CN106381268A - Method for continuously hydrolyzing cellobiose in straws by using immobilized enzyme microreactor - Google Patents
Method for continuously hydrolyzing cellobiose in straws by using immobilized enzyme microreactor Download PDFInfo
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- CN106381268A CN106381268A CN201610781815.7A CN201610781815A CN106381268A CN 106381268 A CN106381268 A CN 106381268A CN 201610781815 A CN201610781815 A CN 201610781815A CN 106381268 A CN106381268 A CN 106381268A
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- cellobiose
- microreactor
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- immobilized enzyme
- flow velocity
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- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 title claims abstract description 71
- 239000010902 straw Substances 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 24
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 23
- 230000003301 hydrolyzing effect Effects 0.000 title abstract 2
- 239000010453 quartz Substances 0.000 claims abstract description 55
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 55
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000000243 solution Substances 0.000 claims abstract description 44
- 102000004190 Enzymes Human genes 0.000 claims abstract description 35
- 108090000790 Enzymes Proteins 0.000 claims abstract description 35
- 230000007062 hydrolysis Effects 0.000 claims abstract description 28
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 28
- 239000007864 aqueous solution Substances 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 16
- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 16
- 239000000758 substrate Substances 0.000 claims abstract description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 5
- 239000008103 glucose Substances 0.000 claims abstract description 5
- 239000007822 coupling agent Substances 0.000 claims abstract description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 36
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 33
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000008213 purified water Substances 0.000 claims description 21
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 14
- YUYCVXFAYWRXLS-UHFFFAOYSA-N trimethoxysilane Chemical compound CO[SiH](OC)OC YUYCVXFAYWRXLS-UHFFFAOYSA-N 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 9
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 9
- 230000015556 catabolic process Effects 0.000 claims description 4
- 238000006731 degradation reaction Methods 0.000 claims description 4
- 239000000835 fiber Substances 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000012494 Quartz wool Substances 0.000 claims 1
- 150000002016 disaccharides Chemical class 0.000 claims 1
- 210000005239 tubule Anatomy 0.000 claims 1
- 150000002772 monosaccharides Chemical class 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- UUEWCQRISZBELL-UHFFFAOYSA-N 3-trimethoxysilylpropane-1-thiol Chemical compound CO[Si](OC)(OC)CCCS UUEWCQRISZBELL-UHFFFAOYSA-N 0.000 abstract 1
- 238000005265 energy consumption Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 28
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 239000003708 ampul Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 238000012545 processing Methods 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000000376 reactant Substances 0.000 description 6
- 229920002678 cellulose Polymers 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 239000004810 polytetrafluoroethylene Substances 0.000 description 5
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 5
- 108010059892 Cellulase Proteins 0.000 description 4
- 229940106157 cellulase Drugs 0.000 description 4
- 229920002488 Hemicellulose Polymers 0.000 description 2
- 238000011953 bioanalysis Methods 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 229920005610 lignin Polymers 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- -1 3- sulfydryl Propyl Chemical group 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- MGCQZNBCJBRZDT-UHFFFAOYSA-N midodrine hydrochloride Chemical compound [H+].[Cl-].COC1=CC=C(OC)C(C(O)CNC(=O)CN)=C1 MGCQZNBCJBRZDT-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- QUNSENANWRESNO-UHFFFAOYSA-N toluene trimethoxysilane Chemical compound CO[SiH](OC)OC.Cc1ccccc1 QUNSENANWRESNO-UHFFFAOYSA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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- Chemical & Material Sciences (AREA)
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- General Chemical & Material Sciences (AREA)
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- Inorganic Chemistry (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
The invention discloses a method for continuously hydrolyzing cellobiose in straws by using an immobilized enzyme microreactor, which is characterized in that beta-glucosidase is immobilized in the microreactor, and a substrate cellobiose aqueous solution is injected into the microreactor for micro-reaction to degrade the cellobiose solution into glucose; wherein, in the microreactor, a carrier for fixing enzyme is a quartz capillary, and a coupling agent for fixing enzyme is a toluene solution of (3-mercaptopropyl) trimethoxysilane. According to the invention, the micro-reactor is selected to carry out enzymolysis on cellobiose in the straw to generate monosaccharide, the enzyme consumption is less, the reaction efficiency is greatly improved, and the energy consumption is reduced; meanwhile, the hydrolysis rate of cellobiose can be obviously improved by changing the control of the operating conditions, and the capability of treating straws is greatly improved. The device is a capillary hollow tube, has simple structure, can proportionally expand the production capacity by simply amplifying the number, and has good industrialization prospect.
Description
Technical field
The present invention relates to biocatalysis field is and in particular to a kind of utilize fiber two in enzyme microreactor continuous hydrolysis straw
The method of sugar.
Background technology
China, as large agricultural country, can generate more than 700,000,000 ton of straw every year, become " use is little " but must dispose
" garbage ".If to be processed by individual completely, occur as soon as the phenomenon of a large amount of burnings, not only waste of resource but also pollute environment.If
Straw is combined with modern biotechnology, is greatly improved its utilization rate.Agricultural crop straw is mainly by cellulose, half fiber
Element and lignin composition.Although lignin content is not high, it wraps in the outside of cellulose, and has with cellulose, hemicellulose
Stronger being connected chemically, hamper the degraded of cellulose and hemicellulose, thus largely effect on the degradation rate of straw.
The main method of straw processed has Physical, chemical method, bioanalysises at present.With scientific and technical development, bioanalysises because
Its advantage is increasingly taken seriously.
Biological method：This method has that action condition is gentle, specificity strong, the low advantage of non-environmental-pollution, processing cost.
Biological treatment had both been not required to too complicated equipment and excessive energy expenditure, was also not required to the conditions such as high temperature, high pressure, highly basic and strong acid,
There is small investment, simple to operate, the advantages of free from environmental pollution, thus it is straw processing method of greatest concern at present.
When producing bioenergy using straw for fermenting raw materials, needing for polysaccharide to be degraded to monosaccharide could be that how sharp microorganism is
With.Cellulase is the general name that lignocellulose degradation becomes one group of enzyme needed for glucose monomer, and it is not single enzyme, but
A kind of multi-component compound enzyme system.Easily cause the accumulation of cellobiose during cellulase hydrolysis straw, cellobiose removes
Strong feedback suppression can be formed to the catalytic action of cellulase, ethanol can not be converted in follow-up fermentation again.Cause
This, add appropriate beta-glucosidase during enzyme hydrolysiss, improves the synergism of each component in cellulase system, is to improve
Cellulose hydrolyzes one of effective measures of yield and glucose yield, is smoothed out in order to follow-up fermentation energy.
Content of the invention
The technical problem to be solved is to provide a kind of utilizing in immobilized enzyme micro-reactor continuous hydrolysis straw
The method of cellobiose, to solve the suppression for whole enzymolysis process for the generation of cellobiose in straw enzymolysis process, realizes
Continuous prodution.
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows：
A kind of method of cellobiose in utilization immobilized enzyme micro-reactor continuous hydrolysis straw, beta-glucosidase is solid
It is scheduled in microreactor, the circulation of substrate cellobiose solution is continuously injected in microreactor and carries out micro- reaction, make fiber two
Sugar juice is degraded to glucose；
Wherein, in microreactor, the carrier of immobilized enzyme is quartz capillary, and the coupling agent used by immobilized enzyme is (3- sulfydryl
Propyl group) trimethoxy silane toluene solution.
Wherein, described quartz capillary internal diameter is 530～630 microns, length range 60～480cm (preferably 240～
480cm, most preferably 300cm).
Wherein, the concentration of the described toluene solution of (3- mercaptopropyi) trimethoxy silane is 10%～15%V/V.
Wherein, method beta-glucosidase being fixed in microreactor comprises the steps：
(1) aqueous solution of Fluohydric acid. is continuously poured into quartz capillary 30～40min (preferably 30min), purified water is rinsed
To neutrality, then hydrochloric acid is continuously poured into quartz capillary 100～150min (preferably 100min), purified water is rinsed to neutrality, so
Afterwards NaOH aqueous solution is continuously poured into quartz capillary 100～150min (preferably 100min), purified water is rinsed to neutrality, blows
Dry, the toluene solution of (3- mercaptopropyi) trimethoxy silane is continuously poured into quartz capillary 12～14 hours, and (preferably 12 is little
When)；Finally dehydrated alcohol is continuously poured into quartz capillary 60～100min, dry up；
(2) enzyme liquid containing beta-glucosidase is passed through with the flow velocity circulation of 0.7～1 μ L/min (preferably 1 μ L/min)
In the quartz capillary in the microreactor that step (1) is processed, the set time is 8～16 hours (preferably 14 hours).Even
Continuous perfusion pH4.8 citric acid solution 2～2.5 hours, 5 μ L/min, rinse loose enzyme.
The successful microreactor of immobilization is saved in 4 DEG C of refrigerators, standby.
In step (1), the concentration of aqueous solution of Fluohydric acid. is 15～20%V/V (preferably 20%V/V), the aqueous solution of Fluohydric acid.
Flow velocity 10～20 μ L/min；The concentration of hydrochloric acid is 1～1.5mol/L (preferably 1mol/L), flow velocity 15～30 μ L/ of hydrochloric acid
min；The concentration of NaOH aqueous solution is 1～1.5mol/L (preferably 1mol/L), flow velocity 15～30 μ L/min of NaOH aqueous solution.
In step (1), the concentration of the toluene solution of (3- mercaptopropyi) trimethoxy silane is that 10%～15%V/V is (excellent
Select 10%V/V), the flow velocity of the toluene solution of (3- mercaptopropyi) trimethoxy silane is 0.5～1 μ L/min (preferably 1 μ L/
min)；The flow velocity of dehydrated alcohol is 15～20 μ L/min.
In step (2), the described enzyme liquid containing beta-glucosidase, the concentration of enzyme is 0.5～1.2mg/mL, preferably
1mg/mL, solvent is the citric acid solution of pH4.8.
The enzyme activity definition of beta-glucosidase：At 50 DEG C, pH is the enzyme consuming cellobiose per minute under conditions of 4.8
Amount.
The enzyme activity scope 6～9U/mg of beta-glucosidase of the present invention.
The assay method of immobilized enzyme is：500 microlitres of cellobiose solution are taken to be passed through in microreactor, coutroi velocity
5 microlitres per minute, reacts at 50 DEG C.It is immediately placed in boiling water after reaction and be incubated with terminating reaction, and by liquid chromatograph
(HPLC) measure the decrement of cellobiose in reactant liquor.The degradation rate that reaction terminates rear cellobiose reaches 50%, preserves one
After individual month, microreactor remnant enzyme activity exceeds 50% compared with resolvase, substantially increases the service life of enzyme.
Wherein, described cellobiose solution, the concentration of solute cellobiose is 3-12wt%, and solvent is pH4.0-5.5
Dilute sulfuric acid aqueous solution, the preferably dilute sulfuric acid aqueous solution of pH4.2～5.0.
Wherein, the flow velocity of substrate cellobiose solution is 1-5 μ L/min, and reaction time is 4～8h, in micro- reaction
Reaction temperature is 50-60 DEG C.
Beneficial effect：The present invention first sulfydryl is coupled on quartz capillary, and using HF increase roughness from
And increase the supported quantity of enzyme.Improve enzymolysis efficiency, decrease the usage amount of enzyme, extend the use time of enzyme.The present invention's
Microreactor operating process is simple, and operational approach is simple.Selection utilization microreactor of the present invention digests the cellobiose in straw
Generate monosaccharide, few with enzyme amount, reaction efficiency greatly improves, power consumption reduces；Meanwhile, controlled by changing operating condition, can be notable
Improve the percent hydrolysiss of cellobiose, the ability processing straw greatly improves.Assembly of the invention is capillary tube blank pipe, structure letter
Single, and only need simply number amplification to be got final product expanding production capacity of equal proportion, there is good industrial prospect.
Brief description
Fig. 1 is the structural representation of microreactor of the present invention.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, it is as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, and should not be also without limitation on basis described in detail in claims
Invention.
Embodiment 1：
Choose 300 centimetres of quartz capillary, 530 microns of internal diameter.It is connected with injection needle with PTFE tube, be installed to syringe pump
On.First with the HF aqueous solution of 20%V/V, quartz capillary is continuously poured into 30 minutes with flow velocity 20 μ L/min, purified water rinse in
Property, 1mol/L hydrochloric acid continuously pours into quartz capillary 100 minutes with 30 μ L/min, and purified water is rinsed to neutrality, 1mol/L's
NaOH aqueous solution continuously pours into quartz capillary 100 minutes with 30 μ L/min, and purified water is rinsed to neutrality.The quartz ampoule processing
Logical nitrogen dries up.The toluene solution of logical 10%V/V (3- mercaptopropyi) trimethoxy silane is continuously poured into the speed of 1 μ L/min
12 hours in quartz capillary.Finally continuously pour into 100 minutes in quartz capillary with dehydrated alcohol with the speed of 20 μ L/min,
Dry up.
By the enzyme liquid containing 0.5mg/mL beta-glucosidase (solvent is the citric acid solution of pH4.8) with 1 μ L/
The flow velocity circulation of min is passed through and is injected in quartz ampoule, is continuously injected into 12 hours, is more continuously passed through Fructus Citri Limoniae with the flow velocity of 5 μ L/min
Acid buffering solution (pH4.8) 2 hours, the microreactor being successfully prepared is saved in 4 DEG C of refrigerators, standby.
It is passed through cellobiose solution (solvent is the aqueous sulfuric acid of pH4.8) the 500 μ L that concentration of substrate is 6wt%, flow velocity
For 5 μ L/min, circulate under the conditions of 50 DEG C and be passed through microreactor reaction 4 hours.Reaction is collected effluent after terminating and is boiled 5 minutes,
Measure the decrement of cellobiose in reactant liquor by liquid chromatograph (HPLC).
Liquid chromatography results show, the decrement of cellobiose reaches 50%, and (in initial substrate, the amount of cellobiose deducts
The amount of the cellobiose in effluent, the ratio of both differences and initially middle cellobiose amount is the decrement of cellobiose,
Following examples are identical).Illustrate to be 300 centimetres in immobilized enzyme micro-reactor, enzyme concentration 0.5mg/mL, the immobilization time is 8
Hour, hydrolysis time be 4 little in the case of 60% is reached to cellobiose hydrolysis effect.
Embodiment 2：
Choose 300 centimetres of quartz capillary, 530 microns of internal diameter.It is connected with injection needle with PTFE tube, be installed to syringe pump
On.First with the HF aqueous solution of 20%V/V, quartz capillary is continuously poured into 30 minutes with flow velocity 20 μ L/min, purified water rinse in
Property, 1mol/L hydrochloric acid continuously pours into quartz capillary 100 minutes with 30 μ L/min, and purified water is rinsed to neutrality, 1mol/L's
NaOH aqueous solution continuously pours into quartz capillary 100 minutes with 30 μ L/min, and purified water is rinsed to neutrality.The quartz ampoule processing
Logical nitrogen dries up.The toluene solution of logical 10%V/V (3- mercaptopropyi) trimethoxy silane is continuously poured into the speed of 1 μ L/min
8 hours in quartz capillary.Finally continuously pour into 100 minutes in quartz capillary with dehydrated alcohol with the speed of 20 μ L/min,
Dry up.
By the enzyme liquid containing 1mg/mL beta-glucosidase (solvent is the citric acid solution of pH4.8) with 1 μ L/min
Flow velocity circulation be passed through and be injected in quartz ampoule, be continuously injected into 14 hours, then citric acid be continuously passed through with the flow velocity of 5 μ L/min and delay
Rush solution (pH4.8) 2 hours, the microreactor being successfully prepared is saved in 4 DEG C of refrigerators, standby.
It is passed through cellobiose solution (solvent is the aqueous sulfuric acid of pH4.8) the 500 μ L that concentration of substrate is 6wt%, flow velocity
For 5 μ L/min, circulate under the conditions of 50 DEG C and be passed through microreactor reaction 4 hours.Reaction is collected effluent after terminating and is boiled 5 minutes,
Measure the decrement of cellobiose in reactant liquor by liquid chromatograph (HPLC).
Liquid chromatography results show, the decrement of cellobiose reaches 70%, and (in initial substrate, the amount of cellobiose deducts
The amount of the cellobiose in effluent, the ratio of both differences and initially middle cellobiose amount is the decrement of cellobiose,
Following examples are identical).Illustrate to be 300 centimetres in immobilized enzyme micro-reactor, enzyme concentration 1mg/mL, the immobilization time is little for 14
When, hydrolysis time be 4 little in the case of 60% is reached to cellobiose hydrolysis effect.
Embodiment 3：
Choose 300 centimetres of quartz capillary, 530 microns of internal diameter.It is connected with injection needle with PTFE tube, be installed to syringe pump
On.First with the HF aqueous solution of 20%V/V, quartz capillary is continuously poured into 30 minutes with flow velocity 20 μ L/min, purified water rinse in
Property, 1mol/L hydrochloric acid continuously pours into quartz capillary 100 minutes with 30 μ L/min, and purified water is rinsed to neutrality, 1mol/L's
NaOH aqueous solution continuously pours into quartz capillary 100 minutes with 30 μ L/min, and purified water is rinsed to neutrality.The quartz ampoule processing
Logical nitrogen dries up.The toluene solution of logical 10%V/V (3- mercaptopropyi) trimethoxy silane is continuously poured into the speed of 1 μ L/min
10 hours in quartz capillary.Finally continuously pour into 100 minutes in quartz capillary with dehydrated alcohol with the speed of 20 μ L/min,
Dry up.
By the enzyme liquid containing 0.8mg/mL beta-glucosidase (solvent is the citric acid solution of pH4.8) with 1 μ L/
The flow velocity circulation of min is passed through and is injected in quartz ampoule, is continuously injected into 14 hours, is more continuously passed through Fructus Citri Limoniae with the flow velocity of 5 μ L/min
Acid buffering solution (pH4.8) 2 hours, the microreactor being successfully prepared is saved in 4 DEG C of refrigerators, standby.
It is passed through cellobiose solution (solvent is the aqueous sulfuric acid of pH4.8) the 500 μ L that concentration of substrate is 6wt%, flow velocity
For 5 μ L/min, circulate under the conditions of 50 DEG C and be passed through microreactor reaction 8 hours.Reaction is collected effluent after terminating and is boiled 5 minutes,
Measure the decrement of cellobiose in reactant liquor by liquid chromatograph (HPLC).
Liquid chromatography results show, the decrement of cellobiose reaches 70%, and (in initial substrate, the amount of cellobiose deducts
The amount of the cellobiose in effluent, the ratio of both differences and initially middle cellobiose amount is the decrement of cellobiose,
Following examples are identical).Illustrate to be 300 centimetres in immobilized enzyme micro-reactor, enzyme concentration 0.8mg/mL, the immobilization time is 10
Hour, hydrolysis time be 8 little in the case of 70% is reached to cellobiose hydrolysis effect.
Embodiment 4：
Choose 300 centimetres of quartz capillary, 530 microns of internal diameter.It is connected with injection needle with PTFE tube, be installed to syringe pump
On.First with the HF aqueous solution of 20%V/V, quartz capillary is continuously poured into 30 minutes with flow velocity 20 μ L/min, purified water rinse in
Property, 1mol/L hydrochloric acid continuously pours into quartz capillary 100 minutes with 30 μ L/min, and purified water is rinsed to neutrality, 1mol/L's
NaOH aqueous solution continuously pours into quartz capillary 100 minutes with 30 μ L/min, and purified water is rinsed to neutrality.The quartz ampoule processing
Logical nitrogen dries up.The toluene solution of logical 10%V/V (3- mercaptopropyi) trimethoxy silane is continuously poured into the speed of 1 μ L/min
12 hours in quartz capillary.Finally continuously pour into 100 minutes in quartz capillary with dehydrated alcohol with the speed of 20 μ L/min,
Dry up.
By the enzyme liquid containing 1mg/mL beta-glucosidase (solvent is the citric acid solution of pH4.8) with 1 μ L/min
Flow velocity circulation be passed through and be injected in quartz ampoule, be continuously injected into 14 hours, then citric acid be continuously passed through with the flow velocity of 5 μ L/min and delay
Rush solution (pH4.8) 2 hours, the microreactor being successfully prepared is saved in 4 DEG C of refrigerators, standby.
It is passed through cellobiose solution (solvent is the aqueous sulfuric acid of pH4.8) the 500 μ L that concentration of substrate is 6wt%, flow velocity
For 5 μ L/min, circulate under the conditions of 50 DEG C and be passed through microreactor reaction 12 hours.Reaction is collected effluent after terminating and is boiled 5 minutes,
Measure the decrement of cellobiose in reactant liquor by liquid chromatograph (HPLC).
Liquid chromatography results show, the decrement of cellobiose reaches 70%, and (in initial substrate, the amount of cellobiose deducts
The amount of the cellobiose in effluent, the ratio of both differences and initially middle cellobiose amount is the decrement of cellobiose,
Following examples are identical).Illustrate to be 300 centimetres in immobilized enzyme micro-reactor, enzyme concentration 0.8mg/mL, the immobilization time is 10
Hour, hydrolysis time be 8 little in the case of 75% is reached to cellobiose hydrolysis effect.
Embodiment 5：
Choose 60～480 centimetres of quartz capillary, 530 microns of internal diameter.It is connected with injection needle with PTFE tube, be installed to injection
On pump.Two groups of parallel tests.First with the HF aqueous solution of 20%V/V, 30 points of quartz capillary is continuously poured into flow velocity 20 μ L/min
Clock, purified water is rinsed to neutrality, and 1mol/L hydrochloric acid continuously pours into quartz capillary 100 minutes with 30 μ L/min, and purified water is rinsed
To neutral, the NaOH aqueous solution of 1mol/L continuously pours into quartz capillary 100 minutes with 30 μ L/min, purified water rinse in
Property.The quartzy Gutron nitrogen processing dries up.The toluene solution of logical 10%V/V (3- mercaptopropyi) trimethoxy silane is with 1 μ L/
The speed of min continuously pours into 12 hours in quartz capillary.Finally with dehydrated alcohol, stone is continuously poured into the speed of 20 μ L/min
100 minutes in English capillary tube, dry up.
By the enzyme liquid containing 1mg/mL beta-glucosidase (solvent is the citric acid solution of pH4.8) with 1 μ L/min
Flow velocity circulation be passed through and be injected in quartz ampoule, be continuously injected into 14 hours, then citric acid be continuously passed through with the flow velocity of 5 μ L/min and delay
Rush solution (pH4.8) 2 hours, the microreactor being successfully prepared is saved in 4 DEG C of refrigerators, standby.
It is passed through cellobiose solution (solvent is the aqueous sulfuric acid of pH4.8) the 500 μ L that concentration of substrate is 6wt%, flow velocity
For 5 μ L/min, circulate under the conditions of 50 DEG C and be passed through microreactor reaction 4 hours.Reaction is collected effluent after terminating and is boiled 5 minutes,
Measure the decrement of cellobiose in reactant liquor by liquid chromatograph (HPLC).
Liquid chromatography results show and are shown in Table 1, the different length of reactor and the relation of hydrolysis result, reach in length
During 480cm, the decrement of cellobiose reaches 78.01%, and reactor is good to cellobiose hydrolysis effect.Table 1 display is solid
Surely changing enzyme microreactor hydrolysis result with the length of reactor is not simple linear relationship.
Table 1
Claims (9)
1. in a kind of utilization immobilized enzyme micro-reactor continuous hydrolysis straw the method for cellobiose it is characterised in that by β-Portugal
Polyglycoside enzyme is fixed in microreactor, substrate cellobiose solution is injected in microreactor and carries out micro- reaction, make fiber
Disaccharide solution degradation is glucose；
Wherein, in microreactor, the carrier of immobilized enzyme is quartz capillary, and the coupling agent used by immobilized enzyme is (3- mercaptopropyi)
The toluene solution of trimethoxy silane.
2. in utilization immobilized enzyme micro-reactor continuous hydrolysis straw according to claim 1 cellobiose method, its
It is characterised by, described quartz capillary internal diameter is 530～630 microns.
3. in utilization immobilized enzyme micro-reactor continuous hydrolysis straw according to claim 1 cellobiose method, its
It is characterised by, the concentration of the described toluene solution of (3- mercaptopropyi) trimethoxy silane is 10%～15%V/V.
4. in utilization immobilized enzyme micro-reactor continuous hydrolysis straw according to claim 1 cellobiose method, its
It is characterised by, method beta-glucosidase being fixed in microreactor comprises the steps：
(1) aqueous solution of Fluohydric acid. is continuously poured into quartz capillary 30～40min, purified water is rinsed to neutrality, then by hydrochloric acid
Continuously pour into quartz capillary 100～150min, purified water is rinsed to neutrality, then NaOH aqueous solution is continuously poured into quartz wool
Tubule 100～150min, purified water is rinsed to neutrality, dries up, by the toluene solution of (3- mercaptopropyi) trimethoxy silane even
Continue and pour into quartz capillary 12～14 hours；Finally dehydrated alcohol is continuously poured into quartz capillary 60～100min, dry up；
(2) enzyme liquid containing beta-glucosidase is passed through with the circulation of the flow velocity of 0.7～1 μ L/min and processes through step (1)
In quartz capillary in microreactor, the set time is 8～16 hours, then with the flow velocity continuous pouring pH4.8 of 5 μ L/min
Citric acid solution 2～2.5 hours.
5. in utilization immobilized enzyme micro-reactor continuous hydrolysis straw according to claim 4 cellobiose method, its
It is characterised by, in step (1), the concentration of aqueous solution of Fluohydric acid. is 15～20%V/V, the flow velocity 10～20 of the aqueous solution of Fluohydric acid.
μL/min；The concentration of hydrochloric acid is 1～1.5mol/L, flow velocity 15～30 μ L/min of hydrochloric acid；The concentration of NaOH aqueous solution be 1～
Flow velocity 15～30 μ L/min of 1.5mol/L, NaOH aqueous solution.
6. in utilization immobilized enzyme micro-reactor continuous hydrolysis straw according to claim 4 cellobiose method, its
It is characterised by, in step (1), the concentration of the toluene solution of (3- mercaptopropyi) trimethoxy silane is 10%～15%V/V,
The flow velocity of the toluene solution of (3- mercaptopropyi) trimethoxy silane is 0.5～1 μ L/min；The flow velocity of dehydrated alcohol is 15～20
μL/min.
7. in utilization immobilized enzyme micro-reactor continuous hydrolysis straw according to claim 4 cellobiose method, its
It is characterised by, in step (2), the described enzyme liquid containing beta-glucosidase, the concentration of enzyme is 1～1.2mg/mL, and solvent is
The citric acid solution of pH4.8.
8. in utilization immobilized enzyme micro-reactor continuous hydrolysis straw according to claim 1 cellobiose method, its
It is characterised by, described cellobiose solution, the concentration of solute cellobiose is 3-12wt%, and solvent is the dilute of pH4.0-5.5
Aqueous sulfuric acid.
9. in utilization immobilized enzyme micro-reactor continuous hydrolysis straw according to claim 1 cellobiose method, its
It is characterised by, the flow velocity of substrate cellobiose aqueous solution is 1-5 μ L/min, and reaction time is 4～8h, anti-in micro- reaction
Temperature is answered to be 50-60 DEG C.
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| CN114437927A (en) * | 2022-02-25 | 2022-05-06 | 重庆大学 | Bionic continuous hydrolysis reaction system and method for carrying out enzymolysis |
| CN114456928A (en) * | 2022-03-22 | 2022-05-10 | 合肥学院 | Immobilized enzyme reactor and reaction system |
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| CN109239182A (en) * | 2018-09-04 | 2019-01-18 | 南京林业大学 | A method of with cellulase in-situ modification gold chip |
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| CN114437927A (en) * | 2022-02-25 | 2022-05-06 | 重庆大学 | Bionic continuous hydrolysis reaction system and method for carrying out enzymolysis |
| CN114456928A (en) * | 2022-03-22 | 2022-05-10 | 合肥学院 | Immobilized enzyme reactor and reaction system |
| CN114456928B (en) * | 2022-03-22 | 2023-10-31 | 合肥学院 | Immobilized enzyme reactor and reaction system |
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