcttcta cgaccagccc gagtgggaca tcttctggca gacttgcgtc 480

gatctcgacg ttccattcta cctccacccc gagcctccct tcggctcgta cctccgcaac 540

cagtacgagg gacgcaagta ccttattggt gctcccgtgt cttttgccaa cggcgtctcg 600

ctccacgtcc tcggcatgat cgtcaacggt gtctttgacc gcttccccaa gctcaaggtc 660

atcctcggcc accttggcga gcacattccc ggagacttct ggcgcatcga gcactggttc 720

gagcactgct cccgccctct cgccaagtcg cgcggagacg tcttcgctga gaagcccctc 780

ctccactact tccgcaacaa catctggctc accacctcgg gcaacttctc caccgagact 840

ctcaagttct gcgtcgagca cgtcggcgcc gagcgcatcc tcttctccgt cgactcgcct 900

tacgagcaca tcgacgtcgg atgcggatgg tacgacgaca acgccaaggc tatcatggag 960

gccgttggcg gtgagaaggc ctacaaggac attggccgtg acaacgccaa gaagctcttc 1020

aagctcggca agttctacga ctcggaggct tag 1053

<210> 5

<211> 1053

<212> DNA

<213> nucleotide sequence of mutant enzyme Sdc-Y64T Gene (Unknown)

<400> 5

atgcgcggaa aggtttctct cgaggaggcg ttcgagcttc ccaagttcgc tgcccagacc 60

aaggagaagg ccgagctcta catcgccccc aacaaccgcg accggtactt tgaggagatt 120

ctcaacccgt gcggcaaccg tctcgagctt tcgaacaagc acggtatcgg ctacaccatc 180

tactctatca cctcgcctgg tccgcaggga tggaccgagc gcgccgagtg tgaggagtac 240

gcgcgcgagt gcaacgacta catctcgggc gagattgcca atcacaagga ccggatgggt 300

gcctttgccg ctctgtcgat gcacgacccc aagcaggcgt ccgaggagct tacccgctgc 360

gttaaagagc tcggtttcct cggcgcgctc gtcaacgacg tgcagcacgc cggacccgaa 420

ggcgagaccc acatcttcta cgaccagccc gagtgggaca tcttctggca gacttgcgtc 480

gatctcgacg ttccattcta cctccacccc gagcctccct tcggctcgta cctccgcaac 540

cagtacgagg gacgcaagta ccttattggt cctcccgtgt cttttgccaa cggcgtctcg 600

ctccacgtcc tcggcatgat cgtcaacggt gtctttgacc gcttccccaa gctcaaggtc 660

atcctcggcc accttggcga gcacattccc ggagacttct ggcgcatcga gcactggttc 720

gagcactgct cccgccctct cgccaagtcg cgcggagacg tcttcgctga gaagcccctc 780

ctccactact tccgcaacaa catctggctc accacctcgg gcaacttctc caccgagact 840

ctcaagttct gcgtcgagca cgtcggcgcc gagcgcatcc tcttctccgt cgactcgcct 900

tacgagcaca tcgacgtcgg atgcggatgg tacgacgaca acgccaaggc tatcatggag 960

gccgttggcg gtgagaaggc ctacaaggac attggccgtg acaacgccaa gaagctcttc 1020

aagctcggca agttctacga ctcggaggct tag 1053

<210> 6

<211> 38

<212> DNA

<213> F_E8A(Unknown)

<400> 6

ggtttctctc gctgaggcgt tcgagcttcc caagttcg 38

<210> 7

<211> 34

<212> DNA

<213> R_E8A(Unknown)

<400> 7

ctcgaacgcc tcagcgagag aaacctttcc gcgc 34

<210> 8

<211> 39

<212> DNA

<213> F_Y64T(Unknown)

<400> 8

tctactctat cacctcgcct ggtccgcagg gatggaccg 39

<210> 9

<211> 40

<212> DNA

<213> R_Y64T(Unknown)

<400> 9

ccaggcgagg tgatagagta gatggtgtag ccgataccgt 40

<210> 10

<211> 40

<212> DNA

<213> F_P191A(Unknown)

<400> 10

tattggtgct cccgtgtctt ttgccaacgg cgtctcgctc 40

<210> 11

<211> 40

<212> DNA

<213> R_P191A(Unknown)

<400> 11

gacacgggag caccaataag gtacttgcgt ccctcgtact 40

Claims

1. The application of mutant salicylic acid decarboxylase in efficiently degrading ginkgolic acid is characterized in that: the mutant salicylic acid decarboxylase is mutant enzyme Sdc-P191A and mutant enzyme Sdc-Y64;

the amino acid sequence of the mutant enzyme Sdc-P191A is shown as SEQ ID NO.2, and the 191 th amino acid of the salicylic acid decarboxylase Sdc with the amino acid sequence of SEQ ID NO.1 is changed from proline to alanine; the 64 th amino acid of salicylic acid decarboxylase Sdc with the amino acid sequence of SEQ ID NO.1 of the mutant enzyme Sdc-Y64T is changed from tyrosine to threonine, and the amino acid sequence of the mutant enzyme Sdc-Y64T is shown as SEQ ID NO. 3;

the optimum action pH of the mutant enzyme Sdc-P191A is 5.5, the optimum action temperature is 50 ℃, the mutant enzyme Sdc-P191A still has the activity of degrading ginkgolic acid after 4 hours, the temperature stability of the mutant enzyme Sdc-P191A is slightly improved compared with that of a wild type Sdc, and the degradation capacity of ginkgolic acid of the mutant enzyme Sdc-P191A is improved by 116.74 percent compared with that of the mutant enzyme Sdc;

the optimal action pH of the mutant enzyme Sdc-Y64T is 5.5, the optimal action temperature is 40 ℃, the optimal action temperature is reduced by 10 ℃ compared with the optimal action temperature of the wild type Sdc, and the capacity of degrading ginkgolic acid can be maintained within 1-4h at 40 ℃; and the degradation capacity of ginkgolic acid is improved by 105.18% compared with that of the non-mutated wild type sdc;

the ginkgolic acid is a 2-hydroxy-6-alkyl benzoic acid compound, and concretely is ginkgolic acid R=C13:0, ginkgolic acid R=C15:0, ginkgolic acid R=C15:1, ginkgolic acid R=C17:1, ginkgolic acid R=C17:2.