JPS6222591A - Method of immobilizing biocatalyst by polyurethane emulsion - Google Patents

Abstract

PURPOSE:To mold a biocatalyst to a desired shape without deactivating it, by blending nonionic polyurethane emulsion with a biocatalyst aqueous solution, etc., while stirring and removing water, etc. CONSTITUTION:Since polyurethane emulsion contains a hydrophilic polyol, a hydrophobic polyol and a diisocyanate as its resin components and, if necessary, a chain extender, etc., and is self-emulsifying type emulsion, it has improved dispersibility and is suitable as an immobilizing agent. Each component is properly selected depending upon uses, a chemical equivalent ratio of NCO/ active hydrogen is preferably about 0.8-4 and a urethanization catalyst, etc., are added to give necessary nonionic urethane emulsion. The emulsion is blended with a biocatalyst, an enzyme, a microorganism, etc., while stirring, molded into a shape suitable for aim and dried so that they are included and immobilized in the resin.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for immobilizing biocatalysts with nonionic polyurethane emulsions.

(Prior art) Methods for immobilizing biologically tactile VLM such as enzymes include methods using natural polymers, methods using black supports such as porous glass, metal hydroxides, metal oxides, and ion exchange resins. In addition, recently used synthetic polymer carriers include polyacrylamide, polyvinyl alcohol, polyhydroxyalkyl methacrylate,
Ethylene-maleic anhydride copolymer, polystyrene,
Examples include nylon and polyisobutylene, and in terms of immobilization methods, there are carrier bonding methods, entrapment methods, crosslinking methods, and the like.

When the carrier is a polymer, there are monomer methods, polymer methods, and prepolymer methods depending on the degree of polymerization of the starting material used for immobilization. These include the use of the target substance,
It is selected as appropriate depending on the seed head of the biocatalyst.

Immobilization methods using polyurethane as a carrier are described, for example, in Japanese Patent Publications No. 56-25118 and Japanese Patent Publication No. 56-42908. These are polycrethane prebomers with hydrophilic properties. In this case, hydrophilicity is imparted by increasing the content of ethylene oxide in the polyol component to 50% or more.

This conventional urethane prepolymer is so-called NC○
Since it is a urethane intermediate with a large number of products, when immobilizing microorganisms and enzymes, even if comprehensive immobilization is performed, this activity NG
It is presumed that O reacts with active groups of microorganisms and enzymes to cause chemical bonding, and as a result, microorganisms and enzymes may be slightly inactivated after immobilization. Although this is unavoidable, the causes that reduce the effectiveness of microorganisms and enzymes in their original intended functions should be eliminated as much as possible.

As a means to compensate for these drawbacks, a method based on self-emulsifying turethane emulsion was tried, and the drawbacks of the prepolymer method described above could be improved (Japanese Patent Application No. 88879/1983). However, this self-emulsifying urethane emulsion is ionic, and when attempts were made to immobilize biocatalysts using this method, good results were obtained when ordinary aqueous solutions or suspensions were used; If an S solution is required to obtain a solution or suspension, depending on one of the components of this buffer,
Urethane polymers may lose their self-emulsifying ability.

In such cases, when the Turethane emulsion liquid and the biocatalyst solution or suspension are mixed, the emulsion collapses, making it difficult to form it into a desired shape such as a film, sheet, or block after mixing. Met.

(Problems to be Solved by the Invention) The purpose of the present invention is to immobilize biocatalysts such as microorganisms, enzymes, and animal and plant cells so that they can be molded into desired shapes without losing their activity as much as possible. The purpose is to provide a method.

(Means for Solving the Problems) The present invention involves mixing and stirring an aqueous solution or suspension of a biocatalyst into a nonionic polyurethane emulsion, mixing the biocatalyst into a nonionic polyurethane emulsion, and then adding water to the nonionic polyurethane emulsion. The present invention relates to a method for immobilizing biocatalysts using a polyurethane emulsion, which is characterized by removing and immobilizing biocatalysts.

The poly-9-rethane emulsion of the present invention is the same! It contains a hydrophilic polyol, a hydrophobic polyol, a noisocyanate as a fat component, and if necessary a chain extender, etc., and is a self-emulsifying emulsion, so it has good dispersibility.
It is suitable as immobilization M for enzymes, microorganisms, animal and plant cells, etc.

In the present invention, as the hydrophilic polyol, polyethylene glyfur, polyvinyl alcohol, polysiloxane, vinyl acetate copolymer, etc. can be used. Further, as the hydrophobic polyol, polyester polyol, polyether polyol, and other polyols can be used.

Examples of polyester polyols include those having 4 to 2 carbon atoms, such as adipic acid, superric acid, sebacic acid, and brassylic acid.
0 aliphatic dicarboxylic acid, terephthalic acid, isophthalic acid, etc. as the acid component, aliphatic alcohols having 1 to 6 carbon atoms such as ethylene glycol, propylene glycol, neopentyl glycol, hexamethylene glycol, diethylene glycol, dipropylene glycol, etc. Polyester polyols or polycaprolactone polyols having polyol components such as ether glycols, spiroglycols, N-furkyldial amines such as N-methylnoethanolamine, etc. can be used.
Specific examples include 7dipate polyols such as polyethylene adipate polyol, polybutylene anovate polyol, and polyethylene propylene adipate polyol, terephthalic acid polyols, and polyols (e.g., Toyobo Co., Ltd., trade names: Vylon RUX, Vylon RV-20OL, etc.)
, polycaprolactone polyol (e.g., Daicel Chemical,
Examples include product names Praxel 212 and Praxel 220).

Specific examples of polyether polyols include polyoxypropylene polyols, polyoxytetramethylene polyols, and the like.

Other polyols include polycarbonate polyol (e.g., Bayer AG, West Germany, trade name Desmoreen 2020E), polybutadiene polyol (e.g., Nippon Soda, trade names G-1000, G-2000, G-30).
00, Idemitsu Petrochemical, trade name Poly bdR-45H
T), polypentadiene polyol, castor oil polyol, and the like. Furthermore, epoxy resins having hydroxyl groups such as bisphenol A type epoxy resin, Novo2 type epoxy resin, and epoxidized olefin type epoxy resin can also be used. These polyols can be used alone or in combination of two or more. In addition,
These hydrophilic polyols and hydrophobic polyols can be used as a mixture, or a copolymer of both can be used.

As the diisocyanate of the present invention, various polyfunctional well-known aliphatic, alicyclic and aromatic isocyanates in the field of polyurethane production can be used, such as hexamethylene diinocyanate (HDI), inholonoisocyanate (IPDI), etc. ), 4.4-dicyclohexylylene diisocyanate (HMDI), 2.4-) lylene diisocyanate (2,4-T DI ), 2.6-)
lylene diisocyanate (2,6-TDI), 4
, 4-diphenylmethane diinsyanate (MDI),
Ortho-toluidine diisocyanate (TODI), naphthylene diisocyanate (MDI), xylylene diisocyanate (XDI), lysine diisocyanate) (LD
I), etc.

Furthermore, in the present invention, a conventional chain extender can also be used if necessary. As the chain extender, for example, a 2- to 6-functional polyol with a molecular weight of 500 or less and a molecular weight of 50
Examples include diamines having 0 or less 1a or secondary terminal amide groups. Suitable head extenders include, for example: (a) ethylene glycol, diethylene glyfur, propylene glycol, dipropylene glycol, butanediol, hexanediol, glycerin, trimethylolpropane, pentaerythritol, sorbitol, 1,4-cyclohexanediol; , 1. Polyols such as 4-cyclohexane dimeta/-ol and xylylene glycol (b) hydrazine, ethylenediamine, tetramethylene diamine, hexamethylene diamine, 1.
Diamines such as 4-cyclohexanediamine (C) Flukanolamines such as ethyl-7-lamine, jetanolamine, triethanolamine, etc. (d) Hydroquinone, pyrogarole, 4,4-impropylidenophenol, aniline, and The above polyols, diamines,
Examples include polyols with a molecular weight of 500 or less obtained by adding propylene oxide and/or ethylene oxide to alkanolamines in any order.

In the present invention, each component may be appropriately selected from a wide range depending on the application, and the chemical equivalent ratio of NGO/active hydrogen (NGO index) is about 0.8 to 4.0, preferably about 1.0. It is preferable to carry out the reaction within a range of 0 to 3.0. The reaction is usually preferably carried out at a temperature of about 50 to 150°C. It is also possible to add a known urethanization catalyst. The urethane prepolymer synthesized in this way is easily dispersible in water, and the required nonionic urethane emulsion can be obtained. To suppress foaming during emulsification, use an aqueous antifoaming agent (e.g., Toray Silicone, SM-5512, San Nopco, SN Deformer 1).
33, 432, etc.) is also optional.

During prepolymer synthesis or emulsification, NGO
Using an organic solvent that is inert to groups, soluble in water, and dissolves the prepolymer, or recovering this organic solvent by distillation, depressurization, etc. at the end of prepolymer synthesis or after emulsification. Things are also optional.

In the present invention, the biocatalyst can be mixed and stirred into the nonionic polyurethane emulsion, molded into a shape suitable for the purpose, and dried to achieve comprehensive fixation within the resin. Drying can usually be carried out at a temperature of about 40°C or lower, but it is generally preferable to cool and dry under reduced pressure at around 0°C. There is no particular restriction on the shape; for example, it may be coated, cast or laminated on the surface of a plastic plate, metal plate, synthetic resin plate, etc., or it may be filled into a container and immersed in the MIL fibrous proboscis-like object.

Examples of the biocatalyst of the present invention include microorganisms, enzymes, and animal and plant cells. Filamentous fungi as microorganisms,
Examples include eukaryotic cells such as yeast, and prokaryotic cells such as bacteria and released bacteria. Specific examples are shown below, but these are just one example. Bacillus subtilis, Streptomyces limosus, Escherichia flea, Mucor pusillus, Rhizopus and Delemer, Aspergillus oryzae, Satucharomyces cerevisiae, etc.

In addition, enzymes include various oxidation/reductases, transferases,
Examples include hydrolase, elimination enzyme, isomerase, and slagase. Specific examples are shown below, but these are just one example. Catalase, gluten oxidase, glutamate dehydrodenase, aspartate acetyltransferase, histamine methyltransferase, invertase, lipase, chymotrypsin, aminoacylase, esterase, lactase, papain, pepsin, isin, trypsin, urokinase, urease, amylase, β-〃 Lactosiguse, aspartase, 7marase, gluten isomerase, methionine racemase, glutathione synthase, etc.

Furthermore, it is optional to include these biocatalysts combined with a carrier such as glass beads, celite, activated carbon, silica sol, etc. in the urethane emulsion of the present invention.

(Example) Examples of the present invention will be described below.

Two types of nonionic polyurethane emulsions were prepared using the following methods.

Production Example 1 Polytetramethylene glycol (PTMG, #1ooo
) 60g, polyethylene glycol (PEG #
6000) 25g, diethylene glycol (DEC)
15 g and tolylene diisocyanate (T D I -
80) 52g were mixed and reacted at 120°C for 3 hours. After further cooling to 80° C., 50 g of MEK was added and thoroughly stirred to obtain a urethane prepolymer solution. 228 g of water was added to the obtained prepolymer solution and mixed and emulsified at room temperature using a homomixer. The emulsion thus obtained was heated (or under reduced pressure) to remove MEK and obtain the desired polyurethane emulsion. The solid content of the emulsion was 40%, the viscosity was 1200 eps/25°C, and the pH was 5.5.

! ! Preparation Example 2 Epicor) 1001 (165g), polyethylene glycol (PEG #1540) 385g and isophorone diisocyanate) (IPDI)
194.71? were mixed and reacted at 120°C for 3 hours. After cooling to 80° C., 248.2 g of nooxane was added and thoroughly stirred to obtain a urethane prepolymer solution. 200 g of water was added to 100 g of the obtained urethane prepolymer solution and thoroughly stirred to obtain the desired polyurethane emulsion. Emulsion solid content 27%, viscosity 400cps/
The temperature was 25°C and the pH was 6.3.

Example 1 Add lipase OF 360 (Meito Sangyo Co., Ltd.) to a 10 ml beaker! :1 30+mg is suspended in 1g of deionized water. This suspension and 1 g of the polyurethane emulsion obtained in Production Example 1 were mixed and left to stand for 12 hours to air dry. This del piece was cut into 3 x 3 x 1 mm pieces and subjected to reaction.

(Reaction) 20 g of water, 20 g of olive oil, and the above-mentioned immobilized enzyme were placed in a 10 lnl Erlenmeyer flask with a stopper, and shaken back and forth 160 times/min in a constant temperature water bath at 30°C.

(Quantitative method) Collect 1+ol of the reaction liquid, boil it for 10 minutes, and then completely separate the oil and water by centrifugation.

Take a part of the oil in a 50ml Erlenmeyer flask (its weight is ffisg) and add a neutral ethanol/ether mixture (volume ratio, ethanol/ether = 2/3) 10
Add n+1. This is an indicator of 7 enols and 7 thalein! - and titrate with 0.05N-KOH (full=r-ru) (titration amount a lll l).

f: 0.05N-K OH strength value 2.8054
: Amount of KOH in 1 liter (g) 202.8% Saponification value of olive oil Calculate the maximum slope (enzyme activity) of the hydrolysis rate-reaction time curve, and calculate the enzyme activity when immobilized and when not immobilized. When we calculated the ratio (activity yield) to the enzyme activity, the activity yield was 68%.
Met.

Example 2 In place of the polyurethane emulsion obtained in Production Example 1, the polyurethane emulsion obtained in Production Example 2 was used to immobilize lipase in the same manner as in Example 1, and the reaction and quantification were carried out in the same manner. When the activity yield was determined from the relationship between the hydrolysis rate and the reaction time, the activity yield was 76%.

Comparative Example 1 An example using a hydrophilic urethane prepolymer instead of a non-ionized urethane emulsion is shown.

Take 30 IIg of lipase 0F360 (Meito Sangyo Co., Ltd. gJ!) into a 10ml beaker and dissolve it in 1g of deionized water.

Then, PU-3 (0.5 g) was added to this solution, mixed quickly, and reacted to obtain an immobilized Del. This was cut into 5 mm cubes to obtain an immobilized enzyme. This was used for reaction.

The activity yield determined in the same manner as in Example 1 was 19%.

Note 1) PU-3 NC obtained from tolylene diisocyanate (TDI) and polyoxyethylene polyoxypropylene polyol
It is an O-terminal prepolymer, the average molecular weight of the polyether polyol is 12,529, and the ethylene glycol content in the polyether polyol is 57%.

(that's all)

Claims (1)

[Claims] (1) The biocatalyst is mixed into the nonionic polyurethane emulsion by mixing and stirring an aqueous solution or suspension of the biocatalyst into the nonionic polyurethane emulsion, and then water etc. are removed and the biocatalyst is immobilized. A method for immobilizing biocatalysts using polyurethane emulsion.