CN101531732A - Immobilized porcine pancreatic lipase carrier, preparation method and application thereof - Google Patents

Abstract

The invention discloses an immobilized porcine pancreas lipase carrier, which is a macroporous bead-like cross-linked polymer containing succinimide ester groups and has a structure represented by general formula I. The carrier forms a polymer by using acrylic acid as a reactive monomer and N,N'-methylenebisacrylamide as a crosslinking agent, and introduces a succinimide ester active group to immobilize porcine pancreas. The time required for lipase is short, only 1 hour, and the immobilization efficiency is high, and the stability of the immobilized enzyme activity is good; the apparent activity of the immobilized enzyme is high, which can reach 750U/g and above. The invention also discloses a preparation method and application of the immobilized porcine pancreatic lipase carrier.

Description

A kind of immobilized porcine pancreatic lipase carrier and its preparation method and application

technical field

The invention belongs to the technical field of preparation of immobilized enzyme carriers, in particular to an immobilized porcine pancreatic lipase carrier and a preparation method thereof.

Background technique

Enzymes are biocatalysts with remarkable characteristics such as strong specificity, high catalytic efficiency and mild action conditions. Therefore, it is widely used and can be used in many fields such as light industry, food, feed, medicine, chemical industry, environmental protection and energy, analysis and detection. However, due to the shortcomings of free enzymes that are difficult to separate from the product and are easily inactivated, people have tried various methods to modify enzyme molecules, one of which is the study of immobilized enzymes, thereby changing some of the properties and functions of enzymes. Make it overcome the shortcoming of free enzyme. The study of immobilized enzymes began in the 1950s. After more than 50 years of research and development, various carrier materials and immobilization methods have been developed in terms of enzyme immobilization. Not many, the reason is that the cost of reagents and carriers used for enzyme immobilization is high, the immobilization efficiency is low, and the stability is poor. Today, when our country is vigorously advocating the establishment of a resource-saving and environment-friendly society, further development of carrier materials with better performance and lower price will make the immobilization of enzymes easier and more practical, so that more immobilized enzymes can be obtained on an industrial scale. Application is still the goal pursued today.

In terms of enzyme immobilization carrier materials, it mainly includes inorganic carrier materials, organic carrier materials and polymer carrier materials, among which polymer carrier materials are more important carrier materials. Polymer carrier materials are further divided into natural polymer carrier materials and synthetic polymer carrier materials. Synthetic polymer carrier materials are currently the most actively studied immobilized enzyme carrier materials, and they are also the most widely used and reported immobilized enzyme carrier materials, mainly due to the high chemical stability and good mechanical properties of synthetic polymer carrier materials. , It is determined by the large loading capacity of the immobilized enzyme.

Enzyme immobilization methods mainly include embedding method, cross-linking method and carrier binding method; among them, carrier binding method includes ion binding method, physical adsorption method and covalent binding method. For the immobilization of synthetic polymer carrier materials and enzymes, covalent bonding is the main method. The covalent binding method is to form a chemical covalent bond connection between the functional group on the enzyme protein molecule and the active group on the surface of the solid phase support (the non-essential group of the enzyme protein forms an irreversible connection with the carrier through a covalent bond), method for immobilizing the enzyme. Because the connection between the enzyme and the carrier is firm, the enzyme is not easy to fall off, and has good stability and reusability, it is the most active type of enzyme immobilization method currently being studied.

For the study of covalent binding of enzymes, when the carrier immobilizes the enzyme, it is required that the carrier contains a certain group (such as amino group, carboxyl group, mercapto group, hydroxyl group, phenol group, imidazole group, etc.) in the enzyme molecule. Group) reactive active group, and the active group does not destroy the active center of the enzyme molecule after reacting with the enzyme molecule. These reactive groups generally include diazo groups, azide groups, imidocarbonate groups, dichloro-s-triazine groups, epoxy groups and the like.

At present, in terms of synthesizing polymer carrier materials, the most representative foreign ones include: Kotha et al. used cyclohexanol as porogen and divinylbenzene as crosslinking agent to prepare GM-trimethylolpropane triacrylate copolymer. Ponrathnam et al. studied the preparation of macroporous cross-linked glycidyl methacrylate (GMA) copolymer carrier, and ROM company in Germany produced EupergitC and other immobilized enzyme carriers containing epoxy groups. In China, research in this area mainly includes: Lu Guanzhong of East China University of Science and Technology and his disciples Wuyun Gaowa, Xue Ping and others synthesized immobilized enzyme carriers containing epoxy groups, and Wuyun Gaowa and others selected reactive monomer A Glycidyl acrylate (GMA), using N, N'-methylenebis(acrylamide) (MBAA) as crosslinking agent, and formamide as porogen, prepared a series of beads by reverse phase suspension polymerization method. Copolymer carrier; Xue Ping and others selected glycidyl methacrylate (GMA) containing active epoxy groups and hydrophilic N-vinylpyrrolidone (NVP) two monomers, and N, N'-methylene Bisacrylamide (MBAA) was used as cross-linking agent, methanol aqueous solution was used as porogen, and liquid paraffin was used as the main medium. The hydrophilic macroporous GMA-NVP-MBAA terpolymer carrier was successfully synthesized by reverse phase suspension polymerization ( GNM), and designed a reversed-phase suspension polymerization system, prepared polyglycidyl methacrylate (GMA)-hydroxyethyl methacrylate (HEMA)-N, N'-methylenebisacrylamide (MBAA) hydrophilic Magnetic polymer GHM microspheres, and mesoporous molecular sieves containing cobalt MCM-48 and MCM-41 were also prepared as carriers for enzyme immobilization; Enzyme carrier, this synthetic carrier is based on vinylene carbonate (VCA) as reactive monomer, hydrophilic N, N'-methylenebisacrylamide (MBAA) as cross-linking agent, respectively choose N-vinylpyrrolidone (NVP) and β-hydroxyethyl acrylate (HEMA) are two hydrophilic co-monomers used as the components of the synthetase carrier. For the first time, dimethylformamide and linear polymer polyethylene glycol 400 were used as composite porogens agent, and at the same time through inverse suspension polymerization, with liquid paraffin as the medium, VCA as the reactive monomer, β-hydroxyethyl methacrylate (HEMA) and hydroxypropyl acrylate (HPA) as the hydrophilic comonomer , synthesized a series of cross-linked resin polymers as immobilized enzyme carriers; Li Yuanxun of Hebei University and others used acrylic acid reactive monomers and divinylbenzene as cross-linking agents and petroleum ether as porogens to prepare immobilized enzymes by suspension polymerization In addition, other researchers have made different levels of research on immobilized enzyme carriers.

From the above synthetic polymer carrier materials, the active groups therein are mainly active groups such as epoxy groups and cyclocarbonate groups.

Lipase (lipase, EC3.1.1.3) can also be defined as carboxylesterase, which catalyzes the hydrolysis of long-chain fatty acylglycerols into glycerol, free fatty acids, and mono- and diglycerides. It can be used in various fields where enzymes can be applied, such as food processing and quality improvement, bread improver, cheese flavoring, etc.; it can be used in degreasing treatment in textile processing, and can be used as a detergent additive to achieve better Decontamination purposes; more importantly, it can also be used to produce biodiesel. Biodiesel is a form of biomass energy, which has the advantages of green, renewable, and low energy consumption. Compared with ordinary diesel, it is environmentally friendly. The emission of toxic organic matter in diesel vehicle exhaust is only 10%, particulate matter is 20%, and _CO2 and CO emissions are only 10%. According to the analysis, the impact on the entire atmosphere, biodiesel emits about 20 _% less CO2 than mineral diesel 50%, it can be seen that using immobilized lipase to produce biodiesel will be a very promising career, which can alleviate the crisis caused by the imminent depletion of oil and environmental pollution to human beings.

The research reports on the immobilization and application of lipase mainly include: Abroad, Kanwar of Himachal Pradesh University in India synthesized acrylic carrier immobilized lipase research, Yunas of Putra University in Malaysia used poly(methacrylic acid) ester, methyl methacrylate, divinylbenzene) microspheres immobilized lipase, Mahiran Basri immobilized lipase with hydrophobic organic carrier, Toshio Hayashi of Kyoto University in Japan immobilized lipase to porous polyvinyl alcohol beads, polyacrylaldehyde beads In China, Xin Jiaying from Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Kou Xiufen from Institute of Microbiology, Chinese Academy of Sciences, Xu Huixian from Nankai University immobilized lipase with acrylic carrier, Sheng Mei from Jiangsu Institute of Petrochemical Technology, Beijing Chemical Industry Co., Ltd. The immobilization and application of lipase by Tan Tianwei and his disciple Peng Lifeng.

Porcine pancreatic lipase, as a kind of lipase, also has the structural characteristics of lipase in structure. First, it includes a homologous segment: His-X-Y-Gly-Z-Ser-W-Gly or Y-Gly -His-Ser-W-Gly (X, Y, W, Z are variable amino acid residues); the second is that the active center is a serine residue, normally protected by an a-helical cap. It has the advantages and disadvantages of enzymes in nature, and has many specific applications, such as catalyzing the polymerization of achiral carboxylic acid derivatives represented by divinyl adipate and 1,4-butanediol, and also catalyzing For the polymerization of chiral carboxylic acid derivatives, Dafha Knani et al. used porcine pancreatic lipase (PPL) as a catalyst to polymerize photoactive side-substituted hydroxyl esters, resulting in optically active oligomers.

At present, there are not many studies on the immobilization and application of porcine pancreatic lipase by synthetic polymer carriers. Arieva V Paula from the University of Sao Paulo in Brazil immobilized porcine pancreatic lipase on polysiloxane-vinyl alcohol to prepare biodiesel He Feng of Wuhan University and Xie Zhidong of Nankai University have also studied the immobilization of porcine pancreatic lipase.

But so far, there is no immobilized porcine pancreatic lipase that can be commercialized. The main reason is as mentioned above, due to the shortcomings of complex preparation methods, harsh reaction conditions, and high costs, it is difficult to meet the requirements of industrialization, and the efficiency of enzyme immobilization of the obtained product is low, and the stability of the immobilized enzyme activity is poor. .

Contents of the invention

The purpose of the present invention is to provide a new immobilized porcine pancreas lipase carrier, which has high efficiency of immobilizing porcine pancreas lipase and good stability in maintaining the activity of the immobilized enzyme.

Another object of the present invention is to provide a method for preparing the above-mentioned immobilized porcine pancreatic lipase carrier, which can overcome the disadvantages of complicated carrier preparation, harsh reaction conditions, high cost, and difficulty in industrialization in the prior art.

The technical solution adopted by the present invention to solve its technical problems is:

An immobilized porcine pancreatic lipase carrier, which is a macroporous bead-shaped cross-linked polymer containing succinimide ester groups, has a structure represented by the following general formula I:

通式I

Formula I

in, It is the part other than the carboxyl group in the high polymer formed with acrylic acid (AA) as the reactive monomer and N,N'-methylenebisacrylamide (MBAA) as the crosslinking agent.

The above-mentioned immobilized porcine pancreatic lipase carrier can be prepared by the following method: using surfactants Span (Span) 40 and Span80 as composite dispersants, kerosene as dispersed phase, acrylic acid (AA) as reactive monomers, N, N'-methylenebisacrylamide (MBAA) is used as the crosslinking agent, ethylene glycol is used as the porogen, and the macroporous bead crosslinked polymer is prepared by reverse phase suspension polymerization technology, and then N-hydroxysuccinyl amine (NHS) activation, that is, to prepare the immobilized porcine pancreatic lipase carrier.

The HLB values (Hydrophily and Lipophily Balance, hydrophilic lipophilic balance value) of Span40 and Span80 are respectively 6.7 and 4.3, when being used as compound dispersant in the preparation method of above-mentioned immobilized porcine pancreas lipase carrier of the present invention, the two are mixed The final HLB value can be adjusted to 4.5-6, so as to control the dosage ratio of Span40 and Span80; the total dosage of the composite dispersant can be calculated as 10%-30% of the mass of kerosene in the dispersed phase.

The quality of the crosslinking agent N, N'-methylenebisacrylamide (MBAA) can preferably be 40% to 60% of the quality of the monomeric acrylic acid (AA);

The amount of porogen ethylene glycol can be 70% to 100% of the mass of monomer acrylic acid (AA);

In addition, in the preparation method of the above-mentioned immobilized porcine pancreatic lipase carrier of the present invention, ammonium persulfate (AP) can also be added as a catalyst for free radical reaction, and the addition amount of catalyst AP can be 1.5% of the mass of monomeric acrylic acid (AA) ～4%; Can also add tetramethylethylenediamine (TMEDA) simultaneously as the accelerator that accelerates AP to produce free radical (because TMEDA itself easily produces free radical thereby accelerates AP to produce free radical to react, so the adding amount of TMEDA and actual It is related to the reaction of the catalyst, as long as the reaction can occur, considering the cost, etc., it can be calculated as 7 to 10 times the mass of the catalyst AP).

The above-mentioned preparation method may preferably comprise the following major steps:

(1), preparation of oil phase:

Dissolve the composite dispersant composed of Span40 and Span80 in the kerosene of the organic dispersed phase, and mix well; then add tetramethylethylenediamine (TMEDA), mix well, and form the oil phase;

(2), preparation of water phase:

Dissolve acrylic acid (AA), N,N'-methylenebisacrylamide (MBAA), and ethylene glycol in water (calculated by volume ratio, the amount of water used can be 1 to 4 times the volume of acrylic acid), and add persulfuric acid Ammonium (AP), mixed to form the water phase;

(3), preparation of macroporous bead-shaped cross-linked polymer:

Add the water phase prepared in step (2) dropwise at a speed of 0.5 ~ 2d/s (dropping with a dropping funnel or other methods to ensure that the size of the water phase droplets added to the oil phase is basically similar) to (1 ) in the oil phase prepared in the first step (the volume ratio of the oil phase to the water phase can be controlled at ≥ 4, and from the perspective of cost saving, it can be preferably 4:1 ~ 10:1), and the stirring speed is controlled to be 100 ~ 350r/min; the reaction is carried out at room temperature of 10-30°C under the protection of nitrogen, and the reaction time is 1-2 hours;

After the reaction finishes, reclaim the upper layer solution (can be recovered by pouring the upper layer liquid, etc.), add volume concentration ≥ 65% ethanol (its consumption is to immerse the product) in the reaction product, leave standstill more than 5 hours after mixing (considering The problem of the production cycle can be left to stand for 5 to 24 hours), remove the upper ethanol solution; then repeatedly wash with water until there is no kerosene smell and alcohol smell, and filter with suction to obtain a macroporous bead-shaped cross-linked polymer, which is placed in a vacuum drying oven for 35 Dry at ~60°C to constant weight to obtain an unactivated carrier for use;

(4), preparation of activated carrier:

Take the unactivated carrier dried to constant weight in step (3), under the condition that tetrahydrofuran (THF) is used as solvent, dicyclohexylcarbodiimide (DCC) is used as dehydrating agent, and N-hydroxysuccinimide (NHS) React at 10-30°C for 2-4 hours, stirring continuously during the reaction (because this is an exothermic reaction, the purpose of stirring is to dissipate heat); after the reaction, recover the solvent; then dissolve with absolute ethanol to remove the by-product dicyclohexyl Urea (DCU), suction-filtered, dried to constant weight in a vacuum oven at 35-60°C to obtain an activated carrier, which is the above-mentioned immobilized porcine pancreatic lipase carrier of the present invention (a kind containing succinimide ester group group of macroporous bead-like crosslinked polymers).

In the process of preparing the activated carrier in the above step (4), the amount of the reactant (unactivated carrier, NHS) and dehydrating agent DCC can be adjusted according to the reactive monomer in the unactivated carrier: N-hydroxysuccinimide: dicyclohexyl carbon Diimine (molar ratio) = 1: (1 ~ 1.2): (1 ~ 1.2) calculation (the molar amount of the reactive monomer in the unactivated carrier can be calculated according to the reaction rate); NHS and DCC can be slightly excessive to ensure The carboxyl group in the unactivated carrier fully reacts; the amount of solvent THF can dissolve DCC and NHS.

In the reaction process of above-mentioned preparation method, the main chemical reaction formula that takes place is as follows:

In the above preparation method of the present invention, the immobilization method of synthesizing macroporous bead-shaped cross-linked polymer and porcine pancreatic lipase is a covalent bonding method.

According to the structural characteristics of porcine pancreatic lipase, the present invention uses acrylic acid (AA) as a reactive monomer, N, N'-methylenebisacrylamide (MBAA) as a crosslinking agent, and ethylene glycol as a porogen. Kerosene is the organic dispersed phase. The water-insoluble support cross-linked acrylic acid porous microspheres are synthesized by reverse phase suspension polymerization technology, and the polymer is activated with N-hydroxysuccinimide (NHS) to introduce succinimide ester active groups. The active group can react with the amino acid residue in porcine pancreatic lipase at room temperature without destroying the active center to achieve the purpose of immobilizing the enzyme.

When the immobilized porcine pancreatic lipase carrier of the present invention immobilizes porcine pancreatic lipase, the above-mentioned activated carrier reacts with the H ₂ N-group in porcine pancreatic lipase through the succinimide ester active group to achieve immobilized porcine pancreatic lipase. The action of lipase.

The above-mentioned immobilized porcine pancreatic lipase carrier can be used not only as an immobilized carrier of porcine pancreatic lipase, but also as an immobilized carrier of other lipases, neutral proteases, alpha-amylases and the like.

In the process of immobilizing porcine pancreatic lipase etc., its main reaction formula can be expressed as follows:

Compared with prior art, the beneficial effect of the present invention is:

The immobilized porcine pancreatic lipase carrier of the present invention forms a high polymer by using acrylic acid as a reactive monomer and N, N'-methylenebisacrylamide as a crosslinking agent, and simultaneously introduces a succinimide ester active group , the time required to immobilize porcine pancreatic lipase is short, only 1 hour, and the immobilization efficiency is high, and the stability of immobilized enzyme activity is good; the apparent activity of the immobilized enzyme is high, up to 750U /g and above. Moreover, in addition to immobilizing porcine pancreatic lipase, it can also be used to immobilize other lipases, neutral proteases, α-amylases, etc.

In addition, the preparation method of the above-mentioned immobilized porcine pancreatic lipase carrier provided by the present invention can overcome the disadvantages of complicated carrier preparation, harsh reaction conditions, high cost and difficult industrialization in the prior art, and its main advantages are as follows:

First, the raw materials are cheap and easy to obtain, and they are all low-toxic or non-toxic reagents, and the cost is low;

Second, the reaction conditions are mild, and there is no need for heating all the year round in southern my country, and the operation is simple;

Third, the required device is simple, which can further reduce the cost;

Fourth, the reaction time is short, which can shorten the production cycle, ensure high annual output and improve economic benefits.

Based on the above advantages, the preparation method has simple operation, mild reaction conditions, low cost, high annual output, easy realization of industrial production, and good economic benefits.

Description of drawings

Fig. 1 is the infrared spectrum of the unactivated carrier prepared in step (3) of Example 1 of the present invention.

Fig. 2 is the infrared spectrum of the activated carrier prepared in step (4) of Example 1 of the present invention.

Detailed ways

The present invention will be further described in detail below in combination with specific embodiments.

However, it should not be construed that the scope of the above-mentioned subject matter of the present invention is limited to the following examples.

Example 1

This embodiment is the immobilized porcine pancreatic lipase carrier prepared by the following method:

Surfactants Span (Span) 40 and Span80 are used as composite dispersants, kerosene is used as dispersed phase, acrylic acid (AA) is used as reactive monomer, and N,N'-methylenebisacrylamide (MBAA) is used as crosslinking agent , Ethylene glycol is the porogen, and the macroporous bead-shaped cross-linked polymer is prepared by reverse-phase suspension polymerization technology, and then activated by N-hydroxysuccinimide (NHS), to obtain the immobilized porcine pancreatic fat Enzyme carrier.

The raw materials used include:

Reactive monomer acrylic acid (AA) 5mL (about 5.3g),

Cross-linking agent N, N'-methylenebisacrylamide (MBAA) 2.1g (about 40% of AA quality),

Porogen ethylene glycol 4.0mL (about 4.5g, about 85% of AA mass),

The consumption ratio of dispersant Span40 and Span80 is adjusted to 5 with the HLB value after the two are mixed and is controlled, and its total consumption is 12g,

Dispersed phase kerosene 100mL,

Catalyst ammonium persulfate (AP) 0.1g (about 1.9% of AA mass),

Accelerator tetramethylethylenediamine (TMEDA) 1mL (about 0.77g, about 7.7 times the mass of AP);

In addition, during the preparation of the activated carrier, the amounts of the reactants (unactivated carrier, N-hydroxysuccinimide) and the dehydrating agent dicyclohexylcarbodiimide are respectively:

Unactivated carrier 2g (wherein the reactive monomer is about 0.02 mol),

N-hydroxysuccinimide (NHS) 2.2g (0.02mol),

Dehydrating agent dicyclohexylcarbodiimide (DCC) 3.9g (0.02mol).

Concrete preparation method comprises following main steps:

(1), preparation of oil phase:

First use a small beaker to weigh the composite dispersant composed of Span40 and Span80, add a small amount of organic dispersing kerosene into the beaker, because Span40 is solid, it needs to be heated slightly to dissolve it, then transfer it to a four-necked bottle, and then wash the beaker with kerosene 2 Transfer to the four-necked bottle once, stir and mix evenly with nitrogen gas; then add tetramethylethylenediamine (TMEDA), mix well, and form the oil phase;

(2), preparation of water phase:

Dissolve acrylic acid (AA), N,N'-methylenebisacrylamide (MBAA), and ethylene glycol in 10ml of water, add ammonium persulfate (AP), and mix well to form an aqueous phase;

(3), preparation of macroporous bead-shaped cross-linked polymer:

Using a dropping funnel, drop the water phase prepared in step (2) into the oil phase prepared in (1) at a speed of 1d/s to disperse, and control the stirring speed to 250r/min; under nitrogen protection, about 18 ℃ to react, the reaction time is 1 hour;

After the reaction, the upper layer solution was recovered by pouring, 100ml of 95% ethanol was added to the reaction product, and the mixture was left to stand overnight to remove the upper layer ethanol solution; then repeatedly washed with water until there was no kerosene smell and alcohol smell, and suction filtration was obtained. The porous bead-shaped cross-linked polymer was dried in a vacuum drying oven at 40-45°C to constant weight to obtain a total of 5.92 g of unactivated carriers, and the calculated reaction rate was about 80%, which was set aside;

The inventor adopts KBr tabletting method (with reference to Wang Kunhua, Luo Chuanqiu, Zhou Xiao. Polymer Modern Instrument Analysis [M]. Beijing: Tsinghua University Press, 1991: 20～44) to detect this unactivated carrier, obtained such as Infrared (IR) spectrum shown in Fig. 1; As can be seen from Fig. 1, 3431.9 place is a broad peak, explanation has-COOH exists, and the characteristic peak of acrylic acid cross-linking polymerization occurs at 1172.7 and 1115.1 simultaneously, and 1721.5 is- The peak of -C=O in COOH; 1650.2 and 1452 are the characteristic peaks of the amide bond in MBAA, 1650.2 is the peak of -C=O, 1452 is the vibration peak of the C-N bond; the rest are skeleton vibration peaks, from the IR analysis spectrum It can be drawn that AA and MBAA have indeed undergone cross-linking polymerization to obtain PAA-MBAA polymer.

(4), preparation of activated carrier:

Take tetrahydrofuran (THF) and dissolve dicyclohexylcarbodiimide (DCC) (3.9g, 0.02 mole) and N-hydroxysuccinimide (NHS) (2.2g, 0.02 mole) respectively, (the amount of THF is 40mL, 20mL), to obtain DCC solution and NHS solution;

Take another unactivated carrier (2g, of which the reactive monomer is about 0.02mol) dried in step (3) to constant weight, add the above NHS solution, and then add the above DCC solution dropwise; react in a closed round bottom flask at about 18°C 2.5 hours, constantly stirring during the reaction (the reaction process is carried out on a magnetic stirrer, using a magnetic stirrer to stir); after the reaction, the solvent is reclaimed; then the by-product dicyclohexylurea (DCU) is removed by dissolving with absolute ethanol, and suction filtered , dried in a vacuum oven at 40-45° C. to constant weight to obtain an activated carrier, which is the immobilized porcine pancreatic lipase carrier.

The inventor has detected this activated carrier by using the aforementioned KBr tablet method, and obtained the infrared (IR) spectrum as shown in Figure 2; as can be seen from Figure 2, more than 3000 in the spectrum is a sharp peak, indicating that there is no -COOH at 1738.2, 1208.8, and 1075.9, the characteristic peaks of ester groups appeared, proving the formation of active esters; at the same time, at 1820.7, 1782.3, new peaks of -C=O on NHS appeared, indicating that NHS was indeed involved in the reaction. of esters.

In addition, the inventor also used the olive oil emulsion method to measure the apparent activity of the activated carrier-immobilized porcine pancreatic lipase on the hydrolysis of the olive oil emulsion, and the measurement result was 850 U/g (expressed by the dry weight of the carrier).

Example 2

This embodiment is the immobilized porcine pancreatic lipase carrier prepared by the following method:

Surfactants Span (Span) 40 and Span80 are used as composite dispersants, kerosene is used as dispersed phase, acrylic acid (AA) is used as reactive monomer, and N,N'-methylenebisacrylamide (MBAA) is used as crosslinking agent , Ethylene glycol is the porogen, and the macroporous bead-shaped cross-linked polymer is prepared by reverse-phase suspension polymerization technology, and then activated by N-hydroxysuccinimide (NHS), to obtain the immobilized porcine pancreatic fat Enzyme carrier.

The raw materials used include:

Reactive monomer acrylic acid (AA) 10mL (about 10.5g),

Cross-linking agent N, N'-methylenebisacrylamide (MBAA) 6.2g (about 59% of AA quality),

Porogen ethylene glycol 6.7mL (about 7.5g, about 71% of AA mass),

The consumption ratio of dispersant Span40 and Span80 is adjusted to 4.5 with the HLB value after the two are mixed and is controlled, and its total consumption is 8g,

Dispersed phase kerosene 100mL,

Catalyst ammonium persulfate (AP) 0.16g (about 1.5% of AA quality),

Accelerator tetramethylethylenediamine (TMEDA) 2mL (about 1.5g, about 9.4 times the mass of AP);

In addition, during the preparation of the activated carrier, the amounts of the reactants (unactivated carrier, N-hydroxysuccinimide) and the dehydrating agent dicyclohexylcarbodiimide are respectively:

Unactivated carrier 5g (wherein the reactive monomer is about 0.047 mol),

N-hydroxysuccinimide (NHS) 5.5g (0.048mol),

Dehydrating agent dicyclohexylcarbodiimide (DCC) 9.9g (0.048mol).

Concrete preparation method comprises following main steps:

(1), preparation of oil phase:

Add the composite dispersant composed of Span40 and Span80 into the organic dispersed phase kerosene, heat and dissolve appropriately, add in a four-necked bottle equipped with an air guide tube and a stirrer, stir and mix evenly with nitrogen gas; then add tetramethylethylenediamine ( TMEDA), mixed to form the oil phase;

(2), preparation of water phase:

Dissolve acrylic acid (AA), N,N'-methylenebisacrylamide (MBAA), and ethylene glycol in 25ml of water, add ammonium persulfate (AP), and mix well to form an aqueous phase;

(3), preparation of macroporous bead-shaped cross-linked polymer:

Using a dropping funnel, drop the water phase prepared in step (2) into the oil phase prepared in (1) at a speed of 1d/s to disperse, and control the stirring speed to 350r/min; under nitrogen protection, about 30 ℃ to react, the reaction time is 2 hours;

After the reaction was over, the upper layer solution was recovered by pouring, and 220ml of 65% ethanol was added to the reaction product, and after mixing, it was allowed to stand overnight, and the upper layer ethanol solution was removed; then, it was repeatedly washed with water until there was no kerosene smell and alcohol smell, and suction filtration was obtained. The porous bead-shaped cross-linked polymer was dried in a vacuum drying oven at 55-60°C to constant weight, and a total of 12.86 g of unactivated carriers were obtained. The calculated reaction rate was about 77%, and it was set aside;

(4), preparation of activated carrier:

Take tetrahydrofuran (THF) and dissolve dicyclohexylcarbodiimide (DCC) (9.9g, 0.048 mole) and N-hydroxysuccinimide (NHS) (5.5g, 0.048 mole) respectively, (the amount of THF is 70ml, 30ml), to obtain DCC solution and NHS solution;

Take another unactivated carrier (5 g, of which the reactive monomer is about 0.047 moles) dried in step (3) to constant weight, add the above NHS solution, and then add the above DCC solution dropwise; react in a closed round bottom flask at about 30 ° C 4 hours, constantly stirring during the reaction (the reaction process is carried out on a magnetic stirrer, using a magnetic stirrer to stir); after the reaction is finished, the solvent is reclaimed;

(DCU), suction-filtered, and dried in a vacuum oven at 55-60° C. to constant weight to obtain an activated carrier, which is the immobilized porcine pancreatic lipase carrier.

Similarly, the inventors used the olive oil emulsion method to measure the apparent activity of the activated carrier-immobilized porcine pancreatic lipase on the hydrolysis of the olive oil emulsion, and the measurement result was 750 U/g (expressed by the dry weight of the carrier).

Example 3

This embodiment is the immobilized porcine pancreatic lipase carrier prepared by the following method:

Surfactants Span (Span) 40 and Span80 are used as composite dispersants, kerosene is used as dispersed phase, acrylic acid (AA) is used as reactive monomer, and N,N'-methylenebisacrylamide (MBAA) is used as crosslinking agent , Ethylene glycol is the porogen, and the macroporous bead-shaped cross-linked polymer is prepared by reverse-phase suspension polymerization technology, and then activated by N-hydroxysuccinimide (NHS), to obtain the immobilized porcine pancreatic fat Enzyme carrier.

The raw materials used include:

Reactive monomer acrylic acid (AA) 2.5mL (about 2.6g),

Cross-linking agent N, N'-methylenebisacrylamide (MBAA) 1.3g (about 50% of AA quality),

Porogen ethylene glycol 2.3mL (about 2.6g, about 100% of AA quality),

The consumption ratio of dispersant Span40 and Span80 is adjusted to 6 with the HLB value after the two are mixed and is controlled, and its total consumption is 24g,

Dispersed phase kerosene 100mL,

Catalyst ammonium persulfate (AP) 0.1g (about 3.8% of AA quality),

Accelerator tetramethylethylenediamine (TMEDA) 1mL (about 0.77g, about 7.7 times the mass of AP);

In addition, during the preparation of the activated carrier, the amounts of the reactants (unactivated carrier, N-hydroxysuccinimide) and the dehydrating agent dicyclohexylcarbodiimide are respectively:

1 g of unactivated carrier (wherein the reactive monomer is about 0.01 mol),

N-hydroxysuccinimide (NHS) 1.3g (0.011mol),

Dehydrating agent dicyclohexylcarbodiimide (DCC) 2.3g (0.011mol).

Concrete preparation method comprises following main steps:

(1), preparation of oil phase:

Add the composite dispersants Span40 and Span80 into the organic dispersed phase kerosene, heat and dissolve properly, add it into a four-necked bottle equipped with an air guide tube and a stirrer, stir and mix well with nitrogen gas; then add tetramethylethylenediamine (TMEDA) , mix well to form the oil phase;

(2), preparation of water phase:

Dissolve acrylic acid (AA), N,N'-methylenebisacrylamide (MBAA), and ethylene glycol in 10ml of water, add ammonium persulfate (AP), and mix well to form an aqueous phase;

(3), preparation of macroporous bead-shaped cross-linked polymer:

Using a dropping funnel, drop the water phase prepared in step (2) into the oil phase prepared in (1) at a speed of 2d/s to disperse, and control the stirring speed to be 100r/min; under nitrogen protection, about 10 ℃ to react, the reaction time is 2 hours;

After the reaction was finished, the upper layer solution was recovered by pouring, 80ml of 70% ethanol was added to the reaction product, and the mixture was left to stand overnight to remove the upper layer ethanol solution; then repeatedly washed with water until there was no kerosene smell and alcohol smell, and suction filtration was obtained. The porous bead-shaped cross-linked polymer was dried in a vacuum drying oven at 35-40°C to constant weight to obtain a total of 3.16 g of unactivated carriers, and the calculated reaction rate was about 80.9%, which was set aside;

(4), preparation of activated carrier:

Take tetrahydrofuran (THF) and dissolve dicyclohexylcarbodiimide (DCC) (2.3g, 0.011 mole) and N-hydroxysuccinimide (NHS) (1.3g, 0.011 mole) respectively, (the amount of THF is 40mL, 20mL), to obtain DCC solution and NHS solution;

Take another unactivated carrier (1 g, of which the reactive monomer is about 0.01 mole) dried in step (3) to constant weight, add the above NHS solution, and then add the above DCC solution dropwise; react in a closed round bottom flask at about 10°C 2 hours, constantly stirring during the reaction (the reaction process is carried out on a magnetic stirrer, using a magnetic stirrer to stir); after the reaction, the solvent is recovered, and then the by-product dicyclohexylurea (DCU) is removed by dissolving with absolute ethanol, and suction filtered , dried in a vacuum oven at 35-40° C. to constant weight to obtain an activated carrier, which is the immobilized porcine pancreatic lipase carrier.

Similarly, the inventor measured the apparent activity of the activated carrier-immobilized porcine pancreatic lipase on the hydrolysis of the olive oil emulsion by the olive oil emulsion method, and the measurement result was 830 U/g (expressed by the dry weight of the carrier).

Example 4

This embodiment is the immobilized porcine pancreatic lipase carrier prepared by the following method:

Surfactants Span (Span) 40 and Span80 are used as composite dispersants, kerosene is used as dispersed phase, acrylic acid (AA) is used as reactive monomer, and N,N'-methylenebisacrylamide (MBAA) is used as crosslinking agent , Ethylene glycol is the porogen, and the macroporous bead-shaped cross-linked polymer is prepared by reverse-phase suspension polymerization technology, and then activated by N-hydroxysuccinimide (NHS), to obtain the immobilized porcine pancreatic fat Enzyme carrier.

The raw materials used include:

Reactive monomer acrylic acid (AA) 5mL (about 5.3g),

Cross-linking agent N, N'-methylenebisacrylamide (MBAA) 2.1g (about 40% of AA quality),

Porogen ethylene glycol 4.6mL (about 5.2g, about 100% of AA mass),

The consumption ratio of dispersant Span40 and Span80 is adjusted to 5 with the HLB value after the two are mixed and is controlled, and its total consumption is 5.4g,

Dispersed phase kerosene 45mL,

Catalyst ammonium persulfate (AP) 0.11g (about 2% of AA quality),

Accelerator tetramethylethylenediamine (TMEDA) 1mL (about 0.77g, 7 times the mass of AP);

In addition, during the preparation of the activated carrier, the amounts of the reactants (unactivated carrier, N-hydroxysuccinimide) and the dehydrating agent dicyclohexylcarbodiimide are respectively:

Unactivated carrier 2g (wherein the reactive monomer is about 0.02 mol),

N-hydroxysuccinimide (NHS) 2.8g (0.024mol),

Dehydrating agent dicyclohexylcarbodiimide (DCC) 5.0 g (0.024 mol).

Concrete preparation method comprises following main steps:

(1), preparation of oil phase:

Add the composite dispersants Span40 and Span80 into the organic dispersed phase kerosene, heat and dissolve properly, add it into a four-necked bottle equipped with an air guide tube and a stirrer, stir and mix well with nitrogen gas; then add tetramethylethylenediamine (TMEDA) , mix well to form the oil phase;

(2), preparation of water phase:

Dissolve acrylic acid (AA), N,N'-methylenebisacrylamide (MBAA), and ethylene glycol in 5ml of water, add ammonium persulfate (AP), and mix well to form an aqueous phase;

(3), preparation of macroporous bead-shaped cross-linked polymer:

Using a dropping funnel, drop the water phase prepared in step (2) into the oil phase prepared in (1) at a speed of 0.5d/s to disperse, and control the stirring speed to 250r/min; The reaction was carried out at 10°C, and the reaction time was 2 hours;

After the reaction was finished, the upper layer solution was recovered by pouring, and 80 ml of 95% ethanol was added to the reaction product, left to stand overnight after mixing, and the upper layer ethanol solution was removed; then repeatedly washed with water until there was no kerosene smell and alcohol smell, and suction filtration was obtained. The porous bead-shaped cross-linked polymer was dried in a vacuum drying oven at 35-40°C to constant weight to obtain a total of 5.1 g of unactivated carrier, and the calculated reaction rate was about 68.7%, which was set aside;

(4), preparation of activated carrier:

Dicyclohexylcarbodiimide (DCC) (5.0 g, 0.024 moles) and N-hydroxysuccinimide (NHS) (2.8 g, 0.024 moles) were dissolved in tetrahydrofuran (THF), respectively (THF dosages were 60 mL, 20mL), to obtain DCC solution and NHS solution;

Take another unactivated carrier (2 g, of which the reactive monomer is about 0.02 moles) dried in step (3) to constant weight, add the above NHS solution, and then add the above DCC solution dropwise; react in a closed round bottom flask at about 10 ° C 2 hours, constantly stirring during the reaction (the reaction process is carried out on a magnetic stirrer, using a magnetic stirrer to stir); after the reaction, the solvent is recovered, and then the by-product dicyclohexylurea (DCU) is removed by dissolving with absolute ethanol, and suction filtered , dried in a vacuum oven at 35-40° C. to constant weight to obtain an activated carrier, which is the immobilized porcine pancreatic lipase carrier.

Similarly, the inventors used the olive oil emulsion method to measure the apparent activity of the activated carrier-immobilized porcine pancreatic lipase on the hydrolysis of the olive oil emulsion, and the measurement result was 800 U/g (expressed by the dry weight of the carrier).

In above-mentioned each embodiment, measure the olive oil emulsion method of immobilized porcine pancreatic lipase carrier apparent activity, all adopt literature (Zhao Yahua editor-in-chief. Biochemical experiment technology tutorial [M]. Guangzhou: South China University of Technology Press, 2000 : 157-159) record the classic olive oil emulsion method. According to the above measurement results, it can be seen that the apparent activity can reach 750U/g or more (expressed by the dry weight of the carrier).

Claims (10)

1. A preparation method of an immobilized porcine pancreatic lipase carrier is characterized by comprising the following steps:

it is a macroporous bead-like crosslinked polymer containing succinimide ester groups, having a structure represented by the following general formula I:

in the general formula I, the compound is shown in the specification,

whereinThe part except carboxyl in the high polymer formed by taking acrylic acid as a reactive monomer and N, N' -methylene bisacrylamide as a cross-linking agent.

2. A method for preparing the immobilized porcine pancreatic lipase vector of claim 1, which comprises:

the preparation method comprises the steps of preparing a macroporous bead-shaped cross-linked polymer by using surfactants Span40 and Span80 as composite dispersants, kerosene as a dispersion phase, acrylic acid as a reactive monomer, N' -methylene bisacrylamide as a cross-linking agent and ethylene glycol as a pore-forming agent through a reverse phase suspension polymerization technology, and then activating by using N-hydroxysuccinimide to prepare the immobilized porcine pancreatic lipase carrier; the method specifically comprises the following main steps:

(1) preparing an oil phase:

dissolving a composite dispersing agent consisting of Span40 and Span80 in organic dispersed phase kerosene, and uniformly mixing; adding tetramethyl ethylenediamine, and mixing to obtain oil phase;

(2) and preparing a water phase:

dissolving acrylic acid, N' -methylene bisacrylamide and ethylene glycol in water, adding ammonium persulfate, and uniformly mixing to form a water phase;

(3) preparation of macroporous bead-like crosslinked polymers:

dropwise adding the water phase prepared in the step (2) into the oil phase prepared in the step (1) at a speed of 0.5-2 d/s for dispersion, and controlling the stirring speed to be 100-350 r/min; reacting at 10-30 ℃ under the protection of nitrogen for 1-2 hours;

after the reaction is finished, recovering the upper-layer solution, adding ethanol with the volume concentration of more than or equal to 65% into the reaction product, uniformly mixing, standing for 5-24 hours, and removing the upper-layer ethanol solution; repeatedly washing with water until no kerosene smell or alcohol smell is produced, performing suction filtration to obtain macroporous bead-shaped cross-linked polymer, and drying in a vacuum drying oven at 35-60 ℃ until the weight is constant to obtain unactivated carrier for later use;

(4) and preparing an activation carrier:

taking the unactivated carrier dried to constant weight in the step (3), taking dicyclohexylcarbodiimide as a dehydrating agent under the condition that tetrahydrofuran is taken as a solvent, reacting with N-hydroxysuccinimide at 10-30 ℃ for 2-4 hours, and continuously stirring in the reaction process; recovering the solvent after the reaction is finished; dissolving the reaction product by using absolute ethyl alcohol to remove a byproduct of dicyclohexylurea, performing suction filtration, and drying the reaction product in a vacuum drying oven at the temperature of 35-60 ℃ to constant weight to prepare an activated carrier, namely the immobilized porcine pancreatic lipase carrier.

3. The method of claim 2, wherein: in the composite dispersant, the proportion of Span40 and Span80 is adjusted according to the HLB value of the mixture of the Span40 and the Span80 being 4.5-6; the total dosage of the composite dispersant is 10-30% of the mass of the dispersed phase kerosene.

4. The method of claim 2, wherein: the mass of the cross-linking agent N, N' -methylene bisacrylamide is 40-60% of the mass of the monomer acrylic acid.

5. The method of claim 2, wherein: the dosage of the pore-foaming agent glycol is 70-100% of the mass of the monomer acrylic acid.

6. The method of claim 2, wherein: the adding amount of the ammonium persulfate is 1.5 to 4 percent of the mass of the monomer acrylic acid; the adding amount of the tetramethyl ethylene diamine is 7-10 times of the mass of the ammonium persulfate.

7. The method of claim 2, wherein: in the step (2), the amount of water is 1-4 times of the volume of acrylic acid.

8. The method of claim 2, wherein: in the step (3), the volume ratio of the oil phase to the water phase is 4: 1-10: 1.

9. The method of claim 2, wherein: in the step (4), the dosage of the non-activated carrier, the N-hydroxysuccinimide and the dicyclohexylcarbodiimide is as follows according to the reactive monomer in the non-activated carrier: n-hydroxysuccinimide: the dicyclohexylcarbodiimide molar ratio is 1: 1-1.2; the solvent THF is used in an amount capable of dissolving DCC and NHS.

10. The use of the immobilized porcine pancreatic lipase carrier according to claim 1 as an immobilized carrier for porcine pancreatic lipase, other lipases, neutral protease, alpha-amylase.

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