JP2676099B2 - Synthesis of fatty acid ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Object of the Invention) It is an object of the present invention to provide an improved method for industrially and advantageously carrying out the previously developed method for enzymatically producing a fatty acid ester.

(Prior Art) Sterol fatty acid esters have hitherto been used as various substances such as cholesteric liquid crystals (see JP-A-52-24992) and hydrophilic bases for pharmaceuticals and cosmetics (see JP-A-52-79030). Widely used in the field.

In addition, fatty acid esters of higher alcohols have been used for a long time as hydrophilic bases for pharmaceuticals and cosmetics.

Conventionally, such fatty acid esters have been produced exclusively by an organic synthesis method, but generally, in the organic synthesis method, harsh reaction conditions are adopted, and the adverse effect that a side reaction occurs is inevitable. The isolation and purification operation of is required. In particular, the hydroxyl group of sterol is secondary, and since this hydroxyl group is close to the steroid skeleton, its reactivity is lower than that of a normal aliphatic secondary alcohol. In producing a sterol fatty acid ester, very harsh conditions have been required.

The present inventors require the above-mentioned harsh reaction conditions, do not require expensive and unstable reaction reagents and the like, and perform desired synthesis of desired esters under milder conditions with favorable quality and yield. As a result of intensive studies aimed at providing a method capable of producing efficiently, it was found that a specific enzyme catalyzes the synthesis of sterol esters, and based on this finding, an invention of the method for synthesizing a fatty acid ester by the enzyme was invented. And applied for a patent. (JP-A-61-204197, JP-A-61-2009
97, JP-A-62-48391, JP-A-62-166895) However, these have the following problems.
Improvement was necessary for industrial implementation.

1. In the case of packed column method using immobilized enzyme, since it is a fixed bed type, the pressure loss of the device is high and it cannot be used at a substrate concentration above the saturation concentration. That is, the substrate concentration is low.

2. It is necessary to use a low substrate concentration with a large amount of solvent added, and the productivity per device and time is low.

3. Furthermore, since the initial millimoles of alcohol component per unit enzyme is low, the productivity is low and the amount of synthesis per enzyme is small.

(Problems to be Solved by the Invention) The present inventors have advanced research and development to solve these problems of the prior art, and solved these problems.

(Solution of Problems of Prior Art) These problems are caused by using a lipase and an enzyme selected from cholesterol esterase to react a substrate solution containing an alcohol component and an acid component with the above enzyme to form a fatty acid ester. In the reaction for synthesis, the concentration of the substrate solution was 2.5 times or more the saturation concentration at 37 ° C, and the initial mmol number of alcohol component per enzyme synthesis unit was 0.
It can be solved by setting it to 1 or more.

The ester synthesis reaction as referred to in the present invention is a reaction in which the product is a fatty acid ester.

The enzymes used in the present invention are selected from lipases and cholesterol esterases.

As the alcohol component which is a substrate, sterols and long chain aliphatic alcohols are used.

Examples of the acid component serving as the other substrate in the present invention include fatty acids, glycerin esters of fatty acids, and carbon atoms of 1 to 32.
The aliphatic alcohol esters of are used.

For these enzymes, alcohol components as substrates, acid components, etc., see JP-A-61-204197, JP-A-61-200997,
All of those described in JP-A-62-48391 and JP-A-62-166895 can be used.

The present invention is a method of synthesizing a fatty acid ester by using a lipase and an enzyme selected from cholesterol esterase to bring a substrate solution consisting of an alcohol component, an acid component and a solvent into contact with the above enzyme to synthesize a fatty acid ester. The concentration of the solution is 2.5 times or more the saturation concentration at 37 ° C, and the initial number of mmol of alcohol component per enzyme synthesis unit is 0.1 or more.

When one of the substrates is a fatty acid ester, water is required for the hydrolysis of the raw material fatty acid ester at the initial stage of the reaction, but this is supplied from the aqueous enzyme solution.

In the present invention, when the substrate as a raw material is a solid at room temperature, a solvent that does not inhibit enzyme activity, such as hexane, heptane, octane, isooctane, cyclohexane, decane, hexadecane, liquid paraffin, squalane, squalene, pristane, and It is necessary to add these mixed solvents, for example, IP Solvent 1016 (manufactured by Idemitsu Petrochemical Co., Ltd.) and Isopar E (manufactured by Exxon Chemical Co., Ltd.) to improve the fluidized state.

The substrate used in the present invention may simply flow and is used in the form of a supersaturated slurry having an undissolved portion having a saturation concentration or higher.

The reactor used in this method is not particularly limited in its shape and may be of any type in which the enzyme and the liquid substrate are sufficiently mixed.

The present invention is particularly suitable for carrying out at a high substrate concentration, and the reaction is carried out in a slurry state which is 2.5 times or more the saturation concentration (about 4% in the case of cholesterol and oleic acid or isooctane).

The substrate consumed by the reaction is replenished by dissolving the undissolved substrate existing in excess in the reaction system as a slurry.

Substrate inhibition was not observed even at concentrations as high as 85%.

Regarding the molar ratio of the substrate, either one may be large, and the larger the ratio, the faster the reaction rate. However, it should be noted that if the molar ratio is too large, the burden on the purification step will increase.

When the substrate ratio (initial mmol of alcohol component / synthetic unit of enzyme) is less than 0.1, the rate of synthesis is sufficiently high, but the frequency of substrate exchange increases and non-production time increases.

On the other hand, the enzyme is unstable in an aqueous solution, and the increase of the non-production time within the duration of the activity lowers the productivity per unit enzyme.

When the substrate ratio is 0.1 or more, it is considered that there is a large excess of substrate in the system, and the turnover of the enzyme-substrate complex proceeds sufficiently quickly. In this case, the time required to reach a predetermined synthesis rate becomes slower as the substrate mass increases, but the number of substrate exchanges decreases, and the non-production time during the activity duration decreases.

What is the substrate ratio?
In consideration of time and cost, it should be determined so that the amount of ester synthesized per enzyme, per hour, per unit device is maximized, but usually 0.1 to 4 is preferable.

(Operation) By stirring and mixing a reactor in which a substrate suspension having a saturation concentration or higher and an enzyme coexist, the enzyme in the system and the dissolved substrate come into contact with each other, and the reaction proceeds. As a result, the dissolved substrate is consumed, but an appropriate amount of the substrate is automatically dissolved and replenished in the reaction system. In addition, by increasing the substrate ratio, non-productive time such as substrate exchange can be reduced, and effective use can be achieved during the active period until deactivation of the short-lived enzyme.

The reaction system becomes a suspension due to the increase in the substrate concentration, and the viscosity increases, but the reaction proceeds promptly until the synthesis rate (W%) reaches about 90%.

When a sterol ester is used as a hydrophilic base for pharmaceuticals and cosmetics, the presence of a small amount of free sterol is preferable, so a synthesis rate of 90% is sufficient, but if a higher purity ester is desired, a further dehydration step is required. By adding, it is possible to easily increase the synthesis rate.

Dehydration is easily achieved by reducing the pressure of the reaction system or blowing an inert gas. In any case, the solvent that is distilled off at the same time as water is condensed in a condenser, left standing in a settling tank to separate the water layer, and then returned to the reaction system by an appropriate means such as using a reflux pipe. It is preferable.

In the following examples, unless otherwise stated, the reaction was carried out at 37 ° C.
The following substrates were used.

Cholesterol (Cho); Cholesterol USP, manufactured by Yoshikawa Oil & Oil Co., Ltd., Oleic acid (OA); Extra olein # 80, manufactured by NOF CORPORATION, acid value = 206 oxygen; Candida syringdracia derived lipase
OF (2076 synthesis unit / g, manufactured by Meito Sangyo Co., Ltd.) The synthesis rate (A%) is TLC-FID (Iatroscan,
(Manufactured by Yatron) to determine the peak area of each component,
The synthesis rate was calculated by the following formula.

A% = ARs × 100 / (ARs + ARc) However, A%; Area standard synthesis rate ARs; Peak area of ester ARc; Peak area of unreacted cholesterol Also, by measuring the standard solution of known concentration in the same manner, the area standard Synthesis rate (A%)-Mole standard synthesis rate (M%)
I calculated the conversion formula.

M% = 0.763 × A% + 5.31 5.5% ≤ A% ≤ 93% Correlation coefficient = 0.996 TLC-FID quantitative method Add an appropriate amount of isooctane to about 1 ml of the collected reaction solution to adjust the concentration appropriately. After that, Chromarod S2 (manufactured by Yatron) is charged with about 10 to 30 μg as a lipid component. This is developed in a solvent system of hexane / ether / formic acid = 56/14 / 0.3 to separate each component. After development, the product is dried at 110 ° C for several minutes to remove the development solvent, and the peak area of each lipid component in the reaction solution is determined by using Iatroscan TH-10.

Further, the weight% (W%) was measured by using HPLC with cholesterol propionate as an internal standard substance under the following conditions.

Column: Microbonder pack C18 3.9φ-15cm Mobile phase: Acetonitrile / IPA / water = 49/49/2, 1.0ml / min (IPA; isopropyl alcohol) Detector: UV # 481 (Waters) 210nm, 1.0AUFS Pump: M600 (Waters) Integrator: D2000 (Hitachi) W% = Ws × 100 / (Ws + Wc) However, W%: Weight-based synthesis rate Ws; Ester weight by HPLC Wc; Unreacted cholesterol by HPLC The enzymatic activity of the enzyme was defined as follows.

Under standard conditions, the titer for synthesizing 1 μmol of cholesterol oleate per minute is 1 synthetic unit (E
U).

Standard conditions Cholesterol 100mg, Oleic acid 219mg, Isooctane
Using 3 ml as the substrate solution, the synthesis rate (A%) is 20% to 60
Phosphate buffer (0.05M pH = 7.0)
Add 8 ml of the enzyme solution prepared in step 2, and incubate at 37 ℃ for 40 minutes. A% is converted into M%, and a synthesis unit is calculated from M%.

Example 1 Using a glass round reactor equipped with a stirrer with an internal volume of 10 L,
Cholesterol 400g, Oleic acid 423g, Isooctane (iO
c) Add 3000 ml as a slurry and add 1.0 g of OF to water 1
It was added after being dissolved in 000 ml.

A half-moon stirring blade (chord length 10 cm) was used to stir at 540 rpm for reaction. The synthesis rate (W%) after 44 hours was 90.0%. After 44 hours, the reaction solution was allowed to stand and after phase separation, the substrate solution layer was removed leaving the aqueous layer and the interface layer. Similarly, the interface layer was washed twice with isooctane, and then renewed with 1/2 amount of the substrate solution and reacted. The synthesis rate (W%) after 68 hours was 86.4%.

The substrate saturation concentration under the above conditions is approximately 4% (the substrate concentration is 37.2
%), And the substrate ratio is 0.498, but it is understood that there is no substrate inhibition under these conditions and that the synthesis proceeds in a sufficiently short time. In addition, the enzyme is oil /
It was adsorbed at the water interface, and it was clear that the remaining water layer retained the activity, and 823 times as many esters were synthesized per unit weight of enzyme in the total of 1 and 2 reactions. I understand.

Example 2 Using the same reactor as in Example 1, cholesterol 400
g, oleic acid 338g, and isooctane (iOc) 1000ml in a slurry state (substrate concentration = 51wt%). OF 0.25g
(Substrate ratio = 1.99) was dissolved in 3000 ml of water and added to react. After 120 hours, the synthesis rate (W%) was 80.2%, and 470 g of ester was synthesized. 1880 times per enzyme weight, per hour,
The synthesis amount per enzyme was 15.7 g / g OF, hr.

To 140 g of the ester synthesized above, 100 ml of isooctane was added, and 100 mg of OF was dissolved in 6 ml of water and added. The temperature of the entire reactor was kept at 45 ° C., and nitrogen gas was blown in to carry out the reaction while stirring and dehydrating. Isooctane, which was distilled off at the same time as water, was condensed by a condenser and then allowed to stand to separate the aqueous layer, and the upper layer solvent was returned to the reactor through a reflux tube.

The synthesis rate (W%) after 96 hours was 95.0%. on the other hand,
When the reaction was performed in the same manner except that the dehydration was not performed, the synthesis rate after 96 hours was 90.0%.

(Effect of the invention) From these examples, per unit enzyme by the method of the present invention,
It is clear that the amount of synthesis per unit of time can be dramatically increased, and the reaction equilibrium can be further promoted by continuously removing the water produced from the reaction system. Obviously, higher final synthesis rates can be obtained if this is possible and required.

Claims (1)

(57) [Claims] 1. A reaction in which a substrate solution containing an alcohol component and an acid component is contact-reacted with the above enzyme using an enzyme selected from lipase and cholesterol esterase to synthesize a fatty acid ester, and the concentration of the substrate solution is 37. A method for synthesizing a fatty acid ester, wherein the concentration is 2.5 times or more the saturation concentration at ℃, and the initial mmol number of alcohol component per enzyme synthesis unit is 0.1 or more.