CN104046608A - Method for immobilizing papain - Google Patents

Method for immobilizing papain Download PDF

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CN104046608A
CN104046608A CN201410265922.5A CN201410265922A CN104046608A CN 104046608 A CN104046608 A CN 104046608A CN 201410265922 A CN201410265922 A CN 201410265922A CN 104046608 A CN104046608 A CN 104046608A
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enzyme
carrier
pan
reaction
immobilized
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CN104046608B (en
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熊春华
贾倩
姚彩萍
周苏果
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Zhejiang Gongshang University
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Zhejiang Gongshang University
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Abstract

The invention discloses a method for immobilizing papain. The method comprises the following steps: (1) soaking and swelling polyacrylonitrile resin by using a reaction solvent, then adding glucosamine hydrochloride and K2CO3, reacting in the presence of nitrogen, and then filtering; (2) firstly soaking and washing obtained filter residues by using absolute ethyl alcohol, then sequentially pickling and washing the obtained filter residues, and then carrying out vacuum drying so as to obtain a PAN-GA carrier; (3) adding an Na104 solution to the PAN-GA carrier, oxygenizing by keeping away from light, washing, then adding a phosphate buffer with the pH of 6.5-8.5 and the papain for immobilization reaction, then carrying out filtering separation, and washing and drying an obtained filter cake so as to obtain the immobilized papain. According to the method, the adopted PAN-GA carrier used for immobilizing the papain can be used for overcoming the defect of enzyme activity loss, which is caused by rigid collision in an enzyme immobilizing process, of a carrier synthesized in the prior art.

Description

The method of immobilized papain
Technical field
The present invention relates to a kind of method of immobilized papain, comprised the synthetic method for immobilized papain fixation support, be specially the synthetic method of polyacrylonitrile-glucosamine flexible resin carrier (PAN-GA).
Background technology
Along with the development of polymer material science, in recent years, polymer modification material was more and more subject to people's attention.Polymer modification is exactly to be overcome polymer materials inherent defect, improve material property, reduce material cost, given a kind of simple method of new function of material etc. by physics, chemistry or mechanical method.Compared with other carrier, the modified support with specific function has great potential, is widely used in every field and utilizes polymer modification material to carry out the immobilization of enzyme, is also a trend of fixed enzyme vector development.
Macro porous crosslinking resin microsphere has the features such as specific tenacity is high, relative density is low, specific surface area is large as fixed enzyme vector, makes it in immobilized enzyme, show unique superiority.For example, there is person to use polystyrene macroporous resin as enzyme immobilization carrier, improve the wetting ability of carrier by the method for graft modification, being used for fixing enzyme, but this class material has used chloromethyl ether in building-up process, and in modifying process, have hydrogenchloride to generate, not only atom utilization is not high, unfriendly to environment yet.
Patent 201310182565.1 discloses " a kind of preparation method of polyacrylonitrile immobilized enzyme ", prepares polyacrylonitrile nanofiber film, for immobilized enzyme by method of electrostatic spinning.The fibre strength that this method is made is low, and the life-span is short, yields poorly, and fibrous texture is also bad, also will control the jet of mutual repulsion at micro, and process more complicated, condition are difficult to control.
Yuan Chuntao, in " preparation of chitosan and acrylonitrile graft copolymer and state change the research of enzyme surely ", adopts grafting copolymerization process to synthesize the immobilization of a kind of chitosan-g-vinyl cyanide carrier for enzyme.The synthetic polymer graft rate of this kind of method compared with low, side reaction is many, due to shortcomings such as the use of initiator are easily degraded of products therefrom, poor stability, fusing point is low, physical strength is low, and subsequent disposal is loaded down with trivial details, therefore makes its application be subject to certain restrictions.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of immobilized papain.Carrier for immobilized papain of the present invention (PAN-GA), can overcome the carrier of prior art synthesized in enzyme immobilization process, and the enzyme activity causing due to rigid collision loses.
In order to solve the problems of the technologies described above, the invention provides a kind of method of immobilized papain, comprise the following steps:
1), take 0.1mg polyacrylonitrile resin (PAN), add the reaction solvent of 100～300ml, soak after swelling 10～14h; Add glucosamine hydrochloride (GA) and the pulverous K of 0.85～0.9g (being preferably 0.88g) of 0.65～0.7g (being preferably 0.68g) 2cO 3, the stirring velocity with 150～250rpm under nitrogen protection first stirs 1～3h; Then be warming up to 90～110 DEG C (being preferably 100 DEG C), continue at nitrogen protection lower magnetic force stirring reaction 11～13h; After reaction finishes, filter, collect filter residue;
2), by step 1) filter residue of gained first washs to washings by soaked in absolute ethyl alcohol is colourless, then carries out successively pickling and washing; Then to constant weight, obtain PAN-GA carrier in 45～55 DEG C of (being preferably 50 DEG C) vacuum-dryings;
3), take the PAN-GA carrier of 10mg, adding concentration is the Nal0 of 2.5～3.5mg/mL (being preferably 3mg/mL) 4solution 2.8～3.2mL (being preferably 3mL), lucifuge oxidation 28～32min (being preferably 30min), then (remarks explanation: washing times is generally 3-4 time after washing successively with dehydrated alcohol and water, at every turn, the consumption of dehydrated alcohol is 30ml, and the consumption of deionized water is 50ml); The phosphoric acid buffer (PBS) that the pH that adds again 8～12ml (being preferably 10mL) is 6.5～8.5, then adds 0.18～0.22mg (being preferably 0.2mg) papoid; Be that under 100～150rpm, constant temperature oscillation is fixed after 3.5～4.5h (being preferably 4h) in immobilization temperature, the rotating speed of 20～30 DEG C (being preferably 25 DEG C), filtering separation (can collect filtrate);
The phosphoric acid buffer (PBS) that gained filter cake is 6.5～8.5 with pH rinses after (generally rinsing 8～10 times) repeatedly, in the vacuum drier inner drying 4.5～5.5h of 36～38 DEG C (being preferably in 37 DEG C of dry 5h), obtain immobilized papain.
Improvement as the method for immobilized papain of the present invention: described step 1) in reaction solvent be ethylene glycol (ED).
Further improvements in methods as immobilized papain of the present invention: the phosphoric acid buffer that described pH is 6.5～8.5 is the phosphoric acid buffer (PBS) of 0.1mol/L pH7.
Further improvements in methods as immobilized papain of the present invention: described Nal0 4solution is with pH=4.0, and the HAc-NaAc damping fluid of 0.1mol/L is prepared as solvent.
Further improvements in methods as immobilized papain of the present invention: step 2) in carry out successively pickling and washing be specially: soaking 2～4 hours with the hydrochloric acid soln (consumption is about 20mL) of 1mol/L, is then neutral with deionized water wash to washings.
In the present invention, clearly do not inform temperature, all refer under conventional (20～30 DEG C) and carry out.
In the present invention:
Polyacrylonitrile resin (PAN, also name polyacrylonitrile resin microballoon, polyacrylonitrile microballoon etc.) is selected D-160 type macroporous adsorbent resin, degree of crosslinking 7%DVB, nitrogen content 22.18%, functional group content: 15.83CN mmol/g; For example can be purchased from Zhonglan Chenguang Chemical Inst.
The present invention selects that to have specific surface area large, and physical strength is high, good stability, low, the easy preparation of price, segregative macro porous crosslinking polyacrylonitrile resin microballoon be as immobilized enzyme modified support, compares other carriers and has stronger superiority.And polyacrylonitrile molecular surface contains a large amount of active cyano group, easily carry out chemical modification, make it with special functional group, the immobilization that can be enzyme provides effective binding site and simple, a gentle immobilization process.
Glycosylated surface can active adsorption specific proteins, and this is " impact of glucosides " of generally acknowledging.In view of this, the present invention uses ligand field theory and Quantum chemical calculation, choosing polyacrylonitrile is parent, carry out surface chemical modification with the glucosamine material that contains glycosyl, improve its surperficial wetting ability and biocompatibility, and then prepare the immobilization of novel glycosylated polyethylene nitrile resin carrier for papoid, be expected to realize " collection cluster effect cui ".
Enzyme immobilization carrier prepared by the present invention has the space of mesoporous diameter, can provide larger outer surface area and modification rear surface to contain compared with features such as polysaccharide-baseds, therefore be conducive to be beneficial to the absorption of the macromolecular substance such as papoid, compared with traditional biologic treating technique, this technological investment is few, efficiency is high, cost is low, there is significant economic benefit and social benefit, be expected to promote the use of in fields such as enzyme immobilization technologies, there is important theory value and good application prospect.
Particularly:
The present invention, select glucosamine hydrochloride (GA) as function base, be incorporated on macroporous resin polyacrylonitrile parent through chemical modification, thereby make novel flexible resin carrier (PAN-GA), build flexibility function base decorative layer at carrier surface, can be immobilized enzyme effective binding site and simple, a gentle immobilization process are provided.
The present invention has inquired into reaction solvent, temperature of reaction, reactant quality ratio and the impact of reaction times on resin rate of body weight gain, and has determined the optimum process condition of building-up reactions.Use elemental analysis method, infrared analysis and thermogravimetric analysis to characterize synthetic flexible resin carrier structure, infer that flexibility function group is grafted on polyacrylonitrile microballoon with the form of addition reaction.
Use infrared technique method, synthetic polyacrylonitrile-glucosamine flexible resin carrier (PAN-GA) is carried out to Structural Identification, inquire into response path and resin structure, comprise the steps:
1. the polyacrylonitrile microballoon that takes a morsel mixes with KBr, and grinding, compressing tablet are measured its infrared spectra, spectral range 4000cm on NICOLET-380 infrared spectrometer -1～400cm -1, resolving power is 4cm -1, scanning times is 32 times.
2. according to above-mentioned steps, the method in is 1. measured respectively the infrared spectra of carrier (PAN-GA) and part glucosamine sugar hydrochloride.
3. pass through the comparative analysis of the infared spectrum of the carrier (PAN-GA) to polyacrylonitrile microballoon, part and after synthesizing, infer the possible response path and the resin structure that resin building-up reactions.
Use thermogravimetric analysis (TGA) to carry out Structural Identification to above-mentioned synthetic carrier (PAN-GA), and further illustrate the successful introducing of flexibility function base.
Specific as follows:
Accurately take fully dried above-mentioned carrier (PAN-GA) sample and be respectively 6.0～8.0mg, use plum Teller TGA/DSC1 type simultaneous thermal analysis instrument to carry out thermogravimetric analysis.
Analysis condition: carrier gas: nitrogen; Carrier gas flux: 20mL/min; Heating schedule: 20 DEG C/min, start-stop temperature: 50 DEG C～1000 DEG C.
The calculation formula of flexible carrier (PAN-GA) weightening finish that adopts the inventive method preparation and obtain is as follows:
ΔG ( % ) = W 1 - W 0 W 0 × 100
In formula: the rate of body weight gain/% of carrier (PAN-GA) after △ G-reaction;
W 0-for reacting the quality/g of front polyacrylonitrile resin (PAN);
W 1-for reacting the quality/g of rear carrier (PAN-GA).
By step 2 of the present invention) preparation and carrier (PAN-GA), according to step 3) described method carries out the fixing of papoid.
Get a certain amount of enzyme solution, filtrate and water lotion, join respectively 2% casein solution and the 1mL0.1mol/L PBS damping fluid (pH=7.0 that contain 4mL, the L-Cys of 5mmol/L and 1mmol/L EDTA) in test tube, in 37 DEG C of water-baths, react 30min, then add immediately trichoroacetic acid(TCA) (TCA) termination reaction of 5mL10%, then after reactant being mixed, in water-bath, leave standstill 10min and filter, filtrate is measured light absorption value at 275nm place.Blank group, before adding enzyme, first adds 5mL10%TCA, and all the other steps are identical.
Enzyme activity determination method:
Enzyme activity unit is defined as under condition determination (37 DEG C of temperature, pH7.2), and it is 1 enzyme activity unit that per minute catalysis casein hydrolysis generates the required enzyme amount of l ug tyrosine.
The activity recovery of immobilized enzyme refers to that immobilized enzyme total activity and immobilization process add the ratio of solution enzyme total activity, are expressed as a percentage.
The relative activity (%) of immobilized enzyme or resolvase: refer in test on the same group taking vigor the highest as 100, the ratio with the vigor of remaining immobilized enzyme or solution enzyme, is expressed as a percentage conventionally.
The residual enzyme vigor (%) of immobilized enzyme or resolvase: refer to that in experiment on the same group vigor taking the immobilized enzyme before untreated or solution enzyme is as 100, with process after the ratio of (comprising heat, acid, alkali, reagent, immobilization, refrigeration etc.) shown vigor, be expressed as a percentage.
The carrier (PAN-GA) that adopts the inventive method preparation and obtain has following technical superiority:
1, the present invention uses elemental analysis method and infrared spectrum technology to characterize synthetic flexible resin carrier structure, result shows cyano group generation core addition reaction in amino in flexibility function group and polyacrylonitrile parent and is grafted on polyacrylonitrile parent this reaction of atomic utilization ratio high (atom in raw material is transformed in product as much as possible); Not to or few in environment, discharge poisonous and hazardous by product, embodied preferably the feature of Green Chemistry, there is obvious economic benefit and environmental benefit.
2,, by the TGA tracing analysis to polyacrylonitrile microballoon, flexibility function base and synthetic rear carrier (PAN-GA), obtained the changing conditions of carrier thermostability.Result shows: carrier just starts to decompose at 325 DEG C and 400 DEG C, and physical strength is high, has higher thermostability.
3, the present invention is taking hydrophilic amino glucose function base as flexible chain, utilize chemical graft process that polyacrylonitrile resin microballoon is carried out to modification, the successful introducing of hydrophilic flexibility chain makes its normal conformation that can effectively maintain zymoprotein in the immobilization process of enzyme, reduces the inactivation of enzyme.The immobilized enzyme rate of recovery of gained is up to 80.6%, is 21.9 times of immobilization level that adopt unmodified polypropylene nitrile resin microsphere.
4, adopt the thermostability, storage stability of polyacrylonitrile-glucosamine flexible resin carrier (PAN-GA) immobilized papain of making of the present invention all apparently higher than resolvase.Illustrate and use the carrier (PAN-GA) that makes of the inventive method to provide a kind of close friend's microenvironment as the fixation support of enzyme for papoid, biological activity and the catalytic activity that can keep preferably zymoprotein molecule, have certain using value.
5, the carrier (PAN-GA) that adopts the inventive method to make, for immobilized papain, its preparation technology is simple, easy and simple to handle, reaction conditions is gentle, raw material is easy to get, cost is low, does not need specific installation, and common process just can carry out.
6, the immobilized papain that the present invention prepares gained has higher thermostability, storage stability, organic solvent tolerance and repetition stability, has certain actual application value.
Brief description of the drawings
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the infrared spectrogram of PAN, GA and PAN-GA;
Fig. 2 is PAN-GA resin synthetic route chart;
Fig. 3 is the thermogravimetric curve figure of PAN, GA and PAN-GA;
Fig. 4 is the immobilization process of papoid on carrier PAN-GA;
Fig. 5 is the impact of temperature on immobilized enzyme and resolvase relative activity;
The repeat performance of Fig. 6 immobilized enzyme;
The storage stability of Fig. 7 immobilized enzyme and resolvase.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, but content of the present invention is not limited to this.
Remarks: the washing in following examples is washs with deionized water.
The synthetic method of embodiment 1, a kind of carrier for immobilized papain, taking polyacrylonitrile resin (PAN) as parent, glucosamine hydrochloride (GA) is part, and polyacrylonitrile microballoon is carried out to chemical graft, is specially and carries out successively following steps:
1., accurately take 0.1mg polyacrylonitrile resin (PAN), move in the three-necked bottle of 100mL, add again the solvent of 100ml---ethylene glycol (ED), soak after swelling 12h, add glucosamine hydrochloride (GA) and the 0.88g K of 0.68g 2cO 3powder, the stirring velocity with 200rpm under nitrogen protection first stirs 2h at normal temperatures; Then be warming up to rapidly 100 DEG C, magnetic agitation reaction 12h; After reaction finishes, filter, collect filter residue.
2. it is colourless that the filter residue of, 1. step being collected first washs to washings by soaked in absolute ethyl alcohol, then first pickling (soaking 3 hours with the hydrochloric acid soln of 20mL, 1mol/L), wash with water again until washings is neutrality, under 50 DEG C of conditions, vacuum-drying is to constant weight, obtain polyacrylonitrile-glucosamine flexible resin carrier (PAN-GA), for subsequent use.
Experiment 1, utilization infrared technique method, carry out Structural Identification to above-mentioned synthetic polyacrylonitrile-glucosamine flexible resin carrier (PAN-GA), inquires into its response path and resin structure.Concrete steps are as follows:
1. the polyacrylonitrile microballoon that takes a morsel mixes with KBr, and grinding, compressing tablet are measured its infrared spectra, spectral range 4000cm on NICOLET-380 infrared spectrometer -1～400cm -1, resolving power is 4cm -1, scanning times is 32 times.
2. according to above-mentioned steps, the method in is 1. measured respectively the infrared spectra of novel carriers (being PAN-GA) and part glucosamine hydrochloride (GA).
3. by the comparative analysis of the infared spectrum (seeing Fig. 1) to polyacrylonitrile microballoon, part and synthetic rear novel carriers (being PAN-GA), infer the response path and the resin structure (seeing Fig. 2) that resin building-up reactions.
Fig. 1 is the infrared spectrogram of PAN, GA and PAN-GA.Analyzed from Fig. 1, at 3500～3000cm -1in scope there is 3344cm in GA -1, 3292cm -1, 3097cm -1, 3039cm -1four significantly strong absorption peaks of division, this is because there are multiple hydroxyls in GA molecule, they belong to hydroxyl (OH) and amino (NH in glucosamine molecule 2) stretching vibration absorption peak.And contrast PAN-GA spectrum, the peak type after reaction within the scope of this has become broad peak, illustrates that reaction has occurred the amino in glucosamine molecule.At 1700～1500cm -1in scope, GA molecule has 1619cm -1and 1584cm -1, 1539cm -1three absorption peaks, they are respectively NH 2angle vibration absorption peak with N-H.And after reaction, at 1627cm -1and 1580cm -1near there is a pair of broad peak, explanation-NH 2participate in reaction.Obviously weakening of charateristic avsorption band (C ≡ N) after simultaneous reactions in polyacrylonitrile, and 1539cm -1the disappearance at peak and 1121cm -1, 1062cm -1(secondary hydroxyl of C-O-H and primary hydroxyl absorption peak in glucosamine molecule), 896cm -1the appearance of (sugar ring absorption peak), can prove that glucosamine molecule has been grafted on polyacrylonitrile parent.
Infer the synthesis path of PAN-GA carrier as shown in Figure 2.
Experiment 2, utilization thermogravimetric analysis (TGA) are carried out Structural Identification to above-mentioned synthetic novel carriers (being PAN-GA), and are further illustrated the successful introducing of flexibility function base.Experiment condition is: N 2for shielding gas, temperature rises to 1000 DEG C by 25 DEG C, and heat-up rate is 20 DEG C/min.Experimental result as shown in Figure 3.
Embodiment 2, the PAN-GA carrier of embodiment 1 gained is carried out to papoid immobilization, specific as follows:
Take the PAN-GA carrier of 10mg, adding concentration is the Nal0 of 3mg/mL 4solution (pH=4.0, the HAc-NaAc damping fluid preparation of 0.1mol/L) 3.00mL, lucifuge oxidation 30min, washs (at every turn, the consumption of dehydrated alcohol is 30ml, and the consumption of deionized water is 50ml) 3-4 time successively with dehydrated alcohol and water; Add again the phosphoric acid buffer (PBS) of the 0.1mol/L pH7 of 10mL, then add the papoid of 0.2mg, it is 25 DEG C in immobilization temperature, rotating speed is under 100rpm, after the fixing 4h of constant temperature oscillation, filter, collect filtrate, and isolate filter cake (for the immobilized papain that makes), gained filter cake rinses after 8～10 times repeatedly with the phosphoric acid buffer (PBS) of 0.1mol/L pH7, under 37 DEG C of conditions, put into after the dry 5h of vacuum drier, obtain immobilized papain, enzyme immobilization process is shown in Fig. 4.Recording its enzyme fixed rate is 67.9%.
Comparative example 1, glucosamine hydrochloride (GA) by embodiment 1 step in 1. make respectively 2-Acetamido-2-deoxy-D-glucose, chitosan into, and all the other are equal to embodiment 1.The carrier of gained is called carrier A, carrier B.Immobilization experiment according to the method described in above-described embodiment 2 for papoid, the correlation data of the carrier PAN-GA of itself and embodiment 1 gained is as shown in table 1:
The impact of the synthetic carrier of table 1, different ligands on enzyme fixed rate
Comparative example 2, reaction solvent by the step in embodiment 1 in 1. make N into by ethylene glycol (ED respectively), N dimethyl formamide (DMF), toluene (Toluol), l, 4-dioxane (Dioxane), methyl-sulphoxide (DMSO), all the other are equal to embodiment 1.
Carrier PAN-GA to 4 of above-mentioned gained kinds of carriers and embodiment 1 gained measures its rate of body weight gain and enzyme immobilization rate, inquires into rate of body weight gain and the enzyme fixed rate impact experiment of different solvents on synthetic vectors.Result is as shown in table 2 below:
Table 2, the reaction solvent rate of body weight gain on synthetic vectors and the impact of enzyme fixed rate
Solvent Resin rate of body weight gain △ W (%) Enzyme fixed rate (%)
Toluene (Toluol) 14.8 17.1
L, 4-dioxane (Dioxane) 9.7 12.9
Methyl-sulphoxide (DMSO) 23.5 36.4
DMF (DMF) 19.1 29.4
Ethylene glycol (ED) (the present invention) 41.7 67.9
From the results shown in Table 2, resin PAN-GA respectively in solvent ED rate of body weight gain and enzyme fixed rate the highest, its value is respectively 35.9% and 67.9%, and reason is the abundant swelling property of solvent to matrix, has also shown thus that reaction solvent is to affect one of synthetic important factor of resin.So the optimum response solvent of the selected PAN-GA resin carrier of the present invention is ethylene glycol (ED).
Comparative example 3, temperature of reaction by the step in embodiment 1 in 1. make respectively 60 DEG C into by 100 DEG C, and 80 DEG C, 120 DEG C, 140 DEG C, all the other are equal to embodiment 1.
Carrier PAN-GA to 4 of above-mentioned gained kinds of carriers and embodiment 1 gained measures its rate of body weight gain and enzyme immobilization rate, inquires into rate of body weight gain and the enzyme fixed rate impact experiment of differing temps on synthetic vectors.Result is as shown in table 3 below:
Table 3, the impact of temperature of reaction on synthetic resins rate of body weight gain and enzyme fixed rate
Temperature of reaction Resin rate of body weight gain △ W (%) Enzyme fixed rate %
60℃ 8.5 19.1
80℃ 13.4 29.9
100 DEG C (the present invention) 41.7 67.9
120℃ 31.1 41.9
140℃ 27.2 31.7
In the situation that other conditions are constant, by changing temperature of reaction, inquire into different temperature and synthetic resins rate of body weight gain and enzyme state are determined to the impact of rate.As can be seen from Table 3, within the scope of test temperature, in the time that temperature is 100 DEG C, it is maximum that the rate of body weight gain of resin PAN-GA reaches, and it is maximum that enzyme fixed rate now also reaches.This is because temperature is too low, is unfavorable for the attack of function base, makes reaction be difficult to carry out completely; Along with the rising of temperature, the solubleness of compound in organic solvent increases, and reactant activity increases, reaction rate accelerates, and rate of body weight gain also increases; But excess Temperature, easily there is side reaction in building-up reactions, so can cause the decline of its rate of body weight gain when temperature exceedes 100 DEG C, can cause solvent evaporates and lose and continue intensification, be unfavorable for the synthetic of resin, consider simultaneously when temperature of reaction too high, the macromolecular scaffold of resin is easily damaged, so test and Selection top temperature is 100 DEG C.The consideration of our Comprehensive Experiment condition and combined coefficient, the optimum synthesising temperature of determining PAN-GA resin is 100 DEG C.
Comparative example 4, by step in embodiment 1 1. in reactant consumption GA (g): K 2cO 3(g) change respectively 0.17:0.22,0.34:0.44,1.02:1.32,1.36:1.76 into by 0.68:0.88, all the other equal same embodiment 1.
Carrier PAN-GA to 4 of above-mentioned gained kinds of carriers and embodiment 1 gained measures its rate of body weight gain and enzyme immobilization rate, inquires into rate of body weight gain and the enzyme immobilization rate impact experiment of differential responses thing consumption on synthetic vectors.Result is as shown in table 4 below:
Table 4, GA and K 2cO 3the impact of consumption on resin PAN-GA rate of body weight gain and enzyme fixed rate
GA(g):K 2CO 3(g) Resin rate of body weight gain △ W (%) Enzyme fixed rate %
0.17:0.22 21.6 23.6
0.34:0.44 31.4 36.8
0.68:0.88 (the present invention) 41.7 67.9
1.02:1.32 38.1 41.2
1.36:1.76 34.9 39.2
For a certain amount of resin microsphere, along with the increase of reactant concn, resin rate of body weight gain also increases, be increased to a timing and work as reactant concn, its rate of body weight gain decreases, be mainly to belong to nonhomogeneous system because of reaction system for this reason, transformation efficiency is not only subject to the impact of reactant concn, is also subject to the impact of space steric effect.So in the time that the amount of the reactant adding is less, reactant fully reacts with polyacrylonitrile, increases weight more obvious; And in the time that the binding site of resin surface reaches capacity, then increase reactant consumption and can not make too much it in conjunction with getting on, on the contrary because reactant concn is excessive, closely flocking together is unfavorable for diffusion, hinders reaction and carries out.So in the time of 0.10g polyacrylonitrile microballoon, glucosamine (GA) and salt of wormwood (K 2cO 3) optimal addn be respectively 0.68g and 0.88g.
Test 1: the optimal reaction temperature of immobilized enzyme and resolvase (immobilized papain of embodiment 2 gained and conventional papoid):
Correspondence is got immobilized enzyme and the resolvase of appropriate 10mg, in different enzymic catalytic reaction temperature (30 DEG C～80 DEG C) scope, measures corresponding enzyme live according to enzyme activity determination method respectively.
It is also one of feature of enzyme to envrionment temperature sensitivity.Temperature not only can affect the speed of enzymic catalytic reaction, also can affect the stability of enzyme, makes zymoprotein molecule degeneration, and can also affect the space structure of enzyme molecule and the catalytic effect of enzyme, under these several combined factors impacts, has produced an optimal reactive temperature.
As seen from Figure 5, the optimum temperuture of resolvase is 50 DEG C, and it is comparatively precipitous that curve moves towards trend, the catalyzed reaction temperature influence that resolvase is described is larger, and after immobilization, optimum temperuture has improved 10 DEG C, and within the scope of 50 DEG C～80 DEG C, all keep higher enzyme to live, optimum temperature range broadens, and may, because papoid has obtained one stable structure more after carrier immobilized, make sex change speed reduced by the impact of envrionment temperature.The rising of optimal reactive temperature after enzyme immobilization, is because the multiple spot existing between enzyme molecule and carrier is connected, and has stablized the conformation of enzyme, has prevented that enzyme from the distortion of peptide chain folding extension occurring because being heated, and causes the decline of enzymic activity.The raising of enzyme optimum temperuture, has not only improved enzyme catalysis efficiency, also makes enzymic catalytic reaction under the environment of comparatively high temps, carry out, significant in processing and practical application.
Test 2: the thermostability of immobilized enzyme and resolvase
Correspondence is got immobilized enzyme and the resolvase of appropriate 10mg, be that in 25 DEG C, 35 DEG C, 55 DEG C, 65 DEG C, different time (30min, 60min, 90min, 120min, 150min, 180min) is processed in insulation in envrionment temperature, then be cooled to 4 DEG C with frozen water rapidly, then according to enzyme activity determination method, at 37 DEG C, measure corresponding enzyme (with reference to the enzyme activity determination method described in the present invention) alive.
The stability of enzyme refers to that enzymic activity is in certain environment condition, such as the ability maintaining vigour under the factor impacts such as time, temperature, organic solvent, mechanical effect.
Experimental result draws: resolvase and immobilized enzyme all can keep higher stability under 25 DEG C of low temperature and 35 DEG C of processing, in the time that temperature rises to 55 DEG C, along with the prolongation of soaking time, enzyme activity all presents obvious downtrending, and in the time of 180min, the residual vigor of resolvase and immobilized enzyme is 40% and 69%.In the time that temperature is 65 DEG C, after insulation 60min, the residual activity of immobilized papain is about 85%, and resolvase only has 57%; After insulation 180min, the residual vigor of immobilized enzyme is surplus 57% left and right also, and now the vigor of resolvase only remains 21%.May be due to after papoid be combined with resin carrier, there is some and changed in the microenvironment of the space conformation of enzyme active center position and enzyme molecule, and thermal denaturation resistant can strengthen.As can be seen here, the papoid specific ionization papoid after fixing there is higher thermostability.This has great importance in actual applications to enzyme.
Test 3: the organic solvent stability of immobilized papain and free papoid
Correspondence is got immobilized enzyme and the resolvase of appropriate 10mg, adding respectively concentration is in acetonitrile, ethanol and the DMF organic reagent mixed solution 5ml of 10% and 90% (V/V), under normal temperature, place and process 1h, then according to enzyme activity determination method, at 37 DEG C, measure corresponding enzyme (with reference to the enzyme activity determination method described in the present invention) alive.
Remarks explanation: above-mentioned organic reagent mixed solution is to prepare with phosphoric acid buffer (PBS) damping fluid of 0.1mol/L pH7.
Table 5, the impact of organic reagent on immobilized enzyme and resolvase
Data from table 5 clearly can find out, resolvase is after 3 kinds of organic solvents of different concns are processed, and enzyme activity all has larger loss, has shown poor stability.And concentration more high enzymatic activity loss is more serious.And papoid is after immobilization, no matter be after processing under the organic solvent of high density and lower concentration, all show higher residual enzyme and lived, stability is apparently higher than resolvase.Organic reagent affect the activity of enzyme and stability be mainly because: on the one hand, organic reagent can be directly and enzyme effect, the space conformation of destructive enzyme, thereby affect active centre and the stability of enzyme; On the other hand, organic reagent can be by affecting the activity of enzyme with the direct effect of product or substrate.And enzyme is after carrier is fixing, carrier has certain provide protection to enzyme, and organic appearance agent cannot directly be contacted with enzyme, and after enzyme is combined with carrier, makes the space structure of enzyme more stable, is difficult for destroyedly, and stability is improved.
Test 4: the repetition stability of immobilized enzyme and resolvase
Get respectively the immobilized enzyme of appropriate 10mg, add 2.00mL2% (quality %) casein solution, 37 DEG C of reaction 30min, filtering separation immobilized enzyme, with PBS damping fluid (phosphoric acid buffer of 0.1mol/L pH7) washing.Repeat under the same conditions above step and measure corresponding enzyme and live, obtaining the immobilized enzyme of reaction repeated 8 times.
Fig. 6 has represented the relation between number of times and relative activity that recycles of immobilized enzyme.Immobilized enzyme is through after 5 times reuse as shown in Figure 6, and activity can retain more than 80%, uses after 8 times, active reservation still more than 60%.May be that part enzyme comes off in use in addition, and in removal process, enzyme also has loss in various degree, so caused the part inactivation of enzyme because some enzyme molecules are combined with carrier and are obtained undertighten by adsorption.For resolvase can not be reused, immobilized enzyme can be repeatedly used, and has obviously improved the service efficiency of enzyme, reduces costs.Good repeat performance, not only can reduce costs effectively, also makes continuous catalytic reaction technological design become possibility, has actual application value.
Test 5: respectively immobilized enzyme and resolvase are preserved some days in the PBS of 4 DEG C of pH7.0 damping fluid, then according to enzyme activity determination method, measured corresponding enzyme and live within the different timed intervals.
As can be seen from Figure 7, the papoid being fixed on carrier has shown higher storage stability compared with resolvase, under identical preservation condition, (4 DEG C) place 7d, resolvase is along with the prolongation of shelf time, and enzyme activity declines very fast, and preservation effect is bad, its residual activity is only left 17%, and two kinds of immobilized enzyme are being preserved after 7d in damping fluid, its residual activity all, more than 80%, has shown good storage stability.
Above-mentioned test has illustrated that utilization the inventive method makes polyacrylonitrile-glucosamine flexible carrier (PAN-GA) and provides a kind of close friend's microenvironment as the fixation support of enzyme for papoid, biological activity and the catalytic activity that can keep preferably zymoprotein molecule, have certain using value.
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.

Claims (6)

1. the method for immobilized papain, is characterized in that comprising the following steps:
1), take 0.1mg polyacrylonitrile resin, add the reaction solvent of 100～300ml, soak after swelling 10～14h; Add glucosamine hydrochloride and the pulverous K of 0.85～0.9g of 0.65～0.7g 2cO 3, the stirring velocity with 150～250rpm under nitrogen protection first stirs 1～3h; Then be warming up to 90～110 DEG C, continue stirring reaction 11～13h under nitrogen protection; After reaction finishes, filter, collect filter residue;
2), by step 1) filter residue of gained first washs to washings by soaked in absolute ethyl alcohol is colourless, then carries out successively pickling and washing; Then in 45～55 DEG C of vacuum-dryings to constant weight, obtain PAN-GA carrier;
3), take the PAN-GA carrier of 10mg, adding concentration is the Nal0 of 2.5～3.5mg/mL 4solution 2.8～3.2mL, lucifuge oxidation 28～32min, after then washing successively with dehydrated alcohol and water; The phosphoric acid buffer that the pH that adds again 8～12ml is 6.5～8.5, then adds 0.18～0.22mg papoid; Be that under 100～150rpm, constant temperature oscillation is fixed after 3.5～4.5h in the immobilization temperature of 20～30 DEG C, rotating speed, filtering separation;
After the phosphoric acid buffer that gained filter cake is 6.5～8.5 with pH rinses repeatedly, in the vacuum drier inner drying 4.5～5.5h of 36～38 DEG C, obtain immobilized papain.
2. the method for immobilized papain according to claim 1, is characterized in that: described step 1) in reaction solvent be ethylene glycol.
3. the method for immobilized papain according to claim 2, is characterized in that:
Described pH is that 6.5～8.5 phosphoric acid buffer is the phosphoric acid buffer of 0.1mol/L pH7.
4. according to the method for the immobilized papain described in claim 2 or 3, it is characterized in that:
Described Nal0 4solution is with pH=4.0, and the HAc-NaAc damping fluid of 0.1mol/L is prepared as solvent.
5. the method for immobilized papain according to claim 4, it is characterized in that: described step 2) in carry out successively pickling and washing be specially: soaking 2～4 hours with the hydrochloric acid soln of 1mol/L, is then neutral with deionized water wash to washings.
6. the method for immobilized papain according to claim 5, is characterized in that:
Described step 1) in: glucosamine hydrochloride is 0.68g, K 2cO 3for 0.88g, be warming up to 100 DEG C of reaction 12h.
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