CN113801900A - Method for preparing pyruvic acid by using forest trees and application thereof - Google Patents
Method for preparing pyruvic acid by using forest trees and application thereof Download PDFInfo
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- CN113801900A CN113801900A CN202111138577.5A CN202111138577A CN113801900A CN 113801900 A CN113801900 A CN 113801900A CN 202111138577 A CN202111138577 A CN 202111138577A CN 113801900 A CN113801900 A CN 113801900A
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- pyruvic acid
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- acid
- enzymolysis
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- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 title claims abstract description 118
- 229940107700 pyruvic acid Drugs 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000000855 fermentation Methods 0.000 claims abstract description 68
- 230000004151 fermentation Effects 0.000 claims abstract description 68
- 102000004190 Enzymes Human genes 0.000 claims abstract description 45
- 108090000790 Enzymes Proteins 0.000 claims abstract description 45
- 238000004880 explosion Methods 0.000 claims abstract description 32
- 239000002699 waste material Substances 0.000 claims abstract description 32
- 238000010335 hydrothermal treatment Methods 0.000 claims abstract description 18
- 241000588724 Escherichia coli Species 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 238000010353 genetic engineering Methods 0.000 claims abstract description 7
- 241000894006 Bacteria Species 0.000 claims abstract description 5
- 229940088598 enzyme Drugs 0.000 claims description 42
- 230000000694 effects Effects 0.000 claims description 34
- 239000002245 particle Substances 0.000 claims description 25
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 18
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 18
- 108010059820 Polygalacturonase Proteins 0.000 claims description 17
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 17
- 108010059892 Cellulase Proteins 0.000 claims description 16
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 16
- 108010029541 Laccase Proteins 0.000 claims description 16
- 239000002253 acid Substances 0.000 claims description 15
- 101710112457 Exoglucanase Proteins 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 238000011081 inoculation Methods 0.000 claims description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 5
- 229940106157 cellulase Drugs 0.000 claims description 4
- 229940059442 hemicellulase Drugs 0.000 claims description 4
- 108010002430 hemicellulase Proteins 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 239000011573 trace mineral Substances 0.000 claims description 4
- 235000013619 trace mineral Nutrition 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 150000003863 ammonium salts Chemical class 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 2
- 235000013379 molasses Nutrition 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 229920002488 Hemicellulose Polymers 0.000 abstract description 11
- 150000002972 pentoses Chemical class 0.000 abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 5
- 229910052799 carbon Inorganic materials 0.000 abstract description 5
- 230000015556 catabolic process Effects 0.000 abstract description 4
- 238000006731 degradation reaction Methods 0.000 abstract description 4
- 150000002402 hexoses Chemical class 0.000 abstract description 4
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 30
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 29
- 235000011613 Pinus brutia Nutrition 0.000 description 29
- 241000018646 Pinus brutia Species 0.000 description 29
- 150000002772 monosaccharides Chemical class 0.000 description 28
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 27
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 24
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 24
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 18
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 18
- 239000002023 wood Substances 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 15
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 15
- 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 14
- 239000008103 glucose Substances 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 12
- 239000012452 mother liquor Substances 0.000 description 12
- 238000012258 culturing Methods 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- NAOLWIGVYRIGTP-UHFFFAOYSA-N 1,3,5-trihydroxyanthracene-9,10-dione Chemical compound C1=CC(O)=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1 NAOLWIGVYRIGTP-UHFFFAOYSA-N 0.000 description 9
- 244000166124 Eucalyptus globulus Species 0.000 description 9
- 241000219000 Populus Species 0.000 description 9
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 9
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 239000006227 byproduct Substances 0.000 description 9
- 239000001530 fumaric acid Substances 0.000 description 9
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 229920001542 oligosaccharide Polymers 0.000 description 8
- 150000002482 oligosaccharides Chemical class 0.000 description 8
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000011218 seed culture Methods 0.000 description 7
- 239000002028 Biomass Substances 0.000 description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 6
- 150000007524 organic acids Chemical class 0.000 description 6
- LGQKSQQRKHFMLI-SJYYZXOBSA-N (2s,3r,4s,5r)-2-[(3r,4r,5r,6r)-4,5,6-trihydroxyoxan-3-yl]oxyoxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)OC1 LGQKSQQRKHFMLI-SJYYZXOBSA-N 0.000 description 5
- LGQKSQQRKHFMLI-UHFFFAOYSA-N 4-O-beta-D-xylopyranosyl-beta-D-xylopyranose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(O)OC1 LGQKSQQRKHFMLI-UHFFFAOYSA-N 0.000 description 5
- SQNRKWHRVIAKLP-UHFFFAOYSA-N D-xylobiose Natural products O=CC(O)C(O)C(CO)OC1OCC(O)C(O)C1O SQNRKWHRVIAKLP-UHFFFAOYSA-N 0.000 description 5
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 229940076788 pyruvate Drugs 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 239000003054 catalyst Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000010298 pulverizing process Methods 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 3
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 3
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 3
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 3
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 239000011565 manganese chloride Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 239000011684 sodium molybdate Substances 0.000 description 3
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 239000011592 zinc chloride Substances 0.000 description 3
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- -1 copper oxychloride phosphate Chemical compound 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000013138 pruning Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- INZDTEICWPZYJM-UHFFFAOYSA-N 1-(chloromethyl)-4-[4-(chloromethyl)phenyl]benzene Chemical compound C1=CC(CCl)=CC=C1C1=CC=C(CCl)C=C1 INZDTEICWPZYJM-UHFFFAOYSA-N 0.000 description 1
- QGEPAYHOJBDUEB-UHFFFAOYSA-B P(=O)([O-])([O-])[O-].[Zr+4].O(Cl)Cl.P(=O)([O-])([O-])[O-].P(=O)([O-])([O-])[O-].P(=O)([O-])([O-])[O-].[Zr+4].[Zr+4] Chemical compound P(=O)([O-])([O-])[O-].[Zr+4].O(Cl)Cl.P(=O)([O-])([O-])[O-].P(=O)([O-])([O-])[O-].P(=O)([O-])([O-])[O-].[Zr+4].[Zr+4] QGEPAYHOJBDUEB-UHFFFAOYSA-B 0.000 description 1
- 229910026551 ZrC Inorganic materials 0.000 description 1
- OTCHGXYCWNXDOA-UHFFFAOYSA-N [C].[Zr] Chemical compound [C].[Zr] OTCHGXYCWNXDOA-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000003178 carboxy group Chemical class [H]OC(*)=O 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 229910001567 cementite Inorganic materials 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229910000398 iron phosphate Inorganic materials 0.000 description 1
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- 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
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
-
- 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
- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for preparing pyruvic acid by using forest trees and application thereof, wherein the method comprises the following steps: performing steam explosion treatment and hydrothermal treatment on forest trees, performing enzymolysis on the treated products by using a complex enzyme, and finally fermenting the products after the enzymolysis by using escherichia coli genetic engineering bacteria producing pyruvic acid to obtain pyruvic acid. The invention adopts a pretreatment mode combining steam explosion treatment and hydrothermal treatment to improve the degradation degree of hemicellulose, so that the structure becomes loose, more enzyme binding sites are improved for subsequent enzymolysis, and the yield of fermentable sugar is improved. Then carrying out enzymolysis on the treated product by using complex enzyme to form a high-concentration fermentable sugar solution mixed by pentose and hexose. And the carbon source is used, and the escherichia coli engineering strain producing pyruvic acid is used as a fermentation strain to ferment so as to obtain high-concentration pyruvic acid. The preparation method can reduce the cost of pyruvic acid and realize high-valued waste forest resources.
Description
Technical Field
The invention belongs to the technical field of pyruvic acid preparation, and relates to a method for preparing pyruvic acid by using forest resources and application thereof.
Background
Forestry waste refers to waste generated in forestry production, forest renewal and forest product processing processes, and comprises cutting residues, forest cleaning and tending residues and wood processing residues. According to statistics, about 1.4 million tons of forestry waste is generated by forest felling and wood processing every year, and 1 million tons of forestry waste is generated by forest pruning. If a large amount of forestry waste can realize high-value utilization, the economic nature of forest resources is greatly improved, and the forest industry is promoted.
Pyruvate, also known as a-oxopropanoic acid, is an important intermediate in sugar metabolism in all biological cells and in the interconversion of various substances in the body. The molecule of the material contains activated ketone and carboxyl group, so the material is widely applied to the fields of chemistry, pharmacy, food, agriculture, environmental protection and the like as a basic chemical raw material. The preparation method comprises chemical synthesis, enzyme conversion, and microbial fermentation.
For example, CN106111173A discloses a catalyst for preparing pyruvate from lactate and a method for preparing pyruvate: the catalyst is selected from one or more than two of iron carbide, zirconium carbide, vanadium carbide, iron phosphate, copper oxychloride phosphate, zirconium oxychloride phosphate and vanadium oxychloride phosphate, lactate is taken as a raw material, oxygen or air is taken as an oxidant, and pyruvate is prepared through catalytic oxidation reaction under the action of the catalyst; the catalytic reaction can be carried out under normal pressure, a solvent is not required, the product yield is high, and the catalyst can be recycled.
Compared with chemical synthesis and enzyme conversion, the microbial fermentation method for synthesizing pyruvic acid has the advantages of wide raw material source, low energy consumption, less pollution and low cost. The strategy for synthesizing pyruvic acid by microbial fermentation is limited, and it is very significant to develop a new method for synthesizing pyruvic acid by microbial fermentation.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for preparing pyruvic acid by using forest resources and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing pyruvic acid from forest trees, which comprises: performing steam explosion treatment and hydrothermal treatment on forest trees, performing enzymolysis on the treated products by using a complex enzyme, and finally fermenting the products after the enzymolysis by using escherichia coli genetic engineering bacteria producing pyruvic acid to obtain pyruvic acid.
Because the forest biomass has a more compact structure compared with the herbaceous biomass and is difficult to degrade, the invention adopts a pretreatment mode combining steam explosion treatment and hydrothermal treatment, and firstly adopts weak steam explosion treatment to break the compact structure of the forest biomass cell wall, so that the structure becomes loose, and the subsequent hydrothermal pretreatment is favorably strengthened; and then, the hydrothermal treatment is adopted, so that the degradation degree of the hemicellulose is improved, the structure is loosened, more enzyme binding sites are improved for subsequent enzymolysis, and the yield of fermentable sugar is improved.
After steam explosion treatment and hydrothermal treatment, the components of the forest are greatly changed, and especially the content of hemicellulose is obviously reduced. Then carrying out enzymolysis on the treated product by using complex enzyme to form a high-concentration fermentable sugar solution mixed by pentose and hexose. And the carbon source is used, and the escherichia coli engineering strain producing pyruvic acid is used as a fermentation strain to ferment so as to obtain high-concentration pyruvic acid. The preparation method can reduce the cost of pyruvic acid and realize high-valued waste forest resources.
In addition, most of the existing pyruvate fermentation strains are yeasts, and since the yeasts lack a pentose utilization way, the pentose is difficult to be efficiently utilized. Therefore, the invention adopts the escherichia coli engineering bacteria which can simultaneously utilize pentose and hexose to produce pyruvic acid, and can realize the saccharic acid conversion to the maximum extent.
The above-mentioned all links are mutually buckled and promoted to obtain a low-cost high-yield method for preparing pyruvic acid.
Preferably, the forest includes forest waste.
As forestry waste generated by forest felling and wood processing and forestry waste generated by forest pruning are numerous every year, the preparation raw material has great practical significance if the preparation raw material related by the invention can realize high-valued forest waste resources by using the forest waste.
Preferably, the complex enzyme comprises cellulase and hemicellulase.
Preferably, the cellulase comprises any one or a combination of at least two of β -glucosidase, endoglucanase, exoglucanase, or pectinase; the combination of at least two of the above-mentioned compounds, such as the combination of beta-glucosidase, endoglucanase, exoglucanase, beta-glucosidase, pectinase, etc., can be selected in any combination manner, and will not be described herein.
Preferably, the hemicellulase comprises a xylanase and/or a laccase.
Further preferably, the complex enzyme comprises beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase.
If the complex enzyme used in the method disclosed by the invention simultaneously contains beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, the enzymolysis effect is better, and the concentration of the fermentable sugar solution obtained by enzymolysis is higher.
Preferably, the ratio of the filter paper enzyme activity, the beta-glucosidase activity, the pectinase activity, the xylanase activity and the laccase activity in the complex enzyme is (3-5): 1-2): 0.5-1.
When the enzyme components of the complex enzyme used in the method are mixed according to the specific proportion, the enzymolysis effect is better, and the concentration of the fermentable sugar solution obtained by enzymolysis is higher.
The specific point value in the above (3-5) may be 3, 3.5, 4, 4.5, 5, etc.
Specific points in the above (1-2) may be 1, 1.2, 1.5, 1.8, 2, etc.
Specific points in the above (0.5-1) may be 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.
Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
Preferably, the solid content in the enzymatic hydrolysis system is 15-30%, for example, 15%, 16%, 18%, 20%, 22%, 23%, 24%, 26%, 28%, 30%, etc., and other specific values in the numerical range may be selected, which is not described herein again. Preferably 20-25%.
Preferably, the temperature of the enzymolysis is 40-60 ℃, for example, 40 ℃, 42 ℃, 45 ℃, 47 ℃, 50 ℃, 52 ℃, 55 ℃, 58 ℃, 60 ℃ and the like, the time of the enzymolysis is 20-40h, for example, 20h, 25h, 30h, 35h, 40h and the like, and other specific point values in the numerical range can be selected, and are not described in detail herein.
Preferably, the forest is subjected to crushing treatment before steam explosion treatment to obtain forest particles.
Preferably, the particle size of the particles is 0.1-50mm, such as 0.1mm, 5mm, 10mm, 20mm, 30mm, 40mm, 50mm, etc., and other specific values in the value range can be selected, which is not described in detail herein. Further preferably 5 to 10 mm.
Preferably, the solid content in the steam explosion treatment system is 50-80%, for example, 50%, 60%, 70%, 75%, 80%, etc., and other specific values in the value range can be selected, which is not described in detail herein. Further preferably 70 to 75%.
Preferably, the system pressure of the steam explosion treatment is 0.8-2.0MPa, such as 0.8MPa, 1.0MPa, 1.2MPa, 1.4MPa, 1.5MPa, 1.6MPa, 2.0MPa, and the like, and other specific values in the numerical range can be selected, which is not described in detail herein. Further preferably 1.4 to 1.6 MPa.
Preferably, the duration of the steam explosion treatment is 1-10min, for example, 1min, 2min, 3min, 4min, 5min, 6min, 8min, 10min, and the like, and other specific values in the numerical range can be selected, which is not described in detail herein. Further preferably 3-5 min.
The parameters of the steam explosion treatment are mutually matched, so that the steam explosion effect is better, the cell wall structure becomes loose, and the subsequent hydrothermal pretreatment is more favorably strengthened.
Preferably, the solid content in the system of the hydrothermal treatment is 30-50%, such as 30%, 35%, 40%, 45%, 50%, etc., and other specific values of the value range can be selected, which is not described herein again. Further preferably 40 to 45%.
Preferably, the system of the hydrothermal treatment contains an acid, the concentration of the acid is 0.1-3.0%, for example, 0.1%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, etc., and other specific values of the value range can be selected, which is not described herein again. Further preferably 1.0 to 1.5%.
Preferably, the temperature of the hydrothermal treatment is 140-. Further preferably at a temperature of 160 ℃ and 180 ℃ for a period of 30-40 min.
The parameters of the hydrothermal treatment are matched with each other, so that the hydrothermal effect is better, the degradation degree of hemicellulose can be improved, more enzyme binding sites are improved for subsequent enzymolysis, and the yield of fermentable sugar is improved.
Preferably, the initial fermentable sugar concentration in the fermentation system is more than 10-40g/L, such as 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L and the like, and other specific values in the numerical range can be selected, which is not described in detail herein. Preferably 25-35 g/L.
During the fermentation process, sugar liquor is supplemented according to the residual sugar content in the fermentation tank, and when the residual sugar content is lower than 5.0g/L, the sugar liquor is supplemented to maintain the sugar concentration in the fermentation tank to be more than 10 g/L. And the pH value of the fermentation liquor is regulated and controlled by adding sodium hydroxide.
Preferably, the pH value of the fermentation system is 6.5-7.5, such as 6.5, 7.0, 7.5, and the like, and other specific values of the value range can be selected, which is not described in detail herein.
Preferably, the fermentation temperature is 30-40 ℃, such as 30 ℃, 32 ℃, 34 ℃, 36 ℃, 38 ℃, 40 ℃ and the like, and the fermentation time is 24-90h, such as 24h, 36h, 48h, 52h, 60h, 70h, 80h, 90h and the like, and other specific values in the numerical range can be selected, and are not described in detail herein.
The fermentation treatment parameters are matched with each other, so that the fermentation effect is better, and the pyruvic acid with higher concentration is obtained.
Preferably, the inoculation amount of the strains in the fermentation system is 1-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc., and other specific values of the numerical range can be selected, which is not described herein again. Further preferably 5 to 7%.
Preferably, the fermentation system also contains a nitrogen source, inorganic salts and trace elements.
Preferably, the nitrogen source comprises any one of yeast extract, peptone, corn steep liquor, molasses, ammonia water, ammonium salt or urea or a combination of at least two of them. The combination of at least two of the above-mentioned compounds, such as the combination of yeast extract and peptone, the combination of ammonium salt and urea, etc., other specific values of the numerical range can be selected, and thus detailed description thereof is omitted.
The inorganic salt may be, for example, (NH)4)2HPO4、NH4H2PO4、KCl、NH4Cl、MgSO4·7H2O, etc.; the micro-odorant may be FeCl, for example3·6H2O、CoCl2·6H2O、CuCl2·2H2O、ZnCl2、Na2MoO4·2H2O、H3BO3、MnCl2·4H2O, and the like.
In a second aspect, the present invention provides the use of a method as described in the first aspect for the preparation of pyruvic acid.
Compared with the prior art, the invention has the following beneficial effects:
because the forest biomass has a more compact structure compared with the herbaceous biomass and is difficult to degrade, the invention adopts a pretreatment mode combining steam explosion treatment and hydrothermal treatment, and firstly adopts weak steam explosion treatment to break the compact structure of the forest biomass cell wall, so that the structure becomes loose, and the subsequent hydrothermal pretreatment is favorably strengthened; and then, the hydrothermal treatment is adopted, so that the degradation degree of the hemicellulose is improved, the structure is loosened, more enzyme binding sites are improved for subsequent enzymolysis, and the yield of fermentable sugar is improved.
After steam explosion treatment and hydrothermal treatment, the components of the forest are greatly changed, and especially the content of hemicellulose is obviously reduced. Then carrying out enzymolysis on the treated product by using complex enzyme to form a high-concentration fermentable sugar solution mixed by pentose and hexose. And the carbon source is used, and the escherichia coli engineering strain producing pyruvic acid is used as a fermentation strain to ferment so as to obtain high-concentration pyruvic acid. The preparation method can reduce the cost of pyruvic acid and realize high-valued waste forest resources.
The above-mentioned all links are mutually buckled and promoted to obtain a low-cost high-yield method for preparing pyruvic acid.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
The pyruvic acid producing colibacillus genetic engineering bacteria related to the following embodiments uses colibacillus YP01 strain with the preservation number of CGMCC No.10691, the preservation unit is China general microbiological culture Collection center, the preservation time is 2015, 4 months and 7 days, and the addresses are as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The following examples relate to seed culture media having the formula: carbon source: 1% of glucose; ammonium chloride: 2 g/L; macroelements (mmol/L): (NH)4)2HPO4 19.92，NH4H2PO4 7.56，KCl 2.00，NH4Cl 7.50，MgSO4·7H2O1.50; trace elements (μmol/l) FeCl3·6H2O 8.88，CoCl2·6H2O 1.26，CuCl2·2H2O 0.88，ZnCl2 2.20，Na2MoO4·2H2O 1.24，H3BO3 1.21，MnCl2·4H2O 2.50。
The following examples relate to fermentation media formulations: carbon source: hydrolyzing the sugar mother liquor; ammonium chloride: 5 g/L; macroelements (mmol/L): (NH)4)2HPO4 19.92，NH4H2PO4 7.56，KCl 2.00，NH4Cl 7.50，MgSO4·7H2O1.50; trace elements (μmol/L) FeCl3·6H2O 8.88，CoCl2·6H2O 1.26，CuCl2·2H2O 0.88，ZnCl2 2.20，Na2MoO4·2H2O 1.24，H3BO3 1.21，MnCl2·4H2O 2.50。
The following examples relate to beta-glucosidase from Sigma, model G0395; endoglucanase was purchased from Sigma, model E2164; the exoglucanase is purchased from Aladdin company and has the model number D298997; pectinase was purchased from Aladdin, Inc. under model number P128776; xylanase was purchased from Sigma, model X3876; laccase was purchased from Aladdin, model L304691.
Example 1
The embodiment provides a method for preparing pyruvic acid by using pine processing waste, which comprises the following steps:
(1) pulverizing pine processing waste to 10 mm; adding water into pine wood particles, blending until the water content is 30%, soaking for 2h, loading into a steam explosion machine for steam explosion treatment under the condition of setting the pressure to be 1.6MPa, maintaining for 3min, and collecting the pine wood particles after steam explosion; adding water into the steam exploded pine wood particles until the water content is 60%, adding acetic acid to make the concentration of the acetic acid reach 1% (v/v), and treating at 180 deg.C for 30 min; obtaining pretreated pine wood particles and a pretreatment solution through solid-liquid separation, and determining component change of the pine wood particles and contents of monosaccharide and oligosaccharide in the solution;
the determination shows that the components of the pine are greatly changed, particularly the content of hemicellulose is reduced from 25 percent to 17 percent; the concentration of xylose in the solution reaches 5.8g/L, and the concentration of xylobiose reaches 3.2 g/L. This data demonstrates that after pretreatment, the pine structure is destroyed and the easily degradable components have been degraded to monosaccharides or oligosaccharides.
(2) Carrying out enzymolysis on a pretreatment solution with a solid content of 25% by using a complex enzyme, wherein the complex enzyme is a mixture of beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, and the ratio of the filter paper enzyme activity, the beta-glucosidase activity, the pectinase activity, the xylanase activity and the laccase activity in the complex enzyme is 4:2:1:2: 1;
firstly, adjusting the pH value of an enzymolysis system to 5.0 by using sodium hydroxide; adding complex enzyme with 20FPU/g of filter paper enzyme activity, carrying out enzymolysis at 50 ℃ for 36h, settling and filtering to obtain hydrolyzed sugar mother liquor, and determining the concentration of monosaccharides such as glucose, xylose and the like in the solution;
through determination, the concentration of glucose in the solution reaches 95g/L, the concentration of xylose reaches 35g/L, and the concentration of the sugar completely meets the requirement of fermentation of pyruvic acid.
(3) Producing pyruvic acid by using Escherichia coli genetic engineering strain YP 01;
(3.1) seed culture: 100mL of the seed medium was sterilized in a 250mL Erlenmeyer flask at 115 ℃ for 30 min. After cooling, inoculating strain YP01 in an inoculum size of 1% (v/v) to a seed culture medium, and culturing at pH 7.0, 37 deg.C and 200rpm for 12h to obtain a seed solution;
(3.2) fermentation culture: and (3) sterilizing the 5L fermentation tank at 121 ℃ for 20min with the volume of the fermentation medium, and adding the hydrolyzed sugar mother liquor obtained in the step (2) which is sterilized at high temperature to ensure that the monosaccharide concentration in the fermentation tank is 5%. Inoculating the seed solution into a fermentation medium according to the inoculation amount of 5% (v/v), culturing at pH 7.0, 37 deg.C and 400rpm for 24h, adding hydrolyzed sugar mother liquor to make the monosaccharide concentration in the fermentation tank reach 5%, and culturing for 48h to obtain fermentation liquor. During the culture period, the pH value of the fermentation system is adjusted by using 20 percent sodium hydroxide to keep the pH value at about 7.0;
the components in the fermentation liquor are quantitatively analyzed by adopting high performance liquid chromatography, and the concentration of glucose and organic acid in the fermentation liquor is measured by adopting an Aminex HPX-87H organic acid analytical column. Through analysis, 75g/L of pyruvic acid can be synthesized, the saccharic acid conversion rate reaches 0.69g/g of monosaccharide, and the concentration of the by-product acid is very low, wherein the alpha-ketoglutaric acid is 1.5g/L, the fumaric acid is 0.3g/L, and the citric acid is 0.5 g/L.
Example 2
The embodiment provides a method for preparing pyruvic acid by using eucalyptus processing waste, which comprises the following steps:
(1) pulverizing the eucalyptus processing waste to 5mm in particle size; adding water into the eucalyptus particles, blending until the water content is 25%, soaking for 2h, loading into a steam explosion machine for steam explosion treatment under the condition of setting the pressure to be 1.4MPa, maintaining for 5min, and collecting the eucalyptus particles after steam explosion; adding water into the vapor-exploded eucalyptus particles until the water content is 55%, adding acetic acid to make the concentration of the acetic acid reach 1.5% (v/v), and treating at 160 deg.C for 40 min; obtaining pretreated eucalyptus particles and a pretreated solution through solid-liquid separation, and measuring component change of the eucalyptus particles and monosaccharide and oligosaccharide content in the solution;
the determination shows that the components of the eucalyptus wood are greatly changed, particularly the content of hemicellulose is reduced from 21% to 18%; the concentration of xylose in the solution reaches 4.9g/L, and the concentration of xylobiose reaches 1.2 g/L. This data demonstrates that after pretreatment, the eucalyptus structure is destroyed and the easily degradable components have been degraded to monosaccharides or oligosaccharides.
(2) Carrying out enzymolysis on the pretreatment solution with the solid content of 20% by using a complex enzyme, wherein the complex enzyme is a mixture of beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, and the ratio of the filter paper enzyme activity, the beta-glucosidase activity, the pectinase activity, the xylanase activity and the laccase activity in the complex enzyme is 3:1:0.5:1: 0.5;
firstly, adjusting the pH value of an enzymolysis system to 5.0 by using sodium hydroxide; adding complex enzyme with 20FPU/g of filter paper enzyme activity, carrying out enzymolysis at 60 ℃ for 33h, settling and filtering to obtain hydrolyzed sugar mother liquor, and measuring the concentration of monosaccharides such as glucose, xylose and the like in the solution;
through determination, the concentration of glucose in the solution reaches 81g/L, the concentration of xylose reaches 33g/L, and the concentration of the sugar completely meets the requirement of fermentation of pyruvic acid.
(3) Producing pyruvic acid by using Escherichia coli genetic engineering strain YP 01;
(3.1) seed culture: 100mL of the seed medium was sterilized in a 250mL Erlenmeyer flask at 115 ℃ for 30 min. After cooling, inoculating strain YP01 in an inoculum size of 1% (v/v) to a seed culture medium, and culturing at pH 7.0, 37 deg.C and 200rpm for 12h to obtain a seed solution;
(3.2) fermentation culture: and (3) sterilizing the 5L fermentation tank at 121 ℃ for 20min with the volume of the fermentation medium, and adding the hydrolyzed sugar mother liquor obtained in the step (2) which is sterilized at high temperature to ensure that the monosaccharide concentration in the fermentation tank is 5%. Inoculating the seed solution into a fermentation medium according to the inoculation amount of 5% (v/v), culturing at pH 6.5, 35 deg.C and 400rpm for 36h, adding hydrolyzed sugar mother liquor to make the monosaccharide concentration in the fermentation tank reach 5%, and culturing for 48h to obtain fermentation liquor. During the culture period, the pH value of the fermentation system is adjusted by using 20 percent sodium hydroxide to keep the pH value at about 7.0;
the components in the fermentation liquor are quantitatively analyzed by adopting high performance liquid chromatography, and the concentration of glucose and organic acid in the fermentation liquor is measured by adopting an Aminex HPX-87H organic acid analytical column. Through analysis, 74.1g/L of pyruvic acid can be synthesized, the saccharic acid conversion rate reaches 0.65g/g of monosaccharide, and the concentration of by-product acid is very low, wherein the alpha-ketoglutaric acid is 1.3g/L, the fumaric acid is 0.3g/L, and the citric acid is 0.5 g/L.
Example 3
The embodiment provides a method for preparing pyruvic acid by using poplar processing waste, which comprises the following steps:
(1) crushing poplar processing waste to 8mm in granularity; adding water into poplar granules, blending until the water content is 50%, soaking for 2h, loading into a steam explosion machine for steam explosion treatment under the condition that the pressure is set to be 1.2MPa, maintaining for 8min, and collecting the poplar granules subjected to steam explosion; adding water into the steam exploded poplar granules until the water content is 65%, adding acetic acid to enable the concentration of the acetic acid to reach 2.0% (v/v), and treating at 140 ℃ for 40 min; obtaining pretreated poplar particles and a pretreated solution through solid-liquid separation, and determining the component change of the poplar particles and the content of monosaccharide and oligosaccharide in the solution;
the determination shows that the components of the poplar are greatly changed, particularly the content of hemicellulose is reduced from 25% to 21%; the concentration of xylose in the solution reaches 4.6g/L, and the concentration of xylobiose reaches 2.9 g/L. This data demonstrates that after pretreatment, the poplar structure is destroyed and the easily degradable components have been degraded to monosaccharides or oligosaccharides.
(2) Carrying out enzymolysis on a pretreatment solution with the solid content of 30% by using a complex enzyme, wherein the complex enzyme is a mixture of beta-glucosidase, endoglucanase, exoglucanase, pectinase, xylanase and laccase, and the ratio of the filter paper enzyme activity, the beta-glucosidase activity, the pectinase activity, the xylanase activity and the laccase activity in the complex enzyme is 5:1:1:2: 0.5;
firstly, adjusting the pH value of an enzymolysis system to 5.0 by using sodium hydroxide; adding complex enzyme with 20FPU/g of filter paper enzyme activity, carrying out enzymolysis at 40 ℃ for 40h, settling and filtering to obtain hydrolyzed sugar mother liquor, and measuring the concentration of monosaccharides such as glucose, xylose and the like in the solution;
through determination, the concentration of glucose in the solution reaches 91g/L, the concentration of xylose reaches 29g/L, and the concentration of the sugar completely meets the requirement of fermentation of pyruvic acid.
(3) Producing pyruvic acid by using Escherichia coli genetic engineering strain YP 01;
(3.1) seed culture: 100mL of the seed medium was sterilized in a 250mL Erlenmeyer flask at 115 ℃ for 30 min. After cooling, inoculating strain YP01 in an inoculum size of 1% (v/v) to a seed culture medium, and culturing at pH 7.0, 37 deg.C and 200rpm for 12h to obtain a seed solution;
(3.2) fermentation culture: and (3) sterilizing the 5L fermentation tank at 121 ℃ for 20min with the volume of the fermentation medium, and adding the hydrolyzed sugar mother liquor obtained in the step (2) which is sterilized at high temperature to ensure that the monosaccharide concentration in the fermentation tank is 5%. Inoculating the seed solution into a fermentation medium according to the inoculation amount of 5% (v/v), culturing at pH 6.5, 35 deg.C and 400rpm for 36h, adding hydrolyzed sugar mother liquor to make the monosaccharide concentration in the fermentation tank reach 5%, and culturing for 48h to obtain fermentation liquor. During the culture period, the pH value of the fermentation system is adjusted by using 20 percent sodium hydroxide to keep the pH value at about 7.0;
the components in the fermentation liquor are quantitatively analyzed by adopting high performance liquid chromatography, and the concentration of glucose and organic acid in the fermentation liquor is measured by adopting an Aminex HPX-87H organic acid analytical column. Through analysis, 70g/L of pyruvic acid can be synthesized, the saccharic acid conversion rate reaches 0.65g/g of monosaccharide, and the concentration of the by-product acid is very low, wherein the alpha-ketoglutaric acid is 1.6g/L, the fumaric acid is 0.4g/L, and the citric acid is 0.5 g/L.
Example 4
This example provides a method for preparing pyruvic acid from pine wood processing waste, which is different from example 1 only in the composition of complex enzyme, which does not contain pectinase, and the amount of reduction is divided into the mass of beta-glucosidase, endoglucanase and exoglucanase according to the ratio of example 1. All other conditions remained unchanged.
In the step (2), the glucose concentration in the solution reaches 81g/L and the xylose concentration reaches 25g/L through determination.
Through analysis in the step (3), 64.1g/L pyruvic acid can be synthesized, the saccharic acid conversion rate reaches 0.60g/g monosaccharide, and the concentration of the by-product acid is very low, wherein the alpha-ketoglutaric acid is 1.1g/L, the fumaric acid is 0.3g/L, and the citric acid is 0.5 g/L.
Example 5
This example provides a method for preparing pyruvic acid using pine wood processing waste, which is operated differently from example 1 only in the composition of complex enzyme, which does not contain β -glucosidase, endoglucanase, exoglucanase, and the reduced amount thereof is distributed to the quality of pectinase. All other conditions remained unchanged.
In the step (2), the concentration of glucose in the solution reaches 30g/L and the concentration of xylose reaches 15g/L through determination.
Through analysis in the step (3), 27g/L pyruvic acid can be synthesized, the saccharic acid conversion rate reaches 0.61g/g monosaccharide, and the concentration of the by-product acid is very low, wherein the alpha-ketoglutaric acid is 0.3g/L, the fumaric acid is 0.1g/L, and the citric acid is 0.1 g/L.
Example 6
This example provides a method for preparing pyruvic acid from pine processing waste, which is operated differently from example 1 only in the composition of complex enzyme, which does not contain laccase, and the reduced amount thereof is distributed to the quality of xylanase. All other conditions remained unchanged.
In the step (2), the glucose concentration in the solution reaches 78g/L and the xylose concentration reaches 31g/L through determination.
Through analysis in the step (3), 71.0g/L pyruvic acid can be synthesized, the saccharic acid conversion rate reaches 0.65g/g monosaccharide, and the concentration of the by-product acid is very low, wherein the alpha-ketoglutaric acid is 1.3g/L, the fumaric acid is 0.5g/L, and the citric acid is 0.4 g/L.
Example 7
This example provides a method for preparing pyruvic acid from pine processing waste, which is operated differently from example 1 only in the composition of complex enzyme, which does not contain xylanase, and the reduced amount thereof is distributed to the quality of laccase. All other conditions remained unchanged.
In the step (2), the glucose concentration in the solution reaches 85g/L and the xylose concentration reaches 15g/L through determination.
Through analysis in the step (3), 62.0g/L of pyruvic acid can be synthesized, the saccharic acid conversion rate reaches 0.62g/g of monosaccharide, and the concentration of by-product acid is very low, wherein the alpha-ketoglutaric acid is 1.2g/L, the fumaric acid is 0.3g/L, and the citric acid is 0.5 g/L.
Example 8
This example provides a method for preparing pyruvic acid from pine processing waste, which is different from example 1 only in the technical parameters of fermentation:
(3.2) fermentation culture: and (3) sterilizing the 5L fermentation tank at 121 ℃ for 20min with the volume of the fermentation medium, and adding the hydrolyzed sugar mother liquor obtained in the step (2) which is sterilized at high temperature to ensure that the monosaccharide concentration in the fermentation tank is 5%. Inoculating the seed solution into a fermentation medium according to the inoculation amount of 5% (v/v), culturing at pH 5.5, 20 deg.C and 400rpm for 24h, adding hydrolyzed sugar mother liquor to make the monosaccharide concentration in the fermentation tank reach 5%, and culturing for 48h to obtain fermentation liquor. During the culture period, the pH value of the fermentation system is adjusted by using 20 percent sodium hydroxide to keep the pH value at about 5.5;
according to analysis in the step (3), 69g/L pyruvic acid can be synthesized, the sugar-acid conversion rate reaches 0.63g/g monosaccharide, and the concentration of the by-product acid is low, wherein the alpha-ketoglutaric acid is 1.3g/L, the fumaric acid is 0.5g/L, and the citric acid is 0.6 g/L.
Comparative example 1
This comparative example provides a method for preparing pyruvic acid using pine processing waste, which is different from example 1 only in that the pine processing waste is not subjected to steam explosion treatment, and other conditions are kept unchanged:
(1) pulverizing pine processing waste to 10 mm; adding water into pine wood particles until the water content is 60%, adding acetic acid to make the concentration of acetic acid reach 1% (v/v), and treating at 180 deg.C for 30 min; obtaining pretreated pine wood particles and a pretreatment solution through solid-liquid separation, and determining component change of the pine wood particles and contents of monosaccharide and oligosaccharide in the solution;
the determination shows that the content of hemicellulose in the pine components is reduced from 25% to 21%; the concentration of xylose in the solution reaches 5.1g/L, and the concentration of xylobiose reaches 2.0 g/L.
Comparative example 2
This comparative example provides a method for preparing pyruvic acid using pine processing waste, which is different from example 1 only in that the pine processing waste is not subjected to hydrothermal treatment, and other conditions are kept unchanged:
(1) pulverizing pine processing waste to 10 mm; adding water into pine wood particles, blending until the water content is 30%, soaking for 2h, loading into a steam explosion machine for steam explosion treatment, setting the treatment condition to be 1.6MPa, maintaining for 3min, obtaining pretreated pine wood particles and a pretreatment solution through solid-liquid separation, and measuring the component change of the pine wood particles and the content of monosaccharide and oligosaccharide in the solution;
the determination shows that the content of hemicellulose in the pine tree component is reduced from 25% to 20%; the concentration of xylose in the solution reaches 4.9g/L, and the concentration of xylobiose reaches 2.1 g/L.
Comparative example 3
This comparative example provides a method for preparing pyruvic acid using pine wood processing waste, which is different from example 1 only in that escherichia coli genetically engineered strain YP01 is replaced with yeast (CCTCC M202019) in an equal amount and other conditions are maintained.
Through analysis in the step (3), 60g/L pyruvic acid can be synthesized, the saccharic acid conversion rate reaches 0.46g/g monosaccharide, and the concentration of the by-product acid is very low, wherein the alpha-ketoglutaric acid is 1.9g/L, the fumaric acid is 0.8g/L, and the citric acid is 0.5 g/L.
The applicant states that the present invention is illustrated by the above examples, but the present invention is not limited to the above examples, i.e. the present invention is not limited to the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A method for preparing pyruvic acid by using forest trees, which is characterized by comprising the following steps: performing steam explosion treatment and hydrothermal treatment on forest trees, performing enzymolysis on the treated products by using a complex enzyme, and finally fermenting the products after the enzymolysis by using escherichia coli genetic engineering bacteria producing pyruvic acid to obtain pyruvic acid.
2. The method of claim 1, wherein the forest trees comprise forest waste.
3. The method of claim 1 or 2, wherein the complex enzyme comprises cellulase and hemicellulase;
preferably, the cellulase comprises any one or a combination of at least two of β -glucosidase, endoglucanase, exoglucanase, or pectinase;
preferably, the hemicellulase comprises a xylanase and/or a laccase.
4. The method of claim 1 or 2, wherein the complex enzyme comprises β -glucosidase, endoglucanase, exoglucanase, pectinase, xylanase, and laccase;
preferably, the ratio of the filter paper enzyme activity, the beta-glucosidase activity, the pectinase activity, the xylanase activity and the laccase activity in the complex enzyme is (3-5): 1-2): 0.5-1.
5. The method according to any one of claims 1 to 4, wherein the enzymatic hydrolysis system has a solids content of 15 to 30%, preferably 20 to 25%;
preferably, the enzymolysis temperature is 40-60 ℃, and the enzymolysis time is 20-40 h.
6. The method according to any one of claims 1 to 5, wherein the forest trees are subjected to a comminution treatment to obtain forest tree particles prior to the steam explosion treatment;
preferably, the particle size of the particles is from 0.1 to 50mm, further preferably from 5 to 10 mm;
preferably, the solid content in the steam explosion treatment system is 50-80%, and further preferably 70-75%;
preferably, the system pressure of the steam explosion treatment is 0.8-2.0MPa, and further preferably 1.4-1.6 MPa;
preferably, the duration of the steam explosion treatment is 1-10min, and further preferably 3-5 min.
7. The method according to any one of claims 1 to 6, wherein the hydrothermal treatment system has a solids content of 30 to 50%, more preferably 40 to 45%;
preferably, the system of the hydrothermal treatment contains acid, and the concentration of the acid is 0.1-3.0%, more preferably 1.0-1.5%;
preferably, the temperature of the hydrothermal treatment is 140-200 ℃ and the time is 20-60min, and further preferably the temperature is 160-180 ℃ and the time is 30-40 min.
8. The method according to any one of claims 1 to 7, wherein the initial fermentable sugar concentration in the fermented system is 10 to 40g/L or more, preferably 25 to 35 g/L;
preferably, the pH value of the fermentation system is 6.5-7.5;
preferably, the fermentation temperature is 30-40 ℃, and the fermentation time is 24-90 h;
preferably, the inoculation amount of the strain in the fermentation system is 1-10%, and more preferably 5-7%.
9. The method according to any one of claims 1 to 8, wherein the system of the fermentation further comprises a nitrogen source, inorganic salts and trace elements;
preferably, the nitrogen source comprises any one of yeast extract, peptone, corn steep liquor, molasses, ammonia water, ammonium salt or urea or a combination of at least two of them.
10. Use of a method according to any one of claims 1 to 9 for the preparation of pyruvic acid.
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