JP2816470B2 - New lipase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention acts on monoglyceride (hereinafter, referred to as MG), diglyceride (hereinafter, referred to as DG), and completely acts on triglyceride (hereinafter, referred to as TG). No new lipase (hereinafter referred to as partial glyceride lipase)
It is used in the production of MG and / or DG (hereinafter referred to as "partial glyceride"), particularly in the field of fats and oils such as production of MG and purification of fats and oils, or as an enzyme for analysis, and is particularly important in industry.

[Conventional technology]

Enzymes related to partial glyceride lipase include:
Monoglyceride lipase and diglyceride lipase derived from animal organs such as rat small intestine and pig adipose tissue are well known. In addition, among microorganisms, those derived from strains belonging to the genus Staphylococcus and those derived from strains belonging to the genus Bacillus are known. However, the enzyme derived from animal tissues acts not only on MG but also on DG and TG (JP-A-63-245672). The former derived from microorganisms also acts on TG as a substrate specificity (JP-A-63-245672). 55-42532) and the latter act only on MG and do not act on TG and DG (JP-A-63-245672).

On the other hand, Okumura et al.
One strain of Cillium cyclopium has been reported to produce enzymes that act well on MG and DG [Journal of Biochem., 87, pp. 205-211 (1980)]. . However, it is stated that this enzyme acts not only on MG, DG, but also on TG.

[Problems to be solved by the invention]

As described above, the enzyme related to the conventional partial glyceride lipase acts not only on the partial glyceride but also on the TG.For example, when used in the above-described method for producing a partial glyceride, TG is generated as a by-product. Further, in the method for purifying fats and oils, not only partial glycerides but also TG, which is the main body of fats and oils, is decomposed, resulting in a poor purification yield. Also, when used as an enzyme for analysis,
A side reaction called degradation of TG occurs, and there is a problem in using the conventional partial glyceride lipase for the above purpose.

[Means for solving the problem and its operation]

Means for Solving the Problems In view of the above-mentioned problems, the inventors of the present invention have conducted intensive studies and have extensively searched for strains producing lipase. As a result, they have found that one of them produces partial glyceride lipase. Further, the present enzyme was isolated and purified, and various properties were examined. As a result, it was found that the enzyme acts only on MG and DG and does not act on TG at all, and thus completed the present invention.

The mycological properties of the microorganism used in the present invention are shown below.

(A) Form Conidia 100-700μ × 3.5-4.5μ, rough surface.

Peniciri asymmetrical branch 13-30μ × about 4μ Metre 10-15μ × about 4μ, 3-5 colonies.

Fialide 10 ~ 13μ × about 3μ, 3 ~ 8 plants rotate.

Conidium Fiaro-type conidia, spherical to subspherical, 3.0-4.0μ, smooth to slightly rough, molecular chains tangled.

(B) Growth condition Tuapec agar medium Growth is rather rapid and conidium formation is small. The conidium forming part is pale blue-green to light greenish-grey, and the back of the colony is yellowish-white to yellow-brown.

Malt extract agar medium Growth is fairly rapid and forms conidia well. The conidium formation part is pale bluish green to bright greenish grey, and the back of the colony is yellowish white.

(C) Physiological properties Optimal growth conditions Grow well at pH 3-7. It grows well at temperatures of 20-30 ° C.

Growth range Grow at pH 2-9. Does not grow at 37 ° C.

Based on the above properties, mainly Barron's The Genera of Hyphom
ycetes from Soil (Williams & Wilkins, Baltimore (1
968)), a conidium is formed, does not form a conidium / conidia, conidiophores develop well, forms many phialides, and forms a penicillium. It is classified into the genus Penicillium because it is linked almost spherically. The present inventors named this strain Penicillium sp. U-150. In addition, this strain is a micro-organism germ strain No. 10452 (FERM P-104
52) deposited at the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.

Culture of a microorganism that produces the partial glyceride lipase of the present invention can be performed by a known method, and may be selected so that the target partial glyceride lipase is produced in a significant amount. Specifically, any of a solid medium and a liquid medium may be used, and the medium components may be any of those used in general media such as a carbon source and a nitrogen source. Culture is aerobically 4-33
C., preferably at 20-30.degree. C. for 1-4 days.

When cultivated aerobically in a solid medium, water is added to the culture to extract partial glyceride lipase, and when cultivated in a liquid medium in the same manner, the culture is filtered and cultured containing partial glyceride lipase. A filtrate can be obtained. Next, a precipitate obtained by adding a large amount of ethanol to the partial glyceride lipase-containing liquid thus obtained is separated by filtration and the ethanol is volatilized, whereby a crude partial glyceride lipase enzyme powder can be obtained.

Further, the crude enzyme powder can be purified by appropriately combining known methods, for example, various types of chromatography such as ion exchange and gel filtration.

The properties of the partial glyceride lipase obtained by culturing and purifying Penicillium sp. U-150 as described above are described in detail below.

The activity of the present enzyme was measured by a shaking method as described below unless otherwise specified.

1.0 g of vinyl laurate 50 mM acetate buffer (pH 5.6) 5.0 ml 1.0 ml of enzyme solution and 30 glass beads (φ5 mm) in an Erlenmeyer flask at 30 ° C, shaking counter-width (140 reciprocations per minute, 3 cm amplitude) I do. After one hour, 20 ml of an acetone: ethanol mixture (1:
The reaction is stopped by adding 1), and the released lauric acid is titrated with a 0.1N KOH solution using phenolphthalein as an indicator.

Under these conditions, the amount of the enzyme that liberates 1 microequivalent of fatty acid per minute was defined as 1 unit.

(1) Enzyme chemical properties a. Substrate specificity Table 1 shows the substrate specificities for various glycerides.

In addition, 1-RAS-monolaurin (Funakoshi), 1-SN-monopalmitin (Funakoshi), 3-SN-monopalmitin (Funakoshi), 2-MG
Monopalmitin (Funakoshi), 1-RAS-monoolein (Sigma), 2-monoolein (Sigma)
DG was used as 1,2-RAS-dilaurin (Funakoshi), 1,3-dilaurin (Funakoshi), 1,3-RAS
-Dipalmitin (Funakoshi), 1,2-SN-dipalmitin (Funakoshi), 1,3-dipalmitin (Funakoshi), 1,2-RAS-diolein (Sigma), 1, 2-S
N-Diolein (Sigma) and 1,3-Diolein (Sigma) are used. TG is trilaurin (Funakoshi), tripalmitin (Funakoshi), and triolein (Sigma). did. The reaction time is MG and
30 minutes for DG and 90 minutes for TG. still,
The measuring method is as follows.

(A) Composition of reaction solution Substrate 11.3 mM 0.2 M acetate buffer (pH 5.6) 0.5 ml Dimethylformamide 0.1 ml Enzyme solution (0.18 units) 0.1 ml Purified water 0.3 ml (b) Operation Centrifuge test tube And allowed to stand at 37 ° C. for each reaction time. After the reaction, 2 ml of a chloroform: methanol mixture (2: 1) is added, and after centrifugation, glycerol in the aqueous layer is quantified by a color development method using glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase.

Thus, this enzyme acts only on MG and DG, and has no effect on TG. As for the regioisomer of DG, 1,3-diolein and 1,2-RAS-diolein are decomposed to the same extent. M
As for the positional isomer of G, 1-RAS-monoolein is more easily decomposed, but also decomposed to 2-monoolein.

In addition, the present enzyme has no stereospecificity for glyceride. That is, 1,2-SN-diolein, 2,3-SN-diolein, and 1-SN-monopalmitin and 3-SN-monopalmitin are decomposed to the same extent.

Furthermore, in order to clarify the difference from known enzymes, a method similar to that of Okumura et al. [Journal of Biochemistry (J. Biochem.), Vol. 87, p. 206 (1980)]
Table 2 shows the results obtained by examining the substrate specificity.

The method of measuring the enzyme activity by Okumura et al. Is as follows.

Substrate 0.5 g, 50 mM acetate buffer (pH 5.6) 5.0 ml, enzyme solution 1.0
The reaction solution containing ml is stirred at 500C (500 rotations per minute). After one hour, 20 ml of an acetone: ethanol mixture (1:
1) is added to stop the reaction, and the liberated fatty acid is titrated with a 0.05N KOH solution using phenolphthalein as an indicator.

In Table 2, the amount of the enzyme used was measured and set by the method of Okumura et al. Specifically, in the above method, 0.5 g of substrate
Was replaced by 1.0 g of methyl laurate.

Data from Okumura et al. (Table, page 208)
3 and Table IV on page 209, it is clear that the partial glyceride lipase of the present invention does not act on TG at all even if the enzyme activity used is 5 times that of the enzyme of Okumura et al.

b. Optimal pH As a result of measurement by the shaking method, the optimal pH is 5, and the results are shown in FIG.

c.Optimum temperature As a result of measurement by the shaking method, the optimal temperature is 30 ° C,
The result is shown in FIG.

d.pH stability After treating the enzyme in a buffer at each pH at 35 ° C. for 30 minutes, its residual activity was measured by a shaking method.
It is stable in the range of 7.07.0, and the results are shown in FIG.

e. Thermostability This enzyme was dissolved in 50 mM acetate buffer (pH 5.6), treated at each temperature for 15 minutes, and the residual activity was measured by the shaking method. As a result, it was stable up to 40 ° C. As shown in FIG.

(2) Physicochemical properties a. Molecular weight: determined by SDS electrophoresis using 12.5% polyacrylamide gel. This resulted in a molecular weight of about 40,000. At this time, ovaltransferrin (molecular weight 76000-78000), albumin (molecular weight 66250), ovalbumin (ovalbumin: molecular weight 450) are used as molecular weight markers.
00), carbonic anhydrase (molecular weight 30,000) and myoglobin (molecular weight 17,200) were used. The results are shown in FIG.

In addition, it was determined by a gel filtration method using a TSK G2000SW column (manufactured by Tosoh Corporation). This gave a molecular weight of about 38000. At this time, adenylate kinase (molecular weight 32000), enolase (molecular weight 6)
7000), lactate dehydrogenase (molecular weight 140,000)
And glutamate dehydrogenase (molecular weight 290,000) were used. The results are shown in FIG.

b. Isoelectric point After energizing for 45 hours at 4 ° C and a constant voltage of 400 V by isoelectric point fractionation using a carrier ampholine, the enzyme activity of each fraction was measured to obtain a value of pI 4.4 ± 0.4. Was.

 Next, the present invention will be described with reference to examples.

Example 1 Culture of cells and preparation of filtrate Penicillium sp. U-150 was inoculated into a medium (pH 6.0) consisting of rice bran 2% and corn steep liquor 1.5%, and cultured with aeration and agitation at 26 ° C for 2 days.

After filtering the cells from the obtained culture solution (20): The filtrate was concentrated by ultrafiltration.

Purification of enzyme Ammonium sulfate was added to the above concentrated liquid (250 ml) so as to be 0.75 saturated, and the resulting precipitate was collected by centrifugation. The obtained precipitate was dissolved in 10 mM phosphate buffer (pH 6.0) (hereinafter referred to as PB), and sufficiently dialyzed against 10 mM PB.

The dialysate was applied to a DEAE-Sepharose CL-6B column equilibrated with 10 mM PB, and the adsorbed protein was eluted with a linear concentration gradient of 0 to 50 mM NaCl. Next, the fractions of the partial glyceride lipase were collected, desalted and concentrated using an ultrafiltration membrane.

Next, the concentrate was loaded on a column containing Sephadex G-75 gel equilibrated with 10 mM PB, and the protein was eluted with the same buffer.

The eluted partial glyceride lipase fraction was collected, and 10
Dialysis was performed sufficiently against mM PB.

This dialysate was loaded onto a column containing a hydroxyapatite gel equilibrated with 10 mM PB, and 10-500 mM PB was added.
The adsorbed protein was eluted with a linear concentration gradient of Next, the partial glyceride lipase fractions were collected, desalted and concentrated using an ultrafiltration membrane.

Next, the above concentrated solution was subjected to isoelectric focusing chromatography using a carrier ampholine having a pH of 3 to 10 to obtain a pH 4.3.
A partial glyceride lipase fraction concentrated to a range of 4.54.5 was obtained and sufficiently dialyzed against 10 mM PB to obtain a purified partial glyceride lipase solution (containing 6250 units / 0D ₂₈₀ , 7 mg). The fractionation pattern at this time is shown in FIG.

〔The invention's effect〕

As described above, the enzyme of the present invention acts on MG and DG but does not act on TG at all, and is an entirely new enzyme that has never existed before.

[Brief description of the drawings]

1 to 4 show the optimal pH, optimal temperature, pH stability and temperature stability of the partial triglyceride lipase of the present invention, respectively. 5 and 6 are diagrams showing the molecular weight of the present enzyme measured by SDS-electrophoresis and gel filtration, respectively. FIG. 7 shows the chromatogram obtained by the purification operation of the present enzyme and the isoelectric point of the present enzyme.

Continuation of front page (58) Field surveyed (Int.Cl. ⁶ , DB name) C12N 9/18

Claims (1)

(57) [Claims] 1. A novel lipase which acts on monoglycerides and diglycerides, does not act on triglycerides, and has the following physicochemical properties. (A) Molecular weight: 40000 ± 4000 (b) Optimum pH: around pH 5 (c) Optimum temperature: around 30 ° C (d) pH stability: pH 5.0 to 7.0