DK144425B - BARE-BASED ENZYME SUBSTANCE FOR USE IN ENZYMATIC REACTIONS - Google Patents

Description

(19) DENMARK ί 'ill

mj 02) PUBLICATION <n) 144425 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 2231/74 (51) IntCI.3 C 12 Q 1/00 (22) Filing date 25 Apr. 1974 // G 01 N 33/02 (24) Running day 23 Apr 1974 (41) Aim. available Oct 26 1974 (44) Submitted Mar 8 1982 (86) International application # - (86) International filing day - (85) Continuation day - (62) Master application no -

(30) Priority 25-Apr. 1973, 354412, US

(71) Applicant KOCKUMS CHEMICAL AKTIEBO LAG, S-21 3 75 Malraoe, SE.

(72) Inventor Kjell Gunnar Blixt, SE: Sven Ivan Arvid Toerntnarck, SE:

Rolf Juhlin, SE: Karl Rune Salenstedt, SE: Mandayam Tiru, SE.

(74) Clerk of the International Patent Office.

(54) Berries are bottom an enzyme substrate for use in enzymatic reactions. -

The present invention relates to a carrier-bound enzyme substrate for use in enzymatic reactions.

In most enzymatic reactions, i.e. reactions in which a substrate under catalytic influence of an enzyme undergoes a change (usually degradation), both the substrate and the enzyme are in the form of a solution and in some cases as a suspension. Several advantages can be achieved, not least with regard to transport and handling, if one of the components, ie. the enzyme or

The substrate could be applied to a carrier. As far as the enzyme is concerned, it is known to combine this with a carrier, e.g. a gel or cellulose.

_-J. It is also known to combine substrate with a carrier, but to date combinations have been made so that the substrate has been covalently bonded to the carrier through a coupling agent or the substrate has only been used.

ISLAND

Is applied loosely to a support so that it can be easily removed from it, e.g. by the action of water. As an example of the prior art, mention may be made of the article "TrSger-bound biologically active substances and their ancestry" by H.D. Orth and W. Brummer in Angewandte Chemie, Vol. 84 (1972), No. 8, pp. 319-368. This article is primarily concerned with carrier enzyme lower enzyme, but in Table 1, several references refer to carrier-bound substrates. Common to these references is that they relate to substrates which are chemically bonded to a carrier, i.e. by covalent bonding or ionic bonding (anionic). The intention is to utilize the carrier-bound substrates for pure enzyme production. In this case, only enzyme-substrate complex formation occurs without degradation of substrate. Furthermore, U.S. Patent No. 2,677,917 and French Patent No. 2,071,058 disclose the application of nutrient medium for fungal mycelium to carriers such as charcoal or expanded pearlite. The nutrient media used are water-soluble and allow the fungal mycelium to absorb the nutrient medium in a moist medium to support growth. Similarly, U.S. Patent No. 3,251,749 discloses bacterial conversion of carbohydrate-loaded carriers into polysaccharides. In this case, too, the "substrate" is water-soluble and only loosely disposed on the support.

As another example of the prior art may be mentioned French Patent Specification No. 1,306,640, which discloses the enclosure of enzyme or substrate in a permeable material such as a polymeric material.

Contrary to what is thus known, the present invention relates to a particular type of carrier-bound enzyme substrate in which the substrate is not covalently bound to the carrier, be it directly or through coupling agents, but by the interaction of the substrate with a particular carrier with a large specific surface, firmly bound to the support by adsorption. Furthermore, it is a condition of the invention that the substrate has low solubility in water and does not undergo hydrolysis or dissolution in the absence of the corresponding enzyme. Thus, in contrast to the products of some of the aforementioned patents, in which the coating is made up of loosely applied, water-soluble material, the substrate of the present invention is firmly adsorbed to the carrier and, moreover, heavily soluble in water. Hereby, according to the invention, an enzyme substrate bound to a carrier is obtained in a very simple and inexpensive manner. To effect the carrier-bound enzyme substrate of the invention, an aqueous phase enzyme is used, and due to the nature and properties of the carrier-bound enzyme substrate of the invention, a high and controllable affinity between the enzyme and the substrate is obtained.

Thus, the carrier-bound enzyme substrate according to the invention is characterized in that it consists of 1) a solid, inert to inert substrate with a specific surface of at least 50 m / g, and 2) a layer which is bonded to the carrier by adsorption containing a substrate having a solubility of less than 0.1% by weight in water at 20 ° C and is substantially stable in the absence of the corresponding enzyme.

According to an embodiment of the invention, the layer further contains a diluent miscible with the substrate.

The diluent is preferably selected from several hydrocarbon mineral oils, higher aliphatic alcohols and polyglycols, but may also be selected from ketones and ethers. In particular, if the diluent is a mineral oil, it is spindle oil.

According to another embodiment of the invention, the layer may additionally contain a viscosity altering agent.

The viscosity altering agent may be metal salts of fatty acids, e.g. aluminum t earat.

According to a further preferred embodiment of the invention, the carrier 2 has a specific surface of at least 200 m / g.

The carrier is preferably selected from organic / inorganic polymers, inorganic oxides, acids and salts.

In particular, it is preferred to select the carrier from alumina, calcium carbonate, magnesium carbonate, silica aerogel and calcium phosphate.

According to a particular embodiment of the invention, the substrate is glyceryl tri-capronate, tributyrin, tripropionine, bis-3,5,5-trimethylhexyl adipate or tripelargonine.

The solid bonding (adsorption) found between the substrate and the carrier in accordance with the present invention is provided using a special carrier which, as mentioned above, is solid and inert above 2 for the substrate and having a specific surface of at least 50 m / g, preferably 2 at least 200 m / g. Through the solid bond obtained, any unwanted separation of the substrate from the support is avoided. Although not fully clarified, it is believed that the (markedly increased affinity occurring between the carrier-bound substrate of the invention and the corresponding enzyme) can be attributed to the combination with the particular carrier and, above all, its large specific surface. In the invention, it has been found that, with increasing specific surface area of the support, an increasing activity or reaction rate between substrate and enzyme is obtained, thereby increasing an activity up to 10 times the normal activity.

Any substrate that meets the above requirements, i.e. the substrate must be heavily soluble or substantially insoluble in water, which means that the substrate must have a solubility of less than 0.1% by weight in water at 20 ° C, and the substrate must also be stable and not undergo hydrolysis or dissolution in the absence of the corresponding enzyme. Preferred substrates of the present invention are those selected from glyceryl trica pronate (tricaproin), tripelargonine, tributyrin and bis-3,5,5-trimethylhexyl adipate, as well as mixed esters of polyhydric alcohols and organic and inorganic acids.

144425 4

The carrier material particularly preferred is silica dioxide in the form of o 2 aerogel with a specific surface of 200-300 m / g. Such silica aerogel is commercially available under the designations "AerosiJ®," Cab-O-Si 2 ', "Silica Gel G" and "Fluosil".

The carrier-bound substrate of the present invention is preferably prepared by applying the substrate to the carrier by impregnation, whereby the finished product is obtained in the form of an easy-to-handle, storage-stable powder which is free-flowing. Thus, the carrier is suspended in a liquid containing substrate or substrate and substrate diluent with stirring. After this treatment, the supplied liquid is evaporated. The residue is comminuted to obtain particles of the desired size and size distribution.

According to one embodiment of the invention, the layer applied to the support is not only of substrate but of substrate in combination with a diluent. Thus, in the layer on the support, the substrate will be dissolved in the diluent. In contrast to the previously described embodiment, wherein the entire substrate is directly available for the action of enzyme, in this last embodiment, only the substrate present in the outer portion of the layer is directly available for the action of the enzyme.

The diluent's properties, such as solubility of substrate, activating or inhibiting enzyme effects, viscosity and propensity for separation under the influence of low temperature, affect the availability of the substrate for the influence of enzyme. Suitable diluents are mineral oils, higher aliphatic alcohols such as decanol, dodecanol, stearyl alcohol and polyglycols, as well as other volatile hydrocarbon compounds which are miscible with the substrate.

A further effect on the availability of the substrate for the action of enzyme is obtained by the addition of a viscosity altering agent, such as aluminum stearate, to the diluent.

The carrier-bound enzyme substrate of the present invention is useful for any suitable purpose for reaction under the action of the corresponding enzyme. It is particularly advantageous to use this carrier-bound substrate for standardization and characterization of enzymes. A particular area of the art in which the present invention has proved particularly useful are indicators of time-temperature dependent indication, e.g. of foods and, in particular, refrigerated and frozen foods. Such indicators are known from, first of all, U.S. Patent No. 2,671,028.

Thus, if an indicator comprising an enzyme and a substrate as well as an indicator for indicating the reaction product formed by the enzymatic reaction is subjected to the same storage conditions as a nutrient, it is possible by appropriate calibration of the indicator by means of the color of the indicator. stroke to determine the state of storage of the food.

An indicator utilizing the carrier-bound enzyme substrate of the invention, as active components, comprises said carrier-bound substrate in combination with a substrate-adapted enzyme and an indicator substance, e.g. a pH indicator for indicating reaction products formed by the enzymatic reaction.

By using the carrier-bound substrate of the invention and the corresponding enzyme in aqueous suspensions, thickeners such as water-soluble, high molecular weight compounds, e.g. agar or carboxycellulose, for the suspension. Hereby an advantage is obtained because the thickener, e.g. agar, gives some change in reaction rate.

The invention is further explained by the following examples.

Example 1

Preparation of substrate coated carrier.

To 2500 ml of methylene chloride are added in small portions and with stirring 100 g of fine-grained silica ("Fluosil") until a homogeneous slurry is obtained.

The slurry is then mixed with 100 g of glyceryl tricapronate (tricaproin, TC) and stirring is continued for an additional hour at room temperature.

Then the methylene chloride is removed by air blowing through filter nozzles and by heating to 30 ° C. After reducing the volume of the mixture to 1500 ml, heating was stopped to avoid shock boiling, and the continued evaporation of methylene chloride was carried out by air blowing only with continuous stirring.

The final evaporation of the methylene chloride was carried out in a heating cabinet at 50 ° C. After drying in the heating cabinet, a white crispy mass was obtained, which was pulverized in mortar and sieved through a sieve of desired mesh width to obtain a fine-grained powder. A similar result was obtained when the dried, white, brittle mass was suspended in water and ground in a colloid mill.

Example 2

Preparation of substrates and diluents coated.

Fine-grained silica with a specific surface area> 50 m 2 / g was slurried in methylene chloride in the same manner as in Example 1. To the slurry was added a mixture of 50 g tricaproin and 50 g spindle oil with stirring and stirring was continued for 1 hour at room temperature.

In the same way as in Example 1, the solvent was then removed and the mass obtained was ground in mortar or colloid mill. A fine-grained carrier coated substrate and diluent were obtained.

Example 3

Example 2 was repeated with the difference that aluminum stearate was added in an amount of 10% to the spindle oil. A similar result was obtained as in Example 2.

6 14442 5

Example 4

Example 2 was repeated except that the spindle oil was replaced with stearyl alcohol. This required heating to allow the mixture of tricaproin and stearyl alcohol to be homogeneous, as in Example 2, and a similar result was obtained.

Example 5

Experiments were made with the substrate coated substrates of Examples 1-3. In the experiments, the time-temperature-dependent indicating properties of indication in combination with the enzyme pancreatic lipase were determined. The acid formed by the enzymatic reaction was indicated by a pH indicator. The weight ratio of carrier to the substrate-containing layer on the carrier was in all cases 1: 1. The results are given in Table 1, using the following abbreviations in the tables: "Fluosil" * »F; tricaproin = TC; spindle oil = Sj aluminum stearate = Alst.

Table 1

Relative cover time

Temp. TC / F TC / S / F TC / S / 1C% Alst / F

(° C) + 20 1.0 1.0 1.0 + 4 2.4 2.0 1.7 _ 5 15.5 20.0 33 - 10 40.0 108 417 - 15 321 856 970 - 20 1396

The test results confirm the foregoing, namely that on the basis of a certain time-temperature dependence ratio of a substrate-coated carrier alone, this dependency ratio is increased when the substrate is combined with diluent, ie. the relative turnaround times are extended. The dependency ratio is further increased if a substrate diluent is added to the combination viscosity enhancer such as aluminum stearate.

Example 6

To study the effect of thickener in the aqueous suspension of enzyme substrate, 0.25% agar was added to aqueous suspensions of enzyme (pancreatic lipase) and substrate-coated carrier of Example 1. The indicated indication times were noted and are reported as relative wrapping times in Table 2, 144425 7

Table 2

Relative cover time

Temp. TC / F TC / F

(° C) 0.25 1 agar + 20 1.0 1.0 + 4 2.4 2.4 - 5 12.7 15.5 - 84.2 40.0 - 15 1336 321 - 20> 16000 1396

In the experiment, the effect of the agar could be clearly felt at temperatures below -10 ° C.

Example 7

Experiments were made to compare the activity of the action of enzyme (pancreatic lipase) on substrate (tricaproin), both without carrier and partly on carrier according to Example 1. The experiment used varying concentrations of enzyme and as a measure of the activity indicated by 20 ° C formed reaction product using ρΗ indicator. The results are listed in Table 3.

Table 3

Wrap time in minutes Lipase concentration TC TC / F

(Ug / ml) 3 7.5 <1 1 15 1.3 0.3 37 4.5 0.1 140 11.5 0.03 380 32

Table 3 shows that the combination of substrate and carrier with large specific surface area results in approximately one-fold increase in activity compared to activity using substrate alone.

Example 8

As in Example 7, attempts were made to compare the activity of the action of enzyme on substrate, both without carrier and partly on carrier.

As the enzyme, pancreatic lipase was used, the substrate was tripelargonine (TP), 2 and the carrier was "Fluosil" V 300, which had a specific surface of approx. 300 m / g.

As a measure of activity, reaction product formed at 20 ° C was indicated. The results 8 1U & 25 are listed in Table 4.

Table 4

Wrap time in hours

Relative Enzyme Concentrat ion TP TP / F

32 21 4.25 16 28 12 8 77 25 4 240 46 2 524 100 1 -240

Example 9

Example 8 was repeated but with the difference that tripropionine (TPP) was used as a substrate instead of tripelargonine. The enzyme was pancreatic lipase and the carrier "Fluosil". The results are given in Table 5.

Table 5

Wrap time in hours

Relative enzyme concentration TPP TPP / F

32 1.25 0.5 16 4.75 1.25 8 21 2.5 4 28 <21 2 70 21 1 142 25

Example 10

Example 8 was repeated but with the difference that bis-3,5,5-trimethylhexyl adipate (THA) was used as a substrate instead of tripelargonine. The enzyme was, like before, pancreatic lipase and the carrier "Fluosil". The results are listed in Table 6.

Table 6

Wrap-in hours ·

Relative enzyme concentration THA THA / F

16 70 21 "" 8 384 35 4 - 71

Claims (2)

Table 6 (Continued) Wrap Time in Hours Relative Enzyme Concentration THA THA / F 2 - 192 1 _ 240-284 Examples 8-10 show that the combination of substrate and carrier with a large specific surface results in a sharp increase in activity compared to the activity of using substrate without carrier. Example 11 The procedure was followed as in Example 7, but with the difference that instead of "Fluosil", "Silica gel G" was used as the carrier. This carrier has the composition: 13% CaSO2, 0.02% chloride, 0.03% Fe and the rest S1O2. The carrier grains have a diameter of 5-25 µm. A 1% solution of tricaproin in acetone was used for the unbound substrate, while a suspension containing a similar amount of silica gel coated with 30% tricaproin was used. After addition of enzyme (pancreatic lipase), pH 5.4 was noted at 28 minutes for the carrier-bound substrate and 220 minutes for the non-carrier-bound substrate at 20 ° C. Thus, it is apparent that by choosing appropriate combinations of substrate, substrate diluent, substrate diluent viscosity altering agent, time-dependent indicator indicators can be produced which indicators are adapted to the conditions applicable to storage of a certain item. It will also be appreciated that, in the manageable form of the carrier-bound enzyme substrate of the invention, the indicator can be further adjusted by mixing substrate material of various kinds, i. mixing material coated with substrate only and material coated with a combination of substrate-diluent viscosity altering agent. The possibilities obtained for adjusting or controlling the time-temperature dependence of the indicator are endless, and by using the carrier-bound enzyme substrate of the invention, indicators which are adapted to any particular purpose, be it indicative, can be produced. of photographic material, pharmaceuticals, foodstuffs or other sensitive goods which, for reasons of durability, require certain specific storage conditions. A carrier-bound enzyme substrate for use in enzymatic reactions, characterized in that it consists of 1) a solid, opposite to the substrate, in 2 different supports with a specific surface of at least 50 m / g, and 2) one for the carrier by adsorption. bonded layer containing a substrate having a soluble