JP2724474B2 - Decomposition method of fats and oils by enzymes - Google Patents

Description

The present invention relates to a method for decomposing fats and oils with an enzyme. More specifically, Candida cylindrase E-118 (Candida c
ylindracea E-118), a method for decomposing fats and oils containing a large amount of short-chain or medium-chain fatty acids, for example, lipase having high specificity for palm oil, palm kernel oil and the like.

(Prior art)

In general, a method of decomposing fats and oils by lipase is studied by a batch stirring method using an aqueous solution of lipase. In addition, lipase generally decomposes fats and oils containing a large amount of long-chain fatty acids such as oleic acid, stearic acid and linoleic acid having 18 carbon atoms (for example, olive oil and beef tallow). It does not easily act on fats and oils rich in medium-chain fatty acids from (14) (for the fatty acid composition of various fats and oils, see the Oil Chemistry Handbook edited by the Japan Oil Chemists' Society). Therefore, no enzymatic decomposition of these fats and oils has been carried out.

[Problems to be solved by the invention]

Beef tallow, lard, coconut oil, and the like are used as raw materials for decomposing fats and oils. Particularly, coconut oil is important in its fatty acid as an industrial raw material such as soap. However, conventional lipases produced by Candida cylindracese are highly specific to fats and oils containing relatively long-chain fatty acids such as oleic acid, stearic acid and linoleic acid, such as beef tallow and olive oil.
Although it decomposed well, its decomposition activity was inhibited in the presence of short-chain and medium-chain fatty acids, and a sufficient decomposition rate was not obtained with palm oil or the like. The fragrance of cheese and butter produced from milk fat is derived from short-chain and medium-chain fatty acids, and the present lipase action enables the creation of flavors of cheese and butter.

As described above, the present invention relates to short chains such as palm oil, palm kernel oil, etc.
An object of the present invention is to provide a method that enables efficient enzymatic decomposition of fats and oils containing a large amount of medium-chain fatty acids.

[Means for solving the problem and its operation]

Conventionally, the enzyme produced by Candida cylindracese is relatively specific for fats and oils containing a large amount of long-chain fatty acids, but not for fats and oils containing a lot of short- and medium-chain fatty acids. Has low specificity and is a degraded short chain,
In some cases, the activity is inhibited by medium-chain fatty acids, and the use of the latter was unsuitable for the decomposition of fats and oils.

The present inventors have conducted intensive studies, and as a result, one mutant strain of Candida cylindrase produces a lipase that exhibits a higher decomposing action on fats and oils containing short-chain and medium-chain fatty acids than conventional lipases. That is, the present invention has been completed.

 Next, the present invention will be described in detail.

The lipase used in the present invention is Candida cylindracea ATCC 14830.
A strain obtained by mutating the strain (this strain is
Deposited as No. 10282 with the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology. ) Can be cultured in a suitable medium and collected from the resulting culture. Specifically, a deep aeration agitation culture is performed, and as a culture source used as a culture medium, in addition to carbon sources, nitrogen sources, inorganic salts and other micronutrient sources generally used for microorganism culture, use of microorganisms belonging to the genus Candida. Any available nutrient source can be used.

As a carbon source of the medium, glucose, sucrose, rastergen, glycerin, dextrin, starch, etc., fatty acids,
Oils and fats, organic acids and the like are used alone or in combination.
As the nitrogen source, any of an inorganic nitrogen source and an organic nitrogen source can be used. Examples of the inorganic nitrogen source include ammonium nitrate, ammonium sulfate, urea, sodium nitrate, and ammonium chloride. Examples of organic nitrogen sources include soybean, rice, corn, wheat and other flours, bran, defatted meal, corn steep liquor, peptone, meat extract, casein, amino acids, yeast extract and the like. Inorganic salts and micronutrients include salts such as magnesium phosphate, potassium phosphate, iron, calcium, and zinc, and other vitamins, nonionic surfactants, defoamers, and other bacteria that promote the growth of bacteria and lipase production. It can be used as needed. The cultivation is performed under aerobic conditions, and the culturing temperature is within a range in which the bacteria grow and lipase is produced, and preferably 20 to 30 ° C. The culturing time varies depending on the conditions, but it is sufficient that the lipase is cultured until the time when lipase is most produced, and is usually about 2 to 4 days.
Lipase is dissolved in the culture solution, and is collected from the culture filtrate from which the solid content has been removed after the culture. To purify lipase from the culture filtrate, any method usually used for enzyme purification can be used. For example, treatment with an organic solvent such as ethanol, acetone, or isopropyl alcohol, salting out with sulfuric acid or salt, dialysis, ultrafiltration, ion exchange chromatography, adsorption chromatography,
Methods such as gel filtration, adsorbents, and isoelectric focusing can be used.
In addition, when the degree of purification of lipase is increased by appropriately combining these methods, they can be appropriately combined. Enzymes obtained by these methods can be added to or without various salts, sugars, proteins, lipids, surfactants, etc. as stabilizers, and can be concentrated by ultrafiltration, reverse osmosis, vacuum drying, freeze drying, spray drying. A liquid or solid lipase can be obtained by the method described above.

The enzymatic chemical properties of the lipase obtained by the above method are as follows.

a) Action Hydrolyzes ester bonds at all positions of triglyceride to produce fatty acids and glycerin.

b) Substrate specificity It acts widely on natural fats and oils, especially on short-chain and medium-chain fatty acid esters, and its substrate specificity is shown in Table 1 as a comparison of activities in various assay methods. . In Table 1, olive oil is used as a substrate in the standard method, dimercaprol tributyrate is used in the kit method, and p-nitrophenyl lauric acid is used in the pNPL method.

The details of the various measurement methods in Table 1 are shown below.

Standard method Add 5 ml of phosphate buffer of pH 7.0 to 5 ml of olive oil emulsion, add 1 ml of enzyme solution, react at 37 ° C for 30 minutes, and quantify the liberated fatty acid by neutralization titration with sodium hydroxide . The amount of the enzyme that produces 1 microequivalent fatty acid per minute under the above conditions is defined as one unit.

Kit method Using Lipase Kit S (Dainippon Pharmaceutical Co., Ltd.), 37
The increase in absorbance at 412 nm after incubation at 15 ° C for 15 minutes is measured. The amount of the enzyme that increases the absorbance by 1 is defined as 1 unit.

pNPL method 0.05 ml of 50 mM acetate buffer containing 2.5 mM p-nitrophenyllauric acid and 2.0% Triton X-100 are mixed, incubated at 37 ° C. for 15 minutes, and the increase in absorbance at 410 nm is measured. The amount of the enzyme that releases 1 micromol of P-nitrophenol per minute under the above conditions is defined as one unit.

c) Optimum pH about 7.0 (shown in FIG. 1) d) Stable pH about 4-7 (shown in FIG. 2) e) Optimum temperature about 50 ° C. (shown in FIG. 3) f) Temperature stability Stable up to 50 ° C at pH 7.0 (shown in Fig. 4) g) Fatty acid specificity (shown in Fig. 5) The above pNPL method was applied mutatis mutandis to determine the specificity in various fatty acid esters. It was measured.

h) Isoelectric point pI 5.1 to 5.3 (shown in Fig. 6) As lipase obtained from Candida syrup, lipase OF (manufactured by each sugar industry), lipase manufactured by Sigma, and the present inventors. Lipase (Japanese Patent Application Akira)
62-142724) and the like, but when compared with the enzyme used in the present invention, a large difference is observed in substrate specificity. Table 2 shows a comparison of the enzyme activities by the various measurement methods in the column of substrate specificity.

As is clear from FIG. 5 and Table 2, the lipase used in the present invention has a high action on short-chain and medium-chain fatty acid esters.

As is clear from FIGS. 3 and 4, the temperature stability is better and the optimum temperature is shifted to a higher temperature range as compared with the conventional lipase of Candida cylindrace.

For decomposing fats and oils using lipase obtained from Candida cylindrase E-118, any lipase in a crude or purified state can be used, and it may be used alone or in combination with other lipases. The fats and oils to be decomposed are not only fats and oils containing a large amount of short-chain and medium-chain fatty acids but also have a high optimum temperature, so that they can be applied to fats and oils which are solid at room temperature, such as beef tallow and palm kernel oil.

Hereinafter, Examples, Comparative Examples, and Reference Examples are shown, but the present invention is not limited thereto.

Example 1 5 g of coconut oil, 4 ml of 0.5 M pine kilbine buffer (pH 7.0),
Candida cylindr E-118
acea E-118), 1 ml of various concentrations of the enzyme solution and 30 glass beads having a diameter of 5 mm were placed in a 100 ml flask,
Incubated for 22 hours with shaking at 35 ° C and 45 ° C. After the reaction, the oil layer in the reaction mixture was extracted with petroleum ether to remove the ether, and the acid value (AV) and saponification value (SV) were measured. Calculated.

The results are shown in Table 3.

Example 2 Using 5 g of olive oil, the same operation as in Example 1 was performed to calculate the decomposition rate of fats and oils.

 The results are shown in Table 3.

Comparative Example 1 In Example 1, Candida cylindrase E-
The decomposition rate of fats and oils when a commercially available enzyme lipase OF (manufactured by Meito Sangyo Co., Ltd.) was used instead of the enzyme No. 118 was calculated.

 The results are shown in Table 3.

Comparative Example 2 In Example 2, Candida cylindrase E-
The decomposition rate of fats and oils when a commercially available enzyme lipase OF (manufactured by Meito Sangyo Co., Ltd.) was used instead of the enzyme No. 118 was calculated.

 The results are shown in Table 3.

When compared to Lipase OF, the lipase of the present invention works well not only on olive oil but also on coconut oil, and also works well at high temperatures.

Comparative Example 3 To 1 g of fatty acid methyl ester having various fatty acid chain lengths, 5 ml of McKilbain buffer at pH 6.0 was added, and the lipase of the present invention was added.
1 ml of each enzyme solution of lipase OF and lipase manufactured by Sigma was added, and the mixture was shake-reacted at 37 ° C. for 30 minutes, and the free fatty acids were titrated with a 0.1N potassium hydroxide reagent using a phenol indicator and quantified. The results are shown in FIG. 7, FIG. 8 and FIG.

As is clear from FIGS. 7 to 9, the lipase of the present invention has higher specificity for short- and medium-chain fatty acid esters than other lipases.

〔The invention's effect〕

According to the present invention, the conventional lipase derived from Candida cylindracese has a high specificity to fats and oils containing relatively long-chain fatty acids, and is not suitable for decomposing fats and oils containing a lot of lower and middle fatty acids such as coconut oil. However, the present invention makes this possible and brings a great contribution to the oil and fat industry.

[Brief description of the drawings]

1 and 2 show the enzymatic chemical properties of the lipase used in the present invention, and show the optimum pH and pH stability, respectively. FIGS. 3 and 4 show the optimum temperature and temperature, respectively. Indicating the stability,-●-indicates the lipase of the present invention,-○-
Indicates lipase OF. FIG. 5 shows a comparison of the activity of the lipase of the present invention depending on the type of constituent fatty acids of the fats and oils. FIG. 6 shows the isoelectric point fractionation patterns of various lipases, where -−- indicates the lipase of the present invention, and-●-indicates the lipase OF.
And-▽-indicates the lipase of Japanese Patent Application No. 62-142724. still,
In FIG. 6,... Indicates pH. FIGS. 7 to 9 show the comparison of the specificities of the various lipases depending on the difference in the number of fatty acid carbon atoms. FIG. 7 shows the lipase of the present invention, FIG. 8 shows the lipase manufactured by Sigma, and FIG. Indicates lipase OF.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:72)

Claims (1)

(57) [Claims] An oil or fat using a lipase produced by Candida syrup, which has a higher specificity for a fatty acid ester having less than 18 carbon atoms as compared with a fatty acid ester having 18 or more carbon atoms. Decomposition method.