CN106480130B - A kind of method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application - Google Patents
A kind of method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application Download PDFInfo
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- C12N9/2405—Glucanases
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Abstract
The present invention discloses a kind of method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application, the buffer solution that concentration is 0.1mol/L is added in stalk after the pre-treatment, reaction is hydrolyzed, the solidliquid mixture that solid-to-liquid ratio is 1: 31: 20 is generated, the pH value of the solidliquid mixture is 4-6；Cellulase is added in solidliquid mixture, the cellulase includes the cellulase of the cellulase that additive amount is stalk dry weight 0.1-0.5% and vigor in 1-100IU/g；In 40-60 DEG C of temperature, 24-72 hours hydrolysis are carried out, final hydrolysis solution is obtained.Immobilized enzyme is recycled using magnet after hydrolysis, the rate of recovery can achieve 10 times or more between 80-100% and still keep certain vigor, and the vigor for obtaining enzyme is maintained between 75-100%.Glucose solution after hydrolysis is used for fermenting alcohol after measurement.
Description
Technical field
The present invention relates to the technologies that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in a kind of application, belong to biological material and answer
Use field.
Background technique
As large agricultural country, the every annual meeting in China generates hundred million tons of all kinds of stalks of 7-9, wherein corn, wheat and three kinds of rice works
The stalk of object accounts for 80% or so.Since stalk yield is huge, from 2005, stalk integrated treatment project became state key branch
It holds project, the industry of production glucose then fermenting alcohol is especially hydrolyzed using stalk as raw material.However, can be hydrolyzed in stalk
Main component for glucose is cellulose, need just be hydrolyzed into monomer under the action of cellulase, and cellulose makes
With the cost is relatively high, so that the practical application of the technology is by severely restricts.According to practical measuring and calculating, enzyme preparation is in cellulose second
Totle drilling cost in alcohol industry accounts for nearly one third, is the main bottleneck of industry development.
A kind of magnetic immobilized cellobiase disclosed in 2015-04-08 application No. is 201410637435.7 is received
Rice mesoporous material and preparation method thereof.The material realizes the multiple utilization of enzyme, and pass through magnetic material by the way that enzyme is fixed
Material improves the operability of recycling.The invention experiments prove that, immobilized cellobiase is directed to fiber two in the lab
The process that sugar is hydrolyzed into glucose is effectively, can be effectively enriched with by magnet.But this kind of material is used for practical stalk
Processing, there is no practical data, the method that also can not adopt at present.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of applications, and the fixed enzyme hydrolysis straw of magnetic Nano can be recycled
The method of stalk establishes the processing mode and hydrolysising condition of stalk, solves the practical problem of stalk hydrolysis, and magnetic immobilized enzyme can be with
Reusability, activity are maintained, by the cost control of this production technology under proportional space.
To achieve the above object, technical solution of the present invention are as follows:
A kind of method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application, this method implementation steps are as follows: step 1:
The buffer solution that concentration is 0.1mol/L is added in stalk after the pre-treatment, reaction is hydrolyzed, generating solid-to-liquid ratio is 1: 3-1:
20 solidliquid mixture, the pH value of the solidliquid mixture are 4-6；The buffer solution includes NaAc_HAc buffer solution,
Or citric acid-sodium citrate buffer；Step 2: cellulase, the cellulose being added in the solidliquid mixture of step 1
Enzyme includes the cellulase of the cellulase that additive amount is stalk dry weight 0.1-0.5% and vigor in 1-100IU/g；Step 3:
In 40-60 DEG C of temperature, 24-72 hours hydrolysis are carried out, final hydrolysis solution is obtained；Step 4: detecting final water
Solve the concentration of glucose in reaction solution；Step 5: final hydrolysis solution is after glucose concentration determination for second of fermenting
Alcohol.
Above-mentioned technical proposal has the beneficial effect that
It is that stalk is being passed through into pretreatment in the method for the recyclable magnetic Nano immobilized enzyme hydrolyzing straw of present invention application
Afterwards, a small amount of cellulase is added, is hydrolyzed under constant temperature conditions.After hydrolysis, immobilized enzyme is recycled using magnet, the rate of recovery exists
Between 80-100%, it can achieve 10 times or more and still to keep certain vigor, and the vigor for obtaining enzyme is maintained between 75-100%.
Glucose solution after hydrolysis is used for fermenting alcohol after measurement.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is equipment schematic diagram used in the production method embodiment of magnetic immobilized enzyme of the present invention；
Fig. 2 is the schematic diagram that the vitality retaining percentege of enzyme is recycled in the embodiment of the present invention one；
Fig. 3 is the schematic diagram that the vitality retaining percentege of enzyme is recycled in the embodiment of the present invention two；
Fig. 4 is the schematic diagram that the vitality retaining percentege of enzyme is recycled in the embodiment of the present invention three.
Specific embodiment
In order to be clearer and more clear technical problems, technical solutions and advantages to be solved, tie below
Drawings and examples are closed, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used
To explain the present invention, it is not intended to limit the present invention.
The method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in a kind of application disclosed by the invention, the stalk include
But be not limited only to corn stover, wheat stalk, rice straw, sweet potato stalk, soybean stalk etc., stalk involved in this method is
By pretreated stalk, preconditioning technique includes but are not limited to cutting and grinding, boiling, steam, ball milling, explosion, microwave etc.
Physical means, and the including but not limited to all kinds of chemical means such as pickling, alkali cleaning, organic solvent also include enzymatic hydrolysis, fungal biodegradation etc.
Thus biological means remove wherein the by-product for having inhibiting effect, such as polyphenols.
Cellulose measuring method of the present invention is as follows:
1.0000~1.0500g sample (G1) is accurately weighed in clean 250mL ground conical flask, 25mL is added
20% nital loads onto reflux condensing tube and heats 1h in boiling water bath, small in the glass sand core filter of preparatory constant weight
Supernatant is poured out in the heart, and filter residue is carefully moved back in former conical flask by vacuum filtration, repeats aforesaid operations for several times, until fiber becomes
Until white.Entire contents in conical flask are finally moved into filter, vacuumizing filtration, with 20% nital of 10mL
Residue is washed, reusable heat water washing to washing lotion methyl orange reagent test is not in acidity, finally washed with 10mL ethyl alcohol point 2 times,
Drying to constant weight (G2) in 105 DEG C for filter residue.Content of cellulose formula:
The method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in the application, implementation steps are as follows:
Step 1: corn stover, wheat after taking the physical means such as cutting and grinding, boiling, steam, ball milling, explosion, microwave
Stalk, rice straw, sweet potato stalk, soybean stalk etc.,.
Step 2: the buffer solution that concentration is 0.1mol/L being added in stalk after the pre-treatment, reaction is hydrolyzed, generate
The solidliquid mixture that solid-to-liquid ratio is 1: 3-1: 20, the pH value of the solidliquid mixture are 4-6；The buffer solution includes acetic acid-
Sodium acetate buffer or citric acid-sodium citrate buffer；
Step 3: cellulase being added in the solidliquid mixture of step 1, the cellulase includes that additive amount is stalk
The cellulase of the cellulase and vigor of dry weight 0.1-0.5% in 1-100IU/g；
Step 4: in 40-60 DEG C of temperature, solidliquid mixture Hybrid Heating, stirring or concussion being carried out 24-72 hours
Hydrolysis obtains final hydrolysis solution；The speed of stirring therein is 60-180 revs/min, and the frequency of concussion is 25-
150 revs/min.The stirring or concussion form includes and is not limited to agitating paddle, magneton, concussion case.Reaction in hydrolysis
Vessel include and are not limited to glass reaction container, plastics reaction vessel and metal reaction container.
Step 5: detecting the concentration of glucose in final hydrolysis solution.
Step 5: detecting in final hydrolysis solution and concentration of glucose is measured using enzyme activity instrument.
Step 6: final hydrolysis solution is used for fermenting alcohol after measurement.
The cellulase that additive amount described in above-mentioned steps 2 is stalk dry weight 0.1-0.5% be available commercially from by enzyme, it is described
Vigor 1-100IU/g cellulase be magnetic immobilized enzyme；Incorporated by reference to shown in Fig. 1.
The production method of the magnetic immobilized enzyme, includes the following steps:
Step 1: following component: 100 parts of reaction substrate, 100 parts of template solution, ferroso-ferric oxide powder is weighed by mass parts
2 parts, 20 parts of cellulase of end；Reaction substrate is tetraethyl orthosilicate and dimethyldisiloxane, wherein tetraethyl orthosilicate matter
Measure accounting 80%, dimethyldisiloxane quality accounting 20%；Template solution is glucose solution, and glucose quality percentage is
30%；
Step 2: hydrochloric acid is weighed by the amount of substance of reaction substrate weighed in step 1, and by following molar ratio, reacts bottom
The molar ratio of object and hydrochloric acid is 150: 1, and the concentration of hydrochloric acid is 0.17mol/L；
Step 3: reaction substrate weighed in step 1 is placed in storage tank 1, and is added in reaction kettle 5 by pump 11；
Step 4: reaction substrate is cooled to 10 DEG C by cold bath；
Step 5: the hydrochloric acid in step 2 is placed in storage tank 2, and the reaction substrate drop by pump 21 into reaction kettle 5
Add hydrochloric acid and stirred with blender 52 until obtained solution becomes clear, control reaction temperature is no more than 30 DEG C, stirring
Speed is 100 revs/min；
Step 6: template solution weighed in step 1 is placed in storage tank 3, and by pump 31 obtained to step 5 it is molten
Template solution is added in liquid, and with the stirring of blender 52 to being uniformly mixed, mixing speed is 100 revs/min；
Step 7: vacuum distillation is carried out to step 6 acquired solution in reaction kettle 5 until not by vacuum pump 6 and cooler 7
Volatilization is generated again, to remove the coproduct ethanol in reaction solution, obtains colloidal sol；The vacuum distillation pressure is
0.005MPa, temperature are 25 DEG C, and coproduct ethanol is cooled back in recycling can 8
Step 8: shutting down vacuum pump 6 and cooler 7, and storage tank 4 is added in the sodium hydroxide solution that concentration is 0.4mol/L
In, above-mentioned sodium hydroxide solution is added dropwise by the colloidal sol that pump 41 is obtained to step 7 and is stirred with blender 52, drop rate is
1L/min, and real-time monitoring pH value, until pH value reaches 3.5, it is weighed into the addition step 1 of reaction kettle 5 by feed opening 52
Ferroferric oxide powder and cellulase continue that sodium hydroxide solution is added dropwise, until pH value reaches 4.2, mixing speed 100
Rev/min；
Step 9: the reaction product that step 8 obtains is transferred in open containers 9 from reaction kettle 5, is cooled to 0 DEG C and is protected
It holds 12 hours, obtains gel；
Step 10: using pulverizer to be ground into granularity as 0.2mm particle below the gel, and be dried,
Using magnetic immobilized enzyme of the packaging of aluminium foil bag after dry.
In above-described embodiment, in step 1, final produce is adjusted by adding dimethyldisiloxane in reaction substrate
The characteristics of product, makes magnetic immobilized enzyme have better toughness, avoids breakable problem in use.In step 4
Reaction substrate temperature is controlled at 10 DEG C by cold bath, effectively preventing reaction under room temperature leads to production caused by heat aggregation
It is dangerous, it is ensured that industrial operability.In step 5, hydrochloric acid is added by dropwise addition mode, avoids being added at one time and lead
It causes reaction excessively violent, in order to preferably control reaction rate, improves reaction safety.Pass through vacuum distillation in step 7
Byproduct of reaction ethyl alcohol is removed, ethyl alcohol is avoided and the vigor of cellulase is impacted, while also can avoid subsequent gel
Rate is excessively slow.In step 8 by way of ferroso-ferric oxide and cellulase is added among N-process, avoid
It reacts and is added early period, since viscosity is small, acid height, ferroso-ferric oxide and cellulase are easy to happen asking for reunion when being directly added into
Topic, while also having avoided phase addition after the reaction, due to system viscosity height, what ferroso-ferric oxide and cellulase were not easy to disperse
Problem, to fundamentally solve cladding problem of non-uniform.In step 9 using gel is carried out under acid condition, avoid
Gel is not easy the problem of shifting under the conditions of property, and avoids the non-uniform problem of gel that gel under neutrallty condition occurs.In step
In rapid ten, the magnetic immobilized enzyme produced using packaging of aluminium foil bag can avoid enzyme inactivation, to improve depositing for magnetic immobilized enzyme
Store up stability.Since the embodiment of the present invention key step is realized in a kettle, this production method has to equipment
The advantages such as of less demanding, simple process, operability height.It, can be with after 4 hydrolysis of above-mentioned stepsIt is magnetic solid using magnet recycling Surely change enzyme, give over to and use next time.The magnet used is permanent magnet or electromagnet, and the permanent magnet uses neodymium-iron-boron
Iron；Hydrolysis magnet being placed in step 4In the equipment of reaction, stirringFinal hydrolysisSolution, can be inhaled by magnet
It is attached to recycle out magnetic immobilized enzyme.The recovery method of the magnetic immobilized enzyme can also be using magnetic field strength in 0.2-
Electromagnet between 0.4T is by final hydrolysisMagnetism in solutionAfter immobilised enzymes is drawn, power-off is i.e. recyclable.
Before magnet recycles magnetic immobilized enzyme, the vigor of enzyme needs first to be measured, and an IU refers to that catalysis 1 in one minute is micro-
Enzyme amount needed for mol substrate.Specific mensuration mode is, using 1 gram of filter paper as substrate, to put into m grams of enzyme (free enzyme or fixation
Enzyme), after reaction t minutes, there is c grams of filter paper transformation for glucose, the molal weight of glucose is the M grams/mol of (work of enzyme
Power formula):
After the enzyme amount for obtaining IU representative, according to the quality of substrate, required enzyme amount is calculated.
As a further improvement, the magnetic immobilized enzyme recovery method in the present invention is magnetic recovery method, the magnetic of use
Iron is permanent magnet or electromagnet.Permanent magnet generallys use ndfeb magnet, is placed in consersion unit and stirs, i.e., recyclable magnetic out
Property immobilised enzymes.It is more convenient compared with permanent magnet to be recycled using electromagnet, and magnetic field strength inhales immobilised enzymes between 0.2-0.4T
After taking, power-off is i.e. recyclable.
Magnetic immobilized enzyme after the recovery, the rate of recovery is between 80-100%.The calculating side of the magnetic immobilized enzyme rate of recovery
Formula:
By the enzyme of recycling according to same reaction condition, reaction rate is measured, the vigor for obtaining enzyme is maintained at 75-100%
Between.Measuring method is to react for the first time, and the cellulose conversion ratio in reaction time is x1, after recycling, in same reaction time
Cellulose conversion ratio be x2, then the vitality retaining percentege formula of enzyme:
The present embodiment recycles immobilised enzymes with magnet, and is used for next secondary response, other reaction conditions are constant.
The invention demonstrates that magnetic immobilized enzyme is hydrolyzed for stalk, it can achieve 10 times or more and still to keep certain vigor.
Embodiment one
100 grams of corn stover after taking steam blasting, 150 DEG C of explosion temperature, pressure 15atm.By test, after explosion
After stalk drying, content of cellulose 41%.After hot water washs, content of cellulose rises to 50%.
10 grams of dry stalk is taken to be put into round-bottomed flask, it is molten that 100 grams of Acetic acid-sodium acetate bufferings are added according to solid-to-liquid ratio 1: 10
Liquid makes pH be maintained at 4.8.Mixed solution is heated to 50 DEG C, is reacted in the form of magneton stirring, and according to 100IU/g
Ratio, 4g magnetic immobilized enzyme is added, for the another 10 μ g that are added available commercially from by enzyme, i.e. adding proportion is the 0.1% of stalk dry weight.Instead
After answering 24 hours, determining concentration of glucose is 2,430mg/dL, and the conversion ratio for converting into glucose is 44%.
Magnetic immobilized enzyme is recycled with magnet, and is used for next secondary response, other reaction conditions are constant.Recycling
The vitality retaining percentege of enzyme is illustrated in fig. 2 shown below.
Embodiment two
A small amount of rice straw is taken, is ground using ball mill, milling time 24 hours.By test, the fiber of stalk
Cellulose content is 32%, and after hot water washs, content of cellulose rises to 38%.
3 grams of dry stalk powder is taken, is added in plastic centrifuge tube, puts into acetic acid sodium acetate buffer by solid-to-liquid ratio 1: 3,
PH value is adjusted to 6, is put into constant temperature oscillation box, oscillation frequency is 40 revs/min, and temperature is controlled at 60 DEG C, according to substrate vigor
0.15 gram of immobilised enzymes of Standard entertion of 1IU/g, the another 15 μ g that are added are available commercially from by enzyme, that is, the 0.5% of stalk dry weight, instead
It answers 72 hours, concentration of glucose is 3,310mg/dL after determining reaction, and converting into inversion rate of glucose is 26%.
Immobilised enzymes is recycled with magnet, and is used for next secondary response, other reaction conditions are constant.Recycle enzyme
Vitality retaining percentege is as shown in Figure 3.
Embodiment 3
100 grams of wheat stalk are taken, the sodium hydroxide solution of 0.4mol/L is added, boils 1 hour, lye is filtered off, is handled
Stalk afterwards after measured, content of cellulose 36%.
It weighs in 10 grams of addition stainless cylinder of steel of stalk, puts into citric acid-sodium citrate buffer according to solid-to-liquid ratio 1: 20,
It is reacted in the form of stirring paddle stirring, stirring rate is 100 revs/min, and pH value is controlled 4.0, starts stirring, temperature control
System is at 40 DEG C, and according to the Standard entertion 1g immobilised enzymes of substrate vigor 20IU/g, for the another 20 μ g that are added available commercially from by enzyme, i.e. stalk is dry
The 0.2% of weight reacts 48 hours, and the concentration of glucose after determining reaction is 550mg/dL, and equivalent inversion rate of glucose is
30.6%.
Immobilised enzymes is recycled with magnet, and is used for next secondary response, other reaction conditions are constant.Recycle enzyme
Vitality retaining percentege is as shown in Figure 4.
The preferred embodiment of the present invention has shown and described in above description, as previously described, it should be understood that the present invention is not office
Be limited to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification and
Environment, and can be changed within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge
It is dynamic.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, then it all should be appended by the present invention
In scope of protection of the claims.
Claims (6)
1. a kind of method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application, it is characterised in that: this method implementation steps are such as
Under:
Step 1: the buffer solution that concentration is 0.1mol/L being added in stalk after the pre-treatment, reaction is hydrolyzed, generate solid-liquid
Than the solidliquid mixture for 1:3-1:20, the pH value of the solidliquid mixture is 4-6, and the buffer solution includes acetic acid-acetic acid
One of sodium buffer solution or citric acid-sodium citrate buffer；
Step 2: cellulase being added in the solidliquid mixture of step 1, the cellulase includes that additive amount is stalk dry weight
Magnetic immobilized enzyme of the free enzyme and vigor of 0.1-0.5% in 1-100IU/g；
Step 3: in 40-60 DEG C of temperature, carrying out 24-72 hours hydrolysis, obtain final hydrolysis solution；
Step 4: detecting the concentration of glucose in final hydrolysis solution；
Step 5: final hydrolysis solution is used for fermenting alcohol after glucose concentration determination.
2. the method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in a kind of application described in claim 1, it is characterised in that: step
Solidliquid mixture in rapid 3 can Hybrid Heating, stirring or concussion reaction is hydrolyzed；The speed of stirring therein is 60-180
Rev/min, the frequency of concussion is 25-150 revs/min.
3. the method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application described in claim 1, it is characterised in that: step 2
Described in magnetic immobilized enzyme production method, include the following steps:
Step 1: following component: 100 parts of reaction substrate, 50-200 parts of template solution, ferroferric oxide powder is weighed by mass parts
1-2 parts, 2-30 parts of cellulase, the reaction substrate include tetraethyl orthosilicate or positive quanmethyl silicate；The template
Solution is mass percent in the glucose of 28-32%, maltose, fructose, dibenzoyl-L-tartaric, cyclodextrin, urine
The solution of any one of element, glycerol, soluble starch, citric acid, ascorbic acid or oligopeptides；
Step 2: weighing hydrochloric acid by the amount of substance of reaction substrate weighed in step 1, and by following molar ratio, reaction substrate with
The molar ratio of hydrochloric acid is 100:1-200:1, and the concentration of hydrochloric acid is equal to or less than 0.2mol/L；
Step 3: reaction substrate weighed in step 1 is added in reaction kettle；
Step 4: reaction substrate is cooled to 15 DEG C or less；
Step 5: weighed hydrochloric acid and stirring are until obtained solution becomes in the reaction substrate a dropping step two into reaction kettle
Clear, control reaction temperature are less than or equal to 30 DEG C；
Step 6: it in the solution that template solution weighed in step 1 addition step 5 is obtained, and stirs to uniformly mixed；
Step 7: coproduct ethanol or methanol in vacuum distillation removal reaction solution obtain colloidal sol；The vacuum distillation pressure
Power is 0.005MPa hereinafter, temperature is 30 DEG C or less；
Step 8: the sodium hydroxide solution that concentration is 0.38-0.42mol/L and stirring, drop is added dropwise in the colloidal sol obtained to step 7
Rate of acceleration is less than or equal to 1L/min, until pH value is greater than or equal to 3.5, weighed ferroferric oxide powder in step 1 is added
After cellulase, continue that sodium hydroxide solution is added dropwise, until pH value is greater than or equal to 4.2；
Step 9: the reaction product that step 8 is obtained is transferred in open containers, be cooled to 0 DEG C or less and keep 10 hours with
On, obtain gel.
4. the method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application as claimed in claim 3, it is characterised in that: magnetism is solid
Surely the recovery method for changing enzyme is magnetic recovery method, and the magnet used is permanent magnet or electromagnet, and the permanent magnet uses
Ndfeb magnet；Magnet is placed in the final hydrolysis solution & stir in step 3, can be gone out by magnet adsorption recovery
Magnetic immobilized enzyme.
5. the method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application as claimed in claim 3, it is characterised in that: magnetism is solid
Surely the recovery method for changing enzyme is magnetic recovery method, will finally be hydrolyzed instead using electromagnet of the magnetic field strength between 0.2-0.4T
After answering the magnetic immobilized enzyme in solution to draw, power-off is i.e. recyclable.
6. the method that magnetic Nano immobilized enzyme hydrolyzing straw can be recycled in application described in claim 4 or 5, it is characterised in that: magnetic
It needs first to be measured before the recycling of property immobilised enzymes, enzyme amount needed for an IU refers to one minute 1 micromole substrate of catalysis；Tool
Body mensuration mode is, using 1 gram of filter paper as substrate, to put into m grams of enzyme, after reaction t minutes, has c grams of filter paper transformation for grape
The molal weight of sugar, glucose is M grams/mol:
After the enzyme amount for obtaining IU representative, according to the quality of substrate, required enzyme amount is calculated.
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| "Immobilized cellulase on Fe304 nanoparticles as a magnetically recoverable biocatalyst for the decomposition of corncob";Qikun Zhang等;《Chinese Journal of Catalysis》;20160612;第37卷(第3期);全文 |
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