CN108410746A - It is a kind of can efficient-decomposition, convert cellulose saccharomyces cerevisiae and stalk fermentation method - Google Patents
It is a kind of can efficient-decomposition, convert cellulose saccharomyces cerevisiae and stalk fermentation method Download PDFInfo
- Publication number
- CN108410746A CN108410746A CN201810113730.0A CN201810113730A CN108410746A CN 108410746 A CN108410746 A CN 108410746A CN 201810113730 A CN201810113730 A CN 201810113730A CN 108410746 A CN108410746 A CN 108410746A
- Authority
- CN
- China
- Prior art keywords
- saccharomyces cerevisiae
- gene expression
- cellulose
- fermentation
- expression cassette
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
- C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
-
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Sustainable Development (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to it is a kind of can efficient-decomposition, convert cellulose saccharomyces cerevisiae, it is transferred to endo glucanase gene expression cassette, exoglucanase gene expression frame and β glucosidase gene expression frames in the saccharomyces cerevisiae, realizes coexpression of three kinds of cellulases in same saccharomyces cerevisiae；The method using above-mentioned fermentation by saccharomyces cerevisiae cellulose is further related to, is included the following steps：The saccharomyces cerevisiae is inoculated in YPD fluid nutrient mediums, in 28 DEG C of overnight incubations to activate；The saccharomyces cerevisiae of activation is forwarded in YPD fluid nutrient mediums, 28 DEG C of cultures obtain fermentation seed liquid to suitable concentration；The fermentation seed liquid is added to 25 DEG C of fermentations in the round equipped with the biomass containing cellulose.By using above-mentioned yeast strain and fermentation process, can high-efficiency fermenting cellulose obtain ethyl alcohol and high protein feed additive.
Description
Technical field
The present invention relates to cellulose field of bioconversion, more specifically it relates to which one kind can efficient-decomposition and trans-utilization fibre
The method for tieing up the saccharomyces cerevisiae and fermented cellulosic biolobic material of element.
Background technology
Cellulose is a kind of reproducible organic resource, it is widely distributed, obtains and is easy.It can be from timber, weeds, agriculture
The acquisitions such as industry residue and municipal solid waste.Currently, fossil fuel as the main energy real life and production in quilt
It utilizes extensively.With increasing for population, the demand of fuel is growing, and the exhaustion of fossil resource is on the rise.In addition, fossil provides
CO is generated after the burning of source2It influences caused by global climatologic change that people is allowed to get over clean reproducible energy with other greenhouse gases
More to pay close attention to.Since cellulose has the advantages that widely distributed and inexpensive, it is considered as, and a kind of be used for producing well can be again
The resource of the raw energy such as ethyl alcohol.
Due to the design feature of itself, cellulose is difficult to be degraded, this becomes its a great problem as bioenergy.This
Outside, the feed matter that prepared by the stalk rich in the non-starch substance for being not easy to be digested such as cellulose, hemicellulose and lignin etc.
Ground is thick and stiff, palatability is poor, livestock feed intake is small, digestibility is low.
In order to improve the nutritive value and utilization rate of stalk, generally improved using the method for microbial fermentation single in feed
Cellular protein content is to achieve the purpose that straw feed.Therefore the degradation efficiency of native cellulose is improved for wood fibre
Application of the element in terms of bioenergy and straw feed has vital meaning.
Cellulase system in organism is mainly made of the different enzyme of three classes function：(1) endoglucanase or Isosorbide-5-Nitrae-
Callose -4- glucan hydrolases；(2) exoglucanase includes Isosorbide-5-Nitrae-callose hydrolase (including fibre
Tie up dextromase) and Isosorbide-5-Nitrae-callose cellobiohydrolase；(3) beta-glucosidase or beta-glucosidase hydrolase.
There is studies have shown that cellulose that can be degraded by synchronous synergetic effect by endoglucanase and exoglucanase
At cellobiose and cell-oligosaccharide (wherein endoglucanase provides for exoglucanase the free chain end of attack), β-Portugal
The soluble cellodextrin of glucosides enzyme hydrolysis and cellobiose finally generate glucose.Therefore, this three classes cellulase is added simultaneously
To hydrolysis efficiency can be improved in hydrolyzation system, has the more document reports phenomenons.
The engineering bacteria of existing structure simple function cellulase at present, then by mixed fermentation to improve natural fiber
The degradation of element and the method for Transform efficiency.Although mixed fermentation improves degradation and the transformation efficiency of native cellulose,
But the difficulty of zymotechnique control is also increased simultaneously, application also has significant limitation.
The present invention is obtained by the cellulase of three kinds of different function in the coexpression cellulase system in same saccharomyces cerevisiae
To multi-functional genetic engineering saccharomyces cerevisiae, single bacterial strain fermentation is realized, improving its degradation and conversion to native cellulose
The difficulty of zymotechnique control is not increased while efficiency yet.
Invention content
Inventor has found that (GenBank is numbered by the eg2 from Stachybotrys chartarum in the course of the research：AM180511), come
From the cbh2 of yellow limb reticulitermes flavipe, (GenBank is numbered：KC751534) and the bg2 from Sphingol single-cell (GenBank compile
Number：JF308485 it) is series on a PGK plasmid, and after being transferred to saccharomyces cerevisiae, cellulase filter paper enzyme activity and wine when fermentation
Smart fermentation efficiency is above the saccharomyces cerevisiae mixed fermentation for being only transferred to individual gene.
Based on this, the present invention provides a kind of saccharomyces cerevisiae of decomposable asymmetric choice net cellulose, inscribe is transferred in the saccharomyces cerevisiae
Glucanase gene expression cassette, exoglucanase gene expression frame and β-glucosyl enzym gene expression frame.
In a preferred embodiment, the endo glucanase gene expression cassette, exoglucanase gene expression
Frame and β-glucosyl enzym gene expression frame are located on the same plasmid.
In a preferred embodiment, the endo glucanase gene expression cassette, exoglucanase gene expression
Frame and β-glucosyl enzym gene expression frame share a promoter, and the promoter can be such as PGK promoters.
In a preferred embodiment, the endo glucanase gene is the eg2 from Stachybotrys chartarum,
GenBank numbers are AM180511；The exoglucanase gene is the cbh2 from yellow limb reticulitermes flavipe, GenBank numbers
For KC751534；The β-glucosyl enzym gene is the bg2 from Sphingol single-cell, and GenBank numbers are JF308485.Through
Several genes combination experiment shows that the synergy of these three assortments of genes coexpression is best.
In a preferred embodiment, the saccharomyces cerevisiae is I NVSc bacterial strains.We attempt during the experiment
Multiple bacterial strains, as a result, it has been found that INVSc bacterial strains are most suitable for for expressing above three gene, and the biology containing cellulose that ferments
Matter.
The present invention also provides a kind of methods of biomass of the fermentation containing cellulose, including above-mentioned saccharomyces cerevisiae is used to send out
Ferment contains the step of biomass of cellulose.
In one embodiment, include the following steps：
S1：The saccharomyces cerevisiae is inoculated in YPD fluid nutrient mediums, in 28 DEG C of overnight incubations to activate, obtains yeast
Activation culture object；
S2：The saccharomyces cerevisiae activation culture object is forwarded to by 0.5% volume ratio in YPD fluid nutrient mediums, 28 DEG C
Culture 2 days, obtains fermentation seed liquid；
S3：The fermentation seed liquid is added in the round equipped with the biomass containing cellulose, in 25 DEG C of hairs
Ferment, 4-5 days alcohol fermentation period, straw feedization are fermented 8-15 days.
In one embodiment, the biomass containing cellulose is stalk.
In a preferred embodiment, the length of the stalk is 0.5-1cm, the ratio of the stalk and fermentation seed liquid
Example is 200g stalks/100ml fermentation seed liquids.
By using above-mentioned yeast strain and fermentation process, can high-efficiency fermenting cellulose, obtain ethyl alcohol and high protein feed
Additive.
Description of the drawings
Fig. 1 is the collection of illustrative plates of co-expression plasmid；
Fig. 2 is the SDS-PAGE electrophoresis dying photos for the supernatant for co-expressing strain culture；
Fig. 3 is Wine brewing yeast strain using filter paper as the growth curve in the culture medium of sole carbon source.
Specific implementation mode
Principles and features of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
1. co-expressing strain construction
Synthesize following gene：(GenBank is numbered eg2 from Stachybotrys chartarum：AM180511 yellow limb reticulitermes flavipe), is come from
Cbh2 (GenBank number：KC751534) and the bg2 from Sphingol single-cell (GenBank number：JF308485).
These three genes are cloned into respectively on the pHBM368-pgk plasmids in this laboratory, 3 recombinant bacterial strain pHBM368- are built into
Pgk-EG2, pHBM368-pgk-CBH2 and pHBM368-pgk-BG2.Using these three plasmids as template, 3 are cloned respectively by PCR
Kind cellulose enzyme gene simultaneously imports Xba I and EcoR I restriction enzyme sites, passes through corresponding restriction enzyme and T4DNA
Ligase successfully builds coexpression recombinant plasmid pHBM368-pgk-ECB (plasmid is as shown in Figure 1).
By in plasmid pHBM368-pgk-ECB electrotransformations to saccharomyces cerevisiae INVSc bacterial strains, the saccharomyces cerevisiae after conversion is applied
Cloth is on the SC culture mediums without uracil.Saccharomyces cerevisiae INVSc bacterial strains are uracil-deficient, and are co-expressed on recombinant plasmid
Containing uracil selection markers, if in recombinant plasmid successful conversion to saccharomyces cerevisiae, recombinant bacterium can be in the SC without uracil
It is grown on culture medium.In order to further verify recombinant bacterial strain, using weight of the panel function identification method verification with cellulase
Group bacterial strain.Obtained conversion bacterial strain energy normal growth on the SC tablets using CMC-Na as sole carbon source, over time also
It can see hydrolysis circle, illustrate that these recombinant bacterial strains being capable of secreting, expressing cellulase.By obtained coexpression endoglucanase,
The Wine brewing yeast strain of exoglucanase and β-glucosyl enzym is named as INVSc-pHBM368-pgk-ECB.Collect the bacterial strain
The supernatant of culture carries out SDS-PAGE electrophoresis, it is seen that three bands (Fig. 2) of 36.5kD, 48kD and 59kD or so, with
The size of tri- enzymes of EG2, CBH2 and BG2 is corresponding, it is seen that the bacterial strain can secrete these three enzymes.
2. Enzyme activity assay is tested
Simulation fermentation is carried out by sole carbon source of filter paper, detects enzyme activity.Steps are as follows：
1) the recombination yeast single bacterium colony obtained after picking screening, is inoculated into the 250mL equipped with 50mL YPD fluid nutrient mediums
It is activated in conical flask, 28 DEG C, 200rpm is incubated overnight；
2) bacterium solution after activation is taken (to replace the YPD Liquid Cultures of glucose with filter paper to equipped with the corresponding culture mediums of 100mL
Base) 500mL conical flasks in, keep bacterium solution initial concentration consistent, continuous culture a period of time, timing sampling；
3) take in 150 μ L bacterium solutions to ELISA Plate, at 600nm measure absorbance value, do every time 3 it is parallel.
(cellulase single-gene is expressed respectively using same way with wild type INVSc and term single gene recombinant bacterial strain
The saccharomyces cerevisiae of eg2, cbh2 and bg2) it is control.The results are shown in Figure 3, and wild type INVSc is hardly grown in culture solution,
The growth curve of coexpression bacterial strain is substantially better than the growth curve of single-gene expression bacterial strain.The bacterial strain is in 50 DEG C, 5.5 conditions of pH
Under, showed highest enzyme activity when using filter paper as substrate at 36 hours, filter paper enzyme activity reaches 68.3U/ml, and single-gene recombinant bacterium
The highest filter paper enzyme activity of strain is respectively 23.2U/ml, 21.6U/ml and 30.3U/ml.
Filter paper enzyme activity co-expresses bacterium as important parameter index of the cellulase to native cellulose degradation validity is judged
The filter paper enzyme activity of strain shows co-expressed in the saccharomyces cerevisiae three kinds of fibers far above the filter paper enzyme activity of single-gene expression bacterial strain
There is plain enzyme significant synergistic effect, degradation to be substantially better than the engineering bacteria of expression Single Fiber element enzyme gene.
3. stalk fermentation is tested
1) it activates：Wine brewing yeast strain is inoculated into respectively on YPD fluid nutrient mediums, 28 DEG C of insulating box overnight incubations；
2) expand culture：Activated bacterial strain is inoculated into respectively in 100mLYPD fluid nutrient mediums, shaking table 220r/min,
28 DEG C are cultivated 2 days, and OD is surveyed600Value, obtains fermentation seed liquid；
3) it is inoculated with：Cultured bacterial strain is inoculated into the rice straw that the length dried equipped with 200g is 0.5-1cm respectively
Or in the jar fermenter of maize straw so that initial inoculum is consistent；
4) it ferments：Jar fermenter is placed in 25 DEG C of cultures, 4-5 days alcohol fermentation period, straw feedization is fermented 8-15 days.
3.1 INVSc-pHBM368-pgk-ECB ethanol productions measurement results and analysis
Since ethyl alcohol does not have absorption peak, this experiment to be given by carrying out pre-column derivatization to ethyl alcohol under UV detector
It adds the group with ultraviolet absorption peak, and measures the ethanol content in zymotic fluid by high performance liquid chromatograph and change, color
Spectrum column is 5 μm of 4.6 × 150mm of Agilent companies C18 columns, and mobile phase is 0.01M potassium dihydrogen phosphates：Acetonitrile=65:35
(pH2.5), flow velocity 0.5mL/min, column temperature are 25 DEG C, select the detection of Agilent UV detector, are detected at wavelength 227nm.
Ethanol content testing result such as table 1, INVSc wild-type strains are using maize straw as in fermenting substrate, 24 hours highest ethyl alcohol
Content 615mg/L, coexpression recombinant bacterial strain INVSc-pHBM368-pgk-ECB reached in 24 hours highest ethanol contents
1040mg/L, and utilize in the saccharomyces cerevisiae mixed fermentation system of expression cellulase single-gene eg2, cbh2 and bg2 respectively,
24 hours highest ethanol content 703mg/L.INVSc bacterial strains theoretically directly cannot utilize maize straw to ferment, but in this experiment
Not pretreated maize straw is used in system, there are part fermentable sugars to lead to having a small amount of ethyl alcohol during the fermentation
It generates, but the later stage bacterial strain that ferments is gradually dead, subsequent performance goes out ethanol content decline.
The ethanol content statistics of 1 maize straw of table fermentation
As shown in table 1, coexpression recombinant bacterial strain INVSc-pHBM368-pgk-ECB being capable of 3 kinds of different fibres of secreting, expressing
The plain enzyme of dimension, sustainable degrading maize straws generate the carbon source that can be utilized by saccharomyces cerevisiae so that recombinant bacterial strain obtains further
Growth, to generate more ethyl alcohol.Ethanol content improves 69.11% compared with INVSc groups, the mixing with expression term single gene
Bacterium fermentation compares ethanol content and improves 47.93%.The recombinant Saccharomyces cerevisiae of three kinds of cellulases of coexpression shows better second
Alcohol generates ability, and operating procedure is simple compared with mixed fermentation.
Crude protein content measurement result and analysis in 3.2 tunnings
After maize straw ferments 15 days, tunning is dried, accurately weighs 1.000g (being accurate to 0.002g) fermentation productions
Object measures crude protein content after digesting and being catalyzed reaction.
Assay method is as follows：Nitrogenous organic compound such as protein passes through Sulfuric-acid-hydrogen-peroxide in fermentation product samples
Disappear and be hydrolyzed into amino acid after boiling under the action of the concentrated sulfuric acid, amino acid is reduced ammonification under the deamination of sulfuric acid again, finally
It is combined with sulfuric acid and generates ammonium sulfate.For the ammonium sulfate of generation after hypochlorite and phenol reactant under alkaline condition, formation can
The dye indophenol of dissolubility is blue, finally by the depth of solution blue at wavelength 630nm colorimetric estimation ammonia nitrogen concentration, according to ammonium
The conversion coefficient of state nitrogen and crude protein obtains crude protein content.
Measurement result is as shown in table 2, and the crude protein content of blank control group is 1.92%, the crude protein content of wild type group
It is 3.24%, the crude protein content of coexpression group is 4.39%, relative to using for INVSc host strains, co-expresses cellulose
The unicellular comparison content raising 35.49% in being converted to corn straw feedstuffization of the bacterial strain of enzyme.
Crude protein content after 2 maize straw of table ferments 15 days counts
It can be seen that the bacterial strain of coexpression cellulase is also significantly high to the raising effect of protein content in straw feed
In wild-type strain.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Claims (10)
1. it is a kind of can efficient-decomposition, convert cellulose saccharomyces cerevisiae, which is characterized in that be transferred to inscribe in the saccharomyces cerevisiae
Glucanase gene expression cassette, exoglucanase gene expression frame and β-glucosyl enzym gene expression frame.
2. saccharomyces cerevisiae according to claim 1, which is characterized in that the endo glucanase gene expression cassette, circumscribed
Glucanase gene expression cassette and β-glucosyl enzym gene expression frame are located on the same plasmid.
3. saccharomyces cerevisiae according to claim 2, which is characterized in that the endo glucanase gene expression cassette, circumscribed
Glucanase gene expression cassette and β-glucosyl enzym gene expression frame share a promoter.
4. saccharomyces cerevisiae according to claim 3, which is characterized in that the promoter is PGK.
5. saccharomyces cerevisiae according to claim 1, which is characterized in that the endo glucanase gene is from paper grape
Fringe mould eg2, GenBank number is AM180511；The exoglucanase gene is the cbh2 from yellow limb reticulitermes flavipe,
GenBank numbers are KC751534；The β-glucosyl enzym gene is the bg2 from Sphingol single-cell, and GenBank numbers are
JF308485。
6. saccharomyces cerevisiae according to any one of claims 1-5, which is characterized in that the saccharomyces cerevisiae is INVSc bacterium
Strain.
7. a kind of method of biomass of the fermentation containing cellulose, which is characterized in that described in any one of claim 1-6
Fermentation by saccharomyces cerevisiae the step of containing the biomass of cellulose.
8. the method according to the description of claim 7 is characterized in that including the following steps：
S1：The saccharomyces cerevisiae is inoculated in YPD fluid nutrient mediums, in 28 DEG C of overnight incubations to activate, obtains activated yeast
Culture；
S2：The saccharomyces cerevisiae activation culture object is forwarded to by 0.5% volume ratio in YPD fluid nutrient mediums, 28 DEG C of cultures 2
It, obtains fermentation seed liquid；
S3：The fermentation seed liquid is added in the round equipped with the biomass containing cellulose, in 25 DEG C of fermentations.
9. according to the method described in claim 8, it is characterized in that, the biomass containing cellulose is stalk.
10. according to the method described in claim 9, it is characterized in that, the length of the stalk be 0.5-1cm, the stalk with
The ratio of fermentation seed liquid is 200g stalks/100ml fermentation seed liquids.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810113730.0A CN108410746A (en) | 2018-02-05 | 2018-02-05 | It is a kind of can efficient-decomposition, convert cellulose saccharomyces cerevisiae and stalk fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810113730.0A CN108410746A (en) | 2018-02-05 | 2018-02-05 | It is a kind of can efficient-decomposition, convert cellulose saccharomyces cerevisiae and stalk fermentation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108410746A true CN108410746A (en) | 2018-08-17 |
Family
ID=63127819
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810113730.0A Pending CN108410746A (en) | 2018-02-05 | 2018-02-05 | It is a kind of can efficient-decomposition, convert cellulose saccharomyces cerevisiae and stalk fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108410746A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564631A (en) * | 2019-08-09 | 2019-12-13 | 辽宁省农业科学院 | yeast for producing cellulase at low temperature and screening method thereof |
| CN110574825A (en) * | 2019-09-25 | 2019-12-17 | 湖北大学 | Straw mixed fermentation feed and production method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101311271A (en) * | 2007-05-25 | 2008-11-26 | 青岛科技大学 | Process for producing cellulosic ethanol by recombining saccharomyces cerevisiae |
| CN101319196A (en) * | 2008-07-22 | 2008-12-10 | 暨南大学 | Transgenic wine brewing saccharomycete for degradation cellulose synthetic ethyl alcohol and construction method thereof |
| CN103103221A (en) * | 2013-01-29 | 2013-05-15 | 广州分子生物技术有限公司 | Method for converting cellulose into ethanol by use of mixed culture of genetically recombinant yeast |
| CN103382444A (en) * | 2013-01-29 | 2013-11-06 | 广州分子生物技术有限公司 | Gene recombinant saccharomyces cerevisiae capable of degrading crystalline cellulose |
| CN105018517A (en) * | 2015-07-16 | 2015-11-04 | 湖北大学 | Method for using natural cellulose as only carbon source to perform simultaneous saccharification and fermentation |
| CN105795120A (en) * | 2016-04-13 | 2016-07-27 | 湖北大学 | Method for producing high-protein straw feed with engineered Yarrowia lipolytica |
-
2018
- 2018-02-05 CN CN201810113730.0A patent/CN108410746A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101311271A (en) * | 2007-05-25 | 2008-11-26 | 青岛科技大学 | Process for producing cellulosic ethanol by recombining saccharomyces cerevisiae |
| CN101319196A (en) * | 2008-07-22 | 2008-12-10 | 暨南大学 | Transgenic wine brewing saccharomycete for degradation cellulose synthetic ethyl alcohol and construction method thereof |
| CN103103221A (en) * | 2013-01-29 | 2013-05-15 | 广州分子生物技术有限公司 | Method for converting cellulose into ethanol by use of mixed culture of genetically recombinant yeast |
| CN103382444A (en) * | 2013-01-29 | 2013-11-06 | 广州分子生物技术有限公司 | Gene recombinant saccharomyces cerevisiae capable of degrading crystalline cellulose |
| CN105018517A (en) * | 2015-07-16 | 2015-11-04 | 湖北大学 | Method for using natural cellulose as only carbon source to perform simultaneous saccharification and fermentation |
| CN105795120A (en) * | 2016-04-13 | 2016-07-27 | 湖北大学 | Method for producing high-protein straw feed with engineered Yarrowia lipolytica |
Non-Patent Citations (1)
| Title |
|---|
| 洪解放: ""表达纤维素酶的酿酒酵母工程菌株构建及其利用木质纤维素发酵产醇研究"", 《中国博士学位论文全文数据库 工程科技I辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564631A (en) * | 2019-08-09 | 2019-12-13 | 辽宁省农业科学院 | yeast for producing cellulase at low temperature and screening method thereof |
| CN110564631B (en) * | 2019-08-09 | 2022-11-15 | 辽宁省农业科学院 | Yeast for producing cellulase at low temperature and screening method thereof |
| CN110574825A (en) * | 2019-09-25 | 2019-12-17 | 湖北大学 | Straw mixed fermentation feed and production method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bhati et al. | Cost‐effective cellulase production, improvement strategies, and future challenges | |
| Lin et al. | Ethanol fermentation from biomass resources: current state and prospects | |
| Krogh et al. | Screening genus Penicillium for producers of cellulolytic and xylanolytic enzymes | |
| Apiwatanapiwat et al. | Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and β-glucosidase | |
| Castro et al. | Selection of a thermotolerant Kluyveromyces marxianus strain with potential application for cellulosic ethanol production by simultaneous saccharification and fermentation | |
| Khalil et al. | Production of cellulase by Pleurotus ostreatus and Pleurotus sajor-caju in solid state fermentation of lignocellulosic biomass | |
| JP2013515482A5 (en) | ||
| US20110171710A1 (en) | Method for producing cellulosic ethanol | |
| Singh et al. | Fusarium oxysporum: status in bioethanol production | |
| Tomás-Pejó et al. | Effect of different cellulase dosages on cell viability and ethanol production by Kluyveromyces marxianus in SSF processes | |
| CN102174433A (en) | Clostridium beijerinckii with high stress resistance and application thereof | |
| Tran et al. | Developing co-culture system of dominant cellulolytic Bacillus sp. THLA0409 and dominant ethanolic Klebsiella oxytoca THLC0409 for enhancing ethanol production from lignocellulosic materials | |
| CN102925365B (en) | Trichoderma atroviride strain and application thereof in preparation of cellulase | |
| CN101481676A (en) | Preparation of composite bacteria | |
| Liu et al. | Producing bioethanol from cellulosic hydrolyzate via co-immobilized cultivation strategy | |
| Shokrkar et al. | Exploring strategies for the use of mixed microalgae in cellulase production and its application for bioethanol production | |
| CN108410746A (en) | It is a kind of can efficient-decomposition, convert cellulose saccharomyces cerevisiae and stalk fermentation method | |
| Zheng et al. | Comparison of bioethanol production by Candida molischiana and Saccharomyces cerevisiae from glucose, cellobiose, and cellulose | |
| CN104024399A (en) | Method for the continuous production of cellulases by a filamentous fungus using a carbon substrate obtained from an acid pretreatment | |
| CN102776242A (en) | Process method for producing ethanol by utilizing low-temperature cellulase to perform simultaneous saccharification and fermentation | |
| CN103805673B (en) | A kind of method utilizing transgenic yeast mixed fermentation to produce straw ethanol | |
| Gomathi et al. | Production of bio-ethanol from pretreated agricultural byproduct using enzymatic hydrolysis and simultaneous saccharification | |
| CN103103221B (en) | Method for converting cellulose into ethanol by use of mixed culture of genetically recombinant yeast | |
| CN105018517A (en) | Method for using natural cellulose as only carbon source to perform simultaneous saccharification and fermentation | |
| CN109402094B (en) | A kind of preparation method and applications of cellulase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180817 |