CH617458A5 - Use of filamentary structures containing encapsulated fumarase for the enzymatic production of l-malic acid - Google Patents

Description

617458

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1. Use of filamentary structures containing fumarate incorporated and obtained starting from polymers selected from esterified or nitrated cellulosic polymers, polyolefins, polymers and copolymers of acrylonitrile, acrylates, methacrylates, vinyl esters, vinyl chloride, vinylidene chloride, styrene, polyamide -mide or polyvinyl buttirral for the enzymatic production of 1-malic acid, characterized by the fact of bringing saline solutions of fumaric acid into contact with said structures.

Use according to claim 1, characterized in that the filamentous structure is obtained starting from cellulose triacetate.

The object of the present invention is the use of filamentous structures containing fumarate in globate and obtained starting from polymers selected from esterified or nitrated cellulosic polymers, polyolefins, polymers and copolymers of acrylonitrile, acrylates, methacrylates, vinyl esters, vinyl chloride, chloride of vinylidine, styrene, polyamide or polyvinyl butyral for the enzymatic production of 1-malic acid.

The enzyme extracted from various sources is used as a catalyst in a heterogeneous form as it is immobilized in filamentous structures from the inside of which it cannot escape while still being accessible to the substrate.

The interest developed around organic acids such as citric acid, tartaric etc. is now known. used in the food industry as acidulants.

We have recently looked at malic acid to be used in applications of the same type and even if the use of the racemic compound has been proposed, it is clear that being able to dispose of the natural isomer, i.e. L-malic which is moreover produced in considerable quantities in the human body, as an intermediate of one of the most important metabolic cycles, it constitutes a safety especially when food is intended for children.

Various methods have been proposed for the production of acid, malic. On the one hand, there are chemical synthesis methods that lead to the racemic compound with the disadvantages mentioned above; moreover the malic acid coming from the chemical synthesis always contains as impurity of the maleic acid that must be eliminated, with relatively expensive procedures, given the strong toxic character.

Other methods are the microbiological ones which, if they have the advantage of producing the natural levo compound, are expensive and laborious; the maintenance of the microbial strain, the fermentation that uses bulky and expensive plants as well as the use of raw materials, compressed air and steam are as many disadvantages of the methods mentioned above. Furthermore, the separation of the malic acid from the fermentation broths involves some difficulties due to the fact that the product is found together with several impurities. One could think of an enzymatic method using the soluble enzyme, however this has several disadvantages: pollution of the final product, the need to inactivate the enzyme, use of considerable quantities of enzyme that is used only once and therefore strongly affects the cost.

The use of filamentary structures containing fumarate in globate for the enzymatic production of 1-malic acid is defined in claim 1.

The use of the present invention allows to obtain the enzymatic hydration of fumaric acid to 1-malic acid with remarkable simplicity; the system consists of a simple column; the enzyme, being immobilized, can be reused for an unknown number, however large in number of times, and does not pollute the product; the latter can be easily separated from the reaction mixture.

The filamentous structures that can be used and the method of incorporation of the enzyme are those described in Italian patent no. 836 462 according to which the enzyme incorporating fibers can be prepared starting from polymer solutions capable of giving fibers in which enzymatic preparations are dispersed in the form of minute droplets of the order of magnitude of the emulsions.

The emulsion thus obtained can be spun wet or dry to give a fiber which has very small cavities inside which the enzymes are arranged which are separated from the environment by a very thin membrane, which prevents the enzyme from escaping and its dispersion in the reaction mass and yet allows the enzyme to carry out its catalytic activity.

Among the polymers that can incorporate enzymes, the esterified cellulosic polymers, nitrates, polyolefins, the polymers and copolymers obtained from acrylonitrile, acrylates, methacrylates, vinyl esters, vinyl chloride, vinylidene chloride, styrene, and also polyamides, can be mentioned. butyral polyvinyl and the like.

With the enzymatic fibers thus obtained, arranged in a column, it is possible to proceed with the transformation of fumaric acid into 1-malic with a continuous system that can be easily automated.

A pump takes the sodium fumarate solution, (whose concentration can vary between 0.1 and 1.2 moles / lt and whose pH between 6.0 and 9.0) and sends it to the column containing the catalyst. Depending on the flow and temperature at which one operates, a concentration of 1-malic is obtained in the effluent which at most reaches the values of the thermodynamic equilibrium.

The unprocessed fumaric acid is separated from the effluent by acidification and is recycled; 1-malic can be recovered with various methods and in the pure crystalline state or in the concentrated syrup state.

The following examples serve to illustrate the present invention, but are not limitative of it.

Example 1

They are dissolved under stirring, in a reactor, gr. 10 of cellulose triacetate in gr. 140 of methylene chloride.

The enzyme solution is prepared separately by dissolving the fumarase in a phosphate buffer pH 7.2 at a concentration of 20 mg of protein per ml.

10 ml of enzymatic solution are added to the organic triacetate solution and the emulsion of the two phases is favored with stirring energy at 0 ° C for 30 min.

The emulsion is poured into a small fuser kept at -6 ° C and spun under nitrogen pressure.

The thread coagulates in toluene at room temperature and is collected in a winder. The yarn obtained to remove the organic solvents is dried under vacuum.

A sample of 2 gr. of thread, after washing with phosphate buffer, pH 7.2, is immersed in 50 ml of 0.10 molar solution of sodium fumarate.

The mixture is stirred at a controlled temperature of 25 ° C.

After 90 minutes, 20.5% of the initial fumaric acid is dosed in the reaction mixture. The remaining 79.5% was transformed into 1-malic.

The same sample of thread after being used for 10 consecutive times retains about 40% of the initial activity.

Example 2

Following the procedure described in example no. 1 incorporates a crude enzyme fumarase preparation obtained from Pa-racolobactrum aerogenoides microbial cells into threads of cellulose triacetate. 450 grams of wet cells obtained with traditional submerged aerobic fermentation methods are subjected to the action of ultrasound for

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20 '; nucleic acids are removed with MnCl2 and the enzyme is precipitated with ammonium sulphate between 40 and 60% saturation. The precipitate is redissolved in 0.01 molar phosphate buffer, pH 7.0. The solution contains 49 mg of protein per ml and an activity corresponding to 45 U / ml (the quantity of enzyme that transforms 1 jj, M of substrate at min. At 25 ° C is defined as a unit of activity).

100 ml of this enzymatic solution are incorporated in thread as described in Example 1.

A sample of thread equal to 85 gr is placed in a column (65 X 3 cm), thermostated at 25 ° C, a solution of 0.5 molar sodium fumarate in phosphate buffer is sent from the bottom upwards, pH 7.0 , at a flow of 50 ml / h.

In the effluent the conversion of 54% of fumaric acid into 1-malic is obtained.

After 35 days of uninterrupted use of the same fiber, the conversion of fumaric acid to malic drops to 38%.

Example 3

Following the procedure described in example no. 1 incorporates a crude enzyme fumarase preparation obtained from microbial cells of Pseudomonas sp. In threads of cellulose triacetate. 315 grams of wet cells obtained with traditional submerged aerobic fermentation methods are subjected to ultrasound for 20 '; nucleic acids are removed with protamine sulfate and the enzyme is precipitated between 30 and 60% saturation with ammonium sulphate. The precipitate containing the activity is dissolved in 0.1 molar phosphate buffer, pH 7.0.

The solution contains 61.5 mg of protein per ml and an activity corresponding to 80 U / ml (the quantity of enzyme that transforms 1 jimole of substrate per min. At 25 ° C is defined as a unit of activity). 100 ml of this enzymatic solution are incorporated in thread as described in Example 1.

A 70 g wire sample is placed in a column (60 X 3 cm), thermostated at 25 ° C.

A 0.5 molar sodium fumarate solution, in phosphate buffer pH 7.0, is sent from the bottom upwards at a flow of 50 ml / h. In the effluent, 60% conversion of fumaric acid to 1-malic is obtained. The enzyme fiber-containing reactor was used continuously for 60 days without appreciable loss of activity.

The fumaric acid is separated from the reaction mixture by acidification, which is put back into the cycle, obtaining a 90% recovery. It is concentrated in vacuo to a syrupy consistency and the inorganic salts are removed.

Malic acid is extracted with alcohol and precipitated with the addition of chloroform. 1-malic acid has a purity of 100%. The yield is 60%.

10 Example 4

According to the technique already described in example 1, an enzymatic solution obtained from microbial cells of Pseudomonas sp. Is incorporated in a cellulose triacetate thread. 500 gr of wet cells obtained from submerged aerobic fermentation 15 are subjected to ultrasound for 20 minutes; nucleic acids are removed with MnCl2; the protein solution • is heated for 10 'at 50 ° C; the enzyme is precipitated with ammonium sulphate at 40% saturation. The precipitate is solubilized with 0.01 M phosphate pH 8.

20 The solution contains 60 mg / ml of protein and an activity corresponding to 150 U / ml. 50 ml of this solution are incorporated in cellulose triacetate according to what has already been described.

A sample of the 80 g wire is placed in a column (60 25 x 3 cm) thermostated at 25 ° C. It is fed from below with a 0.5 M ammonium fumarate solution in phosphate buffer at pH 7.0 ad a flow of 50 ml / h.

A 75% conversion of fumaric acid to 1-malic occurs in the effluent.

Example 5

An enzymatic solution, obtained from microbial cells 35 of Pseudomonas sp., As described in example 2 is incorporated in cellulose triacetate.

A sample of 80 g of this wire is placed in a column (60 X 3 cm), thermostated at 25 ° C. A solution of fumarate of Ca 0.06 M in buffer 40 phosphate pH flows from the bottom upwards 7.0 with a flow rate of 50 ml / h. In the, the effluent is dosed an amount of 1-malate corresponding to 70% of the conversion of fumaric acid.

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