CN103014098B - Method for removing inhibition of reclaimed water to enzymolysis of lignocellulose through reducing agent - Google Patents
Method for removing inhibition of reclaimed water to enzymolysis of lignocellulose through reducing agent Download PDFInfo
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- CN103014098B CN103014098B CN201210571262.4A CN201210571262A CN103014098B CN 103014098 B CN103014098 B CN 103014098B CN 201210571262 A CN201210571262 A CN 201210571262A CN 103014098 B CN103014098 B CN 103014098B
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000003638 chemical reducing agent Substances 0.000 title abstract description 3
- 230000005764 inhibitory process Effects 0.000 title abstract 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims abstract description 40
- 102000004190 Enzymes Human genes 0.000 claims abstract description 39
- 108090000790 Enzymes Proteins 0.000 claims abstract description 38
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 230000002829 reductive effect Effects 0.000 claims abstract description 14
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 51
- 239000012978 lignocellulosic material Substances 0.000 claims description 27
- 238000007781 pre-processing Methods 0.000 claims description 25
- 239000001913 cellulose Substances 0.000 claims description 19
- 235000010980 cellulose Nutrition 0.000 claims description 19
- 229920002678 cellulose Polymers 0.000 claims description 19
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 claims description 14
- 239000008399 tap water Substances 0.000 claims description 13
- 235000020679 tap water Nutrition 0.000 claims description 13
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 10
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 10
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 10
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 239000010902 straw Substances 0.000 claims description 8
- 230000015556 catabolic process Effects 0.000 claims description 6
- 238000006731 degradation reaction Methods 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 239000008213 purified water Substances 0.000 claims description 4
- 240000007594 Oryza sativa Species 0.000 claims description 3
- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- 235000014676 Phragmites communis Nutrition 0.000 claims description 3
- 241000209140 Triticum Species 0.000 claims description 3
- 235000021307 Triticum Nutrition 0.000 claims description 3
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 3
- 235000013339 cereals Nutrition 0.000 claims description 3
- 239000010903 husk Substances 0.000 claims description 3
- 235000009973 maize Nutrition 0.000 claims description 3
- 235000009566 rice Nutrition 0.000 claims description 3
- 239000011534 wash buffer Substances 0.000 claims description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 2
- 239000008351 acetate buffer Substances 0.000 claims description 2
- 238000013016 damping Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 150000003016 phosphoric acids Chemical class 0.000 claims description 2
- 239000002893 slag Substances 0.000 claims description 2
- 235000015099 wheat brans Nutrition 0.000 claims description 2
- 229940088598 enzyme Drugs 0.000 abstract description 37
- 108010059892 Cellulase Proteins 0.000 abstract description 17
- 229940106157 cellulase Drugs 0.000 abstract description 14
- 239000000758 substrate Substances 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 9
- 238000001035 drying Methods 0.000 abstract description 6
- 230000003301 hydrolyzing effect Effects 0.000 abstract description 2
- 230000003647 oxidation Effects 0.000 abstract description 2
- 238000007254 oxidation reaction Methods 0.000 abstract description 2
- 238000007598 dipping method Methods 0.000 abstract 3
- 238000011010 flushing procedure Methods 0.000 abstract 1
- 239000000413 hydrolysate Substances 0.000 abstract 1
- 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 32
- 239000008103 glucose Substances 0.000 description 32
- 230000000052 comparative effect Effects 0.000 description 17
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 13
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- 230000007062 hydrolysis Effects 0.000 description 10
- 238000006460 hydrolysis reaction Methods 0.000 description 10
- 150000002500 ions Chemical class 0.000 description 6
- 229910052742 iron Inorganic materials 0.000 description 5
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 239000007974 sodium acetate buffer Substances 0.000 description 4
- 229910001385 heavy metal Inorganic materials 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000000452 restraining effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000985535 Penicillium decumbens Species 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000012752 auxiliary agent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- -1 iron ion Chemical class 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 210000004158 stalk cell Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Abstract
The invention relates to a method for removing inhibition of reclaimed water to enzymolysis of lignocellulose through a reducing agent. The method comprises the following steps: (1) dipping the lignocellulose raw material to be degraded in reclaimed water, so as to obtain the pretreated lignocellulose; (2) dipping the pretreated lignocellulose into dithiothreitol solution, flushing by water or buffer liquid, and drying to obtain the lignocellulose raw material; and (3) dipping the lignocellulose raw material into the cellulase liquid, and saccharifying to obtain the hydrolysate. According to the method, the lignocellulose pretreated by reclaimed water is dipped in dithiothreitol (DTT), and dithiothreitol can remove the oxidation of Fe ion to the substrate, and also can remove the influence of the Fe ion to the enzyme activity, so that the amount of used enzymes can be greatly increased, the industrial cost can be reduced, and the basis is provided for hydrolyzing the lignocellulose by utilizing reclaimed water.
Description
Technical field
The present invention relates to a kind of utilization and utilize the method that in reductive agent releasing, water suppresses enzymolysis lignocellulose, particularly remove method enzymolysis lignocellulose suppressed containing water in iron ion, belong to cellulose utilization technical field.
Background technology
Day by day exhausted along with non-renewable petrochemical material, and the global warming issue that causes of the greenhouse gases of the harm to environment, particularly burning and exhausting more and more receives the concern of the mankind.With reproducible resource for raw material, through carrying out alternative petrochemical material to environment without the process of harm, become the direction that the whole world is all being tried to explore.At present, with lignocellulose biomass, such as, the raw material such as maize straw (corn cob), wheat straw, rice husk, cereal, reed, timber carries out energy development capable of circulation just becomes one of developing direction of the future biological energy, receives global common concern.Can give birth in energy industry process, the enzymolysis process of cellulase have played very large effect in C circulation and energy transformation.But the subject matter that cellulose enzyme solution preocess faces is that enzymolysis efficiency is low, particularly along with the quickening of industrialization of the country paces, problem of environmental pollution is more and more serious, the particularly heavy metal contamination of underground water.This just cause used in actual industrial in water or former water be water containing various heavy metal ion.And the activity of cellulase and lignocellulose are all easy to the interference being subject to heavy metal ion, cause cellulase can not play maximum effect in lignocellulose hydrolytic process, or lignocellulosic material is subject to middle water or former underwater gold and belong to ionization and cause enzymolysis efficiency to reduce.
Chinese patent literature CN101092447A(application number 200610090131.9) disclose the cellulase enzymolysis auxiliary agent deriving from straw material prepared with the following method: straw powder is carried out water cleaning, filters to obtain cell walls filter residue; The 0.1-0.5MOL/L NAOH solution containing 20-50MM xitix VC, mass percent 0.1-0.5% mercaptoethanol, 1-5MM PMSF, 5-10MM EDTA is added by proportioning; Be incubated 10-30 hour at 30-70 DEG C of temperature, and to add PMSF to final concentration every 3-5 hour be that 1-5MM extracts; After extraction completes, centrifugal and collect supernatant liquor; Supernatant liquor hydrochloric acid is adjusted PH, and adds alcohol and fully shake up, centrifugally again obtain throw out through placing, then carry out low-temperature vacuum drying, the cellulase enzymolysis auxiliary agent of stalk cell wall dry weight 10% must be accounted for.This patent documentation does not relate to middle water or raw water quality to the impact of cellulosic substrate or the impact on cellulase degradation effect.
Chinese patent literature CN101899488A(application number 201010228581.6) disclose a kind of lignocellulose enzymic hydrolysis and be coupled with film separation unit and produce the method for high-concentration reduced sugar.The method utilizes lignocellulose substrate, lytic enzyme and enzymolysis product reducing sugar in micro-filtration and ultra-filtration and separation enzymolysis solution, realizes the Reusability of substrate and enzyme, and concentrates enzymolysis solution by nanofiltration, the reducing sugar solution of obtained high density.Although the method achieve the industrialization that cellulose hydrolysis produces reducing sugar, but water loss is larger, and higher to the requirement of water quality, the middle water after industrial treatment or former water cannot obtain reducing sugar by aforesaid method, do not relate to the restraining effect of middle water to lignocellulose enzymolysis.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of utilize reductive agent to remove in water method that enzymolysis lignocellulose is suppressed
Term explanation
Middle water of the present invention refers to the process water reuse water after treatment suffering metal serious pollution, and water quality meets GB/TT18920-2002 standard, and wherein iron concentration is 3 ~ 10mM.
Technical scheme of the present invention is as follows:
The method that in utilizing reductive agent to remove, water suppresses enzymolysis lignocellulose, step is as follows:
(1) will treat that lignocellulose degradation raw material soaks 20 ~ 28h under 40 ~ 50 DEG C of conditions in middle water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in dithiothreitol (DTT) (DTT) solution of 2 ~ 10mM, 20 ~ 28h is soaked under 40 ~ 50 DEG C of conditions, then through water or wash buffer to pH to 4.5 ~ 5.5, dry obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 3 ~ 8%, enzyme concn is 20 ~ 40mg/g, under 40 ~ 60 DEG C of conditions, carry out saccharification, saccharification 80 ~ 100h, obtained hydrolyzed solution.
Preferred according to the present invention, treat in described step (1) that lignocellulose degradation raw material is Microcrystalline Cellulose, maize straw, wheat straw, rice husk, cereal, reed, timber, wheat bran, xylose residue and/or delignification slag.
Preferred according to the present invention, in described step (2), water is tap water or purified water.
Preferred according to the present invention, in described step (2), damping fluid is by tap water or purified water or the concentration 0.01 ~ 0.05M by its preparation, the Citrate trianion of pH4.5 ~ 5.5, phosphoric acid salt or acetate buffer.
Preferred according to the present invention, in described step (3), lignocellulosic material addition is 5% of saccharification system weight per-cent.
Described step (3) cellulase enzyme liquid can adopt marketed cellulose enzyme to prepare in proportion, and preferably purchased from the cellulase of Ningxia Sunson Industrial Group Co., Ltd., this cellulase comes from Penicillium decumbens (Penicillum decumbens).
Beneficial effect
Find after deliberation, in middle water the restraining effect of Fe ion pair hydrolysis of lignocellulose be by the oxidation to substrate reducing end and with enzyme in conjunction with two aspects, at present conventional reductant Vc or metal chelator EDTA can only from the restraining effect removing Fe ion pair hydrolysis of lignocellulose on the one hand; The present invention by by through in lignocellulose after water pretreatment soak through dithiothreitol (DTT) (DTT), dithiothreitol (DTT) both can eliminate the oxygenizement of Fe ion pair substrate, the impact that Fe ions enzyme is lived can be eliminated again, significantly improve the consumption of enzyme, reduce industrialization cost, thus be that in utilizing, water hydrolysis of lignocellulose is laid a good foundation.
Accompanying drawing explanation
Fig. 1 is that microcrystalline cellulose is in different treatment Water Under solution experiment column result comparison chart.
Fig. 2 is that xylose residue washes post-drying hydrolysising experiment column Comparative result figure with process under different treatment condition.
Fig. 3 is without processing rear direct hydrolysis column results contrast figure after xylose residue adds DTT.
Embodiment
Below by specific examples, the present invention will be further elaborated, should be noted that following explanation is only to explain the present invention, not limiting its content.
Raw material sources
Microcrystalline Cellulose is purchased from Chemical Reagent Co., Ltd., Sinopharm Group; Xylose residue is from Shandong dragon Lik-Sang thing company limited.
Dithiothreitol (DTT) is purchased from Beijing Ding Guo biotechnology limited liability company.
Embodiment 1 ~ 3 cellulase enzyme liquid is prepared as follows:
With the cellulase purchased from Ningxia Sunson Industrial Group Co., Ltd., this cellulase is that Penicillium decumbens obtains after fermentation, and during saccharification, enzyme dosage is 20mg-40mg/g.
Embodiment 4 cellulase enzyme liquid will be dissolved in sodium-acetate buffer purchased from the acidic cellulase in Zaozhuang Jienuo Enzyme Co., Ltd., obtains after centrifugal segregation impurity.The action pH scope of above-mentioned acidic cellulase is 4.5 ~ 6.2, the suitableeest action pH 4.8; Temperature range 50 ~ 65 DEG C, optimum temperature 60 DEG C.
Middle water in embodiment 1 is the process water meeting GB/TT18920-2002 standard, and wherein iron concentration is at 3mM.
Middle water in embodiment 2 is the process water meeting GB/TT18920-2002 standard, and wherein iron concentration is at 5mM.
Middle water in embodiment 3,4 is the process water meeting GB/TT18920-2002 standard, and wherein iron concentration is at 10mM.
Embodiment 1
The method that in utilizing reductive agent to remove, water suppresses enzymolysis lignocellulose, step is as follows:
(1) Microcrystalline Cellulose is soaked 24h under 45 DEG C of conditions in middle water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in dithiothreitol (DTT) (DTT) solution of 6mM, under 45 DEG C of conditions, soak 20h, then rinses to pH4.8 through sodium-acetate buffer, dries obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 3%, enzyme concn is 40mg/g, under 45 DEG C of conditions, carry out saccharification, saccharification 96h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 11.1g/L.
Embodiment 2
The method that in utilizing reductive agent to remove, water suppresses enzymolysis lignocellulose, step is as follows:
(1) xylose residue is soaked 20h under 40 DEG C of conditions in middle water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in dithiothreitol (DTT) (DTT) solution of 2mM, under 50 DEG C of conditions, soak 28h, then through tap water to pH to 5.0, dry obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 5%, enzyme concn is 20mg/g, under 40 DEG C of conditions, carry out saccharification, saccharification 72h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 8.1g/L.
Embodiment 3
The method that in utilizing reductive agent to remove, water suppresses enzymolysis lignocellulose, step is as follows:
(1) xylose residue is soaked 26h under 45 DEG C of conditions in middle water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in dithiothreitol (DTT) (DTT) solution of 6mM, under 45 DEG C of conditions, soak 24h, then through tap water to pH to 4.8, dry obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 5%, enzyme concn is 40mg/g, under 60 DEG C of conditions, carry out saccharification, saccharification 80h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 9.8g/L.
Embodiment 4
The method that in utilizing reductive agent to remove, water suppresses enzymolysis lignocellulose, step is as follows:
(1) xylose residue is soaked 22h under 50 DEG C of conditions in middle water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in dithiothreitol (DTT) (DTT) solution of 10mM, under 40 DEG C of conditions, soak 24h, then rinses to pH to 4.5 through citrate buffer solution, dries obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 8%, enzyme concn is 30mg/g, under 50 DEG C of conditions, carry out saccharification, saccharification 80h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 10.4g/L.
Comparative example 1
Microcrystalline Cellulose is soaked 24h under 45 DEG C of conditions in tap water, and then immerse in cellulose enzyme liquid in the ratio of saccharification system weight per-cent 3%, enzyme concn is 40mg/g, under 45 DEG C of conditions, carry out saccharification, saccharification 96h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 11.9g/L.
Comparative example 2
Treatment step is as follows:
(1) Microcrystalline Cellulose is soaked 24h under 45 DEG C of conditions in middle water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) is rinsed to pH4.8 through sodium-acetate buffer, dry obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 3%, enzyme concn is 40mg/g, under 45 DEG C of conditions, carry out saccharification, saccharification 96h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 9.4g/L.
Comparative example 3
Treatment step is as follows:
(1) microcrystalline cellulose is soaked 24h under 45 DEG C of conditions in tap water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) is added 1mM DTT40 DEG C and soak 20h, dry obtained lignocellulosic material.
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 5%, enzyme concn is 40mg/g, under 45 DEG C of conditions, carry out saccharification, saccharification 96h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 12.1g/L.
Comparative example 4
Treatment step is as follows:
(1) Microcrystalline Cellulose is soaked 24h under 45 DEG C of conditions in middle water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in the Vc solution of 6mM, under 45 DEG C of conditions, soak 20h, then rinses to pH4.8 through sodium-acetate buffer, dries obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 5%, enzyme concn is 40mg/g, under 45 DEG C of conditions, carry out saccharification, saccharification 96h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 9.0g/L.
Comparative example 5
Treatment step is as follows:
(1) xylose residue tap water is cleaned to pH5.0 post-drying, obtained preprocessing lignocellulose;
(2) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (1) in the ratio of saccharification system weight per-cent 5%, enzyme concn is 40mg/g, under 45 DEG C of conditions, carry out saccharification, saccharification 96h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 7.2g/L.
Comparative example 6
Treatment step is as follows:
(1) xylose residue is cleaned to pH5.0 post-drying at 50 DEG C with tap water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in dithiothreitol (DTT) (DTT) solution of 6mM, under 40 DEG C of conditions, soak 24h, dries obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 8%, enzyme concn is 30mg/g, under 50 DEG C of conditions, carry out saccharification, saccharification 80h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 8.8g/L.
Comparative example 7
Treatment step is as follows:
(1) xylose residue tap water is cleaned to pH5.0 post-drying, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in the Vc solution of 6mM, under 40 DEG C of conditions, soak 24h, dries obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 8%, enzyme concn is 30mg/g, under 50 DEG C of conditions, carry out saccharification, saccharification 80h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 8.0g/L.
Comparative example 8
Treatment step is as follows:
(1) xylose residue tap water is cleaned to pH5.0 post-drying, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in the Vc solution of 6mM, under 40 DEG C of conditions, soak 24h, then through tap water to pH to 4.8, dry obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 8%, enzyme concn is 30mg/g, under 50 DEG C of conditions, carry out saccharification, saccharification 80h, obtained hydrolyzed solution.
Adopt HPLC to measure glucose concn, in hydrolyzed solution, glucose content is 6.9g/L.
Interpretation of result
By embodiment 1 and comparative example 1, 2, 3, 4 can find out, in the hydrolyzed solution of embodiment 1, glucose content is 11.1g/L, in the hydrolyzed solution of comparative example 1, glucose content is 11.9g/L, in comparative example 2 hydrolyzed solution, glucose content is 9.4g/L, glucose content in comparative example 3 is 12.1g.L, in the hydrolyzed solution of comparative example 4, glucose content is 9.0g/L, therefore, can find out and pass through sewerage disposing, substrate hydrolysis efficiency can be reduced by 20%, after DTT process Microcrystalline Cellulose and process, DTT is cleaned or with after lower concentration DTT process without washing before hydrolysis efficiency is increased to sewerage disposing, and before being better than sewerage disposing, absolutely prove the reductive action of DTT to substrate, also contribute to improving the hydrolysis efficiency of enzyme and Vc group effect slightly poor (Fig. 1) simultaneously.
As can be seen from embodiment 2 ~ 4 and comparative example 5, in the hydrolyzed solution of embodiment 2 ~ 4, glucose content is respectively 8.1g/L, 9.8g/L, 10.4g/L, in the hydrolyzed solution of comparative example 5, glucose content is 7.2g/L, therefore, adopt DTT process xylose residue to the reductive action of substrate, it is 12.5%, 36%, 44%(Fig. 2 which improves hydrolysis effect).
Glucose content in hydrolyzed solution is had in embodiment 3 and comparative example 6,7,8 to be 9.8g/L, 8.8g/L, 8.0g/L, 6.9g/L can find out, xylose residue substrate after dithiothreitol (DTT) (DTT) solution soaking of 6mM or 10mM, if without water or wash buffer, directly adopt cellulose treatment, can comparatively significantly affect cellulase to cellulosic degradation effect, and Vc group is not through washing and washing effect not good (Fig. 3).
Claims (2)
1. the method that in utilizing reductive agent to remove, water suppresses enzymolysis lignocellulose, step is as follows:
(1) will treat that lignocellulose degradation raw material soaks 20 ~ 28h under 40 ~ 50 DEG C of conditions in middle water, obtained preprocessing lignocellulose;
(2) preprocessing lignocellulose obtained for step (1) being immersed concentration is in the dithiothreitol (DTT) solution of 2 ~ 10mM, 20 ~ 28h is soaked under 40 ~ 50 DEG C of conditions, then through water or wash buffer to pH to 4.5 ~ 5.5, dry obtained lignocellulosic material;
(3) immersed in cellulose enzyme liquid by lignocellulosic material obtained for step (2) in the ratio of saccharification system weight per-cent 3 ~ 8%, enzyme concn is 20 ~ 40mg/g, under 40 ~ 60 DEG C of conditions, carry out saccharification, saccharification 80 ~ 100h, obtained hydrolyzed solution;
Damping fluid is the concentration 0.01 ~ 0.05M prepared by tap water or purified water in described step (2), the Citrate trianion of pH4.5 ~ 5.5, phosphoric acid salt or acetate buffer.
2. the method for claim 1, is characterized in that, treats that lignocellulose degradation raw material is Microcrystalline Cellulose, maize straw, wheat straw, rice husk, cereal, reed, timber, wheat bran, xylose residue and/or delignification slag in described step (1).
3
.the method of claim 1, is characterized in that, in described step (2), water is tap water or purified water.
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