CN108004229A - A kind of preparation of immobilized cellulase and its method for enzyme hydrolysis cellulose - Google Patents
A kind of preparation of immobilized cellulase and its method for enzyme hydrolysis cellulose Download PDFInfo
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- CN108004229A CN108004229A CN201711416657.6A CN201711416657A CN108004229A CN 108004229 A CN108004229 A CN 108004229A CN 201711416657 A CN201711416657 A CN 201711416657A CN 108004229 A CN108004229 A CN 108004229A
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- 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
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- 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)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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Abstract
The present invention provides a kind of method of the preparation and its enzyme hydrolysis cellulose of immobilized cellulase.The method for preparing immobilized cellulase is crosslinking investment, using sodium alginate as carrier, utilizes OH in sodium alginate‑And COOH‑Oxygen atom and Ca2+The principle that ion-exchange reactions occurs forms gel, glutaraldehyde as cross linker, polyethylene glycol is pore-foaming agent and hydrophilizing agent, the enzyme activity fixed rate and enzyme activity, reduction Michaelis constant, the hole density for improving carrier surface of immobilized cellulase can be improved by adding polyethylene glycol, increase content of reducing sugar, enzyme hydrolysis yield and the TS degradation rates of enzymatic hydrolysis reaction.During immobilized enzyme hydrolysis cellulose, it is possible to achieve the recycled for multiple times of cellulase.Preparation and application process of the invention is easy to operate, rigid condition is gentle, investment is low, environmentally friendly.
Description
Technical field
The invention particularly relates to a kind of preparation method of immobilized cellulase, and immobilized cellulase to be used for fibre
The method of dimension element hydrolysis.
Background technology
Cellulose is that the most wide, natural polymer of reserves maximum is distributed in nature, is the basic substance for forming cell membrane.
Annual plant produces hundreds billion of tons of cellulose by photosynthesis, is a kind of not nexhaustible renewable resource taken.Closely
Nian Lai, it is related fine with progressively exhausted and environmental problem the getting worse of the non-renewable resources such as coal, oil, natural gas
Dimension element becomes one of Disciplinary Frontiers of current international scientific research as the research on utilization of renewable resource.
Cellulose is that the unbranched homogeneous of long-chain formed by β-D- glucopyranoses with (1-4)-β-glucosides key joint is more
Sugar.Strand has C1 chair conformations.3 hydroxyls on monomer are located on 2,3,6 carbon atoms on glucose ring unit respectively,
It is wherein secondary hydroxyl group, secondary hydroxyl group and primary hydroxyl group respectively on C-2, C-3, C-6 position.Since itself has higher crystallization
There are good hydrogen bond, native cellulose to melt for degree, intermolecular and intramolecular, it is also difficult to is dissolved in Conventional solvents, this is very big
Ground limits the development and utilization of cellulose.
For cellulose in biotransformation, cellulase can be hydrolyzed to the soluble small molecule reduction for being easy to utilize
Sugar, to realize that the efficient of lignocellulosic utilizes.Cellulase can repeatedly be used for enzymatic hydrolysis reaction in theory as catalyst
In.Cellulase is water soluble preparation, during enzyme hydrolysis cellulose, can on the one hand be caused together with resulting fusion
The loss of cellulase, on the other hand makes the purity of product reduce.Cellulase is expensive, it, which is circulated, repeatedly utilizes and improve
Product purity is the main problem that enzyme hydrolysis process need to solve.
Cellulose enzyme immobilizatio is to realize the effective ways that cellulase is recycled for multiple times.Document：Chen Wei soldier seaweed
Sour sodium/SiO2The preparation of composite hydrogel and as being studied in research [D] the Huaqiao University texts of immobilized cellulase carrier
Show, using sodium alginate as main carriers, after the carboxyl on NHS and EDC activation sodium alginates, with KH-550 coupling agents, add
Enter cellulase to be fixed.But due to enzyme molecule after fixation limitation of movement so that the space between substrate
Steric hindrance increase.The Michaelis constant of immobilised enzymes increases, and compared with resolvase, is increased by 0.122mg/ml to 1.10mg/ml, increase
8 times.The increasing of Michaelis constant shows that its affinity with substrate of enzyme molecule reduces, this is unfavorable for cellulase, and to play its effective
Hydrolysis.
The content of the invention
Further to solve problem above, it is an object of the present invention to provide a kind of fixing means of cellulase, improves
The affinity of enzyme molecule and substrate, and use it in enzymatic hydrolysis reaction, realize recycling for cellulase.
The present invention is using fixed form of the crosslinking investment as cellulase.In order to improve between substrate and enzyme molecule
Affinity, reduce space resistance, using sodium alginate as the carrier of immobilized cellulase, the basis of glutaraldehyde as cross linker
On, add polyethylene glycol；On the one hand polyethylene glycol is used as pore-foaming agent, on the other hand improve the hydrophily of immobilised enzymes；After crosslinking
Cellulase be added in calcium chloride solution and form gel.Reaction principle is：The aldehyde radical of glutaraldehyde can be with cellulase
Schiff base cross-linking reaction, which occurs, for amino makes enzyme molecule be cross-linked with each other；Sodium alginate G units OH-And COO-Oxygen atom and Ca2+
Generation ion-exchange reactions forms " Egg-box " structure, and enzyme molecule is fixed；Polyethylene glycol is in sodium alginate-Ca2+Fixed system
Middle part dissolution, forms uniform pore structure in carrier surface, reduces resistance to mass tranfer；Its hydroxyl and polyethylene glycol for containing at the same time
Base forms hydrogen bond respectively with carrier and cellulase, increases hydrophily.
The immobilized cellulase prepared using above method, for the enzymatic hydrolysis reaction of cellulose, hydrolysis effect compared with
It is not added with polyethylene glycol to compare, is significantly improved.
Comprise the following steps that：
(1) preparation of immobilized cellulase：Accurately weigh cellulase powder to be dissolved in pure water, it is 2.5- to make its concentration
It is 12.5mg/ml stand-by.
Accurately weigh CaCl2It is 0.5-6.0%CaCl to be configured to mass fraction2Solution, it is stand-by；
It is 1% glutaraldehyde solution accurately to weigh glutaraldehyde to be configured to mass fraction, stand-by；
The accurate sodium alginate and polyethylene glycol addition pH of weighing is in 4.8 acidic buffer solution, is uniformly mixed at room temperature
Afterwards, sodium alginate-polyglycol solution is formed, it is stand-by；Wherein the mass ratio of sodium alginate and polyethylene glycol is 1:0.5~1:
2.5, the mass fraction of sodium alginate soln is 1.0-6.0%, molecular weight polyethylene glycol 1000-6000；
Above-mentioned sodium alginate-polyglycol solution is accurately measured, above-mentioned cellulase solution is sequentially added and glutaraldehyde is molten
Liquid, after mixing, in 150r/min vibrate 30min, after 4 DEG C stand reaction 4h；Sodium alginate in mixed liquor after reaction-
Polyglycol solution：Cellulase solution：The volume ratio of glutaraldehyde solution is 5:1:0.5~5:1:2.5；By above-mentioned mixed liquor by
It is added dropwise to the CaCl that mass fraction is 0.5-6.0%2In solution, in 30-80 DEG C of fixed 0.5-7.5h, filtering；More renew
CaCl2After solution, the CaCl that more renews2After solution, 0-6 DEG C stands hardening 1-3h；Washed afterwards with NaCl solution, filter paper blots
Surface moisture, less than 4 DEG C preservations.It is preferred that 4 DEG C stand hardening 2h；Washed afterwards with the NaCl solution of mass fraction 0.9%, filter paper
Blot surface moisture, 4 DEG C of preservations.
Wherein, preferred cellulose enzyme concentration is 5mg/ml, and sodium alginate soln mass fraction is 3.5%, polyethylene glycol point
Son amount is 4000, mass ratio=1 of sodium alginate and polyethylene glycol:1.5, CaCl2Liquid quality fraction is 2%, and immobilization is anti-
It is temperature 50 C to answer condition, time 2.5h, sodium alginate-polyglycol solution：Cellulase solution：Glutaraldehyde solution：CaCl2
The volume ratio of solution is 5:1:1.5:1.
(2) method of immobilized enzyme hydrolysis cellulose：Immobilized cellulase prepared by above-mentioned process for fixation is used for water
Cellulose is solved, cellulose is added in buffer solution so that cellulose mass fraction is 3.0-17.0%, and buffer solution pH is
3.0-6.0；Immobilized cellulase is added in above-mentioned cellulose solution, and dosage is every gram of cellulose 0.5-4.0g；Hydrolysis
Temperature is 40-60 DEG C, and 12-72h is hydrolyzed under the conditions of 120-180r/min.
Wherein, the preferred immobilised enzymes dosage of preferably sodium alginate-polyethylene glycol immobilized enzyme hydrolysis cellulose is every
Gram cellulose 3.55g, cellulose mass fraction are 13.16%, pH=5.11,60 DEG C of hydrolysis temperature, under the conditions of 150r/min
Hydrolyze 48h.
Compared with prior art, the invention has the advantages that：
1) compared with being not added with polyethylene glycol, the present invention can improve the enzyme activity of immobilized cellulase and enzyme activity is fixed
Rate, is respectively increased 25% and 16%.
2) immobilized cellulase can realize being used repeatedly for cellulase.Compared with being not added with polyethylene glycol,
The present invention can improve the recycling effect of immobilized cellulase, and remaining enzyme activity can improve after reaction three times
56%.
3) Michaelis constant of Immobilization in Sodium Alginate enzyme is 2.964mg/ml, sodium alginate-polyethylene glycol immobilised enzymes
Michaelis constant is 1.850mg/ml.Compared with being not added with polyethylene glycol, the present invention can reduce the Michaelis of immobilized cellulase
Constant, reduces 21%.So as to improve the affinity of immobilization enzyme-to-substrate.
4) present invention can improve the hole density on fixed enzyme vector surface, and the immobilization than being not added with polyethylene glycol improves
8%.
5) present invention can improve the enzyme hydrolysis yield and TS degradation rates of enzymatic hydrolysis reaction, with being not added with polyethylene glycol phase
Than 133% and 72% being respectively increased, content of reducing sugar improves nearly 4 times.
Embodiment
Embodiment 1
1) accurately weigh cellulase powder to be dissolved in pure water, it is 5mg/ml to make its concentration, stand-by.Cellulase originates from green
Color trichoderma.
2) preparation of immobilized cellulase：The accurate 2.625g molecular weight that weighs burns for 4000 polyethylene glycol in 100ml
In cup, citric acid-sodium citrate buffer solution of the pH=4.8 of 48.25g 0.05mol/L is added, is stirred at room temperature with glass bar
After dissolving, 1.75g sodium alginates are added so that sodium alginate mass fraction is 3.5%, sodium alginate and polyethylene glycol mass ratio
For 1:1.5.At room temperature after mixing, sodium alginate-polyglycol solution is formed, it is stand-by.
Above-mentioned sodium alginate-the polyglycol solutions of 5ml are drawn in the vial of 20ml with 10ml syringes, are added successively
Enter 1ml 5mg/ml cellulase solutions and 1.5ml mass fractions for 1% glutaraldehyde solution, after mixing, in 150r/
Min vibrates 30min, and 4 DEG C stand crosslinking 4h.Above-mentioned mixed liquor is added to 5ml syringes, is added dropwise to 10ml mass fractions
For 2% CaCl2In solution, in 50 DEG C of fixed 2.5h.Filtering, replace CaCl2, 4 DEG C stand hardening 2h.It is with mass fraction
0.9% NaCl solution washing, filter paper blot surface moisture, 4 DEG C of preservations.
3) Immobilization in Sodium Alginate cellulose enzyme hydrolyzing microcrystalline cellulose：Weigh the addition of 0.5g TS microcrystalline celluloses
In 50ml shaking flasks, immobilised enzymes dosage is 1.785g, adds citric acid-sodium citrate buffer solution of pH=5.13, makes substrate
Mass fraction be 4.60%.Sealed membrane is sealed to prevent moisture evaporation.The enzyme hydrolysis 48h under 60 DEG C, 150r/min, reaction knot
Filtered after beam, survey the content of reducing sugar in filtrate to obtain enzyme hydrolysis yield；After solid residue is dried, the front and rear matter of reaction is surveyed
Amount change.
4) sodium alginate-polyethylene glycol immobilized cellulase enzyme hydrolysis microcrystalline cellulose：Using 50ml shaking flasks.Add
0.5g TS microcrystalline celluloses, immobilised enzymes dosage are 1.775g, add citric acid-buffered sodium citrate of pH=5.11
Liquid, it is 13.16% to make substrate mass fraction.Sealed membrane is sealed to prevent moisture evaporation.The enzyme hydrolysis under 60 DEG C, 150r/min
48h, filters after reaction, surveys the content of reducing sugar in filtrate to obtain enzyme hydrolysis yield；After solid residue is dried, survey anti-
Answer front and rear mass change.
Addition polyethylene glycol is with being not added with the effect of polyethylene glycol anchoring fiber element enzyme and the effect of enzyme hydrolysis microcrystalline cellulose
Fruit analysis in table 1 and table 2.As shown in Table 1, cellulase is after sodium alginate-polyethylene glycol immobilization, enzyme activity and enzyme activity
25% and 16% is respectively increased in fixed rate；Remaining enzyme activity can improve 56% after reaction three times；Michaelis constant reduces
21%.
As shown in Table 2, the cellulase through sodium alginate-polyethylene glycol immobilization, for enzyme hydrolysis microcrystalline cellulose, instead
Nearly 4 times of content of reducing sugar increase after answering, 133% and 72% is respectively increased in enzyme hydrolysis yield and TS degradation rates.
The property analysis of 1 immobilized cellulase of table
| Project | Sodium alginate | Sodium alginate-polyethylene glycol |
| Enzyme activity (U/g) | 6.891 | 8.629 |
| Enzyme activity fixed rate (%) | 44.44 | 51.71 |
| Hole density (%) | 86.55 | 93.44 |
| Michaelis constant (mg/ml) | 2.964 | 1.850 |
| Recycle 3 remaining enzyme activities (%) | 31.66 | 49.41 |
2 anchoring fiber element enzyme of table is used for the effect analysis of cellulose hydrolyzation
| Project | Sodium alginate | Sodium alginate-polyethylene glycol |
| Content of reducing sugar (mg/ml) | 0.61 | 4.05 |
| Enzyme hydrolysis yield (%) | 1.33 | 3.10 |
| TS degradation rates (%) | 3.79 | 6.52 |
Embodiment 2
The preparation of sodium alginate-polyethylene glycol immobilized cellulase, operates same embodiment 1 (1) (2), rigid condition point
Minimum and peak of each factor described in " content of the invention (1) " are not taken, and analysis is optimized to the condition of immobilised enzymes,
The results are shown in Table 3.
Sodium alginate-polyethylene glycol immobilized cellulase is used for hydrolyzing microcrystalline cellulose, operates same embodiment 1 (3)
(4).Enzyme hydrolysis condition takes minimum and peak of each factor described in " content of the invention (2) " respectively, to enzymatic hydrolysis reaction
Condition optimize analysis, as shown in table 4.
The condition optimizing analysis of 3 immobilized cellulase of table
The optimization analysis of 4 immobilized cellulase enzymatic hydrolysis reaction condition of table
Claims (8)
1. a kind of preparation method of immobilized cellulase, it is characterised in that comprise the following steps that：Accurately weigh cellulose enzyme powder
End is dissolved in pure water, and it is 2.5-12.5mg/ml to make its concentration, stand-by；
It is 1% glutaraldehyde solution accurately to weigh glutaraldehyde to be configured to mass fraction, stand-by；
Accurately weigh sodium alginate and polyethylene glycol is added in the acidic buffer solution that pH is 4.8, at room temperature after mixing, shape
It is stand-by into sodium alginate-polyglycol solution；Wherein the mass ratio of sodium alginate and polyethylene glycol is 1:0.5~1:2.5, sea
The mass fraction of solution of sodium alginate is 1.0-6.0%, molecular weight polyethylene glycol 1000-6000；
Above-mentioned sodium alginate-polyglycol solution is accurately measured, sequentially adds above-mentioned cellulase solution and glutaraldehyde solution, is mixed
Close uniformly after, in 150r/min vibrate 30min, after 4 DEG C stand reaction 4h；Sodium alginate-poly- second in mixed liquor after reaction
Glycol solution：Cellulase solution：The volume ratio of glutaraldehyde solution is 5:1:(0.5~2.5)；Above-mentioned mixed liquor is added dropwise
To the CaCl that mass fraction is 0.5-4%2In solution, in 30-80 DEG C of fixed 0.5-7.5h, filtering；The CaCl more renewed2Solution
Afterwards, hardening 1-3h is stood for 0-6 DEG C；Washed afterwards with NaCl solution, filter paper blots surface moisture, less than 4 DEG C preservations.
2. the process for fixation of cellulase according to claim 1, it is characterised in that the enzyme liquid of the cellulase
Concentration is 5mg/ml.
3. the method for cellulase immobilization according to claim 1, it is characterised in that the sodium alginate-poly- second
Glycol is dissolved in pH=4.8 buffer solutions, is configured to sodium alginate-polyglycol solution；Wherein sodium alginate soln mass fraction
For 3.5%, molecular weight polyethylene glycol 4000；Mass ratio=1 of sodium alginate and polyethylene glycol:1.5.
4. the method for cellulase immobilization according to claim 1, it is characterised in that-CaCl2Liquid quality fraction is
2%.
5. the method for cellulase immobilization according to claim 1, it is characterised in that the sodium alginate-poly- second
Glycol, cellulase, glutaraldehyde mixed solution are added dropwise to the CaCl that mass fraction is 2%2After solution, immobilized reactant bar
Part is temperature 50 C, time 2.5h.
6. the method for cellulase immobilization according to claim 1, it is characterised in that sodium alginate-polyethylene glycol is molten
Liquid：Cellulase solution：Glutaraldehyde solution：CaCl2The volume ratio of solution is 5:1:1.5:1.
7. the method for immobilized enzyme hydrolysis cellulose：Comprise the following steps that：Immobilization fibre element prepared by above-mentioned process for fixation
Enzyme is used for hydrocellulose, and cellulose is added in buffer solution so that cellulose mass fraction is 3.0-17.0%, and buffering is molten
Liquid pH is 3.0-6.0；Immobilized cellulase is added in above-mentioned cellulose solution, and dosage is every gram of cellulose 0.5-
4.0g；Hydrolysis temperature is 40-60 DEG C, and 12-72h is hydrolyzed under the conditions of 120-180r/min.
8. the method for immobilised enzymes enzyme hydrolysis cellulose according to claim 7, it is characterised in that wherein sodium alginate-
The immobilised enzymes dosage of polyethylene glycol immobilized enzyme hydrolysis cellulose is every gram of cellulose 3.55g, and cellulose mass fraction is
13.16%, pH=5.11,60 DEG C of hydrolysis temperature, hydrolyze 48h under the conditions of 150r/min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113106604A (en) * | 2021-03-16 | 2021-07-13 | 上海水星家用纺织品股份有限公司 | Preparation method of alginate fiber home textile fabric and prepared fabric |
| CN113388896A (en) * | 2021-07-13 | 2021-09-14 | 基准(北京)生物技术研究院有限公司 | Preparation method of hydrophobic oleophylic nanocellulose based on immobilized enzyme chemistry-oxidative degumming |
| CN113604461A (en) * | 2021-08-13 | 2021-11-05 | 合肥卡迪尔生物科技有限公司 | Immobilized enzyme and preparation method and application thereof |
| CN115232803A (en) * | 2022-08-02 | 2022-10-25 | 周口师范学院 | Immobilized cellulase, preparation method and preparation device of immobilized cellulase |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113106604A (en) * | 2021-03-16 | 2021-07-13 | 上海水星家用纺织品股份有限公司 | Preparation method of alginate fiber home textile fabric and prepared fabric |
| CN113106604B (en) * | 2021-03-16 | 2022-08-26 | 上海水星家用纺织品股份有限公司 | Preparation method of alginate fiber home textile fabric and prepared fabric |
| CN113388896A (en) * | 2021-07-13 | 2021-09-14 | 基准(北京)生物技术研究院有限公司 | Preparation method of hydrophobic oleophylic nanocellulose based on immobilized enzyme chemistry-oxidative degumming |
| CN113388896B (en) * | 2021-07-13 | 2022-08-02 | 基准(北京)生物技术研究院有限公司 | Preparation method of hydrophobic oleophylic nanocellulose based on immobilized enzyme chemistry-oxidative degumming |
| CN113604461A (en) * | 2021-08-13 | 2021-11-05 | 合肥卡迪尔生物科技有限公司 | Immobilized enzyme and preparation method and application thereof |
| CN115232803A (en) * | 2022-08-02 | 2022-10-25 | 周口师范学院 | Immobilized cellulase, preparation method and preparation device of immobilized cellulase |
| CN115232803B (en) * | 2022-08-02 | 2024-05-07 | 周口师范学院 | Immobilized cellulase, preparation method and preparation device of immobilized cellulase |
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