JP5648783B2 - Novel lipase and method for synthesizing functional lipids using the same - Google Patents

Description

  The present invention relates to a novel lipase and a method for synthesizing functional lipids using the same.

  Metazoans including humans have molecular species of fatty acids that are essential for their physiological metabolic processes but cannot be synthesized by themselves. For humans, polyunsaturated fatty acids are essential fatty acids and supplemented by ingesting other organisms that synthesize polyunsaturated fatty acids that cannot be synthesized by themselves as food.

  Polyunsaturated fatty acids are abundant in animal fats and oils such as fish oil, and fats and oils in which polyunsaturated fatty acids are bonded to the 2-position of glycerol give biological activity to living organisms. It is desired to industrially synthesize oils and fats with highly unsaturated fatty acids bound to the 2-position, and to synthesize fats and oils with highly unsaturated fatty acids bound to the 2-position of glycerol using an enzyme such as lipase. Has been tried.

  Lipases are known as enzymes that act on the degradation and synthesis of fats and oils, and lipases derived from microorganisms are widely used in the production and processing fields of various fats and oils. However, the lipase used so far has been one that specifically acts on the 1st and 3rd positions among the three fatty acids of triglycerides, or one that acts nonspecifically. For this reason, it has been difficult to exchange and modify the 2-position where many of the highly unsaturated fatty acids having particularly high bioactive functions are located.

International Publication No. 98/39468

  The present invention has been made in view of the above-mentioned matters, and the object of the present invention is to preferentially act on the 2-position of glycerol and to function by binding a highly unsaturated fatty acid to the 2-position. It is to provide a lipase that contributes to the synthesis of sex lipids.

The lipase according to the present invention is
Produced by Mortierella SAM2197 strain (FERM BP-6261), preferentially have a catalytic effect on the 2-position of glycerol, a molecular weight of 11 kDa.

  Moreover, it is desirable that optimal pH is 8-10.

  Further, it is desirable that the optimum temperature is 45 to 55 ° C.

  The Mortierella sp. SAM2197 strain is preferably cultured and then collected after 120 to 168 hours and purified.

The method for synthesizing a functional lipid according to the present invention includes:
It includes a step of synthesizing 2-monoglyceride by interposing the lipase described above in glycerol and a highly unsaturated fatty acid.

  The polyunsaturated fatty acid may be selected from arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and dihomo-γ-linolenic acid (Dihomomo-gamma-linolenic acid). Is preferably used.

  The lipase according to the present invention has a property of acting preferentially at the 2-position of glycerol by intervening between glycerol and fatty acid, and can synthesize functional lipids having high physiological functions.

It is a graph which shows the follow-up result of lipase activity. It is a graph which shows the gel filtration result by Superdex200. It is a MALDI-TOF-MS analysis figure. It is a graph which shows the relationship between lipase activity and pH. It is a graph which shows the relationship between lipase activity and temperature. It is a graph which shows the substrate specificity of lipase. It is a graph which shows the synthesis reaction course of acylglycerol.

  The lipase according to the present embodiment is a lipase produced by the SAM2197 strain (FERM BP-6261) belonging to the genus Mortierella (Mortierella). This lipase preferentially exerts a catalytic action on the 2-position of triglycerol (1,2,3-propanetriol).

Moreover, this lipase has the following characteristics.
Molecular weight: 11 kDa
Optimum pH: 8-10
Optimal temperature: 45-55 ° C

  The lipase according to the present embodiment is produced by culturing the SAM2197 strain. The SAM2197 strain can be cultured by the following culture conditions and culture methods as an example.

  The spore, mycelium of SAM2197 strain, or a culture solution obtained by culturing in advance can be inoculated into a liquid medium or a solid medium and cultured. In the case of a liquid medium, any carbon source commonly used such as glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol, citric acid, corn starch, etc. can be used. Glucose, fructose, maltose, glycerol, citric acid and corn starch are preferred. Nitrogen sources include natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid, corn steep liquor, and soy protein, organic nitrogen sources such as urea, and inorganics such as sodium nitrate, ammonium nitrate, and ammonium sulfate. Commonly used materials such as a nitrogen source can be used. Other inorganic salts such as potassium phosphate, potassium dihydrogen phosphate, etc., ammonium sulfate, sodium sulfate, magnesium sulfate, iron sulfate, copper sulfate, magnesium chloride, potassium chloride, etc. In addition, all commonly used vitamins can be used.

  The lipase according to the present embodiment is preferably purified by culturing the SAM2197 strain in a medium and collecting it after 120 to 168 hours. The culture time is preferably 132 to 156 hours, more preferably 144 hours.

  The lipase can be purified as follows as an example. After culture, the culture is filtered through a filter to obtain bacterial cells and culture supernatant. The bacterial cells are washed lightly with hexane to remove oils and fats on the surface, suspended in 20 mM Tris-HCl, pH 9.0 (hereinafter referred to as TB), homogenized with glass beads on ice and crushed. Insoluble components were removed by centrifugation (10,000 rpm, 15 min), the supernatant was transferred to a new test tube, cold acetone was added to 50%, cooled for 4 hours, and centrifuged (10,000 rpm, 10 minutes), the active ingredient is recovered by precipitation. The culture supernatant is treated in the same manner, and the active ingredient is recovered in the precipitate. These are dissolved in TB and dialyzed against the same buffer. Insoluble material is removed by centrifugation and then applied to a DEAE-Sepharose column (5 ml, GE Healthcare) equilibrated with TB. Gradient elution is performed using TB containing 0-2M NaCl, and the active fraction is collected. This is applied to a phenyl-Sepharose Fast Flow column (1 ml, GE Healthcare) equilibrated with TB containing 1M ammonium sulfate, and the column is washed thoroughly, and then gradient elution is performed with TB containing 0-1M ammonium sulfate. If the active ingredient is still adsorbed, it is eluted with TB containing 1-1.5% CHAPS (3-[(3-cholamidopropyl) dimethylaminosulfonate) sulfonate). The collected active fraction is dialyzed against TB, centrifuged, applied to a Superdex 200 column (HR 10/30, GE Healthcare), and eluted with TB containing 0.15 M NaCl. In this way, the lipase can be purified.

(Method for synthesizing functional lipids)
The method for synthesizing a functional lipid according to the present embodiment includes a step of synthesizing 2-monoglyceride by interposing the above-described lipase between glycerol and a fatty acid.

  As a functional lipid, for example, a 2-unsaturated fatty acid such as arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and the like is bonded to the 2-position of glycerol. A monoglyceride is mentioned.

  By interposing lipase between glycerol and the above highly unsaturated fatty acid, the lipase preferentially generates an ester bond between the hydroxyl group located at the 2-position of glycerol and the carboxyl group of the highly unsaturated fatty acid. Thereby, the 2-monoglyceride mentioned above is compoundable.

  A functional lipid in which a highly unsaturated fatty acid is bonded to the 2-position of glycerol can impart physiological activity to an organism.

  In addition, when a fatty acid is bonded to the 1st and 3rd positions in addition to the 2nd position of glycerol, the fatty acid bonded to the 1st and 3rd positions is hydrolyzed with an enzyme such as 1,3-lipase specifically, Monoglycerides may be obtained.

  In addition, when a highly unsaturated fatty acid is bonded to the 2-position of glycerol at a low temperature using lipase, the resulting monoglyceride is precipitated and discharged out of the lipase reaction system, so that 2-monoglyceride can be obtained.

  Further, in the same reaction, when the water content is limited to a certain range under reduced pressure, the activity of synthesizing diglyceride from monoglyceride is reduced, so that 2-monoglyceride can be selectively obtained (Watanabe Y. et al.,). Journal of American Oil Chemistry Society, 82, 619-623, 2005).

  Hereinafter, based on an Example, the lipase which concerns on this Embodiment, and the synthesis method of a functional lipid using the same are demonstrated in detail.

  In the following examples, monocaprylin, monopamitin, monoolein, monolinolein, monolinolein, manufactured by Sigma-Aldrich Corporation was used. As for dicaprylin, diolein and dilinolein, those manufactured by Funakoshi Co., Ltd. were used. Other triacylglycerols and fatty acid esters were manufactured by Wako Pure Chemical Industries, Ltd.

First, SAM2197 strain was added to 50 mL of basic medium (0.5% glucose, 1% soybean powder, 0.3% KH ₂ PO ₄ , 0.1% Na ₂ SO ₄ , 0.05% CaCl ₂ .2H ₂ O). , 0.05% MgCl ₂ · 6H ₂ O, 0.01% antifoam, pH 7.0), and cultured at 28 ° C. and 160 rpm.

  Linseed oil was added to 1% 72 hours after the start of the culture, and then the lipase activity of the culture supernatant, the soluble fraction in the cell and the membrane fraction was followed.

  The lipase activity was measured by the copper acetate method in an oil-water emulsion system as follows. First, a 50 mM phosphate buffer solution (pH 7.4) containing 5% olive oil was sonicated for 10 minutes. 1 mL of this emulsion was added to an appropriate volume (about 4 mL) of culture supernatant, cell extract or purified enzyme solution (20 mM Tris-HCl, pH 9.0) and incubated at 37 ° C. for 1 hour with shaking. . The reaction was stopped by adding 1 mL hydrochloric acid and 3.5 mL isooctane and boiling for 5 minutes.

  The upper isooctane phase (3.5 mL) containing free fatty acid was transferred to a new test tube, and 1 mL of 5% (w / v) aqueous copper acetate solution (adjusted to pH 6.1 with pyridine) was added and mixed well. After centrifuging for 1 minute, the upper layer was transferred to a new test tube, and the solvent was removed by evaporation.

  The fatty acid was dissolved in 100 μL of isooctane, the absorbance at 715 nm was measured, and the case where 1 μmole of fatty acid was produced in 1 hour was defined as 1 U in comparison with the oleic acid standard substance.

  FIG. 1 shows the results of tracking lipase activity. As shown in FIG. 1, after 96 hours, all the activities of the culture supernatant, the intracellular soluble fraction and the intracellular membrane fraction were lost, but after 144 hours, the supernatant was 753 U / ml and the bacterial cell soluble. A maximum activity of 276 U / ml was obtained in the fraction. Only a small amount of activity was detected in the cell membrane fraction.

  Focusing on the intracellular lipase, the bacterial cells were collected at the culture time showing the maximum activity, and the lipase was purified.

  Purified lipase was obtained as follows. The culture on the 6th day of culture (144 hours from the start of culture) was filtered through a filter (Toyo Filter Paper No. 2) to obtain cells and culture supernatant.

  The cells were lightly washed with hexane to remove oils and fats on the surface, suspended in 20 mM Tris-HCl, pH 9.0 (hereinafter referred to as TB), homogenized with glass beads on ice and crushed. Insoluble components were removed by centrifugation (10,000 rpm, 15 min).

  The supernatant was transferred to a new test tube, cold acetone was added to a concentration of 50%, the mixture was cooled for 4 hours, and the active ingredient was collected by centrifugation (10,000 rpm, 10 min).

  The culture supernatant was treated in the same manner, and the active ingredient was recovered in the precipitate.

  These were dissolved in TB and dialyzed against the same buffer. Insoluble material was removed by centrifugation and then applied to a DEAE-Sepharose column (5 mL, GE Healthcare) equilibrated with TB. Gradient elution was performed using TB containing 0-2M NaCl, and the active fractions were collected.

  This active fraction was applied to a phenyl-Sepharose Fast Flow column (1 mL, GE Healthcare) equilibrated with TB containing 1M ammonium sulfate, and the column was thoroughly washed, and then eluted with TB containing 0-1M ammonium sulfate. At this time, since the active ingredient was still adsorbed, it was eluted with TB containing 1-1.5% CHAPS (3-[(3-cholamidopropylo) dimethylaminosulfonate).

  The collected active fractions were dialyzed against TB and centrifuged. Then, it was applied to a Superdex 200 column (HR 10/30, GE Healthcare) and eluted with TB containing 0.15 M NaCl.

The purification steps are shown in Table 1. At each stage of purification, no lipase activity was observed other than the recovered active fraction.

  The purified lipase obtained as described above was subjected to gel filtration. The result is shown in FIG. In FIG. 2, Vo is the void volume, Tv is the total column volume, and each numerical value is the molecular weight (kDa) of the marker protein. When FIG. 2 is seen, the active peak which corresponds with the absorption peak of UV absorption (280 nm) shown with a broken line is recognized by a void fraction (> 1,300 kDa).

Moreover, MALDI-TOF-MS (Ultraflex TOF / TOF; Bruker Daltonics) analysis was performed. MALDI-TOF-MS analysis was performed using 1 ng of purified lipase under the following conditions.
Linear mode / positive,
Acceleration voltage = 25 kV,
MALDI matrix = 2,5-dihydroxybenzoic acid,
Calibration = external

  The result is shown in FIG. When FIG. 3 is seen, the mass signal of 10,965 Da is recognized with a minor peak.

  From the above gel filtration and MALDI-TOF-MS analysis results, it can be seen that the molecular weight of the purified lipase is about 11 kDa.

  Subsequently, the effect of pH on the activity and stability of purified lipase was verified.

  The lipase activity of purified lipase (34 μg / mL) at 37 ° C. was determined using various 50 mM buffers (glycine-HCl, pH 2; acetate buffer, pH 3-6; phosphate buffer, pH 6-7; Tris-HCl, olive oil as a substrate). pH 8-9; glycine-NaOH, pH 9-12).

  In order to verify the stability of the purified lipase with respect to pH, the purified lipase was incubated at 4 ° C. for 24 hours in the respective buffers, and then the lipase activity was measured.

  The result is shown in FIG. As shown in FIG. 4, the purified lipase maintains the enzyme activity in the range of pH 2-12. Further, the optimum pH was 8 to 10, and the best value was especially at pH 9. The property of maintaining activity in such a wide pH range is P.I. caseicolum, P.M. crustosum, P.M. Unusual except for a few examples such as roqueforti and Bysochochlamys verrucosa, most lipases have an optimum pH on the acidic side. Thus, purified lipase can be used under a wide range of pH conditions. In addition, the enzyme activity (stability) after 24 hours of incubation also shows a tendency similar to that described above, indicating that the activity is maintained over a wide pH range.

  Subsequently, the effect of temperature on the activity and stability of purified lipase was verified.

  Lipase activity in purified lipase (34 μg / mL) in 50 mM phosphate buffer (pH 7.4) was measured at various temperatures using olive oil as a substrate.

  Further, in order to verify the stability of purified lipase with respect to temperature, the purified lipase was incubated at each temperature for 1 hour, and then lipase activity was measured.

  FIG. 5 shows the result. Referring to FIG. 5, the purified lipase maintained activity under the reaction conditions of 20-80 ° C. The optimum temperature is 45 to 55 ° C, and the best activity is shown at 50 ° C. In addition, when it kept at 70 degreeC or more for 1 hour or more, it deactivated. Most conventional lipases have an optimum temperature of 40 ° C or lower, and no lipase activity at 50 ° C or 60 ° C or higher. Thus, purified lipase is characteristic and can be used under a wide range of temperature conditions. Can be used.

  In order to examine the substrate specificity of purified lipase, monoacylglycerol (hereinafter also referred to as MAG), diacylglycerol (hereinafter also referred to as DAG), triacylglycerol (hereinafter referred to as DAG) having fatty acids having different acyl chain lengths and unsaturated bond numbers. And also the hydrolysis activity of fatty acid esters.

  Hydrolysis activity for 1 mM triacylglycerol, monoacylglycerol, natural vegetable oil, phospholipid, and 2 mM fatty acid methyl ester was performed by quantifying free fatty acids released by hydrolysis. In addition, about each substrate, the activity on 30 degreeC and 50 degreeC conditions was measured.

  The result is shown in FIG. Although activity against various MAGs and TAGs was observed at 30 ° C. and / or 50 ° C., 1,3-dicaprylin, 1,3-dipalmitin, 1,2-diolein not shown for DAG No activity was detected at any temperature in 1,3-diolein and 1,2-dilineolein.

  The reason why there was no activity at 30 ° C. with respect to TAG having saturated fatty acid is considered to be because it was not dissolved at the same temperature because the melting point of the substrate was low. The release activity of unsaturated fatty acids was higher in MAG than in TAG and was dependent on the degree of unsaturation of the acyl chain.

  Among them, 1-monoolinolenin showed the highest value. 2-monoolein and 2-mononolinein gave higher activities than 1-monoolein and 1-mononolinein, respectively.

  On the other hand, fatty acid methyl esters such as methylbutyrate, methyllaureate, methylpalmateate, methylpalylateate, methylesterate, methyllinoleate, methyllinolenate, and thiylamineate, which are not shown in the figure, are in the form of 50 degrees centigrade. Only C was detected.

  Among natural vegetable oils, camelia oil gave the highest activity. This is considered to be because it contains a lot of unsaturated fatty acids (˜86% oleic acid).

  There was little difference between olive oil and linseed oil. Interestingly, it showed high activity against egg yolk phosphatidylcholine. This is extremely rare in fungal lipases, and lipases from other sources do not act on both acylglycerols and phospholipids.

(Acylglycerol hydrolysis)
Lipid compositions of hydrolysates by lipase action from various acylglycerols having only oleic acid were examined.

  A reaction solution consisting of 100 mg of various substrates, 1 mL of purified lipase (10 U), and 1 mL of 50 mM phosphate buffer (pH 7.4) was incubated at 30 ° C. for 1 hour with stirring at 170 rpm. The product was separated and quantified as follows.

  Lipid composition was identified and quantified by thin layer chromatography using silica gel plates (Kieselgel 60; Merck). For better separation of MAG isomers, plates were soaked in 3% oxalic acid and dried overnight. As developing solvents, hexane / ether / acetic acid (70: 30: 1, v / v) or chloroform / acetone / acetic acid (96: 4: 1, v / v) was used, respectively. Lipids were visualized by spraying with 2% copper sulfate / 13% sulfuric acid solution or 50% methanol sulfate solution and heating at 140 ° C.

The results are shown in Table 2.

  When Triolein was used as a substrate, 1,2-DAG and 1,3-DAG (8.5% and 1.9%, respectively) and free fatty acid (FFA) (9.8%) were produced. There was no MAG. This suggests that purified lipase acts efficiently on the 1/3 position of TAG and that DAG hydrolysis is rate limiting.

  In the hydrolysis of 1,2-Diolein and 1,3-Diolein, fatty acids as shown by Triolein were not liberated and no isomerization was observed between both DAGs. However, TAG generation (41%) was observed only for 1,2-Diolein.

  1-Monoolein was hydrolyzed better than 2-Monoolein, and the fatty acid release ratio was approximately 2: 1. This almost coincided with the result shown in FIG. In either case, DAG was not detected, but only 2-monoolein produced TAG (29%).

  The above results suggest that the fatty acid released from 2-Monoolein was re-incorporated into 2-MAG to produce TAG.

(Synthesis of acylglycerol)
Using purified lipase as a catalyst, acylglycerol was synthesized from glycerol and oleic acid as follows. A reaction solution consisting of 1.8 ml of a glycerol / oleic acid mixture (7: 1, v / v) and 0.2 ml of purified lipase (20 U) was incubated for 120 min at 37 ° C. with shaking.

  The product was extracted with chloroform, the lipid composition was analyzed by thin layer chromatography, and the time course of the lipid composition was followed. The result is shown in FIG.

  Referring to FIG. 7, at the initial stage of the reaction, approximately the same molar amounts of 1-MAG and 2-MAG were produced, but 1,2-DAG and 1,3-DAG accumulated to a small amount and then turned to decrease. 1-MAG finally reached 14.5%.

  On the other hand, TAG increased corresponding to the decrease of 2-MAG and finally reached 22%. This is thought to be due to the increase in TAG due to the addition of fatty acids at positions 1 and 3 of 2-MAG.

  The total composition of 2-MAG, 1,2-DAG, 1,3-DAG and TAG (26%) is about twice as high as that of 1-MAG (15%), and the purified lipase is glycerol. This indicates that the activity of directly adding a fatty acid at the 2-position is high.

  Purified lipase acts in the biosynthetic pathway mainly via 2-MAG and 1,2-DAG to produce TAG, and acts strongly on position 2 even in the absence of a solvent for glycerol. It can be said that it has extremely characteristic properties. And purified lipase tends to promote synthesis more strongly than hydrolysis. For this reason, it is considered useful for the synthesis of structured lipids produced by structural design to impart functionality.

  The lipase produced by Mortierella SAM2197 strain has a catalytic action that preferentially promotes the ester bond between a hydroxyl group located at the 2-position of glycerol and a fatty acid. Since it is easy to synthesize a functional lipid having a highly physiological function having a highly unsaturated fatty acid at the 2-position, it is expected to be used in the production field of foods, feeds, pharmaceuticals and the like having a high physiological function.

Claims (6)

Produced by Mortierella SAM2197 strain (FERM BP-6261), preferentially have a catalytic effect on the 2-position of glycerol, lipase molecular weight of 11 kDa. The lipase according to claim 1, wherein the optimum pH is 8-10. The lipase according to claim 1 or 2 , wherein the optimum temperature is 45 to 55 ° C. The lipase according to any one of claims 1 to 3 , which is obtained by culturing the Mortierella sp. SAM2197 strain, collecting it after 120 to 168 hours, and purifying it. A method for synthesizing a functional lipid comprising a step of synthesizing 2-monoglyceride by interposing the lipase according to any one of claims 1 to 4 between glycerol and a highly unsaturated fatty acid. As the polyunsaturated fatty acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and dihomo-gamma-linolenic acid are used. The method for synthesizing a functional lipid according to claim 5 .