CN106497909B - A kind of method of modifying of laccase - Google Patents
A kind of method of modifying of laccase Download PDFInfo
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Abstract
The invention belongs to biological laccase modification technical fields, it is related to method of modifying that is a kind of while improving laccase (laccase) catalytic activity and stability, copper ion and enzyme can be fixed simultaneously using polyacrylic acid (PAA), poly(ethylene glycol) methyl ether acrylate (PEGA) can improve the principle of the stability of laccase, it polymerize synthetic segmented copolymer PAA/PPEGA by RAFT (Reversible Addition Fragmentation Chain Transfer), then the copolymer p AA/PPEGA of preparation is improved into laccase activity in conjunction with copper ion, realize the catalytic activity of laccase and significantly improving for stability after modifying, this method operating procedure is simple, required chemicals additional amount is few, required reaction condition is mild, preparation efficiency is high, after the laccase of preparation is fixed in the graphene paper of large area, it can be realized laccase Recycling in degradation catechol pollutant.
Description
Technical field:
The invention belongs to biological laccase modification technical fields, are related to a kind of while improving laccase (laccase) catalysis
The method of modifying of activity and stability, can fix copper ion and laccase, poly- (poly(ethylene glycol) using polyacrylic acid (PAA) simultaneously
Methyl ether acrylate) (PPEGA) can improve laccase stability principle, it is poly- by Reversible Addition Fragmentation Chain Transfer (RAFT)
Synthesis poly propenoic acid (poly(ethylene glycol) methyl ether acrylate) block copolymer (PAA/PPEGA), and combine copper ion
Improve laccase activity, the Laccase Catalyzed activity and stability after realizing modification significantly improve.
Background technique:
Currently, laccase is a kind of more copper oxidoreducing enzyme, be widely present in plant, fungi and bacterium, biodegrade,
Chemical synthesis, red wine preservation and biosensor etc. have a very wide range of applications, but since its stability is poor, paint
The application of enzyme is very restricted, therefore improving laccase stability is suffered from a problem that in laccase research.The prior art
The middle method for improving laccase stability has fixation, capsule method, hydrogel complexometry, polymer-modified method, however passes through above-mentioned
After method modification, all there is apparent decline in the activity of enzyme.Therefore while improving laccase stability, if laccase can be improved
Activity will to laccase application have very important meaning, currently used for improve laccase activity there are many ways to, such as to
Copper ion, photochemical catalyst or microorganism etc. are added in laccase solution, wherein it is one that copper ion, which is added in laccase solution,
Highly effective and common method, but since copper ion is a kind of heavy metal ion, the introducing of copper ion can give entire enzyme
Reaction system brings secondary pollution.Therefore seek to design method that is a kind of while improving Laccase Catalyzed activity and stability, the party
Method tries to fix copper ion, and can significantly improve the catalysis of laccase simultaneously while improving enzymatic activity using copper ion
Activity, this method preparation process is simple, high-efficient, the good product quality of preparation, and working environment is friendly, before great market
Scape.
Summary of the invention:
It is an object of the invention to overcome disadvantage of the existing technology, design provides a kind of can improve Laccase Catalyzed simultaneously
The method of activity and stability, this method operating procedure is simple, and required chemicals additional amount is few, and required reaction condition is mild,
Preparation efficiency is high, and after the laccase of preparation is fixed in the graphene paper of large area, it is dirty in degradation catechol to can be realized laccase
Contaminate the recycling in object.
To achieve the goals above, the concrete technology for preparing of the laccase of high activity of the present invention, high stability includes
Following steps:
One, synthetic segmented copolymer PAA/PPEGA (poly propenoic acid (poly(ethylene glycol) methyl ether third is polymerize by RAFT
Olefin(e) acid ester):
(1), by 20-60mg RAFT reagent (such as pyrene functionalized thio valeric acid of 4- cyano -4- ethyl three), 2-10mg
Azodiisobutyronitrile (AIBN), 0.2-0.5g acrylic acid (PAA) use 2-5mL ultrapure water and 3-7mL dioxy six in round-bottomed flask
Ring dissolution, round-bottomed flask is sealed with rubber stopper, is passed through nitrogen 30-60 minutes to remove the oxygen in round-bottomed flask, then will justify
Bottom flask oil bath is heated at 20-90 DEG C, and magnetic agitation is reacted 5-36 hours；
(2), round-bottomed flask and 1-5g poly(ethylene glycol) methyl ether acrylate (PEGA) is added in opening steps (1), it will
Round-bottomed flask seals with rubber stopper, and is passed through nitrogen 10-60 minutes, then the oil bath at 30-150 DEG C, and magnetic agitation reacts 10-24
Hour, mixture ether obtained will be reacted and precipitated 1-6 times, obtained sediment is block copolymer PAA/PPEGA, will
Sediment obtained is placed in baking oven, is stored for future use to constant weight within drying 24-48 hours at 20-70 DEG C；
Two, laccase is modified with block copolymer PAA/PPEGA:
(1), 0.3-0.6g copolymer p AA/PPEGA, 7-11mg 1- (3- dimethylamino-propyl) -3- ethyl carbon two is sub-
Amine hydrochlorate (EDC) is dissolved in the round-bottomed flask of 50mL with 15-25ml methylene chloride, is stirred to react at room temperature 20-70 minutes；
(2), 4-9g n-hydroxysuccinimide (NHS) is added in the reagent made from step (1), reacts 24 at room temperature
Hour, three times with ether precipitating, the polymer P AA/PPEGA-NHS of activation is made, then PAA/PPEGA-NHS obtained is placed in
In baking oven, at 25-80 DEG C dry 24-72 hours it is spare to constant weight；
(3), the acetate buffer (ABS buffer) of compound concentration 0.1-0.9mol/L, pH=5-9, weigh 10-
The laccase stock solution that 60mg laccase is dissolved in preparation 0.5-3mg/mL in 20mLABS buffer is spare, weighs 100-400mgPAA/
PPEGA-NHS, the PAA/PPEGA-NHS stock solution for being dissolved in preparation 10-40mg/mL in 10mLABS buffer are spare；
(4), the laccase stock solution for taking 10-15mL step (3) to prepare is added dropwise to the 0.5-11mL PAA/ of stirring
In the stock solution of PPEGA-NHS, solution obtained above is stirred to react 4-12 hours under ice bath, then uses molecular cut off
It dialyses 1-3 days for the dialysis membrane ice bath of 20000-50000, to remove excessive PAA/PPEGA-NHS, obtained block copolymerization
Laccase (PAA/PPEGA-laccase) after object PAA/PPEGA modification；
Three, PAA/PPEGA-laccase is modified with copper ion:
(1), with the copper-bath of ABS buffer 8-20mmol/l, then by PAA/ obtained in step 2
PPEGA-laccase is cultivated 10-20 hours at 0-37 DEG C in copper-bath, be made block copolymer PAA/PPEGA and
Laccase (Cu after copper ion modification2+/ PAA/PPEGA-laccase) mixed solution；
It (2), is saturating in the dialysis membrane ice bath of 500-1000 using molecular cut off by mix reagent obtained in step (1)
Analysis removes the copper ion of absorption loosely, the laccase (Cu after block copolymer PAA/PPEGA and copper ion modification is made2+/PAA/
PPEGA-laccase), by Cu obtained2+It is stored under the conditions of/PAA/PPEGA-laccase freeze-drying.
The present invention is to Cu2+The catalytic activity and stability of/PAA/PPEGA-laccase is measured, respectively to former laccase
And Cu2+The catalytic activity of/PAA/PPEGA-laccase and half-life period have carried out characterization test, the results showed that Cu2+/PAA/
The activity and stability of PPEGA-laccase is improved, and wherein catalytic activity improves 347%, and stability improves 137%,
Show the modification to former laccase, successfully improves the catalytic activity and stability of former laccase.
The present invention relates to the laccase (Cu after block copolymer PAA/PPEGA and copper ion modification2+/PAA/
PPEGA-laccase application and catalytic activity and Stability Determination), the specific process steps are as follows:
One, by Cu2+/ PAA/PPEGA-laccase is fixed to graphene paper surface:
(1), with the Cu of ABS buffer 1-10mg/mL2+/ PAA/PPEGA-laccase solution.
(2), by 1.0cm2-1.0m2Graphene paper be soaked in step (1) preparation solution in, impregnated under the conditions of 0-50 DEG C
6-20 hour
(3), with the ultrapure water graphene paper surface of flowing, to remove the Cu that surface is not fixed firmly2+/PAA/PPEGA-
laccase。
(4), it with graphene paper is dried with nitrogen, is saved backup at 0-60 DEG C.Two, former laccase is fixed on graphene paper
Surface, to compare Cu2+Catalytic activity, stability after/PAA/PPEGA-laccase is fixed, and recycling situation:
Two, former laccase is fixed on to the surface of graphene paper, to compare Cu2+After/PAA/PPEGA-laccase is fixed
Catalytic activity, stability, and recycling situation:
(1), 20mg EDC, 5mg NHS, 20mg original laccase are dissolved in 9mLABS buffer；
(2), 1mg pyrene butyric acid is dissolved into 1mL dimethylformamide；
(3), the solution prepared in step (2) is added dropwise in the solution in step (1), is stirred to react 8 at 4 DEG C
Hour；
(4), with molecular cut off be 1000 bag filter, dialyse 24 hours at 4 DEG C, with remove excessive EDS with
NHS；
(5) laccase that pyrene is modified will be fixed on by graphene paper surface using technique identical with step 1；
To the Cu being fixed in graphene paper2+The activity of/PAA/PPEGA-laccase is tested, the results showed that 25 DEG C
Cu at 30 DEG C2+The activity of/PAA/PPEGA-laccase is improved；To Cu2+/ PAA/PPEGA-laccase is recycled through 8 times
Activity after is tested, as the result is shown the Cu after 8 times recycle2+/ PAA/PPEGA-laccase activity from
317% is reduced to 189%, and the activity of former laccase shows Cu from 100% to complete deactivation2+/ PAA/PPEGA-laccase's
Activity is significantly improved, and after 8 times recycle, Cu2+The activity of/PAA/PPEGA-laccase is still more living than former laccase
Property it is high, it was demonstrated that Cu2+/ PAA/PPEGA-laccase has application potential to can be applied in degradation catechol pollutant, and can
Recycling.
Compared with prior art, the present invention can be according to actual needs using RAFT polymerization, synthesizing has different function
Block copolymer, and by the fixation to copper ion, secondary pollution of the metal copper ion to reaction system is avoided, is mentioned simultaneously
The high catalytic activity and stability of laccase, the laccase of invention preparation are fixed in the graphene paper of large area, can be realized
Recycling of the laccase in degradation catechol pollutant, preparation process is simple, and preparation efficiency is high, product quality
Good, stability is strong, and application environment is friendly.
Detailed description of the invention:
Fig. 1 is the synthesis process schematic diagram of block copolymer PAA/PPEGA of the present invention.
Fig. 2 is the nuclear-magnetism phenogram of block copolymer PAA/PPEGA of the present invention.
Fig. 3 laccase (Cu of the present invention after block copolymer PAA/PPEGA and copper ion modification2+/PAA/
PPEGA-laccase) active testing figure.
Fig. 4 laccase (Cu of the present invention after block copolymer PAA/PPEGA and copper ion modification2+/PAA/
PPEGA-laccase) stability test figure.
The work of former laccase and Cu2+/PAA/PPEGA-laccase after being fixed at 25 DEG C and 30 DEG C of the present invention of Fig. 5
Property measurement chart.
Former laccase and Cu after Fig. 6 fixation of the present invention2+/ PAA/PPEGA-laccase is living after recycling
Property variation diagram.
Specific embodiment:
Below by example with reference, the invention will be further described.
Embodiment 1:
The method of modifying for the laccase that the present embodiment is related to comprising the following three steps:
Step 1: polymerizeing synthetic segmented copolymer PAA/PPEGA by RAFT (Reversible Addition Fragmentation Chain Transfer):
(1), as shown in Figure 1, first by the 23.9mg pyrene functionalized thio valeric acid of 4- cyano -4- ethyl three, 2.8mg azo two
Isobutyronitrile (AIBN), 0.44g acrylic acid (PAA) use the mixing of 3.5mL ultrapure water and 3.5mL dioxane in round-bottomed flask
Solvent dissolution, round-bottomed flask is sealed with rubber stopper, and is passed through high pure nitrogen 30 minutes to remove the oxygen in reaction system, then will
Round-bottomed flask oil bath at 70 DEG C, magnetic agitation are reacted 10 hours；
(2), round-bottomed flask and 1.75g poly(ethylene glycol) methyl ether acrylate (PEGA) is added in opening steps (1), it will
Round-bottomed flask is passed through nitrogen 30 minutes after sealing with rubber stopper, then the oil bath at 70 DEG C, and magnetic agitation is reacted 10 hours, reaction
Obtained mixture ether precipitates 3-6 times, obtains white depositions, as shown in Fig. 2, gained white depositions pass through nuclear-magnetism table
Sign, it was demonstrated that obtained polymer is PAA/PPEGA copolymer, and sediment obtained is placed in baking oven, dry 48 at 30 DEG C
Hour to constant weight, stores for future use；
Step 2: being modified with block copolymer PAA/PPEGA laccase:
(1), by 0.468g PAA/PPEGA block copolymer, 9.6mg 1- (3- dimethylamino-propyl) -3- ethyl carbon two
Inferior amine salt hydrochlorate (EDC) is dissolved in the round-bottomed flask of 50mL with 20ml methylene chloride, is stirred to react 60 minutes；
(2), 5.8g n-hydroxysuccinimide (NHS) is added in the reagent made from step (1), reacts 24 at room temperature
Hour, then three times with ether precipitating, the polymer P AA/PPEGA-NHS of activation is made, then by PAA/PPEGA-NHS obtained
Be placed in baking oven, at 80 DEG C dry 24 hours it is spare to constant weight；
(3), the acetate buffer (ABS buffer) of compound concentration 0.1mol/L, pH=5, it is molten to weigh 20mg laccase
The laccase stock solution that 1mg/mL is prepared in 20mLABS buffer is spare, weighs 200mgPAA/PPEGA-NHS, is dissolved in
The PAA/PPEGA stock solution that 20mg/mL is prepared in 10mLABS buffer is spare；
(4), the laccase stock solution for taking 10mL step (3) to prepare is added dropwise to the PAA/PPEGA stock solution of 1mL stirring
In, it is stirred to react under ice bath 8 hours, the dialysis membrane that above-mentioned solution molecular cut off is 50000 is dialysed 1 day in ice bath,
To remove excessive PAA/PPEGA, it is made with the laccase (PAA/PPEGA- after block copolymer PAA/PPEGA modification
laccase)。
Step 3: being modified with copper ion PAA/PPEGA-laccase:
The copper-bath for first using ABS buffer 8mmol/l, then by laccase (PAA/ obtained in step 2
PPEGA-laccase) in copper-bath, cultivated 10 hours at 4 DEG C, be made block copolymer PAA/PPEGA and copper from
Laccase (Cu after son modification2+/ PAA/PPEGA-laccase) mixed solution, recycle molecular cut off be 500 dialysis membrane
Dialysis removes the copper ion of absorption loosely, the laccase (Cu after obtaining block copolymer PAA/PPEGA and copper ion modification2+/
PAA/PPEGA-laccase), and freeze-drying stores for future use.
Embodiment 2:
The present embodiment is to former laccase and the laccase (Cu after block copolymer PAA/PPEGA and copper ion modification2+/
PAA/PPEGA-laccase catalytic activity and stability) is measured, and specific test process is as follows:
(1), the catalytic activity of laccase is measured using 2'- hydrazine-bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid (ABTS) method,
With the solution of ABS buffer 0.5mmol/l ABTS, the laccase stock solution of 1mg/mL is added, uses ultraviolet spectrometry at 25 DEG C
Then thus the variation of ultraviolet absorption peak at photometer measurement 420nm wavelength calculates the activity of laccase, calculated result is as schemed
Shown in 3, Cu is found2+The activity of/PAA/PPEGA-laccase is increased to original 447%；
(2), to former laccase and Cu at 70 DEG C2+The catalytic activity of/PAA/PPEGA-laccase is measured, and passes through work
Property variation characterize the stability of laccase, as a result as shown in figure 4, after from being heated to 70 DEG C for 10 DEG C, Cu2+/PAA/PPEGA-
The activity of laccase drops to 99% from 410%, and the activity of former laccase is from 100% to complete deactivation, it was demonstrated that Cu2+/PAA/
The activity of PPEGA-laccase improves；
(3), as shown in table 1, to former laccase and Cu2+/ PAA/PPEGA-laccase partly declining under different breeding conditions
Phase is measured, it can be seen that Cu at 25 DEG C2+The half-life period of/PAA/PPEGA-laccase is 23.7, compared to former laccase
10, half-life period improves 137%.
Table 1
Embodiment 3:
The present embodiment is related to the laccase (Cu after block copolymer PAA/PPEGA and copper ion modification2+/PAA/
PPEGA-laccase application and catalytic activity and Stability Determination), the specific process steps are as follows:
Step 1: by Cu2+/ PAA/PPEGA-laccase is fixed to graphene paper surface:
(1), with the Cu of ABS buffer 1mg/mL2+/ PAA/PPEGA-laccase solution；
(2), the graphene paper of 1cm × 1cm size is placed in step (1) solution, is impregnated 8 hours under the conditions of 4 DEG C；
(3), the graphene paper impregnated in step (2) is taken out, with the ultrapure water graphene paper surface of flowing, to remove
The Cu that surface is not fixed firmly2+/PAA/PPEGA-laccase；
(4), it with graphene paper is dried with nitrogen, is saved backup at 4 DEG C；
Step 2: former laccase to be fixed on to the surface of graphene paper, to compare Cu2+/ PAA/PPEGA-laccase is fixed
Catalytic activity, stability afterwards, and recycling situation:
(1), 20mg EDC, 5mg NHS, 20mg original laccase are dissolved in 9mLABS buffer；
(2), 1mg pyrene butyric acid is dissolved into 1mL dimethylformamide；
(3), the solution prepared in step (2) is added dropwise in the solution in step (1), is stirred to react 8 at 4 DEG C
Hour；
(4), with molecular cut off be 1000 bag filter, dialyse 24 hours at 4 DEG C, with remove excessive EDS with
NHS；
(5) laccase that pyrene is modified will be fixed on by graphene paper surface using technique identical with step 1；
Step 3: to fixed former laccase and Cu2+The catalytic activity of/PAA/PPEGA-laccase and recycling property
It can be carried out test characterization:
Fig. 5 is the Cu being fixed in graphene paper2+The active testing of/PAA/PPEGA-laccase and former laccase, as a result table
Cu at bright 25 DEG C and 30 DEG C2+The activity of/PAA/PPEGA-laccase is improved compared to former laccase；Fig. 6 is 8 Cu2+/
PAA/PPEGA-laccase recycle after active testing result, the results showed that Cu2+/ PAA/PPEGA-laccase is fixed
Afterwards, it is recycled by 8 times, activity is reduced to 189% from 317%, and the activity of former laccase is from 100% to complete deactivation, table
Bright laccase (the Cu after block copolymer PAA/PPEGA and copper ion modification2+/ PAA/PPEGA-laccase) activity have
It significantly improves, and after 8 times recycle, the activity of laccase is still higher than unmodified former laccase activity, shows Cu2+/
PAA/PPEGA-laccase has application potential to can be applied in degradation catechol pollutant, and can reuse.
Claims (5)
1. a kind of method of modifying of laccase, it is characterised in that concrete technology includes the following steps:
One, 1 object poly propenoic acid (poly(ethylene glycol) methyl ether of block copolymerization is synthesized by Invertible ideal
Acrylate):
(1), by 20-60mg Reversible Addition Fragmentation Chain Transfer reagent, 2-10mg azodiisobutyronitrile, 0.2-0.5g acrylic acid,
It is dissolved in round-bottomed flask with 2-5mL ultrapure water and 3-7mL dioxane, round-bottomed flask is sealed with rubber stopper, nitrogen 30- is passed through
It is heated at 20-90 DEG C to remove the oxygen in round-bottomed flask, then by round-bottomed flask oil bath within 60 minutes, magnetic agitation reacts 5-36
Hour；
(2), round-bottomed flask and 1-5g poly(ethylene glycol) methyl ether acrylate is added in opening steps (1), round-bottomed flask is used
Rubber stopper sealing, is passed through nitrogen 10-60 minutes, then the oil bath at 30-150 DEG C, and magnetic agitation is reacted 10-24 hours, will be anti-
Mixture ether obtained is answered to precipitate 1-6 times, obtained sediment is poly propenoic acid (poly(ethylene glycol) methyl ether third
Olefin(e) acid ester), sediment obtained is placed in baking oven, is stored for future use to constant weight within drying 24-48 hours at 20-70 DEG C；
Two, laccase is modified with poly propenoic acid (poly(ethylene glycol) methyl ether acrylate):
(1), by 0.3-0.6g poly propenoic acid (poly(ethylene glycol) methyl ether acrylate), 7-11mg 1- (3- dimethylamino
Propyl) -3- ethyl-carbodiimide hydrochloride in the round-bottomed flask of 50mL with 15-25ml methylene chloride dissolve, stir at room temperature
Reaction 20-70 minutes；
(2), 4-9g n-hydroxysuccinimide is added in the reagent made from step (1), reacts 24 hours at room temperature, uses second
Ether precipitates three times, and polymer poly propenoic acid (poly(ethylene glycol) methyl ether acrylate)-N- hydroxysuccinimidyl acyl of activation is made
Imines, then poly propenoic acid obtained (poly(ethylene glycol) methyl ether acrylate)-n-hydroxysuccinimide is placed in baking oven
In, at 25-80 DEG C dry 24-72 hours it is spare to constant weight；
(3), the acetate buffer of compound concentration 0.1-0.9mol/L, pH=5-9 weigh 10-60mg original laccase and are dissolved in
The former laccase stock solution that 0.5-3mg/mL is prepared in 20mL acetate buffer is spare, weighs 100-400mg poly propenoic acid
(poly(ethylene glycol) methyl ether acrylate)-n-hydroxysuccinimide, is dissolved in 10mL acetate buffer and prepares 10-
Poly propenoic acid (poly(ethylene glycol) methyl ether acrylate)-n-hydroxysuccinimide stock solution of 40mg/mL is spare；
(4), the laccase stock solution for taking 10-15mL step (3) to prepare is added dropwise to the 0.5-11mL poly propenoic acid of stirring
In the stock solution of (poly(ethylene glycol) methyl ether acrylate)-n-hydroxysuccinimide, by solution obtained above in ice bath
Under be stirred to react 4-12 hours, then dialysed 1-3 days with the dialysis membrane ice bath that molecular cut off is 20000-50000, to remove
Remove excessive poly propenoic acid (poly(ethylene glycol) methyl ether acrylate)-n-hydroxysuccinimide, obtained polypropylene
Laccase after acid/poly- (poly(ethylene glycol) methyl ether acrylate) modification；
Three, it is modified with copper ion with the laccase after poly propenoic acid (poly(ethylene glycol) methyl ether acrylate) modification:
(1), the copper-bath that 8-20mmol/l is prepared with acetate buffer, then by polypropylene obtained in step 2
Laccase after acid/poly- (poly(ethylene glycol) methyl ether acrylate) modification cultivates 10-20 at 0-37 DEG C in copper-bath
Hour, the mixed solution of the laccase after poly propenoic acid (poly(ethylene glycol) methyl ether acrylate) and copper ion modification is made；
(2), mix reagent obtained in step (1) is utilized in the dialysis membrane ice bath that molecular cut off is 500-1000 and is dialysed,
The copper ion of absorption loosely is removed, after poly propenoic acid (poly(ethylene glycol) methyl ether acrylate) and copper ion modification is made
Laccase, and be freeze-dried under the conditions of store.
2. the method for modifying of laccase according to claim 1, it is characterised in that the Reversible Addition Fragmentation Chain Transfer reagent
For the pyrene functionalized thio valeric acid of 4- cyano -4- ethyl three.
3. the method for modifying of laccase according to claim 1, it is characterised in that respectively to former laccase and poly propenoic acid
The catalytic activity of laccase after the modification of (poly(ethylene glycol) methyl ether acrylate) and copper ion and half-life period carry out characterization test,
The result shows that the activity and stabilization of the laccase after poly propenoic acid (poly(ethylene glycol) methyl ether acrylate) and copper ion modification
Property is improved, and wherein catalytic activity improves 347%, and stability improves 137%, it was demonstrated that the modification to former laccase, successfully
Improve the catalytic activity and stability of former laccase.
4. the method for modifying of laccase according to claim 1, it is characterised in that utilize repairing for laccase described in claim 1
The poly propenoic acid (poly(ethylene glycol) methyl ether acrylate) and the laccase after copper ion modification of decorations method preparation are fixed to stone
After on black alkene paper, it can be reused in degradation catechol pollutant, and catalytic activity and stability are higher than former laccase.
5. the method for modifying of laccase according to claim 4, it is characterised in that fixed in graphene paper and being catalyzed
The testing procedure of activity and stability is as follows:
One, the laccase after poly propenoic acid (poly(ethylene glycol) methyl ether acrylate) and copper ion modification is fixed to graphite
Alkene paper surface:
(1), with acetate buffer prepare 1-10mg/mL poly propenoic acid (poly(ethylene glycol) methyl ether acrylate) and
Laccase solution after copper ion modification；
(2), the graphene paper of 1.0cm2-1.0m2 is soaked in the solution of step (1) preparation, 6- is impregnated under the conditions of 0-50 DEG C
20 hours；
(3), with the ultrapure water graphene paper surface of flowing, to remove the poly propenoic acid (poly- (second that surface is not fixed firmly
Glycol) methyl ether acrylate) and copper ion modification after laccase；
(4), it with graphene paper is dried with nitrogen, is saved backup at 0-60 DEG C；
Two, former laccase is fixed on to the surface of graphene paper, to compare poly propenoic acid (poly(ethylene glycol) methyl ether propylene
Acid esters) and copper ion modification after laccase it is fixed after catalytic activity, stability, and recycling situation:
(1), by 20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride, 5mg n-hydroxysuccinimide,
20mg original laccase is dissolved in 9mL acetate buffer；
(2), 1mg pyrene butyric acid is dissolved into 1mL dimethylformamide；
(3), the solution prepared in step (2) is added dropwise in the solution in step (1), it is small to be stirred to react 8 at 4 DEG C
When；
(4), the bag filter for being 1000 with molecular cut off, dialyses 24 hours at 4 DEG C, to remove excessive 1- (3- diformazan ammonia
Base propyl) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide；
(5) laccase that pyrene is modified is fixed on by graphene paper surface using technique identical with step 1；
Three, the poly propenoic acid (poly(ethylene glycol) methyl ether acrylate) and copper ion being fixed in graphene paper are modified
The activity of laccase afterwards is tested, the results showed that poly propenoic acid (poly(ethylene glycol) methyl ether acrylic acid at 25 DEG C and 30 DEG C
Ester) and copper ion modification after the activity of laccase be improved；To poly propenoic acid (poly(ethylene glycol) methyl ether acrylate)
It is tested with activity of the laccase after copper ion modification after 8 times recycle, is gathered after 8 times recycle as the result is shown
The activity of laccase after acrylic acid/poly- (poly(ethylene glycol) methyl ether acrylate) and copper ion modification is reduced to from 317%
189%, and the activity of former laccase shows poly propenoic acid (poly(ethylene glycol) methyl ether acrylic acid from 100% to complete deactivation
Ester) and copper ion modification after the activity of laccase be significantly improved, and after 8 times recycle, poly propenoic acid is (poly-
(ethylene glycol) methyl ether acrylate) and copper ion modification after laccase activity it is still higher than former laccase activity, it was demonstrated that polypropylene
It is adjacent that laccase after acid/poly- (poly(ethylene glycol) methyl ether acrylate) and copper ion modification has application potential to can be applied to degradation
In benzenediol pollutant, and it can reuse.
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| Simultaneous Enhancement of Bioactivity and Stability of Laccase by Cu2+/PAA/PPEGA Matrix for Efficient Biosensing and Recyclable Decontamination of Pyrocatechol;Tao Chen et al;《Anal. Chem.》;20161228;2065-2072页 |
| 交联聚乙二醇二丙烯酸酯的制备及对漆酶的固定化研究;赵敏 等;《中国造纸学报》;20091231;第24卷(第3期);61-66页 |
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