JPH0673453B2 - Heat resistant lipase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a thermostable lipase.

(Prior Art) Lipase is a general term for an enzyme that hydrolyzes fat or reverse reaction thereof, and is conventionally extracted and produced from animal organs, microorganisms and the like. There are various uses of lipase for medical purposes (digestive agents, clinical tests), detergents, fatty acid production, food applications, and various types of lipases have been developed.

As a method of using lipase in foods, first of all, production of milk flavor can be mentioned, which is commonly called a flavor enzyme. Animal-derived lipases such as pancreatin lipase and oral lipase were used in the production of milk flavor, but lower fatty acids are often produced,
It has a cheese-flavored appearance, especially butyric acid, etc., which causes unpleasant odor. Therefore, the use of microbial lipase can be avoided. For example, Oil Chemistry Vol. 31, No. 744.
Page (1982), Rhizopus del Remar
emar) was said to have been optimal for the above purpose,
It is described that an unpleasant odor is generated when it is operated for a long time. In addition, Rhizopus deremer has a wide substrate specificity and releases acetic acid and butyric acid rather than fats and oils as a result of a hydrolysis reaction, which is not preferable as a flavor enzyme from the viewpoint of flavor and aroma.

On the other hand, from the viewpoint of food hygiene, there is a strong demand for food processing under conditions where contamination by various bacteria does not occur. Therefore, it is desired to carry out the enzyme reaction at a high temperature where there is little risk of contamination by various bacteria, but most commercially available lipases are unstable to heat and are not practical. Therefore, a heat-resistant lipase is proposed in Japanese Patent Publication No. 53-45394 and Japanese Patent Publication No. 59-156282.

(Problems to be Solved by the Invention) Since these thermostable lipases are heat-stable, their merits are great for industrial applications such as fatty acid production, but they also have wide substrate specificity, and as described above, Since it produces a lot of lower fatty acids, it becomes cheese-flavor-like, and especially has the property of producing butyric acid, etc., so it was not suitable as a flavor enzyme.

(Means for Solving Problems) In view of these circumstances, the present inventors are suitable as a flavor enzyme, have a low possibility of contamination by various bacteria from the viewpoint of food hygiene, and have sufficient enzyme activity at high temperatures to exhibit thermostability. As a result of diligent research aimed at providing lipase, Thermomyces (Thermomyces sp.) Isolated from hot spring soil in Iguchi Town, Komatsu City, Ishikawa Prefecture
The present invention has been completed by finding that a lipase that achieves the above-mentioned object exists in a microorganism belonging to the genus (myces).

That is, the present invention is a thermostable lipase having the following physicochemical properties.

(A) Action Hydrolyzes fat to release fatty acid and glycerol.

(B) Substrate specificity It has a substrate specificity that does not act on triacetin or tributyrin.

(C) Optimum pH and stable pH range At 45 ° C, the reaction has an optimum pH around pH 7 and is stable at pH 7-8.

(D) The suitable working temperature range is 60 to 70 ° C (pH 8.0).

(E) Molecular weight: about 35,000 by SDS electrophoresis or about 25,000 by gel filtration using Sephadex G-100.

The lipase of the present invention is extremely thermostable and has the following physicochemical properties. The thermostable lipase referred to in the present invention means a lipase shown in FIG. 5 in which the activity is not substantially reduced in the thermostability test shown in (e) below.

(1) Action As described above.

(2) Substrate specificity As described above.

The results of Table 2 are shown in FIG. As seen from Table 2 and FIG. 1, the lipase of the present invention has a remarkable characteristic that it does not act on triacetin or tributyrin at all.

(3) Optimum pH and stable pH range As described above.

That is, it has an optimum pH around pH 7 (see Fig. 2).
It is stable at pH 7-8 (see Figure 3).

(4) Appropriate temperature range and temperature stability As described above.

That is, there is a suitable temperature for action at 60 to 70 ° C (see Fig. 4). Further, it was not inactivated at all at 60 ° C. for 1 hour (see FIG. 5), and showed 20% residual activity at 65 ° C. for 15 minutes (see FIG. 6).

(5) Activator and Inhibitor Some activation is observed with sodium chloride. Strongly inhibited by mercuric chloride and zinc chloride. It is also slightly inhibited by magnesium chloride, calcium chloride and ferrous sulfate.

(6) Molecular weight As described above.

(7) Method for measuring titer (a) Method for measuring lipase titer Olive oil emulsion (polyol 10%, w / v) 4 ml, 50 mM Tris-HCl buffer (pH 8.0) dispersed in water using polyvinyl alcohol as an emulsifier. ) Add 1 ml of appropriately diluted enzyme solution to the reaction mixture containing 5 ml, react at 45 ° C for 20 minutes, and then add 10 ml of ethanol-acetone mixture (1: 1, v / v) to terminate the reaction and release fatty acid. Was titrated with 0.05 N sodium hydroxide to determine the titer. The amount of enzyme that liberates 1 microequivalent of fatty acid per minute was defined as 1 unit.

(B) Working pH curve measurement conditions pH 4 to 8 are MacIlvain buffer, pH 8 to 11
The fatty acids produced by the reaction in 0.1 M ammonia buffer for 20 minutes at 45 ℃ were measured by the sodium hydroxide titration method.

(C) Measurement conditions for stable pH curve Using the same buffer solution as shown in (b) above, after treating in each buffer solution at 40 ° C for 1 hour, the remaining activity was measured according to the method of (a) above. did.

(D) Measuring conditions of suitable working temperature Among the measuring conditions shown in (a) above, the reaction temperature is 20 ° C to
The temperature was changed in steps of 10 ℃ up to 80 ℃, and the other measurements were performed in the same manner.

(E) Conditions for measuring thermostability curve The enzyme solution was kept in 50 mM Tris-HCl buffer (pH 8.0) at each temperature of 50 ° C and 60 ° C and sampled every 10 minutes to determine the remaining activity as described above ( It was measured by the method a). The result is shown in FIG.

Meanwhile, the enzyme solution was added to 50 mM Tris-HCl buffer (pH 8.0) for 30,
After keeping at 40, 50, 60, 70 and 80 ° C for 15 minutes, the remaining activity was measured by the method (a). The result is shown in FIG.

(8) Method for Purifying Enzyme The lipase of the present invention can be purified singly by the following method, but can also be purified by other methods.

First, bacterial cells were removed from the bacterial culture solution by filtration, and cold acetone was added to the resulting filtrate to a concentration of 60 v / v%,
The resulting precipitate is collected. The obtained precipitate is suspended in 20 mM Tris-HCl buffer (pH 8.0), adsorbed on a DEAE-cellulose column, and then eluted with a linear gradient of KCl. The active fractions (KCl20 to 100 mM) obtained are collected, concentrated by ultrafiltration, and subjected to gel filtration chromatography using a Sephadex G75 column to obtain a single lipase preparation.

In order to obtain the lipase of the present invention, for example, a thermophilic mold may be cultured and collected from the culture. The thermophilic molds preferably used in the present invention include molds of the genus Thermomyces. As a specific strain thereof, Thermomyces lanuginosus UKW-510 strain (Microtechnology Research Institute No. 8319) can be mentioned. This strain is a new strain isolated from the hot spring soil of Iguchi Town, Komatsu City, Ishikawa Prefecture by the agar plate method using olive oil as a substrate. The mycological properties of this strain are shown below.

(1) Growth condition on each medium It grows well on wort agar medium and the agar stains reddish brown.

 It grows well on Sabouraud agar.

It grows well on oatmeal agar, and the fungus turns grayish white to dark green. Spore formation is vigorous, and the agar medium is black gray.

It grows well on YpSs agar. The aerial hyphae have septa and are 1.5 to 4 μm wide. The conidia spores are not branched, emerge at right angles from the basal hyphae, and are 10 to 16 μm long. One conidia exists in each conidia stalk, and the conidia formation method is the Aleuro type. Conidia are spherical and have wart-like projections on the surface.

It grows well on potato glucose agar and changes from white to dark green. The Aleur conidia spores emerge at right angles from the basal hyphae. Aleur conidia have a diameter of 5.5-10 μm
It has a spherical shape and has wart-like protrusions.

(2) Physiological characteristics Optimal growth conditions pH 6.5 to 8.5, temperature around 45 ° C, aerobic growth range pH 4.0 to 10.0, temperature 30 to 60 ° C, aerobic From the above results, this strain is Thermomyces langinosas ( Thermomyces lanuginosus), but because it is considered to be a variant having the lipase-producing ability of the present invention, which has the characteristic that it does not act on the lower fatty acid chains of acetic acid and butyric acid in fat, it was named Thermomyces langinosus UKM-510, It was deposited on June 19, 1985 at the Institute for Microbial Engineering, Ministry of International Trade and Industry, Ministry of International Trade and Industry (Microtechnology Research Institute No. 8319).

Regarding the culture of the strain used in the present invention, the culture temperature is set to 45 ° C. and the addition of fat to the medium for inducing lipase production is the same as that for culturing ordinary fungi. The medium and culture method may be used. An example of such a suitable medium is olive oil 0.5 wt%, Tween
80 0.5wt%, Peptone 0.4wt%, Yeast extract 0.20wt%, KH
₂ PO ₄ 0.10wt%, Na ₂ HPO ₄・ 12aq 0.10wt%, MgSO ₄・ 7H ₂ O
0.05wt% can be raised. As trace elements, Mn, F
You may add e, Ca, Cu, Zn, Mo, Co. The pH of the culture is
4.0-8.5, temperature is suitable 40-55 ℃, aerobically about 20-30
After culturing for a long time and reaching the stationary phase of bacterial cell growth, lipase is substantially accumulated in the culture solution, so if the bacterial cells are removed by a conventional method such as filtration and centrifugation, the culture is stopped. It is possible to obtain the supernatant of the culture broth containing it.

To recover the lipase from the enzyme solution thus obtained, for example, the enzyme powder may be concentrated with an ultrafiltration membrane and then dried to obtain a crude enzyme powder. However, salting out, organic solvent precipitation, or resin Various purification methods that are already widely used in industry, such as adsorption and elution, can be used.

(Example) Next, the present invention will be specifically described with reference to Examples.

Example 1 Olive oil 0.5 wt%, peptone 0.4 wt%, yeast extract 0.2 w
t%, Tween 80 0.5wt%, KH ₂ PO ₄ 0.1wt%, Na ₂ HPO ₄・ 12H ₂ O
_{0.1wt%, MgSO 4 · 7H 2} O 0.05wt medium containing% a (pH 4.5) 5
Add 100 ml to a 00 ml Erlenmeyer flask and pressurize at 121 ° C under 1 atmosphere pressure 10
Sterilized in an autoclave for minutes. Thermomyces Langinosas UKW grown on agar plates of the same composition on this medium
-510 strain (Microtech Lab. No. 8319) was inoculated with 1 platinum loop, 45 ℃
Rotational shaking culture (Takasaki Seisakusho RGR-No. 2 type) was performed. When the lipase activity of the culture after 24 hours of culture was measured, it was 15 units / ml.

The thus obtained lipase had the physicochemical properties described above.

Example 2 The culture 2 containing the bacterial cells obtained in Example 1 was inoculated into one 30-volume jar fermenter (MSJ-30U manufactured by Maruhishi Rika) containing the same medium 20 as in Example 1, and the temperature was increased. 45 ° C, ventilation rate 1
After culturing for 30 hours at vvm and stirring speed of 200-400 rpm, 18.3 units / ml of culture filtrate 10.7 was obtained.

Ammonium sulfate was added to the filtrate so that the final concentration was 30%, the precipitate formed was removed by centrifugation, and ammonium sulfate was further added.
The supernatant was removed by centrifugation at 70% saturation.
50 mM of the obtained precipitate and pasty suspension were added.
Dissolved in Tris-HCl buffer (pH8.0) and cold acetone 1.2
The resulting precipitate was collected and collected in 20 mM Tris-HCl buffer (pH
After being dissolved in 8.0), it was dialyzed against the same buffer solution to remove acetone.

The obtained crude enzyme solution was used as DEAE-cellulose (Whatman DE
-52) column (150 ml, adjusted to 20 mM Tris-HCl buffer, pH 8.0) and eluted with a linear KCl concentration gradient.
The lipase activity was eluted in the fraction with KCl concentration of 20-100 mM. This active fraction was collected, concentrated by ultrafiltration, and subjected to gel filtration chromatography using Sephadex G-75 to collect the active fraction.

The obtained lipase preparation was electrophoretically single, with an activity of 1750 units / mg and a yield of 11.2%. Further, this lipase had the above-mentioned physicochemical properties.

Reference Example 1 Milk fat was hydrolyzed using the purified lipase preparation obtained in Example 1.

That is, 1 ml of purified lipase preparation (350 units) was added to a reaction solution consisting of 3 g of milk fat and 1 ml of distilled water, and the mixture was heated at 30 ° C for 3 hours at 500 rpm.
It was confirmed that a good quality butter flavor was generated when stirred at.

Further, after the reaction time was extended and the reaction was continued for 8 hours, no offensive odor was observed, and a good butter flavor was maintained.

(Effect of the invention) Since the thermostable lipase of the present invention does not act on triacetin or tributyrin, acetic acid or butyric acid is not liberated even when oil or fat is hydrolyzed. Therefore, it can be used as a flavor enzyme for food, and because it is heat-resistant, it is possible to produce a good milk flavor in a high temperature range where there is little possibility of contamination by various bacteria.

[Brief description of drawings]

FIG. 1 shows the substrate specificity of the thermostable lipase of the present invention.
The figure shows the optimum pH, Fig. 3 shows the pH stability, Fig. 4 shows the optimum action temperature, and Figs. 5 and 6 show the heat resistance.

Claims (1)

[Claims] 1. A thermostable lipase having the following physicochemical properties. (A) Action Hydrolyzes fat to release fatty acid and glycerol. (B) Substrate specificity It has a substrate specificity that does not act on triacetin or tributyrin. (C) Optimum pH and stable pH range At 45 ° C, the reaction has an optimum pH around pH 7 and is stable at pH 7-8. (D) The suitable working temperature range is 60 to 70 ° C (pH 8.0). (E) Molecular weight: about 35,000 by SDS electrophoresis or about 25,000 by gel filtration using Sephadex G-100.