CN103436520A - Preparation method of novel immobilized enzyme with brewer yeast spore as carrier - Google Patents
Preparation method of novel immobilized enzyme with brewer yeast spore as carrier Download PDFInfo
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- CN103436520A CN103436520A CN2013104225688A CN201310422568A CN103436520A CN 103436520 A CN103436520 A CN 103436520A CN 2013104225688 A CN2013104225688 A CN 2013104225688A CN 201310422568 A CN201310422568 A CN 201310422568A CN 103436520 A CN103436520 A CN 103436520A
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
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- 102000004190 Enzymes Human genes 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 14
- OQALFHMKVSJFRR-UHFFFAOYSA-N dityrosine Chemical compound OC(=O)C(N)CC1=CC=C(O)C(C=2C(=CC=C(CC(N)C(O)=O)C=2)O)=C1 OQALFHMKVSJFRR-UHFFFAOYSA-N 0.000 claims abstract description 9
- 230000007547 defect Effects 0.000 claims abstract description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 76
- 241000894006 Bacteria Species 0.000 claims description 21
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 17
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 17
- 101150025279 DIT1 gene Proteins 0.000 claims description 16
- 230000002950 deficient Effects 0.000 claims description 16
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- 238000000034 method Methods 0.000 claims description 14
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- 238000011144 upstream manufacturing Methods 0.000 claims description 12
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- 238000012408 PCR amplification Methods 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 210000002421 cell wall Anatomy 0.000 claims description 7
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 7
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
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- 238000013461 design Methods 0.000 claims description 6
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- 229940088594 vitamin Drugs 0.000 claims description 6
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- 150000003722 vitamin derivatives Chemical class 0.000 claims description 6
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- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 235000015097 nutrients Nutrition 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
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- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 claims description 3
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- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
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- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
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Abstract
The invention discloses a preparation method of a novel immobilized enzyme with a brewer yeast spore as a carrier and the immobilized enzyme. A brewer yeast defect type strain deltadit1 is used, a spore wall produced by the brewer yeast defect type strain deltadit1 does not contain an outermost dityrosine layer, an exposed outermost layer is chitosan, and the enzyme is directly fixed on a defect type spore, so that the immobilized enzyme is obtained. According to the preparation method of the novel immobilized enzyme with the brewer yeast spore as the carrier, massive brewer yeast ascus spores are obtained by directly cultivating the brewer yeast defect type strain deltadit1, then wall breaking and purifying are carried out to obtain the immobilized enzyme target product, and corresponding application can be carried out without a carrier used for acquiring a common immobilized enzyme; the preparation method is simple and easy to operate, and cost is saved.
Description
Technical field
The present invention relates to microbial technology field and enzyme immobilization technology, specifically, the present invention relates to a kind of preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier.
Background technology
The unicellular fungi of one large class of yeast general reference energy fermenting carbohydrate, most typical yeast is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).Yeast saccharomyces cerevisiae is under good nutrition and growth conditions, and growth rapidly, is carried out reproduction in the mode of the breeding of sprouting.But take acetate as unique or main carbon source, and be aided with (as only had the potassium acetate existence) under specified conditions such as lacking nitrogenous source, yeast saccharomyces cerevisiae will be bred in sporiparous mode, forms spore in born of the same parents.Cell now is called ascus, and ascus, after nature or artificial broken wall, can discharge thecaspore wherein.
Chitosan is obtained through deacetylation by chitin, and its chemical name is Chitosan (1-4)-2-amino-beta--D-Glucose.Once had bibliographical information to cross chitosan and can be used to immobilized enzyme, this is that amino due to chitosan can easily be activated by glutaraldehyde, thereby is combined with enzyme.And yeast saccharomyces cerevisiae can produce thecaspore take acetate under the state of unique or main carbon source, its conidial cell wall forms by four layers, is followed successively by from inside to outside: seminose, beta-glucan, chitosan, o,o-Dityrosine.In the present invention, we use a kind of yeast saccharomyces cerevisiae deficient strain △ dit1, and the conidial cell wall that this bacterial strain produces is not containing the o,o-Dityrosine layer of outermost, and being exposed to outmost one deck is chitosan, therefore we can directly be fixed on enzyme on the spore of this defective type, obtains the spore immobilized enzyme.
Summary of the invention
The purpose of this part is to summarize some aspects of embodiments of the invention and briefly introduces some preferred embodiments.May do a little simplification or omit to avoid the making purpose of this part, specification digest and denomination of invention fuzzy in this part and the application's specification digest and denomination of invention, and this simplification or omit can not be for limiting the scope of the invention.
Problem in view of existing in above-mentioned and/or existing preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier and application, proposed the present invention.
Therefore, the purpose of this invention is to provide a kind of preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier, to reach the purpose of the curing enzyme for preparing specific end use.
For solving the problems of the technologies described above, the invention provides following technical scheme: a kind of preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier, comprise, the cultivation of yeast saccharomyces cerevisiae list bacterium colony, adopt the method for homologous recombination to knock out responsible coding Saccharomyces Cerevisiae in S K1 conidial cell wall outermost layer o,o-Dityrosine layer DIT1 gene, the yeast saccharomyces cerevisiae defect bacterial strain obtained is rule on YPAD solid medium flat board, and 28 ℃～32 ℃ are cultured to and grow single bacterium colony; The preparation of spore, picking list bacterium colony is in the YPAD liquid nutrient medium, and shaking table is transferred to bacterium liquid in the YPAce substratum after cultivating, and collecting cell after cultivating forwards in 1%～3% liquor kalii acetici and cultivates, and the concentration that makes cell in liquor kalii acetici is 2 * 10
7/ ml～4 * 10
7/ ml, then centrifugal, by PBS damping fluid washing for the yeast saccharomyces cerevisiae thalline after centrifugal, process 0.5h～1.5h with a certain amount of lyticase under 36 ℃～38 ℃ after, carry out ultrasonic disruption, obtain the yeast saccharomyces cerevisiae spore of unbound state and spore is carried out to purifying and lyophilize; The immobilization of enzyme, be fixed to beta-galactosidase enzymes on the spore of yeast saccharomyces cerevisiae DIT1 gene defection type.By purified yeast saccharomyces cerevisiae △ dit1 spore, with 2%～3% glutaraldehyde solution, processed, then wash the yeast saccharomyces cerevisiae spore, add the beta-galactosidase enzymes solution prepared in the yeast saccharomyces cerevisiae spore, under 3 ℃～5 ℃, be fixed, centrifugal, washing, the beta-galactosidase enzymes of being fixed.
As a kind of preferred version of preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier of the present invention, wherein: the cultivation of described yeast saccharomyces cerevisiae list bacterium colony, comprise,
Design of primers: as follows according to yeast saccharomyces cerevisiae DIT1 gene order design primer:
Upstream primer P1
AATTTGTTAATATCCTAATTCGGTAAAGCTTTGTCGAGACATTAACAAAACGGATCCCCGGGTTAATTAA
Downstream primer P2
TGTTTAAGTAAAAGAACAAAAAGGTAGACCAATGTAGCGCTCTTACTTTAGAATTCGAGCTCGTTTAAAC；
Pcr amplification, utilize upstream primer P1 and downstream primer P2 to carry out pcr amplification to plasmid pFA6a-His3MX6, obtains the fragment that knocks out containing yeast saccharomyces cerevisiae DIT1 gene upstream and downstream sequence;
Knocking out of DIT1 gene, the his fragment that the method for transformation by Lithium Acetate/PEG obtains pcr amplification imports to respectively Saccharomyces Cerevisiae in S K1 and obtains in haploid cell 4B and 16D, the row filter of going forward side by side;
The screening of defective bacterial strain, the conversion bacterial strain obtained after the knocking out of DIT1 gene is applied on SD-His defective flat board, after growing bacterium colony, the bacterium colony of picking certain number extracts genome, the performing PCR of going forward side by side checking, contrasted with the genome with wild type strain, if be proved to be successful, again monoploid is merged on the SD-Leu-Arg flat board, obtain the diploid bacterial strain of defective;
The checking primer is as follows:
Upstream primer: CATAAATTGTGCTCCTCCGC
Downstream primer: CATTGCAGTGTCTCGAAACC.
A kind of preferred version as preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier of the present invention, wherein: the cultivation of described yeast saccharomyces cerevisiae list bacterium colony, the YPAD solid medium be formulated as yeast extract 10g, peptone 20g, VITAMIN B4 30mg, agar 20g, constant volume is in 900mL distilled water, after 121 ℃ of sterilizing 15min, add 20% glucose of the independent sterilizing of 100mL.
A kind of preferred version as preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier of the present invention, wherein: the preparation of described yeast saccharomyces cerevisiae spore, the YPAce substratum be formulated as yeast extract 10g, peptone 20g, VITAMIN B4 30mg, constant volume, in 900mL distilled water, after 121 ℃ of sterilizing 15min, is added 20% Potassium ethanoate of the independent sterilizing of 100mL.
A kind of preferred version as preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier of the present invention, wherein: described the yeast saccharomyces cerevisiae spore is carried out to purifying, comprise, preparation Percoll solution, by the 0.5%Triton X-100 solution washing for cell of processing, to remove the impurity such as lyase wherein, then by cell suspension in 0.5%Triton X-100 solution, prepare the Percoll solution of different gradient 50%～80% different concns gradients; The purifying of yeast saccharomyces cerevisiae spore, according to joining successively from the high density to the lower concentration in centrifuge tube, finally add brewing yeast cell suspension by described Percoll solution, centrifugal, and supernatant liquid is removed, and obtains the yeast saccharomyces cerevisiae spore of purifying.
Another object of the present invention is to provide a kind of novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier of take, and the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore of take is carrier adopts the preparation method in the present invention to make.
For solving the problems of the technologies described above, the invention provides following technical scheme: a kind ofly take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier, described novel immobilized enzyme be by the beta-galactosidase enzymes immobilization to the yeast saccharomyces cerevisiae defective type spore that does not contain the o,o-Dityrosine layer.
The thecaspore that the purpose of this invention is to provide a kind of defective type yeast saccharomyces cerevisiae, can being used for fixing enzyme, and shows stronger immobilization ability, has wide practical use.The present invention directly obtains a large amount of thecaspores by cultivating yeast saccharomyces cerevisiae △ dit1 bacterial strain, more in addition the broken wall purifying obtains the purpose product, just can do corresponding application, and without using the required carrier of conventional immobilized enzyme, method is easy, easy handling, cost saving.
The accompanying drawing explanation
Fig. 1 is the fixed amount schematic diagram of yeast saccharomyces cerevisiae spore to beta-galactosidase enzymes;
Fig. 2 is the vitality test of immobilized enzyme and compares schematic diagram.
Embodiment
For above-mentioned purpose of the present invention, feature and advantage can be become apparent more, below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in detail.
A lot of details have been set forth in the following description so that fully understand the present invention, but the present invention can also adopt other to be different from alternate manner described here and implement, those skilled in the art can be in the situation that do similar popularization without prejudice to intension of the present invention, so the present invention is not subject to the restriction of following public specific embodiment.
Secondly, alleged " embodiment " or " embodiment " refers to special characteristic, structure or the characteristic that can be contained at least one implementation of the present invention herein.Different local in this manual " in one embodiment " that occur not all refer to same embodiment, neither be independent or the embodiment mutually exclusive with other embodiment optionally.
Below in conjunction with embodiment, the present invention will be further described, and xylose isomerase derives from the gene of thermus thermophilus, according to the Preference synthetic of yeast, but is not limited only to this enzyme.
Wherein, the method adopts following steps:
A adopts the method for homologous recombination to knock out and is responsible for coding Saccharomyces Cerevisiae in S K1 conidial cell wall outermost layer o,o-Dityrosine layer DIT1 gene, the yeast saccharomyces cerevisiae defect bacterial strain obtained is rule on YPAD solid medium flat board, and 30 ℃ are cultured to and grow single bacterium colony.1L YPAD substratum be formulated as yeast extract 10g, peptone 20g, VITAMIN B4 30mg, constant volume is (solid medium need add agar 20g) in 900mL distilled water, after 121 ℃ of sterilizing 15min, adds 20% glucose of the independent sterilizing of 100mL.
Wherein, the structure of yeast saccharomyces cerevisiae Δ dit1 mutant strain adopts following method to carry out:
1) design of primers: as follows according to yeast saccharomyces cerevisiae DIT1 gene order (GenBank) design primer:
Upstream primer P1
AATTTGTTAATATCCTAATTCGGTAAAGCTTTGTCGAGACATTAACAAAACGGATCCCCGGGTTAATTAA
Downstream primer P2
TGTTTAAGTAAAAGAACAAAAAGGTAGACCAATGTAGCGCTCTTACTTTAGAATTCGAGCTCGTTTAAAC；
Pcr amplification, utilize upstream primer P1 and downstream primer P2 to carry out pcr amplification to plasmid pFA6a-His3MX6, obtains the fragment that knocks out containing yeast saccharomyces cerevisiae DIT1 gene upstream and downstream sequence;
Knocking out of DIT1 gene, the his fragment that the method for transformation by Lithium Acetate/PEG obtains pcr amplification imports to respectively Saccharomyces Cerevisiae in S K1 and obtains in haploid cell 4B and 16D, the row filter of going forward side by side;
The screening of defective bacterial strain, the conversion bacterial strain obtained after the knocking out of DIT1 gene is applied on SD-His defective flat board, after growing bacterium colony, the bacterium colony of picking certain number extracts genome, the performing PCR of going forward side by side checking, contrasted with the genome with wild type strain, if be proved to be successful, again monoploid is merged on the SD-Leu-Arg flat board, obtain the diploid bacterial strain of defective;
The checking primer is as follows:
Upstream primer: CATAAATTGTGCTCCTCCGC
Downstream primer: CATTGCAGTGTCTCGAAACC.
The preparation of b spore: single bacterium colony that picking step a obtains, in the YPAD liquid nutrient medium, is cultivated 24h in 30 ℃ of shaking tables.Bacterium liquid is transferred in the YPAce substratum, and collecting cell after incubated overnight, forward in 2% Potassium ethanoate and cultivate, and the concentration that makes cell in Potassium ethanoate is 3 * 10
7/ ml, after cultivating 20h, after the centrifugal 10min of 5,000rpm, PBS damping fluid washing for thalline is processed 0.5h with lyticase under 37 ℃, and ultrasonication obtains the spore of unbound state and spore is carried out to purifying and lyophilize.1L YPAce liquid nutrient medium be formulated as yeast extract 10g, peptone 20g, VITAMIN B4 30mg, constant volume, in 900mL distilled water, after 121 ℃ of sterilizing 15min, is added 20% Potassium ethanoate of the independent sterilizing of 100mL.Wherein, purifying is the cell that first will process with 0.5%Triton X-100 solution washing three times, to remove the impurity such as lyase wherein, then by cell suspension in 1mL0.5%Triton X-100 solution.
Then prepare the Percoll solution of different gradient 50%-80% different concns gradients:
(1) 8mL Percoll, 1mL0.5%Triton-X, 1mL2.5M sucrose;
(2) 7mL Percoll, 2mL0.5%Triton-X, 1mL2.5M sucrose;
(3) 6mL percoll, 3mL0.5%Triton-X, 1mL2.5M sucrose;
(4) 5mL Percoll, 4mL0.5%Triton-X, 1mL2.5M sucrose.
By above-mentioned Percoll solution 1mL according to join successively the centrifuge tube of 10mL from the high density to the lower concentration, finally add cell suspension, 15,000g, after 4 ℃ of centrifugal 1h, gradient solution will layering, and pure spore will be positioned at the bottom of centrifuge tube, supernatant liquid is removed to the lyophilize in freeze drier of the spore after purifying.
The immobilization of c enzyme, be fixed to beta-galactosidase enzymes (buying the company in sigma) on the spore of △ dit1.Take purified spore that the b step of certainweight obtains in centrifuge tube, add 2% glutaraldehyde solution, be placed on 1h in 30 ℃ of shaking tables, then wash spore three times with distilled water, add the beta-galactosidase enzymes solution prepared in spore, shake 4h under 4 ℃ after, centrifugal, washing, the beta-galactosidase enzymes of being fixed.
Embodiment 1: the yeast saccharomyces cerevisiae spore is fixing to beta-galactosidase enzymes
As shown in Figure 1, after △ dit1 bacterial strain produces spore, after preceding method purifying lyophilize, take the spore powder of 1～10mg in centrifuge tube, and add 2% glutaraldehyde solution, fully shake 1h under 30 ℃ after, remove glutaraldehyde solution, and with distilled water, spore is washed three times, and then add beta-galactosidase enzymes solution, 4h vibrates under 4 ℃, 15, the centrifugal 10min of 000rpm, supernatant is remaining beta-galactosidase enzymes solution, precipitation is the spore after immobilization, with distilled water, spore is washed three times equally, to remove remaining beta-galactosidase enzymes solution.Finally get the content of supernatant solution enzyme wherein with kit measurement, thereby calculate the content that is fixed to the enzyme on conidial cell wall, the data that obtain are as shown in the table.
Embodiment 2: the vitality test of immobilized enzyme and comparison
Referring to Fig. 2, use the spore of 5mg to be fixed beta-galactosidase enzymes, simultaneously in order to determine the immobilization effect of enzyme, use the spore (outermost layer of conidial cell wall is beta-glucan) of wild-type spore (WT), nutritional type cell (veg) and △ chs3 deficient strain to compare, after immobilization, add after ortho-nitrophenyl β-D-synthesis (ONPG) solution reacts 30min under 30 ℃ of the 5mg/ml of 500 μ L, measure the variation of OD value under 410nm, and the calculating enzyme activity, the data that obtain are as shown in the table.
It should be noted that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment, the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (6)
1. take the preparation method of the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier for one kind, it is characterized in that: comprise,
The cultivation of yeast saccharomyces cerevisiae list bacterium colony, adopt the method for homologous recombination to knock out responsible coding Saccharomyces Cerevisiae in S K1 conidial cell wall outermost layer o,o-Dityrosine layer DIT1 gene, the yeast saccharomyces cerevisiae defect bacterial strain obtained is rule on YPAD solid medium flat board, and 28 ℃～32 ℃ are cultured to and grow single bacterium colony;
The preparation of spore, picking list bacterium colony is in the YPAD liquid nutrient medium, and shaking table is transferred to bacterium liquid in the YPAce substratum after cultivating, and collecting cell after cultivating forwards in 1%～3% liquor kalii acetici and cultivates, and the concentration that makes cell in liquor kalii acetici is 2 * 10
7/ ml～4 * 10
7/ ml, then centrifugal, by PBS damping fluid washing for the yeast saccharomyces cerevisiae thalline after centrifugal, process 0.5h～1.5h with a certain amount of lyticase under 36 ℃～38 ℃ after, carry out ultrasonic disruption, obtain the yeast saccharomyces cerevisiae spore of unbound state and the yeast saccharomyces cerevisiae spore is carried out to purifying and lyophilize;
The immobilization of enzyme, be fixed to beta-galactosidase enzymes on the spore of yeast saccharomyces cerevisiae DIT1 gene defection type.By purified yeast saccharomyces cerevisiae △ dit1 spore, with 2%～3% glutaraldehyde solution, processed, then wash the yeast saccharomyces cerevisiae spore, add the beta-galactosidase enzymes solution prepared in the yeast saccharomyces cerevisiae spore, under 3 ℃～5 ℃, be fixed, centrifugal, washing, the beta-galactosidase enzymes of being fixed.
2. preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier according to claim 1 is characterized in that: the cultivation of described yeast saccharomyces cerevisiae list bacterium colony, comprise,
Design of primers: as follows according to yeast saccharomyces cerevisiae DIT1 gene order design primer:
Upstream primer P1
AATTTGTTAATATCCTAATTCGGTAAAGCTTTGTCGAGACATTAACAAAACGGATCCCCGGGTTAATTAA
Downstream primer P2
TGTTTAAGTAAAAGAACAAAAAGGTAGACCAATGTAGCGCTCTTACTTTAGAATTCGAGCTCGTTTAAAC；
Pcr amplification, utilize upstream primer P1 and downstream primer P2 to carry out pcr amplification to plasmid pFA6a-His3MX6, obtains the fragment that knocks out containing yeast saccharomyces cerevisiae DIT1 gene upstream and downstream sequence;
Knocking out of DIT1 gene, the his fragment that the method for transformation by Lithium Acetate/PEG obtains pcr amplification imports to respectively Saccharomyces Cerevisiae in S K1 and obtains in haploid cell 4B and 16D, the row filter of going forward side by side;
The screening of defective bacterial strain, use SD-His defective flat board to be screened, the conversion bacterial strain obtained is applied on SD-His defective flat board, after growing bacterium colony, the bacterium colony of picking certain number extracts genome, the performing PCR of going forward side by side checking, with the genome with wild type strain, contrasted, if be proved to be successful, then monoploid is merged on the SD-Leu-Arg flat board, obtain the diploid bacterial strain of defective;
The checking primer is as follows:
Upstream primer: CATAAATTGTGCTCCTCCGC
Downstream primer: CATTGCAGTGTCTCGAAACC.
3. preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier according to claim 1, it is characterized in that: the cultivation of described yeast saccharomyces cerevisiae list bacterium colony, the YPAD solid medium be formulated as yeast extract 10g, peptone 20g, VITAMIN B4 30mg, agar 20g, constant volume is in 900mL distilled water, after 121 ℃ of sterilizing 15min, add 20% glucose of the independent sterilizing of 100mL.
4. preparation method of take the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier according to claim 1, it is characterized in that: the preparation of described yeast saccharomyces cerevisiae spore, the YPAce substratum be formulated as yeast extract 10g, peptone 20g, VITAMIN B4 30mg, constant volume, in 900mL distilled water, after 121 ℃ of sterilizing 15min, is added 20% Potassium ethanoate of the independent sterilizing of 100mL.
5. the preparation method of yeast saccharomyces cerevisiae spore according to claim 1 is characterized in that: described the yeast saccharomyces cerevisiae spore carried out to purifying, comprises,
Preparation Percoll solution, by the 0.5%Triton X-100 solution washing for cell of processing, to remove the impurity such as lyase wherein, then by cell suspension in 0.5%Triton X-100 solution, prepare the Percoll solution of different gradient 50%～80% different concns gradients;
The purifying of yeast saccharomyces cerevisiae spore, according to joining successively from the high density to the lower concentration in centrifuge tube, finally add brewing yeast cell suspension by described Percoll solution, centrifugal, and supernatant liquid is removed, and obtains the yeast saccharomyces cerevisiae spore of purifying.
6. an employing take as prepared by claim 1～5 either method the novel immobilized enzyme that the yeast saccharomyces cerevisiae spore is carrier, it is characterized in that: described novel immobilized enzyme be by the beta-galactosidase enzymes immobilization to the yeast saccharomyces cerevisiae defective type spore that does not contain the o,o-Dityrosine layer.
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