CN109943608A - The method of rapid enzymolysis and the quantitative enzyme recycling of stalk - Google Patents
The method of rapid enzymolysis and the quantitative enzyme recycling of stalk Download PDFInfo
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Abstract
The invention discloses the methods that a kind of rapid enzymolysis of stalk and quantitative enzyme recycle.The method is by being added hydrolase and water in pretreated stalk, adjust the optimal pH range of pH to enzyme, carry out enzyme digestion reaction for 24 hours, and after the 1st wheel enzyme digestion reaction, it is separated by solid-liquid separation, isolated enzymolysis liquid is separated by solid-liquid separation to obtain solid for fermentative production of ethanol and wherein 75% solid is taken to be directly entered the 2nd wheel enzyme digestion reaction, the enzyme amount that 2nd wheel enzymolysis process is added is the 50% of the 1st wheel enzyme digestion reaction institute enzyme concentration, remaining condition is identical as the 1st wheel enzyme digestion reaction, carries out reaction in 24 hours；Remaining 25% solid is separately taken to be used for producing enzyme as the substrate that trichoderma reesei ferments, the enzyme of production is used for the enzyme hydrolysis of stalk, and the ethyl alcohol of generation is collected in the operation after the above enzyme digestion reaction of 4 wheel of repetition and enzyme digestion reaction.The present invention realizes the regeneration of enzyme while reducing enzyme dosage, largely save the cost of enzyme hydrolysis.
Description
Technical field
The invention belongs to lignocellulosic hydrolysis technology field, it is related to rapid enzymolysis and the quantitative enzyme recycling of a kind of stalk
Method.
Background technique
Biomass energy is to solve reality and one of future source of energy crisis and the most potential approach of environmental pollution.Based on shallow lake
There is the problem of " striving grain with people, strive ground with grain " in the first generation Fuel Ethanol that powder and carbohydrate fermentation produce alcohol fuel, therefore
Using cellulose as one of the mainstream of the second generation Fuel Ethanol of raw material necessarily new century new energy.In addition, remaining at present
The a large amount of stalk biomass energy is mainly incinerated, and utilization rate is extremely low.And cellulosic ethanol technology both can use discarded agriculture
Crop material can produce renewable and clean energy resource again --- alcohol fuel, thus advantage is had more than first generation Fuel Ethanol.
But due to the limitation of the prematurity of key technology and economy, existing cellulosic ethanol technology carries out extensive quotient not yet
Industry application.
The biggest problem that cellulosic ethanol technology faces at present is the processing of lignocellulosic.Lignocellulosic is by fiber
Element, hemicellulose and lignin are constituted, and wherein cellulose and hemicellulose can obtain biological capable of being utilized via enzyme hydrolysis
Carbohydrate, and lignin is then difficult to be biodegradable utilization, and it can also to the package action of cellulose and the crystalline texture of itself
Hinder the degradation of cellulose.Traditional enzymatic hydrolysis process directly presses the water that 40mg enzyme/g glucan enzyme dosage carries out 72 hours
Solution, since cellulase stability is poor, the service life is shorter, and activity is lower, and efficiency when enzyme hydrolysis is lower, leads to Enzymatic hydrolysis
The cost of process also correspondinglys increase.Therefore, seeking efficient lignocellulosic enzymatic hydrolysis process is to solve cellulosic ethanol technology
The breach of problem.
Summary of the invention
The purpose of the present invention is to provide the methods that a kind of rapid enzymolysis of stalk and quantitative enzyme recycle.The method passes through
The enzyme hydrolysis time for controlling stalk, rapid enzymolysis is carried out in batches, and solid recycling is carried out between each batch.
Realize that the technical solution of the object of the invention is as follows:
The method of rapid enzymolysis and the quantitative enzyme recycling of stalk, the specific steps are as follows:
Step 1, by hydrolase and water are added in pretreated stalk, the optimal pH range of pH to enzyme is adjusted, is carried out
Enzyme digestion reaction for 24 hours；
Step 2, it after the 1st wheel enzyme digestion reaction of step 1, is separated by solid-liquid separation, isolated enzymolysis liquid is for sending out
Ferment production ethyl alcohol is separated by solid-liquid separation to obtain solid and wherein 75% solid is taken to be directly entered the 2nd wheel enzyme digestion reaction, the 2nd wheel enzymolysis process
The enzyme amount of addition is 50% of institute's enzyme concentration in step 1, remaining condition is identical as step 1, carries out reaction in 24 hours；It separately takes solid
Remaining 25% isolated solid of liquid is used for producing enzyme as the substrate that trichoderma reesei ferments, and the enzyme of production is for stalk
Enzyme hydrolysis；
Step 3, after the 2nd wheel enzyme digestion reaction, the operation after the 1st wheel enzyme digestion reaction of step 2 is repeated, repeats 4
The operation after the above enzyme digestion reaction and enzyme digestion reaction is taken turns, the ethyl alcohol of generation is collected.
In the present invention, pretreatment described in step 1 can be diluted alkaline pretreatment or dilute acid pretreatment.
In the present invention, hydrolase described in step 1 use hydrolase commonly used in the art, can for cellulase,
The mixture of one or more of zytase and trichoderma reesei etc..
In the present invention, pH described in step 1 be enzyme optimal pH range, generally 4~6.
In the present invention, in step 1, the enzymolysis temperature is 50 DEG C, and enzyme digestion reaction is placed in dynamic shaking table, revolving speed
For 250rpm.
In traditional wood fibre enzymatic hydrolysis, in the enzyme hydrolysis of the high concentration of substrate of lignocellulosic (dry matter concentration >=18%)
Cheng Zhong, enzymatic hydrolysis rate can reach maximum within a very short time.Cellulose and half fiber in about 23h, in pretreated straw
The conversion ratio of dimension element can reach 70% or so.But enzymatic hydrolysis rate meeting sharp fall later, needs other 48-96 hour
Cellulose conversion ratio could be increased to 85% or so from 70%.Rate decline is digested mainly due to pretreated wooden fibre
Dimension element still some be more difficult to degrade, and this part lignocellulosic difficult to degrade was just dropped in the later period of enzymatic hydrolysis
Solution, in addition the strong feedback inhibition of later period enzymolysis product (sugar), causes the slow of later period enzyme hydrolysis.
The present invention utilizes enzyme digestion reaction kinetic characteristics, only carries out enzyme hydrolysis in 24 hours, makes full use of high enzymatic hydrolysis rate
Stage avoids low enzymatic hydrolysis rate.After 24 hours enzymatic hydrolysis, it is separated by solid-liquid separation, by 75% unhydrolysed solid lignocellulosic
It is recycled to the enzyme hydrolysis of next group, so that this part lignocellulosic difficult to degrade avoids to a certain extent in hydrolysis
The feedback inhibition of product.Since enzyme at this time >=60% is adsorbed on unhydrolysed solid lignocellulosic, non-water is recycled
Enzyme has also been recycled while the lignocellulosic of solution.In addition, enzyme recycling be after 24 hours rapid enzymolysis, therefore recycle enzyme
Remain most activity.In order to prevent due to recycling solid lignocellulosic remain in enzymolysis reactor it is excessive tire out
Product, a part of solid lignocellulosic (25%) are regularly transferred in lesser enzymolysis reactor, and water and Richter scale wood is added
Producing enzyme fermentation is carried out after mould, the cellulase of production is used for the enzyme hydrolysis of stalk.The present invention realizes enzyme while reducing enzyme dosage
Regeneration, largely save enzyme hydrolysis cost.
Detailed description of the invention
Fig. 1 is diluted alkaline (a), and (c) and diluted acid (b), (d) pretreated corn stover is 7% (a), (c) and 1% (b), (d)
Enzyme hydrolysis result figure under glucan.Enzyme hydrolysis carries out in 50 DEG C, 250rpm shaking table, corn stover dosage be 1% (w/w) and
7% (w/w) glucan, the enzyme dosage 40mg protein/g glucan of diluted acid corn stover, the enzyme dosage of diluted alkaline corn stover
For 30mg protein/g glucan, the protein content of cellulase and zytase is 1:1, and buffer is 50mMpH4.8 lemon
Phthalate buffer.
Fig. 2 is (a) of diluted acid, the pretreated corn stover of diluted alkaline under 7% (w/w) glucan during enzyme hydrolysis, (c)
Enzyme adsorbs situation and (b), (d) the situation of change result figure of substrate component.Enzyme hydrolysis carries out in 50 DEG C, 250rpm shaking table, beautiful
Rice stalk dosage be 7% (w/w) glucan, the enzyme dosage 40mg protein/g glucan of the corn stover of dilute acid pretreatment,
The enzyme dosage of the pretreated corn stover of diluted alkaline is 30mgprotein/g glucan, the protein content of cellulase and zytase
For 1:1, buffer is 4.8 citrate buffer of 50mMpH.Supernatant protein concentration is measured using improved ninhydrin method.
Fig. 3 is enzyme take-back strategy schematic diagram.
Fig. 4 is the hydrolyzate sugared content result figure that the stalk of dilute acid pretreatment is obtained through enzyme recovery experiment headpin.
Fig. 5 is the hydrolyzate sugared content result figure that the stalk of dilute acid pretreatment is obtained through No. 2 bottles of enzyme recovery experiment.
Fig. 6 is the hydrolyzate sugared content result figure that the stalk of dilute acid pretreatment is obtained through No. 3 bottles of enzyme recovery experiment.
Specific embodiment
Below with reference to embodiment and attached drawing, the invention will be further described.
Embodiment 1
Diluted alkaline and dilute acid pretreatment are carried out to corn stover in optimal conditions respectively, with research pretreatment to enzyme hydrolysis and
The influence of enzyme absorption.Dilute acid pretreatment eliminates the xylan in corn stover, and glucan and lignin (containing ash content) contain
Amount has increased respectively to 50.8% and 40.1%.Glucan relative to dilute acid pretreatment, in the pretreated corn stover of diluted alkaline
Content is close (54.6%), and xylan content is higher (21.8%), and content of lignin is then much lower (10.9%).
Diluted alkaline, dilute acid pretreatment difference of the corn stover in component its enzyme hydrolysis and enzyme absorption can be had an impact.
As shown in Figure 1, corn stover pretreated for diluted alkaline, in 7% (w/w) glucan concentration, glucan and xylan are in enzyme
The hydrolysis initial stage is rapidly converted into glucose and xylose, and hydrolysis rate is begun to decline after 24 hours.At 24 hours, glucose and
The concentration of xylose has respectively reached 68.3g/L and 20.4g/L, and corresponding inversion rate of glucose and xylose rate are respectively
76.4% and 56.0%.At 96 hours, the concentration of glucose and xylose has respectively reached 71.5g/L and 24.2g/L, respectively corresponds
80.7% glucan conversion ratio and 66.4% xylan conversion ratio.As can be seen that initial hydrolysis in 24 hours just reaches
Most of conversion ratio at final 96 hours.The enzyme hydrolysis of the corn stover of dilute acid pretreatment also has identical trend, 7%
(w/w) when glucan concentration, concentration of glucose has reached 62.4g/L at 24 hours, and corresponding inversion rate of glucose is 69.5%,
In next 72 hours, concentration of glucose merely adds 3.5g/L (shown in such as Fig. 1 (b)).
Under high concentration of substrate (7% (w/w) glucan concentration), one of the reason of the decline of enzyme hydrolysis later period hydrolysis rate
It may be that the accumulation of high glucose concentration inhibits the vigor of enzyme.However, in lower concentration of substrate (1% (w/w) glucan concentration)
In hydrolysis, the reduction (Fig. 1 (c) (d)) of hydrolysis later period rate is equally had also discovered.Thus, it may be possible to which pretreated stalk contains
Some opposite substrate for being difficult degradation, this may be another reason for hydrolysis rate reduces.Furthermore, it is also possible to be part
Partial lignin and cellulose in pretreated substrate, which exist, to interact, and hinders the active site of enzyme and cellulose
Contact, results in the decline of hydrolysis rate.
Experiment further determines under 7% (w/w) glucan concentration, the corn stover enzyme water of diluted alkaline, dilute acid pretreatment
Enzyme characterization of adsorption in solution preocess.As shown in Fig. 2 (a), during diluted alkaline pretreated Factor of Enzymolysis Corn Stalk, in liquid
The concentration of zymoprotein is reduced rapidly to 53% in the incipient stage, and floats between 52% to 61% in initial 24 hours,
This surface has the enzyme of about 39-48% to be adsorbed on solid substrate in initial 24 hours.After 24 hours, enzyme adsorbance
It is maintained at 40% or so.It is analyzed, can clearly be found out initial by the ingredient to unhydrolyzed solids non-in hydrolytic process
The ingredient of remaining solid has great changes in 24 hours, and beta-dextran content is reduced to about 16% from 54.6%, and lignin (contains
Ash content) content increase to 60% from 10.9%, as shown in Fig. 2 (b).Theoretically, with the hydrolysis of cellulose, a large amount of fiber
Plain enzyme will be desorbed from solid.But the enzyme being not associated in supernatant is merely added less than 10%.This may be because
A large amount of enzyme adsorption site in corn stover can be opened after pretreatment for hydrolysis, and with the progress of hydrolytic process, more
Lignin is exposed, and enzyme can be integrated on lignin, therefore the variation of the enzyme adsorbed in hydrolytic process is smaller.Locate in advance in diluted acid
Similar enzyme characterization of adsorption is had also discovered in the hydrolysis of the corn stover of reason, as shown in Fig. 2 (c) (d).The initial stage is being hydrolyzed,
About 50% enzyme has been adsorbed on unhydrolysed solid, and increases to 55% at 24 hours, at this time in non-unhydrolyzed solids
The content of lignin (containing ash content) has reached 70%.Generally speaking, after most of glucans are all hydrolyzed, lignin is in enzyme
Absorption in may play an important role.In addition, compared to the pretreated corn stover of diluted alkaline, the corn stalk of dilute acid pretreatment
Enzyme on stalk adsorbs more.This may be caused by the structure as substrate, composition and hydrolysis conversion are different.Previous
Research points out that the property of lignin will affect characterization of adsorption of the cellulase on substrate.Because of the pretreated corn stalk of diluted alkaline
The lignin that stalk includes is less, and the corn stover of dilute acid pretreatment contains more lignin, therefore lignin is likely to
More enzymes are caused to be adsorbed onto the reason in the corn stover of dilute acid pretreatment.Also, diluted acid and diluted alkaline pretreatment may be right
Lignin is modified in different ways, and two kinds of different lignin is caused to have different enzyme characterization of adsorptions.
Embodiment 2
By 1000g reaction system (sulfuric acid content 1%, corn stover dry weight content 10%), dilute acid pretreatment is used
500g (dry weight) corn stover, to subsequent enzymatic hydrolysis and enzyme recovery experiment.
Enzyme hydrolysis is carried out by 100g reaction system (22% concentration of substrate), parallel three groups, has carried out four-wheel experiment altogether.?
In one wheel experiment, enzyme dosage is calculated by beta-dextran content, consisting of: every group of cellulase 3.08mL, zytase
3.2mL.PH is adjusted to 4.8, is placed in 50 DEG C, is digested in 250rpm shaking table.Experiment uses batch charging, i.e., is first added
The corn stover of 11% concentration of substrate adds the corn stover of other 11% concentration of substrate after hydrolyzing 2 hours.
After hydrolysis 24 hours, three shaking flasks are all taken out, are placed in a centrifuge in 4 DEG C, under the conditions of 10000r/min from
The heart 10 minutes, after being separated by solid-liquid separation, hydrolyzate was placed in 4 DEG C of Storage in refrigerator, and 75% solid takes turns enzyme hydrolysis system for second,
The substrate that other 25% solid is then used as trichoderma reesei fermentation carrys out producing enzyme.The operation of subsequent experimental is identical as the first round, but enzyme
Dosage is only the half of first round experiment, i.e., cellulase dosage is 1.54mL, and zytase dosage is 1.6mL.
After four-wheel is tested, liquid phase measurement is carried out to hydrolyzate, as a result such as Fig. 4, Fig. 5, Fig. 6, it can be seen that first
Wheel experiment is using complete enzyme dosage, and subsequent experimental enzyme dosage is only the half of the first round, and hydrolyze the result shows that,
The sugared concentration for the hydrolyzate that subsequent experimental obtains fully achieves the sugared concentration level of first round experiment, therefore this technique is dilute
Alkali, dilute acid pretreatment Factor of Enzymolysis Corn Stalk on reduce respectively 40% and 50% enzyme dosage.
Claims (5)
1. the method that the rapid enzymolysis and quantitative enzyme of stalk recycle, which is characterized in that specific step is as follows:
Step 1, by hydrolase and water are added in pretreated stalk, pH is adjusted to the optimal pH range of enzyme, is carried out for 24 hours
Enzyme digestion reaction；
Step 2, it after the 1st wheel enzyme digestion reaction of step 1, is separated by solid-liquid separation, isolated enzymolysis liquid is for life of fermenting
Producing and ethanol is separated by solid-liquid separation to obtain solid and wherein 75% solid is taken to be directly entered the 2nd wheel enzyme digestion reaction, and the 2nd wheel enzymolysis process is added
Enzyme amount be 50% of institute's enzyme concentration in step 1, remaining condition is identical as step 1, carries out reaction in 24 hours；Separately take solid-liquid point
It is used for producing enzyme as the substrate that trichoderma reesei ferments from remaining 25% obtained solid, the enzyme of production to be used for the enzyme water of stalk
Solution；
Step 3, the 2nd wheel enzyme digestion reaction after, repeat step 2 the 1st wheel enzyme digestion reaction after operation, repeat 4 wheel with
The ethyl alcohol of generation is collected in operation after upper enzyme digestion reaction and enzyme digestion reaction.
2. the method according to claim 1, wherein pretreatment described in step 1 is diluted alkaline pretreatment or diluted acid
Pretreatment.
3. the method according to claim 1, wherein hydrolase described in step 1 is cellulase, zytase
With the mixture of one or more of trichoderma reesei.
4. the method according to claim 1, wherein pH described in step 1 is 4~6.
5. the method according to claim 1, wherein the enzymolysis temperature is 50 DEG C, enzyme in step 1
Solution reaction is placed in dynamic shaking table, revolving speed 250rpm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060418A (en) * | 2012-12-06 | 2013-04-24 | 南昌大学 | Method of constructing mixed bacteria system for fermenting straw stalks to produce ethanol |
| WO2016145527A1 (en) * | 2015-03-16 | 2016-09-22 | Iogen Corporation | Process comprising acid pretreatment and enzymatic hydrolysis |
| CN107488651A (en) * | 2017-10-13 | 2017-12-19 | 西北农林科技大学 | A kind of method for recycling enzyme hydrolysis solid residue cellulase |
-
2019
- 2019-04-10 CN CN201910285626.4A patent/CN109943608A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060418A (en) * | 2012-12-06 | 2013-04-24 | 南昌大学 | Method of constructing mixed bacteria system for fermenting straw stalks to produce ethanol |
| WO2016145527A1 (en) * | 2015-03-16 | 2016-09-22 | Iogen Corporation | Process comprising acid pretreatment and enzymatic hydrolysis |
| CN107488651A (en) * | 2017-10-13 | 2017-12-19 | 西北农林科技大学 | A kind of method for recycling enzyme hydrolysis solid residue cellulase |
Non-Patent Citations (2)
| Title |
|---|
| YUAN Y等: "Developing fast enzyme recycling strategy through elucidating enzyme adsorption kinetics on alkali and acid pretreated corn stover", 《BIOTECHNOLOGY FOR BIOFUELS》 * |
| 周济铭等: "《酶制剂生产及应用技术》", 30 September 2014 * |
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Application publication date: 20190628 |