CN107227319A

CN107227319A - Method for producing tunning from lignocellulose-containing materials

Info

Publication number: CN107227319A
Application number: CN201710232855.0A
Authority: CN
Inventors: 任海彧; 李冬敏; 王云; 方传记; 黄鸿志
Original assignee: China Petroleum and Chemical Corp; Cofco Corp; Novo Nordisk AS
Current assignee: China Petroleum and Chemical Corp; Cofco Corp; Novo Nordisk AS
Priority date: 2009-12-21
Filing date: 2010-12-21
Publication date: 2017-10-03
Also published as: CN102869780A

Abstract

The present invention relates to the method for producing hydrolysate from lignocellulose-containing materials, it includes with Cold pretreatment, hydrolysis and fermented, wherein hydrolysis is by making the lignocellulose-containing materials contact what is carried out with the enzymatic compositions comprising at least 10% zytase zymoprotein w/w%.

Description

Method for producing tunning from lignocellulose-containing materials

The application is to be based on the applying date on December 21st, 2010, and priority date is on December 21st, 2009, Application No. 201080058461.4, the patent application of entitled " method for being used to produce tunning from lignocellulose-containing materials " Divisional application.

Technical field

Disclose for producing the method for tunning from lignocellulose-containing materials, and more specifically, pass through two The method for the enzymatic hydrolysis that stage pre-processes to strengthen lignocellulose-containing materials.

For referring to for sequence table

The application contains the sequence table of computer-reader form.The computer-reader form is incorporated herein by carrying stating.

Background technology

Lignocellulose-containing materials or biomass can be used for producing fermentable sugars, and it then can be used for producing fermentation production Thing such as recyclable fuel and chemicals.Lignocellulose-containing materials are wrapped around the cellulose in lignin and hemicellulose sheath The labyrinth of fiber.Producing tunning from lignocellulose-containing materials includes pretreatment, hydrolysis and ferments to contain lignin fiber Cellulosic material.

The structure of ligno-ccllulose is not that enzymatic hydrolysis can be immediately adjacent to.Therefore, ligno-ccllulose is located in advance Reason, to rupture the crystal structure of lignin sealing and destruction cellulose.This may cause the solubilized and sugar of hemicellulose fraction Change.Then can be by cellulose fraction enzymatic hydrolysis, such as by cellulolytic enzyme, the cellulolytic enzyme is by carbon aquation Compound depolymerization is into fermentable sugars.These fermentable sugars are described then by fermentative bioconversion into required tunning Product optionally can be reclaimed for example by distilling.

It is currently very expensive to produce tunning from lignocellulose-containing materials.Accordingly, it is desirable to provide for self-contained Lignocellulosic material produces the further technique of tunning.

Summary of the invention

The present invention relates to the method for producing hydrolysate from lignocellulose-containing materials, it is included in relatively low temperature Degree pretreatment lignocellulose-containing materials, are then hydrolyzed with the enzymatic compositions comprising zytase at high proportion.

Correspondingly, in one aspect, the present invention relates to the technique of the hydrolysate for producing lignocellulosic material, its Implement the pretreatment of the temperature between 165 DEG C -175 DEG C to the lignocellulosic material including (a), (b) is to preprocessed Lignocellulosic material implement hydrolase effect to produce hydrolysate, wherein the hydrolase include cellulolytic enzyme (cellulytic enzyme) and zytase, the zytase exist with least 10% amount of total amount hydrolase protein.

The digestibility that an advantage of the invention includes lignocellulose-containing materials is significantly improved, therefore reduction is used In the cost that tunning is produced from lignocellulose-containing materials.

Brief description

The corn that Fig. 1 displays are pre-processed using 12 kinds of different components of the hydrolase shown in table 2 in 2 different temperatures The results of hydrolysis of stalk.

Detailed description of the invention

Lignocellulose-containing materials

" ligno-ccllulose " or " lignocellulose-containing materials " means mainly to be made up of cellulose, hemicellulose and lignin Material.Such material is commonly referred to as " biomass ".

Biomass is wrapped around the labyrinth of the cellulose fibre in lignin and hemicellulose sheath.The structure of biomass So that it is not susceptible for enzymatic hydrolysis.In order to strengthen enzymatic hydrolysis, biomass must be pre-processed, wooden to rupture Element sealing, and dissolving hemicellulose, and the crystal structure of destruction cellulose.Cellulose can then carry out enzymatic hydrolysis, for example By cellulose decomposition ferment treatment, carbohydrate polymer is changed into fermentable sugars, it can be fermented into required fermentation Product, such as ethanol.Hemicellulose, which decomposes ferment treatment, can be used for any remaining half in the biomass of hydrolysis pretreatment Cellulose.

Biomass can be any material containing ligno-ccllulose.In a preferred embodiment, biomass contains At least about 30 weight %, preferably at least about 50 weight %, more preferably at least about 70 weight %, even more preferably at least about 90 weights Measure % ligno-cclluloses.

Biomass is general for example in the stem of plant, leaf, skin, shell (hull, husk) and cob or the leaf of tree, branch and timber It was found that.Biomass includes but is not limited to herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper and slurry And paper mill residue.It should be understood that biomass can be containing lignin, cellulose and hemicellulose in mixed-matrix The form of Plant cell wall material.Biomass can further contain constituent, such as proteinaceous material, starch and sugar Such as fermentable sugars or not fermentable sugars or their mixture.

The other examples of suitable biological matter include zein fiber, straw, pine, wood chip (wood chip), bagasse, paper With slurry processing waste, maize straw, corncob, hardwood such as poplar (poplar) and birch (birch), soft wood, Cereal straw (cereal straw) such as wheat straw, straw (rice straw), switchgrass, awns genus (Miscanthus), rice husk, Municipal solid waste (MSW), industrial organic waste, office paper or their mixture.

In a preferred embodiment, biomass is selected from maize straw, corncob, zein fiber, switchgrass, wheat Straw, straw (rice straw) and bagasse and combinations thereof.

Pretreatment

According to the present invention, biomass carries out chemistry, machinery, biology or its any combination of pretreatment before hydrolysis.This Outside, chemistry, machinery or biological processes can simultaneously be carried out with hydrolysis, such as addition with one or more cellulolytic enzymes Or other enzymatic activitys are simultaneously, to discharge such as fermentable sugars such as glucose or maltose.

The purpose of pretreatment is separation or release cellulose, hemicellulose and lignin, and therefore improve hydrolysis and/or The speed or efficiency of fermentation.Biomass can in preprocessing process with the dry solids wt % between about 10-80, preferably from about The dry solids wt % between dry solids wt %, especially about 30-60 between 20-70 is for example in the about weight % of dry solid 50 The amount of left and right is present.

According to the present invention, pre-process and carried out in relatively low temperature, preferably between 165 DEG C -175 DEG C, particularly from 165 DEG C, 166 DEG C, 167 DEG C, 168 DEG C, 169 DEG C, 170 DEG C, 171 DEG C, 172 DEG C, 173 DEG C or 174 DEG C until 166 DEG C, 167 DEG C, 168 DEG C, 169 DEG C, 170 DEG C, 171 DEG C, 172 DEG C, 173 DEG C, 174 DEG C or 175 DEG C.

In the standard biologic matter preprocessing process of typically about 180 ° of wherein temperature, there is the extensive of hemi-cellulose components Degraded.The xylan degrading discharged from lignocellulose-containing materials is xylose, and xylose can further be degraded to chemical combination Thing, it can be enzymatic hydrolysis and/or the inhibitor of fermentation.The catabolite of xylose includes but is not limited to furfural, methylol chaff Aldehyde (HMF), formaldehyde, formic acid, acetaldehyde, crotonaldehyde, lactic acid, dihydroxyacetone (DHA), glyceraldehyde, pyroracemic aldehyde, pyruvic alcohol and hydroxyl acetaldehyde.No It is bound to any specific theory, it is believed that at the pretreatment with the relatively low temperature of the present invention, the knot of lignocellulose-containing materials Structure is opened, but xylan only Partial digestion.Because less xylan is degraded, produce inhibitor lesser degree, And washing step can be omitted.

Chemical Pretreatment

Term " Chemical Pretreatment " refers to the separation for promoting cellulose, hemicellulose or lignin or any chemistry of release is pre- Processing.The example of suitable process for chemically pretreating includes using such as diluted acid, lime, organic solvent, sulfur dioxide or titanium dioxide Carbon processing.Further, wet oxidation and the aquathermolysis of pH controls are also the Chemical Pretreatment of consideration.

In a preferred embodiment, Chemical Pretreatment is acid treatment, at more preferably continuous diluted acid and/or weak acid Reason, such as with sulfuric acid or another organic acid and/or mineral acid treatment, acid such as acetic acid, citric acid, tartaric acid, the amber Acid, hydrogen chloride or its mixture.Other acid can also be used.Weak acid treatment means that handling pH is in about pH 1-5 scope, Preferably from about pH 1-3.

In a preferred embodiment, pretreatment is sour, excellent with 0.1-2.5 weight % acid, preferably 0.5-2.0 weight % Select the low-kappa number of 0.8-1.5 weight % acid.For second acid pre-processed can be hydrochloric acid, phosphoric acid, sulfuric acid, sulfurous acid, Carbonic acid, formic acid, acetic acid, citric acid, tartaric acid, glucuronic acid, galacturonic acid, butanedioic acid and/or its mixture；Especially sulphur Acid.Acid can be several minutes of periods to the several seconds with biomass and mixture contact range.One in the present invention is preferable to carry out In scheme, pretreatment carries out the period of -60 minutes 1 minute.

According to invention also contemplates that other chemical pretreatment techniques.Also show when ligno-ccllulose is destructurized When, enzymatic hydrolysis can be greatly enhanced.Ozone, organic solvent (organosolv) (use (Al) in aqueous alcohol₂SO₄、FeCl₃, lewis acid), glycerine, dioxanes, phenol or ethylene glycol belongs to known destruction cellulosic structure and promotes water Solvent (Mosier et al., 2005, Bioresource Technology 96 of solution：673-686).

The other examples of appropriate pretreatment method are by described below：Schell et al., 2003, Appl.Biochem and Biotechn. the 105-108 volumes, the 69-85 pages, and Mosier et al., 2005, Bioresource Technology 96： 673-686, and U.S.Application Publication No 2002/0164730, each piece bibliography is incorporated to by carrying stating herein.

Mechanical pretreatment

Term " mechanical pretreatment ", which refers to, promotes that any of cellulose, hemicellulose or lignin is separated or discharged from biomass Mechanically or physically pre-process.For example, mechanical pretreatment includes grinding, irradiation, decatize/vapour explosion and the hydro-thermal of multiple types Solve (hydrothermolysis).

Mechanical pretreatment includes crushing, i.e. the machinery reduction of size.Crushing includes dry grinding, wet-milling and vibratory milling.Machinery Pretreatment can be related to high pressure and/or elevated temperature (vapour explosion)." high pressure " mean in about 300-600psi scopes, excellent Select 400-500psi, e.g., from about 450psi pressure.Elevated temperature mean from about 165 DEG C until in 175 DEG C of scopes, it is excellent The temperature of about 170 DEG C of choosing.In a preferred embodiment, pretreatment of the invention is quick-fried as the steam of the temperature at 170 DEG C It is fried to carry out.In a preferred embodiment, mechanical pretreatment is the batch process that instrument system is hydrolyzed with steam gun, and it is using such as The pressure and temperature of upper definition.Sunds Hydrolyzer (can be obtained from Sunds Defibrator AB (Sweden)) can be used In this.

Biological pretreatment

Term " biological pretreatment " refers to any promote from biomass separation or release cellulose, hemicellulose or lignin Biological pretreatment.Biological pretreatment techniques can be related to using lignin dissolution microorganism.See, for example, Hsu, T.- A., 1996, Pre-treatment of biomass, in Handbook on Bioethanol：Production and Utilization, Wyman, C.E., editor, Taylor＆Francis, Washington, DC, 179-212；Ghosh, P. and Singh, A., 1993, Physicochemical and biological treatments for enzymatic/ Microbial conversion of lignocellulosic biomass, Adv.Appl.Microbiol.39：295- 333；McMillan, J.D., 1994, Pre-treating lignocellulosic biomass：A review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M.E., Baker, J.O. and Overend, R.P., editor, ACS Symposium Series 566, American Chemical Society, Washington, DC, the 15th chapter；Gong, C.S., Cao, N.J., Du, J. and Tsao, G.T., 1999, Ethanol Production from renewable resources, in Advances in Biochemical Engineering/ Biotechnology, Scheper, T., editor, Springer-Verlag Berlin Heidelberg, Germany, 65： 207-241；Olsson, L. and Hahn-Hagerdal, B., 1996, Fermentation of lignocellulosic Hydrolyzates for ethanol production, Enz.Microb.Tech.18：312-331；And Vallander, L. and Eriksson, K.-E.L., 1990, Production of ethanol from lignocellulosic materials：State of the art, Adv.Biochem.Eng./Biotechnol.42：63-95.

Hydrolysis

In hydrolytic process, by pretreated biomass (preferably in the form of biomass slurry) enzymatic hydrolysis and degraded Into fermentable sugars or other useful compounds.As used herein, term " biomass slurry " refers to the water of experience enzymatic hydrolysis Property biological material.Biomass slurry by mixing biomass, such as maize straw, bagasse with water, buffer solution and other Material previously treated is produced.Biomass slurry can be pre-processed before hydrolysis, or the slurry can be by pretreated life Material is formed.

Dry solid content can be in about 5-50 weight %, preferably from about 10-40 weight %, preferably from about 20- in hydrolytic process In 30 weight % scope.In a preferred embodiment, hydrolysis can be carried out as batch feeding technique, wherein will be through pre- Progressively such as solution containing enzyme hydrolysis is given in feed supplement to the biomass (i.e. substrate) of processing.

According to the present invention, hydrolysis and/or fermentation are carried out using cellulolytic enzyme and zytase.

In a preferred embodiment, hydrolysis uses many comprising the one or more with cellulolytic enhancing activity The cellulolytic enzyme prepared product of peptide is carried out.In a preferred embodiment, one kind with cellulolytic enhancing activity Or multiple polypeptides are family GH61A sources.It is suitable to increase with preferred cellulolytic enzyme prepared product and with cellulose decomposition The example of strongly active polypeptide is described further below.

Because biomass may containing constituent in addition to lignin, cellulose and hemicellulose, hydrolysis and/or Fermentation can be carried out in the presence of other enzymatic activity, and the other enzymatic activity is selected from protease, amylase, esterase, fat Fat enzyme, cellulase, hemicellulase, amylase, protease, esterase, endoglucanase, β-glucosyl enzym, cellobiose Hydrolase, cellobiase, zytase, xylose isomerase, alpha-amylase, alpha-Glucosidase, glucoamylase and its mixing Thing.

Enzymatic hydrolysis is preferably in suitable aqueous environment under conditions of it can be readily determined by those skilled in the art It is middle to carry out.In a preferred embodiment, hydrolysis is in the suitable, preferably optimal of one or more enzymes for being discussed Under the conditions of carry out.

Preferably, hydrolyze between 25 DEG C -70 DEG C, preferably between 40 DEG C -60 DEG C, especially about 50 DEG C of temperature is entered OK.PH, the preferably pH 4-6, especially about pH 5 preferably in pH 3-8 scopes are hydrolyzed to carry out.Carried out in addition, hydrolysis is general Between 12-96 hours, preferably 16-72 hours, between more preferably 24-48 hours.

Suitable process time, temperature and pH conditions can be readily determined by those skilled in the art.

Fermentation

Fermentation needed for by can directly or indirectly be fermented into sugared such as glucose, xylose, mannose and galactolipin is produced One or more fermenting organisms of thing, can ferment the fermentable sugars from pretreated and/or hydrolysis biomass.Fermentation Condition depends on required tunning and fermenting organism, and can be readily determined by those of ordinary skill in the art.

Especially in the case of alcohol fermentation, between fermentation can be carried out 1-72 hours.In one embodiment, send out Ferment is between about 20 DEG C -40 DEG C, preferably 25 DEG C -40 DEG C, more preferably from about 30 DEG C-about 38 DEG C, particularly from about 32 DEG C of temperature progress. In one embodiment, pH is more than 5.In another embodiment, pH is about pH 3-7, preferably from about pH 4-6, especially It is between 4-5.

Fermentation can in batches, batch feeding or flow reactor carry out.Fed batch fermentation can be fixed volume or Variable-volume batch feeding.In one embodiment, using fed batch fermentation.The volume and speed of fed batch fermentation take Certainly in the characteristic and concentration and required tunning of such as fermenting organism, fermentable sugars.The speed and volume of such fermentation can be with It is readily determined by those of ordinary skill in the art.

Fermenting organism

Term " fermenting organism " refers to any biology for being suitable for producing required tunning, including bacterium and fungal organism. The fermenting organism being particularly suitable can directly or indirectly ferment sugared such as glucose, that is, change into required tunning.Fermentation Biological example includes fungal organism, especially yeast.It is preferred that yeast include Saccharomyces sp (Saccharomyces Spp.) bacterial strain, particularly saccharomyces cerevisiae or saccharomyces uvarum (Saccharomyces uvarum) bacterial strain；Pichia (Pichia) bacterial strain, preferably pichia stipitis (Pichia stipitis), such as pichia stipitis CBS 5773；False silk Saccharomyces (Candida) bacterial strain, particularly candida utili (Candida utilis), Di Dansi Candidas (Candida ) or Candida boidinii (Candida boidinii) bacterial strain diddensii.Other fermenting organisms include zymomonas (Zymomonas)；Hansenula (Hansenula), particularly Hansenula anomala (H.anomala)；Kluyveromyces (Klyveromyces), particularly Kluyveromyces fragilis (K.fragilis)；And Schizosaccharomyces (Schizosaccharomyces), particularly schizosaccharomyces pombe (S.pombe) bacterial strain.

The yeast being obtained commercially includes, for example, ETHANOL RED^TMYeast (can be obtained from Fermentis/Lesaffre, USA ), FALI^TM(can be obtained from Fleischmann ' s Yeast, USA), SUPERSTART and THERMOSACC^TMFresh yeast (can be obtained from Ethanol Technology, WI, USA), BIOFERM AFT and XR (can be from NABC-North American Bioproducts Corporation, GA, USA acquisition), GERT STRAND (can be obtained) from Gert Strand AB, Sweden, (it can be obtained with FERMIOL from DSM Specialties).

, according to the invention it is preferred to fermenting organism can be by the sugared such as xylose fermenting organisms for needed for of C5.

Tunning

The present invention can be used for producing any tunning.It is preferred that tunning include alcohol (such as ethanol, methanol, fourth Alcohol)；Organic acid (such as citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid)；Ketone (such as acetone)；Amino acid (such as paddy ammonia Acid)；Gas (such as H₂And CO₂)；Antibiotic (such as penicillin and tetracycline)；Enzyme；Vitamin (such as riboflavin, B12, β-recklessly Radish element)；And hormone.

Other products include consumable alcohol industrial products, such as beer and grape wine；Dairy industry product, for example, ferment Dairy products；Leather industry product and tobacco industry products.In a preferred embodiment, tunning is alcohol, especially second Alcohol.Fuel alcohol/ethanol can be preferably used as according to the tunning such as ethanol that the present invention is obtained.However, in the feelings of ethanol Under condition, it is also used as drinking alcohol.

SSF, HHF and SHF

According to the present invention, hydrolysis and fermentation (SHHF techniques) or continuous (SHF techniques) can be carried out simultaneously.

Hydrolysis and fermentation can be carried out as hydrolysis simultaneously and the effect of fermentation step or synchronous glycosylation and fermentation (SSF).One As for, this mean combination/while hydrolysis and ferment it is suitable, preferably most in one or more fermenting organisms for being discussed Carried out under good condition (such as temperature and/or pH).

Hydrolysing step and fermentation step can be carried out as mixing (hybrid) hydrolysis and fermentation (HHF).HHF it is general with point The partial hydrolysis step opened starts and to terminate while hydrolyzing with fermentation step.Separated partial hydrolysis step be that typically in for The enzymatic fiber that one or more hydrolases for being discussed are suitable, preferably carry out under optimal conditions (such as in higher temperature) Plain saccharification step.Follow-up hydrolysis simultaneously and fermentation step are (logical generally under the conditions of suitable for one or more fermenting organisms Often than separated hydrolysing step lower temperature) carry out.

Hydrolysis and fermentation step are also used as separated hydrolysis and fermentation progress, wherein making hydrolysis complete before fermentation starting Into.This is commonly referred to as " SHF ".

Reclaim

After fermentation, tunning can be separated optionally with fermentation medium in any way as suitable.For example, culture medium can To be distilled to extract tunning, or tunning can be carried by micro-filtration or membrane filtration technique from fermentation medium Take.Alternately, tunning can be reclaimed by stripping (stripping).Recovery method be it is well known in the art that 's.

Composition

The present invention relates to the composition comprising cellulolytic enzyme and the hydrolase of zytase, wherein the zytase Exist with least 10% amount that total amount hydrolyzes zymoprotein.Cellulolytic enzyme can include and be derived from trichoderma reesei The cellulase system of (Trichoderma reesei).

Zytase is preferably GH10 zytases.Zytase can with least the 10% of hydrolase protein total amount, extremely Few 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20% or even at least 25% amount is present.

Preferably, hydrolase include with least the 1% of hydrolase protein total amount, at least 2%, at least 3%, at least 4% or Even at least 5%, for example, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20% or The β-glucosyl enzym of even at least 25% amount.

Composition can further include the glycoside hydrolase of family 61, and preferably with least the 1% of hydrolase protein total amount, At least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%th, at least 20% or even at least 25% amount is present.

Composition can be prepared according to methods known in the art, and can be with the shape of liquid or dry composition Formula.For example, peptide composition can be in the form of particle or particulate.Polypeptide in the composition to be included can be according to this area Known method is stablized.

The example of the preferable use of the composition of the present invention is described below.The present invention peptide composition dosage and at it The other conditions of lower use composition can be measured based on methods known in the art.

Purposes

The invention further relates to the method for degrading or converting cellulosic material, including：Effective dose is included with the present invention The compositions-treated cellulosic material of polypeptide with endoglucanase activity.In a preferred aspect, this method is further Cellulosic material including reclaiming degraded or conversion.

The polypeptide and host cell of the present invention can be used as biological from cellulose in the generation of monose, disaccharides and polysaccharide The chemistry or fermentation raw material of matter are used to produce ethanol, plastics or other products or intermediate product.Comprising with endoglucanase The enzyme that the composition of the polypeptide of activity can be purified or purified in the form of removing or not remove the thick hair zymotic fluid of cell or with half The form of prepared product.Alternately, composition can be comprising host cell of the invention as in the zymotechnique with biomass In have endoglucanase activity polypeptide originate.Host cell can also contain in the processing of biomass it is useful above What is referred to encodes the natural or heterologous gene of other protein and enzyme.Especially, polypeptide of the invention and host cell can lead to Cross the partly or completely degradable of cellulose or hemicellulose be used to increasing residual processing thing (distiller's dried grain, from the vinasse, sweet brewageed Sugarcane bagasse etc.) value.

Enzyme

Be not specifically mentioned in the method for the present invention or the background of technique, it is to be understood that one or more enzyme and Other compounds are used with effective dose.One or more enzymes can be used.

Cellulolytic enzyme

As used herein, term " cellulolytic enzyme " or term " cellulolytic enzyme prepared product " or term " have The enzyme of cellulolytic activity ", which is interpreted as including, has cellobiohydrolase activity (EC3.2.1.91), such as fiber two Glycosylhydrolase I and cellobiohydrolase II, and endoglucanase activity (EC 3.2.1.4) and beta-glucosidase activity The enzyme of (EC 3.2.1.21).In a preferred embodiment, the enzyme preparation that cellulolytic enzyme can be comprising originated from fungus Thing, such as trichoderma (Trichoderma) bacterial strain, preferably Li's Trichoderma strains；Humicola (Humicola) bacterial strain, such as it is special Different humicola lanuginosa (Humicola insolens) bacterial strain；Or golden spore category (Chrysosporium) bacterial strain, preferably Chrysosporium lucknowense bacterial strains.

Cellulolytic enzyme prepared product can be included can be from Novozymes A/S, Denmark or ACCELERASE^TM1000 (come From Genencor Inc., USA) obtain the product being obtained commercially1.5L or CELLUZYME^TM。

Cellulolytic enzyme can be with 0.1-100FPU/ grams of total solid (TS), preferably 0.5-50FPU/ grams TS, especially 1- 20FPU/ grams of TS scope input.In another embodiment, will at least 0.1mg cellulolytic enzymes/gram total solid (TS), Preferably at least 3mg cellulolytic enzymes/gram TS, such as 5-10mg one or more cellulolytic enzyme/gram TS is used to hydrolyze.

Endoglucanase (EG)

One or more endoglucanases may reside in hydrolytic process.In term " endoglucanase " means Cut -1,4- (1,3；Isosorbide-5-Nitrae)-callose 4- glucan hydrolases (EC numbering 3.2.1.4), its catalytic cellulose, cellulose 1,4- β-D- glycosidic bonds in derivative (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, mixing β- β -1,4 in 1,3 glucans such as cereal beta-D-glucans or xyloglucan and the other plant material containing fibrous component The interior hydrolysis of key.According to Ghose, 1987, Pure and Appl.Chem.59：257-268 program, endo-glucanase enzyme activity Property can use carboxymethyl cellulose (CMC) hydrolysis be measured.

Endoglucanase can be derived from Trichoderma strain, preferably Li's Trichoderma strains；Humicola strain, such as it is special Different detritus trichoderma strain；Or golden spore belongs to bacterial strain, preferably Chrysosporium lucknowense bacterial strains.

β-glucosyl enzym

One or more β-glucosyl enzyms may reside in hydrolytic process.Term " β-glucosyl enzym " means β-D- glucose Glycosides glucohydralase (EC 3.2.1.21), it is catalyzed the hydrolysis of the non-reduced β in end-D-Glucose residue, with β-D-Glucose Release.For the purposes of the present invention, beta-glucosidase activity is according to by Venturi et al., 2002, J.Basic Microbiol.42：The base program of 55-66 descriptions is measured, in addition to different condition is used as described herein.β-glucose 1 unit definition of glycosides enzymatic activity be by 100mM sodium citrates,4mM pairs of substrate is used as in 20 Nitrobenzophenone-β-D- glucopyranosides are in 50 DEG C, 1.0 micromole's p-nitrophenols of the generations per minute of pH 5

β-glucosyl enzym can be originated from fungus, for example trichoderma, aspergillus (Aspergillus) or Penicillium (Penicillium) bacterial strain.β-glucosyl enzym can be derived from trichoderma reesei, such as by the β-glucosyl enzym of bgl1 gene codes (referring to EP 562003 Fig. 1).β-glucosyl enzym can be derived from aspergillus oryzae (Aspergillus oryzae) (according to WO 2002/095014 in aspergillus oryzae restructuring produce), aspergillus fumigatus (Aspergillus fumigatus) is (according to WO 2002/ 095014 embodiment 22 recombinates what is produced in aspergillus oryzae) or aspergillus niger (Aspergillus niger) (1981, J.Appl. Volume 3, the 157-163 pages).

β-glucosyl enzym can be derived from Brazilian mould (Penicillium brasilianum), such as in WO 2009/ SEQ ID NO are shown as in 111706:2 β-glucosyl enzym.

Cellobiohydrolase (CBH)

One or more cellobiohydrolases may reside in hydrolytic process.Term " cellobiohydrolase " means Isosorbide-5-Nitrae-callose cellobiohydrolase (E.C.3.2.1.91), its catalytic cellulose, cell-oligosaccharide or any containing β -1, The hydrolysis of Isosorbide-5-Nitrae-β-D- glucoside bonds in the polymer of the glucose of 4- connections, reduction or non-reducing end release fiber from chain Disaccharides.

The example of cellobiohydrolase is being mentioned above, including from trichoderma reesei；The CBH I of Humicola insolens and The CBH II and CBH II from autochthonal shuttle spore shell (Thielavia terrestris) cellobiohydrolase (CELL6A).

Cellobiohydrolase activity can be according to by Lever et al., 1972, Anal.Biochem.47：273-279 and Tilbeurgh et al., 1982, FEBS Letters 149：152-156；Van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187：The program of 283-288 descriptions is measured.Lever et al. method is suitable for assessing maize straw The hydrolysis of middle cellulose, and van Tilbeurgh et al. method is suitable for determining the fiber two to the sugar derivatives of fluorescence two Glycosylhydrolase activity.

The glycoside hydrolase of family 61

The glycoside hydrolase of family 61：According to Henrissat B., 1991, A classification of glycosyl Hydrolases based on amino-acid sequence similarities, Biochem.J.280：309-316, with And Henrissat B. and Bairoch A., 1996, Updating the sequence-based classification of Glycosyl hydrolases, Biochem.J.316：695-696 and www.cazy.org, the term " glycoside hydrolysis of family 61 Enzyme " or " family GH61 " is defined herein as belonging to the polypeptide of glycoside hydrolase Families 61.At present, Henrissat is by GH61 Family is classified as non-classified, it is indicated that for the property that the polypeptide that belongs to this family is unknown, and such as mechanism, catalysis nucleophilic group/ Base, Catalytic Proton Donor and 3-D structures.In the background of the invention, the glycoside hydrolase of family 61 has " cellulose decomposition increasing It is strongly active ".

Term " cellulolytic enhancing activity " is defined herein as reinforcing fiber cellulosic material and passed through with cellulose point Solve the biological activity of the hydrolysis of the protein of activity.For the purposes of the present invention, fibre is come from by measuring under the following conditions The increase of the increase or cellobiose and glucose of the reduced sugar of the dimension cellulosic material hydrolysis that passes through cellulase protein altogether is surveyed Determine cellulolytic enhancing activity：1-50mg gross proteins/g celluloses in PCS, wherein gross protein are included in PCS 80-99.5%w/w cellulase proteins/g celluloses and 0.5-20%w/w there is the protein of cellulolytic enhancing activity Carry out 1-7 days, compare with the control hydrolysis with the equal gross protein carrying capacity without cellulolytic enhancing activity at 50 DEG C (1-50mg cellulolytic proteins/g celluloses in PCS).In a preferred aspect, it will be carried in cellulase protein matter The 3% gross protein weight aspergillus oryzae β-glucosyl enzym (according to WO 2002/095014 in aspergillus oryzae recombinate generation) of amount or 3% gross protein weight aspergillus fumigatus β-glucosyl enzym (recombinates production according to WO 2002/095014 embodiment 22 in aspergillus oryzae Raw) in the presence of1.5L (Novozymes A/S, Bagsvaerd, Denmark) mixture is used as The source of cellulolytic activity.

Hemicellulase

Hemicellulose can be decomposed by hemicellulase and/or sour water solution, with discharge its five and hexose component. According to the present invention, the lignocellulosic material of pretreatment includes at least one zytase with one or more hemicellulases (EC 3.2.1.8) is handled.

Zytase for using in the present invention is inscribe-Isosorbide-5-Nitrae-beta-xylanase, and can be glycoside hydrolysis Enzyme family 10 or 11 (GH10 or GH11).Nucl.Acids Res.2009s of the GH10 or GH11 in Cantarel et al. (2008) 37：Defined in D233-D238 and on www.cazy.org.

Zytase can be any source, including mammal, plant or animal origin；It is preferable, however, that xylan Enzyme is microbe-derived.Especially, zytase can be derived from that of filamentous fungi or yeast.Preferably, it is wooden Dextranase is derived from filamentous fungi such as aspergillus bacterium (Aspergillus sp.), Bacillus sp (Bacillus Sp.), Humicola strain (Humicola sp.), myceliophthora strain (Myceliophotora sp.), Poitrasia Sp., Rhizomucor strain (Rhizomucor sp.) or trichoderma.

Preferably zytase GH10 zytases.Suitable GH10 zytases can be derived from microorganism Aspergillus aculeatus (Aspergillus aculeatus), for example, be disclosed in WO 1994/021785 SEQ ID NO as xylanase I I:In 2 Enzyme.Further preferably with WO 1994/021785 SEQ ID NO:In 2 and/or herein as SEQ ID NO:1 shows The amino acid sequence shown has at least 50% homogeneity, at least 60% homogeneity, at least 70% homogeneity, at least 80% same Property, at least 90% homogeneity, at least 95% homogeneity or the even at least zytase of 99% homogeneity.

The example of commercial xylanase includes coming from Novozymes A/S, the SHEARZYME of Denmark^TMAnd BIOFEED WHEAT^TM。

According to the present invention, zytase is with least the 6% of hydrolase protein total amount, at least 7%, at least 8%, at least 9%, At least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%th, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24% or even at least 25% amount Add and/or be present in the hydrolysing step of the present invention.

Zytase with the amount of 0.001-1.0g/kg DS substrates, preferably with the amount of 0.005-0.5g/kg DS substrates and Most preferably add and/or be present in the hydrolysing step of the present invention with the amount of 0.05-0.10g/kg DS substrates.

Hemicellulase must be added with the amount of effective hydrolyzed hemicellulose, such as always solid with about 0.001-0.5 weight % Body (TS), more preferably about 0.05-0.5 weight %TS amount.

Zytase can be with the amount of 0.001-1.0g/kg DM (dry) substrate, preferably with 0.005-0.5g/kg DM The amount of substrate and most preferably 0.05-0.10g/kg DM substrates are added.

Other preferred hemicellulases include arabinofuranosidase, acetyl xylan esterase, feruloyl esterase, Portugal Uronic acid glycosidase, inscribe Galactanase, mannonase inscribe or circumscribed arabinase, exogalactanase and The mixture of two or more in them.Preferably, the hemicellulase for using in the present invention is circumscribed effect Hemicellulase, and it is highly preferred that hemicellulase is the hemicellulase of such circumscribed effect, it has less than pH 7th, the preferred ability of pH 3-7 hydrolyzed under acidic conditions hemicellulose.The hemicellulase for being suitable for using in the present invention Example includes VISCOZYME^TM(can be obtained from Novozymes A/S, Denmark).

Xylose isomerase

Xylose isomerase (D- xylose ketoisomerases) (E.C.5.3.1.5.) is that to be catalyzed D- xyloses reversible to D- xyluloses The enzyme of isomerization reaction.

Xylose isomerase can be used in this method or technique, and can be with any of xylose isomerase activity Enzyme, and any source, preferred bacterium or originated from fungus, such as filamentous fungi or yeast can be derived from.Bacterial xylose isomery The example of enzyme includes belonging to streptomyces (Streptomyces), actinoplanes (Actinoplanes), bacillus With Flavobacterium (Flavobacterium) and species thermotoga (Thermotoga) that, including new Apollo dwells thermobacillus (T.neapolitana) (Vieille et al., 1995, Appl.Environ.Microbiol.61 (5), 1867-1875) and sea Dwell thermobacillus (T.maritime).The example of fungi xylose isomerase is the derivative bacterium of Basidiomycetes (Basidiomycetes) Kind.

It is preferred that xylose isomerase is derived from saccharomyces candida (Candida) bacterial strain, preferably Candida boidinii (Candida boidinii) bacterial strain, especially by such as Vongsuvanlert et al., 1988, Agric.Biol.Chem., 52 (7)：Candida boidinii xylose isomerase disclosed in 1817-1824.It is false that xylose isomerase can be preferably derived from Bo Yiding Silk yeast strain (Kloeckera 2201), as DSM 70034 and the preservations of ATCC 48180, is disclosed in Ogata et al., Agric.Biol.Chem, volume 33,1519-1520 pages or Vongsuvanlert et al., 1988, Agric.Biol.Chem, 52 (2), in the 1519-1520 pages.

In one embodiment, xylose isomerase is derived from streptomyces bacterial strain, such as derived from the grey streptomycete of mouse (Streptomyces murinus) (U.S. Patent number 4,687,742)；Streptomyces flavovirens (S.flavovirens), white Streptomycete (S.albus), not streptomyces chromogenes (S.achromogenus), S.echinatus, Vad mole streptomycete (S.wedmorensis) bacterial strain, is all disclosed in U.S. Patent number 3,616,221.It is special that other xylose isomerases are disclosed in the U.S. Profit number 3,622,463, U.S. Patent number 4,351,903, U.S. Patent number 4,137,126, U.S. Patent number 3,625,828, HU In the patent No. 12,415, DE patents 2,417,642, the JP patent No.s 9,28,473 and WO 2004/044129, it is each via carrying State and be incorporated herein.Xylose isomerase can with immobilization or in the form of liquid.Liquid form is preferred.The wood being obtained commercially The example of sugared isomerase includes coming from Novozymes A/S, the SWEETZYME of Denmark^TMT.Xylose isomerase is to provide in 0.01- The amount of activity level is added in 100IGIU/ grams of total solid scope.

Alpha-amylase

One or more alpha-amylases can be used.It is preferred that alpha-amylase be microorganism such as bacterium or originated from fungus 's.Most suitable alpha-amylase is measured based on process conditions, but can be easily accomplished by those skilled in the art.

It is preferred that alpha-amylase can be acid alpha-amylase, such as Fungal acid alpha-amylase or bacterial acid alphalise starch Enzyme.Term " acid alpha-amylase " means that the alpha-amylase (E.C.3.2.1.1) that is added with effective dose has 3-7, preferably Optimal activity under 3.5-6 or pH more preferably in 4-5 scopes.

According to the present invention, acid alpha-amylase can with 0.1-10AFAU/g DS, preferably 0.10-5AFAU/g DS, especially It is 0.3-2AFAU/g DS amount addition.

Preferred commercial composition comprising alpha-amylase includes MYCOLASE (coming from DSM), BAN^TM、TERMAMYL^TMSC、 FUNGAMYL^TM、LIQUOZYME^TMX and SAN^TMSUPER、SAN^TMEXTRA L (Novozymes A/S) and CLARASE^TML-40, 000、DEX-LO^TM、SPEZYME^TMFRED、SPEZYME^TMAA and SPEZYME^TMDELTA AA (Genencor Int.), and with business The Acid Fungal Alpha-amylase of name of an article SP288 (can be obtained from Novozymes A/S, Denmark) sale.

Protease

Protease can be added in hydrolysis, fermentation or hydrolysis simultaneously and fermentation process.Protease can be in fermentation process It is middle to add, with deflocculated fermenting organism especially yeast.Protease can be any protease.In a preferred embodiment In, protease is microbe-derived, the acid protease of preferred fungi or bacterial origin.Acid fungal protease is preferred , it is also possible to use other protease.

Suitable protease includes microbial protease, such as fungi and bacterialprotease.It is preferred that protease be acid Protease, i.e. be characterised by the protease in the ability of the hydrolyzed under acidic conditions protein less than pH 7.

The acid fungal protease of consideration includes being derived from Eurotium, mucor (Mucor), Rhizopus (Rhizopus), candida, Coriolus Qu61 (Coriolus), inner seat shell category (Endothia), entomophthora category (Enthomophtra), rake teeth Pseudomonas (Irpex), Penicillium, sclerotium (Sclerotium) and Torulopsis (Torulopsis) fungal proteinase.Especially consider be derived from aspergillus niger (see, for example, Koaze et al., 1964, Agr.Biol.Chem.Japan, 28,216), Zuo Shi aspergillus (Aspergillus saitoi) (see, for example, Yoshida, 1954, J.Agr.Chem.Soc.Japan, 28,66), aspergillus awamori (Aspergillus awamori) (Hayashida et al., 1977, Agric.Biol.Chem., 42 (5), 927-933, microorganism Aspergillus aculeatus (WO 1995/02044) or aspergillus oryzae protease, Such as pepA protease；With the acid egg from Mucor pusillus (Mucor pusillus) or conspicuous Mucor of rice (Mucor miehei) White enzyme.

It is also contemplated that neutral or alkali protease, such as protease derived from Bacillus strain.For example, it is contemplated that Protease for the present invention is derived from bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and hasSwissprot is used as registration number P06832Obtainable sequence.It is also contemplated that withSwissprot is used as registration number P06832 Obtainable amino acid sequence have at least 90% homogeneity protease, for example, at least 92%, at least 95%, at least 96%, At least 97%, at least 98% or especially at least 99% homogeneity.

It is further contemplated that with being used as SEQ ID NO in WO 2003/048353:Golden yellow thermophilic ascus disclosed in 1 Bacterium (Thermoascus aurantiacus) protease have at least 90% homogeneity, for example, at least 92%, at least 95%, extremely Few 96%, at least 97%, at least 98% or especially at least 99% homogeneity.

It is also contemplated that papain-like proteases, such as egg in E.C.3.4.22.* (cysteine proteinase) White enzyme, such as EC 3.4.22.2 (papain), EC 3.4.22.6 (chymopapain), EC3.4.22.7 (trailing plants Japonica Protease), EC 3.4.22.14 (Actinidin), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 it is (sweet Aminoacyl endopeptidase) and EC 3.4.22.30 (caricain).

In one embodiment, protease can be prepared by the protease derived from Eurotium bacterial strain such as aspergillus oryzae Thing.In another embodiment, protease can be derived from Rhizomucor bacterial strain, preferably Man Hegen Mucors (Rhizomucor meihei).In another related embodiment, protease can be protease preparation, be preferably derived from aspergillus bacterial strain The mixing of the proteolysis prepared product of such as aspergillus oryzae and protease derived from the preferred Man Hegen Mucors of Rhizomucor bacterial strain Thing.

Aspartic proteases are such as in Hand-book of Proteolytic Enzymes, by A.J.Barrett, N.D.Rawlings and J.F.Woessner are edited, Aca-demic Press, San Diego, the 1998, the 270th chapter) described in. The suitable example of aspartic protease is included e.g., as disclosed in R.M.Berka et al., Gene, 96,313 (1990))； (R.M.Berka et al., Gene, 125,195-198 (1993))；With Gomi et al., Biosci.Biotech.Biochem.57, Those in 1095-1100 (1993), the bibliography is incorporated herein by carrying stating.

The product being obtained commercially includesESPERASE^TM、FLAVOURZYME^TM、PROMIX^TM、NOVOZYM^TMFM 2.0L and NOVOZYM^TM50006 (can be from Novozymes A/S, Denmark obtain) and GC106 from Genencor Int., Inc., USA^TMAnd SPEZYME^TMFAN。

Protease can be with 0.0001-1mg zymoproteins/g DS, preferably 0.001-0.1mg zymoproteins/g DS amount In the presence of.Alternately, protease can with 0.0001-1LAPU/g DS, preferably 0.001-0.1LAPU/g DS and/or 0.0001-1mAU-RH/g DS, preferably 0.001-0.1mAU-RH/g DS amount is present.

Lipolytic enzyme

Lipolytic enzyme is the enzyme for being capable of hydrolysing carboxylic's ester bond, to discharge carboxylic acid or carboxylate (EC 3.1.1).The fat Fat catabolic enzyme mainly includes following 3 hypotypes：Galactolipase (EC 3.1.1.26), phosphatidase (A1 or A2, EC 3.1.1.32 Or 3.1.1.4) and triacylglycerol lipase (EC 3.1.1.3), dominant ground has for galactolipid, phosphatide and sweet respectively The activity of oily three esters.Activity can be measured by any appropriate method, for example, pass through known in the art or this specification In the measure that then describes.

Lipolytic enzyme can have narrow specificity, active and have for other 2 kinds for one of 3 kinds of substrates It is seldom active or inactive, or it can have widely specificity, for a kind of substrate there is predominant activation and for Other 2 kinds of substrates have less activity.The combination of two or more lipolytic enzymes can be used.

Lipolytic enzyme can be protokaryon, particularly bacterium, or eucaryon, such as from fungi or animal origin. Lipolytic enzyme can be derived from for example following category or kind：Thermophilic trichosporon spp (Thermomyces), dredges the thermophilic hyphomycete of cotton like (T.lanuginosus) (also referred to as Humicola lanuginosa (Humicola lanuginosa))；Humicola, Humicola insolens；Sickle Spore belongs to (Fusarium), sharp fusarium (F.oxysporum), fusariun solani (F.solani), different spore fusarium (F.heterosporum)；Aspergillus, Tabin aspergillus (A.tubigensis), aspergillus niger, aspergillus oryzae；Rhizomucor；False silk ferment Mother's category, antarctic candida (C.antarctica), fold candida (C.rugosa), Penicillium, penicillium camembertii (P.camembertii)；Rhizopus, Rhizopus oryzae (Rhizopus oryzae)；Absidia (Absidia), dictyostelium (Dictyostelium), mucor, Neurospora (Neurospora), rhizopus, Rhizopus arrhizus (R.arrhizus), Japan Head mold (R.japonicus), Sclerotinia (Sclerotinia), Trichophyton (Trichophyton), Vickers Sclerotinia (Whetzelinia), bacillus, Citrobacter (Citrobacter), Enterobacter (Enterobacter), love Moral Fahrenheit Pseudomonas (Edwardsiella), Erwinia (Erwinia), Escherichia (Escherichia), Escherichia coli (E.coli), Klebsiella (Klebsiella), Proteus (Proteus), Pu Luofeidengsi Pseudomonas (Providencia), Salmonella (Salmonella), Serratia (Serratia), Shigella (Shigella), streptomyces, Yersinia (Yersinia), the false unit cell of pseudomonas (Pseudomonas), onion Bacterium (P.cepacia).

It is described herein and the claimed present invention is not limited in the scope of specific embodiment disclosed herein, because These embodiments are expected the illustration of several aspects as the present invention.Any equivalent embodiments and one or more The combination of embodiment is expected within the scope of the invention.A variety of modifications of the present invention are plus those roots for showing and describing herein It is will become apparent according to aforementioned specification for those skilled in the art.Such modification is it is also contemplated that belong to appended claims The scope of book.

Multiple bibliography are cited herein, the disclosure of which is generally introduced by carrying stating.The present invention passes through following realities Apply example to further describe, the embodiment should not be construed as limiting the scope of the present invention.

Materials and methods

The measure of homogeneity

Relevance between 2 amino acid sequences or between 2 nucleotide sequences is described by parameter " homogeneity ".

For the purposes of the present invention, using Needleman-Wunsch algorithms (Needleman and Wunsch, 1970, J.Mol.Biol.48：The degree of sequence identity between 2 amino acid sequences 443-453) is determined, such as in EMBOSS bags (EMBOSS：The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16：Performed by Needle programs 276-277), the version of preferred version 3.0.0 or renewal.Use Optional parameters be that gap open penalty 10, gap extension penalty 0.5 and EBLOSUM62 (BLOSUM62 EMBOSS versions) take For matrix.Output labeled as the Needle of " most long homogeneity " (use-nobrief options are obtained) is used as homogeneity percentage Than, and be calculated as below：

(identical residue x 100)/(comparing the breach sum in length-comparison)

For the purposes of the present invention, the degree of sequence identity between 2 deoxyribonucleotide sequences is used Needleman-Wunsch algorithms (Needleman and Wunsch, 1970, ibid) be measured, such as EMBOSS bags (EMBOSS： The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid) in Performed by Needle programs, the version of preferred version 3.0.0 or renewal.The optional parameter used be gap open penalty 10, Gap extension penalty 0.5 and EDNAFULL (NCBI NUC4.4 EMBOSS versions) substitution matrix.Labeled as " most long homogeneity " The Needle of (use-nobrief options are obtained) output is used as homogeneity percentage, and is calculated as below：

(identical deoxyribonucleotide x 100)/(comparing the breach sum in length-comparison)

Enzyme

Comprising the cellulose decomposition multienzyme complex derived from trichoderma reesei, SEQ ID NO are used as in WO2005074656： The golden yellow thermophilic ascomycete polypeptide with cellulolytic enhancing activity (GH61A) that 2 amino acid 23-250 is shown, and SEQ ID NO are used as in WO05047499：It is prepared by the cellulase for the aspergillus fumigatus β-glucosyl enzym that 2 amino acid/11-863 is shown Thing C.

Included in WO9421785 SEQ ID NO:As Xyl II and herein as SEQ ID NO in 2:1 disclosure Microorganism Aspergillus aculeatus zytase prepared product.

SEQ ID NO are used as included in WO2005074656：What 2 amino acid 23-250 was shown has cellulose decomposition The prepared product of the golden yellow thermophilic ascomycete polypeptide of enhancing active (GH61A).

SEQ ID NO are used as included in WO 2009/111706：The prepared product of the Brazilian mould β-glucosyl enzym of 2 displays.

Embodiment

Embodiment 1

Pretreatment：By maize straw in 1.13wt.%H₂SO₄Soaked 4 hours in environment temperature in solution.To maize straw Implement the vapour explosion 5.5 minutes respectively at 170 DEG C and 180 DEG C.Pretreated maize straw (PCS) composition is analyzed, and And result is shown in table 1.

Enzymatic compositions 1-12 (table 2) is tested to 2 crowdes of PCS.In the system with 12.6% starting TS, 20g gross weight It has rated the hydrolysis of the PCS.Enzyme dosage is 5mg zymoproteins/g celluloses.Hydrolysis time is 72 hours, with 50 DEG C and PH 5.0 130rpm.Glucose content in hydrolysate is analyzed by HPLC.Cellulose is converted according to from total Portugal in PCS The percentage of the glucose produced in grape sugar potential (potential) is calculated.All processing are repeated with 3 times.As a result show In Fig. 1.

The improved hydrolysis shown for the biomass in Cold pretreatment is handled with high zytase dosage.Have not seen High zytase dosage is for the effect of the biomass pre-processed in high temperature.

Sequence table

<110>Sinopec Group

Cofco Group Co., Ltd.

Novozymes Company

<120>Method for producing tunning from lignocellulose-containing materials

<130> 11649-WO-PCT

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 406

<212> PRT

<213>Microorganism Aspergillus aculeatus (Aspergillus aculeatus)

<400> 1

Met Val Gly Leu Leu Ser Ile Thr Ala Ala Leu Ala Ala Thr Val Leu

1 5 10 15

Pro Asn Ile Val Ser Ala Val Gly Leu Asp Gln Ala Ala Val Ala Lys

20 25 30

Gly Leu Gln Tyr Phe Gly Thr Ala Thr Asp Asn Pro Glu Leu Thr Asp

35 40 45

Ile Pro Tyr Val Thr Gln Leu Asn Asn Thr Ala Asp Phe Gly Gln Ile

50 55 60

Thr Pro Gly Asn Ser Met Lys Trp Asp Ala Thr Glu Pro Ser Gln Gly

65 70 75 80

Thr Phe Thr Phe Thr Lys Gly Asp Val Ile Ala Asp Leu Ala Glu Gly

85 90 95

Asn Gly Gln Tyr Leu Arg Cys His Thr Leu Val Trp Tyr Asn Gln Leu

100 105 110

Pro Ser Trp Val Thr Ser Gly Thr Trp Thr Asn Ala Thr Leu Thr Ala

115 120 125

Ala Leu Lys Asn His Ile Thr Asn Val Val Ser His Tyr Lys Gly Lys

130 135 140

Cys Leu His Trp Asp Val Val Asn Glu Ala Leu Asn Asp Asp Gly Thr

145 150 155 160

Tyr Arg Thr Asn Ile Phe Tyr Thr Thr Ile Gly Glu Ala Tyr Ile Pro

165 170 175

Ile Ala Phe Ala Ala Ala Ala Ala Ala Asp Pro Asp Ala Lys Leu Phe

180 185 190

Tyr Asn Asp Tyr Asn Leu Glu Tyr Gly Gly Ala Lys Ala Ala Ser Ala

195 200 205

Arg Ala Ile Val Gln Leu Val Lys Asn Ala Gly Ala Lys Ile Asp Gly

210 215 220

Val Gly Leu Gln Ala His Phe Ser Val Gly Thr Val Pro Ser Thr Ser

225 230 235 240

Ser Leu Val Ser Val Leu Gln Ser Phe Thr Ala Leu Gly Val Glu Val

245 250 255

Ala Tyr Thr Glu Ala Asp Val Arg Ile Leu Leu Pro Thr Thr Ala Thr

260 265 270

Thr Leu Ala Gln Gln Ser Ser Asp Phe Gln Ala Leu Val Gln Ser Cys

275 280 285

Val Gln Thr Thr Gly Cys Val Gly Phe Thr Ile Trp Asp Trp Thr Asp

290 295 300

Lys Tyr Ser Trp Val Pro Ser Thr Phe Ser Gly Tyr Gly Ala Ala Leu

305 310 315 320

Pro Trp Asp Glu Asn Leu Val Lys Lys Pro Ala Tyr Asn Gly Leu Leu

325 330 335

Ala Gly Met Gly Val Thr Val Thr Thr Thr Thr Thr Thr Thr Thr Ala

340 345 350

Thr Ala Thr Gly Lys Thr Thr Thr Thr Thr Thr Gly Ala Thr Ser Thr

355 360 365

Gly Thr Thr Ala Ala His Trp Gly Gln Cys Gly Gly Leu Asn Trp Ser

370 375 380

Gly Pro Thr Ala Cys Ala Thr Gly Tyr Thr Cys Thr Tyr Val Asn Asp

385 390 395 400

Tyr Tyr Ser Gln Cys Leu

405

Claims

1. a kind of technique for being used to produce the hydrolysate of lignocellulosic material, it includes

A) lignocellulosic material is implemented between 165 DEG C -175 DEG C, the pretreatment of e.g., from about 170 DEG C of temperature, and

B) effect of hydrolase is implemented to the pretreated lignocellulosic material to produce hydrolysate,

Wherein described hydrolase includes cellulolytic enzyme and zytase, and wherein described zytase is with total amount hydrolase At least 10% amount of albumen is present.

2. technique according to claim 1, wherein the hydrolase includes the cellulase system derived from trichoderma reesei.

3. according to the technique of any one of preceding claims, wherein the zytase is GH10 zytases, preferably have It is shown as SEQ ID NO:1 sequence or with being shown as SEQ ID NO:1 sequence have at least 50%, at least 60%, at least 70%th, the sequence of at least 80% or at least 90% homogeneity.

4. according to the technique of any one of preceding claims, wherein the zytase with hydrolase protein total amount at least 10%th, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20% or even at least 25% amount In the presence of.

5. according to the technique of any one of preceding claims, wherein the hydrolase with least the 5% of hydrolase protein total amount, At least 10%, at least 15%, at least 20% or even at least 25% amount includes the glycoside hydrolase of family 61.

6. according to the technique of any one of preceding claims, wherein the hydrolase with least the 1% of hydrolase protein total amount, At least 2%, at least 3%, at least 4% or even at least 5%, for example, at least 6%, at least 7%, at least 8%, at least 9 or even At least 10% amount includes β-glucosyl enzym.

7. according to the technique of any one of preceding claims, wherein the lignocellulose-containing materials come from the material being selected from the group Material：Maize straw, zein fiber, hardwood such as poplar and birch, soft wood, cereal straw such as wheat straw, switchgrass, rice Shell, municipal solid waste, industrial organic waste, office's paper or its mixture.

8. according to the technique of any one of preceding claims, wherein the pretreatment in the step (a) uses organic acid or nothing Machine acid is carried out, preferably sulfuric acid, acetic acid, citric acid, tartaric acid, butanedioic acid and/or its mixture.

9. according to the technique of any one of preceding claims, wherein the pretreatment in the step (a) uses 0.1-2.5 weights Measure % acid, preferably 0.5-2.0 weight % acid, preferably 0.8-1.5 weight % acid to carry out, the sour preferably sulfuric acid.

10. according to the technique of any one of preceding claims, wherein the pretreatment is carried out in about 170 DEG C of temperature.

11. according to the technique of any one of preceding claims, wherein the pretreatment is carried out as vapour explosion.

12. according to the technique of any one of preceding claims, wherein the hydrolysate of the step (b) is fermented to produce hair Ferment product, preferably uses yeast progress.

13. according to the technique of any one of preceding claims, it is included while hydrolyzing and fermenting.

14. according to the technique of any one of preceding claims, it further comprises comprising the recovery tunning, preferably second The step of alcohol.

15. a kind of composition, it includes the hydrolase as defined in any one of preceding claims.