JPH03183480A - Immobilized lipase and ester exchange reaction of fat or oil with the same - Google Patents

Abstract

PURPOSE:To provide an immobilized lipase shortening times for the ester exchange reactions of fats or oils, depressing the progress of side reactions and useful for modifying the edible fats or oils, etc., by immobilizing lipase and a hydrophilic amino acid, a peptide, etc., as an activity-generating agent. CONSTITUTION:A lipase enzyme originated from a Rhizopus strain fungus, etc., and at least one kind of hydrophilic amino acid and/or peptide (e.g. yeast extract) are dissolved in distilled water and subsequently homogeneously mixed with a carrier (e.g. celite) with stirring to immobilize the enzyme and the activity-generating agent on the carrier, followed by drying the carrier e.g. at 40 deg.C for 16 hours under vacuum to provide the immobilized lipase. When a mixture of palm oil and rapeseed oil is subjected to an ester exchange reaction at 60 deg.C for 3 hours with the immobilized lipase, the fats or oils can be modified in the high ester exchange activity in a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to an immobilized lipase suitable for the modification of edible oils and fats, specifically for transesterification.

(Prior art) The transesterification reaction of oils and fats using lipase is superior to chemical methods due to the enzyme's properties such as reactivity under mild conditions, positional specificity, and alkyl group selectivity. There are many points. However, the transesterification reaction by lipase is different from the original hydrolysis reaction in an aqueous system, and the reaction proceeds only in a system with limited water content.

In order to suppress the hydrolysis reaction as a side reaction, it is generally desirable that the water content during the reaction is about 300 ppm or less, but if the water content is extremely low, the enzyme activity will not be expressed and its deactivation will be promoted. In order to solve this problem, a method has been proposed in which several types of activity-expressing agents are added to lipase, brought into contact with it, and then immobilized, as shown below.

That is, (1) a method of pre-wetting lipase with water, glycerin, etc. (Japanese Patent Application Laid-Open No. 149094/1983);
2) Method of drying lipase with fatty acids or fatty acid derivatives (JP-A-62-134090, JP-A-1-153)
097), (3) Method of immobilizing lipase and phospholipid in a state of contact and bonding (Japanese Patent Application Laid-open No. 63-214184
), (4) A method of immobilizing lipase in the presence of surfactants and polyols (JP-A-64-2588), (5) Method of immobilizing lipase in the presence of oil-soluble compounds such as alcohols, ethers, and carbonyl compounds. How to fix it (Unexamined Japanese Patent Publication Hei!-
153091) etc. have been disclosed.

However, the method presented in (1) uses a water-rich system, which tends to cause hydrolysis of the substrate oil, and
A system with water content below pm has the disadvantage that the reaction rate is significantly reduced.

Although the methods (2) and (5) have a certain degree of activity-increasing effect, they are not sufficient for industrial implementation. There is also a concern that the fatty acid derivatives and fat-soluble compounds used may leak into the substrate oil, causing quality deterioration.

Phospholipids and surfactants (3) and (4) have excellent activity under low moisture conditions, but their effectiveness varies depending on the enzyme lot, and they are highly active and difficult to handle. There are problems such as:

(Problems to be Solved by the Invention) An object of the present invention is to provide an immobilized lipase that does not have such drawbacks and is highly active under low moisture conditions.

(Means for Solving the Problem) The present inventor conducted research on factors that increase the transesterification activity of lipase, and discovered that amino acids and peptides have an activity-promoting effect. The present invention was then completed by immobilizing lipase on various immobilization carriers in the presence of these specific activity promoters. That is, the present invention relates to an immobilized lipase characterized by using a peptide with an activity-expressing agent immobilized in a carrier, and a transesterification reaction of fats and oils using this immobilized lipase.

In the present invention, hydrophilic amino acids are preferred as activity promoters, while hydrophobic amino acids generally have poor effects. For example, histidine, arginine, serine, aspartic acid, asparagine, glutamic acid, lysine,
Hydrophilic amino acids such as threonine are preferred. Further, the amino acid may be in the form of a free form, a salt thereof, an acetylated product, or a derivative such as an amide. The peptide also preferably contains these hydrophilic amino acids. In general, amino acids and peptides may be added alone, or two or more types of amino acids and peptides may be added as a mixture. It may also be a hydrolyzate of protein such as casein. Examples include polypeptone, casitone, soiton, etc. In addition to these, yeast extract (Yeas
malt extract), malt extract
Hygroscopic substances containing amino acids and peptides such as xtract, ) are also effective.

The amount of the activity-enhancing agent used is not limited, but it is usually 20% by weight or more, preferably 20-1000% by weight based on the lipase.

Lipases are also not particularly limited; for example, fungi such as Rhizopus, Aspergillus, Penicillium, and Candida
andida), the genus Mucor, and among bacteria, the genus Pseudomonas,
Lipase from Geotricum can be used.

In the present invention, the immobilization carrier can be either an adsorption type or an entrapping type. Adsorption type carriers include celite, clay, activated carbon, cellulose and its derivatives, chitosan and its derivatives, and various ion exchange resins, and entrapping type carriers include photocurable resins, agar, sodium alginate, and the like.

To produce the immobilized lipase of the present invention, first, lipase and an activity promoter (amino acid or peptide) are dispersed or dissolved in water or an appropriate buffer. Next, in the case of an adsorption type carrier, the above aqueous solution is uniformly sprinkled onto the carrier and mixed.

In the case of an entrapping type carrier, the aqueous solution and the carrier monomer are mixed and then polymerized. Thereafter, moisture may be removed by drying under reduced pressure, if necessary.

Regarding the order of addition of the activity-expressing agent and lipase, the lipase and the activity-expressing agent may be added and immobilized at the same time, or the activity-expressing agent may be immobilized first, and then the lipase may be added.

Examples of the substrate oil for the oil-fat modification reaction using the immobilized lipase of the present invention include vegetable oils, animal oils, processed oils and fats, and mixtures thereof. For example, palm oil, rapeseed oil, soybean oil, sunflower oil, rice oil,
Examples include olive oil, corn oil, cottonseed oil, beef tallow, lard, and fish oil.

Further, if necessary, one or more fatty acids such as myristic acid, balmitic acid, stearic acid, oleic acid, and linoleic acid may be added to the above-mentioned fats and oils.

In order to carry out the transesterification reaction using the immobilized lipase of the present invention, the water concentration of the raw material oil is first adjusted to about 300 ppm or less, preferably 150 ppm or less, and then the immobilized lipase is added to the raw material oil. conduct. The amount of immobilized lipase added is not particularly limited, but it is usually
It may be added in an amount of 2 to 30 parts by weight per 00 parts by weight. The reaction format may be a batch method or a continuous reaction using a column, fluidized tank, etc.

The immobilized lipase of the present invention can of course be used not only for transesterification reactions among oil and fat modification reactions but also for ester synthesis reactions. It can also be used for transesterification of ester compounds other than fats and oils, or for ester synthesis reactions.

The present invention will be specifically described below based on examples.

(Example 1) 10 mg of lipase enzyme derived from Rhizopus (manufactured by Amano Pharmaceutical)
and yeast extract (D i fc) as an active agent.

2.5 mg of each product (manufactured by S.A.) in 0.0 ml of distilled water. Dissolve in 5 ml of each 1 g of Celite (No. manufactured by Manville).

535) and stirred well until uniform.

These were then dried under reduced pressure at 40°C for 16 hours.

In this way, immobilized lipase with a water content of about 1% was prepared.

(Example 2) Immobilized lipase was prepared in the same manner as in Example 1 except that a general amino acid (manufactured by Ajinomoto Co., Ltd.) was used instead of the yeast extract.

(Example 3) Immobilized lipase was prepared in the same manner as in Example 1 except that serine was used instead of the yeast extract.

(Example 4) Immobilized lipase was prepared in the same manner as in Example 1 except that serine was used instead of the yeast extract. However, serine was not immobilized at the same time as lipase, but serine was first immobilized on Celite, and then lipase was immobilized.

(Example 5) Immobilized lipase was prepared in the same manner as in Example 1 except that histidine was used instead of the yeast extract.

(Example 6) Immobilized lipase was prepared in the same manner as in Example 1 except that histidine was used instead of the yeast extract. However, histidine and lipase were not immobilized at the same time; instead, histidine was first immobilized on Celite, and then lipase was immobilized.

(Comparative Example 1) Immobilized lipase was prepared in the same manner as in Example 1 except that gelatin was used instead of yeast extract.

(Comparative Example 2) Immobilized lipase was prepared in the same manner as in Example 1 except that dextran was used instead of the yeast extract.

(Comparative Example 3) Immobilized lipase was prepared in the same manner as in Example 1 except that lecithin (manufactured by Ajinomoto Co., Ltd.) was used instead of the yeast extract.

(Comparative Example 4) Immobilized lipase was prepared in the same manner as in Example 1 except that the activity-expressing agent was removed. 2.5 mg of serine was added to the reaction substrate oil.

(Comparative Example 5) Immobilized lipase was prepared in the same manner as in Example 1 except that the activity-expressing agent was removed. 2.5 mg of histidine was added to the reaction substrate oil.

(Comparative Example 6) Immobilized lipase was prepared in the same manner as in Example 1 except that the activity-expressing agent was removed.

(Example 7) A transesterification reaction was performed using the immobilized lipase obtained in Example 1-6 and Comparative Example 1-6 in the following manner. Mixed oil (palm oil and rapeseed oil squeezed 20:80 (w/w)
) at 80°C under a reduced pressure of about 20 torr.
Dehydration was performed for 0 minutes to bring the water concentration to 1100 pp or less, which was used as a reaction substrate oil. Add reaction substrate IOg to a flask with a cap.
After adding 1 g of molecular sieve to this, each of the above-mentioned immobilized lipases was added and reacted at 60'C for 13 hours. After the reaction, the triglyceride sheath formation (T
G (%)) was measured and the results are shown in Table 1.

Transesterification activity shows a large change in TG with 52 carbon atoms.
It was calculated using the following formula using as an index.

Change in C52 It (%) / 3 (Hr) Activity = Lipase weight jl (g) / Substrate weight (g) Table 1 (Example 8) Amino acids in sections A to F listed in Table 2 were used as activity enhancers. The transesterification activity was measured in exactly the same manner as in Example 1 and Example 7, except that the mixture was used.

The results are shown in Table 2.

Hydrophilic amino acids have a particularly high ability to express activity.

Table 2 1) This value used the value determined by Kyte and Doolittle (Musho Hutan, 157, 105-1
32 (1982)).

The higher the number, the more hydrophobic it is.

4. Effects of the Invention By using the immobilized lipase of the present invention, it is possible to shorten the transesterification reaction time, leading to a reduction in the cost of the enzyme lipase. Furthermore, as a result of the improved reaction rate, the progress of side reactions during the transesterification reaction is suppressed, and the quality of the reformed oil is also improved.

Claims (2)

[Claims] (1) An immobilized lipase characterized by using at least one kind of hydrophilic amino acid and/or at least one kind of peptide as the activity-enhancing agent in an enzyme preparation formed by immobilizing lipase and an activity-enhancing agent on a carrier. (2) An enzyme preparation comprising a lipase and an activity-enhancing agent immobilized on a carrier, characterized in that at least one hydrophilic amino acid and/or at least one peptide is used as the activity-enhancing agent. Transesterification reaction.