CN103740690A - Method for concentrating, stabilizing and separating liquid enzyme - Google Patents

Abstract

The invention provides a method for concentrating, separating and stabilizing liquid enzymes. The components are calculated by weight percentage: 1.0-20.0% of macrocyclic molecular synthesis receptors, 0.1%-50% of non-ionic surfactants, and 0.25% of enzyme stabilizers. %-5%, bio-enzyme 0.01%-80%, balance of polar solvent; mix according to the proportion of the above components, control pH3-12, temperature 0-80℃, react for 1-24h. The supramolecular microspheres containing biological enzymes are formed in the solution, and the particle size is between 10nm and 1000nm. The materials selected in the present invention are environmentally friendly and easily degradable materials. The most important thing is that this method does not damage the activity of the enzyme, and the activity of the enzyme has a high activity for a long period of time. The activity of the enzyme encapsulated in the supramolecular microspheres is 20% higher than that of ordinary liquid enzymes. The activity loss of biological enzyme is less than 10% after four weeks when it is applied in the formula of textile emulsifier or liquid detergent.

Description

A method for concentrating, stabilizing, and isolating liquid enzymes

technical field

The invention relates to a liquid enzyme preparation, in particular to methods for concentrating, stabilizing and separating liquid enzyme preparations.

Background technique

Liquid enzyme preparation is a raw material that is easy to use, environmentally friendly and has good biodegradability. Specific catalytic properties and mild reaction conditions have been more and more used and valued in the industry. However, the concentration, stabilization and separation of liquid enzyme preparations have always been a problem that plagues people.

Of course, there are many people trying to find solutions to the problems of concentration, stabilization and separation of liquid enzyme preparations. And put forward some feasible technologies, including adding enzyme stabilizer, membrane separation technology, microwave vacuum concentration method, reagent concentration, and temperature-sensitive hydrogel method.

The method of adding an enzyme stabilizer is a more traditional method of stabilizing and concentrating enzyme preparations. In US20070134375A1, Andreas Habich et al. relate to a stable solid-state or liquid enzyme preparation in the invention, which comprises at least one enzyme and at least one stabilizer selected from gum arabic, at least one vegetable protein and mixtures thereof, improving Enzyme storage and stability.

Membrane separation technology is a separation technology that emerged in the 1960s. With the help of external energy or chemical potential difference, it realizes the separation of two-component or multi-component mixed gas or liquid through the permeation of a specific membrane. , fractionation, purification and enrichment techniques. Commonly used membrane materials are cellulose acetate (CA), polyacrylonitrile (PAN), polyaluminum (PSF), polyamide (PA), polyvinylidene fluoride (PVDF), etc. Common membrane separation technologies: (1) Microfiltration (MF); (2) Ultrafiltration (UF); (3) Nanofiltration (NF); (4) Reverse osmosis (RO) ). At present, in the enzyme preparation industry, microfiltration and ultrafiltration technology are widely used, especially ultrafiltration technology, and the combination of ultrafiltration technology and other separation technologies has been more and more widely used in recent years.

In 1965, Blatt et al proposed the use of membrane separation technology to concentrate microorganisms and conducted experiments. Wang Pingzhu used an external pressure tubular membrane to concentrate and extract bromelain from pineapple juice. The enzyme activity can be concentrated by nearly 4 times. The enzyme retention rate in the concentrated solution is 95%, and the recovery rate is 79.8%. %. In CN201110389274, Zhou Xing and others used ultrafiltration and nanofiltration technology to carry out a series of treatments on the crude nitrilase enzyme, and finally prepared a higher concentration nitrilase product. In the patent CN103122343A, Wei Xingye et al. used tubular or hollow fiber ultrafiltration membranes to concentrate the fermentation broth of the gel-breaking enzyme complex bacteria, and obtained enzyme products with high purity.

The membrane separation technology is used to concentrate and refine the enzyme, the operation process is simple, the chance of bacterial contamination and enzyme inactivation is reduced, the recovery rate of the enzyme is improved and the quality of the product is improved.

At present, there are also people trying to concentrate the enzyme preparation with microwave vacuum concentration method, and have achieved success. In CN102899303A, Li Bing et al. disclose a microwave vacuum concentration method of papain solution. They use the microwave vacuum concentration method to concentrate papain to obtain a papain concentrate. The concentration process of the method has low temperature and high concentration speed, and the obtained papain concentrate has a high recovery rate of enzyme activity. At the same time, the equipment is not easy to scale and has low energy consumption.

The reagent concentration method uses polyethylene glycol (PEG) to absorb the water in the enzyme solution. This method is simple and efficient, but the reagent price is relatively expensive, and it is not considered as an industrial production due to cost problems.

The decompression evaporation concentration method uses a decompression device to make the enzyme liquid to be concentrated under a certain degree of vacuum and temperature. For heat-sensitive substances such as enzyme liquid, the decompression device mostly uses a centrifugal thin-film evaporator and a scraper evaporator, but the energy consumption of this method Consumption is larger.

Thermosensitive hydrogels can also concentrate and separate enzymes, which design the crosslink density or monomer structure of the gel according to the size and properties of the concentrated separated substances. However, the separation efficiency of the gel for protein and enzyme is very high at low temperature (below the phase transition temperature), and the separation efficiency decreases at high temperature (higher than the phase transition temperature), and the separation efficiency suddenly jumps near the phase transition temperature. (Wang Jintang, Zhonghui, Zhu Hongjun, Zhang Weiguang, Journal of Nanjing University of Chemical Technology, 1998, 20, 2)

At present, there are also some technologies that use supramolecules to encapsulate liquid enzyme preparations. However, due to the characteristics of the three-dimensional structure of biological enzymes, in supramolecular technologies, enzyme inactivation and low content of encapsulated biological enzymes have been encountered to varying degrees.

Invention content:

The purpose of the present invention is to provide a method for concentrating, separating and stabilizing liquid enzymes by using the technology of encapsulating biological enzymes with supramolecular nanospheres to solve the problem of enzyme inactivation commonly found in enzyme preparations.

A method for concentrating, separating and stabilizing liquid enzymes provided by the invention,

Components by weight percentage: 1.0-20.0% macrocyclic molecular synthesis acceptor, 0.1%-50% non-ionic surfactant, 0.25%-5% enzyme stabilizer, 0.01%-80% biological enzyme, polar solvent margin;

Mix according to the proportion of the above components, control pH3-12, temperature 0-80°C, and react for 1-24h. The supramolecular microspheres containing biological enzymes are formed in the solution, and the particle size is between 10nm and 1000nm.

The above preferred component distribution ratio is: macrocyclic molecular synthesis acceptor 2.0-18.0%, non-ionic surfactant 0.5%-40%, enzyme stabilizer 0.5%-4.5%, biological enzyme 0.5%-70%, polar Solvent balance;

The reaction temperature is preferably between 5-70°C, more preferably between 10-50°C.

The pH value is preferably 5-11.

The macrocyclic molecular synthesis acceptor is crown ether, cyclopan, cyclodextrin, cucurbituril, calixarene or pillararene and the like.

The crown ether is 15-crown ether-5, 15-crown ether-6, or 18-crown ether-6, etc.;

Huanfan is a big ring fan or a small ring fan, etc.;

Cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, etc.;

Calixarene is calix [4] arene or calix [5] arene, etc.;

Pillar aromatics are pillar [5] aromatics or pillar [6] aromatics and the like.

Described polar solvent is water or linear alcohol or ketone.

The enzyme stabilizer can be a dibasic alcohol or a tribasic alcohol; the dibasic alcohol can be ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol alcohol, 1,4-butanediol or 1,5-pentanediol; the trihydric alcohol can be glycerol, 1,2,4-butanetriol or 1,2,5-pentanetriol.

The non-ionic surfactant, its structural formula can be PEO _X -PPO _Y -PEO _X , R-C ₆ H ₄ -O-(CH ₂ CH ₂ O) _n -H, R-CO-(OC ₂ H ₄ ) _n -OH, RO-(CH ₂ CH ₂ O) _n -H or R-COO-(CH ₂ CH ₂ O) _n -H; and its molecular weight >10000, HLB value between 5-60.

The biological enzyme is any one or more of protease, pectinase, cellulase, lipase, hemicellulase, lipase, Novozymes and subtilase.

Compared with the methods of separating, concentrating and stabilizing liquid enzyme preparations in the prior art, the present invention proposes to use macrocyclic molecules to synthesize receptors, enzyme stabilizers, non-ionic surfactants and biological enzymes to construct supramolecular microspheres to achieve separation, For the purpose of concentrating and stabilizing the liquid enzyme preparation, the materials selected in the present invention are environmentally friendly, non-hazardous and easily degradable materials. The most important thing is that this method does not damage the activity of the enzyme, and the activity of the enzyme has a high activity for a long period of time. The activity of the enzyme encapsulated in the supramolecular microspheres is 20% higher than that of ordinary liquid enzymes. The activity loss of biological enzyme is less than 10% after four weeks when it is applied in the formula of textile emulsifier or liquid detergent.

Description of drawings:

Figure 1 Example 1 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Figure 2 Example 2 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Figure 3 Example 3 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Figure 4 Example 4 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Figure 5 Example 5 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Figure 6 Example 6 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Figure 7 Example 7 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Figure 8 Example 8 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Figure 9 Example 9 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Figure 10 Example 10 Dynamic Light Scattering (DLS) Supramolecular Microsphere Particle Size Distribution Diagram

Fig. 11 SEM photo of supramolecular microspheres in Example 10

Detailed ways:

Example 1

Dissolve 0.9 g of nonionic surfactant EO ₁₁ PO ₆₉ EO ₁₁ (HLB value 6.0) in 50 g of water, then take 1 g of liquid protease preparation and mix it, add the mixture dropwise to 0.8 g of α-cyclodextrin, and then add 2.4 g1,2-propanediol stabilizer and mix well.

The above-mentioned uniformly mixed liquid was sealed and stirred at 20°C for 20 hours. The enzyme activity of the liquid protease preparation was determined according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of supramolecular microspheres (see Figure 1), the average particle size of the encapsulated enzyme particles is about 270nm.

Example 2

Dissolve 9 g of non-ionic surfactant EO ₁₃₃ PO ₅₀ EO ₁₃₃ (HLB value 26.0) in 50 g of ethanol, then mix 38 g of liquid lipase preparation with it, add the mixture dropwise to 1.5 g of β-cyclodextrin, and then add 0.5g 1,3-propanediol stabilizer and mix well.

The above-mentioned uniformly mixed liquid was sealed and stirred at 20°C for 22 hours. The enzyme activity of the liquid lipase preparation was determined according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of the supramolecular microspheres (see Figure 2), the average particle size of the encapsulated enzyme particles is around 298nm.

Example 3

Dissolve 20g of non-ionic surfactant EO ₃ PO ₄₃ EO ₃ (HLB value 6.0) in 100g of acetone, then take 18g of liquid hemicellulase preparation and mix it, and add the mixture dropwise to 10g of small cyclopan, and then Add 0.55g of 1,2-butanediol stabilizer and mix well.

The above-mentioned uniformly mixed liquid was sealed and stirred at 30°C for 24 hours, and the enzyme activity of the liquid hemicellulase preparation was determined according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of supramolecular microspheres is shown in the figure below (see Figure 3). The average particle size of the encapsulated enzyme particles is about 806nm.

Example 4

Dissolve 1.5g of non-ionic surfactant EO ₃₇ PO ₅₆ EO ₃₇ (HLB value 14.0) in 50g of acetone, then take 10g of liquid cellulase preparation and mix it, add the mixture dropwise to 5g of cucurbituril, and then add 1.2 g1,3-butanediol stabilizer and mix well.

The above-mentioned uniformly mixed liquid was sealed and stirred at 36°C for 23 hours. The enzyme activity of the liquid cellulase preparation was determined according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of supramolecular microspheres is shown in the figure below (see Figure 4). The average particle size of the encapsulated enzyme particles is about 353nm.

Example 5

Dissolve 2.0 g nonylphenol polyoxyethylene ether of nonionic surfactant in 30 g propanol, then get liquid cellulase and hemicellulase composition preparation 8.0 g to mix with it, the mixture is added dropwise to 0.7 g gamma- Add 0.9g of 1,4-butanediol stabilizer to the cyclodextrin and mix well.

The above-mentioned homogeneously mixed liquid was sealed and stirred at 40°C for 24 hours to determine the enzyme activity of the liquid cellulase and hemicellulase composition preparation according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of supramolecular microspheres is shown in the figure below (see Figure 5). The average particle size of the encapsulated enzyme particles is about 431nm.

Example 6

Dissolve 3.0 g of non-ionic surfactant EO ₁₀₃ PO ₃₉ EO ₁₀₃ (HLB value 26.0) in 50 g of butanol, then mix 10 g of liquid cellulase preparation with it, add the mixture dropwise to 2.8 g of calixarene, and then Add 1.5g of 1,5-pentanediol stabilizer and mix well.

The above-mentioned uniformly mixed liquid was sealed and stirred at 25°C for 20 hours to determine the enzyme activity of the liquid cellulase preparation according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of supramolecular microspheres is shown in the figure below (see Figure 6). The average particle size of the encapsulated enzyme particles is about 130nm.

Example 7

Dissolve nonionic surfactant: polyoxyethylene laurate LAE-4, 90.8g in 40g of acetone, then take 0.6g of liquid subtilisin preparation and mix it, and add the mixture dropwise to 0.6g of β-cyclodextrin Add 1.0g of 1,2,4-butanetriol stabilizer and mix well.

The above-mentioned uniformly mixed liquid was sealed and stirred at 20°C for 20 hours. The enzyme activity of the liquid subtilisin preparation was determined according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of supramolecular microspheres is shown in the figure below (see Figure 7), and the average particle size of the encapsulated enzyme particles is about 406nm.

Example 8

Dissolve 0.3 g of nonionic surfactant methallyl polyoxyethylene ether in 10 g of acetone, then get liquid pectinase preparation 0.6 g to mix with it, and the mixture is added dropwise to 0.6 g of column [5] aromatics, Then add 0.5g glycerol stabilizer and mix well.

The above-mentioned uniformly mixed liquid was sealed and stirred at 20°C for 20 hours to determine the enzyme activity of the liquid lipase preparation according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of the supramolecular microspheres is shown in the figure below (see Figure 8). The average particle size of the encapsulated enzyme particles is around 676nm.

Example 9

Dissolve 0.5 g of non-ionic surfactant EO ₄₂ PO ₁₆ EO ₄₂ (HLB value 26.0) in 30 g of propanol, then take 0.5 g of liquid protease and pectinase composition and mix it, and add the mixture dropwise to 0.6 g of β - to the cyclodextrin, add 1.0 g of 1,3-butanediol stabilizer and mix well.

The above-mentioned uniformly mixed liquid was sealed and stirred at 20°C for 20 hours. The enzymatic activity of the liquid protease and pectinase composition preparation was determined according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of supramolecular microspheres is shown in the figure below (see Figure 9), and the average particle size of the encapsulated enzyme particles is around 588nm.

Example 10

Dissolve 0.4g of nonionic surfactant EO ₆ PO ₃₄ EO ₆ (HLB value 5.0) in 30g of ethanol, then take 0.5g of liquid pectinase preparation and mix it, and add the mixture dropwise to 0.6g of γ-cyclodextrin , and then add 1.0g of 1,2,5-pentanetriol stabilizer and mix well.

The above-mentioned uniformly mixed liquid was sealed and stirred at 20°C for 20 hours. The enzyme activity of the liquid pectinase preparation was determined according to the national standard GB/T23527-2009, as shown in the table below.

Dynamic Light Scattering (DLS) is used to test the nanostructure of samples. The particle size distribution of supramolecular microspheres is shown in the figure below (see Figure 10). The average particle size of the encapsulated enzyme particles is about 245nm.

In addition, we also observed the molecular morphology with transmission electron microscope (TEM) and scanning electron microscope (SEM), as shown in the figure below (see Figure 11). Figure 11 (A) is the SEM image of enzyme-coated microspheres. It can be seen that the shape of the microspheres is a relatively standard spherical shape. It can be read from the figure that the particle diameter of this microsphere is about 120nm. Figure 11(B) is the TEM image of the enzyme-encapsulated microspheres. It can be seen from the figure that the microspheres are solid microspheres, which are standard solid enzyme-encapsulated microspheres, with a particle size of about 120nm.

Claims (15)

1. A method for concentrating, separating and stabilizing liquid enzymes, characterized in that: Components by weight percentage: 1.0-20.0% macrocyclic molecular synthesis acceptor, 0.1%-50% non-ionic surfactant, 0.25%-5% enzyme stabilizer, 0.01%-80% biological enzyme, polar solvent margin; Mix according to the proportion of the above components, control pH3-12, temperature 0-80°C, and react for 1-24h. 2. A method for concentrating, separating, and stabilizing liquid enzymes as claimed in claim 1, characterized in that: said components are by weight percentage: 2.0-18.0% of macrocyclic molecular synthesis acceptors, non-ionic surface Active agent 0.5%-40%, enzyme stabilizer 0.5%-4.5%, biological enzyme 0.5%-70%, polar solvent balance. 3. A method for concentrating, separating and stabilizing liquid enzymes as claimed in claim 1, characterized in that: the reaction temperature is 5-70°C. 4. A method for concentrating, separating and stabilizing liquid enzymes as claimed in claim 1, characterized in that: the pH value is 5-11. 5. A method for concentrating, separating and stabilizing liquid enzymes as claimed in claim 1, characterized in that: said macrocyclic molecular synthesis acceptor is crown ether, cyclopan, cyclodextrin, cucurbituril, calixarene or pillar aromatics. 6. A method for concentrating, separating, and stabilizing liquid enzymes as claimed in claim 5, characterized in that: the crown ether is 15-crown ether-5, 15-crown ether-6, or 18-crown ether -6. 7. A method for concentrating, separating, and stabilizing liquid enzymes as claimed in claim 5, characterized in that: said cyclopan is macrocyclic or small cyclopan. 8. A method for concentrating, separating and stabilizing liquid enzymes as claimed in claim 5, characterized in that: said cyclodextrin is α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin. 9. A method for concentrating, separating and stabilizing liquid enzymes as claimed in claim 5, characterized in that: said calixarene is calix[4]arene or calix[5]arene. 10. A method for concentrating, separating and stabilizing liquid enzymes as claimed in claim 5, characterized in that: said pillar aromatics are pillar [5] aromatics or pillar [6] aromatics. 11. A method for concentrating, separating and stabilizing liquid enzymes as claimed in claim 1, characterized in that: the polar solvent is water or straight-chain alcohol or ketone. 12. A method for concentrating, separating, and stabilizing liquid enzymes as claimed in claim 1, characterized in that: said enzyme stabilizer is a dibasic alcohol or a tribasic alcohol. 13. A method for concentrating, separating, and stabilizing liquid enzymes as claimed in claim 12, characterized in that: said glycol is ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1, 2-butanediol, 1,3-butanediol, 1,4-butanediol or 1,5-pentanediol; the trihydric alcohol is glycerol, 1,2,4-butanediol or 1,2,5-pentanetriol. 14. A method for concentrating, separating and stabilizing liquid enzymes as claimed in claim 1, characterized in that: said non-ionic surfactant has a structural formula of PEO _X -PPO _Y -PEO _X , R-C ₆ H ₄ -O-(CH ₂ CH ₂ O) _n -H, R-CO-(OC ₂ H ₄ ) _n -OH, RO-(CH ₂ CH ₂ O) _n -H or R-COO-(CH ₂ CH ₂ O) _n -H, and its molecular weight > 10000, HLB value between 5-60 non-ionic surfactant. 15. A method for concentrating, separating and stabilizing liquid enzymes as claimed in claim 1, wherein said biological enzymes are protease, pectinase, cellulase, lipase, hemicellulase, lipid Any one or more of enzymes, Novozymes and subtilases.

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