JP2008148595A - Method for producing useful substance with immobilized enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a useful substance by supplying a liquid mixture forming two liquid phases to a fixed-bed reaction column packed with an immobilized enzyme and reacting the two phases, and capable of producing the useful substance in improved efficiency by improving the reactivity and the productivity of the useful substance without lowering the flow rate. <P>SOLUTION: The invention relates to a method for producing a useful substance by supplying, to a fixed-bed reaction column packed with the immobilized enzyme and having a diameter of ≥35 mmϕ, a liquid mixture forming two liquid phases and reacting the liquid phases by parallelly flowing in the same direction. The fixed-bed reaction column is packed with the immobilized enzyme in a manner that the ratio (column diameter/average particle diameter) of the column diameter (mm) of the fixed bed reaction column to the average particle diameter (mm) of the immobilized enzyme becomes ≤135 (mm/mm). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing a useful substance by a reaction using a fixed bed type reaction column packed with an immobilized enzyme.

  As a reaction performed by passing the liquid through a fixed bed type reaction tower, a reaction using an immobilized enzyme used for L-aspartic acid production, transesterified oil production, lactose hydrolysis, fats hydrolysis, etc. Are known. Since these reactions have a relatively small calorific value, the simplest drum reactor is usually used.

  Among the reactions using immobilized enzymes, when two or more kinds of liquids are circulated through the reaction tower as in the hydrolysis of fats and oils, the reaction liquids are passed in a uniformly mixed state from the viewpoint of improving reaction efficiency. It is preferable to liquefy. In this case, since the oil phase substrate and the water phase substrate used for the hydrolysis do not become a single phase even if they are originally mixed, it is generally used as an emulsion. On the other hand, since emulsion particles are unlikely to reach the enzyme adsorbed in the pores of the carrier, there is a technique in which the liquid passing speed is within a range where the reaction liquid is not emulsified (see Patent Document 1).

In addition, as a method of circulating the oil phase substrate and the aqueous phase substrate through the fixed bed, there are a method of circulating countercurrent (see Patent Literatures 1 and 2) and a method of circulating in parallel flow (see Patent Literature 3). Since the former requires a special mechanism and operation method, generally, a method of circulating in parallel is adopted.
JP-A-61-85195 JP-A-1-98494 JP 2000-160188 A

  In the method of carrying out the reaction by circulating a liquid mixture that forms a two-liquid phase in the fixed bed type reaction tower packed with the immobilized enzyme, the activity of the immobilized enzyme is effectively expressed when the diameter of the reaction tower is small. However, in particular, when the liquid mixture is circulated without being emulsified, it has been found that there is a problem that as the diameter of the reaction tower increases, the expression activity of the enzyme decreases and, as a result, the reactivity decreases. In this case, in order to increase the reactivity, simply increasing the contact time between the immobilized enzyme and the reaction solution has a problem that the productivity (flow rate) decreases.

  Accordingly, the present invention provides a method for producing a useful substance in which a reaction is carried out by circulating a liquid mixture that forms a two-liquid phase through a fixed bed type reaction column packed with an immobilized enzyme. It is intended to provide a method for producing a useful substance more efficiently by enhancing and improving productivity.

  Therefore, the present inventor has conducted various studies on the enzyme expression activity in a fixed bed type reaction column packed with an immobilized enzyme. When a reaction column having a large tube diameter is used, the tube diameter of the reaction column and the immobilized enzyme particles It has been found that by defining the ratio with the diameter, the enzyme activity can be effectively expressed, and the productivity can be improved while maintaining high reactivity. Usually, when an immobilized enzyme is used, a person skilled in the art reduces the particle diameter of the carrier in order to increase the specific surface area of the immobilized carrier if the expression activity of the enzyme is to be improved. The present inventor is effective when the tube diameter of the fixed bed type reaction column is small. However, when the tube diameter is increased, the present inventor should surprisingly take the opposite means for the carrier particle size. Thus, the present invention has been completed.

  That is, the present invention provides a useful substance by supplying a liquid mixture that forms a two-liquid phase to a fixed bed type reaction tower packed with an immobilized enzyme having a tube diameter of 35 mmφ or more, and carrying out the reaction in the same direction. The ratio of the tube diameter (mm) of the fixed bed type reaction tower to the average particle diameter (mm) of the immobilized enzyme (tube diameter / average particle diameter) is 135 (fixed bed type reaction tower). The present invention provides a method for producing a useful substance using a fixed bed type reaction column packed with an immobilized enzyme so as to be equal to or less than (mm / mm).

  According to the present invention, in a reaction performed by supplying a liquid mixture that forms a two-liquid phase to a fixed bed type reaction tower packed with an immobilized enzyme, the enzyme activity is effectively improved even when a reaction tower having a large tube diameter is used. It can be expressed, and useful substances can be produced efficiently.

  In the present invention, it is necessary to supply a liquid mixture (reaction liquid) that forms a two-liquid phase to a fixed bed type reaction tower packed with an immobilized enzyme. A fixed bed type reaction tower (hereinafter also referred to as “enzyme tower”) is a column in which immobilized enzyme is packed so that the reaction solution can be circulated through the gap between the immobilized enzymes and the pores of the immobilized carrier. Say things. The two-liquid phase means a state in which two types of liquids do not become one phase even after mixing, and includes those in which they are in an emulsified state even if they are uniform from those that are phase-separated.

  As an aspect of the present invention, an enzyme in which an oleolytic enzyme is adsorbed on an immobilization carrier is used as an immobilized enzyme, and an oil phase substrate and an aqueous phase substrate are circulated as two liquid phases in an enzyme tower packed with the enzyme. A method of producing fatty acids as useful substances by a hydrolysis reaction of fats and oils is preferable. In the present invention, the term “immobilized enzyme” refers to the whole including the immobilized carrier after the enzyme is adsorbed on the immobilized carrier.

  In the present invention, it is necessary to cause two liquid phases to flow in the same direction. In this case, the two liquid phases may be mixed in advance and supplied as an emulsified state, or may be supplied while being separated. Further, the two liquid phases may be alternately supplied at regular intervals. Each substrate may be supplied to the enzyme tower in a downward flow from the tower top to the tower bottom or in an upward flow from the tower bottom to the tower top.

  The immobilized enzyme used in the present invention is one in which an enzyme is supported on an immobilized carrier by adsorption or the like. As immobilization carriers, Celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics and other inorganic carriers, ceramic powder, polyvinyl alcohol, polypropylene, chitosan, ion exchange resin, hydrophobic adsorption Examples thereof include organic polymers such as resins, chelate resins, and synthetic adsorption resins, and ion exchange resins are particularly preferable from the viewpoint of high water retention. Of the ion exchange resins, a porous material is preferable because it has a large surface area and can increase the amount of adsorbed enzyme.

  The particle diameter of the resin used as the immobilization carrier is preferably 0.1 to 10 mm, more preferably 0.2 to 6 mm, particularly 0.25 to 4 mm, and particularly preferably 0.3 to 2 mm. The pore diameter is preferably 10 to 150 nm, more preferably 10 to 100 nm. Examples of the material include phenol formaldehyde, polystyrene, acrylamide, divinylbenzene, and the like, and phenol formaldehyde resin (for example, Dulite A-568 manufactured by Rohm and Hass) is preferable from the viewpoint of improving the enzyme adsorptivity. The particle diameter of the immobilized enzyme after immobilizing the enzyme is the same as the carrier particle diameter before immobilizing the enzyme in “mm units”.

  The enzyme used for the immobilized enzyme of the present invention is not particularly limited, but lipase is preferred as an enzyme for decomposing oils and fats from the viewpoint of a large productivity improvement effect. As the lipase, not only those derived from animals and plants but also commercially available lipases derived from microorganisms can be used. Examples of microorganism-derived lipases include the genus Risopus, the genus Aspergillus, the genus Mucor, the genus Pseudomonas, the genus Geotrichum and the genus Penicillium and the genus Penicillium The thing of origin is mentioned.

  The temperature at which the enzyme is immobilized can be determined depending on the properties of the enzyme, but is preferably 0 to 60 ° C., particularly 5 to 40 ° C. at which the enzyme is not deactivated. Moreover, the pH of the enzyme solution used at the time of immobilization may be in a range where no denaturation of the enzyme occurs, and can be determined by the characteristics of the enzyme as well as the temperature, but is preferably pH 3-9. In order to maintain this pH, a buffer solution is used. Examples of the buffer solution include an acetate buffer solution, a phosphate buffer solution, and a Tris-HCl buffer solution. The enzyme concentration in the enzyme solution is preferably not more than the saturation solubility of the enzyme and sufficient from the viewpoint of immobilization efficiency. Moreover, the enzyme solution can also use what was refine | purified by the supernatant obtained by removing the insoluble part by centrifugation, ultrafiltration, etc. as needed. Moreover, although the enzyme mass to be used varies depending on the enzyme activity, it is preferably 5 to 1000 mass%, particularly preferably 10 to 500 mass%, based on the mass of the carrier.

  When the enzyme is immobilized, the carrier and the enzyme may be directly adsorbed. However, in order to obtain an adsorption state that expresses high activity, the carrier may be treated with a fat-soluble fatty acid or a derivative thereof in advance before the enzyme adsorption. preferable. As a method for contacting the fat-soluble fatty acid or derivative thereof with the carrier, these may be added directly to water or an organic solvent, but in order to improve dispersibility, the fat-soluble fatty acid or derivative thereof is once dispersed and dissolved in the organic solvent. And then added to a carrier dispersed in water. Examples of the organic solvent include chloroform, hexane, ethanol, and the like. The used mass of the fat-soluble fatty acid or derivative thereof is preferably 1 to 500 mass%, particularly preferably 10 to 200 mass%, based on the mass of the carrier. The contact temperature is preferably 0 to 100 ° C, particularly preferably 20 to 60 ° C, and the contact time is preferably about 5 minutes to 5 hours. The carrier after this treatment is collected by filtration, but may be dried. The drying temperature is preferably room temperature to 100 ° C., and drying under reduced pressure may be performed.

  Among the fat-soluble fatty acids or derivatives thereof for treating the carrier in advance, the fat-soluble fatty acid has a saturated or unsaturated, linear or branched, hydroxy group having 4 to 24 carbon atoms, preferably 8 to 18 carbon atoms. Fatty acids that may be used are listed. Specific examples include capric acid, lauric acid, myristic acid, oleic acid, linoleic acid, α-linolenic acid, ricinoleic acid, isostearic acid and the like. Examples of the fat-soluble fatty acid derivatives include esters of these fat-soluble fatty acids with mono- or polyhydric alcohols or saccharides, phospholipids, and those obtained by adding ethylene oxide to these esters. Specific examples include methyl esters, ethyl esters, monoglycerides, diglycerides, ethylene oxide adducts thereof, polyglycerin esters, sorbitan esters, and sucrose esters of the above fatty acids. These fat-soluble fatty acids and derivatives thereof are preferably liquid at room temperature in terms of immobilizing the enzyme on the carrier. As these fat-soluble fatty acids or derivatives thereof, two or more of the above may be used in combination, and naturally derived fatty acids such as rapeseed fatty acids and soybean fatty acids may be used.

  The hydrolysis activity of the immobilized enzyme is preferably 20 U / g or more, more preferably 100 to 10,000 U / g, and particularly preferably 500 to 5000 U / g. Here, 1 U of enzyme produces 1 μmol of free fatty acid per minute when hydrolyzed at 40 ° C. for 30 minutes while stirring and mixing a mixture of fats and oils: water = 100: 25 (mass ratio). The resolution of the enzyme is shown. The hydrolysis activity (U / g-oil) of the immobilized enzyme imparted per unit mass of fats and oils and the time required to reach a certain hydrolysis rate are in a substantially inverse relationship.

  When hydrolysis is carried out using a packed bed (enzyme tower) filled with an immobilized enzyme, the decomposition rate varies depending on the liquid feeding conditions (liquid passing speed, temperature, etc.), but the hydrolysis rate of fats and oils at the outlet of the enzyme packed bed, The apparent activity (expression activity) of the immobilized enzyme from the time required for hydrolysis (residence time in the packed bed), the mass of fats and oils (g-oil) present in the packed bed and the packed mass (g) of the immobilized enzyme ( U / g). In order to determine the mass of the fats and oils present in the packed bed, the volume of the immobilized enzyme packed part is multiplied by the porosity of the packed part, the volume ratio of the fats and oils in the reaction solution, and the specific gravity of the fats and oils. Ask.

  A preferred type of the liquid mixture forming the two liquid phases in the present invention is an oil phase substrate. The oil phase substrate mainly means vegetable oils, animal oils or oils and combinations thereof, but the oils and fats may contain diacylglycerol, monoacylglycerol, or fatty acids in addition to triacylglycerol. The fatty acid obtained as a result may be included. Specific examples of the oil phase substrate include vegetable oils such as rapeseed oil, soybean oil, sunflower oil, palm oil and linseed oil, animal oils such as beef tallow, pork tallow and fish oil, or a combination of these. These fats and oils can be deodorized oil and non-deodorized fats and oils that have not been deodorized beforehand. However, the use of non-deodorized fats and oils for a part or all of these fats and oils may be trans Saturated fatty acids are preferred, and the plant sterols, plant sterol fatty acid esters, and tocopherols derived from raw oils and fats are preferably left. In the oil phase substrate, oil-soluble components such as fatty acids may be mixed in addition to the oils and fats. Fatty acids refer to fatty acids obtained as a result of hydrolysis, as well as those containing one or more of the above glycerides.

  Another preferred one of the liquid mixtures forming the two liquid phases in the present invention is an aqueous phase substrate. The aqueous phase substrate is water, but other water-soluble components such as glycerin obtained as a result of hydrolysis may be mixed.

  The shape of the enzyme tower used in the present invention is not particularly limited as long as it can withstand the pushing pressure of the pump used. Moreover, it is preferable that a jacket be provided around the enzyme tower so that the reaction liquid flowing in the enzyme tower can be adjusted to a temperature suitable for the enzyme reaction.

  In the present invention, the reaction is carried out using an enzyme tower having a tube diameter of 35 mmφ or more. When the tube diameter of the enzyme tower is less than 35 mmφ, the enzyme activity is less likely to decrease and the reactivity is good. However, as the diameter of the enzyme tower becomes larger than 35 mmφ, the enzyme expression activity decreases, resulting in a reaction. The problem of decreased sex occurs. The tube diameter of the enzyme tower should be 35 to 1000 mmφ, more preferably 35 to 800 mmφ, particularly 40 to 600 mmφ, and particularly 50 to 300 mmφ, in terms of workability, reactivity, productivity, etc. To preferred.

In the present invention, the ratio of the tube diameter (mm) to the average particle size (mm) of the immobilized enzyme (tube diameter / average particle diameter) needs to be 135 (mm / mm) or less. Even when using an enzyme tower with a large tube diameter, by regulating the ratio between the tube diameter of the enzyme tower and the average particle diameter of the immobilized enzyme, it becomes possible to scale up and prevent a decrease in enzyme activity. Useful substances can be produced efficiently.
The tube diameter / average particle diameter is preferably 5 to 135 (mm / mm), more preferably 15 to 130 (mm / mm), and particularly preferably 30 to 125 (mm / mm) from the viewpoint of improving the reactivity. In addition, the average particle diameter of the immobilized enzyme carrying the enzyme in the present invention is a value measured by a laser scattering diffraction particle size distribution analyzer LS 13 320 (manufactured by Beckman Coulter, Inc.).

  The temperature in the enzyme tower is preferably 0 to 60 ° C., more preferably 20 to 40 ° C., in order to extract the activity of the immobilized enzyme more effectively. The length of the enzyme column may be a length necessary for obtaining a desired decomposition rate, but it is in the range of 0.01 to 10 m, preferably 0.1 to 5 m from the viewpoint of reactivity, pressure loss in the column, etc. It is preferable that

As a method of supplying the reaction liquid to the enzyme tower, it may be carried out separately by piping directly connected to the enzyme tower, or may be supplied by a common pipe, but avoiding emulsification of the water phase and the oil phase. From the viewpoints of operability and operability, it is preferably carried out separately by piping directly connected to the enzyme tower.
The flow rate of the reaction liquid in the enzyme tower is preferably 1 to 400 mm / min, more preferably 5 to 200 mm / min. The liquid flow rate (mm / min) is the amount of liquid fed per minute (mm ³ / min) (or liquid feed speed (10 ⁻³ mL / min)) as the packed layer cross-sectional area (mm ² ). The value expressed by the quotient divided by. As the pressure in the packed column increases by increasing the liquid flow rate, liquid passing becomes difficult and an enzyme packed column with high pressure resistance is required. In addition, the immobilized enzyme is crushed by the increased pressure in the column. Since a case may arise, it is preferable that the liquid passing velocity is 400 mm / min or less. Further, from the viewpoint of productivity, it is preferable that the liquid flow rate is 1 mm / min or more. Since the expression activity of the immobilized enzyme changes depending on the flow rate, the reaction can be performed according to the desired production capacity and manufacturing cost by selecting the optimal flow rate and determining the reaction conditions. it can.

  The residence time of the reaction liquid in the enzyme tower is 30 seconds to 60 minutes, and further 1 minute to 40 minutes from the viewpoint of avoiding the equilibrium state of the hydrolysis reaction, more effectively drawing out the activity of the immobilized enzyme, and improving productivity. Minutes are preferred. The residence time (minute) is represented by a value obtained by multiplying the thickness (mm) of the packed bed by the porosity and dividing this by the liquid linear velocity (mm / minute).

  In the present invention, the reaction solution that has passed through the enzyme tower may be used as it is as a reaction-finished product in consideration of reactivity, productivity, and the like. In addition, the reaction solution is once separated into oil and water, and after separating the oil phase, fresh water is collected. May be added again to the same enzyme tower in the same manner as described above, and may be repeatedly passed until a desired reaction rate is obtained. Alternatively, the reaction solution may be once separated into oil and water, and after separating the oil phase, fresh water may be added and supplied again to another enzyme tower by the same method as described above to perform a continuous reaction. In addition, by separating the reaction liquid into water using multiple enzyme towers, the oil phase is supplied to the next enzyme tower and the aqueous phase is supplied to the previous enzyme tower, so that the oil phase with a higher decomposition rate is fresh. You may carry out by the pseudo countercurrent method made to react with an aqueous phase. As an oil-water separation method of the reaction liquid, an oil-water separator such as a natural sedimentation type or a centrifugal separation type is generally used, but is not particularly limited.

[Preparation of immobilized enzyme]
1 part by weight of Duolite A-568 (manufactured by Diamond Shamrock, particle size distribution: 100 to 1000 μm) was stirred in 10 parts by weight of N / 10 NaOH solution for 1 hour. After filtration, it was washed with 10 parts by mass of ion exchange water, and the pH was equilibrated with 10 parts by mass of 500 mM acetate buffer (pH 7). Thereafter, the pH was equilibrated twice for 2 hours with 10 parts by mass of 50 mM acetate buffer (pH 7). Thereafter, filtration was performed to recover the carrier, and then ethanol substitution was performed with 5 parts by mass of ethanol for 30 minutes. After filtration, 5 parts by mass of ethanol containing 1 part by mass of ricinoleic acid was added and ricinoleic acid was adsorbed on the carrier for 30 minutes. After recovering the carrier by filtration, it was washed with 5 parts by mass of 50 mM acetate buffer (pH 7) four times for 30 minutes, ethanol was removed, and the carrier was recovered by filtration. Thereafter, 1 part by mass of a commercially available lipase (Lipase AY, Amano Amano Pharmaceutical Co., Ltd.) was contacted with an enzyme solution dissolved in 9 parts by mass of 50 mM acetate buffer (pH 7) for 5 hours for immobilization. Filtration was performed, and the immobilized enzyme was recovered and washed with 10 parts by mass of 50 mM acetate buffer (pH 7) to remove unimmobilized enzyme and protein. Thereafter, 4 parts by mass of soybean oil to be actually decomposed was added and stirred for 12 hours. All the above operations were performed at 20 ° C. Then, it filtered and isolate | separated from fats and oils, and was set as the immobilized enzyme (henceforth the immobilized enzyme A). Furthermore, an immobilized enzyme was prepared in the same manner as described above using an immobilized carrier obtained by pulverizing and classifying Duolite A-568 and an immobilized carrier obtained by classifying Duolite A-568 and removing fine particles of 425 μm or less ( Respectively, immobilized enzyme B and immobilized enzyme C). Table 1 shows the hydrolysis activity (activity to be expressed) of the immobilized enzymes A to C and the average particle diameter based on mass.

<Example 1>
A stainless steel column with a jacket (inner diameter 35 mm, height 1600 mm) was packed with 350 g of immobilized enzyme A (dry mass) (packing height 1500 mm) and kept at 35 ° C. with the jacket. A liquid in which rapeseed oil and distilled water were mixed at a mass ratio of 10: 6 was fed at 1.1 kg / Hr from the top of the column to conduct a hydrolysis reaction. The results are shown in Table 2. The decomposition rate in the table was calculated by dividing the acid value obtained by analysis by the saponification value. In addition, an acid value is American Oil Chemist. According to the method described in Society Official Method Ca 5a-40, the saponification value was determined by American Oil Chemists. It was measured by the method described in Society Official Method Cd 3a-94.

<Example 2>
A stainless steel column with a jacket (inner diameter 55 mm, height 1600 mm) was charged with 865 g of immobilized enzyme A on a dry basis (packing height 1500 mm), and kept at 35 ° C. with the jacket. A liquid in which rapeseed oil and distilled water were mixed at a mass ratio of 10: 6 was fed from the top of the column at 2.7 kg / Hr to conduct a hydrolysis reaction. The results are shown in Table 2.

<Example 3>
The hydrolysis reaction was performed in the same manner as in Example 1 except that the immobilized enzyme A in Example 1 was replaced with the immobilized enzyme B. The results are shown in Table 2.

<Example 4>
A stainless steel column with a jacket (inner diameter 70 mm, height 1600 mm) was charged with 1400 g of immobilized enzyme C on a dry basis (packing height 1500 mm), and kept at 35 ° C. with the jacket. A liquid in which rapeseed oil and distilled water were mixed at a mass ratio of 10: 6 was fed from the top of the column at 4.3 kg / Hr to conduct a hydrolysis reaction. The results are shown in Table 2.

<Comparative Example 1>
A hydrolysis reaction was performed in the same manner as in Example 4 except that the immobilized enzyme C in Example 4 was replaced with the immobilized enzyme A. The results are shown in Table 2.

<Comparative example 2>
The hydrolysis reaction was performed in the same manner as in Example 2 except that the immobilized enzyme A in Example 2 was replaced with the immobilized enzyme B. The results are shown in Table 2.

<Comparative Example 3>
The hydrolysis reaction was performed in the same manner as in Example 4 except that the immobilized enzyme C in Example 4 was replaced with the immobilized enzyme B. The results are shown in Table 2.

  From the results shown in Table 2, even when a fixed bed type reaction tower having a tube diameter of 35 mmφ or more was used, the ratio of the tube diameter to the average particle diameter of the immobilized enzyme (tube diameter / average particle diameter) It has been clarified that the degradation rate is improved and the (apparent) activity of the immobilized enzyme is effectively expressed by filling the immobilized enzyme so as to be 135 or less.

Claims (5)

  This is a method for producing useful substances by supplying a liquid mixture that forms two liquid phases to a fixed bed type reaction tower packed with an immobilized enzyme having a tube diameter of 35 mmφ or more, and carrying out reactions in the same direction. As a fixed bed type reaction tower, the ratio of the pipe diameter (mm) of the fixed bed type reaction tower to the average particle diameter (mm) of the immobilized enzyme (tube diameter / average particle diameter) is 135 (mm / mm) or less. A method for producing a useful substance using a fixed bed type reaction column packed with an immobilized enzyme so that   The method for producing a useful substance according to claim 1, wherein one of the liquid mixtures is an oil phase substrate.   The method for producing a useful substance according to claim 1 or 2, wherein one kind of the liquid mixture is an aqueous phase substrate.   The method for producing a useful substance according to any one of claims 1 to 3, wherein the reaction is a hydrolysis reaction of fats and oils using an immobilized lipase.   Useful substances are fatty acids, The manufacturing method of the useful substance of any one of Claims 1-4.