JP2005261284A - Method for immobilizing enzyme and reactor using immobilized enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve problems that sufficient satisfactory immobilized phospholipase D cannot be obtained in aspects of inexpensive and efficient industrial obtaining of the objective product though phospholipase D is conventionally immobilized by various methods using glutaraldehyde or a gel unusable for producing foods. <P>SOLUTION: The phospholipase D can be immobilized in a gel containing konjac mannan, i.e. a carrier to thereby minimize the lowering of enzymic activity when the enzyme is immobilized. The phospholipase D can firmly be immobilized and the leakage of the enzyme during a reaction can be minimized by the method. An industrially excellent reaction product can be provided by a reactor using the immobilized phospholipase D. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to immobilization of an enzyme. More specifically, the present invention relates to an immobilization carrier suitable for industrial use so that phospholipase D, which is an enzyme useful for modifying lipids, can be used repeatedly and efficiently. It is related to the method of immobilization. The present invention also relates to a method for producing phospholipids using the obtained phospholipase D-immobilized enzyme, and particularly to a method for producing phosphatidic acid, phosphatidylglycerol, and phosphatidylserine efficiently.

Phospholipase achieved high functionality by advanced modification of various lipids including fats and oils, utilizing its selectivity and specificity. Regarding lipases, 1) Lipases contained in oil seeds and cereals have high substrate specificity and are involved in the production of specific fats and oils. 2) Plant lipases have few applications because of their small abundance. Oil and fat modification by lipases such as peanut, castor seed, rapeseed, coumin, and cabbage-derived phospholipase D has been studied in the laboratory. 3) Papaya (CPL) is a plant origin of protease, but lipase activity depends on the processing conditions Data on various short- and long-chain triglyceride degradation activities and transesterification have been reported, and fat oil modification applications have been increasing since then. 4) Bromelain obtained from pineapple and papain is used for food processing. However, the lipase activity sufficient for oil and fat modification has not been obtained (Non-patent Document 1).

Among various phospholipases, phospholipase D has attracted attention because it can be used for the production of new compounds by catalyzing not only hydrolysis but also base exchange reaction. Kamada et al. (Non-Patent Document 2) studied the conditions for preparing phosphatidylserine (PS) from soybean phospholipid consisting of phosphatidylcholine 90% and phosphatidylethanolamine 10% using phospholipase D. It was clarified that the conversion activity of phospholipase D was improved by pretreatment of phospholipase D with acetic acid buffer at pH 5.6 for 1 h, and the PS content could be increased to 52% in 30 minutes. Furthermore, Kamada et al. Examined phosphatidyl group conversion reaction conditions for quantitatively preparing phosphatidylglycerin (PG) from soybean phospholipids using phospholipase D crude enzyme (17 IU / g) (Non-patent Document 3). In addition, since suppression of the hydrolysis reaction that occurs competitively with phospholipase D becomes an issue in the preparation of PG, the hydrolysis reaction was also examined prior to the conversion reaction. As a result, in the hydrolysis reaction, phosphatidic acid was quantitatively produced at pH 5.6, which was the optimum condition for phospholipase D, while in the conversion reaction, a slightly acidic pH of 4.8 was used. It is reported that the hydrolysis reaction can be effectively suppressed by phosphatidylcholine and phosphatidylethanolamine can be converted quantitatively to PG. Juneja et al. (Non-Patent Document 4) proposed 1) micellar reaction method 2) as a solution to suppress the formation of phosphatidic acid when phosphatidylcholine is converted to phosphatidylglycerol by acting phospholipase D in the presence of glycerol. Considering a method of reacting by suspending the ether solution of the substrate in water, 2) reports that the rate is slow but there is almost no hydrolysis and 100% conversion is possible. Aurich et al. (Non-Patent Document 5) show that the exchange of choline of octadecylphosphocholine by N- (2-hydroxy-ethyl) -N-methylpiperidine using phospholipase D is kinetically controlled and is maximal in buffer. It is reported that 60%, 43% in buffer / hexane, 82% exchange in buffer / n-hexane / 1-octanol can be obtained. Subramino et al. (Non-Patent Document 6) examined the effects of water content, phospholipase D concentration, and phosphatidylcholine concentration on the degradation of phosphatidylcholine of phospholipase D in diethyl ether containing Triton X-100. Ryu et al. (Non-Patent Document 7) showed that the activity of phospholipase D is inhibited by lysophosphatidylethanolamine and lysophosphatidylinositol. On the other hand, lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphatidylserine had no effect. As a patent document other than the above, a technique for reducing cholesterol using phospholipase (for example, Patent Document 1) is known.

As described above, in order to efficiently use industrially useful phospholipase D, attempts have been made to prepare immobilized enzymes. JUNEJA et al. (Non-Patent Document 8) examined the conversion of phosphatidylcholine to phosphatidylserine by phospholipase D immobilized on an amphiphilic gel in the presence of L- or D-serine. The two-layer reaction was performed at 30 ± 0.5 ° C with an aqueous layer containing serine and phospholipase D and ethyl acetate containing phosphatidylcholine. Ethyl acetate and diethyl ether as solvents showed sufficient transphosphatidylation. MASOOM et al. (Non-patent Document 9) describe that hydrogen peroxide generated in a flow injection system is obtained by linking a column in which phospholipase D is immobilized on glass beads and a column in which alkaline phosphatase and choline oxidase are immobilized. It was reported that phosphatidylcholine could be quantified by amperometric titration. JUNEJA et al. (Non-Patent Document 10) examined a method for immobilizing phospholipase D and an efficient method for producing phosphatidylserine using an immobilized enzyme. The optimum conditions were investigated in the continuous processing system and batch processing system, respectively. The immobilization rate and activity of phospholipase D reached 100%, and very good results were obtained in repeated batch processing. MASOOM et al. (Non-Patent Document 11) immobilized phospholipase D on porous glass beads with glutaraldehyde and packed it in a column reactor. Phosphatidylcholine was dissolved in diethylbarbitone buffer containing calcium and Triton X-100 and circulated through the column for reaction. There was no fatty acid specificity in the reaction of PL-D, and phosphatidic acid was prepared.

As an immobilization technique for an enzyme other than phospholipase, a technique for immobilizing a protease to chitosan or an ion exchange resin (for example, Patent Document 3), a technique for immobilizing maltogenic α-amylase in a porous state (for example, Patent Document 4), β -A technique for immobilizing galactosidase on a polymer or glass (for example, Patent Document 5) is known.

JP-A-5-49414

JP-A-8-291188

JP-A-11-2221049

JP 2000-189161 A

JP-A-6-141822

Eur J Lipid Sci Technol Vol.105, No.6, Page308-317 (2003)

Japan Oil Chemists' Society Vol.47, No.1, Page51-56 (1998)

Petrochemistry Vol.42, No.4, Page297-301 (1993)

Enzyme Microb Technol Vol.9, No.6, Page350-354 (1987)

ULBRICH-HOFMANN R Vol.19, No.9, Page875-879 (1997)

ULBRICH-HOFMANN R Vol.18, No.7, Page815-820 (1996)

Proc Natl Acad Sci USA Vol.94, No.23, Page12717-12721 (1997)

Biochim Biophys Acta Vol.1003, No.3, Page277-283 (1989)

Anal Biochem Vol.187, No.2, Page240-245 (1990)

Abstracts of Annual Meeting of Chemical Engineering Society Vol.53rd, Page27 (1988)

Process Biochem Vol.30, No.8, Page701-704 (1995)

As described above, phospholipase D has been immobilized, but all use glutaraldehyde or a gel that cannot be used for food production. Moreover, what has been sufficiently satisfied so far in terms of industrially obtaining a target product at low cost and efficiently has not been obtained. Then, this invention makes it a subject to provide the bioreactor using the manufacturing method of the immobilized phospholipase D which can be used for foodstuff manufacture, and the immobilized phospholipase D.

In order to overcome these points and obtain an industrially advantageous immobilized enzyme that can be used in the production of food, intensive research was conducted. As a result of various investigations on the immobilization of phospholipase using various gelling agents, it was found that immobilization can be carried out efficiently by using konjac mannan gel. Moreover, when the base exchange reaction was performed using the obtained enzyme and the activities were compared, it was found that the activity hardly increased but rather increased. In addition, the obtained immobilized enzyme is extremely strong in immobilization, and almost no leakage of the enzyme is observed even in a high concentration substrate solution, so that it can be used efficiently for continuous production using a packed column. I found it. And it was discovered that industrially superior reactants could be provided by using the present immobilized phospholipase, and as a result, the present invention was completed.

Thus, the present invention provides: [1] Phospholipase D-immobilized enzyme characterized in that phospholipase D is immobilized on a carrier mainly composed of konjac mannan;
[2] The immobilized enzyme according to [1], wherein the phospholipase D is obtained from cabbage or bacteria;
[3] The bacterium is Actinomadura sp. Or Streptomyces sp. The immobilized enzyme according to [2], characterized in that:
[4] The immobilized enzyme according to [1] to [3], wherein the carrier for enzyme immobilization is konjac ginger grated or gelled product obtained by adding a coagulant to konjac fine powder;
[5] The carrier for enzyme immobilization is a gelled product obtained by adding a coagulant to grated konjac ginger or konjac fine powder, and includes a gelling agent derived from a raw material other than konjac as an additive. 1] to [3] immobilized enzyme;
[6] A bioreactor comprising the immobilized enzyme according to any one of [1] to [5].

By immobilizing phospholipase D on a gel containing konjac mannan, that is, a carrier, a decrease in enzyme activity during enzyme immobilization can be minimized. In addition, phospholipase D is firmly immobilized by the method of the present invention, and leakage of the enzyme during the reaction is minimized. Furthermore, industrially excellent reactants can be provided by the reactor using the immobilized phospholipase of the present invention.

The present invention relates to a phospholipase that uses konjac mannan as a carrier and is immobilized on the carrier. The konjac mannan used as a carrier in the present invention is glucomannan, that is, konjac ginger sliced and dried, and then the by-product flying powder is removed (fine powder). It is preferable to use a refined product obtained by purifying the refined powder and increasing the glucomannan content. As the purified glucomannan, a commercially available product such as supermannan (manufactured by Hadano Shoten) or mannan base (manufactured by Sunflower Sugiyoshi) can be used. The following two methods can be adopted as a method for gelling konjac mannan. That is, after diluting the fine powder or purified product with water, add calcium hydroxide or sodium carbonate to make it alkaline, or make it an aqueous solution mixed with a gelling agent other than fine powder or purified product and konjac under heating, Gelation can be achieved by cooling. As the gelling agent, those recognized as food additives such as carrageenan, xanthan gum, gellan gum, gum arabic, starch and the like can be used alone or in combination. The content of konjac mannan or / and the gelling agent in the gel is 0.02 to 20% by weight, preferably 0.1 to 5% by weight, more preferably 1 to 3% by weight. If the content of konjac mannan or / and gelling agent in the gel is less than 0.02% by weight, the gel strength is insufficient and cannot be applied to the reactor. If the content is more than 20% by weight, the gel strength is high. Thus, it becomes difficult to prepare a fine immobilized enzyme.

The konjac koji used as a raw material for the konjac mannan used in the present invention is Amorphophallus rivieri var. Konjac of the taro family. There are native varieties of konjac varieties (red stalks, white birch, flat, Japanese and large varieties), Chinese varieties and binaka varieties (blue stalks, black leopards, long balls, stone balls, etc.) Harunuroro (Konnyaku Norin 1), Akagi Odama (Konnyaku No. 2), Myo Yutaka (Konnyaku No. 3) and Miyama Sari (Konnyaku No. 4) are known. Any varieties of konjac can be used. Also, one or more of the above varieties are used in combination. The parts from which konjac mannan can be collected are the corms and the offspring. In addition, cultivated or natural konjac can be used in appropriate combination.

The phospholipase D to be immobilized in the present invention may be a naturally derived material such as cabbage, peanut, microorganism or the like alone or in combination. As a bacterium which is a raw material of phospholipase D, Actinomadura sp. And actinomycetes Streptomyces sp. Can be mentioned. In the present invention, the origin is not limited, and proteins having phospholipase D activity can be used alone or in appropriate combination. The enzyme content in the immobilized enzyme of the present invention is prepared so that the enzyme activity is 0.001 to 1000 U / mg. When the enzyme activity is 0.001 U / mg, it takes a long time to obtain a necessary reaction product, resulting in a disadvantage in cost. Further, when the enzyme activity is 1000 U / mg, an increase in reaction rate according to the amount of enzyme added cannot be expected.

Enzyme phospholipase D is immobilized on a carrier under conditions that do not extremely inactivate the enzyme. For example, a predetermined amount of konjac mannan and / or gelling agent is dissolved in a solution, and finally Enzyme is added in such an amount that the enzyme activity is about 0.001 to 1000 U / mg. Next, mix for about 0.1 to 50 hours in the stable range of the enzyme, for example, at a pH of about 3 to 10 and a temperature of about 0 to 80 ° C., and add calcium hydroxide or sodium carbonate to make it alkaline, or cool the solution. To gel. By refining the solution using a nozzle, an extrusion granulator or the like, the surface area can be increased and the efficiency can be increased. Further, it is possible to prepare a fine hand or an enzyme by making a plate-like gel in advance and then finely cutting it with a cutter, a pulverizer or the like.

Reactions catalyzed by the immobilized enzyme of the present invention are hydrolysis and base exchange. In a reaction system using phosphatidylcholine and serine as reaction substrates, a hydrolyzate, that is, phosphatidic acid, and a base exchange reaction product, that is, phosphatidylserine, are obtained as reactants. In a reaction system using glycerin instead of serine, phosphatidylserine Instead, phosphatidylglycerol is obtained. The phosphatidylcholine, serine, and glycerol in the reaction solution can be used alone or in appropriate combination, regardless of whether they are chemically synthesized products or natural products. As a solvent for the reaction, serine, glycerin is dissolved in an aqueous layer, phosphatidylcholine is dissolved in an organic solvent, that is, ether, chloroform, toluene, etc., and both are mixed by stirring. A method of emulsifying an oil-soluble substance such as phosphatidylcholine in the aqueous solution to be reacted can be employed.

The immobilized enzyme of the present invention and the reaction solution can be contacted by a batch method or a continuous method for hydrolysis or base exchange reaction, and a method suitable for industrial implementation is selected as appropriate. Can do. The component concentration in the reaction solution is about 5 to 50 w / v% for oil-soluble components such as phosphatidylcholine, and about 5 to 30% by weight for water-soluble components such as serine and glycerin. The reaction rate can be increased by allowing about 1 to 10% by weight of a calcium salt such as calcium chloride to coexist in the substrate solution. The reaction conditions are such that the enzyme in the immobilized enzyme is stable and can act sufficiently, such as pH of about 3 to 10, more preferably about pH 5 to 10, temperature of about 20 to 80 ° C., more preferably about It is chosen in the range of 40-60 ° C. If the concentration of the oil-soluble component or water-soluble component in the reaction solution is less than 5% by weight, a sufficient reaction rate cannot be obtained. On the other hand, when the concentration of the oil-soluble component or water-soluble component in the reaction solution exceeds 50 or 30% by weight, respectively, the viscosity increases and it becomes difficult to carry out a continuous reaction.

The bioreactor of the present invention has a device that allows an enzyme-supported carrier to come into contact with a fluid to be reacted. Advantageously, the apparatus is selected from a stirred reactor, a basket reactor, a fluidized bed reactor, a backbed reactor, a filter reactor, and the like. The most commonly used form of the immobilized enzyme may be a continuous type with a packed column or a batch type in which the immobilized enzyme can be easily recovered. The bioreactor may likewise consist of a device having a single column or advantageously a plurality of columns. The reaction liquid to be treated is preferably one that flows in the direction of gravity in the column. Another advantageous embodiment of the bioreactor includes, in addition to the apparatus, a tank containing the substrate to be treated, a post-treatment tank for post-treating the effluent from the bioreactor, and a product storage tank Is included.

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

1. As a raw material for preparing cabbage phospholipase (crude enzyme), cabbage (snacks) cultivated in a cold district of Gunma Horticultural Experiment Station in Tashiro, Agatsuma-gun, Gunma Prefecture was used. 300 ml of water was added to the inner bright green leaf (200 g) and homogenized for 5 min. The ground sample was squeezed through silk. The filtrate was centrifuged at 13000 × g for 30 min to obtain 380 ml of a clear supernatant. The supernatant was heat-treated at 55 ° C. for 3 minutes. Rapid cooling was performed, and insoluble components were separated and removed by centrifugation at 4 ° C., 15000 × g, 30 min. 0.4 times volume of ethanol was added to the clear supernatant and cooled at -15 ° C for 20 min or longer. Stir for 15 min at -15 ° C. The precipitate was removed by centrifugation at 3000 × g for 5 minutes. The precipitate was redissolved in water and centrifuged at 15000 × g for 5 min. Resuspension and centrifugation were repeated until no enzyme activity could be detected in the supernatant. Ultrafiltration was performed through an Amicon X100 membrane (Nippon Millipore). Lyophilized to obtain crude enzyme.
2. Purification of phospholipase
2 g of the lyophilized crude enzyme was suspended in 20 ml of 30 mM Pipes buffer (pH 6.2) and 50 mM CaCl ₂ . Centrifuged at 12000 × g for 5 minutes. The yellow supernatant containing the enzyme activity was charged on an Octyl-Sepharose CL-4B column (2.5 cm × 20 cm; manufactured by Amsham). The filler was equilibrated in advance with 30 mM Pipes buffer (pH 6.2), 50 mM CaCl ₂ and a flow rate of 24 ml / h. Rinse at least 60 ml with the same buffer until the yellow material was washed away. The active fraction was eluted with 10 mM Pipes Buffer (pH 6.2) and 30 mM CaCl ₂ . Finally, elution was performed with 10 mM Pipes Buffer (pH 6.2) and 0.1 mM EDTA. 5 ml active fractions were pooled. Dialyzed in 20 mM Tris-HCl (pH 7.5). The dialyzed enzyme solution was charged to Mono-Q HR 5/5 column (trimethylammonium, Pharmacia). Elution was performed with a linear gradient (0 to 0.5 M NaCl flow rate in 90 ml of 20 mM Tris-HCl (pH 7.5): 1 ml / min pressure: maintained between 1.0 and 2.0 Mpa (FPLC, Pharmacia)). Active fractions were pooled. It concentrated 10 time by ultrafiltration in Amicon cell using PM30 membrane. Purified enzyme was dispensed, 30% glycerol was added, and the mixture was stored at -80 ° C.
3. The first reaction mixture into the enzyme activity assay, 0.1M Tris-HCl buffer pH8.0 1.6ml , 0.1M CaCl 2 0.4ml, H 2 O 1.2ml, 25mM substrate solution (Sigma dioleoylphosphatidylcholine 88.5mg Was dissolved in 4.5 ml of a 5% (W / V) Triton X-100 solution, and then 0.8 ml of a mixed solution at 4 ° C. for 10 minutes by ultrasonic emulsification. The second reaction reagent mixture was 0.8 ml of 15 mM 4-aminoantipyrine (4-AA) solution, 0.8 ml of 0.2% (W / V) phenol solution, pH 8.0 0.8 ml of 60 mM EDTA solution, 16 ml of 50 mM Tris-HCl buffer, 90 U / ml peroxidase (POD) solution {POD (Asahi Kasei) 900 U dissolved in 10 ml of purified water} 0.8 ml, 30 U / ml choline oxidase (COD) solution {COD (Asahi Kasei) 300 U in 10 mM Tris-HCl buffer pH 8 0.0 Dissolved in 10 ml} A mixed solution of 0.8 ml was prepared. The enzyme-dissolved diluted solution was 10 mM Tris-HCl buffer pH 8.0 containing 0.05% (W / V) BSA (manufactured by Sigma) and 0.1% (W / V) TritonX-100. The enzyme sample solution was accurately weighed about 20 mg of the test sample, and made up to a total volume of 20 ml with the enzyme-dissolved diluted solution. The solution was appropriately diluted with an enzyme-dissolved diluted solution. 40 ml of the first reaction reagent mixture was accurately dispensed into a small test tube and pre-warmed at 37 ° C. After 5 minutes, 4 ml of the enzyme sample solution was accurately added and mixed, and the first reaction was started at 37 ° C. After 10 minutes, 200 ml of the second reaction reagent mixture was added and mixed, and the second reaction was started at 37 ° C. In the blind test, 4 ml of enzyme sample solution was added at this point. After 20 minutes, the absorbance at 500 nm was measured.
4). Quantitative determination of phosphatidylserine Sampling was performed over time during the base exchange reaction, and the PS content in the oil was measured by high performance liquid chromatography (HPLC). That is, 0.5 ml of an organic solvent phase was collected, 5 ml of water and 5 ml of hexane were added and stirred, and then centrifuged (2500 rpm, 10 minutes), and 1 ml of the supernatant was collected. Further, the solvent was dried, and a dried product was used as a sample. An internal standard solution (1 ml) was added to 10 mg of the sample and subjected to HPLC. For analysis, DEVELOSIL 60-5 (4.6 × 250 mm, Nomura Chemical Co., Ltd.) was mounted on a Waters HPLC LC Module-I type, the column temperature was 45 ° C., and the mobile phase was acetonitrile: methanol: phosphoric acid = 780: 50: 9 ( V / V / V) and a flow rate of 1.5 ml / min. Detection was performed using a UV detector at a wavelength of 202 nm.

Immobilization of enzyme 250 mg of konjac mannan (manufactured by Sagano) and 250 mg of carrageenan (manufactured by Sigma) were added to 100 g of distilled water, and dissolved by stirring at 80 ° C. for 30 minutes. After cooling to 50 ° C., 10 g of cabbage phospholipase obtained in the example was added, and the mixture was further stirred for 30 minutes. It was gelled by dropping it into oil (Panasate 810; manufactured by Nippon Oil and Fats) cooled to 5 ° C. with a peristaltic pump (2 ml / min) and holding it for 30 minutes. The gelled immobilized enzyme was received with a mesh to remove the medium chain fatty acid triglyceride, and then washed with 1 L of ethanol to completely remove the oil. The measurement result of enzyme activity was 1.5 U / mg.

30 g of konjac koji fine powder (manufactured by Hadano Shoten) and distilled water 900 were heated and stirred to obtain konjac paste. After adding 2 g of calcium hydroxide and 1 g of actinomycete-derived phospholipase D (manufactured by Asahi Kasei), the mixture was allowed to stand for 30 minutes, and heated at 60 ° C. for 2 hours to gel. The gel was cut using a meat chopper to obtain an immobilized enzyme having a particle size of about 1 mm. The activity of the immobilized enzyme was 10 U / mg.

 10 g of glycerin and 0.2 g of calcium chloride were added to 100 ml of distilled water to form an aqueous layer. 10 g of phosphatidylcholine (SLP-PC70; manufactured by True Lecithin) was dissolved in 100 ml of diethyl ether to form an organic solvent layer. The aqueous layer, the organic solvent layer and 10 g of the immobilized enzyme of Example 1 were stirred at 8O 0 C for 8 hours. The contents of phosphatidylcholine, phosphatidic acid, and phosphatidylglycerol in the total lipid at the start of the reaction solution were 56 wt%, 3 wt%, and 0.5 wt%, respectively, but phosphatidylcholine in the total lipid 8 hours after the start of the reaction, The contents of phosphatidic acid and phosphatidylglycerol were 20% by weight, 11% by weight, and 28.5%. After the reaction yield, the immobilized enzyme was recovered with a mesh. The activity of the recovered immobilized enzyme was 1.3 U / mg.

To 10 kg of distilled water, 200 g of phosphatidylcholine (95% content; manufactured by Nissin Oil Co., Ltd.), 200 g of L-serine and 0.5 g of calcium chloride were added and emulsified with Polytron (manufactured by Kinematica) for 20 minutes. A column (1.8 cm in diameter, 18 cm in length) was filled with 10 g of the immobilized enzyme of Example 2, and the above emulsion was circulated with a peristaltic pump at a rate of 20 ml / min for 3 hours. The column was placed in a constant temperature bath at 50 ° C., and the reaction was performed at around 50 ° C. The contents of phosphatidylcholine, phosphatidic acid, and phosphatidylserine in the total lipid at the start of the reaction were 95%, 0%, and 0.5% by weight, respectively, but phosphatidylcholine and phosphatidine in the total lipid at 8 hours after the start of the reaction. The contents of acid and phosphatidylserine were 32% by weight, 5% by weight and 58.5%. The activity of the immobilized enzyme after completion of the reaction was 9 U / mg.
(Comparative Example 1)
Enzyme-immobilized agar (1.5 g) was added to 100 g of distilled water, and dissolved by stirring at 80 ° C. for 30 minutes. After cooling to 50 ° C., 10 g of cabbage phospholipase obtained in the example was added, and the mixture was further stirred for 30 minutes. It was gelled by dropping it into oil (Panasate 810; manufactured by Nippon Oil and Fats) cooled to 5 ° C. with a peristaltic pump (2 ml / min) and holding it for 30 minutes. The gelled immobilized enzyme was received with a mesh to remove the medium chain fatty acid triglyceride, and then washed with 1 L of ethanol to completely remove the oil. The measurement result of enzyme activity was 0.5 U / mg.
(Comparative Example 2)
20 g of sodium alginate (manufactured by Shinshin Kaken) and 480 ml of distilled water are dissolved by heating and stirring, 1 g of actinomycetes-derived phospholipase D (manufactured by Asahi Kasei) is added, and after stirring, 500 ml of a 4% by weight calcium chloride solution is added. After adding and stirring, the mixture was left for 30 minutes to gel. The gel was cut using a meat chopper, but it did not become particles but became paste. The enzyme activity was 2 U / mg.
(Comparative Example 3)
The reaction was performed in the same manner as in Example 3 except that the enzyme of Comparative Example 1 was used instead of the immobilized enzyme of Example 1. The contents of phosphatidylcholine, phosphatidic acid, and phosphatidylglycerol in the total lipid at the start of the reaction solution were 56 wt%, 3 wt%, and 0.5 wt%, respectively. The contents of phosphatidic acid and phosphatidylglycerol were 43% by weight, 5% by weight and 11.5%. After the reaction yield, the immobilized enzyme was recovered with a mesh. The activity of the recovered immobilized enzyme was 0.2 U / mg.
(Comparative Example 4)
The reaction was performed in the same manner as in Example 4 except that the enzyme of Comparative Example 2 was used instead of the immobilized enzyme of Example 2. Ten minutes after the start of the reaction, clogging occurred at the outlet of the column, making it impossible to circulate the reaction solution. As a result of recovering the gel and measuring the enzyme activity, no activity was observed because the enzyme flowed out.

From the above Examples and Comparative Examples, it can be seen that the immobilized enzyme of the present invention has little loss of enzyme during immobilization and is excellent in retention of the enzyme during the reaction.

Claims (6)

A phospholipase D-immobilized enzyme, wherein phospholipase D is immobilized on a carrier containing konjac mannan. The immobilized enzyme according to claim 1, wherein the phospholipase D is obtained from cabbage or bacteria. The bacterium is Actinomadura sp. Or Streptomyces sp. The immobilized enzyme according to claim 2, wherein The immobilized enzyme according to claims 1 to 3, wherein the enzyme immobilization carrier is a konjac ginger grated or a gelled product obtained by adding a coagulant to konjac fine powder. The enzyme immobilization carrier is a gelled product obtained by adding a coagulant to grated konjac ginger or konjac fine powder, and contains a gelling agent derived from a raw material other than konjac as an additive. 3. The immobilized enzyme according to 3. A bioreactor comprising the immobilized enzyme according to any one of claims 1 to 5.