JPH0697998B2 - Method for producing immobilized lipase preparation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing an immobilized lipase preparation.

[Prior art]

Immobilized lipase preparations suitable for transesterification of fats are known. That is, in US Pat.
An immobilized lipase preparation is disclosed, in which the lipase is Rhizopups genus Geotricum.
hum) or a species belonging to the genus Aspergillus, obtained by fermenting, and the resulting lipase is then bound to an inert individual carrier of diatomaceous earth or alumina. These carriers have a very high specific surface area. It was believed that it was necessary to use immobilized lipase preparations with very high specific surface areas (ie small and porous carrier particles) in order to obtain high enzyme activity.

Although transesterification can be carried out batchwise without solvent using such immobilized lipase preparations, continuous transesterification on the column does not require the presence of solvent, which must be removed later. It cannot be done on an industrial scale. This is because preparations of small particles during column operation can produce unacceptably high pressure drops.
In European Patent Application Publication 069599 the enzymatic rearrangement of fat is carried on a carrier such as celite, Aspergillus sp., Rhizopus
Seed to be carried out using a lipase of the species Mucor javanicus or Mucor miehei, and all examples of continuous transesterification on columns are solvent-based. Should be noted. Immobilized lipases that are compatible with column operations for transesterification of fats without solvent will be appreciated by those skilled in the art.

Of course, the person skilled in the art has studied numerous immobilizations by ionic, covalent and uptake. For example, the inventors of US Pat. No. 4,170,696 are aware of the following contents.
Thus, the controlled size distribution and physical properties of the macropore ion exchange resin provide advantages for enzyme support purposes. However, these inventors objected to a small amount of an enzyme capable of binding per unit weight of the ion exchange resin carrier, and proposed the use of a resin diethylaminoethyl (DEAE) derivative for the purpose of supporting the enzyme. is doing. The teaching as claimed in US Pat. No. 4,170,696 for the effect that a particular enzyme immobilization method can be applied to a large number of enzyme systems can be criticized as it is misleading to those skilled in the art. The difference between enzyme and enzyme and the difference between substrate and substrate actually make the system of enzyme / substrate / reaction conditions exceptional, and the technology described as being widely applicable is hardly or not applicable at all. Ah

In particular, lipase is an exceptional enzyme in the following points. That is, their enzymatic activities function at the interface between two phases (water and oil), and when they are used alone, immobilization methods that are sufficiently compatible with other enzymes can be expected to have little or no application to lipases. I know there isn't. The person skilled in the art is aware that immobilization of lipases presents particular difficulties. For example, J. Lavayr
"Preparation and Property of Immobilized Lipase" by e) et al., Biotechnology and Bioengineering, 24, 1007-10
See John Willey and Sons, p. 13 (1982). Researchers in the industry have studied the specific immobilization of lipases by adsorption or ionic binding, covalently and by uptake, with adsorption (on celite) and subsequent uptake of celite particles giving much better results. 20-25 September 1981 Kashikojima (Japan) Enz. Eng. 6 poster and European Journa
l of Applied Microbiology and Biotechnology, NO14,
See their corresponding papers listed on pages 1-5 (1982), as well as a series of papers in the same journal, NO17, pages 107-112 (1983).

The prior art described above is exemplary by those skilled in the art to obtain immobilized lipase suitable for industrial use by easy column manipulation. To the best of our knowledge, Danish patent applications 4025/83 and 4167/84
The lipase product described in 1. constitutes a substantially superior lipase product for column operation. It is now possible to extend the teachings described in Danish patent applications 4025/83 and 4167/84, and furthermore the lipase products according to the invention are somewhat different from the lipase products described above and their excellent In terms of sex, it outperforms prior art lipase products.

[Structure and Effect of Invention]

According to the present invention, it has now been surprisingly found that: That is, the preparation of the immobilized lipase preparation can be easily carried out by simply mixing the aqueous lipase solution with the granular macropore phenol formaldehyde adsorbing resin, recovering the resin and then drying. By setting the water content to a specific ratio in the final fixed preparation, continuous transesterification of fat can be performed without using a solvent by an economically easy method. It is advantageous to wash the resin with water after removing the spen trypase solution.

According to a preferred embodiment of the process according to the invention, thermostable microbial lipases are used, thermophilic mucor (Mucor) species, in particular mucor miehei-produced lipases. Mucor miehei is a good producer of 1,3-specific lipase, and thus low cost production can be achieved.

According to a preferred embodiment of the method of the present invention, 90% or more of the macroporous phenol formaldehyde adsorbent resin particles have a particle size of about 100-1000 μm, preferably 400-800 μm. At this particle size interval, a good compromise between high unit activity for transesterification and low column pressure drop is obtained.

According to a preferred embodiment of the process according to the invention, the ratio of the amount of aqueous solution of microbial lipase to the weight of adsorbent resin corresponds to 5,000 to 50,000 LU / g of resin (dry weight). In this range, sufficient lipase for commercially available resins is obtained.

In a preferred embodiment of the method of the present invention, where the microbial lipase is a microbial lipase produced from a hyperthermophilic Mucor species, in particular Mucor miehei, the pH during contact of the adsorbent with an aqueous lipase solution is preferred.
Is between 5 and 7. A strong bond between the lipase and the adsorbent resin can be secured.

According to a preferred embodiment of the method of the present invention, the contact time is from 0.5 to
8 hours. In this time range, the resin approaches lipase saturation. At least 75% of the lipase activity is removed from the lipase solution.

According to a preferred embodiment of the process according to the invention, the separation is carried out by filtration, the simple procedure being well suited for industrial practice.

According to a preferred embodiment of the method of the present invention, the separated immobilized lipase is dried to a water content of about 2-40%, more preferably 5-20%. This gives a final lipase preparation with a high transesterification effect.

Thus, the process according to the invention for producing immobilized lipase preparations is carried out in Danish patent application 4025/83, except for the different resins.
And 4167/84. That is, in the Danish application, an aqueous solution of microbial lipase is contacted with a granular macroporous resin, but in the present invention it is contacted with a phenol formaldehyde matrix adsorbent resin. A relatively large average resin particle size is used to obtain a final product suitable for column operation without excessive pressure drop. The reaction conditions and reaction time for the lipase to bind to the adsorbent are sufficient to bind the desired amount of lipase to the adsorbent resin, after which the immobilized lipase formed is separated from the aqueous phase and then separated from the adsorbent. The resin-containing lipase is preferably dried to the desired moisture content of about 2-20%. The drying operation may be a vacuum, fluid bed drying process or any other drying step suitable for large scale operation such that the immobilized lipase is not at a temperature level such that the lipase is inactivated.

The phrase adsorbent resin as used in the present invention is one selected from the ion exchange resin industry. Ion exchange resin manufacturers have found the following contents. That is, certain morphological resin products that have acted on an active group that provides anion or cation exchange activity have an active group that is not commercially useful. Such a resin form is commercially provided as an "adsorbent resin". These commercial materials and other similar or equivalents in physical and chemical properties are contemplated herein as the phrase adsorbent resin. The organoleptic properties of adsorbent resins are primarily attributed to binding strength rather than ionicity.

Adsorbent resins known to the inventor include various polystyrene and phenol formaldehyde matrices. The practice of the present invention contemplates phenol formaldehyde class adsorbent resins. Polystyrene class adsorbent resins are not intended. This is because they are carriers with little advantage for lipase.

The lipase products of the present invention exhibit comparable thermal and physical stability to the products described in Danish patent applications 4025/83 and 4167/84 and can be used instead.

In order not to inactivate the enzyme, prior art transesterification reactions are carried out at relatively low temperature levels, which is made possible by the presence of a solvent capable of dissolving high melting fats. The immobilized lipase preparation produced according to the present invention is sufficiently stable in molten fat and thus catalyzes transesterification at relatively high temperatures. Also, the transesterification column packed with the immobilized lipase preparation obtained by the method of the present invention is sufficiently low to allow a smooth column operation for transesterification. Also, surprisingly, the following contents were found. Thus, the combination of contact conditions, adsorbent resin with phenol formaldehyde matrix and controlled final water content in the immobilized lipase preparation results in a high specific lipase activity in the molten fat mixture.

The products produced by the practice of the present invention are the same as those described herein in Danish patent applications 4025/83 and 416.
The same praise words used on 7/84 are listed above. Although the products are comparable and generally comparable products, both have shown advantages in the art. However, differences do exist and such differences make one product type preferred for use in a particular lipase-catalyzed process.

The immobilized lipase preparation produced by the practice of the present invention can be prepared with high enzyme recovery, the results of which are important for achieving a low cost (continuous) transesterification process.

Greater than 75% of the initial lipase activity present in the lipase solution can and should be removed from the solution by the adsorbent resin. To some extent, lipase absorption by the resin is time dependent. This is probably because it takes a certain amount of time for the enzyme molecules to disperse in the macroporous resin particles. At least an adsorbent resin equilibrium will be approached after a mixing (reaction) time of 8 hours. Acceptable results are obtained even in short times as short as 0.5 hours. A rational way to select the optimal contact time is to remove the lipase activity from the lipase solution. 75% from lipase solution
Any contact time required to remove the above lipase activity can be the reaction time for large scale practice of the invention.

The following contents are noted. That is, the available experimental evidence shows that the optimum contact time is almost independent of the contact time within the range of 5-35 ° C, and further the temperature range of 5-35 ° C is contemplated for the practice of the present invention. To be done.

The physical and chemical differences between the various phenol formaldehyde adsorbent resins can, of course, be expected to affect both the immobilization reaction and the immobilized lipase preparation. As would be expected, all experimental work done to achieve the present invention involved commercially available macroporous adsorbent resins, but with limitations not known to the inventors. It will be in commercial resin. The final immobilized lipase product obtained by the practice of the present invention is 5,000-50,00.
It shows a lipase activity with 0 LU / gm (dry basis), which constitutes a preferred range. A unit activity of 10 BIU / g or more is obtained.

The feature of washing the still moist resin particles separated from the spentripase solution with water is particularly advantageous when the available lipase comprises a relatively crude lipase.

The prior art process described in US Pat. No. 4,275,081 required purified lipase to provide a ready-to-use immobilized lipase preparation. Surprisingly, the following contents were found. Thus, the immobilized lipase preparations obtained by the practice of the present invention can be produced directly from fairly crude lipase products. Obviously, the impurities that were desired to be removed from lipases in the prior art are not bound to the adsorbent and are therefore removed in the spentlipase solution and / or in the wash water.

Above all, the following procedure yields significantly advantageous results.
That is, basically, it is only necessary to mix the particulate adsorbent resin into a fairly crude lipase solution of pH 5 to 7 at room temperature, not even the use of organic solvents (sometimes proposed), The spent solution is then discarded and the resin preferably washed with water, followed by drying the resin to a controlled water content. Washing the separated immobilized enzyme with water before drying is repeated to significantly improve the product.

The following contents were found. That is, the immobilized lipase preparations obtained by the practice of the present invention do not readily inactivate or leave the preparations except at bad pH and / or temperature conditions. For example, lipase activity does not appear in the wash liquor.

The following contents are mentioned. That is, a preferred embodiment of the invention contemplates immobilizing thermostable lipases, particularly Mucor miehei lipases. Of course, thermal stability to lipases is important for solvent-free transesterification of high melting point fats. If the transesterification is carried out at the most reasonable elevated temperatures, other advantages also occur, such as the possibility of reducing bacterial contamination and the reduction of fat viscosity.

In fact, many of the advantages described above over the prior art are practically impaired for practical implementation of the invention on a large scale. Similar advantages apply to Danish applications 4025/83 and 4
167/84 for the practice of the invention described by the inventor. The invention could instead be implemented.

The decision on the large-scale implementation of the invention is made in Danish application 40
It will depend on its relative advantages over the preferred embodiment for the invention of 25/83 and 4167/84. Accordingly, the preferred form of process and product conditions may be found to be practical, even if arbitrarily limiting the scope of the practice of the invention. Clearly, the removal of lipase activity from the aqueous treatment solution by the adsorbent should exceed 75%. The immobilized enzyme product has a lipase activity unit of 5000 LU / g
m (dry basis) (NOVU method AF 95.1 / 2-GB) should be exceeded. The already mentioned range of 5,000 to 50,000 LU / gm is preferred. BIU / g (NOVO method AF-26) should exceed 10.
The half-life of the product should exceed 2500 hours at 60 ° C.

The above numerical range is also useful for distinguishing the present invention from the methods which are clearly unexpected according to the present invention, such as the use of polystyrene adsorbent resin and the method which follows from the description of the present invention.

Examining the mechanisms by which the advantageous immobilized lipase products described in Danish applications 4025/83 and 4167/84 can be produced, not all of these advantages contribute to the weak anion exchange capacity of the resin on which the lipase is immobilized. I realized that it was not possible. The ability of the resin to bind the lipase may contribute in part to anion exchange, some to other physical forces between the lipase and the resin, and some to the chemical and physical structure of the resin matrix. I think that the.

The test results obtained from immobilizations on three typically commercially available substances exemplify the relationships believed to exist.

Since many relationships are involved, many (arbitrary) criteria for the practice of the invention mirror many relationships. The minimum unit activity of immobilized lipase preparation is 5000 LU
The / gm (dry basis) and 10 BIU / gm (dry basis) reflect the need for constant minimum loading to obtain activity against larger carrier particles.

It can be seen that prior art proposals for the formation of immobilized lipase products are changing due to anionic attractive forces in the carrier, specific interactions and the cumulative effects of the matrix material (e.g. U.S. Pat. 4,4
51,565, example 12). In fact, immobilization of lipase on any carrier material will obviously yield a non-uniform product. However, the standards set by the parameters of the present invention are: 75% yield, 5,000 LU / gm and 10 BIU /
g is a similar carrier, but the invention or its parent patent application number 6
It would not be readily achievable outside the claims of 46,752. The low yields and activities obtained with typical polystyrene-based adsorbent resins are shown in the table above. Strong base polystyrene anion exchange resin in a similar test
12BIU / g product was obtained, but the yield was only 50%.

It has now been found that the resins described in the present invention and in Danish applications 4025/83 and 4167/84 have pore sizes and porosities suitable for the practice of the present invention. Testing with large pore size anion exchange and adsorbent resins resulted in products with low unit activity as predicted from the yield results.
Lipases are believed to penetrate deeply into the pores of very large pore size anion exchange and adsorbents, where they lose their action. Certain physical interactions and anionic bonds appear to be an independent source of binding capacity for the resin. The products shown in Table 1 above are A (1% Triton
X-100) and C (1M sodium acetate, pH 6). Condition A tends to remove only non-ionically bound lipase, and condition C tends to remove only ionically bound lipase. The results are shown below.

In contrast to the results of the partial extractions listed in Table 2 above, the lipase adsorbed on Celite was virtually 100% extracted with pure water within a few minutes.

The broadest process adaptability and / or the best yield and highest unit activity product obtained according to the invention is due to the weak anion exchange resin, ie Danish application 4025/83.
And 4167/84, but with high yields and acceptable high unit activity products, can be obtained with a macroporous adsorbent resin of phenol formaldehyde matrix. An exemplary best mode lipase product for such an adsorbent resin showed a half-life of greater than 3000 hours (Durite S761, an implication for this resin was already made in the previous table). If anything, the inventor will have a reasonable choice for carrying out the invention described in Danish applications 4025/83 and 4167/84, but not all circumstances are equal. The second table demonstrates that there are de facto product differences. Product differences would be more favorable for some lipase-catalyzed reactions of the products obtained consistent with the practice of the invention. The practice of the present invention and the practice of Danish applications 4025/83 and 4167/84 is provided to the industry as a closely related invention in a near or equal position.

The temperature range for carrying out the transesterification according to the process of the invention is 25 ° C to 85 ° C, preferably 50 ° C to 80 ° C, especially 55 ° C to 75 ° C.
℃. Please refer to the attached drawings. In this figure, the important features of the immobilized enzyme of the illustratively preferred embodiment for the packed bed (column) continuous transesterification described below are depicted. FIG. 1 shows the logarithm of the flow rate (g / hr) shown with respect to time (hour) in the operation at 60 ° C.

The lipase activity unit (LU) given in the examples below to illustrate the method of the invention is described in AF95.1 of publication 83-01-03 available from Novo Industry A / S (Novoales, 2880 Basugbaert, Denmark). Measured as described in / 2-GB.

〔Example〕

Example 1 This example illustrates the immobilization of lipase according to the invention and the characteristics of the immobilized enzyme.

Fixed 124,000 LU / g of Mucor miehei 1g 1
Dissolved in 0 ml water and then mixed with 4.25 g (dry matter) of Duolite S-761 resin, adjusted to pH 6.0 beforehand in an aqueous suspension (batch DE 3.12.7002, lot 81312 E4, 85% granules) Diameter 4
25-825 μm). The pH was then adjusted to 6.0 and the bottle containing the mixture was further stirred for 2 hours at room temperature, during which time the pH was readjusted after 1 hour of rotation.

The resin was filtered, washed twice with 10 ml of water and then vacuum dried at room temperature for one day.

31 ml of the combined filtrate at pH 6.3 has 700 LU / ml, which is about 17.5% of the original total enzyme.

The yield of immobilized lipase was 4.91% in 94.3% dried form. The calculated load was 22,000 LU / g (dry).

Batch Transesterification Activity This assay was performed according to International Novo Method AF206, described below. The activity was 20.3 BIU / g.

Column test 30 g of pre-column, Duolite A561, 86% dry matter hydrated with 21 g of water are packed in a 2.5 x 25 cm column with petroleum ether.

Lipase column 3.5 g of the above immobilized lipase together with petroleum ether 1.5 x 2
Pack in a 5 cm column.

A 2.5 to 1 w / w ratio of olive oil (Sigma 0-1500) / decanoic acid (Merck) stabilized with substrate 0.1% BHT is used. The substrate is stirred in the reservoir at 60 ° C and then saturated with water. Introduction into decane D00-triglyceride in triolein (main triglyceride in olive). Pump the column downwards while displacing the substrate with petroleum ether.

Analysis About 65% conversion of OOO + P to DOO + P in the product flow
Adjust the flow rate to obtain 23% DOO. A sample of 7-8 ml is weighed and the flow is calculated in g / h.

For triglyceride (TG) composition analysis, 4 drops of 7 drops of sample
Dissolve in l of heptane and then load the column with 12 g of active Al ₂ O _{3 in} heptane. Elution is carried out with 15 ml of ether, the ether is evaporated and the residue is redissolved in 1.5 ml of heptane. Operating on AF206 HPLC as described below, the DOO and DOD peaks appear at 5.6 and 3.9 minutes, respectively.

The water content of the product stream is checked by Karl Fischer titration.

Column operation Samples were taken at 2 and 3 day intervals. Per hour
The flow rate of 1 g of substrate was recalculated to gTG / hg enzyme (F) units as activity units. The productivity of gTG / g enzyme (unit of P was also calculated. The temperature had a standard deviation of 0.4 ° C and averaged 60.3 ° C.

The water content in the product dropped from 0.36% to 0.28% after 400 hours. The precolumn was then replaced and the water dropped from 0.50 to 0.31% over 400 hours. Results within the conversion range of 23 ± 1% DOO were used for the semilogarithmic plot of flow rate versus time. From this plot, the initial activity is Finit = 1.94gTG / h
It was estimated to be a g-enzyme product catalyst, which has a half-life of 3100 hours at 60 ° C. FIG. 1 is a plot of the test results.

The productivity, with two half-life operating times, should be 6.5 ton TG / kg enzyme product catalyst.

Example 2 This example shows the immobilization of a lipase according to the invention on two similar phenolic resins with or without high ionic strength during immobilization.

The principle of measurement of lipase transesterification activity (BIU) by the Novo method AF206 is the introduction of free palmitic acid (P) in pure triolein (OOO), where the initial rate is calculated for this equilibrium ratio: OOO + PPOO + O POO + PPOP + P If the lipase (for example as rucor mayay lipase) is 1,3 specific, then no PPP will be formed.

The value Pinc is calculated with the reaction time given as the amount of palmitic acid in positions 1 and 3 of the triglyceride: This curve fits an exponential model with an initial activity of 40 ° C to 1 BIU
Calculated as 1 μmole of P introduced into OOO every minute.

678 μmole OOO and 678 μmole P in 12 ml petroleum ether
And are mixed with 250 mg dry matter, hydrated in 10% water. The mixture is shaken for 15 minutes and / or 30 minutes at 40 ° C. and the relevant composition of OOO, POO and POP is determined by HPLC.

Pinc, eq is the equilibrium Pinc (molar fraction) and is ˜0.22.

Pinc is calculated as stated in mole fraction.

M is the molar amount of OOO and P and is ˜678 μmole.

t is time (minutes).

M is the dry weight of the catalyst (0.250 g).

Calculation of conversion, activity, half-life and column test productivity Column or flow activity F = Fo · exp (−ln2-t / t1 / 2) [gTG / h · catalyst (g)] Fo: initial activity, t1 / 2 = half-life triglyceride (g) / hour · catalyst (g Units are derived from the substrate flow in g / hr by knowing the amount of triglyceride in the substrate and the amount of catalyst in the column.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between substrate flow and time.

Claims (5)

[Claims] 1. A method for producing an immobilized enzyme preparation suitable for transesterification of fat, which comprises an aqueous solution of mucor mayay lipase and 90% or more of resin particles of 100 to 1000 μm in the form of granular macroporous phenol. The formaldehyde adsorbent resin is contacted at a pH in the range of 5-7 for a time sufficient to remove at least 75% of the lipase activity from the solution, thereby binding the desired amount of lipase to the resin, after which this The immobilized enzyme thus formed is separated from the aqueous-phase spentripase solution, and then the separated immobilized enzyme is dried to a water content of about 5 to 20%. Such a method having at least 10 BIU and at least 5000 LU per gram dry basis. 2. The method according to claim 1, wherein the immobilized lipase is washed with water after being separated from the spentlipase solution. 3. The method of claim 1 wherein 90% or more of the particles of macroporous adsorbent resin have a particle size of about 400 to about 800 μm. 4. The method according to claim 1, wherein the aqueous solution of mucor mayay lipase has 5,000 to 20,000 LU per gram of dry adsorbent resin. 5. The method according to claim 1, wherein the contact time is 0.5 to 4 hours.