JPH01215294A - Modification of enzyme and hydrolysis of fats and oils - Google Patents

Abstract

PURPOSE:To efficiently provide highly unsaturated fatty acids in a high concentration by hydrolyzing fats and oils with an enzyme having a 2-monoglyceride lipase activity and obtained by modifying Candida-cylindracea lipase with an alkali. CONSTITUTION:Candida-cylindracea lipase is treated with an alkali to modify the lipase into an enzyme having a 2-monoglyceride lipase activity. Fats and oils are subjected to a hydrolysis reaction using the modified enzyme under an alkaline condition of pH7.5-9 to specifically decomposed the ester bonds at the Sn-2 positions of triglycerides to provide highly unsaturated fatty acids.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to Candida cylindracea (Candida cylindracea).
A method for enzyme modification is employed in which lipase (hereinafter simply referred to as Candida cylindracelipase) obtained from No. 91 Yamashiko Shotanri is treated with an alkali.

The present invention also relates to a method of hydrolyzing fats and oils using the lipase.

[Conventional technology]

Traditionally, in the oil and fat industry, a method was used to hydrolyze oils and fats under high temperature and pressure, but recently enzymatic methods at room temperature and pressure have been used to produce heat-unstable substances such as highly unsaturated fatty acids. has started to take place. For example, JP-A-58
-205499, JP-A-57-170193, and the like. These highly unsaturated fatty acids are arachidonic acid,
Eicosapentaenoic acid, docosahexaenoic acid, and other acids have attracted attention for their physiological activities and are considered promising as pharmaceuticals or their precursors.

For hydrolysis of fats and oils by enzymatic methods, various substances such as Candida cylindracelipase, pancreatic lipase, and rhizopus lipase are used.

[Problem to be solved by the invention]

In order to use enzymes industrially, they must be inexpensive and available in large quantities, but all commercially available lipases to date, with the exception of Candida cylindracelipase, are expensive and cannot be produced in large quantities. It is difficult to produce. Therefore, Candida cylindracelipase can be said to be the most suitable for industrial cost. Conventionally, this lipase hydrates all three bonds of triglyceride, that is, the ester bonds at Sn-1, 2, and 3 positions. It was known as an enzyme that decomposes Therefore, it was said that it does not exhibit positional specificity.

Since many highly unsaturated fatty acids exist at the Sn-2 position of triglycerides, in order to obtain higher concentrations of these compounds, there are two methods: hydrolyzing only the Sn-2 position and extracting these compounds in the free form; A conceivable method is to hydrolyze only the -1 and 3-positions and concentrate them in the form of glyceride. However, the normal Candida cylindracelipase
There is a problem in that these site-specific hydrolysis cannot be performed.

It is an object of the present invention to modify Candida cylindracelipase and to provide a method for position-specifically hydrolyzing highly unsaturated fatty acids using Candida cylindracelipase.

[Means to solve the problem]

The first invention of the present invention is a method for modifying Candida cylindracelipase into an enzyme having 2-monoglyceride lipase activity, which is characterized by treating Candida cylindracelipase with an alkali. This is a method for hydrolyzing fats and oils that specifically decomposes the ester bond at the Sn-2 position of triglyceride, which is characterized by carrying out the reaction under alkaline conditions of pH 7.5 to 9 using Candida cylindracelipase. .

The lipase obtained by culturing Candida cylindrace used in the present invention can be a commercially available lipase, for example, Lipase OF (manufactured by Polysaccharide Sangyo ■, obtained by acetone treatment of a culture solution of Candida cylindrace) Enzyme powder) and the like can be preferably mentioned.

The present inventors have been researching the use of Candida cylindracelipase, which is advantageous for industrial costs, and as part of this effort, we attempted to purify this enzyme, and found that 2-monoglyceride lipase and 1,3-diglyceride lipase It was found that it contains two types of enzymes.

Among these, 1.3-diglyceride lipase is sensitive to alkali (and is easily inactivated when treated in the pH range of 7.5 to 12), and 2-monoglyceride lipase was not inactivated at all under the same conditions. That is, Candida cylindracelipase, which does not exhibit positional specificity, can be treated with alkali to inactivate only 1,3-diglyceride lipase, and can be modified into an enzyme that exhibits only 2-monoglyceride activity. .

Various alkalis can be used in the present invention, such as potassium hydroxide, sodium hydroxide,
Aqueous solutions of Tris, ammonia, sodium carbonate, sodium hydrogen carbonate, etc., with a pH in the range of 7.5 to 12, or 20mM to IM sodium phosphate buffer, Tris-hydrochloric acid buffer, glycine-sodium hydroxide buffer , sodium bicarbonate-sodium carbonate buffer, etc., having a pH within the range of 7.5 to 12. If the pH is lower than 7.5, the deactivation of Sn-1,3 diglyceride lipase will be insufficient, and the position specificity of Sn-2 monoglyceride lipase will deteriorate. A particularly preferred pH is 8-12.

The amount of enzyme to alkali ranges from 0.1 to 30 weight percent, preferably from 1 to 10 weight percent.

A treatment time of 1 to 10 hours is sufficient.

The treatment temperature is preferably within the range of 1 to 5°C so that the target 2-monoglyceride lipase is not deactivated.

After the alkaline treatment, the enzyme solution is neutralized to pH 7.0 with hydrochloric acid or the like, desalted by dialysis for 48 hours, and then freeze-dried to obtain the modified enzyme. The present invention is inexpensive and suitable for industrial costs, and can make Candida cylindracelipase, which conventionally did not exhibit position specificity, exhibit position specificity by utilizing its pH dependence.

Moreover, the present invention is a method for efficiently obtaining a target fatty acid by hydrolyzing fats and oils under conditions that express this position specificity. The fats and oils used in the present invention can be either animal oils or vegetable oils, such as beef tallow, liver fat, fish oil, soybean oil, corn oil, evening primrose oil, olive oil, etc. In particular, physiologically active oils can be used. Fish oil, olive oil, evening primrose oil, etc. containing highly unsaturated acids are preferred.

When hydrolyzing fats and oils in the present invention, if commercially available Candida cylindracelipase is used as is, it is preferably carried out under alkalinity of 9H1,5 to 9;
When H exceeds 9, the hydrolyzability of fats and oils decreases, and the pH
When is smaller than 7.5, Sn-1,3 lipase is insufficiently deactivated and the specific decomposition of Sn-2 is reduced. As the alkali used to maintain the pH at 7.5 to 9 in the reaction, the alkali and buffer solution described above can be used.

Since each fat or oil used as a substrate exhibits its own optimum pH, this optimum pH is measured in advance, and then one of the above-mentioned acids is selected and used.

In addition, when decomposing fats and oils using the modifying enzyme obtained in the first aspect of the present invention, it is preferable to carry out the reaction under neutral or slightly alkaline conditions; under acidic conditions, the enzyme is deactivated and cannot be used.

The amount of enzyme added is 0.01■ to 50■ per 1g of fat or oil.
The reaction temperature is preferably 0.1 to 10°C, and the reaction temperature is 10°C to 50°C, preferably 20 to 40°C.
A temperature between ℃ is good.

Since this reaction is carried out in a two-layer system of water and oil, sufficient stirring is required. Usually 100 to 50 Or, p, m.

It is carried out within the range of.

This is done while expelling air by nitrogen bubbling to prevent highly unsaturated fatty acids from coming into contact with air and oxidizing.

The reaction time is usually 1 to 5 hours.

〔Effect of the invention〕

According to the modification method of the present invention, 2-diglyceride lipase that specifically reacts with the Sn-2 position of fats and oils can be selectively obtained from commercially available lipases that exhibit no position specificity.

Furthermore, according to the method for hydrolyzing fats and oils of the present invention, it is possible to specifically decompose the ester bond at the Sn-2 position of triglyceride using commercially available lipase, so highly unsaturated fatty acids with strong physiological activity can be extracted from natural fats and oils. The desired Sn-1,3 diglyceride can be obtained by separation and concentration.

(Example) Hereinafter, the present invention will be specifically described based on Examples and Comparative Examples.

Example 1 Candida cylindrace lipase (polysaccharide industry product, Candida cylindrace (Candida 1)
5 g of lipase produced by Ind. Cea, trade name Lipase OF) was dissolved in 0.1 M glycine-sodium hydroxide buffer, pH 10,0,100, and left at 4°C for 5 hours. Then, adjust the pH to 7 with 0.05N hydrochloric acid.
, 0, dialyzed in water for 48 hours to remove buffers and salts, and freeze-dried to obtain about 5 g of enzyme powder.

1g of this enzyme 5■, water 5-1 fish oil (NOF product)
In addition, the mixture was reacted at 37° C. and 500 r, p, m for 5 hours. When the product was analyzed by thin layer chromatography, no triglyceride was found, and the majority was 1,3-diglyceride and fatty acids, with trace amounts of 1°2-diglyceride and 1- and 3-
Monoglycerides were detected. This makes the enzyme 2-
It turns out to be monoglyceride lipase.

Example 2 In Example 1, the 0.1M glycine-sodium hydroxide buffer, pH 10.0, was changed to 0.2M, sodium bicarbonate-sodium carbonate buffer, pH 11.0, and the treatment time was changed from 5 hours to 3 hours. The rest was done in the same way. When similar hydrolysis was carried out using this modified enzyme, triglycerides were not present in the product, and 1,3
- Diglyceride and fatty acids were the majority, and trace amounts of 1,2-diglyceride and 1- and 3-monoglycerides, which were thought to be generated by rearrangement, were detected.

Example 3 Same as Example 1 except that 0.1 M glycine-sodium hydroxide buffer, pH 10.0 was replaced with sodium hydroxide solution, p) 112.0, and the treatment time was changed from 5 hours to 3 hours. went. When similar hydrolysis was carried out using the obtained modification enzyme, the product contained no triglyceride, but contained mostly 1,3-diglyceride and fatty acids, with a trace amount thought to have been produced by rearrangement. 1.2
-diglycerides and 1- and 3-monoglycerides were detected.

Example 4 0.5 g of Candida cylindracelipase (Lipase 0F, a product of Polysaccharide Industries) was dissolved in 500 g of olive oil (a product of Kiyu Pharmaceutical Industries). Add 500- and heat to 37°C.
During the 0 reaction, which was carried out at 500 r, p, m, for 3 hours, air was expelled by nitrogen bubbling.

After the reaction was completed, the product was analyzed by thin layer chromatography, and the presence of triglycerides was not observed.
-Diglyceride and fatty acids were present as main components, and 1,2-diglyceride and monoglyceride of it, which were thought to be produced by rearrangement, were detected.

Example 5 In Example 4, olive oil was replaced with fish oil (NOF ■ product), 0.1M) Lissu-hydrochloric acid buffer, pH'7.5
to 0.2M) lys-hydrochloric acid buffer, pH 8.0, and 0.5 to 5 g of Candida cylindracelipase.
I did the same thing except change it to . No triglyceride was observed in the product, 1,3-diglyceride and fatty acids were the main components, and trace amounts of 1,2-diglyceride and monoglyceride, which were thought to be produced by rearrangement, were detected. In addition, the composition of the resulting fatty acids was analyzed by gas chromatography, and the results showed that eicosahencitric acid was 19.8% and docosahexaenoic acid was 18.4%, compared to the composition in the raw fish oil of 13.5% and 9.5%. Each was better than the other.

Example 6 The same procedure as in Example 4 was carried out except that the olive oil was replaced with evening primrose seed oil (a product of Summit Oil Manufacturing) and the reaction time was changed from 3 hours to 5 hours. No triglycerides were found in the product;
1,3-diglyceride and fatty acids were present as main components, and trace amounts of 1,2-diglyceride and monoglyceride, which were thought to be produced by rearrangement, were detected. Furthermore, as a result of analyzing the fatty acid composition by gas chromatography, the γ-lylunic acid content was 18.6%, which exceeded 1000% in the raw material.

Comparative Example In Example 4, 0.1M) Li-HCl buffer p
The same procedure was carried out except that n 7.5 was replaced with 0.1 M sodium phosphate pn 7.0. triglycerides in the product,
Diglycerides and monoglycerides were not observed, and only fatty acids were detected. This shows that all positions were resolved, regardless of Sn-1, Sn-2, and Sn-3 positions.

Claims (2)

[Claims] (1) A method for modifying an enzyme, which comprises treating Candida cylindracelipase with an alkali to modify it into an enzyme having 2-monoglyceride lipase activity. (2) It is characterized by carrying out a hydrolysis reaction of fats and oils using Candida cylindracelipase under alkaline conditions of pH 7.5 to 9 to specifically decompose the ester bond at the Sn-2 position of triglyceride. Method of hydrolyzing fats and oils.