CN106480130A - A kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw - Google Patents

A kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw Download PDF

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CN106480130A
CN106480130A CN201611135542.5A CN201611135542A CN106480130A CN 106480130 A CN106480130 A CN 106480130A CN 201611135542 A CN201611135542 A CN 201611135542A CN 106480130 A CN106480130 A CN 106480130A
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immobilized enzyme
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straw
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CN106480130B (en
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危岩
孙亚飞
于伟娜
叶培荣
童龙欢
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Xiamen Geng Nengxin Materials Technology Co Ltd
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    • C12P19/00Preparation of compounds containing saccharide radicals
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    • C12N9/2405Glucanases
    • C12N9/2434Glucanases acting on beta-1,4-glucosidic bonds
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    • C12P7/00Preparation of oxygen-containing organic compounds
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    • C12P7/06Ethanol, i.e. non-beverage
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Abstract

The present invention discloses a kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw, the buffer solution that concentration is 0.1mol/L is added to be hydrolyzed reaction in straw after the pre-treatment, produce the solidliquid mixture that solid-to-liquid ratio is 1: 31: 20, the pH value of this solidliquid mixture is 46;Add cellulase in solidliquid mixture, described cellulase includes the cellulase that addition is straw dry weight 0.1 0.5%, and vigor is in the cellulase of 1 100IU/g;In 40 60 DEG C of temperature, carry out the hydrolysis of 24 72 hours, obtain final hydrolysis solution.Adopt Magnet to reclaim immobilized enzyme after hydrolysis, the response rate, between 80 100%, can reach more than 10 times and still keep certain vigor, and obtain the vigor of enzyme and be maintained between 75 100%.Glucose solution after hydrolysis is used for fermenting alcohol after measuring.

Description

A kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw
Technical field
The present invention relates to a kind of technology applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw, belonging to biological material should Use field.
Background technology
As large agricultural country, the every annual meeting of China produces hundred million tons of all kinds of straws of 7-9, wherein Semen Maydiss, Semen Tritici aestivi and three kinds of works of Oryza sativa L. The straw of thing accounts for 80% about.Because straw yield is huge, from 2005, straw integrated treatment project becomes state key and props up Hold project, especially hydrolyze the industry producing glucose fermenting alcohol then with straw for raw material.However, can hydrolyze in straw Main component for glucose is cellulose, needs just to be hydrolyzed into monomer in the presence of cellulase, and the making of cellulose With cost of a relatively high so that this technology practical application be subject to severely restricts.According to actual measuring and calculating, enzyme preparation is in cellulose second Totle drilling cost in alcohol industry accounts for nearly 1/3rd, is the Main Bottleneck of industry development.
Disclosed in 2015-04-08, a kind of magnetic immobilized cellobiase of Application No. 201410637435.7 is received Rice mesoporous material and preparation method thereof.This material by being fixed the multiple utilization it is achieved that enzyme to enzyme, and passes through magnetic material Material improves the operability of recovery.This invention experiments prove that, immobilized cellobiase is directed to fiber two in the lab The process that sugar is hydrolyzed into glucose is effective, effectively can be enriched with by Magnet.But this kind of material is used for actual straw Process, there is no practical data at present, the method also not having to adopt.
Content of the invention
The problem existing for prior art, the invention provides a kind of recyclable magnetic Nano of application fixes enzyme hydrolysiss straw The method of stalk, establishes processing mode and the hydrolysising condition of straw, solves the practical problem of straw hydrolysis, magnetic immobilized enzyme is permissible Reusability, activity is maintained, by the cost control of this production technology under proportional space.
For achieving the above object, technical solution of the present invention is:
A kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw, the method implementation steps are as follows:Step 1: The buffer solution that concentration is 0.1mol/L is added to be hydrolyzed reaction in straw after the pre-treatment, producing solid-to-liquid ratio is 1: 3-1: 20 solidliquid mixture, the pH value of this solidliquid mixture is 4-6;Described buffer solution includes NaAc_HAc buffer solution, Or citric acid-sodium citrate buffer;Step 2:Cellulase, described cellulose is added in the solidliquid mixture of step 1 Enzyme includes the cellulase that addition is straw dry weight 0.1-0.5%, and vigor is in the cellulase of 1-100IU/g;Step 3: In 40-60 DEG C of temperature, carry out the hydrolysis of 24-72 hour, obtain final hydrolysis solution;Step 4:Detect final water Concentration of glucose in solution reaction solution;Step 5:Final hydrolysis solution is used for second of fermenting after glucose concentration determination Alcohol.
Technique scheme is beneficial in that:
The present invention apply recyclable magnetic Nano immobilized enzyme hydrolyzing straw method be by straw through pretreatment it Afterwards, add a small amount of cellulase, be hydrolyzed under constant temperature.After hydrolysis, immobilized enzyme is reclaimed using Magnet, the response rate exists Between 80-100%, can reach more than 10 times and still keep certain vigor, and obtain the vigor of enzyme and be maintained between 75-100%. Glucose solution after hydrolysis is used for fermenting alcohol after measuring.
Brief description
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this Bright schematic description and description is used for explaining the present invention, does not constitute inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is the equipment schematic diagram that used of production method embodiment of magnetic immobilized enzyme of the present invention;
Fig. 2 is the schematic diagram of the vitality retaining percentege reclaiming enzyme in the embodiment of the present invention one;
Fig. 3 is the schematic diagram of the vitality retaining percentege reclaiming enzyme in the embodiment of the present invention two;
Fig. 4 is the schematic diagram of the vitality retaining percentege reclaiming enzyme in the embodiment of the present invention three.
Specific embodiment
In order that the technical problem to be solved, technical scheme and beneficial effect are clearer, clear, below tie Close drawings and Examples, 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.
A kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw disclosed by the invention, described straw includes But it is not limited only to corn straw, wheat stalk, rice straw, Radix Ipomoeae straw, soybean stalk etc., the straw being related in the method is Through the straw of pretreatment, preconditioning technique includes but are not limited to cutting and grinding, decocting in water, steam, ball milling, explosion, microwave etc. Physical means, and include but is not limited to all kinds of chemical means such as pickling, alkali cleaning, organic solvent, also include enzymolysis, fungal biodegradation etc. Biological means, thus remove the by-product wherein having inhibitory action, such as polyphenols.
Cellulose assay method according to the present invention is as follows:
Accurately weigh 1.0000~1.0500g sample (G1) in clean 250mL ground conical flask, add 25mL 20% nital, loads onto reflux condensing tube and heats 1h in boiling water bath, little in the glass core filter of constant weight in advance The heart pours out supernatant, vacuum filtration, filtering residue is carefully moved back in former conical flask, repeats aforesaid operations for several times, until fiber becomes Till white.Finally entire contents in conical flask are moved into filter, vacuumizing filtration, with 10mL 20% nital Washing residue, reusable heat water washing to washing liquid methyl orange reagent test is not in acid, finally divides 2 washings with 10mL ethanol, Filtering residue is dried to constant weight (G2) in 105 DEG C.Content of cellulose formula:
The method of the described recyclable magnetic Nano immobilized enzyme hydrolyzing straw of application, implementation steps are as follows:
Step 1:Take the corn straw after the physical means such as cutting and grinding, decocting in water, steam, ball milling, explosion, microwave, Semen Tritici aestivi Straw, rice straw, Radix Ipomoeae straw, soybean stalk etc.,.
Step 2:The buffer solution that concentration is 0.1mol/L is added to be hydrolyzed reaction in straw after the pre-treatment, generation Solid-to-liquid ratio is 1: 3-1: 20 solidliquid mixture, and the pH value of this solidliquid mixture is 4-6;Described buffer solution include acetic acid- Sodium acetate buffer, or citric acid-sodium citrate buffer;
Step 3:Add cellulase in the solidliquid mixture of step 1, it is straw that described cellulase includes addition The cellulase of dry weight 0.1-0.5%, and vigor is in the cellulase of 1-100IU/g;
Step 4:In 40-60 DEG C of temperature, 24-72 hour is carried out to solidliquid mixture Hybrid Heating, stirring or concussion Hydrolysis, obtain final hydrolysis solution;The speed of stirring therein is 60-180 rev/min, and the frequency of concussion is 25- 150 revs/min.Described stirring or concussion form include and are not limited to stirring 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:Detect the concentration of glucose in final hydrolysis solution..
Step 5:Detect 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 measuring..
Addition described in above-mentioned steps 2 is the cellulase of straw dry weight 0.1-0.5% is available commercially from by enzyme, described Vigor 1-100IU/g cellulase be magnetic immobilized enzyme;Incorporated by reference to shown in Fig. 1.
The production method of this magnetic immobilized enzyme, comprises the steps:
Step one:Weigh following component by mass parts:100 parts of reaction substrate, 100 parts of template solution, ferroso-ferric oxide powder Last 2 parts, 20 parts of cellulase;Reaction substrate is tetraethyl orthosilicate and dimethyldisiloxane, wherein tetraethyl orthosilicate matter Amount accounting 80%, dimethyldisiloxane quality accounting 20%;Template solution is glucose solution, and glucose quality percentage ratio is 30%;
Step 2:By the amount of substance of the reaction substrate weighing in step one, and weigh hydrochloric acid by following mol ratio, react bottom Thing is 150: 1 with the mol ratio of hydrochloric acid, and described concentration of hydrochloric acid is 0.17mol/L;
Step 3:The reaction substrate weighing in step one is placed in storage tank 1, and is added in reactor 5 by pump 11;
Step 4:By psychrolusia, reaction substrate is cooled to 10 DEG C;
Step 5:Hydrochloric acid in step 2 is placed in storage tank 2, and is dripped to the reaction substrate in reactor 5 by pump 21 Salt adding acid simultaneously becomes clear with agitator 52 stirring up to the solution obtaining, and controlling reaction temperature is less than 30 DEG C, stirring Speed is 100 revs/min;
Step 6:The template solution weighing in step one is placed in storage tank 3, and by pump 31 to step 5 obtain molten Liquid adds template solution, and is stirred to mix homogeneously with agitator 52, and mixing speed is 100 revs/min;
Step 7:Vacuum distillation is carried out up to not by vacuum pump 6 and cooler 7 to step 6 resulting solution in reactor 5 Produce volatilization again, to remove the coproduct ethanol in reaction solution, obtain colloidal sol;Described vacuum distillation pressure is 0.005MPa, temperature is 25 DEG C, and coproduct ethanol is cooled back in recycling can 8
Step 8:Close down vacuum pump 6 and cooler 7, the sodium hydroxide solution for 0.4mol/L for the concentration is added storage tank 4 In, the above-mentioned sodium hydroxide solution of colloidal sol Deca that obtained to step 7 by pump 41 is simultaneously stirred with agitator 52, and drop rate is 1L/min, and real-time monitoring pH value, until pH value reaches 3.5, are weighed in reactor 5 addition step 1 by charge door 52 Ferroferric oxide powder and cellulase, continue Deca sodium hydroxide solution, until pH value reaches 4.2, mixing speed is 100 Rev/min;
Step 9:The product that step 8 is obtained is transferred to open containers 9 from reactor 5, is cooled to 0 DEG C and protects Hold 12 hours, obtain gel;
Step 10:It is 0.2mm particles below that described gel is ground into granularity using pulverizer, and is dried, Using the dried magnetic immobilized enzyme of packaging of aluminium foil bag.
In above-described embodiment, in step one, adjust final producing by adding dimethyldisiloxane in reaction substrate The feature of product, makes magnetic immobilized enzyme have more preferable toughness, it is to avoid breakable problem in use.In step 4 By psychrolusia by reaction substrate temperature control at 10 DEG C, effectively prevent the production that reaction under room temperature leads to heat gathering to cause Dangerous it is ensured that industrial operability.In step 5, hydrochloric acid is added by Deca mode, it is to avoid disposable addition is led Cause reaction excessively violent, in order to better control over reaction rate, improve reaction safety.Pass through vacuum distillation in step 7 Remove byproduct of reaction ethanol, it is to avoid ethanol impacts to the vigor of cellulase, also can avoid follow-up gel simultaneously Speed is excessively slow.In step 8 by way of adding ferroso-ferric oxide and cellulase in the middle of the N-process, it is to avoid Reaction early stage adds, and because viscosity is little, acid height, ferroso-ferric oxide and cellulase are susceptible to asking of reunion when being directly added into Topic, has also avoided the phase after the reaction to add simultaneously, and because system viscosity is high, ferroso-ferric oxide and cellulase are difficult scattered Problem, thus fundamentally solve cladding problem of non-uniform.In step 9 using carrying out gel under acid condition, it is to avoid in Property under the conditions of gel be difficult the problem that shifts, and avoid the uneven problem of the gel that gel under neutrallty condition occurs.In step In rapid ten, using the magnetic immobilized enzyme of packaging of aluminium foil bag production, enzyme can be avoided to inactivate, thus improving depositing of magnetic immobilized enzyme Storage stability.Because embodiments of the invention key step is all realized in a kettle., therefore this production method has to equipment The less demanding, advantage such as simple process, operability height.After above-mentioned steps 4 hydrolysis, permissibleMagnetic is reclaimed using Magnet solid Surely change enzyme, give over to and use next time.Using Magnet be permanent magnet or electric magnet, and described permanent magnet adopts neodymium-iron-boron Ferrum;Magnet is positioned over the hydrolysis in step 4In the equipment of reaction, stirringFinal hydrolysisSolution, you can inhaled by Magnet Attached reclaim out magnetic immobilized enzyme.Described magnetic immobilized enzyme recovery method can also using magnetic field intensity in 0.2- Electric magnet between 0.4T is by final hydrolysisMagnetic in solutionAfter immobilized enzyme is drawn, power-off is recyclable.
Before Magnet reclaims 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.Concrete mensuration mode is, using 1 gram of filter paper as substrate, puts into m gram of enzyme (free enzyme or fixation Enzyme), after reaction t minute, there is c gram of filter paper to change for glucose, the molal weight of glucose is the M gram/mol of (work of enzyme Power formula):
After obtaining the enzyme amount that an IU represents, according to the quality of substrate, calculate required enzyme amount.
As improving further, the magnetic immobilized enzyme recovery method in the present invention is magnetic recovery method, the magnetic of employing Ferrum is permanent magnet or electric magnet.Permanent magnet generally adopts ndfeb magnet, is positioned over stirring in consersion unit, you can reclaim out magnetic Property immobilized enzyme.It is more convenient compared with permanent magnet to be reclaimed using electric magnet, and magnetic field intensity, between 0.2-0.4T, immobilized enzyme is inhaled After taking, power-off is recyclable.
Magnetic immobilized enzyme after recovery, the response rate is between 80-100%.The calculating side of the magnetic immobilized enzyme response rate Formula:
By the enzyme reclaiming according to same reaction condition, measure reaction rate, the vigor obtaining enzyme is maintained at 75-100% Between.Assay method is to react first, and the cellulose conversion ratio in reaction time is x1, after recovery, in same reaction time Cellulose conversion ratio be x2, then the vitality retaining percentege formula of enzyme:
Immobilized enzyme is reclaimed by the present embodiment with Magnet, and is used for next secondary response, and other reaction conditions are all constant.
The invention demonstrates that, magnetic immobilized enzyme is used for straw and hydrolyzes, and can reach more than 10 times and still keep certain vigor.
Embodiment one
Take 100 grams of the corn straw after steam explosion, 150 DEG C of explosion temperature, pressure 15atm.Through test, after explosion After straw is dried, content of cellulose is 41%.After hot wash, content of cellulose rises to 50%.
Take and straw 10 gram is dried puts in round-bottomed flask, add 100 grams of Acetic acid-sodium acetate bufferings molten 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, add 4g magnetic immobilized enzyme, another add 10 μ g available commercially from by enzyme, that is, adding proportion is the 0.1% of straw dry weight.Instead After answering 24 hours, determining concentration of glucose is 2,430mg/dL, and the conversion ratio of equivalent glucose is 44%.
Magnetic immobilized enzyme is reclaimed with Magnet, and is used for next secondary response, other reaction conditions are all constant.Reclaim The vitality retaining percentege of enzyme is illustrated in fig. 2 shown below.
Embodiment two
Take a small amount of rice straw, be ground using ball mill, milling time 24 hours.Through test, the fiber of straw Cellulose content is 32%, and after hot wash, content of cellulose rises to 38%.
Take and stalk powder 3 gram be dried, add in plastic centrifuge tube, put into acetic acid sodium acetate buffer by solid-to-liquid ratio 1: 3, PH value is adjusted to 6, puts in isothermal vibration case, concussion frequency be 40 revs/min, temperature control at 60 DEG C, according to substrate vigor 0.15 gram of immobilized enzyme of the Standard entertion of 1IU/g, 15 μ g are available commercially from by enzyme for another addition, that is, be equivalent to the 0.5% of straw dry weight, instead Answer 72 hours, after determining reaction, concentration of glucose is 3,310mg/dL, equivalent inversion rate of glucose is 26%.
Immobilized enzyme is reclaimed with Magnet, and is used for next secondary response, other reaction conditions are all constant.Reclaim enzyme Vitality retaining percentege is as shown in Figure 3.
Embodiment 3
Take 100 grams of wheat stalk, add the sodium hydroxide solution of 0.4mol/L, boil 1 hour, alkali liquor is filtered off, process After measured, content of cellulose is 36% to straw afterwards.
Weigh 10 grams of straw to add in stainless cylinder of steel, put into citric acid-sodium citrate buffer according to solid-to-liquid ratio 1: 20, Reacted in the form of stirring paddle stirring, stir speed (S.S.) is 100 revs/min, pH value controls 4.0, starts stirring, temperature control , at 40 DEG C, according to the Standard entertion 1g immobilized enzyme of substrate vigor 20IU/g, available commercially from by enzyme, that is, straw is dry for another addition 20 μ g for system The 0.2% of weight, reacts 48 hours, and determining reacted concentration of glucose is 550mg/dL, and equivalent inversion rate of glucose is 30.6%.
Immobilized enzyme is reclaimed with Magnet, and is used for next secondary response, other reaction conditions are all constant.Reclaim enzyme Vitality retaining percentege is as shown in Figure 4.
Described above illustrate and describes the preferred embodiments of the present invention, as previously mentioned it should be understood that the present invention not office Be limited to form disclosed herein, be not to be taken as the exclusion to other embodiment, and can be used for various other combinations, modification and Environment, and can be changed by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein Dynamic.And the change that those skilled in the art are carried out and change without departing from the spirit and scope of the present invention, then all should be appended by the present invention In scope of the claims.

Claims (6)

1. a kind of apply recyclable magnetic Nano immobilized enzyme hydrolyzing straw method it is characterised in that:The method implementation steps are such as Under:
Step 1:The buffer solution that concentration is 0.1mol/L is added to be hydrolyzed reaction in straw after the pre-treatment, generation solid-liquid Than for 1: 3-1: 20 solidliquid mixture, the pH value of this solidliquid mixture is 4-6.Described buffer solution includes acetic acid-acetic acid Sodium buffer solution, or citric acid-sodium citrate buffer;
Step 2:Add cellulase in the solidliquid mixture of step 1, it is straw dry weight that described cellulase includes addition The cellulase of 0.1-0.5%, and vigor is in the cellulase of 1-100IU/g;
Step 3:In 40-60 DEG C of temperature, carry out the hydrolysis of 24-72 hour, obtain final hydrolysis solution;
Step 4:Detect the concentration of glucose in final hydrolysis solution;
Step 5:Final hydrolysis solution is used for fermenting alcohol after glucose concentration determination.
2. a kind of application recyclable magnetic Nano immobilized enzyme hydrolyzing straw described in claim 1 method it is characterised in that:Step Solidliquid mixture in rapid 3 can be hydrolyzed reaction in Hybrid Heating, stirring or concussion;The speed of stirring therein is 60-180 Rev/min, the frequency of concussion is 25-150 rev/min.
3. described in claim 1 application recyclable magnetic Nano immobilized enzyme hydrolyzing straw method it is characterised in that:Step 2 Described in vigor 1-100IU/g cellulase be magnetic immobilized enzyme;The production method of this magnetic immobilized enzyme, including Following steps:
Step one:Weigh following component by mass parts:100 parts of reaction substrate, template solution 50-200 part, ferroferric oxide powder 1-2 part, cellulase 2-30 part.Described reaction substrate includes tetraethyl orthosilicate or positive quanmethyl silicate;Described template Solution is mass percent in the glucose of 28-32%, maltose, Fructose, dibenzoyl-L-tartaric, cyclodextrin, urine The solution of any one in element, glycerol, soluble starch, citric acid, ascorbic acid or oligopeptide;
Step 2:By the amount of substance of the reaction substrate weighing in step one, and weigh hydrochloric acid by following mol ratio, reaction substrate with The mol ratio of hydrochloric acid is 100: 1-200: 1, and described concentration of hydrochloric acid is equal to or less than 0.2mol/L;
Step 3:The reaction substrate weighing in step one is added in reactor;
Step 4:Reaction substrate is cooled to less than 15 DEG C;
Step 5:The hydrochloric acid that weighs in the reaction substrate Deca step 2 in reactor simultaneously stirs until the solution obtaining becomes Clear, controlling reaction temperature is less than or equal to 30 DEG C;
Step 6:In the solution that the template solution weighing in step one addition step 5 is obtained, and stir to mix homogeneously;
Step 7:Vacuum distillation removes coproduct ethanol or methanol in reaction solution, obtains colloidal sol;Described vacuum distillation pressure Power is below 0.005MPa, and temperature is less than 30 DEG C;
Step 8:The colloidal sol Deca concentration obtaining to step 7 is the sodium hydroxide solution of 0.38-0.42mol/L and stirs, and drips Rate of acceleration is less than or equal to 1L/min, until pH value is more than or equal to 3.5, adds the ferroferric oxide powder weighing in step one After cellulase, continue Deca sodium hydroxide solution, until pH value is more than or equal to 4.2;
Step 9:The product that step 8 is obtained is transferred in open containers, be cooled to less than 0 DEG C and keep 10 hours with On, obtain gel.
4. described in claim 3 application recyclable magnetic Nano immobilized enzyme hydrolyzing straw method it is characterised in that:Magnetic is solid Surely change enzyme recovery method be magnetic recovery method, the Magnet of employing is permanent magnet or electric magnet, and described permanent magnet is adopted Use ndfeb magnet;Magnet is positioned over the final hydrolysis solution & stir in step 3, you can reclaim by magnet adsorption Go out magnetic immobilized enzyme.
5. described in claim 3 application recyclable magnetic Nano immobilized enzyme hydrolyzing straw method it is characterised in that:Magnetic is solid Surely change enzyme recovery method be magnetic recovery method, will finally be hydrolyzed using electric magnet between 0.2-0.4T for the magnetic field intensity After magnetic immobilized enzyme in reaction solution is drawn, power-off is recyclable.
6. described in claim 4,5 application recyclable magnetic Nano immobilized enzyme hydrolyzing straw method it is characterised in that:Magnetic Need first to be measured before the recovery of immobilized enzyme, an IU refers to the enzyme amount needed for one minute catalysis 1 micromole substrate;Specifically Mensuration mode is, using 1 gram of filter paper as substrate, puts into m gram of enzyme (free enzyme or immobilized enzyme), after reaction t minute, has c Gram filter paper changes for glucose, and the molal weight of glucose is M gram/mol:After obtaining the enzyme amount that an IU represents, according to The quality of substrate, calculates required enzyme amount.
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CN112746087A (en) * 2019-10-31 2021-05-04 中国石油化工股份有限公司 Method for detoxifying lignocellulose hydrolysate
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