CN1884569A - Xylose enzyme method preparing method - Google Patents
Xylose enzyme method preparing method Download PDFInfo
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- CN1884569A CN1884569A CN 200610040383 CN200610040383A CN1884569A CN 1884569 A CN1884569 A CN 1884569A CN 200610040383 CN200610040383 CN 200610040383 CN 200610040383 A CN200610040383 A CN 200610040383A CN 1884569 A CN1884569 A CN 1884569A
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Abstract
This invention involves a kind of enzymatic production method of xylose. The specific steps are pre-treat wasted crop to produce xylan or steam exploded substance; then add xylanase, bi-activity xylose/arabinosidase and alpha-glucuronidase to buffer to generate multi- enzyme mix liquid. Mixing it and adding the xylan or steam exploded substance to multi- enzyme mix liquid to perform enzymatic hydrolysis. After hydrolysis, adds ethanol to enzymatic hydrolysis liquid and further condese and crystallize to get xylose product. The release rate of xylose can reach as much as two times of using Xylanase and xylosidase so to increase the productivity. The power consuming is low. The product can keep the natural quality. At the same time the xylose produced with enzyme has no harm to microbial species, and can be used directly in biotransformation of xylitol by yeast. It is virus free and with good fermentation performance. It is benefical for the transformation from xylose to xylitol.
Description
Technical field
The present invention relates to a kind of preparation method of wood sugar, relate to a kind of enzymatic-process preparation method of wood sugar particularly, relate to fields such as foodstuffs industry, cleaner production, zymetology and molecular biology.
Background technology
Wood sugar is the aldose (CH of five carbon atoms
2OH (CHOH)
3CHO), white crystalline powder, pleasantly sweet, 144 ℃ of fusing points, water-soluble.Wood sugar is mainly used in diabetic subject's sweeting agent as foodstuff additive, industrial be auxiliary agent in printing and dyeing and the curriery.Along with the widespread use of Xylitol as the diabetic therapy agent, and Xylitol is as the exploitation of sweeting agent as chewing gum for preventing decayed tooth, candy, beverage etc., as the technology of preparing of Xylitol raw materials for production wood sugar, and also people's extensive concern extremely.
Agricultural waste material mainly comprises straw, corn cob, bagasse, wheat bran, rice bran, beet pulp etc., its main chemical is Mierocrystalline cellulose, hemicellulose, xylogen etc., wood sugar just is present in the hemicellulose pentosan of these agricultural waste materials, is the important renewable resources of demanding urgently developing.China is a large agricultural country, and annual stalk output reaches 900,000,000 tons, accounts for 25%～30% of world's stalk ultimate production.Jiangsu Province's level of agricultural production comes out at the top in the whole nation, and annual total output of grain reaches more than 3,500 ten thousand tons, and the output stalk is 4,000 ten thousand tons simultaneously, and wheat bran and rice bran output all surpass 2,700,000 tons, and the content of these tankage hemicelluloses is all more than 50%.The comprehensive utilization ratio of rice bran, wheat bran is less than 20% at present, and all the other are essentially industrial waste.And stalk is most of or burned except that being the direct returning to farmland on a small quantity, or is not piled up and utilize, and causes the waste of resource, simultaneously environment is also polluted.If people can utilize advanced technology that Mierocrystalline cellulose and hemicellulose are fully degraded and effectively conversion, agricultural waste material just can substitute grain as fermentation raw material, becomes the Biological resources of horn of plenty.Along with science and technology development, the method for utilizing of varied agricultural waste material has appearred.
But the relevant at present method of extracting wood sugar from agricultural waste material is generally all used acid-hydrolysis method.Specifically be with the agricultural waste material raw material pulverizing, add raw material weight 4-6 water doubly, after the high temperature steaming pre-treatment; Add acid then in processings that be hydrolyzed of 105-125 ℃ of temperature, hydrolyzed solution obtains xylose product through methods such as vacuum concentration, Crystallization Separation, spraying dryings at last again through the purification of methods such as neutralization, filtration, decolorizing with activated carbon, chromatographic separation, ion-exchange, ultrafiltration.
But acid-hydrolyzed method exists multiple weak point; be mainly reflected in: 1; cellulose not only in the agricultural waste material; pentosan; xylogen etc. have the composition of the value utilized; also contain protein; starch; fat; pectin; pigment; metal ion; inorganic salt; compositions such as tannin and chemical fertilizer and agricultural chemicals; the existing method of utilizing; these compositions can not be removed; and when high temperature and strong acid hydrolysis; these main components and useless impurity are hydrolyzed together; can produce or discharge in a large number to the deleterious material of yeast; mainly comprise furfural (degradation by-products); acetic acid (discharging) by the acetylize xylan; some lignin derivative (as phenolic compound), heavy metal ion etc.This not only causes very big pollution to environment, and is used for also needing hemicellulose hydrolysate is carried out detoxification before the microbial fermentation production.Though 2, dilute acid hydrolysis can reduce toxic content, often contain the oligose that can not ferment in a large number in the hydrolyzate, finally influence wood sugar and Xylitol recovery rate.Adopt the low temperature hydrolysis method simultaneously, not only hydrolysis effect is poor, speed is slow, and the acid solution that must add high density just can be hydrolyzed, and has brought the handling problem of acid solution in the hydrolyzed solution so again, must carry out neutralizing treatment thus after hydrolysis, has increased cost.3,, make that efficiency of post treatment is low, the chemical industry program is many, to the equipment requirements height, the increase environmental burden because acid-hydrolyzed liquid glucose impurity is many.
Though and adopt enzymatic hydrolysis to have advantages such as efficient, single-minded, environmental protection, but because the bacterial strain that much has prozyme system that can degradation of xylan class hemicellulose that nature exists often is suitable for oligotrophic environment, poor growth, cell density is low, can not satisfy the production demand; The substrate-function concentration efficient low, that easy pollution brought of enzymatic hydrolysis xylan existence simultaneously is low and cost is high, so do not adopt enzymatic hydrolysis at present so far in the production of wood sugar.
Summary of the invention
The purpose of this invention is to provide the method that a kind of enzyme process prepares wood sugar.
The enzymatic-process preparation method of wood sugar of the present invention, its step is as follows:
Step 1: agricultural waste material carries out pre-treatment through alkali extraction process or vapour waterfall method, makes xylan or vapour waterfall thing;
Step 2: in damping fluid, add zytase, dual-active wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme and make the multienzyme mixed solution;
Step 3: xylan or the quick-fried thing of vapour are added to carry out enzymolysis in the multienzyme mixed solution while stirring;
Step 4: hydrolysis adds ethanol after finishing in xylan enzymolysis liquid, and the back adds ethanol if the quick-fried thing enzymolysis solution of vapour then removes slag after filtration; Get the further condensing crystal of supernatant liquor then and obtain xylose product.
The contriver is through a large amount of experiments, find to prepare wood sugar, can satisfy need of industrial production with thermotolerance zytase, dual-active wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme, and, adopt the multienzyme effect, to carrying out enzymolysis through pretreated agricultural waste material, make xylan in the raw material reach the purpose of thorough hydrolysis, not only enzymic hydrolysis efficient improves greatly, and can improve the concentration of zymolyte, fast reaction speed reduces risk of pollution, is the most direct valid approach at present.Though the multienzyme effect just can realize the purpose that this is bright, the contriver further studies show that, suitable enzyme dosage ratio can reach better economic benefit.
In preparing the method for wood sugar, described step 2 specifically is with the phosphoric acid buffer of pH4-7 or citrate buffer solution preparation multienzyme mixed solution, each enzyme dosage is according to zytase in the multienzyme mixed solution: wood sugar/arabinofuranosidase/xylosidase: α-Pu Taotang aldehydic acid enzyme enzyme is 1: the ratio of 0.5-1.5: 0.5-1.25 is added, best 1: 1: 0.75.
In preparation process, concrete enzyme dosage is recommended based on zytase 10-20 (U/g) (enzyme dosage described in the literary composition is all with the weight of xylan or vapour waterfall thing).
Because the thermostability enzyme has risk of pollution, the fast reaction speed of minimizing, can improve zymolyte solubleness and higher advantages such as anti-chemical modification are arranged in biological treatment process, the enzyme that uses among the present invention is recommended the thermotolerance enzyme, preferably have a liking for the zytase of high temperature bacterium Thermotoga maritima Thermotoga maritima, dual-active wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme.
Said step 3 among the above-mentioned preparation method, concrete hydrolysis temperature are 55-100 ℃, and the present invention recommends 55-85 ℃, and preferred 60-80 ℃, best is 80 ℃.
In order to reach effect preferably, the step 3 in above-mentioned preparation method, xylan is recommended as 1 with the ratio of enzyme liquid: 3-10 (w/v), the quick-fried thing of vapour is 1 with the ratio of enzyme liquid: 6-20 (w/v).
Among the preparation method of the present invention, described agricultural waste material is plant fiber materials such as wheat straw stalk, straw, corn cob, bagasse or beet pulp.Employed zytase in the inventive method, dual-active wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme are by the genetic engineering bacterium fermentative production that efficiently expresses.
Empirical tests adopts method of the present invention, and the release rate of wood sugar can reach and adopt zytase and more than 2 times of xylosidase, has improved production efficiency greatly.Because enzyme process prepares wood sugar, have efficient, single-minded, the advantage of reaction conditions gentleness, therefore, whole course of processing action condition gentleness is not used chemical substances such as soda acid, and energy consumption is low, pollutes little, easy control simple to operate, the easy purifying of while product, product keeps natural quality; The wood sugar that utilizes the enzyme process preparation simultaneously is to the bacterial classification toxicological harmless, can be directly used in yeast bio-transformation Xylitol, do not need detoxification, leavening property is good, be beneficial to wood sugar and be converted into Xylitol, and the sponge behind the enzymatic hydrolysis is good agriculture bacterial manure raising agent, do not bring pollution to environment, therefore, have important economic benefit and social benefit.
Description of drawings
Fig. 1 is the flow process that enzyme process prepares wood sugar.
The enzymolysis product chromatography of ions figure of Fig. 2 zytase and wood sugar/Arabinoside enzymic hydrolysis birch xylan, HPLC analytical column: Sugarpak 1; 6.5mm the analysis of * 300mm sugar post, chromatographic condition: column temperature: 85 ℃; Moving phase: water; Flow velocity: 0.5mL/min; Differential refraction detector sensitivity: 4, external standard method is demarcated, and sample size is 10 μ L.Standard model is purchased the company in Sigma, according to standard specimen, and 10.7min: xylotriose; 12.5min: xylo-bioses; 15.3min: wood sugar.
The enzymolysis product chromatography of ions figure of Fig. 3 zytase, wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme combined action birch xylan, HPLC analytical column and chromatographic condition are the same, 10.7min among the figure: xylotriose; 12.5min: xylo-bioses; 15.2min: wood sugar.
The enzymolysis product chromatography of ions figure of Fig. 4 zytase and xylobiase hydrolysis oat xylan, HPLC analytical column and chromatographic condition are the same, 10.283min among the figure: xylotriose; 11.447min: xylo-bioses; 15.104min: wood sugar.
The enzymolysis product chromatography of ions figure of Fig. 5 zytase, xylobiase and arabinofuranosidase/xylosidase combined action oat xylan, HPLC analytical column and chromatographic condition are the same, 10.7min among the figure: xylotriose; 11.706min: xylo-bioses; 14.795min: wood sugar.
Fig. 6 is the composition analysis collection of illustrative plates in the xylose product.HPLC analytical column and chromatographic condition are the same, according to standard specimen, and 14.2min: wood sugar; 11.7min: xylo-bioses; 9.9min: xylotriose.
Fig. 7 is the TLC collection of illustrative plates of zytase, xylosidase and arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme combined action straw xylan. wherein: X1 is a wood sugar, and X2 is an xylo-bioses, and X3 is an xylotriose; The 1st, the xylo-oligosaccharide standard specimen; The 2nd, zytase effect straw xylan; The 3rd, zytase and xylosidase effect straw xylan; The 4th, zytase, xylosidase and arabinofuranosidase/xylosidase combined action straw xylan; The 5th, zytase, xylosidase and α-Pu Taotang aldehydic acid enzyme combined action straw xylan; The 6th, zytase, xylosidase, arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme combined action straw xylan.
Embodiment
Further specify the inventive method below in conjunction with accompanying drawing:
Embodiment 1:
(1) preparation thermotolerance xylan degrading enzyme
According to zytase among the Genbank, xylosidase, arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme gene order design primer,
Tm-xynB-N：5’-CCGTTCCATGGACTACAGGATGTGC-3’
Tm-xynB-C：5’-CCGCTCGAGCGGATATATCTTTCTTCCCTT-3’
Tm-xyl-N：5’-CATGCCATGGAACTGTACAGGGATC-3’
Tm-xyl-C：5’-ATAAGAATGCGGCCGCCTCCTCGCAGGCTTCC-3’
Tm-aguA-N：5’-GGAATTCCATATGAAAATATTACCTTCTGTGTTGAT-3’
Tm-aguA-C：5’-CCC?TCGAGTCTTTCTTCTATCTTTTTCTCCAG-3’
Genomic dna with Thmotiga maritima bacterium is that template is carried out pcr amplification.
After the PCR product of zytase, wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme inserted plasmid pET-20b and pET-28a respectively, make up recombinant expression plasmid pET-20b-xynB, pET-28a-xyl and pET-28a-aguA.
Get 0.01 μ g recombinant expression plasmid pET-20b-xynB respectively, after pET-28a-xyl and pET-28a-aguA electricity are transformed in 50 μ L e. coli jm109s (DE3) or BL21-CodonPlus (the DE3)-RIL competent cell, shaking culture 1h in the SOC substratum of 500 μ L, getting 100 μ L bacterium liquid coats LB and contains paraxin Cm (60 μ g/mL) resistance, and penbritin Amp (100 μ g/mL) or kantlex Kna (30 μ g/mL) resistant panel, 37 ℃ of overnight incubation, thereby obtain genetic engineering bacterium E.coli BL21-CodonPlus (DE3)-RIL/pET-20b-xynB, E.coli BL21-CodonPlus (DE3)-RIL/pET-28a-xyl and E.coli BL21-CodonPlus (DE3)-RIL/pET-28a-aguA, note is made EC1, EC and EC3.
The cultivation of Thermotoga maritima is that substratum is driven oxygen, fills nitrogen, adds reductive agent Na after the cooling through heating
2S 0.5g/L, and being sub-packed in the 100mL serum bottle that fills nitrogen in advance, seal and the cooling of sterilizing after, insert kind of a liquid with syringe by 0.5% inoculum size, 80 ℃ leave standstill and cultivate 8-10h and get final product.
Used culture medium prescription is (1L): 10g Tryptones, 5g yeast powder, 27g NaCl, 1mg resin reddish black (resazurin), 15ml trace element trace (Difco Maritima Broth medium), 0.5g Na
2S, pH 7.0.The preparation of trace element: by following prescription, Nitrilotriacetic acid 1.5g, MgSO
47H
2O 3.0g, MnSO
4H
2O 500.0mg, NaCl 1.0g, FeSO
47H
2O 100.0mg, Co (NO
3)
26H
2O 100.0mg, CaCl
2(anhydrous) 100.0mg, ZnSO
47H
2O 100.0mg, CuSO
45H
2O 10.0mg, AlK (SO
4)
2(anhydrous) 10.0mg, Boric acid 10.0mg, Na
2MoO
42H
2O 10.0mg, Na
2SeO
3(anhydrous) 1.0g.Earlier Nitrilotriacetic acid is dissolved in 500mL water, the KOH that uses 2-3M is with pH modulation 6.5.Add other compositions again, be settled to 1L.
Single colony inoculation of picking genetic engineering bacterium EC1, EC and EC3 contains the LB liquid nutrient medium of Amp or Kna resistance in 5mL respectively, 37 ℃ of incubated overnight, insert LB resistance liquid nutrient medium again with 1% inoculum size then, rotating speed 200r/min, 37 ℃ of shaking culture are to OD
600Reach 0.8～1.0 and make liquid seeds, be linked in the fermentor tank.
The LB substratum is formed: 1% Tryptones, 0.5% yeast extract, 1%NaCl, transfer pH 7.0, and after sterilization is cooled to 37 ℃, adds and insert 0.006%Cm, and Amp or 0.003%Kna, liquid amount 70%.The initial pH 7.0 of fermentor tank, final pH 7.9, air flow 1: 1, temperature is 30 ℃, rotating speed 250r/min.Be cultured to OD
600Reach 0.6-0.8, stream adds IPTG to concentration 0.8mmol/L, continues to cultivate 6h, puts jar.
With 4, the centrifugal 10min collecting cell of 800 * g, resuspended with 150mL potassium phosphate buffer (50mmol/L, pH 6.8), through ultrasonic wave or broken instrument (the French Pressure of high pressure cell, Thermo) smudge cells, the centrifugal 20min of 9,600 * g, get supernatant behind 70 ℃ of following thermal treatment 20min, the centrifugal 30min of 9,600 * g, supernatant liquor is enzyme liquid.
(2) be to damping fluid in successively to add zytase, wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme at 1: 1: 0.75 to make the multienzyme mixed solution with adding proportion, wherein the consumption of zytase is 10U/g, wood sugar/arabinofuranosidase/xylosidase 10U/g and α-Pu Taotang aldehydic acid enzyme 7.5U/g, xylan is 1: 3 (w/v) with the ratio of multienzyme liquid.
(3) will go into birch xylan and add in the enzyme liquid and be hydrolyzed while stirring, hydrolysis temperature is 80 ℃, after hydrolysis finishes, after adding 4 times of volume of ethanol and stirring, behind the centrifugal unhydrolysed xylan of removing in the enzymolysis solution completely and zymoprotein molecule, resulting enzymolysis solution adopts the HPLC high pressure liquid chromatography to detect wherein sugared composition and content, and the result shows that wood sugar content is than the obvious increase of adopting zytase and wood sugar/arabinofuranosidase/xylosidase to handle (seeing that accompanying drawing 3 and Fig. 2 compare).The wood sugar analysis on Content is used Sugarpakl on Waters HPLC 246-E high pressure liquid chromatograph, 6.5mm * 300mm sugar post analysis.Chromatographic condition: column temperature: 85 ℃; Moving phase: water; Flow velocity: 0.5mL/min; Differential refraction detector sensitivity: 4, external standard method is demarcated, and sample size is 10 μ L.Wood sugar, xylo-bioses and xylotriose standard model are purchased the company in Sigma.
Embodiment 2: substantially the same manner as Example 1, difference is, used xylan is the oat xylan, purchase company in Sigma, xylan is 1: 10 (w/v) with the ratio of multienzyme liquid, resulting enzymolysis solution shows after adopting HPLC to detect that wood sugar content is handled double many (seeing that accompanying drawing 5 and Fig. 4 are relatively) than employing zytase and wood sugar/arabinofuranosidase/xylosidase.
Embodiment 3: substantially the same manner as Example 1, difference is that adopting through pretreated corn cob is raw material, and concrete operations are as follows:
(1) raw materials pretreatment
Getting the chopping diameter is air-dry corn cob about 3-20mm, soak the centrifugal moisture of removing in back with clear water earlier, press solid-to-liquid ratio 7-10: 1 adds sodium hydroxide solution, final concentration is 1%-3%, at room temperature or lixiviate under the high temperature, collect filtrate then and be neutralized to subacidity, promptly get xylan with 3 times of volume ethanol precipitations then with acid.
(2) be to damping fluid in successively to add zytase, wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme at 1: 1: 0.75 to make the multienzyme mixed solution with adding proportion, wherein the consumption of zytase is 20U/g, wood sugar/arabinofuranosidase/xylosidase 20U/g and α-Pu Taotang aldehydic acid enzyme 15U/g, xylan is 1: 4 (w/v) with the ratio of multienzyme liquid.
(3) will go into xylan and add in the enzyme liquid and be hydrolyzed while stirring, hydrolysis temperature is 80 ℃, after hydrolysis finishes, after adding 4 times of volume of ethanol and stirring, centrifugally remove in the enzymolysis solution not hydrolysis completely behind xylan and the zymoprotein, get the further vacuum concentration of supernatant liquor and obtain xylose product, the gained xylose product adopts the HPLC high pressure liquid chromatography to detect, and the results are shown in accompanying drawing 6.
Embodiment 4: substantially the same manner as Example 1, difference is that what pretreating raw material was selected is wheat straw fiber, and goes out xylan with NaOH solution lixiviate from the wheat straw silk of 2%, carry out multienzyme, two enzyme and single enzymolysis then, the gained enzymolysis solution adopts the TLC thin-layer chromatography to detect.The result shows (seeing accompanying drawing 7), and 3 channel ratios, 2 road wood sugars, xylo-bioses all have increase, illustrates that the adding of xylosidase helps the degraded of zytase to xylan; Xylotriose in 4,6 channel ratios, 2,3 roads and Geng Gao wood oligose poly-and degree significantly reduces, and illustrate that arabinofuranosidase/xylosidase removal Arabinoside side shoot helps the degraded to xylan of zytase and xylosidase; 5 roads and 3, xylo-bioses is compared in 4 roads obviously to be increased, 5 roads are compared wood oligose high poly-and degree and are obviously reduced with 3 roads, illustrate α-Pu Taotang aldehydic acid enzyme go to make behind the glucuronic acid side shoot zytase can in conjunction with and decompose the xylan skeleton of these side shoots of vicinity, thereby xylo-bioses is obviously increased.The result of swimming lane 6 shows, zytase, xylosidase, arabinofuranosidase/xylosidase and the combined action of α-Pu Taotang aldehydic acid enzyme, and xylan thoroughly is degraded to monose the most at last.
Embodiment 5: substantially the same manner as Example 1, difference is that pretreating raw material adopts the quick-fried thing of wheat straw vapour, and concrete operations are as follows:
(1) nothing is gone mouldy wheat straw shrug off dust and dry after wear into the long broken wheat straw silk of 3-20mm;
(2) take by weighing wheat straw silk 10kg, after soaking, extract and abandon water, controlling moisture content is 30%, high temperature steaming step-down suddenly after 10 minutes under the 1.5Mpa then, and cooling is dried to water content 5%-10% and promptly gets the quick-fried thing of vapour.
(3) be to damping fluid in successively to add zytase, wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme at 1: 2: 1 to make the multienzyme mixed solution with adding proportion, wherein the consumption of zytase is 10U/g, wood sugar/arabinofuranosidase/xylosidase 20U/g and α-Pu Taotang aldehydic acid enzyme 10U/g, the quick-fried thing of vapour is 1: 10 (w/v) with the ratio of multienzyme liquid.
(4) while stirring the quick-fried thing of vapour is added in the enzyme liquid 80 ℃ of insulations 12 hours, the hydrolysis end is after behind the filter and remove residue, after adding 4 times of volume of ethanol and stirring, the centrifuging and taking supernatant, keep and carry out evaporation concentration under 75-80 ℃, be cooled to 60 ℃ then, get xylose product.
Embodiment 6: substantially the same manner as Example 1, difference is that zytase and α-Pu Taotang aldehydic acid enzyme are bacstearothermophilus zytase and α-Pu Taotang aldehydic acid enzyme, and hydrolysis temperature is 65 ℃.
Embodiment 7: substantially the same manner as Example 1, difference is, zytase and α-Pu Taotang aldehydic acid enzyme are Thermotoga maritima zytase and α-Pu Taotang aldehydic acid enzyme, and wood sugar/arabinofuranosidase/xylosidase is thermophilic anaerobic ethanol bacterium dual-active Arab/xylosidase, and hydrolysis temperature is 70 ℃.
Embodiment 8: substantially the same manner as Example 1, difference is that the adding proportion of zytase, wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme is 1: 1.5: 0.5, wherein the consumption of zytase is 20U/g, wood sugar/arabinofuranosidase/xylosidase 30U/g and α-Pu Taotang aldehydic acid enzyme 10U/g.
Embodiment 9: substantially the same manner as Example 5, difference is the raw materials used quick-fried thing of corn cob vapour that is; The adding proportion of zytase, wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme is 1: 0.5: 1.25, the consumption that is zytase is 20U/g, wood sugar/arabinofuranosidase/xylosidase 10U/g and α-Pu Taotang aldehydic acid enzyme 25U/g, the quick-fried thing of vapour is 1: 8 (w/v) with the ratio of multienzyme liquid.
Embodiment 10: substantially the same manner as Example 5, difference is the raw materials used quick-fried thing of bagasse vapour that is, the quick-fried thing of vapour is 1: 20 (w/v) with the ratio of multienzyme liquid.
Claims (5)
1, a kind of enzymatic-process preparation method of wood sugar, its step is as follows:
Step 1: agricultural waste material carries out pre-treatment through alkali extraction process or vapour waterfall method, makes xylan or vapour waterfall thing;
Step 2: in damping fluid, add zytase, dual-active wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme and make the multienzyme mixed solution;
Step 3: xylan or the quick-fried thing of vapour are added to carry out enzymolysis in the multienzyme mixed solution while stirring;
Step 4: hydrolysis adds ethanol after finishing in xylan enzymolysis liquid, and the back adds ethanol if the quick-fried thing enzymolysis solution of vapour then removes slag after filtration; Get the further condensing crystal of supernatant liquor then and obtain xylose product.
2, according to the said preparation method of claim 1, it is characterized in that: step 2 is that said damping fluid is the damping fluid with pH4-7; Wherein the amount ratio of zytase, dual-active wood sugar/arabinofuranosidase/xylosidase and α-Pu Taotang aldehydic acid enzyme is 1: 0.5-1.5: 0.5-1.25.
3, according to the said preparation method of claim 2, it is characterized in that: zytase, dual-active wood sugar/arabinofuranosidase/xylosidase and-amount ratio of glycuronidase is 1: 1: 0.75.
4, according to the said preparation method of claim 2, it is characterized in that: the said enzymolysis of step 3 is to carry out under 55-100 ℃; Xylan is 1 with the ratio of enzyme liquid: 3-10w/v, or the ratio of the quick-fried thing of vapour and enzyme liquid is 1: 6-20w/v.
5, according to the said preparation method of claim 2, it is characterized in that: step 4 adds 4-5 times of volume of ethanol of enzymolysis solution in the quick-fried thing enzymolysis solution of the vapour of xylan enzymolysis liquid or filter and remove residue, gets the further condensing crystal of supernatant liquor then and obtains xylose product.
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| CN1132929C (en) * | 2000-07-07 | 2003-12-31 | 无锡轻工大学 | Enzyme method preparation for high-purity oligoxylose |
| CN1640881A (en) * | 2004-01-12 | 2005-07-20 | 邾立能 | Process for preparing xylose |
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| CN101250567B (en) * | 2008-04-16 | 2011-06-15 | 中国石油化工股份有限公司 | Method for preparing monosaccharide by steam blasting lignocellulosic materials and synchronous hydrolysis with soluble oligosaccharide |
| CN102575268A (en) * | 2009-08-06 | 2012-07-11 | 安尼基有限责任公司 | Method for producing carbohydrate cleavage products from a lignocellulosic material |
| CN102051350B (en) * | 2009-10-30 | 2012-06-06 | 复旦大学 | Cryophilic xylosidase/arabinofuranosidase and preparation method and application thereof |
| CN104560846A (en) * | 2014-01-14 | 2015-04-29 | 南京师范大学 | Double-active xylanolytic enzyme engineering bacteria and application thereof |
| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
| US11840500B2 (en) | 2016-02-19 | 2023-12-12 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
| CN110042177A (en) * | 2019-05-08 | 2019-07-23 | 河北曌玉科技有限公司 | A kind of hydrolysis of hemicellulose prepares the method and system of xylose |
| CN110468169A (en) * | 2019-09-04 | 2019-11-19 | 吉林省农业科学院 | A kind of method of corncob production L-arabinose |
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