CN104046608B - The method of immobilized papain - Google Patents

Abstract

The invention discloses a kind of method of immobilized papain, comprise the following steps：1) after, the immersion of polyacrylonitrile resin reaction dissolvent is swelled, aminoglucose hydrochloride and K are added₂CO₃, reacted under nitrogen protection；Then filter；2), gained filter residue is first washed with soaked in absolute ethyl alcohol, and pickling and washing are then carried out successively；Then it is dried in vacuo, obtains PAN GA carriers；3), Nal0 is added in PAN GA carriers₄Solution lucifuge is aoxidized, after washing, is added phosphate buffer and the reaction of being fixed of papain that pH is 6.5~8.5, is then separated by filtration；Gained filter cake is dried after rinsing, and obtains immobilized papain.Carrier (PAN GA) of the present invention for immobilized papain, can overcome the carrier synthesized by prior art during enzyme immobilization, the enzyme activity loss caused due to rigid collision.

Description

The method of immobilized papain

Technical field

The present invention relates to a kind of method of immobilized papain, include for immobilized papain immobilization The synthetic method of the synthetic method of carrier, specially polyacrylonitrile-Glucosamine flexible resin carrier (PAN-GA).

Background technology

With continuing to develop for polymer material science, in recent years, polymer modification material is increasingly by people's Pay attention to.Polymer modification is exactly the method by physics, chemically or mechanically to overcome polymeric material inherent shortcoming, improve material Performance, reduction material cost, a kind of method simple and easy to apply for assigning material New function etc..Compared with other carriers, with spy The modified support of distinguished service energy possesses great potential, is widely used in every field and is carried out using polymer modification material Enzyme immobilizatio, is also a trend of fixed enzyme vector development.

Macro porous crosslinking resin microsphere has specific strength is high, relative density is low, specific surface area is big etc. as fixed enzyme vector Feature, the superiority for making it show uniqueness in immobilised enzymes.For example, someone selects polystyrene macroreticular resin conduct Enzyme immobilization carrier, improves the hydrophily of carrier by the method for graft modification, and for immobilised enzymes, but this kind of material is in conjunction Chloromethyl ether has been used during, and has had hydrogen chloride generation in modifying process, not only atom utilization is not high, to environment also not It is friendly.

Patent 201310182565.1 is disclosed《A kind of preparation method of polyacrylonitrile immobilised enzymes》, pass through electrostatic spinning Method prepares polyacrylonitrile nanofiber film, for immobilised enzymes.The fibre strength that this method is made is low, and short life yields poorly, Fibre structure is also bad, also to control mutually exclusive jet in micro, process is more complicated, condition is difficult to control to.

Yuan Chuntao exists《The preparation of chitosan and acrylonitrile graft copolymer and state change the research of enzyme surely》In, using grafting Copolymerization method, which has synthesized a kind of chitosan-g- acrylonitrile carrier, is used for enzyme immobilizatio.The polymer grafting rate of such a method synthesis Relatively low, side reaction is more, lacks because the use of initiator is that products therefrom is degradable, stability is poor, fusing point is low, mechanical strength is low Point, and subsequent treatment is cumbersome, therefore it is subject to certain restrictions its application.

The content of the invention

The technical problem to be solved in the present invention is to provide a kind of method of immobilized papain.It is of the present invention For the carrier (PAN-GA) of immobilized papain, the carrier synthesized by prior art can be overcome in enzyme immobilization process In, the enzyme activity loss caused due to rigid collision.

In order to solve the above-mentioned technical problem, the present invention provides a kind of method of immobilized papain, including following step Suddenly：

1), weighing 0.1mg polyacrylonitrile resins (PAN), add 100~300ml reaction dissolvent, immersion is swelled 10~ After 14h；Add 0.65~0.7g (preferably 0.68g) aminoglucose hydrochloride (GA) and 0.85~0.9g (preferably 0.88g) powdered K₂CO₃, 1~3h is first stirred with 150~250rpm mixing speed under nitrogen protection；Then heat to 90~110 DEG C (preferably 100 DEG C), continue magnetic agitation under nitrogen protection and react 11~13h；After reaction terminates, filter, receive Collect filter residue；

2), by step 1) obtained by filter residue first washed with soaked in absolute ethyl alcohol to cleaning solution to be colourless, then carry out successively Pickling and washing；Then constant weight is dried under vacuum in 45~55 DEG C (preferably 50 DEG C), obtains PAN-GA carriers；

3) 10mg PAN-GA carriers, are weighed, addition concentration is 2.5~3.5mg/mL (preferably 3mg/mL) Nal0₄ 2.8~3.2mL of solution (preferably 3mL), lucifuge aoxidizes 28~32min (preferably 30min), then with absolute ethyl alcohol and water according to (remarks explanation after secondary washing：Washing times are generally 3-4 times, every time, and the consumption of absolute ethyl alcohol is 30ml, the use of deionized water Measure as 50ml)；The phosphate buffer (PBS) that 8~12ml (preferably 10mL) pH is 6.5~8.5 is added, is then added 0.18~0.22mg (preferably 0.2mg) papain；Immobilization temperature, rotating speed in 20~30 DEG C (preferably 25 DEG C) are Constant temperature oscillation is fixed after 3.5~4.5h (preferably 4h) under 100~150rpm, is separated by filtration (collectable filtrate)；

Gained filter cake for 6.5~8.5 phosphate buffer (PBS) rinse and (typically rinse 8~10 times) repeatedly with pH Afterwards, in drying 4.5~5.5h (preferably in 37 DEG C of dry 5h) in 36~38 DEG C of vacuum desiccator, immobilization Papain is obtained Enzyme.

It is used as the improvement of the method for the immobilized papain of the present invention：The step 1) in reaction dissolvent be second two Alcohol (ED).

It is used as the further improvements in methods of the immobilized papain of the present invention：The pH is 6.5~8.5 phosphoric acid Buffer solution is 0.1mol/L pH7 phosphate buffer (PBS).

It is used as the further improvements in methods of the immobilized papain of the present invention：The Nal0₄Solution is with pH= 4.0,0.1mol/L HAc-NaAc buffer solutions are prepared as solvent.

It is used as the further improvements in methods of the immobilized papain of the present invention：Step 2) in carry out pickling successively It is specially with washing：Soaked 2~4 hours, be then washed with deionized with 1mol/L hydrochloric acid solution (consumption is about 20mL) It is neutrality to cleaning solution.

In the present invention, temperature is not informed clearly, is each meant and is carried out under routine (20~30 DEG C).

In the present invention：

Polyacrylonitrile resin (PAN, also name polyacrylonitrile resin microballoon, polyacrylonitrile microballoon etc.) selects D-160 type macropores Polymeric adsorbent, degree of cross linking 7%DVB, nitrogen content 22.18%, functional group content：15.83CN mmol/g；For example it is purchased from middle indigo plant Morning twilight chemical research institute.

Specific surface area is big from having by the present invention, high mechanical strength, stability are good, price is low, it is easy prepare, it is segregative big Hole crosslinked polypropylene nitrile resin microsphere has stronger superiority compared to other carriers as immobilised enzymes modified support.And Polyacrylonitrile molecular surface contains a large amount of active cyano group, easily carries out chemical modification, it is carried special functional group, can be Enzyme immobilizatio provides effective binding site and a simple, gentle immobilization process.

Glycosylated surface can effective absorption specificity albumen, this is generally acknowledged " influence of glucosides ".In view of this, originally Ligand field theory and Quantum chemical calculation are used in invention, and selection polyacrylonitrile is parent, with the aminoglucose containing glycosyl Sugar substance carries out surface chemical modification, improves the hydrophily and biocompatibility on its surface, and then prepare new glycosylation Polyacrylonitrile resin carrier is used for Papain enzyme immobilizatio, it is expected to realize " collection cluster effect ".

Enzyme immobilization carrier prepared by the present invention has the space of mesopore diameter size, using the teaching of the invention it is possible to provide larger external surface area And modified surface is the features such as contain compared with polysaccharide-based, therefore be conducive to being beneficial to the absorption of the macromolecular substances such as papain, with Traditional biologic treating technique is compared, and the technological investment is few, efficiency high, cost are low, is imitated with significant economic benefit and society Benefit, is expected to promote the use of in fields such as enzyme immobilization technologies, with important theory value and good application prospect.

Specifically：

The present invention, from aminoglucose hydrochloride (GA) as function base, macroreticular resin is incorporated into by chemical modification On polyacrylonitrile parent, so that novel flexible resin carrier (PAN-GA) has been made, building flexibility function base in carrier surface repaiies Layer is adornd, effective binding site and a simple, gentle immobilization process can be provided for immobilised enzymes.

The present invention inquired into reaction dissolvent, reaction temperature, reactant quality than with shadow of the reaction time to resin rate of body weight gain Ring, and the optimum process condition of synthetic reaction is determined.With elemental analysis method, infrared analysis and thermogravimetric analysis to synthesis Flexible resin carrier structure is characterized, and infers that flexibility function group is grafted to polyacrylonitrile microballoon in the form of addition reaction On.

With infrared technique method, polyacrylonitrile-Glucosamine flexible resin carrier (PAN-GA) of synthesis is carried out Structural Identification, inquires into response path and resin structure, comprises the following steps：

1. a small amount of polyacrylonitrile microballoon is taken to be mixed with KBr, grinding, tabletting are determined on NICOLET-380 infrared spectrometers Its infrared spectrum, spectral region 4000cm^-1~400cm^-1, resolution ratio is 4cm^-1, scanning times are 32 times.

2. according to above-mentioned steps 1. in method determine the sugared hydrochloride of carrier (PAN-GA) and part aminoglucose respectively Infrared spectrum.

3. by the infared spectrum to the carrier (PAN-GA) after polyacrylonitrile microballoon, part and synthesis to score Analysis, thus it is speculated that go out the possibility response path and resin structure of resins synthesis reaction.

Structural Identification is carried out to the carrier (PAN-GA) of above-mentioned synthesis with thermogravimetric analysis (TGA), and further illustrated Flexibility function base is successfully introduced into.

It is specific as follows：

Accurate fully dried above-mentioned carrier (PAN-GA) sample that weighs is respectively 6.0~8.0mg, uses plum Teller TGA/DSC1 types synchronous solving carries out thermogravimetric analysis.

Analysis condition：Carrier gas：Nitrogen；Carrier gas flux：20mL/min；Heating schedule：20 DEG C/min, start-stop temperature：50℃ ~1000 DEG C.

The calculation formula for flexible carrier (PAN-GA) weightening being prepared using the inventive method is as follows：

In formula：Rate of body weight gain/% of carrier (PAN-GA) after △ G- reactions；

W₀- it is the quality/g for reacting preceding polyacrylonitrile resin (PAN)；

W₁- for reaction after carrier (PAN-GA) quality/g.

By step 2 of the present invention) carrier (PAN-GA) that is prepared, according to step 3) methods described carries out papain Fixation.

Take a certain amount of enzyme solutions, filtrate and water lotion, be added separately to 2% casein solution containing 4mL and In 1mL0.1mol/L PBSs (pH=7.0,5mmol/L L-Cys and 1mmol/L EDTA) test tube, in 37 DEG C of water-baths Middle reaction 30min, adds 5mL10% trichloroacetic acid (TCA) terminating reaction immediately after, after then reactant is well mixed 10min filterings are stood in water-bath, filtrate determines light absorption value at 275nm.Blank control group then before enzyme is added, first adds 5mL10%TCA, remaining step is identical.

Enzyme activity determination method：

Enzyme activity unit is defined as under condition determination (37 DEG C of temperature, pH7.2), catalysis casein hydrolysis generation per minute Enzyme amount needed for l ug tyrosine is 1 enzyme activity unit.

The activity recovery of immobilised enzymes refer to immobilised enzymes total activity and immobilization process add solution enzyme total activity it Than being expressed as a percentage.

The relative activity (%) of immobilised enzymes or resolvase：Refer in group experiment using vigor highest as 100, with remaining Immobilised enzymes or solution enzyme the ratio between vigor, be generally expressed as a percentage.

The remaining enzyme activity (%) of immobilised enzymes or resolvase：Refer in group experiment with untreated preceding immobilised enzymes or The vigor of solution enzyme be vigor after 100, with processing shown by (including heat, acid, alkali, reagent, immobilization, refrigeration etc.) it Than being expressed as a percentage.

The carrier (PAN-GA) being prepared using the inventive method has following technical advantage：

1st, the present invention is carried out with elemental analysis method and infrared spectrum technology to the flexible resin carrier structure of synthesis Characterize, as a result show that amino occurs core addition reaction with cyano group in polyacrylonitrile parent and is grafted to poly- third in flexibility function group On alkene nitrile parent, reaction of atomic utilization rate height (atom in raw material is transformed into product as much as possible)；Not to or it is few to Poisonous and hazardous accessory substance is discharged in environment, the feature of Green Chemistry is preferably embodied, with obvious economic benefit and ring Border benefit.

2nd, by the TGA tracing analysis to carrier (PAN-GA) after polyacrylonitrile microballoon, flexibility function base and synthesis, obtain The situation of change of carrier heat endurance is arrived.As a result show：Carrier just starts to decompose at 325 DEG C and 400 DEG C, high mechanical strength, With higher heat endurance.

3rd, the present invention is using hydrophilic amino glucose function base as flexible chain, using chemical graft process by polyacrylonitrile resin Microballoon is modified, and being successfully introduced into for hydrophilic flexibility chain makes it effectively to maintain zymoprotein just during enzyme immobilizatio Normal conformation, reduces the inactivation of enzyme.The immobilized enzyme rate of recovery of gained is up to 80.6%, is to use unmodified polypropylene nitrile tree 21.9 times of the immobilization level of lipid microspheres.

4th, using polyacrylonitrile produced by the present invention-Glucosamine flexible resin carrier (PAN-GA) immobilization pawpaw egg The heat endurance of enzyme, storage-stable are all apparently higher than resolvase in vain.Illustrate with carrier (PAN- made from the inventive method GA a kind of friendly microenvironment) is provided for papain as enzyme immobilizatio carrier, zymoprotein can be preferably kept The bioactivity and catalytic activity of molecule, with certain application value.

5th, using carrier made from the inventive method (PAN-GA), for immobilized papain, its preparation technology letter Single, easy to operate, reaction condition is gentle, raw material is easy to get, cost is low, it is not necessary to which special installation, common process can be carried out.

6th, the present invention prepare obtained by immobilized papain there is higher heat endurance, it is storage-stable, organic Solvent tolerance and repetition stability, with certain actual application value.

Brief description of the drawings

The embodiment to the present invention is described in further detail below in conjunction with the accompanying drawings.

Fig. 1 is PAN, GA and PAN-GA infrared spectrogram；

Fig. 2 is PAN-GA resins synthesis route maps；

Fig. 3 is PAN, GA and PAN-GA thermogravimetric curve figure；

Fig. 4 is immobilization process of the papain on carrier PAN-GA；

Fig. 5 is influence of the temperature to immobilised enzymes and resolvase relative activity；

The repeat performance of Fig. 6 immobilised enzymes；

The storage stability of Fig. 7 immobilised enzymes and resolvase.

Embodiment

The present invention is further depicted as with reference to specific embodiment, but present disclosure is not limited to this.

Remarks：Washing in following examples is to be washed with deionized water.

Embodiment 1, a kind of synthetic method of carrier for immobilized papain, with polyacrylonitrile resin (PAN) For parent, aminoglucose hydrochloride (GA) is part, and chemical graft is carried out to polyacrylonitrile microballoon, be specially carry out successively with Lower step：

1. 0.1mg polyacrylonitrile resins (PAN), are accurately weighed, in the three-necked bottle for moving into 100mL, the molten of 100ml are added Agent --- ethylene glycol (ED), immersion is swelled after 12h, adds 0.68g aminoglucose hydrochloride (GA) and 0.88g K₂CO₃Powder End, first stirs 2h at normal temperatures with 200rpm mixing speed under nitrogen protection；Then it is brought rapidly up to 100 DEG C, magnetic force is stirred Mix reaction 12h；After reaction terminates, filter residue is collected in filtering.

2., the filter residue for 1. collecting step is first washed to cleaning solution with soaked in absolute ethyl alcohol to be colourless, and then first pickling is (i.e. Soaked 3 hours with 20mL, 1mol/L hydrochloric acid solution), it is washed with water and washs until cleaning solution is neutral, the vacuum under the conditions of 50 DEG C Dry to constant weight, obtain polyacrylonitrile-Glucosamine flexible resin carrier (PAN-GA), it is standby.

Test 1, with infrared technique method, to polyacrylonitrile-Glucosamine flexible resin carrier of above-mentioned synthesis (PAN-GA) Structural Identification is carried out, its response path and resin structure is inquired into.Comprise the following steps that：

1. a small amount of polyacrylonitrile microballoon is taken to be mixed with KBr, grinding, tabletting are determined on NICOLET-380 infrared spectrometers Its infrared spectrum, spectral region 4000cm^-1~400cm^-1, resolution ratio is 4cm^-1, scanning times are 32 times.

2. according to above-mentioned steps 1. in method determine novel carriers (i.e. PAN-GA) and part glucosamine salt respectively The infrared spectrum of hydrochlorate (GA).

3. by the infared spectrum to novel carriers (i.e. PAN-GA) after polyacrylonitrile microballoon, part and synthesis (see figure 1) comparative analysis, thus it is speculated that go out the response path and resin structure of resins synthesis reaction (see Fig. 2).

Fig. 1 is PAN, GA and PAN-GA infrared spectrogram.Analyzed from Fig. 1, in 3500~3000cm^-1In the range of GA Occur in that 3344cm^-1、3292cm^-1、3097cm^-1、3039cm^-1Four obvious strong absworption peaks of division, because GA molecules In there are multiple hydroxyls, hydroxyl (- OH) and amino (- NH that they belong in glucosamine molecules₂) stretching vibration inhale Receive peak.And PAN-GA spectrum are contrasted, the peak type after reaction in the range of this becomes for broad peak, illustrates the ammonia in glucosamine molecules Base is reacted.In 1700~1500cm^-1In the range of, GA molecules have 1619cm^-1And 1584cm^-1、1539cm^-1Three absorptions Peak, they are NH respectively₂With N-H angle vibration absorption peak.And after the reaction, in 1627cm^-1And 1580cm^-1Nearby occur in that A pair of broad peaks, explanation-NH₂It take part in reaction.The obvious of characteristic absorption peak (C ≡ N) after simultaneous reactions in polyacrylonitrile subtracts It is weak, and 1539cm^-1The disappearance at peak and 1121cm^-1、1062cm^-1(C-O-H secondary hydroxyl and primary hydroxyl in glucosamine molecules Base absworption peak), 896cm^-1The appearance of (sugared ring absworption peak), can prove that glucosamine molecules have been grafted to polyacrylonitrile On parent.

Speculate that the synthesis path of PAN-GA carriers is as shown in Figure 2.

Test 2, Structural Identification is carried out to the novel carriers (i.e. PAN-GA) of above-mentioned synthesis with thermogravimetric analysis (TGA), and Further illustrate being successfully introduced into for flexibility function base.Experiment condition is：N₂For protective gas, temperature rises to 1000 by 25 DEG C DEG C, programming rate is 20 DEG C/min.Experimental result is as shown in Figure 3.

Embodiment 2, the PAN-GA carriers progress Papain enzyme immobilization by the gained of embodiment 1, it is specific as follows：

10mg PAN-GA carriers are weighed, the Nal0 that concentration is 3mg/mL is added₄Solution be (pH=4.0,0.1mol/L's HAc-NaAc buffers) 3.00mL, lucifuge oxidation 30min, washed successively with absolute ethyl alcohol and water 3-4 time (it is each, it is anhydrous The consumption of ethanol is 30ml, and the consumption of deionized water is 50ml)；Add 10mL 0.1mol/L pH7 phosphate buffer (PBS) 0.2mg papain, is then added, is 25 DEG C in immobilization temperature, rotating speed is under 100rpm, constant temperature oscillation is solid Determine after 4h, filter, collect filtrate, and isolate filter cake (for obtained immobilized papain), gained filter cake 0.1mol/ L pH7 phosphate buffer (PBS) is carried out after rinsing 8~10 times repeatedly, and vacuum desiccator is put under the conditions of 37 DEG C and is dried after 5h, Immobilized papain is obtained, enzyme immobilization process is shown in Fig. 4.It is 67.9% to measure its enzyme fixed rate.

Comparative example 1, by the step of embodiment 1 1. in aminoglucose hydrochloride (GA) make N- acetamido glucoses into respectively Sugar, chitosan, remaining is equal to embodiment 1.The carrier of gained is referred to as carrier A, carrier B.According to the side described in above-described embodiment 2 Method is tested for Papain enzyme immobilizatio, and the carrier PAN-GA of its 1 gained with embodiment correction data is as shown in table 1：

The influence of table 1, the carrier of different ligands synthesis to enzyme fixed rate

Comparative example 2, by the step in embodiment 1 1. in reaction dissolvent N, the diformazans of N mono- made into by ethylene glycol (ED difference) Base formamide (DMF), toluene (Toluol), l, 4- dioxane (Dioxane), dimethyl sulfoxide (DMSO), remaining is equal to reality Apply example 1.

The carrier PAN-GA of 4 kinds of carriers and the gained of embodiment 1 to above-mentioned gained determines its rate of body weight gain and enzyme immobilization Rate, inquire into different solvents influences to test on the rate of body weight gain and enzyme fixed rate of synthetic vectors.As a result it is as shown in table 2 below：

Table 2, reaction dissolvent are to the rate of body weight gain of synthetic vectors and the influence 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
			Dimethyl sulfoxide (DMSO)	23.5	36.4
DMF (DMF)	19.1	29.4
			Ethylene glycol (ED) (present invention)	41.7	67.9

From the results shown in Table 2, resin PAN-GA rate of body weight gain and enzyme fixed rate highest, its value in solvent ED respectively Respectively 35.9% and 67.9%, reason is abundant swellability of the solvent to matrix, and it is shadow to be also indicated above reaction dissolvent Ring one of key factor of resins synthesis.So the optimum response solvent of the selected PAN-GA resin carriers of the present invention is ethylene glycol (ED)。

Comparative example 3, by the step in embodiment 1 1. in reaction temperature make 60 DEG C, 80 DEG C, 120 into respectively by 100 DEG C DEG C, 140 DEG C, remaining is equal to embodiment 1.

The carrier PAN-GA of 4 kinds of carriers and the gained of embodiment 1 to above-mentioned gained determines its rate of body weight gain and enzyme immobilization Rate, inquire into different temperatures influences to test on the rate of body weight gain and enzyme fixed rate of synthetic vectors.As a result it is as shown in table 3 below：

The influence of table 3, reaction temperature to synthetic resin rate of body weight gain and enzyme fixed rate

Reaction temperature	Resin rate of body weight gain △ W (%)	Enzyme fixed rate %
			60℃	8.5	19.1
80℃	13.4	29.9
			100 DEG C (present invention)	41.7	67.9
120℃	31.1	41.9
			140℃	27.2	31.7

In the case where other conditions are constant, by changing reaction temperature, inquire into different temperature and synthetic resin is increased weight Rate and enzyme state determine the influence of rate.From table 3 it is observed that in the range of test temperature, when temperature is 100 DEG C, resin PAN- GA rate of body weight gain reaches maximum, and enzyme fixed rate now also reaches maximum.Because temperature is too low, it is unfavorable for entering for function base Attack, reaction is difficult to completely；With the rise of temperature, the solubility increase of compound in organic solvent, reactant is lived Property increase, reaction rate accelerates, and rate of body weight gain also increases；But temperature is too high, side reaction easily occurs for synthetic reaction, so temperature exceedes The decline of its rate of body weight gain can be caused at 100 DEG C, and continuing heating can cause solvent to volatilize and lose, and be unfavorable for the synthesis of resin, Simultaneously too high in view of working as reaction temperature, the macromolecular scaffold of resin is easily damaged, so experiment selection maximum temperature is 100℃.The consideration of our Comprehensive Experiment conditions and combined coefficient, the optimum synthesising temperature for determining PAN-GA resins is 100 DEG C.

Comparative example 4, by step in embodiment 1 1. in reactant consumption GA (g):K₂CO₃(g) by 0.68:0.88 difference It is changed to 0.17:0.22、0.34:0.44、1.02:1.32、1.36:1.76, remaining is equal to be the same as Example 1.

The carrier PAN-GA of 4 kinds of carriers and the gained of embodiment 1 to above-mentioned gained determines its rate of body weight gain and enzyme immobilization Rate, inquire into differential responses thing consumption influences to test on the rate of body weight gain and enzyme immobilization rate of synthetic vectors.As a result it is as shown in table 4 below：

Table 4, GA and K₂CO₃Influence of the consumption to resin PAN-GA rates of body weight gain and enzyme fixed rate

GA(g):K2CO3(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 (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, with the increase of reactant concentration, resin rate of body weight gain also increases, and when anti- Thing concentration is answered to increase to a timing, its rate of body weight gain decreases, is primarily due to this reaction system and belongs to heterogeneous system, converts Rate is not only influenceed by reactant concentration, is also influenceed by space steric effect.So when add reactant amount compared with When few, reactant fully reacts with polyacrylonitrile, increases weight more apparent；And when the binding site of resin surface reaches saturation, then Increase reactant consumption can not be such that it combines up too much, and on the contrary because reactant concentration is excessive, tight clusters are together It is unfavorable for diffusion, hinders reaction to carry out.So when 0.10g polyacrylonitrile microballoons, Glucosamine (GA) and potassium carbonate (K₂CO₃) optimal addn be respectively 0.68g and 0.88g.

Experiment 1：Immobilised enzymes and the resolvase (immobilized papain and conventional Papain of the gained of embodiment 2 Enzyme) optimal reaction temperature：

Correspondence takes appropriate 10mg immobilised enzymes and resolvase, respectively in different enzymic catalytic reaction temperature (30 DEG C~80 DEG C) in the range of, determine corresponding enzyme activity according to enzyme activity determination method.

One of the characteristics of being also enzyme to environment temperature sensitivity.Temperature can not only influence the speed of enzymic catalytic reaction, also can shadow The stability of enzyme is rung, makes zymoprotein molecule degeneration, and the space structure of enzyme molecule and the catalytic effect of enzyme can also be influenceed, at this An optimal reactive temperature is generated under the influence of several combined factors.

As seen from Figure 5, the optimum temperature of resolvase is 50 DEG C, and to move towards trend more precipitous for curve, illustrate to dissociate The catalytic reaction of enzyme is affected by temperature larger, and after immobilization, optimum temperature improves 10 DEG C, and in 50 DEG C~80 DEG C scopes Higher enzyme activity is inside all maintained, optimum temperature range broadens, probably due to papain is obtained after carrier immobilized A kind of structure more stablized so that denaturation speed is influenceed to reduce by environment temperature.Optimal reactive temperature after enzyme immobilization Rise, be due to the Mulit-point Connection existed between enzyme molecule and carrier, stabilize the conformation of enzyme, it is therefore prevented that because heated peptide occurs for enzyme Chain folding extensional deformation, causes the decline of enzymatic activity.The raising of enzyme optimum temperature, not only increases enzymatic efficiency, also makes enzyme Catalytic reaction can be carried out in the environment of higher temperature, significant in processing and practical application.

Experiment 2：The heat endurance of immobilised enzymes and resolvase

Correspondence takes appropriate 10mg immobilised enzymes and resolvase, is protected in environment temperature is 25 DEG C, 35 DEG C, 55 DEG C, 65 DEG C Temperature processing different time (30min, 60min, 90min, 120min, 150min, 180min), is then cooled to 4 with frozen water rapidly DEG C, then according to enzyme activity determination method, corresponding enzyme activity is determined at 37 DEG C (with reference to heretofore described enzyme activity determination Method).

The stability of enzyme refers to enzymatic activity in certain environment condition, such as time, temperature, organic solvent, mechanism etc. The ability maintained vigour under the influence of factor.

Experimental result is drawn：Resolvase and immobilised enzymes can keep higher stabilization under 25 DEG C and 35 DEG C processing of low temperature Property, when temperature rises to 55 DEG C, with the extension of soaking time, obvious downward trend is presented in enzyme activity, in 180min When resolvase and immobilised enzymes residual vigor be 40% and 69%.When temperature is 65 DEG C, after insulation 60min, immobilization pawpaw The residual activity of protease is about 85%, and resolvase only has 57%；It is incubated after 180min, the residual vigor of immobilised enzymes is also remained 57% or so, and now the vigor of resolvase only remains 21%.After may being combined due to papain with resin carrier, enzymatic activity The microenvironment of the space conformation of centre and enzyme molecule there occurs some changes, and thermal denaturation resistant can strengthen.As can be seen here, it is fixed Papain specific ionization papain afterwards have higher heat endurance.This has important in actual applications to enzyme Meaning.

Experiment 3：The organic solvent stability of immobilized papain and free papain

Correspondence takes appropriate 10mg immobilised enzymes and resolvase, be separately added into acetonitrile that concentration is 10% and 90% (V/V), Processing 1h is placed in ethanol and DMF organic reagent mixed liquors 5ml, under normal temperature, then according to enzyme activity determination method, at 37 DEG C Determine corresponding enzyme activity (with reference to heretofore described enzyme activity determination method).

Remarks explanation：Above-mentioned organic reagent mixed liquor is entered with 0.1mol/L pH7 phosphate buffer (PBS) buffer solution What row was prepared.

The influence of table 5, organic reagent to immobilised enzymes and resolvase

After the data in table 5 clearly can be seen that 3 kind organic solvents processing of the resolvase through various concentrations, enzyme activity Power has larger loss, shows poor stability.And concentration more high enzymatic activity loss is more serious.And Papain Enzyme is after immobilization, after either being handled under the organic solvent of high concentration and low concentration, is demonstrated by higher residue Enzyme activity, stability is apparently higher than resolvase.The activity and stability of organic reagent influence enzyme are primarily due to：On the one hand, it is organic Reagent can directly and enzyme effect, the space conformation of destructive enzyme, so as to influence activated centre and the stability of enzyme；On the other hand, Organic reagent can influence the activity of enzyme by the direct effect with product or substrate.And after enzyme is fixed through carrier, carrier is to enzyme With certain protective effect so that after organic appearance agent can not be contacted directly with enzyme, and enzyme is combined with carrier so that the sky of enzyme Between structure more stablize, be difficult to be destroyed, stability is improved.

Experiment 4：The repetition stability of immobilised enzymes and resolvase

Appropriate 10mg immobilised enzymes is taken respectively, adds 2.00mL2% (quality %) casein solution, 37 DEG C of reactions 30min, is separated by filtration immobilised enzymes, is washed with PBS (0.1mol/L pH7 phosphate buffer).Under the same conditions Repeat above step and determine corresponding enzyme activity, obtain the immobilised enzymes that repetition reacts 8 times.

Fig. 6 illustrates the relation recycled between number of times and relative activity of immobilised enzymes.Immobilised enzymes as shown in Figure 6 After the reuse of 5 times, activity can retain more than 80%, after 8 times, and activity retains still more than 60%. It is probably that part enzyme comes off when in use in addition because some enzyme molecules are combined to obtain defective tightness by suction-operated with carrier, and And in removal process, enzyme also has different degrees of loss, so result in the partial inactivation of enzyme.It can not be weighed relative to resolvase For multiple use, immobilised enzymes can be repeatedly used, hence it is evident that improved the service efficiency of enzyme, reduced cost.Well Repeat performance, can not only be effectively reduced cost, also make it possible continuous catalytic reaction technological design, with reality Border application value.

Experiment 5：Immobilised enzymes and resolvase are preserved into some days in 4 DEG C of pH7.0 PBS respectively, then pressed According to enzyme activity determination method, in determining corresponding enzyme activity in different time intervals.

From figure 7 it can be seen that the papain being fixed on carrier shows higher storage compared with resolvase Stability, (4 DEG C) the placement 7d under identical preservation condition, resolvase is with the extension of holding time, and enzyme activity declines quickly, protects Deposit effect bad, its residual activity only be left 17%, and two kinds of immobilised enzymes in buffer solution preserve 7d after, its residual activity More than 80%, preferable storage-stable is shown.

Above-mentioned experiment illustrates that polyacrylonitrile-Glucosamine flexible carrier (PAN-GA), which is made, with the inventive method makees A kind of friendly microenvironment is provided for papain for enzyme immobilizatio carrier, zymoprotein molecule can be preferably kept Bioactivity and catalytic activity, with certain application value.

Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure All deformations for directly exporting or associating, are considered as protection scope of the present invention.

Claims (1)

1. the method for immobilized papain, it is characterized in that comprising the following steps：

1) 0.1mg polyacrylonitrile resins, are weighed, 100~300ml reaction dissolvent is added, immersion is swelled after 10~14h；Add The powdered K of 0.68g aminoglucose hydrochloride and 0.88g₂CO₃, under nitrogen protection with 150~250rpm stirring speed Degree first stirs 1~3h；100 DEG C are then heated to, continues stirring reaction 12h under nitrogen protection；After reaction terminates, filter, receive Collect filter residue；

The reaction dissolvent is ethylene glycol；

2), by step 1) obtained by filter residue first washed with soaked in absolute ethyl alcohol to cleaning solution to be colourless, pickling is then carried out successively And washing；Then constant weight is dried under vacuum in 45~55 DEG C, obtains PAN-GA carriers；

The pickling is to be soaked 2~4 hours with 1mol/L hydrochloric acid solution, and the washing is：It is washed with deionized to washing Liquid is neutrality；

3) 10mg PAN-GA carriers, are weighed, the Nal0 that concentration is 2.5~3.5mg/mL is added₄2.8~3.2mL of solution, lucifuge 28~32min is aoxidized, after then being washed successively with absolute ethyl alcohol and water；0.1mol/L, the pH for adding 8~12ml are 7 phosphorus Acid buffer, then adds 0.18~0.22mg papains；Immobilization temperature, rotating speed in 20~30 DEG C for 100~ Constant temperature oscillation is fixed after 3.5~4.5h under 150rpm, is separated by filtration；The Nal0₄Solution with pH=4.0,0.1mol/L's HAc-NaAc buffer solutions are prepared as solvent；

Gained filter cake is carried out after rinsing repeatedly with pH for 6.5~8.5 phosphate buffer, in 36~38 DEG C of vacuum desiccator 4.5~5.5h is dried, immobilized papain is obtained.