CN109486867B - Composite cellulase system and application thereof in starch fuel ethanol production - Google Patents
Composite cellulase system and application thereof in starch fuel ethanol production Download PDFInfo
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Abstract
The invention discloses a composite cellulase system prepared by the following method, which comprises the following steps: fermenting and culturing Aspergillus niger transformed with a ferulic acid esterase gene derived from Penicillium juniperi and a dehydrogenase gene derived from Penicillium juniperi to obtain a fermentation culture solution I; fermenting and culturing trichoderma reesei to obtain a fermentation culture solution II; and compounding the fermentation culture solution I and the fermentation culture solution II according to a certain volume ratio to obtain the composite cellulase system. The composite cellulase system can effectively improve the trichoderma reesei enzyme system, and further effectively improve the speed of degrading the crystalline cellulose by the trichoderma reesei enzyme system. The invention also provides an application of the composite cellulase system, and the composite cellulase is added in the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, so that the viscosity of corn mash is reduced, and the residual cellulose and starch are further hydrolyzed, and the high-efficiency conversion from corn to fuel ethanol is realized, thereby bringing remarkable economic benefit for the ethanol fermentation industry.
Description
Technical Field
The invention relates to the technical field of ethanol fermentation production, and particularly relates to a composite cellulase system and application thereof in starch fuel ethanol production.
Background
Ethanol is an important bulk chemical product, has wide application, is commonly used as fuel, solvent and disinfectant, and is also used for preparing other compounds. Ethanol is also the largest fermentation product in the world at present, and the development plan of emerging industries in China indicates that the annual utilization amount of the biological liquid fuel reaches 1200 ten thousand tons by 2020. Although the production process of starch ethanol fermentation is mature, the production process has problems. In the production process of the corn fuel ethanol, the corn cellulose is not effectively utilized, the corn starch is not fully degraded, and the problems of high sugar solution viscosity, more solid matters and the like appear in the later period, so that the growth and the fermentation of the yeast are seriously influenced.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a composite cellulase system to improve the trichoderma reesei enzyme system and further effectively improve the speed of degrading the crystalline cellulose by the trichoderma reesei enzyme system.
The invention also aims to provide an application of the composite cellulase system, in the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, the composite cellulase is added, so that the viscosity of corn mash is reduced, residual cellulose and starch are further hydrolyzed, the efficient conversion from corn to the fuel ethanol is realized, and therefore, the obvious economic benefit is brought to the ethanol fermentation industry.
To achieve these objects and other advantages in accordance with the present invention, there is provided a complex cellulase system prepared by a method comprising the steps of:
inoculating Aspergillus niger which is transformed with a ferulic acid esterase gene derived from Penicillium juniperi and a dehydrogenase gene derived from Penicillium juniperi into a fermentation culture medium II according to the inoculation amount of 3-5% for fermentation culture to obtain a fermentation culture solution I, wherein the fermentation culture solution I is an Aspergillus niger transformant enzyme system containing a ferulic acid esterase derived from Penicillium juniperi and a dehydrogenase derived from Penicillium juniperi, and is beta-glucosidase derived from Aspergillus niger;
inoculating 3-5% of trichoderma reesei into a fermentation culture medium I according to the inoculation amount, and performing fermentation culture to obtain a fermentation culture solution II, wherein the fermentation culture solution II is cellulase containing trichoderma reesei sources; and
and compounding the fermentation culture solution I and the fermentation culture solution II according to the volume ratio of 1:7-10 to obtain the composite cellulase system.
Preferably, the Aspergillus niger transformant enzyme system and the Trichoderma reesei enzyme system are compounded according to the volume ratio of 1:8.5 to obtain the composite cellulase system.
Preferably, the fermentation medium I comprises the following components:
2.8g/L of ammonium sulfate, 50g/L of corncob, 30g/L of bran, 0.9g/L of magnesium sulfate, 0.9g/L of calcium chloride, 4g/L of monopotassium phosphate and 1L of distilled water;
the fermentation medium II comprises the following components: micro-meter33g/L of crystalline cellulose and 17g/L of corn steep liquor dry powder, wherein the mass percentage of (NH) in the fermentation medium II is 0.5 percent4)2SO4MgSO accounting for 0.1 percent of the mass of the fermentation medium II40.25 percent of glycerin in the mass percent of the fermentation medium II and 0.25 percent of CaCO in the mass percent of the fermentation medium II3And the pH was adjusted to 5.0.
Preferably, the conditions for the fermentation culture of trichoderma reesei are as follows: the fermentation temperature is 28 ℃, the rotating speed of a shaking table is 18rpm, and the fermentation time is 120 hours;
the Aspergillus niger fermentation culture conditions are as follows: the fermentation temperature was 30 ℃ and the cultivation time was 7 days.
The application of the composite cellulase system in the production of fuel ethanol.
During the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, the composite cellulase system is added into fermentation mash, and the addition amount is such that the enzyme activity of the composite cellulase system in the fermentation mash is 20FPU/L-200 FPU/L.
Preferably, the addition amount of the complex cellulase system is such that the enzyme activity of the complex cellulase system in the fermentation mash is 30-50 FPU/L.
Preferably, the addition amount of the complex cellulase system is such that the enzyme activity of the complex cellulase system in the fermentation mash is 50 FPU/L.
Preferably, the ethanol production process by yeast synchronous saccharification and fermentation specifically comprises the following steps:
inoculating 0.3g yeast into 100ml sterilized distilled water for activation treatment to obtain seed liquid;
adding the seed liquid into the corn mash according to the volume percentage of 1%, adding the composite cellulase system, uniformly mixing, and performing yeast synchronous diastatic fermentation to obtain ethanol fermentation liquid;
wherein, the yeast synchronous saccharification and fermentation conditions are as follows: the fermentation temperature was 32 ℃, the shaker speed was 200rpm, and the fermentation time was 36 hours.
The invention at least comprises the following beneficial effects:
the composite cellulase system provided by the invention can effectively improve the trichoderma reesei enzyme system, and further effectively improve the speed of degrading crystalline cellulose by the trichoderma reesei enzyme system;
in the invention, the composite cellulase is added in the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, so that the viscosity of corn mash is reduced, and the residual cellulose and starch are further hydrolyzed to realize high-efficiency conversion from corn to the fuel ethanol, thereby bringing remarkable economic benefit for the ethanol fermentation industry. In practical application, under the condition that the optimal dosage of the composite cellulase system is 50FPU/L, compared with a control example (without the composite cellulase system), the ethanol yield is improved by 13.44 percent. At the same time, it can be seen that the ethanol yield is increased and the remaining total sugar and viscosity in the mash are also greatly reduced, 64.57% and 46.27% lower than those of the control.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram of a process for constructing a heterologous expression cassette pSIMPLE-19-FAE-SDR of successful ferulic acid esterase and dehydrogenase by using pSIMPLE-19 as an original plasmid and adding juniper-derived ferulic acid esterase and dehydrogenase into the pSIMPLE-19 plasmid through a recombinase one-step method;
FIG. 2 photograph of gel electrophoresis of transformants successfully expressed in A.niger by heterologous expression cassette pSIMPLE-19-FAE-SDR for feruloyl esterase and dehydrogenase;
FIG. 3 photograph of gel electrophoresis of the successful heterologous expression of heterologous expression cassette pSIMPLE-19-FAE-SDR of feruloyl esterase and dehydrogenase in A.niger;
FIG. 4 is a photograph of DDGS of corn mash residues dried at 65 ℃ in a flat plate in the analysis of DDGS of corn mash residues after simultaneous saccharification and fermentation according to the present invention, wherein a is the corn mash residue without the addition of the complex cellulase system, and b is the corn mash residue with the addition of the complex cellulase system.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
1. The preparation method of the Aspergillus niger with the ferulic acid esterase gene and the dehydrogenase gene of the cypress-like penicillium derived by transformation comprises the following steps:
the applicant uses pSIMPLE-19 as original plasmid, ligates the ferulic acid esterase and dehydrogenase from Penicillium juniperi into pSIMPLE-19 plasmid by recombinase one-step method (as shown in FIG. 1), constructs a heterologous expression cassette pSIMPLE-19-FAE-SDR of successful ferulic acid esterase and dehydrogenase and detects the heterologous expression cassette pSIMPLE-19-FAE-SDR by agarose gel electrophoresis (as shown in FIG. 2), wherein lanes 1-8 are the verification of transformant molecules successfully expressing dehydrogenase and ferulic acid esterase in Aspergillus niger, and lane 9 is the blank control of pSIMPLE-19 plasmid.
Wherein, the sequence (polynucleotide sequence) of the ferulic acid esterase gene is shown in SEQ ID NO: 1:
the dehydrogenase gene sequence (polynucleotide sequence) is shown in SEQ ID NO. 2.
2. The constructed heterologous expression cassette pSIMPLE-19-FAE-SDR of the feruloyl esterase and the dehydrogenase is subjected to heterologous expression in Aspergillus niger, and agarose gel electrophoresis detection (shown in figure 3) is carried out, wherein the lane 1 is an expression cassette of the dehydrogenase, the lane 2 is an expression cassette of the feruloyl esterase, the lane 3 is an expression cassette of the dehydrogenase-feruloyl esterase.
3. The source of mash and related parameters used in this application.
The mash source, the middle oil Jilin fuel ethanol research institute, is a raw material from starch fuel ethanol mass production. The glucose concentration reaches 200g/L, and the viscosity reaches 34.8 Pa.s.
Example 1
The invention provides a composite cellulase system prepared by the following method, which comprises the following steps:
inoculating Aspergillus niger which is transformed with a ferulic acid esterase gene derived from Penicillium juniperi and a dehydrogenase gene derived from Penicillium juniperi into a fermentation culture medium II according to the inoculation amount of 3% for fermentation culture to obtain a fermentation culture solution I, wherein the fermentation culture solution I is an Aspergillus niger transformant enzyme system containing ferulic acid esterase derived from Penicillium juniperi and dehydrogenase derived from Penicillium juniperi, and beta-glucosidase derived from Aspergillus niger;
inoculating trichoderma reesei into a fermentation culture medium I according to the inoculation amount of 3% for fermentation culture to obtain a fermentation culture solution II, wherein the fermentation culture solution II contains cellulase derived from trichoderma reesei; and
and (2) compounding (fully mixing) the fermentation culture solution I and the fermentation culture solution II according to the volume ratio of 1:10 to obtain the composite cellulase system.
Wherein the fermentation medium I comprises the following components:
2.8g/L of ammonium sulfate, 50g/L of corncob, 30g/L of bran, 0.9g/L of magnesium sulfate, 0.9g/L of calcium chloride, 4g/L of monopotassium phosphate and 1L of distilled water;
the fermentation medium II comprises the following components: 33g/L of microcrystalline cellulose and 17g/L of corn steep liquor dry powder, wherein the mass percentage of (NH) in the fermentation medium II is 0.5 percent4)2SO4MgSO accounting for 0.1 percent of the mass of the fermentation medium II40.25 percent of glycerin in the mass percent of the fermentation medium II and 0.25 percent of CaCO in the mass percent of the fermentation medium II3And adjusting the pH to 5.0;
the conditions for the fermentation culture of the trichoderma reesei are as follows: the fermentation temperature is 28 ℃, the rotating speed of a shaking table is 18rpm, and the fermentation time is 120 hours;
the Aspergillus niger fermentation culture conditions are as follows: the fermentation temperature was 30 ℃ and the cultivation time was 7 days.
Example 2
The invention provides a composite cellulase system prepared by the following method, which comprises the following steps:
inoculating Aspergillus niger which is transformed with a ferulic acid esterase gene derived from Penicillium juniperi and a dehydrogenase gene derived from Penicillium juniperi into a fermentation medium II according to the inoculation amount of 4% for fermentation culture to obtain a fermentation culture solution I, wherein the fermentation culture solution I is an Aspergillus niger transformant enzyme system containing ferulic acid esterase derived from Penicillium juniperi and dehydrogenase derived from Penicillium juniperi, and beta-glucosidase derived from Aspergillus niger;
inoculating 4% of trichoderma reesei into a fermentation culture medium I according to the inoculation amount, and performing fermentation culture to obtain a fermentation culture solution II, wherein the fermentation culture solution II contains cellulase derived from the trichoderma reesei; and
and (2) compounding (fully mixing) the fermentation culture solution I and the fermentation culture solution II according to the volume ratio of 1:8.5 to obtain the composite cellulase system.
Wherein the fermentation medium I comprises the following components:
2.8g/L of ammonium sulfate, 50g/L of corncob, 30g/L of bran, 0.9g/L of magnesium sulfate, 0.9g/L of calcium chloride, 4g/L of monopotassium phosphate and 1L of distilled water;
the fermentation medium II comprises the following components: 33g/L of microcrystalline cellulose and 17g/L of corn steep liquor dry powder, wherein the mass percentage of (NH) in the fermentation medium II is 0.5 percent4)2SO4MgSO accounting for 0.1 percent of the mass of the fermentation medium II40.25 percent of glycerin in the mass percent of the fermentation medium II and 0.25 percent of CaCO in the mass percent of the fermentation medium II3And adjusting the pH to 5.0;
the conditions for the fermentation culture of the trichoderma reesei comprise: the fermentation temperature is 28 ℃, the rotating speed of a shaking table is 18rpm, and the fermentation time is 120 hours;
the Aspergillus niger fermentation culture conditions are as follows: the fermentation temperature was 30 ℃ and the cultivation time was 7 days.
Example 3
The invention provides a composite cellulase system prepared by the following method, which comprises the following steps:
inoculating Aspergillus niger which is transformed with a ferulic acid esterase gene derived from Penicillium juniperi and a dehydrogenase gene derived from Penicillium juniperi into a fermentation medium II according to the inoculation amount of 5% for fermentation culture to obtain a fermentation culture solution I, wherein the fermentation culture solution I is an Aspergillus niger transformant enzyme system containing ferulic acid esterase derived from Penicillium juniperi and dehydrogenase derived from Penicillium juniperi, and beta-glucosidase derived from Aspergillus niger;
inoculating trichoderma reesei into a fermentation culture medium I according to the inoculation amount of 5% for fermentation culture to obtain a fermentation culture solution II, wherein the fermentation culture solution II contains cellulase derived from trichoderma reesei; and
and (2) compounding (fully mixing) the fermentation culture solution I and the fermentation culture solution II according to the volume ratio of 1:7 to obtain the composite cellulase system.
Wherein the fermentation medium I comprises the following components:
2.8g/L of ammonium sulfate, 50g/L of corncob, 30g/L of bran, 0.9g/L of magnesium sulfate, 0.9g/L of calcium chloride, 4g/L of monopotassium phosphate and 1L of distilled water;
the fermentation medium II comprises the following components: 33g/L of microcrystalline cellulose and 17g/L of corn steep liquor dry powder, wherein the mass percentage of (NH) in the fermentation medium II is 0.5 percent4)2SO4MgSO accounting for 0.1 percent of the mass of the fermentation medium II40.25 percent of glycerin in the mass percent of the fermentation medium II and 0.25 percent of CaCO in the mass percent of the fermentation medium II3And adjusting the pH to 5.0;
the conditions for the fermentation culture of the trichoderma reesei are as follows: the fermentation temperature is 28 ℃, the rotating speed of a shaking table is 18rpm, and the fermentation time is 120 hours;
the Aspergillus niger fermentation culture conditions are as follows: the fermentation temperature was 30 ℃ and the cultivation time was 7 days.
Example 4
A fermentation production method of starch fuel ethanol is characterized in that the composite cellulase system is added into fermentation mash in the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, and the addition amount is such that the enzyme activity of the composite cellulase system in the fermentation mash is 20 FPU/L.
Wherein the complex cellulase system obtained in example 1 was used;
the process for producing ethanol by yeast synchronous saccharification and fermentation specifically comprises the following steps:
inoculating 0.3g yeast into 100ml sterilized distilled water for activation treatment to obtain seed liquid;
adding 1% of seed liquid into corn mash according to the volume percentage, adding the composite cellulase system, uniformly mixing, and performing yeast synchronous diastatic fermentation to obtain ethanol fermentation liquor;
wherein, the yeast synchronous saccharification and fermentation conditions are as follows: the fermentation temperature was 32 ℃, the shaker speed was 200rpm, and the fermentation time was 36 hours.
Example 5
A fermentation production method of starch fuel ethanol is characterized in that the composite cellulase system is added into fermentation mash in the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, and the addition amount is such that the enzyme activity of the composite cellulase system in the fermentation mash is 30 FPU/L.
Wherein the complex cellulase system obtained in example 1 was used;
the process for producing ethanol by yeast synchronous saccharification and fermentation specifically comprises the following steps:
inoculating yeast into the corn mash to carry out activation treatment to obtain seed liquid;
inoculating 0.3g yeast into 100ml sterilized distilled water for activation treatment to obtain seed liquid;
adding the seed liquid into the corn mash according to the volume percentage of 1%, adding the composite cellulase system, uniformly mixing, and performing yeast synchronous diastatic fermentation to obtain ethanol fermentation liquid;
wherein, the yeast synchronous saccharification and fermentation conditions are as follows: the fermentation temperature was 32 ℃, the shaker speed was 200rpm, and the fermentation time was 36 hours.
Example 6
A fermentation production method of starch fuel ethanol is characterized in that the composite cellulase system is added into fermentation mash in the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, and the addition amount is such that the enzyme activity of the composite cellulase system in the fermentation mash is 40 FPU/L.
Wherein the complex cellulase system obtained in example 2 was used;
the process for producing ethanol by yeast synchronous saccharification and fermentation specifically comprises the following steps:
inoculating 0.3g yeast into 100ml sterilized distilled water for activation treatment to obtain seed solution;
adding the seed liquid into the corn mash according to the volume percentage of 1%, adding the composite cellulase system, uniformly mixing, and performing yeast synchronous diastatic fermentation to obtain ethanol fermentation liquid;
wherein, the yeast synchronous saccharification and fermentation conditions are as follows: the fermentation temperature was 32 ℃, the shaker speed was 200rpm, and the fermentation time was 36 hours.
Example 7
A fermentation production method of starch fuel ethanol is characterized in that the composite cellulase system is added into fermentation mash in the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, and the addition amount is such that the enzyme activity of the composite cellulase system in the fermentation mash is 50 FPU/L.
Wherein the complex cellulase system obtained in example 2 was used;
the process for producing ethanol by yeast synchronous saccharification and fermentation specifically comprises the following steps:
inoculating 0.3g yeast into 100ml sterilized distilled water for activation treatment to obtain seed liquid;
adding the seed liquid into the corn mash according to the volume percentage of 1%, adding the composite cellulase system, uniformly mixing, and performing yeast synchronous diastatic fermentation to obtain ethanol fermentation liquid;
wherein, the yeast synchronous saccharification and fermentation conditions are as follows: the fermentation temperature was 32 ℃, the shaker speed was 200rpm, and the fermentation time was 36 hours.
Example 8
A fermentation production method of starch fuel ethanol is characterized in that the composite cellulase system is added into fermentation mash in the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, and the addition amount is such that the enzyme activity of the composite cellulase system in the fermentation mash is 100 FPU/L.
Wherein the complex cellulase system obtained in example 3 was used;
the process for producing ethanol by yeast synchronous saccharification and fermentation specifically comprises the following steps:
inoculating 0.3g yeast into 100ml sterilized distilled water for activation treatment to obtain seed liquid;
adding the seed liquid into the corn mash according to the volume percentage of 1%, adding the composite cellulase system, uniformly mixing, and performing yeast synchronous diastatic fermentation to obtain ethanol fermentation liquid;
wherein, the yeast synchronous saccharification and fermentation conditions are as follows: the fermentation temperature was 32 ℃, the shaker speed was 200rpm, and the fermentation time was 36 hours.
Example 9
In the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, the composite cellulase system is added into fermentation mash, and the addition amount is that the enzyme activity of the composite cellulase system in the fermentation mash is 150 FPU/L.
Wherein the complex cellulase system obtained in example 2 was used;
the process for producing ethanol by yeast synchronous saccharification and fermentation specifically comprises the following steps:
inoculating 0.3g yeast into 100ml sterilized distilled water for activation treatment to obtain seed liquid;
adding the seed liquid into the corn mash according to the volume percentage of 1%, adding the composite cellulase system, uniformly mixing, and performing yeast synchronous diastatic fermentation to obtain ethanol fermentation liquid;
wherein, the yeast synchronous saccharification and fermentation conditions are as follows: the fermentation temperature was 32 ℃, the shaker speed was 200rpm, and the fermentation time was 36 hours.
Example 10
A fermentation production method of starch fuel ethanol is characterized in that the composite cellulase system is added into fermentation mash in the process of producing the starch fuel ethanol by yeast synchronous saccharification and fermentation, and the addition amount is such that the enzyme activity of the composite cellulase system in the fermentation mash is 200 FPU/L.
Wherein the complex cellulase system obtained in example 2 was used;
the process for producing ethanol by yeast synchronous saccharification and fermentation specifically comprises the following steps:
inoculating 0.3g yeast into 100ml sterilized distilled water for activation treatment to obtain seed liquid;
adding the seed liquid into the corn mash according to the volume percentage of 1%, adding the composite cellulase system, uniformly mixing, and performing yeast synchronous diastatic fermentation to obtain ethanol fermentation liquid;
wherein, the yeast synchronous saccharification and fermentation conditions are as follows: the fermentation temperature was 32 ℃, the shaker speed was 200rpm, and the fermentation time was 36 hours.
Comparative example
The ethanol production conditions by simultaneous saccharification and fermentation of yeast are the same as in examples 4-10.
The ethanol yields, total sugar remaining in the mash and viscosities for examples 4-10 and the control are detailed in Table 1 below.
Table 1:
compared with the control example (without cellulase), the ethanol yield is improved by 13.44 percent. Meanwhile, the yield of ethanol is improved, and the residual total sugar and viscosity are greatly reduced, namely reduced by 64.57 percent and 46.27 percent compared with the control.
According to the above embodiments of the present invention, the application of the complex cellulase system to promote ethanol production at different fermentation scales in actual production is compared:
the applicant applied a complex cellulase system (added in an amount such that the enzyme activity of said complex cellulase system in the fermentation mash is 50FPU/L) on different fermentation scales (0.3L shake flask, 1L shake flask, 5L fermentation tank, 70L) to evaluate ethanol yield. As can be seen from the results in Table 2 below, ethanol yields were relatively stable in the 0.3L shake flask, the 1L shake flask, the 5L fermenter, and the 70L fermenter on different fermentation scales. On the fermentation scale of 5L, the yield of the ethanol can reach 120.83 g/L; on a 70L fermentation scale, the ethanol yield can reach 117.04g/L, and compared with a fermentation tank control, the ethanol yield is respectively improved by 16.58% and 12.93%.
Note that: the fermentation tank has reduced ethanol volatilization on the one hand, and on the other hand, the fermentation tank is more uniformly mixed, the yeast is better utilized, and the ethanol yield is higher.
Table 2: application of composite cellulase system in starch ethanol production on different fermentation scales
Application of cellulase in promoting ethanol yield in continuous fermentation
The experiment is that Angel yeast is activated by water for 1 hour according to the method of Jilin ethanol research institute, and then added into corn mash for simultaneous saccharification and fermentation.
In addition, the applicant also analyzed DDGS of corn mash residues after simultaneous saccharification and fermentation. We poured the remaining DDGS residue onto a plate, dried at 65 ℃ and observed the DDGS treated with the cellulase complex (FIG. 4b) and the DDGS treated without the cellulase complex (FIG. 4a) to have no distinct color and no color change (FIG. 4).
And (4) accounting of economic benefits:
jilin fuel ethanol produces 60 ten thousand tons each year, and the amount of mash is about 600 ten thousand tons each year. The price of ethanol is 6000 yuan/ton. We added 5X 10 per ton of corn mash4The yield of the ethanol of the FPU composite cellulase system can be improved by 5 percent.
1 ton of ethanol can be produced by 10 tons of mash; 1 ton of ethanol can improve the yield by 6 percent, and the income is increased by about 360 yuan; 10 tons of mash require a 5X 10 complex cellulase system5FPU, about 0.0125 ton of composite fiberEnzyme system liquid (according to 4X 10)7FPU/ton enzyme solution), the production cost of the compound cellulase system is about 10000 yuan/ton. 1 ton of ethanol needs to increase the cost of the compound cellulase system by 12.5 yuan. One ton of ethanol can simultaneously produce 0.8 ton of DDGS. After the compound cellulase system is added, the residual DDGS amount of the synchronous saccharification and fermentation is reduced by 10.47 percent according to 1400 yuan/ton of DDGS. One ton of ethanol reduces the income of DDGS by 117 yuan (1400X 0.8X 10.47%), 1 ton of ethanol net income can be increased by about 230 yuan, and Jilin fuel ethanol can increase the net income by 1.38 million yuan per year (calculated according to 60 million tons).
While embodiments of the invention have been described above, it is not intended to be limited to the details shown, or described, but rather to be accorded the widest scope consistent with the principles and novel features disclosed herein.
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> composite cellulase system and application thereof in production of starch fuel ethanol
<160>2
<210>1
<211>1500
<212>DNA
<213> Penicillium juniperi (Penicillium piceum)
<220>
<400>1
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ccatcgaaac tggaatgtgc ttccatagca aaagagagaa ccaatcccgc cgttgagata 120
tcccgggctg tcgaggtgcc agcccatggt ctcaacattt catccgttgt gaacgagatt 180
cccttgtgtt gggtacaagg cactatcaag tataatgcga acgataaact cgatgtctcg 240
ggaaacaata cgctcacatg ggagctcttc ctaccaagtc cagatacgta caatggacga 300
tatctaatga ccggagacgg cggttttgcc ggtgccattg agaacaacac catgttgacc 360
tatctgaacc tgggatacgc tgtggccggt tcggacgccg gtcatccaga ggcagccaat 420
ggcgatggta catatgcacc ttttcttcaa aacccagccg aacttcaggc ctggatccat 480
aactccgttg ccatggcgac tcctgttact cggtcccttg ttgcaaagta ctattcaaag 540
catccggact acagttactt ttgggggtgt tcgaccggcg gcgcccaggg atacgccttg 600
gcccagtatc atccagagtt gtttgatggt atctatgccg gaagtcccgg taactggtac 660
agccacctgg ttctgagctt tctctggaac ggactgcaca ccaccggcag cgcgttcatg 720
agtcaacaag tcttgtcgtt tgtgaccgat cgagtggttg cagcgtgtga ccaactggac 780
ggagtcaaag acgggctgat cgagaatcca ctgcgctgct cgttcgatgt gacttcacta 840
cggtgcaaat ctgaacaggc accagacagc ggagagccta catgtctgac ggcggatcaa 900
atcgcagcat tgcagaagat ctatgaggga cccaaggacg ttcggtccgg aaatcagatt 960
tatcccggct tcagccttgg gtctgagaac ggtctgctcg accaagaaca ggtgctttac 1020
ctaaactaca cggcgccgat tctgcgagaa gtcgttcttg acgaccaaga cttcaacata 1080
acccatttca actggggatc tgacgtcgac gctgtggaca aaaaggcatc tccattcata 1140
gacagcctgt ccccgaatct ctcacagttc cagcgacgag gcggcaagtt actcaccacg 1200
caaggctggt ctgatcagta taatgccgca ctgtggccga tccagcatct gcaggagatc 1260
caaaggacaa tgggaccccg atccgatttt gtacaggtgt tcatggtccc cggtggggga 1320
cactgcggtc ctaacccgta ttatccacat gtacctggtg tttatcacgt tatggaggcg 1380
ctggtgccgt gggtcgagga tggcaagaga ccaagggata tgcttgccac ggagcctccg 1440
gatcgttctg atacgaccag aaaactttgc ccgtggcctg caactgccaa gcatgtgggc 1500
ggggacgtag atgactggaa atcatacgac tgtgcctag 1539
<210>2
<211>928
<212> Penicillium juniperi (Penicillium piceum)
<213> Artificial sequence
<220>
<400>2
atggcgacga gcaagccggt ggcacttgtc attggagcat ctcggggcat aggaagacag 60
gtctccatcg acctggctcg caaggggtat gccgttgtcg ttgctgccaa gacaacatct 120
gatgccagca agaccgttcc atttccaccc cctcctaact ctccgcagtc gactatcaac 180
actgtcgagc gtgagatccg ggaagctggt ggcgaagcca ctgccattgc cgtggataca 240
agagacgtcg agcagataaa tcaactcgta aacaaaactg tcagtattta cgggaagctt 300
gacgttctta tctacaactc tggcgcaata tggtggtctt cggtggagaa cacgcccgtg 360
aaacggttcc agctcatgca gagaattaat ccagagggac tttatgctag cgtacaagcg 420
gcacttccac acttcggaaa caatggatgg aaaggccgga taattgttgt ttcgccacca 480
atctattcgc gattcttccg tgggaaaacc gcctatgcga tgggcaaggt cggaatgagc 540
gttttgacca aagggctggc gatggacttt atccggcaag gtcgaaaaga catggccatt 600
acaagtatct ggccagcggt atccatcgag tccgcagcta ccggagacat tgtgaaaaag 660
gaccctacgg ctgagaagga cttgcggaaa gcaactattt tctcagacgc tatcctggaa 720
atgatcaatg ccccagcaga gtccgtcaat ggtctgttag accttgatga ggattttctt 780
agaaatcggt gcggaatcac cgatttttcg aaatactccg tcgtaccagg tgccgatccc 840
cgccgaatta tgcctgcgga atttccaact ctagaggtca aagagcaaga tgacgaaggg 900
cgcagggtgg acagtgccca gcttcgaa 928
Claims (7)
1. The application of the composite cellulase system in the production of starch fuel ethanol, wherein the composite cellulase system prepared by the following method comprises the following steps:
inoculating Aspergillus niger which is transformed with a ferulic acid esterase gene derived from Penicillium juniperi and a dehydrogenase gene derived from Penicillium juniperi into a fermentation culture medium II according to the inoculation amount of 3-5% for fermentation culture to obtain a fermentation culture solution I, wherein the fermentation culture solution I is an Aspergillus niger transformant enzyme system containing ferulic acid esterase derived from Penicillium juniperi and dehydrogenase derived from Penicillium juniperi and beta-glucosidase derived from Aspergillus niger, wherein the sequence of the ferulic acid esterase gene is shown as SEQ ID NO. 1, and the sequence of the dehydrogenase gene is shown as SEQ ID NO. 2;
inoculating 3-5% of trichoderma reesei into a fermentation culture medium I according to the inoculation amount, and performing fermentation culture to obtain a fermentation culture solution II, wherein the fermentation culture solution II is cellulase containing trichoderma reesei sources; and
compounding the fermentation culture solution I and the fermentation culture solution II according to the volume ratio of 1:7-10 to obtain a composite cellulase system;
the fermentation medium I comprises the following components:
2.8g/L of ammonium sulfate, 50g/L of corncob, 30g/L of bran, 0.9g/L of magnesium sulfate, 0.9g/L of calcium chloride, 4g/L of monopotassium phosphate and 1L of distilled water;
the fermentation medium II comprises the following components: 33g/L of microcrystalline cellulose and 17g/L of corn steep liquor dry powder, wherein the mass percentage of (NH) in the fermentation medium II is 0.5 percent4)2SO4MgSO 0.1% in weight percentage of fermentation medium II40.25 percent of glycerin in the mass percent of the fermentation medium II and 0.25 percent of CaCO in the mass percent of the fermentation medium II3And the pH was adjusted to 5.0.
2. The use of the complex cellulase system of claim 1 in the production of starch fuel ethanol, wherein said aspergillus niger transformant enzyme system is
The trichoderma reesei enzyme system is compounded according to the volume ratio of 1:8.5 to obtain the composite cellulase system.
3. The use of the complex cellulase system of claim 1 in the production of ethanol from starch fuel,
the conditions for the fermentation culture of the trichoderma reesei are as follows: the fermentation temperature is 28 ℃, the rotating speed of a shaking table is 18rpm, and the fermentation is carried out
The time is 120 hours;
the Aspergillus niger fermentation culture conditions are as follows: the fermentation temperature was 30 ℃, the shaker speed was 200rpm, and the cultivation time was 7 days.
4. A method for producing starch fuel ethanol by fermentation, which is characterized in that the composite cellulase system as claimed in any one of claims 1-3 is added into fermentation mash in the process of producing starch fuel ethanol by yeast simultaneous saccharification and fermentation, and the addition amount is such that the enzyme activity of the composite cellulase system in the fermentation mash is 20FPU/L-200 FPU/L.
5. The method of claim 4, wherein the complex cellulase enzyme system is added in an amount such that the enzymatic activity of the complex cellulase enzyme system in the fermentation mash is between 30-50 FPU/L.
6. The method of claim 4, wherein the complex cellulase enzyme system is added in an amount such that the enzymatic activity of the complex cellulase enzyme system in the fermentation mash is 50 FPU/L.
7. The method for the fermentative production of starch-fueled ethanol according to claim 4, wherein the ethanol production process by simultaneous saccharification and fermentation of yeast comprises the following steps:
inoculating 0.3g yeast into 100ml sterilized distilled water for activation treatment to obtain seed liquid;
adding the seed liquid into the corn mash according to the volume percentage of 1%, adding the composite cellulase system, uniformly mixing, and performing yeast synchronous diastatic fermentation to obtain ethanol fermentation liquid;
wherein, the yeast synchronous saccharification and fermentation conditions are as follows: the fermentation temperature was 32 ℃, the shaker speed was 200rpm, and the fermentation time was 36 hours.
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| CN113403207B (en) * | 2021-08-23 | 2021-11-05 | 中国科学院天津工业生物技术研究所 | Aspergillus niger strain for high yield of beta-glucosidase and application thereof |
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