CN113637712A - Method for designing enzyme preparation feeding mode in fermentation process through computer and application - Google Patents
Method for designing enzyme preparation feeding mode in fermentation process through computer and application Download PDFInfo
- Publication number
- CN113637712A CN113637712A CN202111189956.7A CN202111189956A CN113637712A CN 113637712 A CN113637712 A CN 113637712A CN 202111189956 A CN202111189956 A CN 202111189956A CN 113637712 A CN113637712 A CN 113637712A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- enzyme
- cellulase
- target product
- concentration
- 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.)
- Granted
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 168
- 230000004151 fermentation Effects 0.000 title claims abstract description 168
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 149
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 149
- 238000000034 method Methods 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 54
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 163
- 229940088598 enzyme Drugs 0.000 claims abstract description 148
- 108010059892 Cellulase Proteins 0.000 claims abstract description 55
- 229940106157 cellulase Drugs 0.000 claims abstract description 54
- 230000000694 effects Effects 0.000 claims abstract description 41
- 238000000329 molecular dynamics simulation Methods 0.000 claims abstract description 22
- 230000008569 process Effects 0.000 claims abstract description 19
- 239000000463 material Substances 0.000 claims abstract description 14
- 230000001502 supplementing effect Effects 0.000 claims abstract description 13
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- 239000013078 crystal Substances 0.000 claims abstract description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 102000005575 Cellulases Human genes 0.000 claims description 101
- 108010084185 Cellulases Proteins 0.000 claims description 101
- 239000002994 raw material Substances 0.000 claims description 36
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 33
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 32
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 28
- 239000006184 cosolvent Substances 0.000 claims description 27
- 240000008042 Zea mays Species 0.000 claims description 25
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 25
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 25
- 235000005822 corn Nutrition 0.000 claims description 25
- 239000000758 substrate Substances 0.000 claims description 22
- 238000002156 mixing Methods 0.000 claims description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- 239000011259 mixed solution Substances 0.000 claims description 15
- 238000002835 absorbance Methods 0.000 claims description 14
- 239000002904 solvent Substances 0.000 claims description 13
- 239000007853 buffer solution Substances 0.000 claims description 12
- 238000005315 distribution function Methods 0.000 claims description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 9
- 239000004202 carbamide Substances 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 238000001228 spectrum Methods 0.000 claims description 9
- 102000004139 alpha-Amylases Human genes 0.000 claims description 8
- 108090000637 alpha-Amylases Proteins 0.000 claims description 8
- 229940024171 alpha-amylase Drugs 0.000 claims description 8
- 238000013461 design Methods 0.000 claims description 8
- 230000005764 inhibitory process Effects 0.000 claims description 8
- 238000007614 solvation Methods 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 6
- 230000009471 action Effects 0.000 claims description 6
- 230000006399 behavior Effects 0.000 claims description 6
- 238000000547 structure data Methods 0.000 claims description 6
- IAYJZWFYUSNIPN-KFRZSCGFSA-N (2s,3r,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-(4-nitrophenoxy)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](OC=2C=CC(=CC=2)[N+]([O-])=O)[C@H](O)[C@H]1O IAYJZWFYUSNIPN-KFRZSCGFSA-N 0.000 claims description 5
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 5
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 5
- 240000006394 Sorghum bicolor Species 0.000 claims description 5
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 5
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 5
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 5
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 5
- 238000004807 desolvation Methods 0.000 claims description 5
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 5
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 5
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- IFBHRQDFSNCLOZ-RMPHRYRLSA-N 4-nitrophenyl beta-D-glucoside Chemical group O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-RMPHRYRLSA-N 0.000 claims description 4
- 240000007594 Oryza sativa Species 0.000 claims description 4
- 235000007164 Oryza sativa Nutrition 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 4
- 235000009566 rice Nutrition 0.000 claims description 4
- 230000001360 synchronised effect Effects 0.000 claims description 4
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 238000000126 in silico method Methods 0.000 claims 1
- 239000002054 inoculum Substances 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 3
- 239000000047 product Substances 0.000 description 49
- 230000036571 hydration Effects 0.000 description 10
- 238000006703 hydration reaction Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 241001052560 Thallis Species 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000004088 simulation Methods 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 235000010980 cellulose Nutrition 0.000 description 3
- 238000005094 computer simulation Methods 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 238000013329 compounding Methods 0.000 description 2
- 230000002478 diastatic effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000009510 drug design Methods 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000019624 protein content Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000002945 steepest descent method Methods 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 241001520808 Panicum virgatum Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 108010052085 cellobiose-quinone oxidoreductase Proteins 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013626 chemical specie Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000011960 computer-aided design Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 239000010907 stover Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical class 0.000 description 1
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q3/00—Condition responsive control processes
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
- G16C10/00—Computational theoretical chemistry, i.e. ICT specially adapted for theoretical aspects of quantum chemistry, molecular mechanics, molecular dynamics or the like
-
- 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
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/59—Biological synthesis; Biological purification
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Theoretical Computer Science (AREA)
- Microbiology (AREA)
- Computing Systems (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to renewable energy and bioengineering technology, and discloses a method for designing an enzyme preparation feeding mode in a fermentation process through a computer and application thereof. The method comprises the following steps: measuring the activity of each enzyme component separately in the presence of a target product of the fermentation process, wherein the concentration of the target product is set to a plurality of different simulated concentrations; obtaining a crystal structure of the enzyme component from a protein database, selecting a model of a target product, and performing molecular dynamics simulation of the enzyme component when the concentration of the target product is a simulated concentration; and (3) obtaining a concentration value of a target product in the fermentation process when no enzyme preparation is added, combining the activity data with a molecular dynamics simulation result, and providing a material supplementing time period and a material supplementing amount of each enzyme component. The method can optimize the adding strategy of the cellulase during ethanol production by fermentation, improve the ethanol yield, reduce the cost and further improve the economic benefit of the whole ethanol process.
Description
Technical Field
The invention relates to renewable energy and bioengineering technology, in particular to a method for designing an enzyme preparation feeding mode in a fermentation process through a computer and application thereof.
Background
Bioethanol is a promising liquid fuel, can meet the ever-increasing energy demand and sustainable economy, and has a positive impact on rural areas, the environment and energy safety. Much research work on bioethanol has been carried out in recent years, and advanced fermentation production technologies have been developed to improve the economy and profitability of the entire bioethanol process. However, there is still an urgent need in the industry for higher biomass utilization and ethanol production.
In the production of bioethanol, hydrolysis is a key step in the conversion of cellulosic biomass (corn fiber, corn stover, wheat straw, switchgrass, etc.) to ethanol, and in addition to hydrolysis of chemical species (primarily acids), enzyme-driven biocatalysis is one of the most potential alternatives to hydrolysis of cellulose to glucose at relatively low temperatures, and is currently the most commonly used process in industrial processes, alleviating the difficulties encountered with acid hydrolysis. When various enzymes such as cellulase, xylanase, polysaccharide monooxygenase and cellobiose dehydrogenase are applied, the hydrolysis efficiency is improved, and the ethanol fermentation performance is better. However, the high cost and single component of cellulase are considered as bottlenecks in the commercialization of bioethanol, and the rational design of cellulase preparation is also a time-consuming and labor-consuming challenge when multiple cellulases are used as cellulase preparation to act on bioethanol production.
Disclosure of Invention
The invention aims to overcome the problem of difficult rational design of the formula of a cellulase preparation in the prior art, and provides a method for designing a feed supplement mode of the cellulase preparation in a fermentation process by a computer and application thereof.
In order to achieve the above object, the present invention provides in a first aspect a method for computer-designing a feeding pattern of an enzyme preparation in a fermentation process, the enzyme preparation comprising a plurality of enzyme components, the method comprising:
(1) separately determining the activity of each of the enzyme components in the presence of a target product of the fermentation process, wherein the concentration of the target product is set to a plurality of different simulated concentrations;
(2) obtaining the crystal structure of the enzyme component from a protein database, selecting a model of the target product, and performing molecular dynamics simulation of the enzyme component when the concentration of the target product is a simulated concentration;
(3) and (3) obtaining a concentration value of a target product in the fermentation process when the enzyme preparation is not added, combining the activity data obtained in the step (1) with the molecular dynamics simulation result obtained in the step (2), and proposing a material supplementing time period and a material supplementing amount of each enzyme component.
Preferably, in the step (1), the method for measuring the activity of the enzyme component comprises: under the condition that the concentration of the target product is increased in a gradient manner, mixing the solution containing the enzyme component, the solution containing the substrate corresponding to the enzyme component and the buffer solution in the same proportion to obtain a mixed solution, carrying out enzymolysis reaction on the mixed solution for the same time, and then determining the absorbance value of the product of the enzymolysis reaction.
Preferably, the concentration of the target product in the mixed solution is 0-20 vol%, and the mass ratio of the enzyme component to the substrate corresponding to the enzyme component is 1: 0.04-50;
preferably, the conditions of the enzymatic hydrolysis reaction include: the temperature is 40-60 deg.C, and the time is 20-120 min; the wavelength of the absorbance value measurement is 400-600 nm.
Preferably, the target product of the fermentation process is ethanol, the enzyme preparation is a cellulase preparation, and the enzyme components are exocellulase I, exocellulase II, endo cellulase and beta-glucosidase;
preferably, the substrate corresponding to the exo-cellulase I is 4-nitrophenyl-beta-D-cellobioside, the substrate corresponding to the exo-cellulase II is microcrystalline cellulose, the substrate corresponding to the endo-cellulase is carboxymethyl cellulose, and the substrate corresponding to the beta-glucosidase is 4-nitrophenyl-beta-D-glucopyranoside;
Preferably, the buffer is a citrate buffer.
Preferably, in step (2), the molecular dynamics simulation process comprises: obtaining the crystal structure of the enzyme component from a protein database, selecting a model of the target product, simulating a basic solvent system and a cosolvent system by utilizing Gromacs software, and performing MD simulation analysis on each enzyme component to obtain energy and structure data of each enzyme component and action behaviors of the cosolvent on an active site of each enzyme component; wherein the cosolvent comprises the target product and a base solvent, and the concentration of the target product in the cosolvent is the simulated concentration;
preferably, the base solvent is water and the concentration of the target product in the co-solvent is 0-20% by volume.
Preferably, in step (3), the process of combining the activity data with the molecular dynamics simulation result comprises:
(a) obtaining a spatial distribution function, a radial distribution function, a contact frequency of a basic solvation shell layer and solvent-amino acid residues and a contact frequency of a target product layer and solvent-amino acid residues of the enzyme component by analyzing energy and structure data of the enzyme component and action behaviors of the cosolvent on active sites of the enzyme component, and further obtaining a specific fingerprint spectrum of the enzyme component in the cosolvent;
(b) Respectively carrying out desolvation and active site inhibition design on the enzyme component according to the specific fingerprint spectrum of the enzyme component, and providing a feeding time period and a feeding amount of each enzyme component by combining a time point when the concentration value of a target product in the fermentation process is the same as the simulated concentration when the enzyme preparation is not added.
The second aspect of the present invention provides a method for producing bioethanol, the method comprising: liquefying starchy raw materials to form fermentation raw materials, inoculating an ethanol fermentation strain into the fermentation raw materials, adding saccharifying enzyme and a nitrogen source to perform synchronous diastatic fermentation, and adding corresponding enzyme components in the material supplementing time period according to the enzyme preparation material supplementing mode in the fermentation process obtained by the method.
Preferably, the enzyme preparation is a cellulase preparation, and the plurality of enzyme components are exocellulase I, exocellulase II, endo cellulase and beta-glucosidase;
preferably, the feeding time periods are 0-2h, 4-12h, 12-16h, 20-30h and 45-55h respectively.
Preferably, the enzyme preparation feeding mode comprises: adding 3-7mg of exocellulase I, 3-7mg of exocellulase II, 3-7mg of endo cellulase and 3-7mg of beta-glucosidase into each liter of fermentation liquor after the fermentation is carried out for 0-2 h; performing the fermentation for 4-12h, and adding 0.3-1.8mg of exocellulase I, 1.8-3.5mg of exocellulase II, 0-0.7mg of endo cellulase and 0-0.7mg of beta-glucosidase into each liter of fermentation liquor; adding 0.3-1.8mg of exo-cellulase I and 0-7mg of beta-glucosidase into the fermentation liquor after the fermentation is carried out for 12-16 h; performing fermentation for 20-30h, and adding 0-0.7mg of exocellulase I, 0-7mg of exocellulase II, 0-3.5mg of endo cellulase and 0-0.7mg of beta-glucosidase into the fermentation liquor; and (3) adding 0-0.7mg of exocellulase I, 0-3.5mg of exocellulase II, 0-0.7mg of endo cellulase and 0-0.7mg of beta-glucosidase into the fermentation liquor after the fermentation is carried out for 45-55 h.
Preferably, the ethanol fermentation strain is saccharomyces cerevisiae, the inoculation amount of the ethanol fermentation strain is 0.3-0.5g dry cell weight/kg fermentation raw material, and the conditions of synchronous saccharification and fermentation comprise: the temperature is 25-35 ℃, the rotating speed is 120-;
preferably, the process of liquefaction treatment comprises: mixing the starchy raw material with water, and then mixing the mixture with alpha-amylase for liquefaction;
preferably, the dry matter concentration of the starchy raw material after the starchy raw material is mixed with water is 25-30 wt%, and the liquefaction treatment conditions comprise: the temperature is 85-90 deg.C, and the time is 60-120 min;
preferably, the starchy material is selected from at least one of corn, sorghum and rice, and the nitrogen source is selected from at least one of ammonium sulfate, urea and ammonium nitrate;
preferably, the dosage of the alpha-amylase is 0.010-0.015%, the dosage of the saccharifying enzyme is 0.0320-0.0330%, and the dosage of the nitrogen source is 0.010-0.015% relative to the dry weight of the starchy raw material.
Through the technical scheme, the invention has the beneficial effects that:
the method provided by the invention is based on the molecular dynamics simulation of a computer, reveals the molecular mechanism of the stability change of the concentration change of a target product to an enzyme component in an enzyme preparation in the fermentation process, and provides an active site inhibition guiding strategy by combining the activity influence of the concentration change of the target product to the enzyme component in the fermentation process, so as to carry out the design of desolvation and active site inhibition of the enzyme component, thereby obtaining the feeding mode of the enzyme preparation in the fermentation process; when the method is applied to the fermentation of the bioethanol, the method is time-saving and labor-saving, the addition amount of cellulase in the fermentation process is effectively reduced, the yield of the ethanol is improved, the production cost is saved, and the economy of the whole ethanol process is improved.
Drawings
FIG. 1 is a graph of residual activity of cellulase in example 1 versus different ethanol concentrations;
FIG. 2 is a schematic overlay of a representative structure of cellulase extracted from the MD traces in example 1;
FIG. 3 is the time-averaged RMSD of cellulase heavy atoms at different ethanol concentrations in example 1;
FIG. 4 is the internal hydrogen bond number >95% in cellulase at different ethanol concentrations in example 1;
FIG. 5 is the average values of the hydration layer and the ethanol layer around cellulase in example 1 at different ethanol concentrations; wherein (a) represents the average of hydration layers around the cellulase at different ethanol concentrations, and (b) represents the average of ethanol layers around the cellulase at different ethanol concentrations;
FIG. 6 is the average number of solvent molecules ethanol and water in the cellulase active sites at different ethanol concentrations in example 1; wherein (a) represents the average number of water molecules in the cellulase active site at different ethanol concentrations, and (b) represents the average number of ethanol molecules in the cellulase active site at different ethanol concentrations.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect, the present invention provides a method for computer-aided design of a feed profile for an enzyme preparation in a fermentation process, said enzyme preparation comprising a plurality of enzyme components, the method comprising:
(1) separately determining the activity of each of the enzyme components in the presence of a target product of the fermentation process, wherein the concentration of the target product is set to a plurality of different simulated concentrations;
(2) obtaining the crystal structure of the enzyme component from a protein database, selecting a model of the target product, and performing molecular dynamics simulation of the enzyme component when the concentration of the target product is a simulated concentration;
(3) and (3) obtaining a concentration value of a target product in the fermentation process when the enzyme preparation is not added, combining the activity data obtained in the step (1) with the molecular dynamics simulation result obtained in the step (2), and proposing a material supplementing time period and a material supplementing amount of each enzyme component.
During the research process, the inventors of the present invention unexpectedly found that in the process of producing a target product (e.g. ethanol) by fermentation, corresponding target product concentrations at different time points are obtained without adding an enzyme preparation (e.g. cellulase); and then, performing dynamic simulation based on the data of the concentrations of the target products, simulating the change of the enzyme preparation under different target product concentrations, and obtaining the reduced percentages (which can be defined as the reduction rate of the enzyme activity) of the residual activity or hydration and the like of the enzyme preparation under different target product concentrations so as to more accurately provide the amount of the enzyme preparation to be supplemented under different time points of fermentation, thereby recovering the enzyme activity of the enzyme preparation as much as possible under the condition of improving the target product concentration, further improving the yield of the target products, obviously reducing the dosage of the enzyme preparation and effectively reducing the production cost.
According to the present invention, in the step (1), the method for measuring the activity of the enzyme component comprises: under the condition that the concentration of the target product is increased in a gradient manner, mixing the solution containing the enzyme component, the solution containing the substrate corresponding to the enzyme component and the buffer solution in the same proportion to obtain a mixed solution, carrying out enzymolysis reaction on the mixed solution for the same time, and then determining the absorbance value of the product of the enzymolysis reaction. That is, in the presence of the target product, under the condition that other conditions are not changed, the activity of the enzyme component is examined to change along with the increase of the concentration of the target product, which can further reflect whether the increase of the concentration of the target product has an inhibitory effect on the activity of the enzyme component.
According to the present invention, the simulated concentration of the target product may be set according to the corresponding target product concentration obtained at different time points when no enzyme preparation is added. Preferably, the concentration of the target product in the mixture is 0 to 20 vol%, for example, the concentration of the target product in the mixture may be 0 vol%, 4 vol%, 8 vol%, 12 vol%, 16 vol%, 20 vol%.
According to the present invention, the amount of the enzyme component to be used in combination with the substrate corresponding to the enzyme component is not particularly limited, and the enzyme component may be capable of catalyzing the hydrolysis reaction of the corresponding substrate. Preferably, the mass ratio of the enzyme component to the substrate corresponding to the enzyme component is 1: 0.04-50. In order to enhance the enzymatic hydrolysis catalytic effect of the enzyme component, it is further preferable that the solution containing the enzyme component is obtained by diluting the enzyme component 0 to 5000 times, and the concentration of the solution containing the substrate corresponding to the enzyme component is 0.1 to 2% by weight.
According to the invention, conditions such as temperature, time, wavelength of absorbance determination and the like of the enzymolysis reaction are determined according to different enzyme components and corresponding substrates. In order to improve the efficiency of the enzymatic hydrolysis reaction, preferably, the conditions of the enzymatic hydrolysis reaction include: the temperature is 40-60 deg.C, and the time is 20-120 min; the wavelength of the absorbance value measurement is 400-600 nm.
According to the invention, preferably, the target product of the fermentation process is ethanol, the enzyme preparation is a cellulase preparation, and a plurality of the enzyme components are exo-cellulase I, exo-cellulase II, endo-cellulase and beta-glucosidase;
further preferably, the substrate corresponding to the exo-cellulase I is 4-nitrophenyl-beta-D-cellobioside (pNPC), the substrate corresponding to the exo-cellulase II is microcrystalline cellulose, the substrate corresponding to the endo-cellulase is carboxymethyl cellulose, and the substrate corresponding to the beta-glucosidase is 4-nitrophenyl-beta-D-glucopyranoside (pNPG).
According to the present invention, the buffer may be any buffer that can be used in an enzymatic reaction. Preferably, the buffer is a citric acid buffer, and the concentration of the citric acid buffer is 0.01-0.1M.
Illustratively, the activity of the exo-cellulase I (CBHI-Tr) is determined by mixing 0-1000 times diluted exo-cellulase I solution, 5-50 mmol/L4-nitrophenyl-beta-D-cellobioside solution, 0.01-0.1M citric acid buffer solution, adding ethanol corresponding to the simulated concentration, reacting at 40-60 deg.C for 20-60min, adding 0.5-2mol/L Na2CO3And water, the absorbance values at 400-450nm were measured.
Illustratively, the activity of the exocellulase II (CBHII-Hi) is determined by mixing a 10-1000-fold diluted exocellulase II solution, a 1-3 wt% microcrystalline cellulose solution, and a 0.01-0.1M citric acid buffer solution, adding ethanol corresponding to the simulated concentration, mixing, reacting at 40-60 deg.C for 20-60min, adding 3, 5-dinitrosalicylic acid (DNS), reacting with boiling water for 3-8min, adding water, and measuring the absorbance at 600nm of 500-.
Illustratively, the activity of the endo-cellulase (EG-An) is measured by mixing a 10-2000-fold diluted endo-cellulase solution, a 0.5-1.5 wt% carboxymethyl cellulose solution, and a 0.01-0.1M citric acid buffer solution, adding ethanol corresponding to the simulated concentration, mixing, reacting at 40-60 deg.C for 20-60min, adding 3, 5-dinitrosalicylic acid (DNS), reacting with boiling water for 3-8min, adding water, and measuring the absorbance at 600nm of 500-.
Illustratively, the activity of beta-glucosidase (BG-Pc) is determined by mixing a 5000-fold diluted solution of exocellulase I, a 5-50mmol/L solution of 4-nitrophenyl beta-D-glucopyranoside, and a 0.01-0.1M citric acid buffer, adding ethanol corresponding to the simulated concentration, reacting at 40-60 deg.C for 20-60min, adding 0.5-2mol/L Na2CO3And water, the absorbance values at 400-450nm were measured.
According to the present invention, preferably, in step (2), the molecular dynamics simulation process includes: obtaining the crystal structure of the enzyme component from a protein database, selecting a model of the target product, simulating a basic solvent system and a cosolvent system by utilizing Gromacs software, and performing MD simulation analysis on each enzyme component to obtain energy and structure data of each enzyme component and action behaviors of the cosolvent on an active site of each enzyme component; wherein the cosolvent contains the target product and a base solvent, and the concentration of the target product in the cosolvent is the simulated concentration.
Preferably, the base solvent is water and the concentration of the target product in the co-solvent is 0-20% by volume.
Illustratively, when the target product is ethanol, the ethanol model is taken from the ATB and the GROMOS96 (54 a 7) force field after optimizing the parameters, the simulation system is filled with 15892 water molecules in the pure water system (i.e., the basic solvent system), and is filled with 146-; energy minimization was performed using the steepest descent method, then 100ps equilibration was performed in the NPT ensemble with positional constraints on the cellulase, three independent computational simulations were performed with different starting atomic velocities, and coordinates, energy and velocity were collected every 500ps for later analysis.
According to the present invention, preferably, in the step (3), the process of combining the activity data with the molecular dynamics simulation result includes:
(a) obtaining a spatial distribution function, a radial distribution function, a contact frequency of a basic solvation shell layer and solvent-amino acid residues and a contact frequency of a target product layer and solvent-amino acid residues of the enzyme component by analyzing energy and structure data of the enzyme component and action behaviors of the cosolvent on active sites of the enzyme component, and further obtaining a specific fingerprint spectrum of the enzyme component in the cosolvent to study the complete solvation phenomenon around the enzyme component in the cosolvent in detail;
(b) Respectively carrying out desolvation and active site inhibition design on the enzyme component according to the specific fingerprint spectrum of the enzyme component, and providing a feeding time period and a feeding amount of each enzyme component by combining a time point when the concentration value of a target product in the fermentation process is the same as the simulated concentration when the enzyme preparation is not added.
According to the invention, when ethanol is used as a target product, the process of combining the activity data and the molecular dynamics simulation result can be as follows:
(a) analyzing a Spatial Distribution Function (SDF), a Radial Distribution Function (RDF), a hydration shell layer, an ethanol layer and solvent-amino acid residue contact frequency by combining the activity and the kinetic simulation result of different cellulases in ethanol, obtaining a specific fingerprint spectrum of each cellulase in a cosolvent, and researching the complete solvation phenomenon around the cellulases in the ethanol cosolvent in detail;
(b) according to the specific fingerprint spectrum of each cellulase in the cosolvent, a specific design method is carried out on the cellulase, and according to the solvation state (hydration layer and ethanol layer) of the surface of the cellulase, the precise number change of water molecules is selected as a design source of a compounding scheme, and a hydration layer guidance strategy and an active site inhibition guidance strategy are respectively provided to solve the problem of activity reduction of the cellulase in the ethanol fermentation process.
According to the invention, when the enzyme preparation is needed in the fermentation process, the method for designing the enzyme preparation feeding mode in the fermentation process through computer rationality provided by the invention can be adopted; for example, biomass raw materials such as corn, sorghum, wheat, cassava, potato, straw and the like are adopted to produce ethanol by fermentation.
The second aspect of the present invention provides a method for producing bioethanol, the method comprising: liquefying starchy raw materials to form fermentation raw materials, inoculating an ethanol fermentation strain into the fermentation raw materials, adding saccharifying enzyme and a nitrogen source to perform synchronous diastatic fermentation, and adding corresponding enzyme components in the material supplementing time period according to the enzyme preparation material supplementing mode in the fermentation process obtained by the method.
The activity determination of cellulase components under different simulated concentrations shows that in all cellulase-ethanol systems, when the ethanol concentration is between 0-16% (v/v), the number of ethanol molecules is gradually increased, more obvious inhibition effect on cellulase can be generated, and the activity of cellulase is gradually reduced along with the increase of the ethanol concentration, which undoubtedly limits the effective hydrolysis of cellulose in the starchy raw material to generate glucose, thereby interfering the growth of saccharomyces cerevisiae and the process of producing ethanol. Therefore, by proposing an active site inhibition-directed method to restore reduced cellulase activity by the above-described method, the ethanol/glycerol ratio can be significantly optimized to obtain better ethanol yield, when the concentration of ethanol produced by fermentation without cellulase addition is about the same as the concentration simulated in the experiment, the feed time for cellulase is determined, lower cellulase consumption can be obtained, ethanol yield is significantly improved, and the computationally designed method requires less experimental work. The process verification of producing ethanol by corn fermentation proves that the feeding mode of the cellulase preparation obtained by the method provided by the invention has good synergistic effect.
According to the present invention, preferably, the enzyme preparation is a cellulase preparation, and the plurality of enzyme components are exo-cellulase I, exo-cellulase II, endo-cellulase and β -glucosidase.
According to the invention, preferably, the feeding time periods are 0-2h, 4-12h, 12-16h, 20-30h, 45-55h, respectively.
According to the present invention, preferably, the enzyme preparation feeding mode comprises: adding 3-7mg of exocellulase I, 3-7mg of exocellulase II, 3-7mg of endo cellulase and 3-7mg of beta-glucosidase into each liter of fermentation liquor after the fermentation is carried out for 0-2 h; performing the fermentation for 4-12h, and adding 0.3-1.8mg of exocellulase I, 1.8-3.5mg of exocellulase II, 0-0.7mg of endo cellulase and 0-0.7mg of beta-glucosidase into each liter of fermentation liquor; adding 0.3-1.8mg of exo-cellulase I and 0-7mg of beta-glucosidase into the fermentation liquor after the fermentation is carried out for 12-16 h; performing fermentation for 20-30h, and adding 0-0.7mg of exocellulase I, 0-7mg of exocellulase II, 0-3.5mg of endo cellulase and 0-0.7mg of beta-glucosidase into the fermentation liquor; and (3) adding 0-0.7mg of exocellulase I, 0-3.5mg of exocellulase II, 0-0.7mg of endo cellulase and 0-0.7mg of beta-glucosidase into the fermentation liquor after the fermentation is carried out for 45-55 h.
According to the invention, preferably, the ethanol fermentation strain is saccharomyces cerevisiae, the inoculation amount of the ethanol fermentation strain is 0.3-0.5g dry cell weight/kg fermentation raw material, and the fermentation conditions comprise: the temperature is 25-35 ℃, the rotating speed is 120-;
according to the invention, the liquefaction process comprises: mixing the starchy raw material with water, and mixing the mixture with alpha-amylase for liquefaction;
according to the invention, preferably, the dry matter concentration of the starchy raw material after the starchy raw material is mixed with water is 25-30 wt%, and the liquefaction treatment conditions comprise: the temperature is 85-90 deg.C, and the time is 60-120 min.
According to the present invention, preferably, the starchy raw material is selected from at least one of corn, sorghum and rice, and the nitrogen source is selected from at least one of ammonium sulfate, urea and ammonium nitrate. Wherein, corn, sorghum, rice and the like can be purchased in the market, and the starch raw materials are preferably selected from corresponding powder.
According to the present invention, preferably, the amount of the α -amylase is 0.010 to 0.015%, the amount of the saccharifying enzyme is 0.0320 to 0.0330%, and the amount of the nitrogen source is 0.010 to 0.015% with respect to the dry weight of the starchy material.
The present invention will be described in detail below by way of examples.
In the following examples, corn flour was given by bioenergy GmbH of Wuning iron Ling, China (glucan content: 82.40%, xylan content: 2.50%, starch content: 77.65%, cellulose content: 4.79%); alpha-amylase is purchased from Genencor biological products, Inc., glucoamylase is purchased from Shandongdong biological products, Inc., exo-cellulase I (CBHI-Tr) is purchased from Sigma Aldrich, exo-cellulase II (CBHII-Hi), endo-cellulase (EG-An) and beta-glucosidase (BG-Pc) are purchased from Megazyme, and the protein contents of CBHI-Tr, CBHII-Hi, EG-An and BG-Pc are respectively 2.97, 1.55, 0.33 and 5.05 mg/mL; saccharomyces cerevisiae was purchased from Angel Yeast Co., Ltd, and other raw materials and reagents were all conventionally commercially available.
In the following examples, absorbance values were measured by aspirating 200. mu.L of the supernatant into an microplate, by means of a microplate reader, and the ethanol concentration was measured by centrifuging the sample at 10000rcf for 10min to obtain a supernatant, diluting 10-fold, immediately subjecting to sterile filtration through a 0.22 μm filter, and measuring by high performance liquid chromatography.
Example 1
(1) Determination of the Activity of exo-cellulase I (CBHI-Tr): 0.1mL of exo-cellulase I solution, 0.7mL of 4-nitrophenyl-beta-D-cellobioside solution with the concentration of 10mmol/L and 0.1mL of 4-nitrophenyl-beta-D-cellobioside solution with the concentration of 0.1 mmol/LMixing with 0.05M citric acid buffer solution, adding ethanol to obtain mixed solution with ethanol concentration of 0 vol%, 4 vol%, 8 vol%, 12 vol%, 16 vol% and 20 vol%, reacting at 50 deg.C for 40min, and adding 2mL Na with concentration of 1mol/L2CO3And 10mL of water, and the absorbance at 400nm was measured, the results are shown in Table 1;
(2) determination of exo-cellulase II (CBHII-Hi) Activity: mixing 0.1mL of 200-fold diluted exocellulase II solution, 0.14mL of 2 wt% microcrystalline cellulose PH101 solution and 0.1mL of 0.05M citric acid buffer solution, adding ethanol to obtain a mixed solution, wherein the ethanol concentrations in the mixed solution are respectively 0 vol%, 4 vol%, 8 vol%, 12 vol%, 16 vol% and 20 vol%, reacting at 50 ℃ for 40min, adding 0.6mL of 3, 5-dinitrosalicylic acid (DNS), reacting with boiling water for 5min, adding 2mL of water, and measuring the light absorption value at 550nm, wherein the results are shown in Table 1;
(3) determination of endo-cellulase (EG-An) Activity: mixing 0.1mL of 200-fold diluted endo-cellulase solution, 0.14mL of 1 wt% carboxymethyl cellulose solution and 0.1mL of 0.05M citric acid buffer solution, adding ethanol to obtain a mixed solution, wherein the ethanol concentrations in the mixed solution are respectively 0 vol%, 4 vol%, 8 vol%, 12 vol%, 16 vol% and 20 vol%, reacting at 50 ℃ for 40min, adding 0.6mL of 3, 5-dinitrosalicylic acid (DNS), reacting with boiling water for 5min, adding 2mL of water, and measuring the light absorption value at 550nm, wherein the results are shown in Table 1;
(4) Measurement of β -glucosidase (BG-Pc) Activity: mixing 0.1mL of 5000-fold diluted exocellulase I solution, 0.7mL of 5 mmol/L4-nitrophenyl beta-D-glucopyranoside solution and 0.1mL of 0.05M citric acid buffer solution, adding ethanol to obtain a mixed solution, wherein the ethanol concentration in the mixed solution is respectively 0 vol%, 4 vol%, 8 vol%, 12 vol%, 16 vol% and 20 vol%, reacting at 50 ℃ for 120min, adding 2mL of 1mol/L Na2CO3And 10mL of water, and the absorbance at 400nm was measured, the results are shown in Table 1;
(5) obtaining crystal structures of CBHI-Tr, CBHII-Hi, BG-Pc and EG-An from a protein database, performing MD simulation using Gromacs v5.1.2, using a GROMOS96 (54 a 7) force field to simulate cellulase in ethanol solvent, placing the cellulase crystal structures in a cubic box of SPCE water with a minimum distance of 1.2 nm; according to experimental conditions, the simulation system is filled with 15892 water molecules in a pure water system and is filled with 146-800 ethanol molecules in a 4-16% (v/v) ethanol solvent system, in order to avoid the most adverse interaction, firstly, the energy minimization is carried out by adopting the steepest descent method, then, under the condition of position limitation on cellulase, 100 ps balance is carried out in an NPT system, three independent MD simulations are carried out at different initial atomic speeds, and coordinates, energy and speed are collected once per 500 ps for MD analysis;
(6) Combining the activity (shown in figure 1) and the overall structure performance (shown in figure 2, figure 3 and figure 4) of different cellulases in the steps (1) to (4) and the MD simulation result of the step (5), analyzing a Space Distribution Function (SDF), a Radial Distribution Function (RDF), a hydration shell layer, an ethanol layer and the contact frequency of solvent-amino acid residues, researching the complete solvation phenomenon around the cellulases in the ethanol cosolvent in detail, carrying out a specific design method on each cellulase according to the specific fingerprint spectrum of the cellulase in the cosolvent, combining the solvation state (including the hydration layer and the ethanol layer, shown in figure 5) of the cellulase surface, selecting the cellulase feeding mode which utilizes the accurate number change of water molecules (shown in figure 6) as the design source of a compounding scheme and respectively proposes the desolvation and the active site derepression, that is, according to the actual study, the amount of each cellulase added under the conventional conditions (taking the amount of each cellulase added as 0.03 wt% of the dry weight of corn as an example) was first determined, 0.16mg of each cellulase protein was added at 0h in consideration of the effective protein content, the percentage reduction of selected factors (e.g., residual activity, global hydration, degree of hydration of the active center) at each time point was calculated based on the above-mentioned residual activity data or simulation results, and the reduction of the latter was defined as the amount of reduction of the enzyme activity, and the cellulase was finally obtained by supplementing the corresponding amount of enzyme at each time point in order to theoretically recover 100% of the enzyme activity, specifically: adding 3-7mg of exocellulase I, 3-7mg of exocellulase II, 3-7mg of endo cellulase and 3-7mg of beta-glucosidase into each liter of fermentation liquor after the fermentation is carried out for 0-2 h; performing the fermentation for 4-12h, and adding 0.3-1.8mg of exocellulase I, 1.8-3.5mg of exocellulase II, 0-0.7mg of endo cellulase and 0-0.7mg of beta-glucosidase into each liter of fermentation liquor; adding 0.3-1.8mg of exo-cellulase I and 0-7mg of beta-glucosidase into the fermentation liquor after the fermentation is carried out for 12-16 h; performing fermentation for 20-30h, and adding 0-0.7mg of exocellulase I, 0-7mg of exocellulase II, 0-3.5mg of endo cellulase and 0-0.7mg of beta-glucosidase into the fermentation liquor; and (3) adding 0-0.7mg of exocellulase I, 0-3.5mg of exocellulase II, 0-0.7mg of endo cellulase and 0-0.7mg of beta-glucosidase into the fermentation liquor after the fermentation is carried out for 45-55 h.
TABLE 1
Example 2
(1) Culturing YPD culture medium containing glucose as seed culture medium of Saccharomyces cerevisiae (glucose concentration is 100 g/L) at 30 deg.C and rotation speed of 150rpm for 24 hr to obtain seed culture solution, and centrifuging the seed culture solution at 4000rpm for 10min to obtain wet seed thallus;
(2) mixing corn flour with water to make corn dry matter concentration be 30 wt%, adding 0.012 wt% (relative to corn dry matter) alpha-amylase, liquefying at 88 deg.C for 90min, and cooling to 60 deg.C to obtain fermentation raw material;
(3) taking 30g of fermentation raw material obtained in the step (2) as fermentation liquor, inoculating the wet seed thalli obtained in the step (1) with 0.44g of dry cell weight/kg of fermentation liquor, adding 0.0325 wt% (relative to the dry weight of corn) of saccharifying enzyme and 0.012 wt% (relative to the dry weight of corn) of urea, and then fermenting under the conditions of pH value of 4.0, rotation speed of 150rpm and temperature of 30 ℃; when the fermentation was carried out for 0h, 0.16mg of CBHI-Tr, 0.16mg of CBHII-Hi, 0.16mg of EG-An and 0.16mg of BG-Pc were added to the fermentation broth; adding 0.04mg of CBHI-Tr, 0.09mg of CBHII-Hi, 0.01mg of EG-An and 0.01mg of BG-Pc into the fermentation broth when the fermentation is carried out for 8 h; adding 0.03mg of CBHI-Tr and 0.10mg of BG-Pc to the fermentation broth when the fermentation is carried out for 13 h; adding 0.01mg of CBHI-Tr, 0.19mg of CBHII-Hi and 0.04mg of EG-An to the fermentation broth at 22h after fermentation; when the fermentation was carried out for 50 hours, 0.07mg of CBHII-Hi and 0.01mg of EG-An were added to the fermentation broth, and the fermentation was continued until 72 hours were reached.
And (4) determining the ethanol concentration in the fermentation liquor obtained in the step (3) to be 119.57 g/L.
Example 3
Bioethanol was prepared according to the method of example 2, except that step (3) was replaced by:
(3) taking 30g of fermentation raw material obtained in the step (2) as fermentation liquor, inoculating the wet seed thalli obtained in the step (1) with 0.44g of dry cell weight/kg of fermentation liquor, adding 0.0325 wt% (relative to the dry weight of corn) of saccharifying enzyme and 0.012 wt% (relative to the dry weight of corn) of urea, and then fermenting under the conditions of pH value of 4.0, rotation speed of 150 rpm and temperature of 30 ℃; when the fermentation was carried out for 0h, 0.16mg of CBHI-Tr, 0.16mg of CBHII-Hi, 0.16mg of EG-An and 0.16mg of BG-Pc were added to the fermentation broth; adding 0.04mg of CBHI-Tr, 0.09mg of CBHII-Hi, 0.01mg of EG-An and 0.01mg of BG-Pc into the fermentation broth when the fermentation is carried out for 8 h; adding 0.05mg of CBHI-Tr and 0.12mg of BG-Pc to the fermentation broth when the fermentation is carried out for 13 h; adding 0.02mg of CBHI-Tr, 0.18mg of CBHII-Hi, 0.04mg of EG-An and 0.01mg of BG-Pc into the fermentation broth when the fermentation is carried out for 22 h; when the fermentation was carried out for 50 hours, 0.01mg of CBHI-Tr, 0.07mg of CBHII-Hi, 0.01mg of EG-An and 0.01mg of BG-Pc were added to the fermentation broth, and the fermentation was continued for 72 hours.
And (4) determining the ethanol concentration in the fermentation liquor obtained in the step (3) to be 119.15 g/L.
Example 4
Bioethanol was prepared according to the method of example 2, except that step (3) was replaced by:
(3) taking 30g of fermentation raw material obtained in the step (2) as fermentation liquor, inoculating the wet seed thalli obtained in the step (1) with 0.44g of dry cell weight/kg of fermentation liquor, adding 0.0325 wt% (relative to the dry weight of corn) of saccharifying enzyme and 0.012 wt% (relative to the dry weight of corn) of urea, and then fermenting under the conditions of pH value of 4.0, rotation speed of 150 rpm and temperature of 30 ℃; when the fermentation was carried out for 0h, 0.16mg of CBHI-Tr, 0.16mg of CBHII-Hi, 0.16mg of EG-An and 0.16mg of BG-Pc were added to the fermentation broth; when fermentation was carried out for 6h, 0.04mg of CBHI-Tr, 0.09mg of CBHII-Hi, 0.01mg of EG-An and 0.01mg of BG-Pc were added to the fermentation broth; adding 0.03mg of CBHI-Tr and 0.10mg of BG-Pc to the fermentation broth when the fermentation is carried out for 13 h; when fermentation proceeded for 24h, 0.01mg of CBHI-Tr, 0.19mg of CBHII-Hi and 0.04mg of EG-An were added to the broth; when the fermentation was carried out for 50 hours, 0.07mg of CBHII-Hi and 0.01mg of EG-An were added to the fermentation broth, and the fermentation was continued for 80 hours.
And (4) measuring the ethanol concentration in the fermentation liquor obtained in the step (3) to be 118.37 g/L.
Comparative example 1
Bioethanol was prepared according to the method of example 2, except that step (3) was replaced by:
(3) taking 30g of fermentation raw material obtained in the step (2) as fermentation liquor, inoculating the wet seed thalli obtained in the step (1) with 0.44g of dry cell weight/kg of fermentation liquor, adding 0.0325 wt% (relative to the dry weight of corn) of saccharifying enzyme and 0.012 wt% (relative to the dry weight of corn) of urea, and then fermenting under the conditions of pH value of 4.0, rotation speed of 150rpm and temperature of 30 ℃ for 96h to finish.
And (4) measuring the ethanol concentration in the fermentation liquor obtained in the step (3) to be 113 g/L.
Comparative example 2
Bioethanol was prepared according to the method of example 2, except that step (3) was replaced by:
(3) taking 30g of fermentation raw material obtained in the step (2) as fermentation liquor, inoculating the wet seed thalli obtained in the step (1) with 0.44g of dry cell weight/kg of fermentation liquor, adding 0.0325 wt% (relative to the dry weight of corn) of saccharifying enzyme and 0.012 wt% (relative to the dry weight of corn) of urea, and then fermenting under the conditions of pH value of 4.0, rotation speed of 150rpm and temperature of 30 ℃; when the fermentation was carried out for 0 hour, 0.20mg of CBHI-Tr, 4.14mg of CBHII-Hi, 0.78mg of EG-An and 0.16mg of BG-Pc were added to the fermentation broth, and the fermentation was completed up to 72 hours.
And (4) determining the ethanol concentration in the fermentation liquor obtained in the step (3) to be 116.74 g/L.
Comparative example 3
Bioethanol was prepared according to the method of example 2, except that step (3) was replaced by:
(3) taking 30g of fermentation raw material obtained in the step (2) as fermentation liquor, inoculating the wet seed thalli obtained in the step (1) with 0.44g of dry cell weight/kg of fermentation liquor, adding 0.0325 wt% (relative to the dry weight of corn) of saccharifying enzyme and 0.012 wt% (relative to the dry weight of corn) of urea, and then fermenting under the conditions of pH value of 4.0, rotation speed of 150 rpm and temperature of 30 ℃; when the fermentation was carried out for 0 hour, 0.30mg of CBHI-Tr, 0.32mg of CBHII-Hi, 0.39mg of EG-An and 0.39mg of BG-Pc were added to the fermentation broth, and the fermentation was completed up to 72 hours.
And (4) determining the ethanol concentration in the fermentation liquor obtained in the step (3) to be 117.73 g/L.
TABLE 2
| Numbering | Total cellulase addition (mg) | Ethanol yield (g/L) |
| Example 2 | 1.24 | 119.57 |
| Example 3 | 1.31 | 119.15 |
| Example 4 | 1.24 | 118.37 |
| Comparative example 1 | 0 | 113.00 |
| Comparative example 2 | 5.28 | 116.74 |
| Comparative example 3 | 1.40 | 117.73 |
From the results in table 2, it can be seen that examples 2, 3 and 4, which were fermented by feeding cellulase preparations obtained by the method of the present invention, gave ethanol yields 5.8% higher than that obtained in comparative example 1 without cellulase preparation. The results of the examples 2, 3 and 4 and the comparative examples 2 and 3 show that compared with the method of adding cellulase once, the method of the invention is based on computer simulation, the yield of ethanol produced by adopting an active site inhibition guiding strategy is higher, and simultaneously, the addition amount of cellulase preparation can be reduced, the production cost is saved, and the economy of the whole ethanol process is improved.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A method for designing in silico feeding patterns of an enzyme preparation in a fermentation process, wherein the enzyme preparation comprises a plurality of enzyme components, the method comprising:
(1) separately determining the activity of each of the enzyme components in the presence of a target product of the fermentation process, wherein the concentration of the target product is set to a plurality of different simulated concentrations;
(2) obtaining the crystal structure of the enzyme component from a protein database, selecting a model of the target product, and performing molecular dynamics simulation of the enzyme component when the concentration of the target product is a simulated concentration;
(3) and (3) obtaining a concentration value of a target product in the fermentation process when the enzyme preparation is not added, combining the activity data obtained in the step (1) with the molecular dynamics simulation result obtained in the step (2), and proposing a material supplementing time period and a material supplementing amount of each enzyme component.
2. The method according to claim 1, wherein in step (1), the method for measuring the activity of the enzyme component comprises: under the condition that the concentration of the target product is increased in a gradient manner, mixing the solution containing the enzyme component, the solution containing the substrate corresponding to the enzyme component and the buffer solution in the same proportion to obtain a mixed solution, carrying out enzymolysis reaction on the mixed solution for the same time, and then determining the absorbance value of the product of the enzymolysis reaction.
3. The method according to claim 2, wherein the concentration of the target product in the mixed solution is 0 to 20 vol%, and the mass ratio of the enzyme component to the substrate corresponding to the enzyme component is 1: 0.04-50;
the conditions of the enzymolysis reaction comprise: the temperature is 40-60 deg.C, and the time is 20-120 min; the wavelength of the absorbance value measurement is 400-600 nm.
4. The method according to claim 2, wherein the target product of the fermentation process is ethanol, the enzyme preparation is a cellulase preparation, and a plurality of the enzyme components are exo-cellulase I, exo-cellulase II, endo-cellulase and β -glucosidase;
the substrate corresponding to the exo-cellulase I is 4-nitrophenyl-beta-D-cellobioside, the substrate corresponding to the exo-cellulase II is microcrystalline cellulose, the substrate corresponding to the endo-cellulase is carboxymethyl cellulose, and the substrate corresponding to the beta-glucosidase is 4-nitrophenyl-beta-D-glucopyranoside;
The buffer solution is a citric acid buffer solution.
5. The method according to any one of claims 1 to 4, wherein in the step (2), the molecular dynamics simulation process comprises: obtaining the crystal structure of the enzyme component from a protein database, selecting a model of the target product, simulating a basic solvent system and a cosolvent system by utilizing Gromacs software, and performing MD simulation analysis on each enzyme component to obtain energy and structure data of each enzyme component and action behaviors of the cosolvent on an active site of each enzyme component; wherein the cosolvent comprises the target product and a base solvent, and the concentration of the target product in the cosolvent is the simulated concentration;
the base solvent is water and the concentration of the target product in the co-solvent is 0-20% by volume.
6. The method of claim 5, wherein the step (3) of combining the activity data with the molecular dynamics simulation results comprises:
(a) obtaining a spatial distribution function, a radial distribution function, a contact frequency of a basic solvation shell layer and solvent-amino acid residues and a contact frequency of a target product layer and solvent-amino acid residues of the enzyme component by analyzing energy and structure data of the enzyme component and action behaviors of the cosolvent on active sites of the enzyme component, and further obtaining a specific fingerprint spectrum of the enzyme component in the cosolvent;
(b) Respectively carrying out desolvation and active site inhibition design on the enzyme component according to the specific fingerprint spectrum of the enzyme component, and providing a feeding time period and a feeding amount of each enzyme component by combining a time point when the concentration value of a target product in the fermentation process is the same as the simulated concentration when the enzyme preparation is not added.
7. A method for preparing bioethanol, comprising: liquefying a starchy raw material to form a fermentation raw material, inoculating an ethanol fermentation strain into the fermentation raw material, adding saccharifying enzyme and a nitrogen source to perform synchronous saccharification and fermentation, and adding corresponding enzyme components in the feeding time period according to the enzyme preparation feeding mode in the fermentation process obtained by the method of any one of claims 1 to 6.
8. The production method according to claim 7, wherein the enzyme preparation is a cellulase preparation, and the plurality of enzyme components are exo-cellulase I, exo-cellulase II, endo-cellulase and β -glucosidase;
the feeding time periods are respectively 0-2h, 4-12h, 12-16h, 20-30h and 45-55 h.
9. The method of claim 8, wherein the enzyme preparation is fed in a manner comprising: adding 3-7mg of exocellulase I, 3-7mg of exocellulase II, 3-7mg of endo cellulase and 3-7mg of beta-glucosidase into each liter of fermentation liquor after the fermentation is carried out for 0-2 h; performing the fermentation for 4-12h, and adding 0.3-1.8mg of exocellulase I, 1.8-3.5mg of exocellulase II, 0-0.7mg of endo cellulase and 0-0.7mg of beta-glucosidase into each liter of fermentation liquor; adding 0.3-1.8mg of exo-cellulase I and 0-7mg of beta-glucosidase into the fermentation liquor after the fermentation is carried out for 12-16 h; performing fermentation for 20-30h, and adding 0-0.7mg of exocellulase I, 0-7mg of exocellulase II, 0-3.5mg of endo cellulase and 0-0.7mg of beta-glucosidase into the fermentation liquor; and (3) adding 0-0.7mg of exocellulase I, 0-3.5mg of exocellulase II, 0-0.7mg of endo cellulase and 0-0.7mg of beta-glucosidase into the fermentation liquor after the fermentation is carried out for 45-55 h.
10. The process according to any one of claims 7 to 9, wherein the ethanol fermentation strain is saccharomyces cerevisiae, the inoculum size of the ethanol fermentation strain is 0.3-0.5g dry cell weight/kg fermentation raw material, and the conditions of simultaneous saccharification and fermentation comprise: the temperature is 25-35 ℃, the rotating speed is 120-;
the process of the liquefaction treatment comprises the following steps: mixing the starchy raw material with water, and then mixing the mixture with alpha-amylase for liquefaction;
the dry matter concentration of the starchy raw material after the starchy raw material is mixed with water is 25-30 wt%, and the liquefaction treatment conditions comprise: the temperature is 85-90 deg.C, and the time is 60-120 min;
the starchy raw material is selected from at least one of corn, sorghum and rice, and the nitrogen source is selected from at least one of ammonium sulfate, urea and ammonium nitrate;
relative to the dry weight of the starchy raw material, the dosage of the alpha-amylase is 0.010-0.015%, the dosage of the saccharifying enzyme is 0.0320-0.0330%, and the dosage of the nitrogen source is 0.010-0.015%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111189956.7A CN113637712B (en) | 2021-10-13 | 2021-10-13 | Method for designing enzyme preparation feeding mode in fermentation process through computer and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111189956.7A CN113637712B (en) | 2021-10-13 | 2021-10-13 | Method for designing enzyme preparation feeding mode in fermentation process through computer and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113637712A true CN113637712A (en) | 2021-11-12 |
| CN113637712B CN113637712B (en) | 2022-01-18 |
Family
ID=78426542
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111189956.7A Active CN113637712B (en) | 2021-10-13 | 2021-10-13 | Method for designing enzyme preparation feeding mode in fermentation process through computer and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113637712B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117229905A (en) * | 2023-11-15 | 2023-12-15 | 山东朝辉生物科技有限公司 | Biological feed fermentation control method and system |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492579A (en) * | 2011-04-29 | 2014-01-01 | 丹尼斯科美国公司 | Use of cellulase and glucoamylase to improve ethanol yields from fermentation |
| CN104797708A (en) * | 2012-08-29 | 2015-07-22 | 拉勒曼德匈牙利流动性管理有限责任公司 | Expression of enzymes in yeast for lignocellulose derived oligomer CBP |
| CN112779296A (en) * | 2019-11-06 | 2021-05-11 | 南京百斯杰生物工程有限公司 | Enzyme preparation adding process for promoting fermentation of starch grains |
-
2021
- 2021-10-13 CN CN202111189956.7A patent/CN113637712B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492579A (en) * | 2011-04-29 | 2014-01-01 | 丹尼斯科美国公司 | Use of cellulase and glucoamylase to improve ethanol yields from fermentation |
| CN104797708A (en) * | 2012-08-29 | 2015-07-22 | 拉勒曼德匈牙利流动性管理有限责任公司 | Expression of enzymes in yeast for lignocellulose derived oligomer CBP |
| CN112779296A (en) * | 2019-11-06 | 2021-05-11 | 南京百斯杰生物工程有限公司 | Enzyme preparation adding process for promoting fermentation of starch grains |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117229905A (en) * | 2023-11-15 | 2023-12-15 | 山东朝辉生物科技有限公司 | Biological feed fermentation control method and system |
| CN117229905B (en) * | 2023-11-15 | 2024-02-06 | 山东朝辉生物科技有限公司 | Biological feed fermentation control method and system |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113637712B (en) | 2022-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Integrated bioethanol production from mixtures of corn and corn stover | |
| Yamada et al. | Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression | |
| Zhang et al. | Energy-saving direct ethanol production from viscosity reduction mash of sweet potato at very high gravity (VHG) | |
| Choudhary et al. | Bioprospecting thermotolerant ethanologenic yeasts for simultaneous saccharification and fermentation from diverse environments | |
| JP5334060B2 (en) | Production method of biofuel using seaweed | |
| Han et al. | Improving cellulase productivity of Penicillium oxalicum RE-10 by repeated fed-batch fermentation strategy | |
| Menon et al. | Biocatalytic approach for the utilization of hemicellulose for ethanol production from agricultural residue using thermostable xylanase and thermotolerant yeast | |
| Srivastava et al. | Application of cellulases in biofuels industries: an overview | |
| Paschos et al. | Simultaneous saccharification and fermentation by co-cultures of Fusarium oxysporum and Saccharomyces cerevisiae enhances ethanol production from liquefied wheat straw at high solid content | |
| Soni et al. | BIOCONVERSION OF SUGARCANE BAGASSE INTO SECOND GENERATION BIOETHANOL AFTER ENZYMATIC HYDROLYSIS WITH IN-HOUSE PRODUCED CELLULASES FROM Aspergillus sp. S 4 B 2 F. | |
| Philippidis et al. | Mathematical modeling of cellulose conversion to ethanol by the simultaneous saccharification and fermentation process | |
| Shukor et al. | Saccharification of polysaccharide content of palm kernel cake using enzymatic catalysis for production of biobutanol in acetone–butanol–ethanol fermentation | |
| Favaro et al. | Processing wheat bran into ethanol using mild treatments and highly fermentative yeasts | |
| Li et al. | In-situ corn fiber conversion improves ethanol yield in corn dry-mill process | |
| Saravanakumar et al. | Bioethanol production by mangrove-derived marine yeast, Sacchromyces cerevisiae | |
| WO2010014631A2 (en) | Methods and compositions for improving the production of products in microorganisms | |
| Das et al. | Bioconversion of rice straw to sugar using multizyme complex of fungal origin and subsequent production of bioethanol by mixed fermentation of Saccharomyces cerevisiae MTCC 173 and Zymomonas mobilis MTCC 2428 | |
| CN102876590B (en) | Penicillium sp. mutant strain and application of penicillium sp. mutant strain to cellulase preparation | |
| Han et al. | Solid-state fermentation on poplar sawdust and corncob wastes for lignocellulolytic enzymes by different Pleurotus ostreatus strains | |
| Li et al. | In situ pretreatment during distillation improves corn fiber conversion and ethanol yield in the dry mill process | |
| Spindler et al. | The simultaneous saccharification and fermentation of pretreated woody crops to ethanol | |
| CN113637712B (en) | Method for designing enzyme preparation feeding mode in fermentation process through computer and application | |
| Radhakumari et al. | Pongamia pinnata seed residue–A low cost inedible resource for on-site/in-house lignocellulases and sustainable ethanol production | |
| CN1986820B (en) | Alcohol output increasing method and device | |
| Baibakova et al. | Preparing bioethanol from oat hulls pretreated with a dilute nitric acid: Scaling of the production process on a pilot plant |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |