JPS59179091A - Enzymatic hydrolysis of lipid - Google Patents

Abstract

PURPOSE:An immobilized lipase in which anion-exchange groups distributes on the carrier surface linking with the lipase is used to enable the enzymatic hydrolysis, even in a high concentration of lipids as much as 50-94% in the reaction mixture. CONSTITUTION:As a carrier, is used a variety of anionic exchange resins, glass beads, silica beads of 15-1,000Angstrom pore radii, which can keep a wide area linking with lipase and anion-exchange groups. Lipase and anion-exchange groups are bonded with the carrier to prepare an immobilized lipase. As an anion-exchange group, is mentioned a variety of amines, ammonium salts or their derivatives. The resultant immobilized lipase can effect readily enzymatic hydrolysis of lipid, even when the lipid concentration is very high as much as 50-94% in the reaction mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION In enzymatically decomposing lipids, the present invention utilizes immobilized iR-ase, which has an anion exchange group on the surface of a carrier to which rhinoξ-ase is bound. Substrate concentration 50-94
The present invention relates to a method for enzymatically decomposing lipids, which is characterized in that the decomposition is performed within the following range.

When lipase hydrolyzes oils and esters, glycerin, alcohol, fatty acids, fatty acid ions, and protons are produced. Fatty acids and fatty acid ions do not inhibit production because they are poorly soluble in water, but glycerin, alcohol, and protons inhibit production because they are soluble in water. Conventionally, in order to eliminate these harmful effects on the product, a method has been adopted in which the water content in the reaction solution is increased to dilute Noricerin, alcohol, and protons. (Iwai et al., J, Gen, Ap
pl.

Mierobiol, 10.13<1964))
If the water content in the L-oxidation reaction solution is high, it takes a lot of energy to recover the glycerin and alcohol obtained in the aqueous layer after the reaction, which is an obstacle to industrially implementing fat and oil decomposition using the enzymatic method. Ta.

The present inventors have conducted intensive research on industrial fat and oil decomposition methods using a carrier bound to lipase. We focused on the fact that the protons that inhibit the production of lipase can be removed from the vicinity of the lipase molecule due to the repulsion of If fat and oil decomposition is carried out using an immobilized solution prepared using a new method, even if the substrate concentration in the reaction solution is increased to 50-94%, which is a high concentration that has never been used before. We have discovered that scales can be hydrolyzed to a certain degree of decomposition, and based on this knowledge, we have accomplished the present invention.

The origin of the lysine/ξ-ase used in the present invention is not particularly selected, but it is preferably an enzyme that has good affinity for fats and oils, has a good reaction start-up, and can decompose fats and oils to a high decomposition rate. When decomposing fats and oils with a high melting point such as beef tallow, it is desirable to use heat-resistant IJA-ze, which reacts at temperatures of 45°C or higher.

The carrier used in the present invention is a carrier to which as many anion exchange groups as possible are bonded, and is usually made by combining various anion exchange resins, glass beads, or silica beads with a silane cup coupling agent. Hydrophobic carriers are used, such as those into which ion exchange groups have been introduced, and those in which as many anion exchange groups as possible have been introduced into metal powder. Hydrophilic vinyl polymers and cellulose with as many anion exchange groups as possible can also be used, but hydrophobic carriers have better affinity for the substrate and can eliminate the generated hydrophilic glycerin. This is a desirable carrier because it reduces the product-inhibiting effects caused by. Even better results can be obtained by using macroporous anion exchange resins, glass beads, or silica beads. In other words, since the radius of a normal glue/couse molecule is 10 Å or more, if a porous carrier with an average pore radius larger than this is used, lipase will also bind to the surface area within the pores, resulting in IJ-R-ase and anion exchange. The bonding area of the group can be widened. Furthermore, when the average pore radius is 1000 Å or more, the pore surface area to which lipase and anion exchange groups bond decreases, so a carrier having an average pore radius of 10 to 1000 is used. A macroporous carrier can provide a wide surface area for bonding lipase and anion exchange groups, and can also prevent carrier particles from becoming finer, resulting in an immobilized enzyme column with less pressure loss.

The anion exchange group used in the present invention includes various amines,
A chemical residue such as ammonium or its derivatives that dissociates in a solvent, becomes positively charged, and adsorbs anions.

If the amount of IJ-R-ase bound to the carrier used in the present invention is too small, the immobilized enzyme activity will be low and immobilization will occur.
The start-up of the reaction due to '-ze becomes difficult.

Furthermore, when a very large amount of lipase is bound, although the start-up of the reaction by the immobilized lipase is improved, the final decomposition rate at a high substrate concentration may be deteriorated. This is because when lipase is bound, it is often bound through an anion exchange group, so the anion exchange group is blocked and there are no anion groups on the surface of the carrier to which lipase is bound. be. In order to obtain the effects of the present invention, it is necessary to use a carrier to which sufficient ion exchange groups, which are generally low molecules, are bound, and to adjust the amount of linose ξ-ase bound.

Regarding the adhesive bonding method of the present invention, since lipase is usually an acidic protein, it easily binds to the anion exchange carrier through an ionic bond. In addition, when a hydrophobic carrier having an anion exchange group is used, it is strongly bound by ionic bonds and hydrophobic bonds. Furthermore, when those bound by ionic bonds and hydrophobic bonds are treated with geltaraldehyde or cal-2-diimide, the bonds become even stronger. In the case of geltaraldehyde treatment, when -H is 7 or more, geltaraldehyde becomes
It increases reactivity and deactivates S-ξ-ze.”
It is better to adjust the pH to around 4.5 to 6.5, keep the temperature below 25°C, and process for a short time of about 10 to 20 minutes. After the reaction, remove excess geltaraldehyde with sodium bisulfite or the like.

The above operation is carried out after pouring an appropriate amount of lipase solution onto the carrier packed in the column and adsorbing it through ionic and hydrophobic bonds.
This is an industrially advantageous binding method because it is easy to prepare an immobilized enzyme column by adjusting the pI (and temperature) and flowing an appropriately diluted gel taraldehyde solution, followed by flowing a sodium bisulfite solution. As mentioned above, if the anion exchange group is used as a functional group for binding lipase protein to gold,
It is also possible to remove the protons from the anion exchange group and weaken the effect of protons to remove the product-inhibiting effect. Therefore, if the anion exchange group is protected and So-Kuse is bound to it via another functional group, Since the effects of the present invention are further enhanced, there is no problem with such a combination method.

pH of reaction conditions in enzymatic decomposition of lipids in the present invention
Regarding the pH and temperature, the optimum pH and temperature for the immobilized/e-se reaction are used. If the desired decomposition rate can be obtained even if the pH and temperature are optimum, there is no problem in using it. Furthermore, if necessary, metal ions, serum albumin, etc. may be added.

Examples of the lipids used in the present invention include various glycerides and esters that serve as substrates for linose ξ-ase. Any lipase can be used as long as it acts on lipase.

The substrate concentration of glue-zoase in the reaction solution used in the present invention is 5
0-94% will be selected. Even if the substrate concentration is lower than 50%, the final decomposition rate does not increase, and the size of the reaction tank must be reduced [7. It is more advantageous to react at a concentration higher than that. In addition, the substrate concentration of 3 molecules of water per 1 molecule of triglyceride, which is the stoichiometric water content of the hydrolysis reaction, is about 94 molecules for long-chain triglycerides such as beef tallow; However, it is possible to achieve a relatively high decomposition rate.

In the present invention, the substrate concentration refers to the weight percent of the substance containing an ester bond to the sum of the weights of the substance containing an ester bond and water, which serve as a substrate. When producing fatty acids industrially, after oil and fat decomposition are completed, oil and water (or glycerin) are separated, and the oil is divided into liquid fatty acids and solid fatty acids for later refining. In addition to oleic acid, linoleic acid, etc., undecomposed triglycerides may remain. In other words, when decomposing about 8 grams of glyceride contained in crude fatty acids, the small amount of water contained in the crude fatty acids can be sufficient to hydrolyze it, so if the crude fatty acids are reacted as they are with immobilized linocuse, the triglycerides present can be removed. It is also possible to remove it.

As the W1 reaction tank for use in the present invention, there are two types: a one-shot type in which the immobilized linosose and the reaction solution are mixed together, and a continuous type in which the immobilized linosose is packed in a column.

Both the No2Tsuchi method and the column method have the advantage that it is easy to recover the immobilized enzyme, there is no need to remove glue/glue proteins at the final stage of the product, and the recovered enzyme can be used repeatedly. has. The column type has further advantages such as less contact with air during the reaction, so unsaturated fatty acids are less likely to be oxidized in the air, and because it can be continuous, it can be easily incorporated into the currently available high-pressure continuous cracking process. has.

According to the present invention, it is possible to decompose fats and oils and esters to a high decomposition rate with a significantly reduced amount of water in the reaction solution. This not only reduces the energy required to recover the glycerin and alcohol obtained after the reaction by distillation, but also reduces the size of the reaction tank when decomposing large amounts of fats and oils. The effect is great.

Furthermore, the use of the immobilized ξ-ze column makes it possible to carry out the cell reaction and prevents air oxidation of unsaturated fatty acids. Moreover, it is an excellent method for industrial enzymatic decomposition of fats and oils, as it is easy to prepare and regenerate the immobilized enzyme column.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1y-Dowex MWA-1 (total exchange capacity 1.2M
Eya JJ / mL of Macrovira Styzo's weakly basic anion exchange resin (manufactured by Dow Chemical Company) with a tertiary amine/exchange group having a parent body made of polystyrene chains crosslinked with divinylbenzene (manufactured by Dow Chemical Company) was mixed with distilled water. After washing with M/15 pine quilvain buffer pH 5,0, 50 μt of Rhinoξ-
A solution (1450 units) and 1 r of Matsukuil Bay/buffer were added, and the mixture was shaken at 8°C overnight. Next, 1-Matsuki Irbain buffer solution and 80 μt of 25% geltal aldehyde solution were added, and the mixture was shaken at 8° C. for 10 minutes to bind to DOWEX MWA-1. Finally, add 20% sodium hydrogen sulfite and shake for 10 minutes at 8°C to remove excess glutaraldenoid, wash thoroughly with water and buffer, and use DOWEX MWA- 1 immobilized lipase was obtained. The lino used here? The amount of enzyme is sufficient to activate the activity of the immobilized lipase, and is also an amount that does not impair the final decomposition rate at high substrate concentrations.

1? DOWEX WGR (total conversion capacity: 1.6Me-1, a gel-type weakly basic anion exchange resin containing polyamine as an exchange group, a condensate of epichlorohydrin and ammonia, manufactured by Dow Chemical Co.) as described above. Process in the same way as
Su/sose was synthesized to have a large number of extremely fine pores to allow a large number of inorganic ions to pass through, and was immobilized on an anion exchange resin of FC GEL TYSO.

death,? Chit CNP80 (total exchange capacity i 4.7 M e
Macroporous weakly acidic cation exchange resin with acrylic base and carboxylic acid exchange group.

) manufactured by Inil Co., Ltd.) 1. P, 1-cyclohexyl-3-
(2-morpholinoethyl)-calcediimide p-)luenemethosulfonic acid (cMc and Kt) under 50 m9
* Add 10 - of water and continue the reaction at room temperature while keeping the pH at 4-5 with 6N HCl. When the pH stabilizes, excess CMC is thoroughly washed away. Next, add 1715M pine quill pain buffer pH 4 to 2-250μ, /= Nori・G-
A solution of CNP80 (1450 units) was added thereto, and the mixture was allowed to react overnight in a cold room to obtain CNP80-immobilized CNP80.

Lipase activity was 3.141 tributyrin, 35-2% polyvinyl alcohol (Kurashiki Bosser 117)
Solution, 40 ml of 0.1 M phosphate buffer pH 7, 12-
Mix 91 nl of tributyrin emulsion prepared by mixing with water and sonicating for 10 minutes and 1z of enzyme suspension,
The reaction was carried out at 60°C for 20 minutes with shaking. A 1:1 mixture of methanol and acetone was added to stop the reaction, and the resulting fatty acid was titrated with 0.05N NaOH to measure its activity. The amount of acid released in 1 minute under the above conditions was defined as 1 unit.

- Guze was used after being freeze-dried after fC, which includes ammonium sulfate salting out, dialysis, and dialysis with chloroform.

In a 100-inch Erlenmeyer flask, add reaction solutions of various substrate concentrations of beef tallow and 0.1 M phosphate buffer pH 7.0.
Add immobilized lipase that has been well removed from water and react at 60°C for 160 to 200 hours with shaking 178 times per minute. Table 1 shows the degradation rate (final degradation rate). The final decomposition rate of tertiary amine or ? Those with anion exchange groups such as reaamine (DOWEX MWA-1 and DOWEX WGR as immobilized carriers) are better than those with carboxylic acid, which is a cation exchange group (Shi/NichitCNP).
80 as an immobilization carrier).

In addition, the decomposition rate is particularly high at high substrate concentrations when rhinoξ-ase is immobilized on a macroporous anion-exchange resin that extends into the pores of the carrier surface where rhino-converting groups are present. It shows good things.

Example 2 Porous glass (CPGO (1500, average hole radius 5
15 people 1 particle size 120-200 mesh (manufactured by Electronucleonics) was activated at 500°C for 2 hours and then placed in a solution of 2% silane coupling agent (3-aminopropyltriethoxysilane) in acetone. If you attach a backflow cooling pipe and leave it at 50℃ for 20 hours, the first layer will appear at the end.
An anion exchanger having primary amine groups is obtained. Aminoalkyl CPG-immobilized IJA-
I got ze.

Calzexialkyl CPG (average pore radius 547 people.

A porous glass having a carboxyl group at the end of the Particle Cyno 20-80 mesh (manufactured by Electronucleonics Co., Ltd.) was mixed with the resin of Example 1) as CNP80.
Cal with the same operation as Tato? A xyalkyl CPG-immobilized nose ξ-ase was obtained.

5? Toyo ξ-L HW60 (manufactured by Toyo Ichida, hydrophilic vinyl polymer) was mixed with 5-60'% NaOH, 12.5 m
of epichlorohydrin was added, and after shaking at 50°C for 2 hours, the mixture was washed with 500°C of distilled water to obtain epoxy toyo A-ru. Epoxy toyo, 12 in o-le? (7) Add hexamethylene diamine and go with 1M caustic soda solution 2-
After the reaction, the mixture was shaken for 2 hours at C.c. and washed with acetone to obtain an aminated toyonol having a primary amine group at the terminal group. An aminoalkyl toyo ξ-ol-immobilized resin was obtained in the same manner as shown for Dowex MWA-1 in Example 1.

For each immobilized enzyme, the final decomposition rate was determined in the same manner as in Example 1.

Table 2 shows the final decomposition rate of fats and oils at high substrate concentrations.
This shows that it is better to have a diamino group present on the surface of the carrier to which -ze is bound than a carzexyl group chain. It is also shown that it is better to have an amino group on the surface of the carrier and to make the carrier itself hydrophobic.

Example 3 Immobilization was performed on various macroporous anion exchange resins in the same manner as shown for DOWEX MWA-1 in Example 1, and the final decomposition rates were compared using 7e-ase. These anion exchange resins have many anion exchange groups on the surface of macroporous pores.

Table 3 shows that when immobilized lipase combined with an appropriate amount of glue and ξ-ase is used with various macroporous type anion exchangers and anion exchange type chelate resin (Dia f-on CR20), This indicates that the decomposition rate of fats and oils is good at high substrate concentrations due to the presence of anion exchange groups on the surface of the carrier to which Su-Guze is bound.

Example 4 DOWEX MWA-1 prepared in the same manner as Example 1
The immobilized a-ze was thoroughly dried and the final and final decomposition rate was compared with that of beef tallow. When immobilized as a control, Table 4 shows that when using soluble lipase, the final decomposition rate was significantly reduced at high substrate concentrations, whereas when anion exchange groups were attached to the surface of the carrier to which Suguse was attached, No. 1. We demonstrate the use of locally prepared immobilized lipase.

Claims (1)

[Claims] When enzymatically decomposing lipids, it uses an immobilized lipase that has an anion exchange group on the surface of the carrier to which lipase is bound, and is characterized by decomposing the substrate concentration of linose within the range of 50 to 94 cm. Enzymatic decomposition method for lipids