JP3024845B2 - Method for producing immobilized lipase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing highly active immobilized lipase .

[0002]

2. Description of the Related Art As a method for producing an immobilized enzyme such as an immobilized lipase used for reforming, decomposing, or synthesizing an oil or fat, a method for producing an immobilized enzyme in the presence of an enzyme activity-expressing agent is used. And a method of obtaining a highly active immobilized enzyme by immobilizing the enzyme on an insoluble carrier. Fatty acids, fatty acid derivatives, phospholipids, aliphatic compounds such as alcohols, and surfactants such as sucrose fatty acid esters have been studied as the activity-expressing agents (JP-A-62-134090, JP-A-63-134090). 214184, JP-A-3-49684, etc.). However, when an immobilized enzyme is produced by these methods, high activation is possible, but there is a problem in its reproducibility.

[0003]

Means for Solving the Problems In order to solve the above problems, the present inventors studied a method for producing an immobilized lipase using an activity developing agent. ,
It was found that the activity of immobilized lipase was highly correlated. That is, when the active developer is oxidized, or if oxidized in the production process of the immobilized lipase preparation of immobilized lipase with high activity found that difficulty, and have completed the present invention . That is, the present invention is, fat
Fatty acids, fatty acid derivatives, alcohols, carbonyl compounds
, Ethers, alkyl halides, phospholipids and
One or more selected from the group consisting of surfactants
Immobilized lipa in the presence of an oil-soluble compound
The present invention provides a method for producing immobilized lipase , which comprises using a peroxide having a peroxide value of 0 to 2 meq / kg, preferably 0 to 1 meq / kg, as an activity developing agent. is there.

As a method for measuring the degree of oxidation of an activity developing agent, a peroxide value is generally measured, and those having a peroxide value of 2 meq / kg or less (0 to 2 meq / kg) are used. ,
It can be said that the agent has not been oxidized. The non-oxidized activity developing agent having a peroxide value of 2 meq / kg or less (0 to 2 meq / kg) used in the present invention can be obtained by a general purification method, that is, treatment with an adsorbent, distillation or the like. . For its storage, it is necessary to avoid contact with oxygen, such as in an inert gas atmosphere, and to manage such as low-temperature storage. The addition of an antioxidant is also effective.

[0005] In the method of the present invention, the immobilized lipa
And it is also important to prevent the oxidation of the active developing agent during manufacture of over zero, a method to manufacture the immobilized lipase Therefore a method of performing at a low temperature, in a substantially oxygen existent systems, active developer Can be used. Here, "low temperature" means room temperature or lower, and "system substantially free of oxygen" means an inert gas atmosphere or a reduced pressure state, and means a partial pressure of oxygen of 10 Torr or less.

As a method for producing an immobilized lipase in the presence of an active developer, a method for immobilizing lipase on a carrier after contacting the lipase and active developing agent was adsorbed in advance carrier activity enhancer How to immobilize lipase afterwards,
There is a method of immobilizing lipase on a carrier and then adding an activity-expressing agent. In the present invention, any method can be used. Also, contact between the activity-expressing agent and lipase ,
Adsorption of the active developing agent to the carrier, no particular limitation is imposed on the method of addition of the active developing agent to lipase immobilization carrier, solvent-free, the solution of water or an organic solvent, Ru can be used as the state of dispersion .

[0007] The activity developing agent used in the present invention includes:
One or two or more oil-soluble compounds selected from the group consisting of fatty acids, fatty acid derivatives, alcohols, carbonyl compounds, ethers, alkyl halides, phospholipids and surfactants. The fatty acid used in the present invention is preferably one having 2 to 36 carbon atoms, more preferably 8 to 18 carbon atoms, for example, linear saturated fatty acids such as capric acid, lauric acid, myristic acid, oleic acid, linoleic acid and the like. Examples include unsaturated fatty acids such as acids, hydroxy fatty acids such as ricinoleic acid, and branched fatty acids such as isostearic acid. Examples of the fatty acid derivative include an ester of a fatty acid having 2 to 36 carbon atoms, preferably 8 to 18 carbon atoms, and a compound having a hydroxyl group. A monohydric alcohol ester, a polyhydric alcohol ester, or ethylene oxide is further added to these esters. Derivatives and the like are exemplified. Monohydric alcohol esters include methyl esters and ethyl esters, and polyhydric alcohol esters include monoglycerides, diglycerides, and derivatives thereof, and fatty acid esters of polyhydric alcohols such as propylene glycol and polyglycerin.

The alcohols used in the present invention include linear or branched aliphatic monohydric alcohols having 4 to 36 carbon atoms, preferably 8 to 24 carbon atoms, for example, saturated alcohols such as octyl alcohol and lauryl alcohol, and oleyl. It may be an unsaturated alcohol such as alcohol, or a branched alcohol such as 5-decanol or isostearyl alcohol. Further, dihydric alcohols such as hexamethylene glycol and polyhydric alcohols are also effective. In addition, phenol compounds such as alkyl-substituted phenols, cholesterol, stigmasterol, brassicasterol,
Sterols such as cambesterol are exemplified. or,
Terpene alcohols such as phytol, geraniol, farnesol and linalool, and fat-soluble vitamins such as retinol and tocopherol are also effective. Examples of carbonyl compounds used in the present invention include 2,4-decadienal, decanal, aliphatic aldehydes such as hexadecanal, terpene aldehydes such as retinal,
Examples thereof include aliphatic ketones such as 2-octanone, 2-decanone and octyldecyl ketone. Examples of the ethers used in the present invention include long-chain ethers such as dioctyl ether, glyceryl ethers such as thymyl alcohol and batyl alcohol, or glyceride-like compounds such as glycidyl ether, triethylene glycol monomethyl ether, and polyethylene. Examples thereof include polyoxy compounds such as glycol monomethyl ether, trimethylsilyl ether derivatives of the alcohol, and silicon compounds such as polymethylsiloxane. Examples of the alkyl halides used in the present invention include long-chain alkyl halides such as oleyl chloride, octyl chloride, and octyl bromide.

Examples of the phospholipid used in the present invention include crude and / or purified mixed lecithin such as commercially available soybean lecithin and egg yolk lecithin, and phosphatidylcholine, phosphatidylserine, phosphatidylethanol obtained by fractionating these. Amine, phosphatidylinositol, phosphatidic acid, cardiolipin and the like may be used alone or as a mixture. Synthetic phospholipids obtained by various synthetic methods and derivatives thereof can also be used. Examples of the surfactant used in the present invention include sucrose fatty acid esters and sorbitan fatty acid esters.

The antioxidants used in the present invention include tocopherol, gallic acid, gallic acid derivatives, flavonoids, polyphenols, sesamoles, citric acid, citric acid derivatives, dibutylhydroxytoluene, butylhydroxyanisole, nordihydrog Alletic acid and the like.

The carrier used in the present invention may be any carrier which is insoluble in water and alcohol, various organic solvents, fats and oils, and may be celite, diatomaceous earth, kaolinite, molecular sieve, porous glass, activated carbon, calcium carbonate. Inorganic carriers such as ceramics, and lipase activities such as organic polymers such as cellulose powder, polyvinyl alcohol, chitosan, ion-exchange resins, and adsorption resins, and are physically and chemically stable from operation. Any can be used. The carrier may be in various forms such as powder, granule, fiber, sponge, etc., and any of them can be used. In particular, from the viewpoint of the process operation, it is preferable to use a porous carrier having a particle size of 400 to 1000 μm and a pore size of 100 to 1500 °.

[0012]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Commercially available weak anion exchange resin ｛phenol formaldehyde resin, trade name: Duolite A-568,
Duolight International Co., Ltd. ¥ 300g 0.1
After adding 3000 ml of N NaOH and washing with water, the pH was equilibrated with 500 mM, 50 mM acetate buffer at pH 5, followed by filtration and drying under reduced pressure.
To 200 g of this resin was added 2 liters of a 10% ethanol solution of oleic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) having a peroxide value of 0.14 meq / kg, and the mixture was stirred at 20 ° C. for 30 minutes. Distilled off. A lipase solution in which a commercially available lipase (lipase preparation derived from Rhizopus japonicas, trade name: lipase A-10, manufactured by Nagase Seikagaku Corporation) was previously dissolved in 150 g of this resin in 50 mM of pH 5 and acetate buffer at 10%. Was added and the mixture was stirred for 2 hours to perform immobilization. Next, after washing with a 50 mM acetate buffer solution having a pH of 5 for 1 hour, the resin was separated by filtration and dried under reduced pressure at normal temperature so that the water content was 5% or less. The activity yield determined from the lipase activity in the filtrate was 92%, and most of the added lipase was immobilized.

[0014] The activity of the obtained immobilized lipase was measured by the following method. The transesterification reaction was performed using a flow tube type continuous reaction apparatus. 38 g of the immobilized enzyme obtained above was packed in an enzyme tower, and commercially available stearic acid was used in a ratio of 1.5 parts by weight to 1 part by weight of palm oil melting point (iodine value: 32.5, diglyceride content: 4.6%). : Lunac S-90,
The raw material obtained by mixing and dissolving stearic acid with a purity of 93% and manufactured by Kao Corporation was adjusted to a water content of 0.10 to 0.11%, and then passed through an enzyme tower (70 ° C). The reaction rate constant ( _Kst ) was determined from the triglyceride composition of the reaction product by GLC and the stearic acid uptake rate. Depending on the activity of the enzyme, the transesterification activity was measured by controlling the flow rate within the range of 0.23 to 1.3 hours for the residence time in the enzyme tower. Table 1 shows the reaction rate constants.

Comparative Example 1 An immobilized enzyme was produced in the same manner as in Example 1 except that oleic acid having a peroxide value of 6.2 meq / kg was used instead of oleic acid having a peroxide value of 0.14 meq / kg. . The activity yield determined from the lipase activity in the filtrate was 93%. The transesterification activity was measured in the same manner as in Example 1. Table 1 shows the reaction rate constants.

Example 2 An immobilized enzyme was produced in the same manner as in Example 1 except that ricinoleic acid having a peroxide value of 0.04 meq / kg was used instead of oleic acid having a peroxide value of 0.14 meq / kg in Example 1. . The activity yield determined from the lipase activity in the filtrate was 92%. The transesterification activity was measured in the same manner as in Example 1. Table 1 shows the reaction rate constants.

Comparative Examples 2-3 The same as Example 1 except that oleic acid having a peroxide value of 0.14 meq / kg was replaced with ricinoleic acid having a peroxide value of 5.3 meq / kg or 15.2 meq / kg. The immobilized enzyme was produced. The activity yields determined from the lipase activities in the filtrate were all 91%. The transesterification activity was measured in the same manner as in Example 1. Table 1 shows the reaction rate constants.

[0018]

[Table 1]

Continuation of the front page (72) Inventor Yasumasa Oki 5-2-3, Chute, Kamisu-cho, Kashima-gun, Ibaraki (72) Inventor Seiji Nomura 1569-3, Omigawa-cho, Omigawa-cho, Katori-gun, Chiba (56) References JP Akira 64-2588 (JP, A) JP-A-1-153309 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 11/00 C12N 9/20 BIOSIS (DIALOG) CA (STN ) WPIDS (STN)

Claims (2)

(57) [Claims] Claims: 1. Fatty acids, fatty acid derivatives, alcohols,
Carbonyl compounds, ethers, alkyl halides
1 selected from the group consisting of phospholipids, phospholipids and surfactants
A method for producing an immobilized lipase in the presence of an activity developing agent comprising one or more oil-soluble compounds, wherein an activity developing agent having a peroxide value of 0 to 2 meq / kg is used. For producing immobilized lipase . 2. A peroxide value of 0 to 1 meq as an activity developing agent.
The process according to claim 1, wherein the use of those / kg.