CN105018364B - One plant of saccharomyces cerevisiae engineered yeast for expressing pectinesterase and its application - Google Patents
One plant of saccharomyces cerevisiae engineered yeast for expressing pectinesterase and its application Download PDFInfo
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- CN105018364B CN105018364B CN201510413914.5A CN201510413914A CN105018364B CN 105018364 B CN105018364 B CN 105018364B CN 201510413914 A CN201510413914 A CN 201510413914A CN 105018364 B CN105018364 B CN 105018364B
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- saccharomyces cerevisiae
- pectinesterase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The saccharomyces cerevisiae engineered yeast for expressing pectinesterase the invention discloses one plant and application, belong to field of fermentation engineering.Saccharomyces cerevisiae engineered yeast provided by the present invention, it is transformed saccharomyces cerevisiae after merging the encoding gene of pectinesterase with α-agglutinin encoding gene, recombinant plasmid is integrated on the chromosome NDA of host, it obtains being anchored the bacterial strain of expression pectinesterase in cell surface, pectinesterase enzyme activity reaches 2.6U/g.Alcoholic fermentation production of the bacterial strain for raw materials such as sweet potato and cassavas, alcoholic fermentation efficiency can not only be improved, and can significantly reduce the viscosity of fermentation liquid in fermentation process, is conducive to the effect and saccharomyces cerevisiae metabolism of enzyme, facility load, energy saving can also be reduced.
Description
Technical field
The saccharomyces cerevisiae engineered yeast for expressing pectinesterase the present invention relates to one plant and its application, especially one are plant in cell table
The Wine brewing yeast strain of face anchoring expression pectinesterase and its application, belong to field of fermentation engineering.
Background technique
Environmental protection requirement in the increasingly exhausted and global range of fossil fuel, brings very well for fuel alcohol industry
Opportunity to develop.Fuel alcohol is a kind of reproducible clean energy resource, adds a certain proportion of alcohol in the oil, can be significant
Reduce pollution of the vehicle exhaust to environment.China is the big fuel alcohol main production country in third place in the world after the U.S., Brazil, annual output
Increase production up to 151.8 ten thousand tons, and year by year.Starchy material is the primary raw material for producing fuel alcohol, colloid, pectic substance etc. in raw material
Stickum is more, and the viscosity of mash is very big when high gravity fermentation, and fermentation liquid mass-and heat-transfer ability is poor, seriously affects the effect of enzyme
Effect and saccharomyces cerevisiae metabolic activity, influence ethanol production.It is negative that mash viscosity height can also block conveyance conduit, increase equipment operation
Load improves the processing difficulty of later period separation of solid and liquid, brings difficulty to the thick mash fermentation of alcohol.
Pectic substance is the important component of plant cell wall, with cellulose, hemicellulose and certain extensin phases
Mutually crosslinking plays the cyto-architectural effect of support, it is also the filler of cytoplasm, influences intercellular adhesion and tissue
Hardness.Pectinesterase is one kind of pectase, has application in industries such as weaving, papermaking, sewage treatments, special in food industry
It is in garden stuff processing using more, pectinesterase can keep the integrality of fruit, be conducive to the filtering and concentration of fruit juice, improve
Crushing juice rate.Pectinesterase can be also used for enzyme process preparation low-methoxyl base pectin, low methoxy pectin food have containing sugar less, heat it is low
The features such as, meet the requirement of healthy diet.
Summary of the invention
The present invention provides the saccharomyces cerevisiae engineered yeast that one plant first is expressed pectinesterase, the pectinesterases that has been amalgamation and expression
Encoding gene PE and α-agglutinin encoding gene AG α 1, obtain cell surface be anchored expression pectinesterase saccharomyces cerevisiae
Engineering bacteria.
In one embodiment of the invention, the nucleotide sequence of the encoding gene PE of the pectinesterase is such as
Shown in GenBank XM_001390469.
In one embodiment of the invention, the nucleotide sequence of the encoding gene AG α 1 of the α-agglutinin is such as
Shown in GenBank M28164.
In one embodiment of the invention, the encoding gene PE of pectinesterase and α-agglutinin encoding gene AG α 1
Pass through saccharomyces cerevisiae phosphoglycerokinase PGK promoter integrant expression in saccharomyces cerevisiae genome；With alpha factor signal peptide
Instruct the positioning of protein.
In one embodiment of the invention, the encoding gene PE of pectinesterase and α-agglutinin encoding gene AG α 1
By with saccharomyces cerevisiae phosphoglycerokinase PGK promoter, G418 resistant gene, for Homologous integration site sequence load
Body pMD18-T, which is integrated into saccharomyces cerevisiae genome, carries out integrant expression.
The pectinesterase enzyme activity of the saccharomyces cerevisiae engineered yeast is 2.6U/g wet thallus.
The present invention also provides a kind of methods using above-mentioned saccharomyces cerevisiae engineered yeast production alcohol, are with starchy material
Fermentation medium is prepared, the saccharomyces cerevisiae engineered yeast, static fermentation are inoculated with.
It in one embodiment of the invention, is that fermentation medium is prepared with tapioca starch or/and corn flour, described in inoculation
Saccharomyces cerevisiae engineered yeast, static fermentation.
In one embodiment of the invention, the method that fermentation medium is prepared with tapioca starch or/and corn flour
Are as follows: tapioca starch or/and corn flour are mixed with deionized water in the ratio of 1:3 (g/mL), adjust pH to 6.0, the resistance to height of 10U/g is added
Warm alpha amylase heats feed liquid to 95 DEG C, maintains 2h；It is cooled to room temperature and adjusts pH to 4.5, add deionized water to make up moisture damage
It loses.The urea of 130U/g carbohydrase and 0.05% is added in high pressure steam sterilization after cooling.
Saccharomyces cerevisiae engineered yeast provided by the present invention is sent out using tapioca starch or/and corn flour as the synchronous saccharification of raw material
When ferment produces alcohol, the speed of growth is higher than starting strain, while alcohol output compares starting strain and improves 2.2%.It is even more important
Be that the pectin esterase activity of surface display being capable of pectin object effectively in degradation material in saccharomyces cerevisiae engineered yeast fermentation process
Matter, to significantly reduce fermentation broth viscosity.By taking the 12h that ferments as an example, the fermentation broth viscosity of recombination yeast is 120mPa.s, compared to out
The fermentation broth viscosity 145mPa.s of bacterium germination strain reduces 20.8%, and the reduction of viscosity is conducive to effect and the saccharomyces cerevisiae generation of enzyme
It thanks, facility load, energy saving can also be reduced.
Detailed description of the invention
Fig. 1 is the pectinesterase gene PCR product and recombinant plasmid pMGK-AG-PE digestion products electrophoretogram of band signal peptide
Fig. 2 is the PCR products electrophoresis map of the chromosomal DNA of Saccharomyces cerevisiae transformant
Fig. 3 is the glucose and alcohol variation diagram in saccharomyces cerevisiae engineered yeast PE fermentation process
Fig. 4 is the viscosity change of fermentation liquid during saccharomyces cerevisiae engineered yeast PE ferments
Specific embodiment
The measurement of the enzyme activity of pectinesterase: alkali titration, concrete operations: by 37 DEG C of pectin solution of 2.5mL 1% are used
10min is preheated, 500 μ L enzyme solutions to be measured are added, 37 DEG C of reaction 30min boil 15min inactivation, with 0.02molL-1NaOH titration
To pH 8.0.Enzyme activity is defined as: under the conditions of 37 DEG C, 5.0 pH, act on enzyme amount needed for pectin generates 1 μm of ol carboxyl per minute
It is defined as 1 enzyme activity unit.
Seed culture based formulas is as follows: 2% glucose, 0.85% yeast powder, 0.13% ammonium chloride, 0.01% magnesium sulfate,
0.006% calcium chloride, 115 DEG C of high pressure steam sterilizations.
Fermentation medium preparation method is as follows: mixing sweet potato flour with deionized water in the ratio of 1:3 (w/v), adjusts pH extremely
6.0, it is added thermostable α-amylase (10U/g).Feed liquid is heated to 95 DEG C, maintains 2h.It is cooled to room temperature and adjusts pH to 4.5, addition is gone
Ionized water is to make up moisture loss.Carbohydrase (130U/g) and urea (final concentration is added in 121 DEG C of high pressure steam sterilizations after cooling
0.05%).
The analysis of the consumption of glucose and alcohol in fermentation process: efficient liquid phase chromatographic analysis, chromatographic column Aminex are used
HPX-87H ion exchange column, mobile phase are 10mmol/L H2SO4, flow velocity 0.8mL/min, 65 DEG C of column temperature；Ferment initial total reducing sugar
It is measured using acid-hydrolysis method, DNS method surveys reduced sugar, with formula: fermentation efficiency=actual measurement fermentation alcohol concentration/(initial total sugar is dense
Degree * 0.511) * 100% calculating fermentation efficiency.
Embodiment 1 constructs saccharomyces cerevisiae engineered yeast (Saccharomyces cerevisiae) PE
1, the building of the recombinant plasmid pMGK-AG-PE containing pectinesterase encoding gene
According to kit operating procedure, the total serum IgE of aspergillus niger is extracted with kit and as template, reverse transcription synthesis one
Chain cDNA, according to PE gene order (No. GenBank is XM_001390469) design primer PEf:
ATGGTTAAGTCAATTCTTGCATCCGT and PEr:TTAGTTGATGTAGCTAG, and using a chain cDNA as template,
PCR amplification is free of the complete pectinesterase gene of native signal peptide, is inserted into the site SnaBI of carrier pPIC9K, is recombinated
PPIC9k-PE.Upstream primer PE1 is designed according to the nucleotide sequence of the alpha factor signal peptide in pPIC9K, according to PE
Gene order (No. GenBank is XM_001390469), designs downstream primer PE2, introduces EcoR I restriction enzyme site, primer sequence
It is as follows
Upstream primer PE1:CCGGAATTCCGATGAGATTTCCTTCAA
Downstream primer PE2:CCGGAATTCTTAGTTGATGTAGCTAG, using recombinant plasmid pPIC9k-PE as template, with
PE1 and PE2 is upstream and downstream primer, carries out PCR amplification, obtains the pectinesterase gene sPE of the factor signal peptide containing alpha.
It will be connected after the pcr amplification product segment of α-agglutinin encoding gene AG α 1 and carrier pMGK I digestion of sal,
Obtain recombinant plasmid pMGK-AG.It is the carrier that sets out that the building of carrier pMGK, which is with pMD18-T, is inserted into wine brewing ferment in I site sal
Female phosphoglycerokinase PGK promoter is inserted into G418 resistant gene in I site Not, is inserted into one section of saccharomyces cerevisiae in I site Nde
RDNA segment as Homologous integration site.The specific building process of carrier pMGK is the same as Zhong-peng Guo et
al.Improving the performance of industrial ethanol-producing yeast by
expressing the aspartyl protease on the cell surface.Yeast.2010Dec；27(12):
The construction method for the pMGKR that the right column of page 1018 of 1017-27 is recorded.
EcoR I digestion is all used to handle sPE and pMGK-AG, the connection overnight of 16 DEG C of T4 ligase, construction of expression vector
pMGK-AG-PE.Digestion verification, the result is shown in Figure 1 are carried out using restriction enzyme Hind III, digestion obtains size about
The band of 1060bp, 3700bp and 5700bp, suitable with expection, digestion verification result proves that the structure of pMGK-AG-PE is correct.Fig. 1
In, swimming lane M is DL 15000DNA Marker, and swimming lane 1 is the PCR product of sPE, and swimming lane 2 is the plasmid after EcoR I digestion
PMGK-AG, swimming lane 3 are the recombinant plasmid pMGK-AG-PE after Hind III digestion.
2, recombinant plasmid pMGK-AG-PE conversion wine brewing ferment
Industrial saccharomyces cerevisiae will be converted after recombinant plasmid pMGK-AG-PE Sac II linearization for enzyme restriction, conversion fluid coating adds
Added the YPD solid plate of 300 μ g/mL G418, random picking transformant extracts chromosomal DNA, using PE1 and PE2 as primer into
Row PCR amplification.PCR product is carried out to 1% agarose gel electrophoresis, electrophoresis result is as shown in Fig. 2, obtain a treaty 1270bp
Specific band, it is identical as the size of pectinesterase gene, show that pectinesterase gene is successfully integrated on yeast chromosomal.
In Fig. 2, swimming lane M is DL 15000DNA Marker, and swimming lane 1-3 is to produce by the PCR of template of the chromosomal DNA of yeast transformant
Object, swimming lane 4 are to go out bacterium germination chromosomal DNA as the PCR product of template, and swimming lane 5 is using plasmid pMGK-AG-PE as the PCR of template
Product.
Picking positive transformant and starting strain are inoculated in YPD culture medium respectively, after cultivating 16h, collect cell, carry out fruit
Glue esterase enzyme activity determination.Enzyme activity determination uses alkali titration, concrete operations: by 37 DEG C of the pectin solution preheatings of 2.5mL 1%
10min, is added 500 μ L bacteria suspensions to be measured, and 37 DEG C of reaction 30min boil 15min inactivation, with 0.02molL-1NaOH is titrated to
pH 8.0.Enzyme activity is defined as: under the conditions of 37 DEG C, 5.0 pH, it is fixed to act on enzyme amount needed for pectin generates 1 μm of ol carboxyl per minute
Justice is 1 enzyme activity unit.
The highest transformant of pectinesterase enzyme activity is saccharomyces cerevisiae engineered yeast PE, and enzyme activity is 2.6U/g (thallus weight in wet base).
Pectinesterase enzyme activity is not measured in starting strain, illustrates pectinesterase gene integration on yeast chromosomal and successful expression；Turn
Beggar's cell re-suspension liquid is after ultrasonic disruption, and enzyme activity is 2.2U/g in precipitating, does not detect enzyme activity in supernatant, illustrates fruit
Glue esterase is anchored in yeast cell surface and expresses.
Embodiment 2 produces alcohol using saccharomyces cerevisiae engineered yeast PE fermentation sweet potato flour
Saccharomyces cerevisiae engineered yeast PE and starting strain are inoculated in 20mL seed culture medium, 30 DEG C of 200r/min cultures respectively
12h, new seed culture medium of being transferred with 1% inoculum concentration, 30 DEG C of 200r/min cultivate 18h, the seed liquor as fermenting experiment.
Fermentation medium liquid amount is 135mL, inoculum concentration 10%, 30 DEG C of static fermentations.To guarantee data reliability, every group of experiment is done
Three parallel, samples every 6h, and ferment 42h.In addition, the effect for verifying pectinesterase in alcoholic fermentation, to starting strain
Alcoholic fermentation has carried out the experiment of external source addition pectinesterase, and fermentation process is same as above.
(1) fermentation situation of the alcohol output saccharomyces cerevisiae engineered yeast PE in sweet potato flour culture medium is as shown in figure 3, fermentation
36h, recombination yeast PE alcohol output are 95g/L, and the alcohol output (93g/L) compared to starting strain improves 2.2%.Fermentation is just
The total sugar concentration of beginning is 211g/L, and the fermentation efficiency of recombination yeast PE and starting strain is respectively 88.11% and 86.25%；Hair
8h before ferment, concentration of glucose have obvious rising, this is because carbohydrase continued hydrolysis amylodextrin, produces glucose, recombinate ferment
Glucose consumption rate in female PE fermentation process is slightly faster than starting strain.
External source adds starting strain fermentation situation and similar, the alcohol output of saccharomyces cerevisiae engineered yeast PE performance of pectinesterase
Starting strain is slightly faster than with glucose consumption rate consumption sugar.
(2) viscosity change of fermentation liquid is as shown in Figure 4 during fermentation liquid viscosity saccharomyces cerevisiae engineered yeast PE fermentation.Hair
Ferment early period, due to the consumption of sugar, fermentation broth viscosity is all declined, identical fermentation time, the fermentation broth viscosity of recombination yeast PE
Significantly lower than starting strain, the fermentation broth viscosity of the starting strain of external source addition pectinesterase is similarly in reduced levels, explanation
Pectinesterase can play the role of viscosity reduction in sweet potato alcoholic fermentation process.The reduction of viscosity is conducive to the abundant of enzyme-to-substrate that be saccharified
Contact, the slightly higher reason of sugared concentration in recombination yeast PE fermentation liquid when this may be fermentation 6h；The reduction of viscosity is also beneficial to ferment
Female metabolism, therefore the phase is all slightly faster than starting strain, fermentation efficiency after fermentation for the sugar consumption of recombination yeast PE and Alcohol Production
Also some higher.
As it can be seen that saccharomyces cerevisiae engineered yeast in the simultaneous saccharification and fermentation using sweet potato flour as raw material, is shown better than bacterium germination out
The fermenting property of strain, growth rate and alcohol output all increase, and the viscosity of fermentation liquid reduces in fermentation process.The drop of viscosity
The low mass transfer for being conducive to fermentation liquid and heat transfer facilitate the effect and saccharomyces cerevisiae metabolism of enzyme, can also reduce facility load, save
About energy consumption.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (5)
1. the saccharomyces cerevisiae engineered yeast of one plant of expression pectinesterase, which is characterized in that the coding for the pectinesterase that has been amalgamation and expression
Gene PE and α-agglutinin encoding gene AG α 1 obtains being anchored the saccharomyces cerevisiae engineering of expression pectinesterase in cell surface
Bacterium；
The nucleotide sequence of the encoding gene PE of the pectinesterase is as shown in GenBank XM_001390469；
The nucleotide sequence of the encoding gene AG α 1 of the α-agglutinin is as shown in GenBank M28164；
Encoding gene PE and α-agglutinin encoding gene AG α 1 of pectinesterase is opened by saccharomyces cerevisiae phosphoglycerokinase PGK
Mover integrant expression in saccharomyces cerevisiae genome；The positioning of protein is instructed with alpha factor signal peptide.
2. application of the saccharomyces cerevisiae engineered yeast described in claim 1 in alcoholic fermentation.
3. a kind of method using the production alcohol of saccharomyces cerevisiae engineered yeast described in claim 1, which is characterized in that be with starchiness
Raw material prepares fermentation medium, is inoculated with the saccharomyces cerevisiae engineered yeast, static fermentation.
4. according to the method described in claim 3, it is characterized in that, being to prepare fermentation medium with tapioca starch or/and corn flour.
5. according to the method described in claim 4, it is characterized in that, using tapioca starch or/and corn flour as the fermented and cultured of substrate
The preparation method of base are as follows: corn flour or/and tapioca starch are mixed with water by the liquid ratio of 1:3, adjust pH to 6.0,10U/g is added
Thermostable α-amylase heats feed liquid to 95 DEG C, maintains 2h；It is cooled to room temperature and adjusts pH to 4.5, add deionized water to make up water
Divide loss；The urea of 130U/g carbohydrase and final concentration 0.05% is added in high pressure steam sterilization after cooling.
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| CN102206688A (en) * | 2011-04-27 | 2011-10-05 | 浙江大学 | Method for catalytically synthesizing conjugated linoleic acid by saccharomyces cerevisiae display linoleic acid isomerase |
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| CN102206688A (en) * | 2011-04-27 | 2011-10-05 | 浙江大学 | Method for catalytically synthesizing conjugated linoleic acid by saccharomyces cerevisiae display linoleic acid isomerase |
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| Simultaneous saccharification and fermentation of solid household waste following mild pretreatment using a mix of hydrolytic enzymes in combination with Saccharomyces cerevisiae;A. Nwobi et al.;《Appl Microbiol Biotechnol》;20140902;第99卷;第929-938页 |
| 酒精高浓发酵过程中果胶酶应用的研究;王晓霞等;《食品与发酵工业》;20010420;第27卷(第3期);第44页左栏第1段，第46页右栏第4段 |
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