CN101376902B - Method for preparing monosaccharide from raw material containing cellulose - Google Patents
Method for preparing monosaccharide from raw material containing cellulose Download PDFInfo
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- CN101376902B CN101376902B CN200710121090XA CN200710121090A CN101376902B CN 101376902 B CN101376902 B CN 101376902B CN 200710121090X A CN200710121090X A CN 200710121090XA CN 200710121090 A CN200710121090 A CN 200710121090A CN 101376902 B CN101376902 B CN 101376902B
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Abstract
The invention relates to a method for preparing monosaccharide with a raw material containing cellulose; the method comprises the following steps: the ram material containing cellulose is blasted by steam; the obtained steam blasting product is mixed with enzyme to have zymohydrolysis; a zymohydrolysis product monosaccharide is obtained; wherein, the method for mixing the steam blasting product and the enzyme comprises mixing the steam blasting product with the enzyme by at least two batches; the amount of each batch of steam blasting product added in is at most 60 percent of the weight of all the steam blasting product; the water content of the raw material containing cellulose is 30 weight percent to 60 weight percent. The yield of the monosaccharide prepared by the method is quite high.
Description
Technical field
The invention relates to the preparation method of sugar, more specifically is the method about the feedstock production monose that adopts cellulose.
Background technology
Prior art can be used the feedstock production monose of cellulose, and gained monose mainly is glucose, and glucose can be used for food, medicine industry, in the printing and dyeing tanning industry, can be used as
Reductive agent, glucose commonly used is made reductive agent in industry of system mirror and the glass liner of a thermos flask silver plating process; Gained monose also comprises wood sugar, can make Xylitol through hydrogenation, as the non-caloric sweetener of food.Said monose also can pass through mikrobe (for example yeast) alcohol prepared by fermenting.
The raw material of cellulose is generally root, stem and the leaf of plant, comprises stalk.Said stalk is root, stem and the leaf that stays behind farm crop (being generally herbage) the results fruit, is mainly stem and leaf, like stalk, the wheat straw of corn straw, broomcorn straw, cereal grass.The staple of stalk is a lignocellulose.Said lignocellulose is the mixture of Mierocrystalline cellulose, semicellulose and xylogen.The wood fibre that Mierocrystalline cellulose, semicellulose and xylogen are combined closely and formed through covalent linkage or non covalent bond in the stalk accounts for the 70-90% of stalk gross weight.The content of Mierocrystalline cellulose, semicellulose and xylogen can the difference to some extent with the different piece of the farm crop of different sources or farm crop in the stalk.
The pretreatment process of the raw material of cellulose is generally steam explosion and/or pre-treatment is carried out in acid hydrolysis, to destroy the embedding of xylogen to Mierocrystalline cellulose and semicellulose.Though acid hydrolysis can destroy xylogen, also can destroy Mierocrystalline cellulose, reduce the productive rate of glucose then, and also need add after the acid hydrolysis in a large amount of alkali and could carry out enzymolysis and fermentation.Therefore more tendency is only accomplished preprocessing process through steam explosion, reaches the embedding effect of xylogen to Mierocrystalline cellulose and semicellulose that destroy.
Mierocrystalline cellulose is commonly referred to enzyme digestion reaction by the reaction of cellulase hydrolysis, can be called saccharification react again, and the primary product of enzymolysis is a monose.Mierocrystalline cellulose can change into glucose under the effect of cellulase; Semicellulose can change into monose such as wood sugar under the effect of hemicellulase; Glucose and wood sugar may be used to fermentative production of ethanol; Can prepare ethanol by glucose fermentation such as yeast saccharomyces cerevisiae, pichia stipitis can prepare ethanol by xylose-fermenting.Cellulosic non-crystalline structure is easy to broken, and it can be degraded into glucose fully, becomes ethanol by glucose fermentation then.But cellulosic crystalline texture is difficult to be destroyed.In addition, xylogen is a kind of non-polysaccharide material that is polymerized by phenyl-propane, by the verivate of aromatic hydrocarbon with-C-C-key ,-the O-key is crosslinked together in length and breadth; Its side chain again with semicellulose with covalent bonds, form a very fine and close network structure, Mierocrystalline cellulose tightly is wrapped in the inside; Be unfavorable for that cellulase is to cellulosic attack; Make cellulase can't contact substrate, therefore, need earlier stalk to be carried out pre-treatment with hemicellulase; Contact with cellulase and/or hemicellulase again after steam explosion or the s.t. as stalk is carried out, Mierocrystalline cellulose is hydrolyzed.
CN1629321A discloses a kind of method of utilizing stalk plant extract system ethanol with glucose and/or wood sugar; This method comprises at room temperature; According to the stalk plant: the weight ratio of dilute acid soln is 1: the ratio of 4.5-8.5 is mixed stalk with dilute acid soln; Be warmed up to 90-100 ℃ of constant temperature then, the semicellulose in the hydrolyzing straw extracts sugar soln, sugar soln alcohol prepared by fermenting fast.This method also is included in and further adds the Mierocrystalline cellulose that does not dissolve in the cellulase hydrolysis sugar soln in the said sugar soln; The add-on of said cellulase and the weight ratio of stalk are 1: 10-1: 100; Adjust pH is at 4.5-5.5; Be warmed up to 45-60 ℃ then, obtain containing the aqueous solution of glucose and wood sugar, alcohol prepared by fermenting.CN1117835C discloses a kind of method of dissolve with ethanol steam burst straw lignin prepare liquid fuel, and this method is a raw material with the steam puffed stalk, in autoclave, with the xylogen in the alcohol extraction steam puffed stalk; Obtain filtrating with vacuum filtration again, and steam puffed stalk is extracted slag carry out the synchronous saccharification solid state fermentation of ethanol through, alcohol extraction liquid and vacuum filtration gained filtrating are the liquid fuel of this method preparation.
The preparation method of said steam puffed stalk raw material comprises plant materials such as wheat straw, straw or the cornstalk steam-explosion jar of packing into of chopping, under 1.2-1.7 MPa pressure, keeps pressure 5-10 minute, and afterwards, instant decompression discharges.
The method of the xylogen in the described alcohol extraction steam puffed stalk is in autoclave, adds steam puffed stalk and ethanol, and steam puffed stalk and alcoholic acid volume ratio are 1: 10-1: 50, in the pressure range of 1.5-2.0 MPa, kept 1-2 hour.
Describedly steam puffed stalk is extracted slag carry out the synchronous saccharification solid state fermentation of ethanol through and be meant: in 33-35 ℃ of scope (fermentation), kept 14-16 hour, in 38-40 ℃ of scope (enzymolysis), keep 68 hours, and fermentation 2-3 days that hockets.
CN1193099C discloses a kind of use heat resistant type yeast and from the lignin fiber cellulosic biomass, has produced the alcoholic acid method, and this method may further comprise the steps:
It is the 15-30 millimeter that the lignin fiber cellulosic biomass is ground into particle size; Lignin fiber cellulosic biomass to pulverizing carries out the vapor explosion pre-treatment, keeps the pressure of 1-3 MPa and 190-230 ℃ temperature 1-10 minute time according to the type of material therefor, causes explosive decompression afterwards; Collect pretreated material and pass through filtering separation liquid and solid part, solid part is introduced in the fermentation storage; Adding concentration to the fermentation storage is that 15FPU/ restrains the cellulosic beta-glucosidase of cellulosic cellulase and 12.6 ius/restrain; Give fermentation storage inoculation Marx Crewe Vickers yeast CECT the suspension-s of 10875 heat resistant type yeast cultures; Mixture was shaken under 42 ℃ 72 hours.
Document " to the introduction of straw biomass to alcohol conversion process ", Zhang Xuefeng, orchid are kindly helped secure the success of, wine brewing, the 29th the 4th phase of volume; The 37-38 page or leaf discloses a kind of method of utilizing straw to produce alcohol in July, 2002, and this method comprises to be removed corn straw after the stone deironing cleans; Cut into 1.5 centimetres segment, water logging 40 minutes feeds 2.5 MPa steam, is incubated 8 minutes; Pressure release, the steam explosion products of washing 1/10th, and this wooden enzyme produces cellulase in said 1/10th steam explosion products, cultivating, and the gained culture is mixed with other 9/10ths steam explosion products; Be incubated 50 ℃ of hydrolysis 24 hours, through filtering 6% liquid glucose, mineral membrane is condensed into the liquid glucose more than 20%, adds Xiu Hata yeast (0.8-1.2 hundred million/milliliter); Fermented 24 hours, and produced wine part 10%, distill 99.5% alcohol.
Adopt aforesaid method all can realize purpose, but the productive rate of monose is lower, is generally about 35% by the feedstock production monose of cellulose.
Summary of the invention
The objective of the invention is to overcome the defective that the monose productive rate is lower, enzymolysis efficiency is lower that the method for the feedstock production monose of existing employing cellulose prepares, the method for the feedstock production monose of the employing cellulose that a kind of monose productive rate is higher, enzymolysis efficiency is higher is provided.
Contriver of the present invention finds; In the method for the feedstock production alcohol fuel of the employing cellulose of prior art with the feedstock production alcohol fuel of monose and employing cellulose; The raw material of cellulose normally exists with the form of liquid slurry like stalk, when the whole stalks with the slurries shape directly mix with enzyme; Enzyme concn is reduced, enzyme activity is descended.Moreover; To pass through pretreated stalk and the method for enzyme mixed enzymolysis is generally in the mixed solution that stalk particle with fragmentation once all joins enzyme and water and mixes with enzyme liquid; Carry out the enzymatic saccharification reaction, perhaps, earlier with used reaction mass---mix with water in retort through pretreated stalk particle; Then under hydrolysis temperature, enzyme directly joined in the retort react.Although above-mentioned mixing is carried out under agitation condition, stalk particle still is not easy to mix with enzyme.Because; Diminish through pretreated stalk granularity, particle and clastic stalk particle are easy to mutual adhesion and reunion, therefore once whole stalks are mixed with enzyme fully; Only there is the part stalk fully to contact with enzyme; Because the adhesion between the stalk has the part stalk with reunion and can not fully contact with enzyme, even under agitation, can not fully stalk be broken up.Therefore, behind the enzymolysis Mierocrystalline cellulose and/or semicellulose be converted into sugar transformation efficiency low, the ethanol yield that causes sugar-fermenting to obtain is also lower.
In addition, prior art is when the raw material of steam blasting cellulose-containing, and the raw material that it has been generally acknowledged that said cellulose is corn straw for example; Fully water logging can obtain the effect of better steam explosion, because fully water logging can make the raw material of said cellulose for example contain a large amount of water in corn straw inside; When steam explosion, the raw material of the said cellulose for example inner water of corn straw can be gasificated into water vapor, is for example gone out corn straw inside by the raw material of said cellulose during pressure release; With the raw material that fully the destroys said cellulose from inside to outside xylogen sheath of corn straw embedding Mierocrystalline cellulose and semicellulose for example; Such as (Zhang Xuefeng, orchid are kindly helped secure the success of, wine brewing, the 29th the 4th phase of volume in above-mentioned " to the introduction of straw biomass to alcohol conversion process "; The 37-38 page or leaf; In July, 2002) put down in writing in the method, need just carry out steam explosion with the corn straw water logging after 40 minutes.But contriver of the present invention is surprised to find that again; After 40 minutes, the water cut of corn straw is all up to more than the 80 weight % with corn straw (sapphire rice stalk and the dry corn straw) water logging of various water cut, and the water cut of the raw material of the cellulose of steam explosion is low; It is no doubt bad for example to be lower than 30 weight %; If but the water cut of the raw material of cellulose, as be higher than 60 weight %, also can reduce the transformation efficiency and the alcoholic acid yield of monose.
The invention provides a kind of method that adopts the feedstock production monose of cellulose, this method comprises the raw material of steam blasting cellulose-containing; With the steam explosion products that obtains mix with enzyme, enzymolysis; Obtain enzymolysis product monose; Wherein, Mix steam explosion products said steam explosion products is comprised with the enzyme method of mixing with enzyme in batches, said number of times in batches is at least 2 times, and the amount of the steam explosion products of every batch of adding is at most 60% of whole steam explosion products weight; The water cut of the raw material of said cellulose is 30-60 weight %.
Method of the present invention is through mix steam explosion products in batches with enzyme; Make the steam explosion products of every batch of adding mix and fully to contact, and in reaction process, the concentration of enzyme remain on higher level always with enzyme; Therefore, steam explosion products can fully be reacted with enzyme; In addition; The strict water cut of controlling the raw material of the cellulose that is used for steam explosion is 30-60 weight %; Can guarantee under the steam explosion condition, to make the inner aqueous vapor of raw material of cellulose to change into water vapor again; Water vapour is gone out by the raw material inside of cellulose when pressure release; Fully destroy the xylogen sheath of embedding Mierocrystalline cellulose and semicellulose in the raw material of cellulose,, thereby improved the sugared transformation efficiency of Mierocrystalline cellulose in the fibrous raw material and/or semicellulose effectively with the cellulose ratios that can contact with enzyme in the raw material that has increased cellulose.For example, under other enzymatic hydrolysis condition and all identical situation of steam explosion condition, the monose productive rate of embodiment 1 reaches 42.0%, and the monose productive rate of Comparative Examples 1 is merely 35.6%, and the monose productive rate of Comparative Examples 2 is merely 35.5%, and increase rate is up to 18.0-18.3%.
Description of drawings
Fig. 1 is the electron scanning micrograph of natural corn stalk;
Fig. 2 is the electron scanning micrograph according to stalk behind the method steam explosion of Comparative Examples 1 of the present invention;
Fig. 3 is the electron scanning micrograph according to stalk behind the method steam explosion of Comparative Examples 2 of the present invention;
Fig. 4 is the electron scanning micrograph according to stalk behind the method steam explosion of the embodiment of the invention 1.
Embodiment
According to the present invention, this method comprises the raw material of steam blasting cellulose-containing; With the steam explosion products that obtains mix with enzyme, enzymolysis; Obtain enzymolysis product monose; Wherein, Mix steam explosion products said steam explosion products is comprised with the enzyme method of mixing with enzyme in batches, said number of times in batches is at least 2 times, and the amount of the steam explosion products of every batch of adding is at most 60% of whole steam explosion products weight; The water cut of the raw material of said cellulose is 30-60 weight %, is preferably 35-50 weight %.
Under the preferable case; For enhance productivity and can guarantee simultaneously steam explosion products and enzyme thorough mixing, obtain the enzyme speed of action of higher steam explosion products concentration and higher level; The amount of first and enzyme blended steam explosion products is the 20-60% of whole steam explosion products weight, more preferably 20-40%; The amount of the steam explosion products that adds after first is the 40-80% of whole steam explosion products weight, more preferably 60-80%.
More preferably under the situation, said number of times in batches is 3 times, and the amount of first and enzyme blended steam explosion products is the 20-60% of whole steam explosion products weight, more preferably 20-40%; The amount of the steam explosion products of second batch of adding is the 20-50% of whole steam explosion products weight, more preferably 20-40%; The amount of the steam explosion products of the 3rd batch of adding is the 20-40% of whole steam explosion products weight, more preferably 20-30%.
The raw material water content difference of the cellulose of different sources is very big; Such as; The water content difference of the corn straw raw material of different times purchase is just very big; The fresh straw water cut that harvesting is no more than a week reaches more than the 80 weight %, but possibly have only 10% through the air-dry storage stalk water cut of airing.Existing method all adopts the similarity condition water logging to handle for the raw material of the cellulose of different moisture content without exception; Cause the water cut of raw material of cellulose excessive; Under the steam explosion condition; The inner moisture of the raw material of cellulose is gone out with liquid form during pressure release, does not reach the effect of the xylogen sheath of embedding Mierocrystalline cellulose and semicellulose in the raw material that destroys cellulose from inside to outside.
According to method provided by the invention, can obtain the raw material that water cut is the cellulose of 30-60 weight % through variety of way.The raw material of the different batches cellulose that there are differences for water cut can carry out different processing, such as for the high stalk of water cut, can 45-90 ℃ of oven dry part moisture, and make it water cut and be reduced to 30-60 weight %; For the low stalk of water cut, can adopt control water logging time method, the water cut that increases stalk is to 30-60 weight %; Just in time at stalk, can directly carry out steam explosion for water cut to 30-60 weight %.The water cut of the raw material of the preferred said cellulose that is used for steam explosion is 35-50 weight %.Only if specified otherwise; The water cut of the raw material of cellulose according to the invention all be meant cellulose raw material initial weight W1 with under 80-110 ℃, dry poor to the dried meter weight W 2 of the raw material of the cellulose of constant weight; With the weight percent of the initial weight W1 of the raw material of cellulose, i.e. water cut (weight %)=(W1-W2)/W1 * 100%.
Method according to the feedstock production monose with cellulose according to the invention; Can adopt the conventional steam explosion condition of prior art to accomplish steam explosion to the raw material of cellulose; For example; The temperature of said steam explosion can be 180-200 ℃, and the pressure of said steam explosion can be the 1.4-2.0 MPa, and holding time of said steam explosion pressure can be 3-7 minute.More preferably the temperature of said steam explosion is 185-195 ℃, and the pressure of said steam explosion is the 1.5-1.7 MPa, and holding time of said steam explosion pressure is 4-5 minute.Under above-mentioned steam explosion condition, can both fully destroy the xylogen sheath of stalk embedding Mierocrystalline cellulose and semicellulose, realize the object of the invention.
In order to guarantee that enzyme has best reactive behavior; With first steam explosion products with before enzyme mixes, the pH value of steam regulation explosion product is greater than 3 less than 7, is preferably 4-5.5; So that steam explosion products is with after enzyme mixes, enzyme has best reactive behavior.The method of said conditioned reaction material pH value can adopt and well known to a person skilled in the art the whole bag of tricks, like the pH value according to the gained steam explosion products, in this steam explosion products, adds acidic substance or alkaline matter.For example, said acidic substance can be one or more in sulfuric acid, hydrochloric acid and the phosphoric acid; Said alkaline matter can be sodium hydroxide and/or Pottasium Hydroxide.
Under the preferable case, also comprise the timed interval between said two adjacent batch mixing steps.In order to make fully hydrolysis steam explosion products of enzyme, improve the transformation efficiency of sugar, the said timed interval is preferably 10-120 minute, more preferably 30-60 minute.
In order to make enzyme and reaction raw materials blended more even, the enzymolysis of said steam explosion products preferably carries out under the condition that water exists, and more preferably in the suspension-s that contains water and steam explosion products, carries out.That is to say; When the present invention preferably mixes first steam explosion products with enzyme, add entry, first steam explosion products and water are mixed with suspension-s after; Again the required whole enzymes of enzymolysis are mixed with suspension-s, and then continue to add steam explosion products in batches according to method of the present invention.Do not limit the amount of the water that added is special, under the preferable case, the add-on of said water is 1-4 with whole weight ratios of steam explosion products: 1.More preferably under the situation; Mixture to obtain after first steam explosion products, water and the enzyme mixing is a benchmark, and with the dry weight basis of steam explosion products, the content of first steam explosion products is at least 1 weight %; Be preferably 1-20 weight %, more preferably 1.2-10 weight %.In the method for existing feedstock production monose by cellulose, when enzymolysis,, cause the production cycle long because the concentration of the raw material of cellulose is too rare, production efficiency is low.And contriver of the present invention finds; The concentration of steam explosion products in the mixture of when enzymolysis, controlling first steam explosion products, water and obtaining after enzyme mixes, with first steam explosion products, water and the mixture that obtains after enzyme mixes be benchmark, with the dry weight basis of steam explosion products; Make the content of steam explosion products be at least 1 weight %; Be preferably 1-20 weight %, more preferably 1.2-10 weight % can either guarantee that the cycle of enzymolysis is short; Further guarantee to have high production efficiency simultaneously, promptly the productive rate of monose is high.
Because the present invention only relates to the improvement of the feed way of the steam explosion products that obtains to the raw material water cut of cellulose and by the raw material of steam blasting cellulose-containing, therefore other step with the method for the feedstock production monose of cellulose is had no particular limits.
The enzyme that said enzymolysis uses comprises cellulase.Said cellulase can obtain through variety of way, for example is commercially available, perhaps through using the microbes producing cellulase secretion to obtain.
Because the enzyme that uses the microbes producing cellulase secretion to obtain can contain various by products, the therefore preferred enzyme that directly adds.The consumption of said enzyme is The more the better, and from cost consideration, preferably with the dry weight basis of the raw material of every gram cellulose, the consumption of said cellulase is the 8-20 enzyme activity unit, more preferably the 10-15 enzyme activity unit.The enzyme activity of cellulase according to the invention is according to American National renewable energy source laboratory (National Renewable Energy Laboratory; NREL) standard method that provides---cellulase activity is measured NREL LAP-006 and is measured; The enzyme activity unit of said cellulase is under the condition determination of this standard method regulation, restrains the micrograms that Whatman No.1 filter paper is converted into the required enzyme of glucose with 1 in 1 minute.
The temperature of said enzymolysis can be any optimum temperature of cellulase, is generally 45-55 ℃, more preferably 48-52 ℃.The longer the better on the time theory of said enzymolysis, considers plant factor, and the time of preferred said enzymolysis is 25-48 hour, more preferably 30-40 hour.Said enzymolysis time begins to calculate from the raw materials mix of enzyme and cellulose the time, that is, said enzymolysis time refer to the raw material with cellulose mixes with enzyme beginning to the feedstock conversion of cellulose for sugar after sugared content no longer increase the used time.Therefore, according to the present invention, the timed interval between said adjacent two batch mixing steps is also included within the said enzymolysis time.
Preferred embodiment said is that the method for the steam explosion products enzymolysis that obtains of the raw material of the cellulose of 30-60 weight % comprises earlier first steam explosion products is mixed with water afterwards in the adding enzymatic vessel with the steam explosion water cut according to one of the present invention, and the pH value of steam regulation explosion product is to less than 7 greater than 3; And be warming up to 48-52 ℃ to reach the optimum response reactive conditions of enzyme; Then, under agitation, in enzymatic vessel, add enzyme; Stir after 30-60 minute; Add second batch of steam explosion products again, after stirring reaction 30-60 minute, add the 3rd batch of steam explosion products.The amount of said water is 1-4 with the weight ratio of whole steam explosion products: 1; The amount of said first and enzyme blended steam explosion products is the 20-60% of whole steam explosion products weight; Second batch of amount with enzyme blended steam explosion products is the 20-50% of whole steam explosion products weight; The 3rd batch of amount with enzyme blended steam explosion products is the 20-40% of whole steam explosion products weight.And with first steam explosion products, water and the mixture that obtains after enzyme mixes be benchmark, with the dry weight basis of steam explosion products, the content of first steam explosion products is 1.2-10 weight %.
Said cellulase is a prozyme, comprises C at least
1The plain enzyme of fiber type, the plain enzyme of Cx fiber type and three kinds of enzymes of cellose.
C
1Enzyme can make the crystalline Mierocrystalline cellulose change amorphous Mierocrystalline cellulose into.
C
xThe plain enzyme of fiber type is divided into C again
X1Plain enzyme of fiber type and C
X2Two kinds of the plain enzymes of fiber type.C
X1The plain enzyme of fiber type is the endo-type cellulase, can be from the plain intramolecular action of hydration amorphous cellulose in β-1, and the 4-glycosidic link generates cellodextrin and cellobiose.C
X2The plain enzyme of fiber type is a kind of circumscribed-type cellulase, can act on β-1 from the non-reducing end of hydration non-cellulose molecule, and the 4-glycosidic link cuts off β-1 one by one, and the 4-glycosidic link generates glucose.
Cellose then acts on cellobiose, generates glucose.
The enzyme that preferred said enzymolysis uses also comprises hemicellulase.Because hemicellulase is degraded into water-soluble wood sugar with semicellulose; So the enzyme that enzymolysis uses comprises hemicellulase; Exposed fibers is plain more fully on the one hand; Increase the contact probability of Mierocrystalline cellulose and cellulase, semicellulose degraded product wood sugar also is important monose product on the other hand.With the dry weight basis of the raw material of every gram cellulose, the consumption of said hemicellulase is the 4.4-8.8 enzyme activity unit.
The enzyme activity unit of hemicellulase according to the invention (U) is under 50 ℃, pH=4.8 condition, and it is that 1 weight % xylan solution produces the required enzyme amount of 1 microgram reducing sugar (in wood sugar) that PM decomposes concentration.
The vigor of hemicellulase according to the invention refers to the unit of activity that every gram hemicellulase is had.It is that hydrolysis 1 weight % xylan produces reducing sugar (in wood sugar) under 4.8 the condition at 50 ℃, pH that the vigor of said hemicellulase utilizes hemicellulase; Gained reducing sugar and excessive 3; Color reaction takes place in 5-dinitrosalicylic acid (DNS), records the proportional mensuration of growing amount of the absorbance value and the reducing sugar (in wood sugar) of reaction solution 550 nanometers with spectrophotometer.Concrete measuring method is following:
Accurately take by weighing 1.000 gram xylans, dissolve, use deionized water constant volume to 100 milliliter then, obtain 1 weight % xylan solution with the 0.1 mol acetate of 0.5 milliliter of pH=4.8-sodium acetate buffer solution;
Take by weighing 30 the gram Rochelle salts put into 500 milliliters of Erlenmeyer flasks, add 16 the gram NaOH after, add 50 ml deionized water; Speed heating in water bath to solid matter with 5 ℃/minute dissolves, and adds 1 gram 3, the 5-dinitrosalicylic acid; To dissolving, be cooled to room temperature, be settled to 100 milliliters with deionized water; Can get 3,5-dinitrosalicylic acid (DNS) solution;
Wood sugar is dried to constant weight for 80 ℃, accurately take by weighing 1.000 grams and be dissolved in 1000 ml waters, it is anticorrosion to add 10 milligrams of sodiumazide, obtains the normal wood sugar soln of 1 mg/ml;
Accurately take by weighing 1.000 gram solid hemicellulases or pipette 1 milliliters of liquid hemicellulase stoste; 0.1 mole of acetic acid-sodium acetate buffer solution dissolving with 0.5 milliliter of pH=4.8; Use deionized water constant volume to 100 milliliter then, obtain diluting 100 times enzyme liquid to be measured;
Respectively will be in 50 ℃ of heating in water bath 2 milliliters of wood sugar gradient standardized solution (0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml and 0..5 mg/ml of 60 minutes; Said wood sugar gradient standardized solution is mixed with the normal wood sugar soln of deionized water and 1 mg/ml) or deionized water (wood sugar blank); With 2 milliliters of DNS mixing boiling water baths 5 minutes; After the cooling, 15 milliliters of deionized water constant volumes, with the absorbance value of spectrophotometer wood sugar gradient standardized solution after distinguishing assaying reaction under 550 nanometers; With the absorbance value is X-coordinate, and xylose concentration is an ordinate zou drawing standard curve.Can get regression equation y=bx+a by this typical curve, wherein, x is an absorbance value, and y is an xylose concentration, and a is the intercept of gained straight-line equation, and b is the slope of gained straight-line equation;
Get 0.1 mol acetate-sodium acetate buffer solution (xylan blank) of 0.2 milliliter of enzyme liquid to be measured and 1.8 milliliters of said 1 weight % xylan solutions or pH=4.8, according to the step test light absorption value identical with above-mentioned wood sugar gradient standardized solution.And according to the vigor of computes hemicellulase:
X is the absorbance value of enzyme liquid to be measured in the formula; B is consistent to b in the regression equation of absorbance value and a with xylose concentration with a; N is the extension rate of enzyme, 60 be expressed as enzymatic reaction time be 60 minutes, 5 for the sampling multiple (from 1 milliliter of enzyme liquid to be measured, having taken out 0.2 milliliter here tests).
Can determine the vigor of concrete hemicellulase according to aforesaid method, and then calculate the consumption of hemicellulase.
In addition; The pressure and temperature of steam explosion can be sterilized; For preventing that in enzymolysis process assorted bacterium (mainly being bacterium) pollutes the toxin of the enzyme activity that exerts an influence; Preferably before enzymolysis, add the bacteria antibiotic such as industrial penicillium mould, said microbiotic does not have effect to yeast but the growth of the bacterium that can suppress to mix.With every milliliter of enzymolysis solution is benchmark, and said antibiotic add-on is a 1-10 unit.Said enzymolysis solution comprises steam explosion products, enzyme and water.
Because under the high-temperature and high-pressure conditions of steam explosion; Semicellulose in the raw material of cellulose can produce the inhibition such as furfural, furans; Said inhibition can reduce the vigor and the zymogenic activity of enzyme; Therefore said method also is included in after the raw material of steam blasting cellulose-containing, before the steam explosion products that enzymolysis obtains, washes said steam explosion products.Said washing is that steam explosion products is mixed with water, stirs, and above-mentioned inhibition is dissolved in the water, spinning steam explosion products then, thus from steam explosion products, remove inhibition.The temperature of said washing is high more in theory, and is can the dissolved inhibition many more, considers energy consumption, and the temperature of preferred said washing is at least 60 ℃, more preferably 60-80 ℃.Because the water that added of washing is many more, the solvent that then dissolves inhibition is many more, but considers the energy consumption of spinning, and preferably with the dry weight basis of the raw material of every gram cellulose, the amount of water of said washing is the 2-10 gram, more preferably the 2-3 gram.
Owing to may contain sandstone impurity and iron contamination in the raw material of cellulose; Can cause damage to steam explosion equipment; So method for preparing monose of the present invention; Can be included in steam explosion and before the raw material of cellulose removed stone deironing routine operation, such as removing the stone deironing with the raw material of " wind send " cellulose and the method for attraction.Sandstone can not be delivered in the steam explosion equipment by wind because quality is big, iron contamination since the attraction of magnet can not get in the steam explosion equipment with raw material yet, thereby can accomplish except that the stone deironing.In addition; Because the raw material of cellulose itself tangles and the occluding device pipeline easily; Therefore; Before getting into steam explosion equipment, the size that preferably makes the raw material of said cellulose is 0.2-1 centimetre of a 0.5-3 cm x 0.2-1 cm x, and the size that more preferably makes the raw material of said cellulose is 0.5-1 centimetre of a 1-2 cm x 0.4-0.6 cm x.
Monose in the enzymolysis product can be through sugar industry method separation and purification commonly used.For example will remove the solid matter (raw material of the cellulose of for example not degraded) in the enzymolysis product through filtration, centrifugation method.Collect filtrating or supernatant, evaporation is removed the portion water branch and is obtained the supersaturation syrup that contents of monosaccharides is 60-65 weight %.In the vacuum crystallization jar, through transpiring moisture the monose crystallization is separated out, crystal and syrup form massecuite; When the crystal volume meets the requirements, massecuite is transferred to crystallizer from the vacuum crystallization jar, cooling is further separated out uncrystallized monose gradually.Carry out solid-liquid separation with whizzer, crystallization monose and molasses get product.Molasses are repeated said process crystallization 2-4 time, can fully extract monose.Crystallisation process can adopt nature graining method, stimulate graining method and nucleus graining method graining, preferably in syrup, adds crystallization with the nucleus graining method graining of kind of paste as crystal seed.
The present invention will be described in more detail below in conjunction with embodiment.
Embodiment 1
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
(1) steam explosion
Test corn straw raw material as shown in Figure 1; The water cut that records corn straw according to above-mentioned method is 20 weight %; Raw material is cut into the segment of 0.5 centimetre of 1.5 cm x, 0.2 cm x; With vertical steeping cell (the auspicious mechanical ltd in river in Shangdong Province, Shandong) and weight is that 1/3rd water of said corn straw raw material weight mixes and soaks, and obtains the corn straw that water cut is 40 weight %.Getting 1000 gram water cut is the corn straw of 40 weight %, under 195 ℃, keeps 5 minutes with the pressure of 1.8 MPas, and steam explosion is accomplished in pressure release then.The steam explosion products of gained and 70 ℃ water were mixed 30 minutes according to weight ratio at 1: 3; Use LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 2000 gram solid steam explosion products (water cut is 70 weight %) altogether.
The mensuration of Mierocrystalline cellulose gross weight and semicellulose gross weight in the gained solid steam explosion products:
Get the steam explosion products of 5 grams after the said washing and under 45 ℃, dry to constant weight 1.5 grams, 300.0 milligrams of this dried steam explosion products of weighing are positioned in heavy 80 100 milliliters of dry Erlenmeyer flasks restraining.Add the sulphuric acid soln that 3.00 ml concns are 72 weight % to said Erlenmeyer flask, stirred 1 minute.Then Erlenmeyer flask was heated 60 minutes in 30 ℃ of water-baths, every at a distance from 5 minutes with stirring once to guarantee even hydrolysis.Water-bath makes the vitriolic concentration dilution to 4 weight % with deionized water after finishing.Filter the steam explosion products acid hydrolysis liquid after the said dilution with B, obtain altogether filtrating 84 milliliters.20 milliliters of filtratings are transferred in the triangular flask of 50 milliliters of dryings.Use 2.5 gram lime carbonate to regulate this pH value of filtrate to 5.5, left standstill 5 hours, collect supernatant liquid.0.2 the supernatant liquid that the micron membrane filtration is collected, the filtrating of gained supernatant liquid is analyzed with Biorad Aminex HPX-87P performance liquid chromatography (HPLC).HPLC condition: sample size 20 microlitres; Moving phase is 0.2 micron membrane filtration, and the HPLC ultrapure water of the sonic oscillation degassing; Flow velocity is 0.6 ml/min; Column temperature 80-85 ℃; Detector temperature 80-85 ℃; Detector is a refractive index detector; Be 35 minutes working time.With D-(+) glucose of 0.1-4.0 mg/ml concentration range and 0.1-4.0 mg/ml concentration range D-(+) wood sugar as standard model.HPLC analyzes and to obtain that glucose concn is 1.73 mg/ml in the steam explosion products acid hydrolysis liquid; The steam explosion products acid hydrolysis that calculating can get after the said washing of 1 gram can obtain the glucose that weight is 0.145 gram; Because being the sulphuric acid soln of 72 weight %, concentration can the Mierocrystalline cellulose of the product of steam explosion all be hydrolyzed into glucose; Therefore the weight of gained glucose is 1.11 times of cellulosic weight in the steam explosion products; Cellulose 0.131 gram in the steam explosion products after the i.e. said washing of 1 gram then is total to cellulose 261 grams in the 2000 gram washing steam explosion products.HPLC analyzes and to obtain that xylose concentration is 0.41 mg/ml in the steam explosion products acid hydrolysis liquid; The steam explosion products acid hydrolysis that calculating can get after the said washing of 1 gram can obtain the wood sugar that weight is 0.042 gram; Because being the sulphuric acid soln of 72 weight %, concentration can semicellulose in the product of steam explosion all be hydrolyzed into wood sugar; Therefore the weight of gained wood sugar is 1.14 times of weight of semicellulose in the steam explosion products; Promptly contain semicellulose 0.037 gram in the 1 gram steam explosion products, then contain semicellulose 73.7 grams altogether in the 2000 gram steam explosion products.
(2) enzymolysis
In enzymatic vessel, add entry, and under agitation, add remaining washing steam explosion products behind first step (1) sampling and testing; The amount of the washing steam explosion products of first adding be behind step (1) sampling and testing remaining whole washing steam explosion products weight 35%; Regulating the pH value then is 4, be heated to 52 ℃ after, with the dry weight basis of every gram stalk; The cellulase that adds 20 enzyme activity units calculates; All the dry weight of stalk is 600 grams, add the cellulase (jade of the He family Bioisystech Co., Ltd) of 12000 enzyme activity units (about 80 gram enzymes) altogether, and insulation mixes 30 minutes under 52 ℃; Add second batch of washing steam explosion products then, the amount of the washing steam explosion products of second batch of adding be behind step (1) sampling and testing remaining whole washing steam explosion products weight 35%, and 52 ℃ down insulation mixed 50 minutes; And then add the 3rd batch the washing steam explosion products; The amount of the washing steam explosion products of the 3rd batch of adding be behind step (1) sampling and testing remaining whole washing steam explosion products weight 30%, and 52 ℃ down the insulation enzymolysis obtain enzymolysis product after 38 hours.The add-on of said water is 2: 1 with the weight ratio of all washing steam explosion products.Enzymolysis product is filtered with B, 20 milliliters of filtratings are transferred in the triangular flask of 50 milliliters of dryings, left standstill 5 hours, collect supernatant liquid.0.2 the supernatant liquid that the micron membrane filtration is collected, according to the said performance liquid condition of above-mentioned steps (1), the glucose of measuring and calculating in the enzymolysis product 276.1 restrains totally.The glucose weight that said enzymolysis obtains promptly totally 248.7 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11, and according to computes cellulose conversion rate and monose productive rate, calculation result is seen table 1.Per hour the cellulosic amount of enzymolysis is 6.33 grams.(with the dry weight basis of steam explosion products, be benchmark with the weight of the mixture that obtains, the content of first steam explosion products is 4.4 weight %.)
Cellulose conversion rate=100% * by the cellulosic weight of enzymolysis/cellulosic gross weight
Glucose weight/stalk dry weight that monose productive rate=100% * enzymolysis obtains
Comparative Examples 1
The reference method of the feedstock production monose of cellulose is adopted in the explanation of this Comparative Examples.
Method according to embodiment 1 prepares monose, and different is that (Zhang Xuefeng, orchid are kindly helped secure the success of according to document " to the introduction of straw biomass to alcohol conversion process "; Wine brewing; The 29th the 4th phase of volume, 37-38 page or leaf, in July, 2002) disclosed method; Stalk was soaked in water earlier before steam explosion 40 minutes, and the water cut that records the corn straw raw material after the immersion reaches 83 weight %.Getting 1000 gram water cut then is that the stalk of 83 weight % carries out steam explosion, obtains 1500 gram solid steam explosion products (water cut is 88.6 weight %) after the washing altogether.Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 1500 gram steam explosion products cellulose 71 grams altogether, semicellulose 20.4 grams.In enzymolysis step (2), remaining washing steam explosion products behind step (1) sampling and testing is once all joined in the aqueous enzymatic vessel, mix with enzyme then, enzymolysis time is 39 hours 20 minutes.According to the said performance liquid condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 59.9 restrains totally then.The glucose weight that said enzymolysis obtains promptly totally 53.96 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11.And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Comparative Examples 2
The reference method of the feedstock production monose of cellulose is adopted in the explanation of this Comparative Examples.
Method according to embodiment 1 prepares monose, and different is, the corn straw of getting 1000 gram water cut and be 20 weight % directly carries out steam explosion, obtains 1600 gram solid steam explosion products (water cut is 50 weight %) after the washing altogether.Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 1600 gram steam explosion products cellulose 344 grams altogether, semicellulose 96 grams.And steam explosion products is carried out enzymolysis according to the method for above-mentioned Comparative Examples 1, then according to the said performance liquid condition of embodiment 1 step (1), measure and calculate glucose totally 284.5 grams in the enzymolysis product.The glucose weight that said enzymolysis obtains promptly totally 256.3 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11.And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
The electron microscopic observation result:
Amplify 1000 times of steam explosion products of observing Comparative Examples 1, Comparative Examples 2 and embodiment 1 with the JSM-35CF ESEM, the result is respectively like Fig. 2, Fig. 3 and shown in Figure 4.As can beappreciated from fig. 2, because stalk inside does not have moisture can be gasificated into water vapor, so steam explosion can not fully destroy stalk from inside to outside; As can beappreciated from fig. 3, the effect of Comparative Examples 1 corn straw steam explosion and bad, Mierocrystalline cellulose still is pencil, and cellulose surface is residual that the xylogen sheath arranged; And the steam explosion effect of embodiment 1 as shown in Figure 4 is fine, can see the free Mierocrystalline cellulose.
Embodiment 2
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
(1) steam explosion
Getting 1000 gram water cut is the corn straw of 50 weight %; Be cut into the segment of 0.8 centimetre of 1.3 cm x, 0.4 cm x, under 190 ℃, kept 4 minutes pressure release then with the pressure of 1.7 MPas; Accomplish steam explosion, obtain 1250 gram steam explosion products (water cut is 60 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 1250 gram steam explosion products cellulose 213 grams altogether, semicellulose 63 grams.
(2) enzymolysis
According to above-mentioned not the steam explosion products of the method enzymolysis of embodiment 1 through washing; Different is; After in enzymatic vessel, adding entry, and under agitation, steam explosion products is mixed with enzyme in three batches; The amount of the steam explosion products of first adding be behind the sampling and testing remaining whole steam explosion products weight 60%, 50 ℃ down insulation mixed 100 minutes; Add second batch of steam explosion products then, the amount of the steam explosion products of second batch of adding be behind the sampling and testing remaining whole steam explosion products weight 20%, 50 ℃ down insulation mixed 30 minutes; And then add the 3rd batch of steam explosion products, the amount of the steam explosion products of the 3rd batch of adding be behind the sampling and testing remaining whole steam explosion products weight 20%, 50 ℃ down the insulation enzymolysis obtain enzymolysis product after 37 hours.According to the said performance liquid condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 213.3 restrains totally.The glucose weight that said enzymolysis obtains promptly totally 192.2 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11, and per hour the cellulosic amount of enzymolysis is 5.0 grams.(with the dry weight basis of steam explosion products, be benchmark with the weight of the mixture that obtains, the content of first steam explosion products is 9.0 weight %.)
And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Embodiment 3
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
(1) steam explosion
Getting 1000 gram water cut is the corn straw of 40 weight %, is cut into the segment of 0.7 centimetre of 1.2 cm x, 0.4 cm x, under 200 ℃, keeps 6 minutes with the pressure of 1.7 MPas, and steam explosion is accomplished in pressure release then.The water of gained steam explosion products and 75 ℃ was mixed 50 minutes according to weight ratio at 1: 2.5; Use impeller pump LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 2000 gram solid steam explosion products (water cut is 70 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 2000 gram steam explosion products cellulose 260 grams altogether, semicellulose 78 grams.
(2) enzymolysis
The above-mentioned steam explosion products of method enzymolysis according to embodiment 1; Different is; After in enzymatic vessel, adding entry, and under agitation, will wash steam explosion products and mix with enzyme in three batches; The amount of the washing steam explosion products of first adding be behind the sampling and testing remaining whole washing steam explosion products weight 20%, 52 ℃ down insulation mixed 20 minutes; Add second batch of washing steam explosion products then, the amount of the washing steam explosion products of second batch of adding be behind the sampling and testing remaining whole washing steam explosion products weight 40%, 52 ℃ down insulation mixed 40 minutes; And then add the 3rd batch of washing steam explosion products, the amount of the washing steam explosion products of the 3rd batch of adding be behind the sampling and testing remaining whole washing steam explosion products weight 40%, 52 ℃ down the insulation enzymolysis obtain enzymolysis product after 40 hours.According to the said performance liquid condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 268.1 restrains totally.The glucose weight that said enzymolysis obtains promptly totally 241.5 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11, and per hour the cellulosic amount of enzymolysis is 5.90 grams.(with the dry weight basis of steam explosion products, be benchmark with the weight of the mixture that obtains, the content of first steam explosion products is 2.7 weight %.)
And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Embodiment 4
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
(1) steam explosion
Getting 1000 gram water cut is the corn straw of 55 weight %, is cut into the segment of 0.8 centimetre of 2.0 cm x, 0.6 cm x, under 190 ℃, keeps 7 minutes with the pressure of 1.55 MPas, and steam explosion is accomplished in pressure release then.The water of gained steam explosion products and 65 ℃ was mixed 40 minutes according to mass ratio at 1: 2; Use impeller pump LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 1800 gram solid washing steam explosion products (water cut is 75 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 1800 gram steam explosion products cellulose 184.5 grams altogether, semicellulose 56.2 grams.
(2) enzymolysis
The above-mentioned steam explosion products of method enzymolysis according to embodiment 1; Different is; After in enzymatic vessel, adding entry, and under agitation, will wash steam explosion products and mix with enzyme in three batches; The amount of the washing steam explosion products of first adding be behind the sampling and testing remaining whole washing steam explosion products weight 10%, 52 ℃ down insulation mixed 10 minutes; Add second batch of washing steam explosion products then, the amount of the washing steam explosion products of second batch of adding be behind the sampling and testing remaining whole washing steam explosion products weight 40%, 52 ℃ down insulation mixed 40 minutes; And then add the 3rd batch of washing steam explosion products, the amount of the washing steam explosion products of the 3rd batch of adding be behind the sampling and testing remaining whole washing steam explosion products weight 50%, 52 ℃ down the insulation enzymolysis obtain enzymolysis product after 40 hours.The add-on of said water is 2.5: 1 with the weight ratio of all washing steam explosion products.According to the said performance liquid condition of embodiment 1 step (1), the glucose of measuring and calculating in the enzymolysis product 188.6 restrains totally.The glucose weight that said enzymolysis obtains promptly totally 169.9 is restrained by the cellulosic weight of enzymolysis in the steam explosion products divided by 1.11, and per hour the cellulosic amount of enzymolysis is 4.2 grams.(with the dry weight basis of cellulosic material, the content of the raw material of first cellulose is 0.95 weight %.)
And calculate cellulose conversion rate and monose productive rate according to the method for embodiment 1 and formula, calculation result is seen table 1.
Embodiment 5
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
(1) steam explosion
Getting 1000 gram water cut is the corn straw of 55 weight %, is cut into the segment of 0.8 centimetre of 2.0 cm x, 0.6 cm x, under 190 ℃, keeps 7 minutes with the pressure of 1.6 MPas, and steam explosion is accomplished in pressure release then.The water of gained steam explosion products and 65 ℃ was mixed 40 minutes according to mass ratio at 1: 2; Use impeller pump LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 1800 gram solid steam explosion products (water cut is 75 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 1800 gram steam explosion products cellulose 184.5 grams altogether, semicellulose 56.2 grams.
(2) enzymolysis
According to the above-mentioned steam explosion products through washing of the method enzymolysis of embodiment 1, different is, after in enzymatic vessel, adding entry; And under agitation; Add remaining washing steam explosion products behind first step (1) sampling and testing, the amount of the washing steam explosion products of first adding be behind step (1) sampling and testing remaining whole washing steam explosion products weight 60%, the pH value of conditioned reaction material is 5 then; And after being heated to 50 ℃; With the dry weight basis of every gram stalk, the cellulase that adds 20 enzyme activity units calculates, and all the dry weight of stalk is 450 grams; Add the cellulase (jade of the He family Bioisystech Co., Ltd) of 9000 enzyme activity units (about 45 grams) and the hemicellulase (Beijing chemical reagents corporation) of 3600 enzyme activity units (about 49.5 grams) altogether, and insulation mixed 80 minutes; Add then second batch the washing steam explosion products, the amount of the washing steam explosion products of second batch of adding be behind step (1) sampling and testing remaining whole washing steam explosion products weight 40%, the insulation enzymolysis obtain enzymolysis product after 40 hours.And according to the said performance liquid condition of embodiment 1 step (1); The glucose of measuring and calculating in the enzymolysis product 188 restrains totally; The weight of the glucose that said enzymolysis obtains is divided by 1.11; Be totally 169.4 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products, the wood sugar of measuring and calculating in the enzymolysis product 44.8 restrains totally, and the weight of the wood sugar that said enzymolysis obtains is divided by 1.14; Be totally 39.3 to be restrained by the weight of the semicellulose of enzymolysis in the steam explosion products, per hour the gross weight of the Mierocrystalline cellulose of enzymolysis and semicellulose is 5.2 grams; And calculate Mierocrystalline cellulose, semicellulose transformation efficiency and monose productive rate according to the following equation, calculation result is seen table 1.(with the dry weight basis of steam explosion products, be benchmark with the blended weight that obtains, the content of said first steam explosion products is 5.66 weight %.)
Cellulose conversion rate=100% * by the cellulosic weight of enzymolysis/cellulosic gross weight
Semicellulose transformation efficiency=100% * by the gross weight of the weight/semicellulose of the semicellulose of enzymolysis
Monose productive rate=100% * (the wood sugar weight that glucose weight+enzymolysis that enzymolysis obtains obtains)/stalk dry weight
Calculation result is seen table 1.
Embodiment 6
Present embodiment is used to explain the method for the feedstock production monose of employing cellulose of the present invention.
(1) steam explosion
Is that the corn straw of 50 weight % carries out steam explosion according to the method for embodiment 2 to 1000 gram water cut; Different is; After accomplishing steam explosion; The water of gained steam explosion products and 60 ℃ was mixed 40 minutes according to mass ratio at 1: 2, use impeller pump LW400 type horizontal screw centrifuge (Zhangjiagang Huada Centrifugal Manufacturing Co., Ltd.) under 900 rev/mins of rotating speeds, to carry out solid-liquid separation then, obtain 1785 gram washing solid steam explosion products (water cut is 72 weight %) altogether.
Analyze the method for the steam explosion products of sulphuric acid hydrolysis according to the said HPLC of embodiment 1, record in the 1785 gram steam explosion products cellulose 215 grams altogether, semicellulose 60 grams.
(2) enzymolysis
Method according to embodiment 5 is carried out enzymolysis to steam explosion products; Different is; After in enzymatic vessel, adding entry, and under agitation, steam explosion products is mixed with enzyme in three batches; The amount of the steam explosion products of first adding be behind the sampling and testing remaining whole steam explosion products weight 40%, 50 ℃ down insulation mixed 50 minutes; Add second batch of steam explosion products then, the amount of the steam explosion products of second batch of adding be behind the sampling and testing remaining whole steam explosion products weight 30%, 50 ℃ down insulation mixed 30 minutes; And then add the 3rd batch of steam explosion products, the amount of the steam explosion products of the 3rd batch of adding be behind the sampling and testing remaining whole steam explosion products weight 30%, 50 ℃ down the insulation enzymolysis obtain enzymolysis product after 38 hours.And; Dry weight basis with every gram stalk; The cellulase that adds 20 enzyme activity units calculates; All the dry weight of stalk is about 500 grams, adds the cellulase (jade of the He family Bioisystech Co., Ltd) of 10000 enzyme activity units (about 60 grams) and the hemicellulase (Beijing chemical reagents corporation) of 4000 enzyme activity units (about 60 grams) altogether.And according to the said performance liquid condition of embodiment 1 step (1); The glucose of measuring and calculating in the enzymolysis product 222.9 restrains totally; The weight of the glucose that said enzymolysis obtains is divided by 1.11; Be totally 200.8 to be restrained by the cellulosic weight of enzymolysis in the steam explosion products, the wood sugar of measuring and calculating in the enzymolysis product 48.4 restrains totally, and the weight of the wood sugar that said enzymolysis obtains is divided by 1.14; Be totally 42.5 to be restrained by the weight of the semicellulose of enzymolysis in the steam explosion products, per hour the gross weight of the Mierocrystalline cellulose of enzymolysis and semicellulose is 6.34 grams; And calculate Mierocrystalline cellulose, semicellulose transformation efficiency and monose productive rate, calculation result is seen table 1.(with the dry weight basis of steam explosion products, be benchmark with the weight of the mixture that obtains, the content of said first steam explosion products is 4.54 weight %.)
And according to the formula calculating Mierocrystalline cellulose of embodiment 5, the transformation efficiency and the monose productive rate of semicellulose, calculation result is seen table 1.
Table 1
| Embodiment or Comparative Examples | Embodiment 1 | Comparative Examples 1 | Comparative Examples 2 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Embodiment 6 |
| Cellulose conversion rate (%) | 95.3 | 76.0 | 74.5 | 90.2 | 92.9 | 92.1 | 91.8 | 93.4 |
| Semicellulose transformation efficiency (%) | - | - | - | - | - | - | 70.0 | 70.8 |
| Monose productive rate (%) | 46.0 | 35.2 | 35.6 | 42.7 | 44.7 | 41.9 | 51.7 | 54.3 |
Data from last table 1 can find out, adopt the productive rate of the monose that the method for the feedstock production monose of cellulose of the present invention obtains and the method for Comparative Examples 1 and Comparative Examples 2 more all to be significantly improved.
Claims (10)
1. method that adopts the feedstock production monose of cellulose, this method comprises the raw material of steam blasting cellulose-containing; With the steam explosion products that obtains mix with enzyme, enzymolysis; Obtain enzymolysis product monose; It is characterized in that; Mix steam explosion products said steam explosion products is comprised with the enzyme method of mixing with enzyme in batches, said number of times in batches is at least 2 times, and the amount of the steam explosion products of every batch of adding is at most 60% of whole steam explosion products weight; The amount of said first and enzyme blended steam explosion products is the 20-60% of whole steam explosion products weight, and the amount of the steam explosion products that adds after first is the 40-80% of whole steam explosion products weight; The water cut of the raw material of said cellulose is 30-60 weight %; The raw material of said cellulose is a stalk; Also comprise the timed interval between two adjacent batch mixing steps; The enzyme that said enzymolysis uses comprises cellulase, and with the dry weight basis of the raw material of every gram cellulose, the consumption of said cellulase is the 8-20 enzyme activity unit.
2. method according to claim 1, wherein, said number of times in batches is 3 times, the amount of first and enzyme blended steam explosion products is the 20-60% of whole steam explosion products weight; The amount of the steam explosion products of second batch of adding is the 20-50% of whole steam explosion products weight; The amount of the steam explosion products of the 3rd batch of adding is the 20-40% of whole steam explosion products weight.
3. method according to claim 1 and 2, wherein, when first steam explosion products is mixed with enzyme; This method also comprises and adds entry; With the dry weight basis of steam explosion products, be benchmark with the weight of the mixture that obtains, the content of first steam explosion products is at least 1 weight %.
4. method according to claim 3 wherein, with the dry weight basis of steam explosion products, is a benchmark with the weight of the mixture that obtains, and the content of first steam explosion products is at least 1.2-10 weight %.
5. method according to claim 1, wherein, the water cut of the raw material of said cellulose is 35-50 weight %.
6. method according to claim 1, wherein, the said timed interval is 10-120 minute.
7. method according to claim 1, wherein, the temperature of said steam explosion is 180-200 ℃, and the pressure of said steam explosion is the 1.4-2.0 MPa, and holding time of said steam explosion pressure is 3-7 minute.
8. method according to claim 1, wherein, the temperature of said enzymolysis is 45-55 ℃, and the time of said enzymolysis is 25-48 hour, and the pH value of said enzymolysis is 3.0-7.0.
9. method according to claim 1, wherein, the enzyme that said enzymolysis uses also comprises hemicellulase, with the dry weight basis of the raw material of every gram cellulose, the consumption of said hemicellulase is the 4.4-8.8 enzyme activity unit.
10. method according to claim 1, wherein, the size of the raw material of said cellulose is 0.2-1 centimetre of a 0.5-3 cm x 0.2-1 cm x.
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| CN1451755A (en) * | 2002-04-12 | 2003-10-29 | 中国农业大学 | Method for producing alcohol by solid fermentation of stalks |
| CN1806945A (en) * | 2005-01-20 | 2006-07-26 | 中国科学院过程工程研究所 | Method for complete enzymolysis of straw cellulose by utilizing pretreatment and enzymolysis process |
| CN1880416A (en) * | 2005-06-14 | 2006-12-20 | 河南农业大学 | Process for preparing fuel ethanol by using straw fiber materials |
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| CN1451755A (en) * | 2002-04-12 | 2003-10-29 | 中国农业大学 | Method for producing alcohol by solid fermentation of stalks |
| CN1806945A (en) * | 2005-01-20 | 2006-07-26 | 中国科学院过程工程研究所 | Method for complete enzymolysis of straw cellulose by utilizing pretreatment and enzymolysis process |
| CN1880416A (en) * | 2005-06-14 | 2006-12-20 | 河南农业大学 | Process for preparing fuel ethanol by using straw fiber materials |
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