CN104450829B - The method that high temperature lignocellulolyticenzymes coordinated enzymatic hydrolysis prepares fermentable sugars - Google Patents
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Abstract
Lignocellulosic material enzymolysis of high cost is the major obstacle for realizing its bioconversion, and Thermophilic Bacterium has unique lignocellulosic enzyme system, has strong degradation to natural wooden fiber's element raw material.The present invention generates lignocellulosic enzyme system with xylan, stalk etc. for substrate for induction Thermophilic Bacterium, extracting intracellular and ectoenzyme has complete hemicellulase and cellulase system, the energy directly raw materials such as degrading straw, it is digested after being mixed in a certain ratio with endocellular enzyme and ectoenzyme with commercialized cellulase synergistic, the enzymatic hydrolyzation of hemicellulose and cellulose reaches more than 80%.Enzyme solution provided by the present invention can directly digest natural wooden fiber's element raw material, remove biomass pre-treatment step from, reduce comprehensive production cost with, the mortifier of preprocessing process generation is avoided to the adverse effect that is converted to subsequent bio, clean environment firendly, without sugar loss, have broad application prospects.
Description
Technical field
The invention belongs to biomass efficients to convert field, prepared by more particularly to a kind of high temperature lignocellulolyticenzymes coordinated enzymatic hydrolysis
The method of fermentable sugars.
Background technology：
Fossil resource production bioenergy and chemicals are substituted using lignocellulosic material, is to realize that human society can be held
The needs of supervention exhibition.However, its bioconversion is limited by cellulose hydrolysis difficulty, this is because the main component of stalk
Together with (cellulose, hemicellulose and lignin) tightly embraces, lignin and carbohydrate complex are formed, is anti-micro-
Biodegradable barrier.Therefore, lignocellulosic is converted into fermentable sugars, is to be met using renewable resource instead of fossil fuel
One significant challenge of growing energy demand.Enzymatic hydrolysis is most common cellulose and hemicellulose degradation Cheng Kefa
The method of ferment sugar such as glucose and xylose.At present, the high cost of enzymolysis process is limitation bio-fuel and biochemicals industry
The major obstacle of metaplasia production.It is to overcome that searching high efficiency cellulose and hemicellulase preparations develop efficient mode of action simultaneously
The main means of this obstacle, it has also become one of hot spot of whole world research.
Many Thermophilic Bacteriums can be converted into biological energy source using the relevant lignocellulosic material of various carbohydrate
Source.These extreme thermophilics are bacteriogenic novel and unique enzyme may be brought to the generation of bio-fuel and chemicals it is new
Chance.Wherein, pyrolysis cellulose Pseudomonas (Caldicellulosiruptor) is the most heat-resisting biolobic material found so far
It using bacterium, can be grown under higher temperature (70-90 DEG C), so far, pyrolysis cellulose Pseudomonas has had 8 kinds
(C.saccharolyticus,C.bescii, C.obsidiansis C.hydrothermalis,C.kristjanssonii,
C.kronotskyensis, C.lactoaceticus, and C. owensensis) in Europe, North America, Asia and Australia etc.
Ground is successively found.Caldicellulosiruptor belongs to Thermophilic Bacterium since it can generate diversified glycoside hydrolase
Hydrolysis of lignocellulose is considered to have the prospect of being widely applied.Bioinformatics method analysis find (Blumer et al.,
Journal of bacteriology.2012), Caldicellulosiruptor genome encodings 106 glycoside hydrolase point
Cloth is in 43 glycoside hydrolase Families.And many cellulases and hemicellulase genes cluster are found that from its genome.
Some novel and high-efficiencies and high lignocellulolyticenzymes of thermostabilization have obtained heterogenous expression.Particularly some enzymes not only have multiple structures
Domain, and possess a variety of catalysis, their structure and mode of action is different from the common cellulase system of other two：
One is to generate free single cellulase and hemicellulase, such as fungi and most of bacteriogenic cellulases
(Martinez, Nature Biotechnology 2008), the other is there is multiple hydrolases to self-assemble to a common egg
White matter stent forms macromolecular polymeric body, referred to as cellulosome cellulosome (Bayer, Annual Review of
Microbiology.2004) for example, the complex cellulase CelA that C.bescii is generated, includes the 9th family's glucosides
Hydrolase and the 48th family's glycoside hydrolase catalyst structure domain, it has been found that the mode of enzyme enzymolysis cellulose is very unique,
In addition to can be with common mode of action other than the nickase solution cellulose of surface, moreover it is possible to be deep into fibrous inside to excavate and digested
(Brunecky,Science.2013).Current commercialized cellulase and hemicellulase are mainly generated by fungi.Due to
Caldicellulosiruptor, which belongs to high temperature bacterium, can generate the cellulase system different from fungi, in enzymolysis lignocellulosic
When, they have complementarity with fungal cellulase, the strain high temperature fiber element enzyme belonged to using Caldicellulosiruptor
Better enzymolysis efficiency will be generated, therefore with potential business application valency with commercialized fungal cellulase coordinated enzymatic hydrolysis
Value.However, its application value is far from being developed, presently mainly some of which lignocellulosic enzyme gene is carried out
Heterogenous expression, and have studied its enzymatic property, different substrate for induction are generated compound cellulose enzyme system as a whole with quotient
Industry cellulose enzyme synergistic effect is studied.The present invention provides a kind of efficiently with Caldicellulosiruptor category generations
The high temperature lignocellulosic complex enzyme method for preparing fermentable sugars with the plain enzyme coordinated enzymatic hydrolysis of commercialization dimension, there are industrial applications
Prospect.
Invention content
(1) purpose of the present invention
Current commercialized cellulase is of high cost, it is difficult to support the large-scale production of biomass product, find new fibre
The plain enzyme of dimension and mode of action are to solve the problems, such as this inevitable approach.By literature survey combine our research teams research into
Fruit finds that Caldicellulosiruptor microorganism belonging to genus has strong degradation to natural wooden fiber's element raw material, they are generated
Lignocellulolyticenzymes there is unique performance, with the current commercialized good complementation of cellulase, coordinated enzymatic hydrolysis energy
Increase substantially enzymolysis efficiency.The lignocellulosic materials enzymolysis such as stalk, which prepares fermentable sugars, needs the synergistic effect of a variety of enzymes
It can complete, the enzymolysis of wherein cellulose needs cellulose restriction endonuclease, excision enzyme and beta-glucosidase collaboration to complete, and hemicellulose
Element enzymolysis needs the synergistic effects such as zytase, xylosidase, arabinofuranosidase, feruloyl esterase that could complete, and half
Cellulase and cellulase synergistic effect could effectively by natural wooden fiber's element feed degradation be fermentable sugars, single enzyme
It can not play a role.Therefore, the lignocellulosic complex enzyme that the present invention is generated with different substrate for induction is (including cellulase and half
Cellulase) it is whole, it acts synergistically with commercial fibres element enzyme, natural wooden fiber's element raw material or pretreated wooden of degrading
Cellulosic material improves enzymolysis efficiency, reduces cellulase hydrolysis cost.
(2) technical solution
The method that high temperature lignocellulolyticenzymes coordinated enzymatic hydrolysis prepares fermentable sugars, includes the following steps：
1) with the lignocellulosic material of 0.1-2% such as stalk, Corncob Xylan, bagasse xylan, microcrystalline cellulose
Element, steam puffed stalk etc. are carbon source, and the Caldicellulosiruptor category Thermophilic Bacteriums for cultivating 20-30h are utilized at 60-90 DEG C
(C. saccharolyticus,C.bescii,C.obsidiansis C.hydrothermalis,C.kristjanssonii,
C.kronotskyensis, C. lactoaceticus, C.owensensis one or more) for seed liquor, anaerobism
Ferment 20-72h；
2) 15min is centrifuged under the conditions of after fermentation at normal temperatures 6,000 × g, detaches cell and supernatant, cell is through super
After sound broken wall, centrifugation removal cell wall, supernatant is intracellular lignocellulolyticenzymes, and fermented supernatant fluid is 70- with saturation degree
80% ammonium sulfate protein precipitation, after centrifugation removes supernatant, with the buffer solution albumen of pH5.0-7.6, protein solution
It sucks in bag filter in the buffer solution dialysed overnight of pH5.0-7.6, is adsorbed outside bag filter with polyethylene glycol after the completion of dialysis dense
Pix protein, protein concentration reach 0.5-2mg/ml, and it is extracellular lignocellulose enzyme to collect protein solution；
3) intracellular and extracellular lignocellulose enzyme obtained with step 2) digests wooden with commercialized cellulase synergistic
Cellulosic material prepares fermentable sugars, and specific enzyme solution is：With ectoenzyme individually or endocellular enzyme and ectoenzyme are according to 1:100-
100:The ratio of 1 (w/w), which is blended at 60-85 DEG C, to be digested naturally or after the lignocellulosic material 24-72h by pretreatment,
Add commercial fibres element enzyme preparation and digest 48-72h at 50-55 DEG C, obtain fermentable sugars, including glucose, xylose, I
Uncle's sugar, mannose, rhamnose etc..Caldicellulosiruptor belongs to the lignocellulosic that strain generates in above-mentioned enzymolysis process
Enzyme dosage is 1-15mg/g dry bottom objects, and commercial fibres element enzyme dosage is 5-30FPU/g dry bottom objects.
(3) advantage and novelty of the invention
The degradation of lignocellulosic needs the coordinated enzymatic hydrolysis of a variety of enzymes, and enzymolysis efficiency depends on ' individual soldier's work of each enzyme
War ' suitable ratio between ability and each enzyme.Commercial cellulose enzyme is stronger to the degradation capability of cellulose at present, hemicellulose
The degradation capability of element is weak, and lignocellulosic material has to pass through pretreatment removal hemicellulose and can be only achieved preferable enzymolysis effect
Fruit, it is poor to natural material or the halfway raw material of pretreatment, hydrolysis result.Caldicellulosiruptor belongs to what strain generated
Lignocellulolyticenzymes include complete hemicellulase and cellulase system, have strong degradation to make to natural wooden fiber's element raw material
With, but they produce the ability of enzyme not as good as fungi, individually prepare enzyme preparation with the enzyme that they are produced, are extremely difficult to commercialized want
It asks, it, can be directly by natural wooden fiber using the composite lignocellulosic element enzyme that they are generated and fungal cellulase coordinated enzymatic hydrolysis
Plain raw material is efficiently degraded to fermentable sugars, removes the pre-treatment steps such as acid, alkali, steam explosion, ionic liquid from, without sugar loss,
Also it is generated without mortifier, good condition is created for subsequent fermentation.For different pretreatments method and pretreatment degree not
Together, the ratio that can adjust Caldicellulosiruptor category lignocellulolyticenzymes and commercial fibres element enzyme is digested, and is reached
Best effect.
Our research finds that Caldicellulosiruptor belongs to the lignocellulolyticenzymes that strain generates, intracellular and born of the same parents
The distributional difference of exoenzyme is very big, and zytase, cellulose restriction endonuclease, excision enzyme are mainly distributed on extracellular, and xylosidase, β-Portugal
Polyglycoside enzyme, arabinofuranosidase are mainly distributed on intracellular, this project is directed to different raw materials by endocellular enzyme and ectoenzyme
With commercial fibres element enzyme coordinated enzymatic hydrolysis after mixing in certain proportion, reach the hydrolysis result of economical and efficient.This is also the present invention
Innovative point.
Specific embodiment
1 C.bescii of embodiment is using corncob as the lignocellulolyticenzymes that carbon source generates and commercial fibres element enzyme coordinated enzyme
Solve maize straw
1) will be cultivated at 80 DEG C C.bescii seed liquors for 24 hours be inoculated by 3% inoculum concentration it is beautiful with 0.1% crushing
Meter Xin is carbon source culture medium, and culture medium other compositions are (every liter)：0.9g NH4Cl,0.9g NaCl,0.75g KH2PO4,1.5g
K2HPO4,0.4g MgCl2·6H2O,2.5mg FeCl3·6H2O, 1ml trace element, 1mg resazurins, 2g tryptoses, 1g ferment
Female cream, 0.75g cysteine hydrochloric acid H2O, anaerobic fermentation 26h at 80 DEG C；
2) 15min is centrifuged under the conditions of after fermentation at normal temperatures 6,000 × g, detaches cell and supernatant, cell is through super
After sound broken wall, centrifugation removal cell wall, supernatant is intracellular lignocellulolyticenzymes, and fermented supernatant fluid is 70- with saturation degree
80% ammonium sulfate protein precipitation, after centrifugation removes supernatant, with the Tris- hydrochloride buffer soluble proteins of pH7.2, albumen
In the Tris- hydrochloride buffer dialysed overnights of pH7.2 in solution sucking bag filter, polyethylene glycol is used after the completion of dialysis in bag filter
Outer Adsorption Concentration albumen, protein concentration reach 2mg/ml, and it is extracellular lignocellulose enzyme to collect protein solution；
3) lignocellulosic is digested with the extracellular lignocellulose enzyme that step 2) obtains and commercialized cellulase synergistic
Raw material prepares fermentable sugars, and specific enzyme solution is as follows：Delayed with ectoenzyme (dosage is 10mg/g dry bottoms object) in the acetic acid of pH6.0
After digesting natural corn stalk (enzymolysis liquid amount of solid is 1%, w/v) 48h in fliud flushing at 75 DEG C, adding Xia Sheng industry group has
The commercial fibres element enzyme preparation (dosage is 10FPU/g dry bottoms object) of limit company production digests (enzymolysis liquid amount of solid at 50-55 DEG C
For 3%, w/v) 72h, obtain fermentable sugars, wherein the enzymatic hydrolyzation of cellulose is 81%, and the enzymatic hydrolyzation of xylan is 83%, Ah
It is 90% to draw primary glycan enzymatic hydrolyzation.
2 C.owensensis of embodiment is using steam puffed stalk as the lignocellulolyticenzymes that carbon source generates and commercial fibres element enzyme
Coordinated enzymatic hydrolysis wheat stalk
1) C.owensensis seed liquors for 24 hours will be cultivated at 75 DEG C by 3% inoculum concentration and are inoculated into the vapour with 0.1%
Quick-fried stalk is carbon source culture medium, and culture medium other compositions are (every liter)：0.9g NH4Cl,0.9g NaCl,0.75g KH2PO4,
1.5g K2HPO4,0.4g MgCl2·6H2O,2.5mg FeCl3·6H2O, 1ml trace element, 1mg resazurins, 2g tryptoses,
1g yeast extracts, 0.75g cysteine hydrochloric acid H2O, anaerobic fermentation is for 24 hours at 80 DEG C；
2) 15min is centrifuged under the conditions of after fermentation at normal temperatures 6,000 × g, detaches cell and supernatant, cell is through super
After sound broken wall, centrifugation removal cell wall, supernatant is intracellular lignocellulolyticenzymes, and fermented supernatant fluid is 70- with saturation degree
80% ammonium sulfate protein precipitation, after centrifugation removes supernatant, with the Tris- hydrochloride buffer soluble proteins of pH7.2, albumen
In the Tris- hydrochloride buffer dialysed overnights of pH7.2 in solution sucking bag filter, polyethylene glycol is used after the completion of dialysis in bag filter
Outer Adsorption Concentration albumen, protein concentration reach 2mg/ml, and it is extracellular lignocellulose enzyme to collect protein solution；
3) lignocellulosic is digested with the extracellular lignocellulose enzyme that step 2) obtains and commercialized cellulase synergistic
Raw material prepares fermentable sugars, and specific enzyme solution is as follows：Delayed with ectoenzyme (dosage is 15mg/g dry bottoms object) in the acetic acid of pH6.0
After digesting natural corn stalk (enzymolysis liquid amount of solid is 1%, w/v) 48h in fliud flushing at 75 DEG C, adding Xia Sheng industry group has
The commercial fibres element enzyme preparation (dosage is 10FPU/g dry bottoms object) of limit company production digests (enzymolysis liquid solid content at 55 DEG C
For 1%, w/v) 72h, obtain fermentable sugars, wherein the enzymatic hydrolyzation of cellulose is 84%, and the enzymatic hydrolyzation of xylan is 88%, Ah
It is 94% to draw primary glycan enzymatic hydrolyzation.
3 C.saccharolyticus of embodiment is using Corncob Xylan as the lignocellulolyticenzymes that carbon source generates and business
Cellulase synergistic digests steam explosion maize straw
1) will be cultivated at 80 DEG C C.saccharolyticus seed liquors for 24 hours by 2% inoculum concentration be inoculated into
0.2% Corncob Xylan is carbon source culture medium, and culture medium other compositions are (every liter)：0.9g NH4Cl,0.9g NaCl,
0.75g KH2PO4, 1.5g K2HPO4,0.4g MgCl2·6H2O,2.5mg FeCl3·6H2O, 1ml trace element, 1mg swords day
Blueness, 2g tryptoses, 1g yeast extracts, 0.75g cysteine hydrochloric acid H2O, anaerobic fermentation is for 24 hours at 80 DEG C；
2) 15min is centrifuged under the conditions of after fermentation at normal temperatures 6,000 × g, detaches cell and supernatant, cell is through super
After sound broken wall, centrifugation removal cell wall, supernatant is intracellular lignocellulolyticenzymes, and fermented supernatant fluid is 70- with saturation degree
80% ammonium sulfate protein precipitation, after centrifugation removes supernatant, with the Tris- hydrochloride buffer soluble proteins of pH7.2, albumen
In the Tris- hydrochloride buffer dialysed overnights of pH7.2 in solution sucking bag filter, polyethylene glycol is used after the completion of dialysis in bag filter
Outer Adsorption Concentration albumen, protein concentration reach 2mg/ml, and it is extracellular lignocellulose enzyme to collect protein solution；
3) intracellular and extracellular lignocellulose enzyme obtained with step 2) digests wooden with commercialized cellulase synergistic
Cellulosic material prepares fermentable sugars, and specific enzyme solution is as follows：10 are pressed with born of the same parents' endocellular enzyme and ectoenzyme:1 ratio mixing, takes
The mixed enzyme (1mg/g dry bottoms object) is blended in pH6.0 with Novi letter commercial fibres element enzyme preparation CTec2 (10FPU/g dry bottoms object)
55 DEG C of citrate buffer solution at after enzymolysis steam explosion corn stalk raw material (enzymolysis liquid amount of solid is 3%, w/v) 72h, obtain to send out
Ferment sugar, the wherein enzymatic hydrolyzation of cellulose are 92%, and the enzymatic hydrolyzation of xylan is 95%.
The lignocellulolyticenzymes and commercial fibres that 4 C.obsidiansis of embodiment is generated using Corncob Xylan as carbon source
Plain enzyme coordinated enzymatic hydrolysis steam explosion maize straw
1) the C.obsidiansis seed liquors cultivated at 85 DEG C for 24 hours are inoculated by 5% inoculum concentration with 0.1%
Corncob Xylan is carbon source culture medium, and culture medium other compositions are (every liter)：0.9g NH4Cl,0.9g NaCl,0.75g
KH2PO4,1.5 g K2HPO4,0.4g MgCl2·6H2O,2.5mg FeCl3·6H2O, 1ml trace element, 1mg resazurins, 2g
Tryptose, 1g yeast extracts, 0.75g cysteine hydrochloric acid H2O, anaerobic fermentation is for 24 hours at 80 DEG C；
2) 15min is centrifuged under the conditions of after fermentation at normal temperatures 6,000 × g, detaches cell and supernatant, cell is through super
After sound broken wall, centrifugation removal cell wall, supernatant is intracellular lignocellulolyticenzymes, and fermented supernatant fluid is 70- with saturation degree
80% ammonium sulfate protein precipitation, after centrifugation removes supernatant, with the Tris- hydrochloride buffer soluble proteins of pH7.2, albumen
In the Tris- hydrochloride buffer dialysed overnights of pH7.2 in solution sucking bag filter, polyethylene glycol is used after the completion of dialysis in bag filter
Outer Adsorption Concentration albumen, protein concentration reach 2mg/ml, and it is extracellular lignocellulose enzyme to collect protein solution；
3) intracellular and extracellular lignocellulose enzyme obtained with step 2) digests wooden with commercialized cellulase synergistic
Cellulosic material prepares fermentable sugars, and specific enzyme solution is as follows：1 is pressed with born of the same parents' endocellular enzyme and ectoenzyme:1 ratio mixing, takes
The mixed enzyme (5mg/g dry bottoms object) is blended in pH5.0 with Novi letter commercial fibres element enzyme preparation CTec2 (15FPU/g dry bottoms object)
50 DEG C of acetate buffer solution at digest the processed rice straw raw material of acid (enzymolysis liquid amount of solid is 2%, w/v) 60h after, obtain
The enzymatic hydrolyzation of fermentable sugars, wherein cellulose is 90%, and the enzymatic hydrolyzation of xylan is 92%.
5 C.kristjanssonii and C.kronotskyensis Mixed Microbes of embodiment are generated by carbon source of wheat stalk
Lignocellulolyticenzymes and commercial fibres element enzyme coordinated enzymatic hydrolysis wheat stalk
1) C.kristjanssonii the and C.kronotskyensis seed liquors cultivated at 70 DEG C for 24 hours are pressed respectively
2% inoculum concentration is inoculated into using 0.1% wheat stalk as carbon source culture medium, and culture medium other compositions are (every liter)：0.9g
NH4Cl,0.9g NaCl,0.75g KH2PO4,1.5g K2HPO4,0.4g MgCl2·6H2O,2.5mg FeCl3·6H2O,1ml
Trace element, 1mg resazurins, 2g tryptoses, 1g yeast extracts, 0.75g cysteine hydrochloric acid H2O, anaerobic fermentation at 80 DEG C
24h；
2) 15min is centrifuged under the conditions of after fermentation at normal temperatures 6,000 × g, detaches cell and supernatant, cell is through super
After sound broken wall, centrifugation removal cell wall, supernatant is intracellular lignocellulolyticenzymes, and fermented supernatant fluid is 70- with saturation degree
80% ammonium sulfate protein precipitation, after centrifugation removes supernatant, with the Tris- hydrochloride buffer soluble proteins of pH7.2, albumen
In the Tris- hydrochloride buffer dialysed overnights of pH7.2 in solution sucking bag filter, polyethylene glycol is used after the completion of dialysis in bag filter
Outer Adsorption Concentration albumen, protein concentration reach 2mg/ml, and it is extracellular lignocellulose enzyme to collect protein solution；
3) lignocellulosic is digested with the extracellular lignocellulose enzyme that step 2) obtains and commercialized cellulase synergistic
Raw material prepares fermentable sugars, and specific enzyme solution is as follows：Delayed with ectoenzyme (dosage is 15mg/g dry bottoms object) in the acetic acid of pH5.0
After digesting native wheat stalk (enzymolysis liquid amount of solid is 3%, w/v) 48h in fliud flushing at 75 DEG C, adding Xia Sheng industry group has
The commercial fibres element enzyme preparation (dosage is 25FPU/g dry bottoms object) of limit company production digests 72h at 55 DEG C, obtains to ferment
The enzymatic hydrolyzation of sugar, wherein cellulose is 86%, and the enzymatic hydrolyzation of xylan is 92%, and araban enzymolysis rate is 95%.
The lignocellulolyticenzymes and commercial fibres that 6 C.obsidiansis Mixed Microbes of embodiment are generated using corncob as carbon source
Plain enzyme coordinated enzymatic hydrolysis corncob
1) the C.obsidiansis seed liquors that 44h is cultivated at 80 DEG C are inoculated by 3% inoculum concentration with 0.1%
Crushing maize core is carbon source culture medium, and culture medium other compositions are (every liter)：0.9g NH4Cl,0.9g NaCl,0.75g
KH2PO4,1.5g K2HPO4,0.4g MgCl2·6H2O,2.5mg FeCl3·6H2O, 1ml trace element, 1mg resazurins, 2g pancreases
Albumen, 1g yeast extracts, 0.75g cysteine hydrochloric acid H2O, anaerobic fermentation 26h at 80 DEG C；
2) 15min is centrifuged under the conditions of after fermentation at normal temperatures 6,000 × g, detaches cell and supernatant, cell is through super
After sound broken wall, centrifugation removal cell wall, supernatant is intracellular lignocellulolyticenzymes, fermented supernatant fluid 70-80% ammonium sulfate
Solution protein precipitation, after centrifugation removes supernatant, with the Tris- hydrochloride buffer soluble proteins of pH7.2, protein solution sucking is saturating
It analyses and uses polyethylene glycol Adsorption Concentration outside bag filter in bag after the completion of the Tris- hydrochloride buffer dialysed overnights of pH7.2, dialysis
Albumen, protein concentration reach 2mg/ml, and it is extracellular lignocellulose enzyme to collect protein solution；
3) lignocellulosic is digested with the extracellular lignocellulose enzyme that step 2) obtains and commercialized cellulase synergistic
Raw material prepares fermentable sugars, and specific enzyme solution is as follows：Delayed with ectoenzyme (dosage is 10mg/g dry bottoms object) in the acetic acid of pH6.0
After digesting natural corn core (enzymolysis liquid amount of solid is 2%, w/v) 48h in fliud flushing at 75 DEG C, it is limited to add Xia Sheng industry group
The commercial fibres element enzyme preparation (dosage is 15FPU/g dry bottoms object) of company's production digests 72h at 50 DEG C, obtains fermentable sugars,
Wherein the enzymatic hydrolyzation of cellulose is 83%, and the enzymatic hydrolyzation of xylan is 89%, and araban enzymolysis rate is 90%.
Claims (4)
1. a kind of method that high temperature lignocellulolyticenzymes coordinated enzymatic hydrolysis prepares fermentable sugars, includes the following steps：
1) using the lignocellulosic material of 0.1-2% as carbon source, Thermophilic Bacterium fermentation 20-72h is utilized at 60-90 DEG C；
2) it centrifuging after fermentation, detaches cell and supernatant, the buffer solution of cellular pH 5.0-7.6 suspends, after ultrasonication,
Centrifugation removal cell wall, supernatant is intracellular lignocellulolyticenzymes, and fermented supernatant fluid is that 70-80% ammonium sulfate is molten with saturation degree
Liquid precipitate albumen, after centrifugation removes supernatant, with the buffer solution albumen of pH5.0-7.6, sucking bag filter is interior in pH5.0-
7.6 buffer solution dialysed overnight is reached after the completion of dialysis with polyethylene glycol Adsorption Concentration albumen outside bag filter, protein concentration
0.5-2mg/ml, it is ectoenzyme to collect protein solution；
3) intracellular and extracellular lignocellulose enzyme obtained with step 2) digests wood fibre with commercialized cellulase synergistic
Plain raw material prepares fermentable sugars, and specific enzyme solution is：With ectoenzyme individually or endocellular enzyme and ectoenzyme are according to 1:100‐100:1
Ratio be blended at 60-85 DEG C digest natural wooden fiber element raw material 24-72h after, add commercial fibres element enzyme preparation and exist
48-72h is digested at 50-55 DEG C, obtains fermentable sugars.
2. method as described in claim 1, the Thermophilic Bacterium is pyrolysis cellulose Pseudomonas
Caldicellulosiruptor strains.
3. method as described in claim 1, the commercial cellulose enzyme preparation can buy fibre on the market to be all
The plain enzyme preparation of dimension.
4. method as described in claim 1, the fermentable sugars is glucose, xylose, arabinose, mannose, sandlwood
One or more of sugar.
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