CN103642788B - A kind of preparation method of cellulase immobilization carrier - Google Patents
A kind of preparation method of cellulase immobilization carrier Download PDFInfo
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- CN103642788B CN103642788B CN201310674347.XA CN201310674347A CN103642788B CN 103642788 B CN103642788 B CN 103642788B CN 201310674347 A CN201310674347 A CN 201310674347A CN 103642788 B CN103642788 B CN 103642788B
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Abstract
The invention discloses a kind of preparation method of cellulase immobilization carrier, comprise the steps: (1) cellulosic charing; (2) modification of carrier; (3) carrier drying is shaping.Advantage of the present invention is: 1, the inventive method adopt Mierocrystalline cellulose be raw material prepare special anchoring fiber element enzyme carrier, this carrier surface area is large, adsorption site is many, can with enzyme stable bond, decrease coming off of enzyme, improve the adsorption rate of enzyme.2, simple, the preparation process environmental protection of the preparation method of this carrier, pollution-free.3, this carrier is repeatedly reusable, still keeps good enzyme to live, and therefore, the rate of utilization of enzyme and carrier thereof improves, and cost reduces.
Description
Technical field
The present invention relates to a kind of preparation method of fixed enzyme vector, particularly relate to a kind of preparation method of cellulose carrier of special anchoring fiber element enzyme, belong to biological chemical field.
Background technology
Cellulase is a kind of prozyme, one that is made up of multiple lytic enzyme complicated enzyme system, and a lot of fungi of occurring in nature, bacterium and animal body can eccrine fiber element enzymes.Usually cellulase is divided into three classes: C1 enzyme, Cx enzyme and β Polyglucosidase.Wherein, C1 enzyme is the enzyme worked at first to Mierocrystalline cellulose, destroys the crystalline texture of cellulose chain; Cx enzyme be act on through C1 enzyme activation Mierocrystalline cellulose, decompose the cellulase of β-Isosorbide-5-Nitrae-glycosidic link; Cellobiose, procellose and other low molecule cellodextrins can be decomposed into glucose by β Polyglucosidase.It is fermentable sugars that cellulase can be used for cellulose conversion, and then produces alcohol fuel.Because natural cellulose enzyme is resolvase, mostly be disposable direct use, use cost is high, hinders the industrialization of this conversion process.
Immobilized enzyme, compared with resolvase, has incomparable advantage, is mainly manifested in: first, and enzyme and product are easy to separate, thus can recycling; Secondly, processing property and the stability of enzyme can be improved to a certain extent; 3rd, can repeatedly Reusability and operate continuously, reduce production cost; 4th, enzyme is not mixed into product, can simplify separation circuit etc.Therefore, by cellulase immobilization, be conducive to the recovery and reuse of enzyme, be expected to the practical problems solving this enzyme production application, become the study hotspot of Ge great institutes in recent years.
The enzyme that existing carrier adsorbs easily comes off, and causes immobilized enzyme stability and activity not high thus, and enzyme immobilization carrier seldom can reuse simultaneously, therefore there is the aftertreatment of solid support material and the idle problem of useless carrier.
Summary of the invention
Goal of the invention: the object of this invention is to provide a kind of can adsorb in a large number and stablize anchoring fiber element enzyme, simultaneously can recycling the preparation method of enzyme immobilization carrier.
Technical scheme: the preparation method of cellulase immobilization carrier of the present invention, comprises the steps:
(1) cellulosic charing: Mierocrystalline cellulose is carbonized under the protection of inert gas condition of 400 DEG C-500 DEG C;
(2) modification of carrier: in the Mierocrystalline cellulose after charing, adds ionic liquid and treatment agent, stirs, and isothermal holding obtains cellulose carrier solution;
(3) carrier drying is shaping: in cellulose carrier solution, add solvent, and stir washing removing impurity, and then cellulase immobilization carrier is made in dry, vacuum outgas.
Wherein, in step (1), described Mierocrystalline cellulose is Microcrystalline Cellulose.Described rare gas element is nitrogen or argon gas.
In step (2), described ionic liquid is acidic ion liquid.Described acidic ion liquid is [DMIM] [DMP], [AMIM] CI or [AMIM] [OAc].Described treatment agent is polyvinyl alcohol, polyoxyethylene glycol or bromo fourth acetimide.Described Mierocrystalline cellulose is 1:1 ~ 5 with the weight ratio of the ionic liquid added.The concentration of the treatment agent added is 0.1-0.2M(mol/L), wherein, the treatment agent added and there is not proportionlity between ionic liquid and Mierocrystalline cellulose, concentration height is good, concentration is low also passable, if can immobilized enzyme and for its provide more multi-contact surface amass.
In step (3), described solvent is water or short chain alcohol.The water added is 1 ~ 5 times of cellulose carrier liquor capacity; The short chain alcohol added is 1 ~ 3 times of cellulose carrier liquor capacity.
Reaction mechanism: pyroprocessing makes cellulose materials carbonize, thus cellulose conversion is become rigidity is strong, the carbon material of good stability, the ionic liquid added can provide condition for subsequent reactions by dissolving cellulos, the treatment agent added is combined with carbon fiber, and being connected to surface and the inside of fibrous carrier, these treatment agents make the surface-area of carrier increase, and the contact site of enzyme increases, amino groups can also be combined on enzyme molecule simultaneously, and then realize enzyme immobilizatio.
Beneficial effect: the present invention compared with prior art, its remarkable advantage is: 1, the inventive method adopt Mierocrystalline cellulose be raw material prepare special anchoring fiber element enzyme carrier, this carrier surface area is large, adsorption site is many, can with enzyme stable bond, decrease coming off of enzyme, improve the adsorption rate of enzyme.2, simple, the preparation process environmental protection of the preparation method of this carrier, pollution-free, with low cost; 3, this carrier is repeatedly reusable, still keeps good enzyme to live, and therefore, the rate of utilization of enzyme and carrier thereof improves, and cost reduces.
Accompanying drawing explanation
Fig. 1 is the XRD(X ray diffraction of cellulase immobilization carrier of the present invention) figure.
Embodiment
Below technical scheme of the present invention is described in detail.
Embodiment 1, to carry out as follows:
(1) Microcrystalline Cellulose is added tube furnace, the lower 400 DEG C of charing 1h of nitrogen protection condition.
(2) Microcrystalline Cellulose getting 6g charing adds the there-necked flask of 250ml, successively add 6g1, the polyoxyethylene glycol 60ml of 3-methylimidazole dimethyl phosphate [DMIM] [DMP] and 0.2M, stir, 100 DEG C of isothermal holding 20min, the surface of carrier and inside are changed, and surface-area increases, and the contact site of enzyme increases.
(3) in cellulose carrier solution, add the water of 2 times of volumes, stir 20min, and washing removing impurity, then by carrier 60 DEG C of vacuum-dryings, vacuum outgas is prepared into carrier.
As the XRD figure spectrum that Fig. 1 is this carrier, cellulose carrier carbonizes, and the position of 30-50 degree occurs that small peak illustrates that carrier has the existence of phenyl ring (i.e. benzaminic acid), and the carboxyl of benzaminic acid can combine with the amino acid whose amino of zymoprotein.
Embodiment 2:
(1) Microcrystalline Cellulose is got, the lower 500 DEG C of charing 1h of argon shield condition.
(2) get the Microcrystalline Cellulose of 1g charing, successively add the polyvinyl alcohol of 5g chlorination allyl group-3-Methylimidazole [AMIM] CI and 100ml0.2M, stir, 100 DEG C of isothermal holding 20min, the surface of carrier and inside are changed, and surface-area increases, and the contact site of enzyme increases.
(3) in cellulose carrier solution, add the ethanol of 1 times of volume, stir 20min, and washing removing impurity, then by carrier 65 DEG C of vacuum-dryings, vacuum outgas is prepared into carrier.
Embodiment 3:
(1) Microcrystalline Cellulose is got, the lower 450 DEG C of charing 1h of argon shield condition.
(2) get the Microcrystalline Cellulose of 5g charing, successively add the bromo fourth acetimide of 15g [AMIM] [OAc] and 75ml0.2M, stir, 80 DEG C of isothermal holding 30min, the surface of carrier and inside are changed, and surface-area increases, and the contact site of enzyme increases.
(3) in cellulose carrier solution, add the methyl alcohol of 1 times of volume, stir 30min, and washing removing impurity, then by carrier 60 DEG C of vacuum-dryings, vacuum outgas is prepared into carrier.
Embodiment 4:
Get the 50mM of 20ml, the Acetic acid-sodium acetate damping fluid of pH5.0, adds the cellulase of 50IU, adds carrier prepared by 1g embodiment 1, in 30 DEG C of shaking baths, reacts 24h.Filter and obtain immobilized enzyme, with Acetic acid-sodium acetate buffer solution till can't detect albumen.Adopt the adsorption rate of Coomassie Brilliant Blue detection fibers element enzyme, record that its adsorption rate 1500mg/g(is former adsorption rate 96%); Adopt DNS method simultaneously, the activity of element enzyme using glucose as standard substance detection fibers, the apparent activity recording this immobilized enzyme is 40IU, and after reusing 15 times, carrier immobilized enzymic activity reaches about 90%.
Embodiment 5: the carrier prepared by embodiment 2 repeats embodiment 4, the adsorption rate recording cellulase is 1600mg/g(is 98% of former adsorption rate); The apparent activity of this immobilized enzyme is 43IU, and after reusing 15 times, this carrier immobilized enzymic activity reaches about 85%.
Embodiment 6: the carrier prepared by embodiment 3 repeats embodiment 4, the adsorption rate recording cellulase is 1800mg/g(is 99% of former adsorption rate); The apparent activity of this immobilized enzyme is 45IU, and after reusing 15 times, this carrier immobilized enzymic activity reaches about 80%.
Claims (6)
1. a preparation method for cellulase immobilization carrier, is characterized in that comprising the steps:
(1) cellulosic charing: Microcrystalline Cellulose is carbonized under the protection of inert gas condition of 400 DEG C-500 DEG C;
(2) modification of carrier: in the Microcrystalline Cellulose after charing, add acidic ion liquid and treatment agent polyvinyl alcohol, polyoxyethylene glycol or bromo fourth acetimide, stir, isothermal holding obtains Microcrystalline Cellulose carrier soln, wherein, described Microcrystalline Cellulose and acidic ion liquid weight ratio are 1:1 ~ 5; The concentration of the treatment agent added is 0.1-0.2M;
(3) carrier drying is shaping: in Microcrystalline Cellulose carrier soln, add aqueous solvent or short chain alcohol, and stir washing removing impurity, and then cellulase immobilization carrier is made in dry, vacuum outgas.
2. the preparation method of cellulase immobilization carrier according to claim 1, is characterized in that: in step (1), and described rare gas element is nitrogen or argon gas.
3. the preparation method of cellulase immobilization carrier according to claim 1, is characterized in that: in step (2), and described acidic ion liquid is [DMIM] [DMP], [AMIM] CI or [AMIM] [OAc].
4. the preparation method of cellulase immobilization carrier according to claim 1, is characterized in that: in step (3), and the water added or short chain alcohol are 1-5 times of Microcrystalline Cellulose carrier soln volume.
5. the preparation method of cellulase immobilization carrier according to claim 1, is characterized in that: in step (3), and described drying is vacuum-drying under the condition of 60 DEG C or 65 DEG C.
6. the preparation method of cellulase immobilization carrier according to claim 1, is characterized in that: in step (3), and described short chain alcohol is methyl alcohol or ethanol.
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| CN201310674347.XA CN103642788B (en) | 2013-12-11 | 2013-12-11 | A kind of preparation method of cellulase immobilization carrier |
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| CN103642788B true CN103642788B (en) | 2016-03-30 |
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| CN104450663A (en) * | 2014-11-02 | 2015-03-25 | 清华大学 | High-activity immobilized cellobiase nano mesoporous material and preparation method thereof |
| CN110144342A (en) * | 2019-04-24 | 2019-08-20 | 湖州硕邦生物科技有限公司 | A kind of method that cellulose carrier prepares immobilised enzymes |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492668A (en) * | 2009-03-10 | 2009-07-29 | 江苏明睿科技实业有限公司 | Process for producing biological modified carbonize stalk |
| CN102504285A (en) * | 2011-11-03 | 2012-06-20 | 中国科技开发院广西分院 | Regenerated cellulose microspheres and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492668A (en) * | 2009-03-10 | 2009-07-29 | 江苏明睿科技实业有限公司 | Process for producing biological modified carbonize stalk |
| CN102504285A (en) * | 2011-11-03 | 2012-06-20 | 中国科技开发院广西分院 | Regenerated cellulose microspheres and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| consercation of cellulase enzyme in biopolishing application of cotton fabrics;V.S.Kumar et al;《The Journal of The Textile Institulte》;20090119;第99卷(第4期);339-346 * |
| 基于纤维素的固体酸催化剂的制备及其催化高酸值废油脂生产生物柴油;娄文勇 等;《催化学报》;20111130;第32卷(第11期);摘要 * |
| 纤维素在离子液体中的溶解性能及水热碳化研究;邹鑫;《中国优秀硕士论文全文数据库 工程科技I辑》;20130715(第7期);全文 * |
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