CN110172487A - The extracting method of withy Ji hemicellulose with arabinose and uronic acid branch - Google Patents
The extracting method of withy Ji hemicellulose with arabinose and uronic acid branch Download PDFInfo
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- CN110172487A CN110172487A CN201910505151.5A CN201910505151A CN110172487A CN 110172487 A CN110172487 A CN 110172487A CN 201910505151 A CN201910505151 A CN 201910505151A CN 110172487 A CN110172487 A CN 110172487A
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- China
- Prior art keywords
- hemicellulose
- withy
- arabinose
- uronic acid
- extracting method
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- 229920002488 Hemicellulose Polymers 0.000 title claims abstract description 87
- 238000000034 method Methods 0.000 title claims abstract description 47
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 title claims abstract description 32
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 239000002253 acid Substances 0.000 title claims abstract description 31
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 title claims abstract description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 66
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000000843 powder Substances 0.000 claims abstract description 28
- 238000012545 processing Methods 0.000 claims abstract description 26
- 108010059892 Cellulase Proteins 0.000 claims abstract description 23
- 229940106157 cellulase Drugs 0.000 claims abstract description 22
- 239000006228 supernatant Substances 0.000 claims abstract description 13
- 238000000746 purification Methods 0.000 claims abstract description 4
- 230000001376 precipitating effect Effects 0.000 claims abstract description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 38
- 239000002994 raw material Substances 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 37
- 239000008367 deionised water Substances 0.000 claims description 25
- 229910021641 deionized water Inorganic materials 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 23
- 239000000284 extract Substances 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- 239000007974 sodium acetate buffer Substances 0.000 claims description 11
- 238000003811 acetone extraction Methods 0.000 claims description 10
- 238000000498 ball milling Methods 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- 229920002678 cellulose Polymers 0.000 claims description 9
- 239000001913 cellulose Substances 0.000 claims description 9
- 238000007710 freezing Methods 0.000 claims description 9
- 230000008014 freezing Effects 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 239000002023 wood Substances 0.000 claims description 9
- -1 Ethyl alcohol Chemical compound 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 8
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 8
- 238000000605 extraction Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 229910000428 cobalt oxide Inorganic materials 0.000 claims description 2
- IVMYJDGYRUAWML-UHFFFAOYSA-N cobalt(ii) oxide Chemical compound [Co]=O IVMYJDGYRUAWML-UHFFFAOYSA-N 0.000 claims description 2
- 125000004122 cyclic group Chemical group 0.000 claims description 2
- RVTZCBVAJQQJTK-UHFFFAOYSA-N oxygen(2-);zirconium(4+) Chemical compound [O-2].[O-2].[Zr+4] RVTZCBVAJQQJTK-UHFFFAOYSA-N 0.000 claims description 2
- 229910001928 zirconium oxide Inorganic materials 0.000 claims description 2
- 150000004823 xylans Chemical class 0.000 abstract description 7
- 239000002028 Biomass Substances 0.000 abstract description 6
- 229920001221 xylan Polymers 0.000 abstract description 6
- 238000000502 dialysis Methods 0.000 abstract description 5
- 125000000524 functional group Chemical group 0.000 abstract description 3
- 230000007062 hydrolysis Effects 0.000 abstract description 3
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 3
- 230000003993 interaction Effects 0.000 abstract description 2
- 238000006116 polymerization reaction Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 25
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 14
- 239000003513 alkali Substances 0.000 description 13
- 239000007864 aqueous solution Substances 0.000 description 13
- 230000007071 enzymatic hydrolysis Effects 0.000 description 9
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 8
- 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 7
- 150000003384 small molecules Chemical class 0.000 description 7
- 238000000944 Soxhlet extraction Methods 0.000 description 6
- 229960000583 acetic acid Drugs 0.000 description 6
- 239000012632 extractable Substances 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 150000002632 lipids Chemical class 0.000 description 6
- 239000006166 lysate Substances 0.000 description 6
- 238000003760 magnetic stirring Methods 0.000 description 6
- 239000011343 solid material Substances 0.000 description 6
- 239000003643 water by type Substances 0.000 description 6
- 238000005303 weighing Methods 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 5
- 229920005610 lignin Polymers 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 239000012362 glacial acetic acid Substances 0.000 description 4
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 3
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical group O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 239000004744 fabric Substances 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 229940097043 glucuronic acid Drugs 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 241000499912 Trichoderma reesei Species 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000004804 polysaccharides Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 150000005846 sugar alcohols Chemical class 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000004299 exfoliation Methods 0.000 description 1
- 229940059442 hemicellulase Drugs 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 210000001724 microfibril Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003457 sulfones Chemical class 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000003809 water extraction 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- 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
Abstract
The present invention provides a kind of extracting methods of withy Ji hemicellulose with arabinose and uronic acid branch, comprising: withy Ji powder is added in peracetic acid soln and carries out obtaining holocellulose after delignification processing；Enzymolysis processing is carried out to holocellulose with cellulase, ethanol solution is added in supernatant, precipitating obtains thick hemicellulose；Thick hemicellulose obtained goes out by dialysis purification obtains the withy Ji hemicellulose with arabinose and uronic acid branch.The method of present invention cellulase hydrolysis releases hemicellulose from holocellulose, and hemicellulose after purification is known as higher molecular weight and the degree of polymerization, remains more functional group and xylan branched structure.For understanding pretreatment to biomass structure, the influence of especially hemicellulose structure has very great help the present invention, is conducive to the research of interaction relationship between the component in biomass cells wall.
Description
Technical field
The invention belongs to biorefinery fields, are related to a kind of withy Ji half fiber with arabinose and uronic acid branch
Tie up the extracting method of element.
Background technique
The separation of the big component of biomass three (cellulose, hemicellulose and lignin) is for grasping biomass structure feature extremely
It closes important.Withy Ji is the raw material of most potential second generation bio-fuel and production alternative energy source.Hemicellulose is biology
Content is only second to the component of cellulose in matter, in order to preferably be used, understands its physical and chemical performance, it is necessary to by half fiber
Dimension element is completely extracted from plant cell wall.Compared to cellulose and lignin, the hemicellulose degree of polymerization is low, and branch is multiple
It is miscellaneous, it is often connected with each other with lignin and cellulose etc., therefore can extract what structure was not damaged completely currently without method
Hemicellulose.Xylan is the most important a kind of polysaccharide of hemicellulose, is widely used in chemistry, biomaterial and medical accessory etc.
Field.The application that characterization is conducive to hemicellulose, functional group are carried out to the complete hemicellulose structure containing branch and functional group
Type and quantity directly affect the effective use of biomass, the physical and chemical performance of hemicellulose can be reflected, while also can
Influence its degree of reaction and dissolubility.Arabinose and uronic acid in xylan branch can pass through the fiber of hydrogen bond and amorphous region
β-Isosorbide-5-Nitrae-glucose chain link on the micro- fibril of element reduces cellulose crystallity, therefore its arabinose and alditol together
The degree of substitution of acid is two key factors for improving biomass enzymolysis efficiency.
The structure of hemicellulose depends primarily on species and other environmental factors.The hemicellulose of withy Ji be typically with
The xylan that β -1,4 couples is the symmetrical structure of main chain.The extracting method of hemicellulose mainly has alkali soluble solution, You Jirong at present
Agent extraction method, Hot water extraction and biochemistry are combined method, these methods have certain limitation.Alkali soluble solution is using most
More hemicellulose separation methods, cellulose swollen in aqueous slkali, cell wall are broken, and uronic acid and acetic acid ester bond are by enzyme
Solution, so that hemicellulose, lignin and colloid dissolution.But alkali process can destroy ester bond, interrupt and be connected to xylan bone
Acetyl group and other branches on frame, reduce the solubility of hemicellulose, then influence its stability.Opposite, dimethyl is sub-
The extent of exfoliation of the hemicellulose that the organic solvents such as sulfone, dioxane extract, ester bond is smaller, but its yield (~16%)
Well below alkali soluble solution (72%~85%).Cellulase and hemicellulase have specificity to the hydrolysis of polysaccharide, have
Research shows that enzymatic hydrolysis and alkali soluble uncoupling usage can separate cellulose and hemicellulose.Pass through zytase/endoglucanase
The two-step method that hydrolysis and alkali soluble solution combine can selectively extract high-purity from leaf wood and (account for about hemicellulose total amount
60%), the xylan of high molecular weight.Nevertheless, these extracting methods all inevitably destroy the knot of hemicellulose
Structure, yield is low or specificity is insufficient.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of withy Jis with arabinose and uronic acid branch half
The extracting method of cellulose.
In order to solve the above-mentioned technical problems, the present invention provides a kind of withy Ji with arabinose and uronic acid branch
The extracting method of hemicellulose, which comprises the following steps:
Step 1: withy Ji raw material being smashed with pulverizer, obtains withy Ji powder；
Step 2: withy Ji powder is continuously used into toluene/ethanol solution and acetone extraction, it is dry, obtain no extract timber
Raw material；
Step 3: delignification processing is carried out to no extract wood raw material with Peracetic acid, is washed, it is dry, withy is made
Ji holocellulose；
Step 4: withy Ji holocellulose ball grinds are digested with cellulase solution；
Step 5: the supernatant being centrifuged after being digested is added in ethanol water and carries out alcohol precipitation, is then centrifuged for being sunk
Forming sediment is thick hemicellulose enzymolysis；
Step 6: by thick hemicellulose enzymolysis by dialysing, freezing, be dried to obtain with arabinose and uronic acid branch
The withy Ji hemicellulose of chain.
Preferably, the smashed withy Ji powder in the step 1 crosses a 0.5mm sieve.
Preferably, method for extracting is soxhlet type in the step 2.
It is highly preferred that extraction conditions in the step 2 are as follows: 85 DEG C of temperature, extract 8h with toluene/ethanol mixed solution
Afterwards, then with acetone extraction 4h.
Further, in the toluene/ethanol mixed solution, VToluene: VEthyl alcohol=2:1.
Preferably, in the step 3 delignification treatment conditions are as follows: peroxide will be added to without extract wood raw material
In acetic acid aqueous solution, treatment temperature is 25 DEG C, and the time is for 24 hours；Mass ratio wherein without extract wood raw material and Peracetic acid is
1:(3.5~5.5), the quality of no extract wood raw material accounts for the 10~15% of reaction system gross mass.
It is highly preferred that the operation of the step 2, step 3 carries out under ventilation condition.
Preferably, it is washed in the step 3 are as follows: filtered, and be washed with deionized water with 0.45 μm of water phase micropore filtering film
Washing to pH value is 7.0.
Preferably, ball milling condition in the step 4 are as follows: select the ball grinder and ball-type zirconium oxide abrasive of cobalt oxide material
The revolving speed of ball, Ball-milling Time 3h, ball mill is set as 600rpm, and it is ball milling 5min that a cyclic control system, which is arranged, cooling
5min。
Preferably, the condition digested in the step 4 are as follows: by the withy Ji holocellulose powder after ball milling be added to containing
The sodium acetate buffer of cellulase carries out enzymolysis processing, and the specification of sodium acetate buffer is 50mM, pH 5.0, herein
Reason carries out in shaking table, and temperature is 37 DEG C, revolving speed 200rpm, and the processing time is 72h.
It is highly preferred that the mass ratio of the withy Ji holocellulose powder and cellulase is 1000:(5.4~30).
It is highly preferred that the cellulase selected is buying in Sigma-Aldrich, the Trichoderma reesei of Inc.
ATCC 26921。
Preferably, in the step 5 alcohol precipitation condition are as follows: supernatant is slowly dropped into 65% (v:v) second of 4 times of volumes
In alcohol solution, the lower ethanol water of concentration, which can screen, is settled out the biggish hemicellulose of molecular weight, removes glucose etc.
Small-molecule substance, the cool overnight in 4 DEG C of refrigerators, is collected by centrifugation precipitating.
It is highly preferred that the condition being centrifuged in the step 5: revolving speed is set as 8000rpm, time 15min.
Preferably, the step 6 specifically: thick hemicellulose enzymolysis is added in deionized water, is transferred to bag filter
In, at least 72h is placed in the deionized water of flowing, and then dialyzate after purification is placed in -20 DEG C of refrigerators or in liquid nitrogen
Freezing is put into freeze dryer dry 48h, the withy Ji hemicellulose with arabinose and uronic acid branch is made.
It is highly preferred that the dialysis selection is purchased in the 3.5K MWCO of Thermo Fisher scientific
Thermo Scientific dialysis machine.
Compared with prior art, the beneficial effects of the present invention are:
1, the method for the novel cellulase degradation of one kind of the invention, in conjunction with the processing skill of Peracetic acid removal lignin
Art extracts the small withy Ji hemicellulose of structural damage, is conducive to the structure feature for grasping natural hemicellulose, especially I
The branch chain information of uncle sugar and uronic acid, so as to more effectively be used.
2, hemicellulose extracting method provided by the invention can retain the function in hemicellulose macromolecular to the maximum extent
Group facilitates the interaction relationship for understanding hemicellulose and other cell-wall components.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
The present invention is suitable for any withy Ji kind, and the withy Ji that various embodiments of the present invention are selected is by Samuel Roberts
Nobel Foundation is provided, and plantation in 2011 was harvested in the area Ardmore, 2012；The cellulase of selection is buying
In Sigma-Aldrich, the Trichoderma reesei ATCC 26921 of Inc.；What dialysis was selected is buying in Thermo
The Thermo Scientific dialysis machine of the 3.5K MWCO of Fisher scientific.
Embodiment 1
Present embodiments provide a kind of withy Ji hemicellulose extracting method of cellulase auxiliary, specific steps are as follows:
(1) withy Ji raw material is crushed, crosses 0.5mm sieve, obtains withy Ji powder；
(2) in draught cupboard, with soxhlet extraction methods (condition are as follows: 85 DEG C of temperature, taken out with the toluene/ethanol that volume ratio is 2:1
After mentioning 8h, then with the extractables such as lipid, wax in acetone extraction 4h) removal raw material, take out the powder extracted be placed in it is logical
It is dry under room temperature in wind cupboard, it obtains without extract solid material；
(3) in draught cupboard, delignification processing is carried out without extracting raw material to dry: weighing 328g Peracetic acid
The aqueous solution that deionized water is configured to Peracetic acid to 360g is added in (concentration 32wt%), and being placed in volume is 1000mL tool plug
In conical flask, it is put into magnet rotor, the 360g peroxide acetate aqueous solution prepared is added without extracting raw material after dry in 30g
In, it is reacted for 24 hours, is filtered by 0.45 μm of water phase micropore filtering film, with a large amount of deionized waters at room temperature using magnetic stirring apparatus
Washing to the pH value of solution is 7.0 or so, obtains withy Ji holocellulose；
(4) the withy Ji holocellulose is filtered out, air-dries, is clayed into power with PA-100 planetary type ball-milling instrument, the time
The revolving speed of 3h, ball mill are set as 600rpm, and the circulation that control system is arranged is ball milling 5min, and cooling 5min takes 3g ball
Holocellulose powder after mill is added to the sodium acetate buffer that 40mL contains 90mg cellulase and carries out enzymolysis processing, acetic acid
The specification of sodium buffer solution is 50mM, pH 5.0, this processing carries out in shaking table, and temperature is 37 DEG C, revolving speed 200rpm, processing
Time is 72h；
(5) supernatant of the enzymatic hydrolysis, centrifugal condition are obtained by centrifuging and taking are as follows: revolving speed is set as 8000rpm, time
15min；The supernatant of above-mentioned enzymatic hydrolysis, is added to the water of 65% ethyl alcohol of 4 times of volumes by the ethanol water for preparing 65% (v:v)
In solution, it is statically placed in cool overnight in 4 DEG C of refrigerators, centrifugation obtains and is precipitated as thick hemicellulose enzymolysis；
(6) 5mL deionized water is added in thick hemicellulose enzymolysis, is transferred in the bag filter of 3.5K, bag filter is put
It sets in 2000mL beaker, at least 72h is placed under the deionized water of flowing, the small molecules such as ethyl alcohol, glucose are constantly set
It swaps out and, dialyzate is then placed in -20 DEG C of refrigerators (or in liquid nitrogen) freezing, be put into freeze dryer at least dry 48h, system
There must be the withy Ji hemicellulose of arabinose and uronic acid branch.
Its yield is tested with weight method, as a result sees attached list 1.
With its monosaccharide of ion chromatography and glucuronic acid content, its molecular weight and molecular weight point are tested with gel permeation chromatography
As a result cloth sees attached list 2~3.
Embodiment 2
Present embodiments provide a kind of withy Ji hemicellulose extracting method of cellulase auxiliary, specific steps are as follows:
(1) withy Ji raw material is crushed, crosses 0.5mm sieve, obtains withy Ji powder；
(2) in draught cupboard, with soxhlet extraction methods (condition are as follows: 85 DEG C of temperature, taken out with the toluene/ethanol that volume ratio is 2:1
After mentioning 8h, then with the extractables such as lipid, wax in acetone extraction 4h) removal raw material, take out the powder extracted be placed in it is logical
It is dry under room temperature in wind cupboard, it obtains without extract solid material；
(3) in draught cupboard, delignification processing is carried out without extracting raw material to dry: weighing 328g Peracetic acid
The aqueous solution that deionized water is configured to Peracetic acid to 360g is added in (concentration 32wt%), and being placed in volume is 1000mL tool plug
In conical flask, it is put into magnet rotor, the 360g peroxide acetate aqueous solution prepared is added without extracting raw material after dry in 30g
In, it is reacted for 24 hours, is filtered by 0.45 μm of water phase micropore filtering film, with a large amount of deionized waters at room temperature using magnetic stirring apparatus
Washing to the pH value of solution is 7.0 or so, obtains withy Ji holocellulose；
(4) the withy Ji holocellulose is filtered out, air-dries, takes 3g holocellulose powder to be added to 40mL and contain 90mg
The sodium acetate buffer of cellulase carries out enzymolysis processing, and the specification of sodium acetate buffer is 50mM, pH 5.0, herein
Reason carries out in shaking table, and temperature is 37 DEG C, revolving speed 200rpm, and the processing time is 72h；
(5) supernatant of the enzymatic hydrolysis, centrifugal condition are obtained by centrifuging and taking are as follows: revolving speed is set as 8000rpm, time
15min；The supernatant of above-mentioned enzymatic hydrolysis, is added to the water of 65% ethyl alcohol of 4 times of volumes by the ethanol water for preparing 65% (v:v)
In solution, it is statically placed in cool overnight in 4 DEG C of refrigerators, centrifugation obtains and is precipitated as thick hemicellulose enzymolysis；
(6) 5mL deionized water is added in thick hemicellulose enzymolysis, is transferred in the bag filter of 3.5K, bag filter is put
It sets in 2000mL beaker, at least 72h is placed under the deionized water of flowing, the small molecules such as ethyl alcohol, glucose are constantly set
It swaps out and, dialyzate is then placed in -20 DEG C of refrigerators (or in liquid nitrogen) freezing, be put into freeze dryer at least dry 48h, system
There must be the withy Ji hemicellulose of arabinose and uronic acid branch.
Its yield is tested with weight method, as a result sees attached list 1.
Embodiment 3
Present embodiments provide a kind of withy Ji hemicellulose extracting method of cellulase auxiliary, specific steps are as follows:
(1) withy Ji raw material is crushed, crosses 0.5mm sieve, obtains withy Ji powder；
(2) in draught cupboard, with soxhlet extraction methods (condition are as follows: 85 DEG C of temperature, taken out with the toluene/ethanol that volume ratio is 2:1
After mentioning 8h, then with the extractables such as lipid, wax in acetone extraction 4h) removal raw material, take out the powder extracted be placed in it is logical
It is dry under room temperature in wind cupboard, it obtains without extract solid material；
(3) in draught cupboard, delignification processing is carried out without extracting raw material to dry: weighing 328g Peracetic acid
The aqueous solution that deionized water is configured to Peracetic acid to 360g is added in (concentration 32wt%), and being placed in volume is 1000mL tool plug
In conical flask, it is put into magnet rotor, the 360g peroxide acetate aqueous solution prepared is added without extracting raw material after dry in 30g
In, it is reacted for 24 hours, is filtered by 0.45 μm of water phase micropore filtering film, with a large amount of deionized waters at room temperature using magnetic stirring apparatus
Washing to the pH value of solution is 7.0 or so, obtains withy Ji holocellulose；
(4) the withy Ji holocellulose is filtered out, air-dries, takes 3g holocellulose powder to be added to 40mL and contain 54mg
The sodium acetate buffer of cellulase carries out enzymolysis processing, and the specification of sodium acetate buffer is 50mM, pH 5.0, herein
Reason carries out in shaking table, and temperature is 37 DEG C, revolving speed 200rpm, and the processing time is 72h；
(5) supernatant of the enzymatic hydrolysis, centrifugal condition are obtained by centrifuging and taking are as follows: revolving speed is set as 8000rpm, time
15min；The supernatant of above-mentioned enzymatic hydrolysis, is added to the water of 65% ethyl alcohol of 4 times of volumes by the ethanol water for preparing 65% (v:v)
In solution, it is statically placed in cool overnight in 4 DEG C of refrigerators, centrifugation obtains and is precipitated as thick hemicellulose enzymolysis；
(6) 5mL deionized water is added in thick hemicellulose enzymolysis, is transferred in the bag filter of 3.5K, bag filter is put
It sets in 2000mL beaker, at least 72h is placed under the deionized water of flowing, the small molecules such as ethyl alcohol, glucose are constantly set
It swaps out and, dialyzate is then placed in -20 DEG C of refrigerators (or in liquid nitrogen) freezing, be put into freeze dryer at least dry 48h, system
There must be the withy Ji hemicellulose of arabinose and uronic acid branch.
Its yield is tested with weight method, as a result sees attached list 1.
Embodiment 4
Present embodiments provide a kind of withy Ji hemicellulose extracting method of cellulase auxiliary, specific steps are as follows:
(1) withy Ji raw material is crushed, crosses 0.5mm sieve, obtains withy Ji powder；
(2) in draught cupboard, with soxhlet extraction methods (condition are as follows: 85 DEG C of temperature, taken out with the toluene/ethanol that volume ratio is 2:1
After mentioning 8h, then with the extractables such as lipid, wax in acetone extraction 4h) removal raw material, take out the powder extracted be placed in it is logical
It is dry under room temperature in wind cupboard, it obtains without extract solid material；
(3) in draught cupboard, delignification processing is carried out without extracting raw material to dry: weighing 328g Peracetic acid
The aqueous solution that deionized water is configured to Peracetic acid to 360g is added in (concentration 32wt%), and being placed in volume is 1000mL tool plug
In conical flask, it is put into magnet rotor, the 360g peroxide acetate aqueous solution prepared is added without extracting raw material after dry in 30g
In, it is reacted for 24 hours, is filtered by 0.45 μm of water phase micropore filtering film, with a large amount of deionized waters at room temperature using magnetic stirring apparatus
Washing to the pH value of solution is 7.0 or so, obtains withy Ji holocellulose；
(4) the withy Ji holocellulose is filtered out, air-dries, takes 3g holocellulose powder to be added to 40mL and contain
The sodium acetate buffer of 16.2mg cellulase carries out enzymolysis processing, and the specification of sodium acetate buffer is 50mM, and pH is
5.0, this processing carries out in shaking table, and temperature is 37 DEG C, revolving speed 200rpm, and the processing time is 72h；
(5) supernatant of the enzymatic hydrolysis, centrifugal condition are obtained by centrifuging and taking are as follows: revolving speed is set as 8000rpm, time
15min；The supernatant of above-mentioned enzymatic hydrolysis, is added to the water of 65% ethyl alcohol of 4 times of volumes by the ethanol water for preparing 65% (v:v)
In solution, it is statically placed in cool overnight in 4 DEG C of refrigerators, centrifugation obtains and is precipitated as thick hemicellulose enzymolysis；
(6) 5mL deionized water is added in thick hemicellulose enzymolysis, is transferred in the bag filter of 3.5K, bag filter is put
It sets in 2000mL beaker, at least 72h is placed under the deionized water of flowing, the small molecules such as ethyl alcohol, glucose are constantly set
It swaps out and, dialyzate is then placed in -20 DEG C of refrigerators (or in liquid nitrogen) freezing, be put into freeze dryer at least dry 48h, system
There must be the withy Ji hemicellulose of arabinose and uronic acid branch.
Its yield is tested with weight method, as a result sees attached list 1.
Comparative example 1
This comparative example provides a kind of method for extracting withy Ji hemicellulose by organic solvent dimethyl sulfoxide, specifically
Step are as follows:
(1) withy Ji raw material is crushed, crosses 0.5mm sieve, obtains withy Ji powder；
(2) in draught cupboard, with soxhlet extraction methods (condition are as follows: 85 DEG C of temperature, taken out with the toluene/ethanol that volume ratio is 2:1
After mentioning 8h, then with the extractables such as lipid, wax in acetone extraction 4h) removal raw material, take out the powder extracted be placed in it is logical
It is dry under room temperature in wind cupboard, it obtains without extract solid material；
(3) in draught cupboard, delignification processing is carried out without extracting raw material to dry: weighing 328g Peracetic acid
The aqueous solution that deionized water is configured to Peracetic acid to 360g is added in (concentration 32wt%), and being placed in volume is 1000mL tool plug
In conical flask, it is put into magnet rotor, the 360g peroxide acetate aqueous solution prepared is added without extracting raw material after dry in 30g
In, it is reacted for 24 hours, is filtered by 0.45 μm of water phase micropore filtering film, with a large amount of deionized waters at room temperature using magnetic stirring apparatus
Washing to the pH value of solution is 7.0 or so, obtains withy Ji holocellulose；
(4) the withy Ji holocellulose is filtered out, is air-dried, take 3g holocellulose powder that the dimethyl of 120mL is added
Sulfoxide (DMSO), nitrogen charging gas shielded come into full contact with holocellulose with DMSO with magnetic agitation, and extraction temperature is 70 DEG C, the time
For 5h；
(5) alkali lysate is obtained by filtration with 0.45 μm of oily phase filter membrane, 20mL deionized water is added to wash solid residue, it will
PH value is adjusted to 3.5 using glacial acetic acid after DMSO lysate and washing lotion mixing, and the DMSO lysate among the above with after is added to 4
In 95% ethanol solution of times volume, it is statically placed in cool overnight in 4 DEG C of refrigerators, centrifugation obtains and is precipitated as thick DMSO hemicellulose
Element, centrifugal condition: revolving speed is set as 8000rpm, time 15min；
(6) 5mL deionized water is added in thick DMSO hemicellulose, is transferred in the bag filter of 3.5K, bag filter is put
It sets in 2000mL beaker, at least 72h is placed under the deionized water of flowing, the small molecules such as ethyl alcohol, glacial acetic acid, DMSO are continuous
Ground cements out, and dialyzate is then placed in -20 DEG C of refrigerators (or in liquid nitrogen) freezing, is put into freeze dryer at least dry
Pure DMSO withy Ji hemicellulose is made in 48h.
With its monosaccharide of ion chromatography and glucuronic acid content, its molecular weight and molecular weight point are tested with gel permeation chromatography
As a result cloth sees attached list 2~3.
Comparative example 2
This comparative example provides a kind of method for extracting withy Ji hemicellulose by traditional alkali soluble solution, specific steps
Are as follows:
(1) withy Ji raw material is crushed, crosses 0.5mm sieve, obtains withy Ji powder；
(2) in draught cupboard, with soxhlet extraction methods (condition are as follows: 85 DEG C of temperature, taken out with the toluene/ethanol that volume ratio is 2:1
After mentioning 8h, then with the extractables such as lipid, wax in acetone extraction 4h) removal raw material, take out the powder extracted be placed in it is logical
It is dry under room temperature in wind cupboard, it obtains without extract solid material；
(3) in draught cupboard, delignification processing is carried out without extracting raw material to dry: weighing 328g Peracetic acid
The aqueous solution that deionized water is configured to Peracetic acid to 360g is added in (concentration 32wt%), and being placed in volume is 1000mL tool plug
In conical flask, it is put into magnet rotor, the 360g peroxide acetate aqueous solution prepared is added without extracting raw material after dry in 30g
In, it is reacted for 24 hours, is filtered by 0.45 μm of water phase micropore filtering film, with a large amount of deionized waters at room temperature using magnetic stirring apparatus
Washing to the pH value of solution is 7.0 or so, obtains withy Ji holocellulose；
(4) the withy Ji holocellulose is filtered out, air-dry, take 3g holocellulose powder be added 36g mass fraction be
17.5% sodium hydroxide (NaOH) solution, nitrogen charging gas shielded come into full contact with holocellulose with lye with magnetic agitation, extracting
Time is 16h；
(5) alkali lysate is obtained by filtration with 0.45 μm of oily phase filter membrane, 20mL deionized water is added to wash solid residue, it will
PH value is adjusted to 5.5 using glacial acetic acid after DMSO lysate and washing lotion mixing, and the alkali lysate among the above with after is added to 4 times
In 95% ethanol solution of volume, it is statically placed in cool overnight in 4 DEG C of refrigerators, centrifugation obtains and is precipitated as thick alkali hemicellulose, from
Heart condition: revolving speed is set as 8000rpm, time 15min；
(6) 5mL deionized water is added in thick alkali hemicellulose, is transferred in the bag filter of 3.5K, bag filter is placed
In 2000mL beaker, at least 72h is placed under the deionized water of flowing, the small molecules such as ethyl alcohol, glacial acetic acid are constantly replaced
Out, dialyzate is then placed in -20 DEG C of refrigerators (or in liquid nitrogen) freezing, is put into freeze dryer at least dry 48h, is made
Pure alkali withy Ji hemicellulose.
With its monosaccharide of ion chromatography and glucuronic acid content, its molecular weight and molecular weight point are tested with gel permeation chromatography
As a result cloth sees attached list 2~3.
The comparison of 1 hemicellulose chemical component test result of subordinate list
* yield is calculated based on the content without hemicellulose in extracting raw material.
The test result of 2 hemicellulose yield of subordinate list, molecular weight and molecular weight distribution compares
* yield is calculated based on the content without hemicellulose in extracting raw material.
The comparison of 3 hemicellulose chemical component test result of subordinate list
* Glc: glucose, Xyl: xylose, Ara: arabinose, Gal: galactolipin, GalA: galacturonic acid, GlaA: Portugal
Grape uronic acid
Contain more arabinose and alditol with the withy Ji hemicellulose that the method that cellulase assists is extracted
Acid, both usual ingredients are to be connected on hemicellulose xylan skeleton as branch, therefore hemicellulose enzymolysis retains
More branches and active group.
The method of cellulase auxiliary of the present invention can be extracted to obtain the hemicellulose of high molecular weight, and polydispersity coefficient
Small, molecular weight homogeneity is higher.
Claims (10)
1. a kind of extracting method of the withy Ji hemicellulose with arabinose and uronic acid branch, which is characterized in that including
Following steps:
Step 1: withy Ji raw material being smashed with pulverizer, obtains withy Ji powder；
Step 2: withy Ji powder is continuously used into toluene/ethanol solution and acetone extraction, it is dry, it is former to obtain no extract timber
Material；
Step 3: delignification processing is carried out to no extract wood raw material with Peracetic acid, is washed, it is dry, it is comprehensive that withy Ji is made
Cellulose；
Step 4: withy Ji holocellulose ball grinds are digested with cellulase；
Step 5: the supernatant being centrifuged after being digested is added in ethanol water and carries out alcohol precipitation, is then centrifuged for being precipitated as
Thick hemicellulose enzymolysis；
Step 6: by thick hemicellulose enzymolysis by dialysing, freezing, be dried to obtain with arabinose and uronic acid branch
Withy Ji hemicellulose.
2. the extracting method of the withy Ji hemicellulose as described in claim 1 with arabinose and uronic acid branch, special
Sign is that method for extracting is soxhlet type, extraction conditions in the step 2 are as follows: 85 DEG C of temperature, with toluene/ethanol mixed solution
After extracting 8h, then with acetone extraction 4h.
3. the extracting method of the withy Ji hemicellulose as claimed in claim 2 with arabinose and uronic acid branch, special
Sign is, in the toluene/ethanol mixed solution, VToluene: VEthyl alcohol=2:1.
4. the extracting method of the withy Ji hemicellulose as described in claim 1 with arabinose and uronic acid branch, special
Sign is, the treatment conditions of delignification in the step 3 are as follows: it is water-soluble will to be added to Peracetic acid without extract wood raw material
In liquid, treatment temperature is 25 DEG C, and the time is for 24 hours；Mass ratio wherein without extract wood raw material and Peracetic acid be 1:(3.5~
5.5), the quality of no extract wood raw material accounts for the 10~15% of reaction system gross mass.
5. the extracting method of the withy Ji hemicellulose as described in claim 1 with arabinose and uronic acid branch, special
Sign is, washs in the step 3 are as follows: is filtered, and is washed with deionized to pH value with 0.45 μm of water phase micropore filtering film
It is 7.0.
6. the extracting method of the withy Ji hemicellulose as described in claim 1 with arabinose and uronic acid branch, special
Sign is, ball milling condition in the step 4 are as follows: selects the ball grinder and ball-type zirconium oxide abrasive ball of cobalt oxide material, ball milling
The revolving speed of time 3h, ball mill are set as 600rpm, and it is ball milling 5min that a cyclic control system, which is arranged, cooling 5min.
7. the extracting method of the withy Ji hemicellulose as described in claim 1 with arabinose and uronic acid branch, special
Sign is, the condition digested in the step 4 are as follows: the withy Ji holocellulose powder after ball milling is added to containing cellulase
Sodium acetate buffer carry out enzymolysis processing, the specification of sodium acetate buffer is 50mM, pH 5.0, this processing is in shaking table
Middle progress, temperature are 37 DEG C, revolving speed 200rpm, and the processing time is 72h.
8. the extracting method of the withy Ji hemicellulose as claimed in claim 7 with arabinose and uronic acid branch, special
Sign is that the mass ratio of the withy Ji holocellulose powder and cellulase is 1000:(5.4~30).
9. the extracting method of the withy Ji hemicellulose as described in claim 1 with arabinose and uronic acid branch, special
Sign is, the condition of alcohol precipitation in the step 5 are as follows: supernatant is slowly dropped into 65% (v:v) ethanol water of 4 times of volumes
In, precipitating is collected by centrifugation in the cool overnight in 4 DEG C of refrigerators.
10. the extracting method of the withy Ji hemicellulose as described in claim 1 with arabinose and uronic acid branch, special
Sign is, the step 6 specifically: thick hemicellulose enzymolysis is added in deionized water, is transferred in bag filter, is flowing
Deionized water in place at least 72h, then dialyzate after purification is placed in -20 DEG C of refrigerators or is freezed in liquid nitrogen, is put into
Dry 48h, is made the withy Ji hemicellulose with arabinose and uronic acid branch in freeze dryer.
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