CN102517225A - Recombinant saccharymyces cerevisiae exhibiting amylase at surface as well as construction method and application - Google Patents
Recombinant saccharymyces cerevisiae exhibiting amylase at surface as well as construction method and application Download PDFInfo
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Abstract
The invention discloses recombinant saccharymyces cerevisiae exhibiting amylase at the surface as well as a construction method and application. The construction method of the recombinant saccharymyces cerevisiae comprises the following steps: inserting an amylase gene between the downstream MF alpha 1 signal peptide of a GAL1 promoter in an expression vector pGMAK of saccharymyces cerevisiae and an alpha lectin gene at the cell wall of the saccharymyces cerevisiae so as to obtain recombinant plasmid by construction; and replacing GAL1 genes of the saccharymyces cerevisiae by GAL4 genes through adopting a homologous recombination technology and transferring the recombinant plasmid into the cell so as to obtain the recombinant saccharymyces cerevisiae. According to the recombinant saccharymyces cerevisiae exhibiting the amylase at the surface as well as the construction method and the application, the recombinant saccharymyces cerevisiae with double GAL4 genes is obtained by the homologous recombination technology, and the expression efficiency of the GAL1 promoter can be effectively enhanced; and meanwhile, the amylase can be expressed at the outer surface of the cell wall of the saccharymyces cerevisiae in a high-density exhibiting way by alpha lectin at the cell wall. By exhibiting the amylase through utilizing the surface of the recombinant saccharymyces cerevisiae, the expression efficiency is higher, the expression quantity is larger, and the amylase with higher enzyme activity can be obtained; and moreover, the recombinant saccharymyces cerevisiae can be directly used as an amylase preparation after fermented culture.
Description
Technical field
The present invention relates to bioengineering field, relate in particular to the diastatic recombinant Saccharomyces cerevisiae of a kind of surface display and construction process and application.
Background technology
Because the mineral fuel resource is non-renewable, be badly in need of seeking substitute energy.With cheap cereal is that raw material can be produced bioenergy.Glycase comprises AMS and glucoamylase, and the former is the starch-liquefying enzyme, and the latter is a diastatic enzyme, and two kinds of enzyme actings in conjunction can thoroughly be hydrolyzed to glucose with starch, and then can further generate bio-ethanol through organism of fermentation such as zymic fermentation.Therefore, develop high vigor glycase for the development bioenergy significant.
Mainly there is following problem in the glycase of having developed at present: the vigor that starts the recombinant expressed promotor of enzyme is not enough, thereby makes the recombinant expressed quantity not sufficient of enzyme; The recombinant expressed mode of enzyme generally is cell inner expression and secreting, expressing.During cell inner expression since enzyme can not contact with the extracellular substrate, thereby greatly influence the catalytic efficiency (of enzyme, and the enzyme molecule is collected at formation feedback inhibition in the cell, also influences expression efficiency.The main drawback of secreting, expressing is that expression efficiency is not high, nor can realize complete secreting, expressing, always has quite a few expression product to be stranded in the cell, thereby can not realize contacting fully of enzyme and extracellular substrate.
In sum, start the recombinant expressed promotor vigor deficiency of enzyme and the cell inner expression and the secreting, expressing mode of enzyme and all have a strong impact on amylase activity.
Summary of the invention
The invention provides the diastatic recombinant Saccharomyces cerevisiae of a kind of surface display, utilize this recombinant Saccharomyces cerevisiae surface display glycase, expression efficiency is higher, and expression amount is bigger, can obtain the glycase that high enzyme is lived.
The diastatic recombinant Saccharomyces cerevisiae of a kind of surface display is the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) that contains recombinant plasmid; The GAL1 gene of said yeast saccharomyces cerevisiae is replaced by the GAL4 gene; Described recombinant plasmid is made up of the amylase gene between the cell walls α agglutinin gene of GAL1 promotor downstream MF α 1 signal peptide and yeast saccharomyces cerevisiae among yeast saccharomyces cerevisiae expression vector pGMAK and the insertion yeast saccharomyces cerevisiae expression vector pGMAK.
Described yeast saccharomyces cerevisiae is yeast saccharomyces cerevisiae ZD-1 (Saccharomyces cerevisiae ZD-1), is to obtain from orange peel surface isolation, screening in March, 2010, is preserved in Zhejiang University's biosystem engineering and Food science institute fermentation engineering laboratory.
Described amylase gene is alpha-amylase gene or glucose amylase gene.Glycase is made up of AMS and glucoamylase, and the former is the starch-liquefying enzyme, and the latter is a diastatic enzyme, and two kinds of enzyme actings in conjunction can thoroughly be hydrolyzed to glucose with starch.Respectively with the enzyme gene of two kinds of enzymes as external source goal gene construction recombination plasmid, can obtain can the corresponding enzyme of surface display recombinant Saccharomyces cerevisiae.
Genbank number of the GAL4 gene order of said yeast saccharomyces cerevisiae is NC 001148.3; Genbank number of the GAL1 gene order of said yeast saccharomyces cerevisiae is NC 001134.7.GAL4 albumen is activation that yeast saccharomyces cerevisiae expression vector pGMAK goes up the GAL1 promotor; The recombinant Saccharomyces cerevisiae of GAL1 genetically deficient can not be carbon source with the lactose; But the recombinant Saccharomyces cerevisiae with dual GAL4 gene can be expressed the GAL4 albumen of double amount; And then can significantly strengthen the vigor of GAL1 promotor, improve the secreting, expressing level of external source goal gene under GAL1 promotor and MF α 1 signal peptide guide.
The expression cassette of said yeast saccharomyces cerevisiae expression vector pGMAK holds 3 ' end to be followed successively by from 5 ': GAL1 promotor (Genbank number: AY428072), MF α 1 signal peptide (Genbank number: M17301), the cell walls α agglutinin gene of yeast saccharomyces cerevisiae (Genbank number: M28164), the ADH1 terminator.Described GAL1 promotor can start the expression of external source goal gene; Described MF α 1 signal Toplink guiding enzyme is to cell exocrine; The cell walls α lectin of described yeast saccharomyces cerevisiae can be anchored in the cell walls of yeast saccharomyces cerevisiae, can make enzyme molecule high-density presenting and expressing at brewing yeast cell wall outside surface as carrier proteins, can effectively improve the activity of enzyme.
Described alpha-amylase gene can be from suis (Streptococcus bovis), and Genbank number is AB000829.1; Described glucose amylase gene can be from aspergillus oryzae (Rhizopus oryzae), and Genbank number is D00049.1.
The present invention also provides the construction process of described recombinant Saccharomyces cerevisiae; May further comprise the steps: amylase gene is inserted between the cell walls α agglutinin gene of GAL1 promotor downstream MF α 1 signal peptide and yeast saccharomyces cerevisiae among the yeast saccharomyces cerevisiae expression vector pGMAK, make up and obtain recombinant plasmid; Adopt homologous recombination technique that the GAL1 gene of said yeast saccharomyces cerevisiae is replaced with the GAL4 gene, change said recombinant plasmid over to this cell, obtain recombinant Saccharomyces cerevisiae.
Adopt described homologous recombination technique; With the GAL1 sequence in the GAL4 sequence replacement yeast saccharomyces cerevisiae genome of yeast saccharomyces cerevisiae; Original GAL4 gene still keeps in this Yeast system genome, thereby can make this Yeast system have dual GAL4 gene and GAL1 genetically deficient (dGAL4 Δ GAL1 type).
The present invention also provides the application of described recombinant Saccharomyces cerevisiae in the preparation diastase; May further comprise the steps: described recombinant Saccharomyces cerevisiae is inoculated in the YPD substratum that contains living starch and semi-lactosi carries out fermentation culture; Fermentation culture is centrifugal collection thalline after 36～72 hours, makes diastase.
But described living starch and the diastatic secreting, expressing of semi-lactosi co-induction.Preferably, by weight percentage, farinose concentration is 3-8% in the said YPD substratum, and the concentration of semi-lactosi is 0.5-2%, helps zymic growth and diastatic expression most.
Preferably, described fermented incubation time is 40-45 hour, and the glycase enzyme of secreting, expressing is lived the highest under this condition.
The present invention replaces with the GAL4 gene through homologous recombination technique with the GAL1 gene in the brewing yeast cell, obtains to have the recombinant Saccharomyces cerevisiae of dual GAL4 gene, can effectively improve the expression efficiency of recombinant plasmid GAL1 promotor, increases diastatic expression amount; Simultaneously, the terminal expressed cell walls α lectin of cell walls α agglutinin gene that connects of recombinant plasmid can significantly improve diastatic enzyme and live with glycase high-density presenting and expressing at brewing yeast cell wall outside surface.Evidence is compared as the presenting and expressing host with using the starting strain yeast saccharomyces cerevisiae, uses the constructed recombinant Saccharomyces cerevisiae presenting and expressing glycase of the present invention, expresses the glycase enzyme that obtains and lives higher.
Embodiment
Embodiment 1 has the structure of the yeast saccharomyces cerevisiae of dual GAL4 gene and GAL1 genetically deficient
Adopt homologous recombination technique; Replace the GAL1 gene (Genbank number: NC 001134.7) in this yeast saccharomyces cerevisiae genome with the GAL4 gene (Genbank number: NC 001148.3) of yeast saccharomyces cerevisiae ZD-1 (Saccharomyces cerevisiae ZD-1); Make this Yeast system have dual GAL4 gene and GAL1 genetically deficient (dGAL4 Δ GAL1 type), concrete steps are following:
(1) (Genbank number: S78175.1), these gene two ends contain GAL1 albumen 5 ' end and 3 ' terminal sequence respectively to synthetic KanMX resistant gene, respectively are 47bp.
(2) the KanMX resistant gene that two ends is contained GAL1 albumen 5 ' end and 3 ' terminal sequence respectively is transformed into yeast saccharomyces cerevisiae ZD-1 through electric shock, to replace the GAL1 gene.The electric shock Transformation Program is:
1. the single yeast colony of picking from the YEPD flat board is inoculated in the 5ml YEPD substratum, after 30 ℃, 200rpm shaking culture spend the night, gets 100 μ l bacterium liquid and is inoculated in the triangular flask that contains 10ml YEPD substratum 30 ℃, 200rpm shaking culture 8-10h to OD
600=1.3-1.5.
2. packing bacterium liquid is placed 4 ℃ of centrifugal 5min collecting cells of 5500rpm behind the 15min on ice in the 2ml centrifuge tube of sterilization.Deionized water wash 1-2 time is abandoned supernatant, and every effective 320 μ l sterilization ultrapure water is resuspended, adds 40 μ l 10 * TE and 40 μ l, 10 * LiAc, mixing, and 30 ℃ of 85rpm vibration 45min, adding 20 μ l DTT is 25mM to final concentration, 30 ℃ of 85rpm vibration 15min.4 ℃ of centrifugal 5min collecting cells of 5500rpm.
3. with the sterilization ultrapure water re-suspended cell of 1ml precooling deposition, 4 ℃ of centrifugal 5min collecting cells of 5500rpm repeat this step 2 time.
4. with the 1M D-sorbyl alcohol re-suspended cell deposition of 1ml precooling, 4 ℃ of centrifugal 5min of 5500rpm with the 1M D-sorbyl alcohol re-suspended cell deposition of 100 μ l precoolings, rotate mixing more gently, and this is the yeast competent cell, places to be used for the electric shock conversion on ice.
5. get 40 μ l yeast competence bacterias, mix with 5 μ l 10mM KanMX resistant gene solution, move into electric shock and transform cup, behind the ice bath 5min, putting shocks by electricity on the electroporation transforms.Condition is: voltage 1.5kV, electric capacity 25 μ F, resistance 200 Ω, 5 milliseconds of times.
6. the YEPD substratum that adds the 1ml ice bath immediately forwards mixture in the 1.5ml Ependorf pipe to, 30 ℃ of vibration 2h, and every therebetween separated 30min puts upside down mixing once gently.
7. the centrifugal 5min of 5500rpm room temperature; Abandon supernatant, after the washing once of sterilization ultrapure water, the centrifugal 5min of 5500rpm room temperature; With 200 μ l sterilization ultrapure water suspension cell; Bacterium liquid is coated the YPD culture plate that contains G418 (200 μ g/ml) or Zeocin (150 μ g/ml), be inverted for 30 ℃ and cultivated 2-3 days, the positive bacterium colony that grows is the GAL1 gene by KanMX resistant gene displacement winner.
(3) synthetic GAL4 protein gene, these gene two ends contain GAL1 albumen 5 ' end and 3 ' terminal sequence respectively, respectively are 47bp.
(4) the GAL4 protein gene that two ends is contained GAL1 albumen 5 ' end and 3 ' terminal sequence respectively is transformed into yeast saccharomyces cerevisiae ZD-1 through electric shock, to replace the KanMX resistant gene.The electric shock Transformation Program is:
1.-6. the relevant portion in the same step (2);
7. the centrifugal 5min of 5500rpm room temperature abandons supernatant, after the washing once of sterilization ultrapure water; The centrifugal 5min of 5500rpm room temperature; With 200 μ l sterilization ultrapure water suspension cell, bacterium liquid coated the YPD culture plate that does not contain G418 (200 μ g/ml) or Zeocin (150 μ g/ml) after, cover flat board with nitrocellulose filter again after; Duplicate the YPD culture plate that contains G418 (200 μ g/ml) or Zeocin (150 μ g/ml), so then 2 kinds of flat boards are xeroxed flat board each other.Be inverted for 30 ℃ and cultivated 2-3 days; If certain position on the YPD culture plate that contains G418 (200 μ g/ml) or Zeocin (150 μ g/ml) does not have bacterium colony and grows; And on the YPD culture plate that does not contain G418 (200 μ g/ml) or Zeocin (150 μ g/ml) the corresponding bacterium colony that grows, then this bacterium colony is the KanMX resistant gene by GAL4 gene substitution success.
Operation through step (1) to step (4); The GAL1 gene of yeast saccharomyces cerevisiae ZD-1 is replaced by the KanMX resistant gene earlier; The KanMX resistant gene is by GAL4 gene substitution then; The final GAL1 gene of realizing is made up the yeast saccharomyces cerevisiae that acquisition has dual GAL4 gene and GAL1 genetically deficient (dGAL4 Δ GAL1 type) by GAL4 gene substitution.
The recombinant Saccharomyces cerevisiae of embodiment 2 surface display AMSs
(1) structure of AMS presenting and expressing frame
With alpha-amylase gene (from suis Streptococcus bovis; Genbank number: (Genbank number: AY428072) downstream MF α 1 signal peptide is (Genbank number: M17301) and brewing yeast cell wall α agglutinin gene (Genbank number: M28164) AB000829.1) to insert GAL1 promotor among the yeast saccharomyces cerevisiae expression vector pGMAK; Make up and obtain AMS presenting and expressing frame (holding the C end) from N: GAL1 promotor+MF α 1 signal peptide+alpha-amylase gene+cell walls α agglutinin gene, concrete steps are following:
1. synthetic MF α 1 signal peptide (5 ' end band EcoR I site, 3 ' end band BamH I site), GAL1 promotor (GAL1p; 5 ' end band BamH I site, 3 ' end band Hind III site), alpha-amylase gene (aAM; 5 ' end band Hind III site; 3 ' end is with Sac I site) and brewing yeast cell wall α agglutinin gene C terminal sequence (α AGc, 5 ' end band Sac I site, 3 ' end band Sal I site).
2. BamH I enzyme is cut MF α 1 signal peptide sequence and GAL1p sequence, will be connected through the T4DNA ligase enzyme with the GAL1p sequence of gluing end with 5 ' BamH I with sticking terminal MF α 1 signal peptide sequence of 3 ' BamH I, becomes MF α 1-GAL1p chimeric sequences.
3. Hind III enzyme is cut MF α 1-GAL1p chimeric sequences and aAM sequence; To be connected through the T4DNA ligase enzyme with the aAM sequence of gluing end with 5 ' Hind III with the sticking terminal MF α 1-GAL1p chimeric sequences of 3 ' Hind III, become MF α 1-GAL1p-aAM chimeric sequences.
4. Sac I enzyme is cut MF α 1-GAL1p-aAM chimeric sequences and α AGc sequence; To be connected through the T4 dna ligase with the α AGc sequence of gluing end with 5 ' Sac I with the sticking terminal MF α 1-GAL1p-aAM chimeric sequences of 3 ' SacI; Become MF α 1-GAL1p-aAM-α AGc chimeric sequences, i.e. AMS presenting and expressing frame (holding the C end): GAL1 promotor+MF α 1 signal peptide+alpha-amylase gene+cell walls α agglutinin gene from N.
(2) structure of the recombinant Saccharomyces cerevisiae of presenting and expressing AMS
The recombinant plasmid transformed that will comprise AMS presenting and expressing frame is gone in the dGAL4 Δ GAL1 type brewing yeast cell, makes up recombinant Saccharomyces cerevisiae, specifically comprises:
1. the AMS presenting and expressing frame that step in the present embodiment (1) is made up is transformed into through electric shock in the dGAL4 Δ GAL1 type yeast of embodiment 1 structure, and the electric shock step of converting is seen embodiment 1.
2. after accomplishing the electric shock conversion; Yeast liquid coated to contain 5% farinose YPD dull and stereotyped; Be inverted for 30 ℃ and cultivated 2-3 days, bacterium colony the transparent circle occurs greatly and on every side, and the person is the dGAL4 Δ GAL1 type yeast (Sc-dGAL4 Δ GAL1-aAM-α AGc) of being with AMS presenting and expressing frame.
(3) presenting and expressing of AMS and enzyme activity determination
The recombinant Saccharomyces cerevisiae Sc-dGAL4 Δ GAL1-aAM-α AGc that above-mentioned steps (2) is made up is inoculated among the 10ml YEPD 30 ℃, 200rpm shaking culture 8-10h to OD
600Behind=the 1.3-1.5, get 1mL bacterium liquid and be inoculated in 5% and give birth in the YPD substratum of starch and 1% semi-lactosi, after 45 hours, alpha-amylase activity reaches 858U/mL in the culture system.
Wherein, the mensuration that the AMS enzyme is lived: AMS makes macromolecules starch be fractured into small molecules starch from giving birth to starch molecule inner cut-out glycosidic link, exposes more reducing end, so can estimate alpha-amylase activity through measuring the reducing sugar increasing amount.The alpha-amylase activity definition: to give birth to starch is substrate, and it is 1 alpha-amylase activity unit that 1mL enzyme liquid PM produces the required enzyme amount of 1 μ mol reducing sugar (with glucose meter), is designated as U.
The comparison that enzyme is lived behind the AMS presenting and expressing:
Embodiment 2 is according to the result of above-mentioned steps (1) to step (3); Present embodiment uses dGAL4 Δ GAL1 type yeast saccharomyces cerevisiae as the presenting and expressing host, and the AMS expression cassette is " a GAL1 promotor+MF α 1 signal peptide+alpha-amylase gene+cell walls α agglutinin gene "; Through measuring, the alpha-amylase activity of presenting and expressing reaches 858U/mL.
Comparative Examples 1 is according to the operation of step (1) to step (3) among the embodiment 2; Wherein, Use starting strain yeast saccharomyces cerevisiae ZD-1 as the presenting and expressing host; The AMS expression cassette is " a GAL1 promotor+MF α 1 signal peptide+alpha-amylase gene+cell walls α agglutinin gene ", and all the other steps are identical; Through measuring, the alpha-amylase activity of presenting and expressing reaches 335U/mL.
Comparative Examples 2 is according to the operation of step (1) to step (3) among the embodiment 2; Wherein, Use starting strain yeast saccharomyces cerevisiae ZD-1 as the secreting, expressing host, the AMS expression cassette is " a GAL1 promotor+MF α 1 signal peptide+alpha-amylase gene ", and all the other steps are identical; Through measuring, the alpha-amylase activity of secreting, expressing reaches 212U/mL.
Comparative Examples 3 is according to the operation of step (1) to step (3) among the embodiment 2; Wherein, Use dGAL4 Δ GAL1 type yeast saccharomyces cerevisiae as the secreting, expressing host, the AMS expression cassette is " a GAL1 promotor+MF α 1 signal peptide+alpha-amylase gene ", and all the other steps are identical; Through measuring, the alpha-amylase activity of secreting, expressing reaches 265U/mL.
The recombinant Saccharomyces cerevisiae of embodiment 3 surface display glucoamylases
(1) structure of glucoamylase presenting and expressing frame
With glucose amylase gene (from aspergillus oryzae Rhizopus oryzae; Genbank number: (Genbank number: AY428072) downstream MF α 1 signal peptide is (Genbank number: M17301) and brewing yeast cell wall α agglutinin gene (Genbank number: M28164) D00049.1) to insert GAL1 promotor among the yeast saccharomyces cerevisiae expression vector pGMAK; Make up and obtain glucoamylase presenting and expressing frame (holding the C end) from N: GAL1 promotor+MF α 1 signal peptide+glucose amylase gene+cell walls α agglutinin gene, concrete steps are following:
1. synthetic MF α 1 signal peptide (5 ' end band EcoR I site, 3 ' end band BamH I site), GAL1 promotor (GAL1p; 5 ' end band BamH I site, 3 ' end band Hind III site), glucose amylase gene (GLU; 5 ' end band Hind III site; 3 ' end is with Sac I site) and brewing yeast cell wall α agglutinin gene C terminal sequence (α AGc, 5 ' end band Sac I site, 3 ' end band Sal I site).
2. BamH I enzyme is cut MF α 1 signal peptide sequence and GAL1p sequence, will be connected through the T4DNA ligase enzyme with the GAL1p sequence of gluing end with 5 ' BamH I with sticking terminal MF α 1 signal peptide sequence of 3 ' BamH I, becomes MF α 1-GAL1p chimeric sequences.
3. Hind III enzyme is cut MF α 1-GAL1p chimeric sequences and GLU sequence; To be connected through the T4DNA ligase enzyme with the GLU sequence of gluing end with 5 ' Hind III with the sticking terminal MF α 1-GAL1p chimeric sequences of 3 ' Hind III, become MF α 1-GAL1p-GLU chimeric sequences.
4. Sac I enzyme is cut MF α 1-GAL1p-GLU chimeric sequences and α AGc sequence; To be connected through the T4 dna ligase with the α AGc sequence of gluing end with 5 ' Sac I with the sticking terminal MF α 1-GAL1p-GLU chimeric sequences of 3 ' SacI; Become MF α 1-GAL1p-GLU-α AGc chimeric sequences, i.e. glucoamylase presenting and expressing frame (holding the C end): GAL1 promotor+MF α 1 signal peptide+glucose amylase gene+cell walls α agglutinin gene from N.
(2) structure of the recombinant Saccharomyces cerevisiae of presenting and expressing glucoamylase
The recombinant plasmid transformed that will comprise glucoamylase presenting and expressing frame is gone in the dGAL4 Δ GAL1 type brewing yeast cell, makes up recombinant Saccharomyces cerevisiae, specifically comprises:
1. the glucoamylase presenting and expressing frame that step in the present embodiment (1) is made up is transformed into through electric shock in the dGAL4 Δ GAL1 type yeast of embodiment 1 structure, and the electric shock step of converting is seen embodiment 1.
2. after accomplishing the electric shock conversion; Yeast liquid coated to contain 5% farinose YPD dull and stereotyped; Be inverted for 30 ℃ and cultivated 2-3 days, bacterium colony the transparent circle occurs greatly and on every side, and the person is the dGAL4 Δ GAL1 type yeast (Sc-dGAL4 Δ GAL1-GLU-α AGc) of being with glucoamylase presenting and expressing frame.
(3) presenting and expressing of glucoamylase and enzyme activity determination
The recombinant Saccharomyces cerevisiae Sc-dGAL4 Δ GAL1-GLU-α AGc that above-mentioned steps (2) is made up is inoculated among the 10ml YEPD 30 ℃, 200rpm shaking culture 8-10h to OD
600Behind=the 1.3-1.5, get 1mL bacterium liquid and be inoculated in 5% and give birth in the YPD substratum of starch and 1% semi-lactosi, after 45 hours, the glucoamylase enzyme activity reaches 763U/mL in the culture system.
Wherein, The mensuration that the glucoamylase enzyme is lived: glucoamylase is through cutting off the saccharification of glycosidic link generation glucose from giving birth to the starch molecule end; The glucose band reducing end that forms is so can estimate the glucoamylase enzyme activity through measuring the reducing sugar increasing amount.The definition of glucoamylase enzyme activity: to give birth to starch is substrate, and it is 1 glucoamylase enzyme activity unit that 1mL enzyme liquid PM produces the required enzyme amount of 1 μ mol glucose, is designated as U.
The comparison that enzyme is lived behind the glucoamylase presenting and expressing:
Embodiment 3 is according to the result of above-mentioned steps (1) to step (3); Present embodiment uses dGAL4 Δ GAL1 type yeast saccharomyces cerevisiae as the presenting and expressing host, and the glucoamylase expression cassette is " a GAL1 promotor+MF α 1 signal peptide+glucose amylase gene+cell walls α agglutinin gene "; Through measuring, the glucoamylase enzyme activity of presenting and expressing reaches 763U/mL.
Comparative Examples 4 is according to the operation of step (1) to step (3) among the embodiment 3; Wherein, Use starting strain yeast saccharomyces cerevisiae ZD-1 as the presenting and expressing host; The glucoamylase expression cassette is " a GAL1 promotor+MF α 1 signal peptide+glucose amylase gene+cell walls α agglutinin gene ", and all the other steps are identical; Through measuring, the glucoamylase enzyme activity of presenting and expressing reaches 445U/mL.
Comparative Examples 5 is according to the operation of step (1) to step (3) among the embodiment 3; Wherein, Use starting strain yeast saccharomyces cerevisiae ZD-1 as the secreting, expressing host, the glucoamylase expression cassette is " a GAL1 promotor+MF α 1 signal peptide+glucose amylase gene ", and all the other steps are identical; Through measuring, the glucoamylase enzyme activity of secreting, expressing reaches 218U/mL.
Comparative Examples 6 is according to the operation of step (1) to step (3) among the embodiment 3; Wherein, Use dGAL4 Δ GAL1 type yeast saccharomyces cerevisiae as the secreting, expressing host, the glucoamylase expression cassette is " a GAL1 promotor+MF α 1 signal peptide+glucose amylase gene ", and all the other steps are identical; Through measuring, the glucoamylase enzyme activity of secreting, expressing reaches 324U/mL.
Claims (7)
1. diastatic recombinant Saccharomyces cerevisiae of surface display, the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) for containing recombinant plasmid is characterized in that the GAL1 gene of said yeast saccharomyces cerevisiae is replaced by the GAL4 gene; Described recombinant plasmid is made up of the amylase gene between the cell walls α agglutinin gene of GAL1 promotor downstream MF α 1 signal peptide and yeast saccharomyces cerevisiae among yeast saccharomyces cerevisiae expression vector pGMAK and the insertion yeast saccharomyces cerevisiae expression vector pGMAK.
2. recombinant Saccharomyces cerevisiae according to claim 1 is characterized in that, described amylase gene is alpha-amylase gene or glucose amylase gene.
3. the construction process of recombinant Saccharomyces cerevisiae as claimed in claim 1; It is characterized in that; May further comprise the steps: amylase gene is inserted between the cell walls α agglutinin gene of GAL1 promotor downstream MF α 1 signal peptide and yeast saccharomyces cerevisiae among the yeast saccharomyces cerevisiae expression vector pGMAK, make up and obtain recombinant plasmid; Adopt homologous recombination technique that the GAL1 gene of said yeast saccharomyces cerevisiae is replaced with the GAL4 gene, change said recombinant plasmid over to this cell, obtain recombinant Saccharomyces cerevisiae.
4. according to claim 1 or claim 2 recombinant Saccharomyces cerevisiae is in the application of preparation in the diastase.
5. application according to claim 4; It is characterized in that; May further comprise the steps: described recombinant Saccharomyces cerevisiae is inoculated in the YPD substratum that contains living starch and semi-lactosi carries out fermentation culture, fermentation culture is centrifugal collection thalline after 36～72 hours, makes diastase.
6. application according to claim 5 is characterized in that, by weight percentage, farinose concentration is 3-8% in the said YPD substratum, and the concentration of semi-lactosi is 0.5-2%.
7. application according to claim 5 is characterized in that, described fermented incubation time is 40-45 hour.
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| WO2003061596A2 (en) * | 2002-01-23 | 2003-07-31 | Gpc Biotech, Inc. | Methods and reagents for generating chimeric serum peptide carri ers |
| CN101864371A (en) * | 2010-05-18 | 2010-10-20 | 浙江大学 | Homologous recombination construction double-GAL4 genotype industrial saccharomyces cerevisiae and application thereof |
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