CN103966196A - Method for preparing dityrosine layer loosening type spore immobilized enzyme from saccharomyces cerevisiae - Google Patents

Abstract

The invention discloses a method for preparing a dityrosine layer loosening type spore immobilized enzyme from saccharomyces cerevisiae, belonging to the technical field of microorganisms and immobilized enzymes. The method comprises the steps of inserting an alpha-galactosidase gene with signal peptide into a plasmid pRS424-TEFpr to obtain a recombinant plasmid pRS424-TEFpr-MEL1, converting OSW2 genetic defect type saccharomyces cerevisiae, culturing recombinant yeasts by taking acetate as a unique or primary carbon source in lack of a nitrogen source, and finally collecting and purifying the yeasts to obtain the immobilized enzyme. The microcapsule immobilized enzyme can wrap a periplasmic space of spores by virtue of a dityrosine layer, and meanwhile, the dityrosine layer is loosened, so that substrate molecules can be conveniently in contact with enzyme molecules, and the reaction efficiency is improved. Due to unique space positioning, the protease stability is improved, the protein purification and immobilization operations are not needed, the production cost is reduced, and the like.

Description

A method for immobilizing enzymes using Saccharomyces cerevisiae dityrosine layer loose type spores

technical field

The invention relates to a method for immobilizing enzymes by using loose spores of Saccharomyces cerevisiae dityrosine layer, and belongs to the technical field of microorganisms and immobilized enzymes.

Background technique

Yeast generally refers to a large class of single-celled fungi that can ferment sugars. The most typical yeast is Saccharomycescerevisiae. Under good nutrition and growth conditions, Saccharomyces cerevisiae grows rapidly and reproduces by budding. However, with acetate as the only or main carbon source, supplemented by specific conditions such as lack of nitrogen source, such as only potassium acetate exists, Saccharomyces cerevisiae will reproduce in the form of sporulation and form spores in the cell. It is called ascus. The ascospores can be released after the ascus wall is broken naturally or artificially.

Although the specific function of the OSW2 gene is not yet clear, it is certain that it is necessary for the correct assembly of the spore wall of Saccharomyces cerevisiae. It has been reported in the literature that the spores of Saccharomyces cerevisiae are sensitive to ether after the OSW2 gene is knocked out, while the wild-type spores are not sensitive to ether. Saccharomyces cerevisiae can produce ascospores in the state of acetate as the sole or main carbon source, and its spore wall consists of four layers, from inside to outside: mannose, β-glucan, chitosan, Dityrosine.

In the present invention, we use a Saccharomyces cerevisiae deficient strain osw2Δ. Although the spore wall produced by this strain contains the outermost dityrosine layer, the structure of the spore wall becomes loose and the gaps become larger, making certain size Moderate substrates can pass. We immobilized the target enzyme in the periplasmic space between the chitosan layer and the dityrosine layer on the defective spore wall by means of molecular biology to obtain the spore-immobilized enzyme. On the one hand, the spore dityrosine can be used The acid layer wraps the enzyme in the periplasmic space of the spore, while the dityrosine layer becomes looser to facilitate the contact between the substrate molecule and the enzyme molecule, improving the reaction efficiency. This unique spatial orientation makes it have many excellent properties compared to free enzymes, such as temperature (organic solvent, protease) stability, reusability, no need for protein purification and immobilization operations, and reduced production costs.

Contents of the invention

The purpose of the present invention is to provide a method for immobilizing enzymes using Saccharomyces cerevisiae dityrosine layer loose spores, which is to construct OSW2 gene-deficient Saccharomyces cerevisiae, use it as a host to construct recombinant Saccharomyces cerevisiae expressing the target enzyme, and cultivate recombinant Saccharomyces cerevisiae The yeast reproduces in the form of sporulation and forms spores in the cell. The target enzyme is expressed in the periplasmic space between the chitosan layer and the dityrosine layer of the Saccharomyces cerevisiae spores, and the immobilized enzyme is obtained by collecting the spores.

The enzyme is preferably alpha-galactosidase, sucrase or phytase.

The method comprises the steps of:

1) Through homologous recombination, the Saccharomyces cerevisiae spore wall synthesis-related gene OSW2 was knocked out, and the Saccharomyces cerevisiae deficient strain osw2Δ was obtained through screening and verification, and its spore wall dityrosine layer was loose;

The nucleotide sequence of OSW2 is shown in SEQ ID NO.1;

2) Insert the α-galactosidase gene (MEL1) with its own signal peptide into the plasmid pRS424-TEFpr to obtain the recombinant plasmid pRS424-TEF _pr -MEL1;

The α-galactosidase gene (MEL1) is from yeast, and its nucleotide sequence is shown in SEQ ID NO.2;

3) transforming the obtained recombinant plasmid pRS424-TEF _pr -MEL1 into Saccharomyces cerevisiae osw2Δ by lithium acetate/PEG transformation method to obtain recombinant Saccharomyces cerevisiae osw2Δ/pRS424-TEF _pr -MEL1;

4) Inoculate recombinant Saccharomyces cerevisiae into SD-Trp liquid medium, culture at 28°C to 32°C overnight; the components of the SD-Trp medium are amino acid-free yeast nitrogen source, deionized water, after sterilization, add Individually sterilized dextrose and tryptophan-free amino acid mixture;

5) Transfer the bacterial solution in step 3) to YPAce medium, and shake it at 28°C to 32°C for 12h to 16h; the components of the YPAce medium are yeast extract, peptone, adenine and potassium acetate;

6) Collect the bacteria in step 4) by centrifugation, wash, and then resuspend with 1.5% to 3% (m/v) potassium acetate, shake at 28°C to 32°C for 24h to 48h;

7) Collect the recombinant Saccharomyces cerevisiae cells, wash them with PBS solution for 2-3 times, and then resuspend them with PBS solution;

8) Add lyticase to the cell suspension in step 7), and sonicate on ice;

9) Wash the broken cells in step 8) for 2 to 3 times with PBS solution, discard the supernatant to obtain the enzyme immobilized in Saccharomyces cerevisiae dityrosine layer loose spore microcapsules.

The culture temperature is preferably 30°C, and the concentration of potassium acetate is preferably 2%.

The step 9) also includes purifying the obtained spore microcapsule immobilized enzyme, washing the obtained spore microcapsule immobilized enzyme with 0.5% Triton X-100 solution, and then suspending the spores in 0.5% Triton X-100 solution Middle; prepare Percoll solutions with different concentration gradients; add Percoll solutions with different concentration gradients to the centrifuge tube in sequence from high concentration to low concentration, and finally add the spore suspension. After centrifugation, the solution will be layered, and the spores will be located in the centrifuge tube At the bottom, the upper liquid and cell debris were removed, the spores were washed 2 to 3 times with 0.5% Triton X-100 solution, and freeze-dried.

The SD-Trp medium preferably has a mass percentage of amino acid-free yeast nitrogen source and deionized water of 0.67%, a final concentration of glucose of 2%, and a final concentration of amino acid mixture of 0.67%. Among them, the composition and ratio of amino acid mixture powder are as follows:

The YPAce medium is preferably: 2% peptone, 1% yeast extract, 2% potassium acetate, 0.003% adenine.

The preparation method of the immobilized enzyme provided by the present invention only needs to clone and express the enzyme through the method of genetic engineering, and it can also be genetically modified and optimized if necessary; as the immobilized carrier cell, Saccharomyces cerevisiae spore is a An environmentally friendly carrier for cheap large-scale cultivation. In addition, by knocking out the gene OSW2 related to the spore wall synthesis of S. Compared with the free enzyme, the immobilized enzyme prepared by the invention can resist the attack of different hydrolytic enzymes and withstand higher temperature, and can be reused for many times at the same time.

Description of drawings

Figure 1 shows the α-galactosidase activity of different spores (wt, osw2Δ, dit1Δ) expressing α-galactosidase and Saccharomyces cerevisiae SK1 vegetative cells expressing α-galactosidase before and after high salt solution elution, Note: veg-vegetative cells; wt-wild-type spores; osw2Δ-dityrosine layer loose type spores; dit1Δ-the outermost layer of the spore wall does not contain the dityrosine layer; chs3Δ-the outermost layer of the spore wall does not contain the dityrosine layer Tyrosine layer and subouter chitosan layer; MEL1-galactosidase gene.

Figure 2 The relative value of α-galactosidase activity of different spores expressing α-galactosidase after 4 washes, note: wt-wild type spore; osw2Δ-dityrosine layer loose spore; dit1Δ-spore wall The outermost layer does not contain a dityrosine layer.

Figure 3 The relative percent activity of the remaining enzyme activity of different spores expressing α-galactosidase after treatment with β-glucanase, note: wt-wild-type spores; osw2Δ-dityrosine layer loose spores; dit1Δ- The outermost layer of the spore wall does not contain a dityrosine layer.

Figure 4 Relative percentage activity of α-galactosidase free enzyme and different spores expressing α-galactosidase after proteinase K treatment, Note: wt-wild type spore; osw2Δ-dityrosine layer loose type Spore; dit1Δ - the outermost layer of the spore wall does not contain a dityrosine layer.

Figure 5 The expression and localization of α-galactosidase on the spore wall of different spores, Note: wt-wild type spore; osw2Δ-loose spore with dityrosine layer; dit1Δ-the outermost layer of the spore wall does not contain dityrosine layer ; chs3Δ-spore wall does not contain the outermost dityrosine layer and the subouter chitosan layer.

Detailed ways

Example 1 Construction of Saccharomyces cerevisiae SK1osw2Δ strain

Primer design: Primers were designed according to the Saccharomyces cerevisiae OSW2 gene sequence as follows:

Upstream primer P1:

TATTCCTAAGCCTTTCTTTCTTTTTTGAAGGCAAGAACTCGCATTAGTTCGGATCCCCGGGTTAATTAA;

Downstream primer P2:

AATTTTGCGCATCCCACCCCTTATTAACAATCACATTTTTTTTTTAATAGAATTCGAGCTCGTTTAAAC.

The plasmid pFA6a-His3MX6 was amplified by PCR using the upstream primer P1 and the downstream primer P2 to obtain a knockout fragment containing the upstream and downstream sequences of the Saccharomyces cerevisiae OSW2 gene.

Knockout of OSW2 gene: The knockout fragment containing the HIS3 marker amplified by PCR was introduced into haploid cells 4B and 16D of Saccharomyces cerevisiae SK1 by lithium acetate/PEG transformation method, and screened.

Screening of defective strains: use SD-His deficient plate for screening, spread the obtained transformed strains on the SD-His deficient plate, after the colonies grow, pick a certain number of colonies to extract the genome, and perform PCR For verification, compare it with the genome of the wild-type strain. If the verification is successful, the haploids are fused on the SD-Leu-Arg plate to obtain the defective diploid strain SK1osw2Δ. The verified primers are as follows: upstream primer: AGCACATAGACGCACGATAC; downstream primer: GTGCAACAACCGCTTTTCTAC.

For the construction method of plasmid pFA6a-His3MX6, please refer to the literature Longtine M S, McKenzie III A, Demarini D J, et al.Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[J].Yeast,1998,14(10):953 -961. Saccharomyces cerevisiae SK1 was deposited in the China University Industrial Microbiology Resource Platform (CICIM, accession number Y0702).

Example 2 Preparation of microcapsule immobilized enzyme based on loose spores of Saccharomyces cerevisiae dityrosine layer

(1) Insert the α-galactosidase gene (MEL1) with its own signal peptide into the plasmid pRS424-TEFpr to obtain the recombinant plasmid pRS424-TEF _pr -MEL1;

(2) The obtained recombinant plasmid pRS424-TEF _pr -MEL1 was transformed into Saccharomyces cerevisiae SK1osw2Δ by lithium acetate/PEG transformation method to obtain recombinant Saccharomyces cerevisiae SK1osw2Δ/pRS424-TEF _pr -MEL1;

(3) Inoculate recombinant Saccharomyces cerevisiae into SD-Trp medium, culture at 30°C overnight; the components of the SD-Trp medium are amino acid-free yeast nitrogen source, deionized water, after sterilization, add separate sterilized Glucose and tryptophan-free amino acid mixture; wherein the mass percentage of amino acid-free yeast nitrogen source and deionized water is 0.67%, the final concentration of glucose is 2%, and the final concentration of amino acid mixture is 0.67%.

The amino acid mixture powder composition and ratio are as follows:

(4) Transfer the bacterial solution in step 3) to YPAce medium at a volume ratio of 1:30, and shake it at 30° C. for 15 hours; said YPAce: 2% peptone, 1% yeast extract, 2% potassium acetate, 0.003% adenine;

(5) Collect the bacteria in step 4) by centrifugation, wash, then resuspend with 2% potassium acetate, shake at 30°C for 36 hours;

(6) Collect the recombinant Saccharomyces cerevisiae cells, wash them with PBS solution for 2 to 3 times, and then resuspend them with PBS solution;

(7) Add 10 μg/mL lyticase to the cell suspension in step (6), and sonicate on ice;

(8) Wash the broken cells in step (7) 2 to 3 times with PBS solution, and discard the supernatant to obtain recombinant Saccharomyces cerevisiae spores.

(9) Purification of Saccharomyces cerevisiae recombinant spores: the obtained Saccharomyces cerevisiae recombinant spores were washed with Triton X-100 solution, and then the spores were suspended in Triton X-100 solution; Percoll solutions with different concentration gradients were prepared; Add the Percoll solution into the centrifuge tube in order from high concentration to low concentration, and finally add the spore suspension. After centrifugation, the solution is layered, and the pure spores will be located at the bottom of the centrifuge tube. Remove the upper liquid and cell debris and wash with PBS. The spores were washed 2 to 3 times with the solution, and then freeze-dried.

Concentration gradient of 50%, 60%, 70%, 80% Percoll solution:

(1) 80% Percoll, 10% 0.5% Triton-X, 10% 2.5M sucrose;

(2) 70% Percoll, 20% 0.5% Triton-X, 10% 2.5M sucrose;

(3) 60% Percoll, 30% 0.5% Triton-X, 10% 2.5M sucrose;

(4) 50% Percoll, 40% 0.5% Triton-X, 10% 2.5M sucrose.

For the construction method of plasmid pRS424-TEFpr, please refer to the literature Longtine M S, McKenzie III A, Demarini D J, et al.Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[J].Yeast,1998,14(10): 953-961. Saccharomyces cerevisiae SK1 was deposited in the China University Industrial Microbiology Resource Platform (CICIM, accession number Y0702).

Example 3 Determination of Enzyme Activity Based on Microcapsule Immobilized Enzymes of Saccharomyces cerevisiae Dityrosine Layer Loose Spores

2 mg of purified spores (or vegetative cells) expressing α-galactosidase were suspended in 1 mL of acetic acid buffer (0.2 M acetic acid-sodium acetate pH 4.6). In order to prevent glucose-induced haploid spores from germination and fusion into diploid cells, resulting in the rupture of the spore wall, the reaction system was added with cycloheximide (cycloheximide) at a final concentration of 40 μg/mL, and the substrate was 5% melibiose. ), dissolved in the above acetic acid buffer. The reaction system is: 1 mL spore or cell suspension, 1 mL 5% melibiose solution. After mixing well, place it at 37°C for 10 minutes, and finally place it in a boiling water bath for 1 minute to terminate the reaction. Centrifuge at 9,000r.min ^-1 for 5min to collect the supernatant of the reaction solution, dilute to an appropriate multiple, and use a glucose detection kit to detect the amount of glucose generated by the reaction. One unit of α-galactosidase activity is defined as follows: the amount (mg) of glucose produced by reacting for 10 minutes at 37°C. For wet cells, the quantification of cells is based on the corresponding OD ₆₆₀ on the following website to calculate the corresponding cell number http://www.pangloss.com/seidel/Protocols/ODvsCells.html. In order to avoid the influence of the original glucose in the medium on the results as much as possible, the glucose-free medium YPAce was used to treat the vegetative cells. The cells were first cultured in YPAD, and then transferred to YPAce for expansion.

As can be seen from Figure 1, compared with the α-galactosidase expressed by wild-type (wt) spores and osw2Δ spores, the activity of α-galactosidase expressed by dit1Δ spores is the highest, and the possible reason is due to dityramine The absence of the acid layer makes the substrate easier to react with the enzyme; the activity of α-galactosidase expressed by osw2Δ spores is higher than that of wild-type spores, the possible reason is that the dityrosine layer of osw2Δ spores has more Deletion to a small extent, results in easier passage of substrates through the dityrosine layer than wild-type spores. Although the α-galactosidase expressed by dit1Δ spores has the highest activity, the enzyme expressed by dit1Δ spores may not be able to tightly anchor to the spore wall due to the absence of the dityrosine layer. In order to test this possibility, various spores expressing α-galactosidase were washed with a high-salt solution (0.6M NaCl and 0.1% Triton X-100) containing detergent, and then the activity after washing was determined , the results are shown in Figure 1: compared with wild-type spores and osw2Δ spores, the enzyme activity expressed by dit1Δ spores decreased most significantly. and osw2Δ spores expressed enzyme activities decreased by about 25% (as shown in Figure 2). It can be inferred that the presence of loose dityrosine layer in osw2Δ spores can prevent the release of α-galactosidase from the spore wall, and make the substrate easier to pass through the dityrosine layer, thereby increasing the enzyme activity.

Example 4 Detection of the protective effect of yeast spore wall on α-galactosidase

Including treating spore microcapsule immobilized enzyme with β-glucanase and proteinase K respectively, the specific process is as follows:

(1) Different spores (wt, osw2Δ, dit1Δ) expressing α-galactosidase were incubated with β-glucanase (β-glucanase) respectively, and the treatment method was as follows:

About 2mg of the above-mentioned purified and dried recombinant spore powder containing α-galactosidase was resuspended in 1mL of protoplast solution (50mM phosphate buffer pH 7.5, 1.4M sorbitol, 40mM β-mercaptoethanol), and mixed thoroughly Add 10 μL of β-glucanase stock solution (1mg β-glucanase dissolved in 500 μL of 50% glycerol), mix thoroughly, place in a shaker at 30°C for 3 hours, and then use 0.6M NaCl solution containing 0.1% TritonX-100 After washing several times, the treated cells were collected by centrifugation, and the α-galactosidase activity was detected according to the above method. The results shown in Figure 3 show that the α-galactosidase activity expressed by wild-type Saccharomyces cerevisiae SK1 spores (wt) and osw2Δ spores was almost unaffected, however, the α-galactosidase activity expressed by dit1Δ spores decreased sharply. (2) Different spores (wt, osw2Δ, dit1Δ) expressing α-galactosidase and free galactosidase in the culture medium were incubated with proteinase K respectively, and the treatment method was as follows:

About 2 mg of recombinant spore powder containing α-galactosidase after the above purification and drying were resuspended in 1 mL of proteinase K buffer (50mM Tris-HCl, pH7.5, 10mM CaCl ₂ ), and proteinase K was added to a final concentration of 500 μg/mL , after mixing well, place it in a shaker at 30°C for overnight treatment, then wash several times with 0.6M NaCl containing 0.1% TritonX-100, collect the treated cells by centrifugation, and detect α-galactosidase activity according to the above method. The treatment of α-galactosidase secreted into the medium is as follows: wild-type spores expressing α-galactosidase were first pre-cultured with 5 mL of SD-TRP selection medium, and then expanded with 30 mL of LYPAce, and the supernatant was collected by centrifugation , the supernatant was concentrated to about 300 μL (final protein concentration 6 mg/mL), and 10 μL was taken for each experiment. Refer to Example 3 for enzyme activity determination. The results are shown in Figure 4, the activity of α-galactosidase expressed by wild-type Saccharomyces cerevisiae SK1 spores and osw2Δ spores was almost unaffected, and the activity of α-galactosidase expressed by dit1Δ spores was slightly decreased. However, the activity of free α-galactosidase in the culture medium decreased sharply after proteinase K treatment.

Example 5 Detection of the expression localization of α-galactosidase on the spore wall

1) Insert the α-galactosidase gene MEL1 (without stop codon) containing its own signal peptide into the plasmid pRS424-TEFpr to obtain the recombinant plasmid pRS424-TEF _pr -MEL1;

2) The RFP gene (SEQ ID NO.3) amplified by PCR was connected to the plasmid pRS424-TEF _pr -MEL1 to obtain the recombinant plasmid pRS424-TEF _pr -MEL1-RFP, and transformed by lithium acetate/PEG respectively Saccharomyces cerevisiae SK1, osw2Δ, dit1Δ were transformed to obtain corresponding recombinant Saccharomyces cerevisiae respectively; the sporulation process and spore purification process were referred to in Example 2.

3) The spores or vegetative cells are centrifuged, washed several times with sterile water, and then resuspended in an appropriate amount of sterile water. Take 5 μL of cell suspension and put it on a clean glass slide, cover it with a cover glass, place it under a Nikon Eclipse Ti-E fluorescence inverted microscope, observe with a 100 times oil lens, and use the software NIS-Element AR for image analysis.

For Saccharomyces cerevisiae SK1 wild-type spores, the fusion protein expressed by osw2Δ spores is located on the spore wall; it is worth noting that the fusion protein expressed by dit1Δ spores is also located on the spore wall, while the fusion protein expressed by chs3Δ spores cannot be expressed and located on the spore wall On the spore wall, it is diffuse, indicating that for α-galactosidase, the existence of the dityrosine layer is not required, and the chitosan layer can express the enzyme on the spore wall, which may be related to the special properties of the enzyme Related, but due to the lack of dityrosine layer, the enzyme expressed by dit1Δ spores may not be tightly fixed on the spore wall. As shown in Figure 2, the activity decreased significantly after repeated washing.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. A method of utilizing Saccharomyces cerevisiae dityrosine layer loose spore-immobilized enzyme, characterized in that, constructing OSW2 gene-deficient Saccharomyces cerevisiae, using it as a host, constructing recombinant Saccharomyces cerevisiae expressing the enzyme of interest, cultivating recombinant Saccharomyces cerevisiae The yeast reproduces in the form of sporulation and forms spores in the cell. The target enzyme is expressed in the periplasmic space between the chitosan layer and the dityrosine layer of the Saccharomyces cerevisiae spores, and the immobilized enzyme is obtained by collecting the spores. 2. preparation method as claimed in claim 1 is characterized in that, described enzyme is α-galactosidase, and the nucleotide sequence of coding described α-galactosidase and its self signal peptide is as SEQ ID NO .2 shown. 3. The preparation method according to claim 1, characterized in that the enzyme is expressed in the periplasmic space under the guidance of a signal peptide. 4. The preparation method according to claim 1, wherein the OSW2 gene-deficient Saccharomyces cerevisiae is obtained by knocking out the Saccharomyces cerevisiae spore wall synthesis-related gene OSW2 through homologous recombination, and screening and verification; the OSW2 gene The nucleotide sequence is shown in SEQ ID NO.1. 5. The preparation method according to claim 1, wherein the α-galactosidase gene with its own signal peptide shown in SEQ ID NO.2 is inserted into the plasmid pRS424-TEFpr to obtain the recombinant plasmid pRS424 -TEF _pr -MEL1, and then transform the OSW2 gene-deficient Saccharomyces cerevisiae osw2Δ, culture the recombinant yeast with acetate as the sole or main carbon source, supplemented by the condition of lack of nitrogen source, and finally collect and purify Saccharomyces cerevisiae dityrosine Lamellar loose spore-immobilizing enzyme. 6. preparation method as claimed in claim 5, is characterized in that, comprises the following steps: (1) Insert the α-galactosidase gene with its own signal peptide into the plasmid pRS424-TEFpr to obtain the recombinant plasmid pRS424-TEF _pr -MEL1; (2) Transforming the obtained recombinant plasmid pRS424-TEF _pr -MEL1 into Saccharomyces cerevisiae osw2Δ to obtain recombinant Saccharomyces cerevisiae osw2Δ/pRS424-TEF _pr -MEL1; (3) Inoculate the recombinant Saccharomyces cerevisiae into SD-Trp medium, culture at 28°C to 32°C overnight; (4) transfer the bacterial liquid in step (3) to YPAce medium, shake the bed at 28°C-32°C for 12h-16h; (5) Collect the thalli in step (4) by centrifugation, wash, and then resuspend with 1.5% to 3% potassium acetate, shake at 28°C to 32°C for 24h to 48h, and make Saccharomyces cerevisiae form spores in the cells; (6) Collecting and crushing the recombinant Saccharomyces cerevisiae cells, collecting and purifying to obtain spores expressing α-galactosidase between the chitosan layer and the dityrosine layer. 7. the preparation method as claimed in claim 6 is characterized in that, step (6) collects recombinant Saccharomyces cerevisiae cells, washes 2～3 times with PBS solution, then resuspends with PBS solution; Add Lyticase to cell suspension, Sonicate on ice; wash the broken cells 2-3 times with PBS solution, discard the supernatant to obtain recombinant Saccharomyces cerevisiae spores. 8. the preparation method as claimed in claim 6 is characterized in that, the recombinant Saccharomyces cerevisiae spore that is collected is washed with TritonX-100 solution, then spore is suspended in Triton X-100 solution; Utilize Percoll layering liquid, centrifugal To obtain pure spores, wash the spores with PBS solution 2-3 times, and freeze-dry. 9. the preparation method as claimed in claim 6 is characterized in that, described SD-Trp culture medium composition is without amino acid yeast nitrogen source, deionized water, after sterilization, add the glucose of independent sterilization and color-free amino acid mixture. 10. The preparation method according to claim 6, wherein the YPAce medium components are yeast extract, peptone, adenine and potassium acetate.