DE2015504A1 - Aqs enzyme prepns stabilised by sugars - Google Patents

Abstract

Protease (I) solns. are stabilised by addition of (a) a water-soluble saccharide (pref. 10-95% and esp. 30-70% w/w of I), e.g. saccharose, glucose or lactose, opt. with (b) a filler and/or (c) a calcium salt. Such solns. are useful for prepn. of washing powders, etc.

Description

Protease Preparations with Improved Stability in Solution, Process on their manufacture and use "Priority: April 9, 1969, Great Britain, No. 18288/69 The invention relates to a method for improving stability of proteases in aqueous media, powder containing a protease and a stabilizer for the protease contained in aqueous media, and aqueous solutions after this Process and are made from such powders.

In recent years, the demand for technical enzymes has been microbiological Originated strongly, especially among the detergent manufacturers, the large ones Use amounts of proteases, and in the textile industry. where on a large scale oC amylase is applied.

In general, industrial enzymes are stored under dry conditions Conditions constant. There are commercially available preparations, e.g. B. from Bacillus species produced proteases that were stored for one year without a noticeable loss of activity as long as their water content does not rise above about 8%.

If such enzymes are dissolved in water, the activity of the decreases resulting solution quickly, the decrease being temperature-dependent. The higher the temperature, the faster the activity decreases. It is known that the decrease in the activity of enzymes in an aqueous medium by the addition of certain Cations can be prevented. Aqueous solutions of many proteases can pass through Adding a certain amount of calcium ions are stabilized while α-amylases stabilized by adding sodium ions. Stabilization can be easy achieved by adding small amounts of the ions in the form of their salts to the enzymes will. Accessories of this type are commonly used and are considered portable.

For practical reasons it is advisable to use technical enzymes with certain Add fillers to give them a pre-set activity pro before selling them To give unit of weight. In the case of proteases, this diluting additive diminishes the risk of accidents caused by handling high concentrations of enzymes untrained personnel can occur. It also enables the addition of Fillers to produce enzyme preparations with a standard activity. To this way it is much easier to determine the dosage to be used when using the preparations to determine. Suitable fillers are generally water-soluble salts and -preferably alkali or alkaline earth metal salts, e.g. B. sodium sulfate, sodium tripolyphosphate or sodium chloride. The salts should meet certain requirements. You should have no adverse effect on the activity of the enzyme, neither in the dry still in the dissolved state. An unfavorable influence on an enzyme in dry state occurs when e.g. B. the Sal :: e are too hygroscopic. z Addition of small amounts of other salts, e.g. B. those with a stabilizing Effect, like the calcium salts with the anions mentioned above, leave in these preparations no difficulties arise.

Technical enzymes such as protease and α-amylase are generally used by precipitation with organic solvents or with organic salts or a Combination of the same from the unsprüngllchen solution, d. H. the filtrate from a bacterial culture or a plant or animal extract. That way you get a impure enzyme product which, in addition to active enzyme protein, has a number of quite contains undefined materials such as other proteins and amino acids. A commercially available one technical enzyme, e.g. B. Proteases from bacterial cultures of Bacilluserten, contains Probably also the enzyme protein protein from the fermentation mash, stabilisierede Salts such as calcium sulfate or calcium acetate and also fillers such as sodium sulfate.

The activity of a solution of an enzyme preparation of this kind decreases depending on the temperature over time. The higher the temperature of the solution, the faster the activity of the enzyme decreases. The influence of calcium ions in this solution causes the activity of the enzyme to be slower than in its absence of these ions decreases. In other words, stability is in the presence of this Ions bigger. Otherwise the stability differs from enzyme to enzyme.

It was found according to the invention that the stability of a protease in aqueous solution by adding a water-soluble saccharic to the protease is improved. An important feature of the invention is that regardless whether calcium ions are present in the aqueous solution or not, an improved one Stability is achieved. Saccharide amounts of 10-95% by weight are preferred and in particular 30-70% by weight, based on the enzymes, are used.

It was found according to the invention that glucose and sucrose are particularly suitable as stabilizing saccharides.

In addition, the saccharides apparently satisfy those given above Claims for a filler.

Another object of the invention is the production of a powder, one protease and one water-soluble saccharide with or without a u iGhCn Contains filler and / or a calcium salt, which is used for the production of stabilized, aqueous solutions of a protease can be used.

The following experiments, in which the activity of enzyme solutions was measured, explain the invention.

The activity of an enzyme is given in units. One enzyme unit is the amount of enzyme that produces a specific chemical in a given period of time Reaction can take place.

The reaction conditions have a strong influence on the activity determinations. The activity is preferably determined under conditions such as those in wash liquors can be expected as some proteases are very commonly used in washing powders will. For this reason, the determination is carried out in the presence of sodium tripolyphosphate as this salt is contained in most washing powders. aside from that the pH and the temperature are adjusted to the same conditions as when Washing prevail. The activity regulations stated in Delft Units (DU) are carried out by measuring the reaction of the enzyme with casein; The stability of aqueous solutions of the enzyme is examined by at certain time intervals Activity determinations of the solutions are carried out.

To determine the activity of the enzyme, as carried out in the example the following reagents are used.

Reagent 1 .: 130 g CaCl2. 6 H20 dissolved in 3000 ml of distilled water.

Reagent 2: 42 g of MgCl2. 6 H20 dissolved in 3000 ml of distilled water.

Reagent 3: 63 g NaHCO3 dissolved in 3000 ml distilled water.

Reagent 4: A mixture of 100 ml of reagent 1, 100 ml of reagent 2 and 100 ml of reagent 3 in 9700 ml of distilled water.

The solution has a hardness of 150.

Reagent 5: 36.34 g of tris-hydroxymethylaminomethane in 1000 ml of reagent 4 solved.

Reagent 6: A mixture of 1000 g sodium tripolyphosphate, 500 ml Reagent 1, 500 ml of reagent 2 and 500 ml of reagent 3 in 48.5 liters of distilled water.

The pH value is set by adding an appropriate amount of 10 Hydrochloric acid adjusted to 8.5.

Reagent 7: 540 g of trichloroacetic acid in 3000 ml of distilled water.

Reagent 8: 900 g sodium acetate. 3 H20 in 3000 ml of distilled water solved.

Reagent 9; Dissolve 900 ml of acetic acid in 3000 ml of distilled water.

Reagent 10: 2.5 ml polyoxyethylene sorbitan monooleate (? Ween-80) in 500 ml of distilled water dissolved.

Reagent 11: 100 ml reagent 7, 100 ml reagent 8, 100 ml reagent 9 and 4 ml of reagent 10 are combined in the order given and made up to 1000 ml with distilled water.

Reagent 12: Casein substrate solution. This solution is based on the following Way made: 36.0 g of casein (Merck, for biochemical purposes) are in 2.5 Liters of reagent 4 dissolved with stirring.

Stirring is continued for 10 minutes, 300 ml of reagent 5 are added, and the mixture is stirred for an additional 10 minutes. The mixture is then on a Heated water bath of 70 ° C with further stirring until a temperature of 400 C is reached. At this temperature, the pH is increased with 1N NaOH solution 8.5 set. After cooling to room temperature, the mixture is filled with reagent 4 brought to a volume of 3 liters and the solution is stirred again.

If stored in the refrigerator, this solution can be used for a day be used.

Protease activity is determined as follows: One weighed An amount of a homogeneous enzyme preparation is dissolved in a suitable amount of reagent 6. The pH of this solution is adjusted at room temperature by adding 1N NaOH or Adjusted to 8.5 in RCl.

The enzyme concentration of the solution is to be chosen so that its activity approximately 20-30 Delft Units (DU) protease per liter. This solution must not stored for longer than 2 hours will. The enzyme solution, the casein substrate and some centrifuge tubes are warmed to 400C + 0.10C on a water bath. 1 ml of the enzyme solution is pipetted into a centrifuge tube. Once this solution When the desired temperature of 400 C has been reached, 5 ml of a casein substrate solution are added pipetted into the centrifuge tube and mixed thoroughly with the enzyme solution. the Response time is exactly 40 minutes.

Now 5 ml of reagent 11 are pipetted into the centrifuge tube and mixed with the solution. The centrifuge tube is on a water bath for 30 minutes heated to cause the precipitate to coagulate. It is then centrifuged for 15 minutes.

A blank test is carried out for each enzyme solution by adding 5 ml Reagent 11 can be pipetted into a centrifuge tube. The solution is warmed up 400 C and then add 1 ml of enzyme solution and 5 ml of casein substrate solution. The content of the centrifuge tube is mixed thoroughly. Then on a water bath 30 Heated minutes and then centrifuged for 15 minutes. The optical density of the clear solution is distilled at 275 mu in a UV spectrophotometer against Water measured. The difference between the optical density of the test solution and the reference solution (# OD) should be approximately 0.4-0.6. Is the difference too big or too small, a new test with a more dilute ocer should be more concentrated Enzyme solution can be carried out. The activity of the enzyme, expressed in DU / g -enzyme major Material can be calculated as follows: / \ OD x -1 x dilution a-ktor x 4.545, where 11 is the dilution of the enzyme solution by the substrate and 11 reagent 11 means. The dilution factor is the quotient of the total number of ml of enzyme solution and the weight of the sample in grams and the number 4.545 is a conversion factor.

Example 1 A certain amount of a obtained by filtering a fermentation mash a protease preparation obtained from a species of bacillus and one under the trade name "Maxatase" available protease preparation was with a certain amount of Ca.lciumionen mixed and diluted with powdered sucrose so that the activity 300,000 DU / g was. This product contained approximately 60% sucrose.

An enzyme solution with an activity of 100,000 DU / ml was obtained from the Product made by dilution with tap water.

The solution contained 180 g sucrose / liter. Parts of this brown colored, but clear liquids were stored at 4, -16 and 20 ° C, respectively. Simultaneously were corresponding solutions are produced in which. an enzyme preparation of the same activity was used. In these cases the sucrose was replaced by sodium sulphate. The latter solutions served as comparison solutions. After 6, 14 or 21 days the protease activity of the fluids was determined by the method described above certainly. In Table I the activities of the samples are as a percentage of the original Activity (100,000 DU / ml) indicated.

Table I Shelf life; test of sucrose-containing protease solutions Residual activity in percent after dilution - G days at 14 days at 21 days medium 4 ° C 16 ° C 25 ° C 4 ° C 16 ° C 25 ° C 4 ° C 16 ° C 25 ° C sodium sulphate 88 70 58 88 71 60 82 70 60 sucrose 97 90 82 92 87 79 90 85 74 In a further test, the Shelf life of solutions with an initial activity of 100,000 DU / ml with 5 and 10% by weight of sucrose compared with the same solutions without sucrose.

The results are given in the following table: Table II Residual activity at 37 ° C in percent after:% sucrose 3 days 8 days 30 days 0 35 28 12 5 43 34 17 10 43 34 20 These results confirm that the addition of Sugar improves the shelf life of an aqueous solution.

Example 2 In a manner analogous to Example 1, an enzyme preparation was produced made with about 60% glucose. In this case there were no stabilizing effects Calcium ions added. The prepared solution had an initial activity of 100 000 DU / g. The shelf life was determined at 5, 22 and 370 ° C. After 6, 12 or Samples were made for 20 days. investigated these solutions. The results of these investigations are summarized in Table III.

Table III Stability test of glucose-containing protease solutions Residual activity in percent after dilution - 6 days for 12 days for 20 days medium 5 ° C 22 ° C 37 ° C 5 ° C 22 ° C 37 ° C 5 ° C 22 ° C 37 ° C sodium sulfate 97 96 45 -91 74 34 87 - -glucose 103 90 64 -105 89 52 97 - -In a further test, the shelf life of solutions with an initial activity of 100,000 DU / ml with 5 and 10 wt .-, o Determined glucose, which was compared with the same solutions without glucose. the The results are shown in the following table: Table IV Residual activity at 370 C in percent after: 5 'glucose 3 days 8 days 30 days 0 55 28 12 5 nQ 38 23 10 53 44 21 Example 3 In this example the shelf life of lactose-containing protease solutions. The shelf life of solutions with an initial activity of 100,000 DU / ml with 5 and 10 wt .-% lactose was with the same Solutions without lactose compared.

The results are given in the following table: Table V Shelf life of lactose-containing protease solutions Residual activity at 370 C in percent after: ° h lactose 3 days 8 days 30 days 0 35 28 12 5 43 32 14 10 48 37 22

Claims (15)

Sat t; an sprü che stabilized aqueous protease solution, d a d u r c h g e ke n n z e.i c h ne s t that they are a protease, one in water or water-soluble saccharide dissolved in an aqueous medium, with or without a filler and / or a calcium salt. 2. Aqueous solution according to claim 1, d a d u r c h g g k e n n z e i c h n e t that k e n n n z e i c h n e t that the saccharide content 10 - 95 k e It should be noted that the saccharide content is 10 - 95 and the saccharide content is 10 - 95 % By weight based on the protease. 3. Aqueous solution according to claim 1, d a d u r c h g e -k e n n z e i c h n e t that the saccharide content is 30-70% by weight, based on. on the protease, amounts to. 4. Aqueous solution according to one of claims 1 to 3, d a -d u r c it should be noted that sucrose is used as the saccharide. 5. Aqueous solution according to one of claims 1 to 3, d a -d a d u r Note that glucose is used as saccharide. 6. Aqueous solution according to one of claims 1 to 3, since -durc hgekisiert that lactose is used as the saccharide. 7. Aqueous solution according to one of the preceding claims, d a d u r c h e k e k e n n n n e i n e t, that they also have at least one filler and / or contains a calcium salt. 8. A method for the preparation of an aqueous solution according to one of the The preceding claims, d a d u r g e k e n n -z e i c h n e t that a water-soluble Saccharide with an aqueous protease solution with or without the addition of a filler and a calcium salt is mixed. 9. The method according to claim 8, d a d u r c h g e k e n n -z e i c h n e t that a saccharide content of 10-95% by weight, based on the protease, is used will. 10. The method according to claim 8, d a d u r c h g e k e n n -z e i c h n e t that a saccharide content of 30 - 70 wt .-%, based on the protease, is used. il. Method according to claim 8, d a d u r c h g e k e n n -z e i c Not that sucrose is used as the saccharide. 12. The method according to claim 8, d a d u r c h g e k e n n -z e i c Note that glucose is used as the saccharide. 13. The method according to claim 8, d a d u r c h g e k e n n -z e i c Note that lactose is used as the saccharide. 14. The method according to any one of claims 8 to 13, d a -d u It is noted that at least one filler and / or a calcium salt is added. 15. Use of protease preparations containing water-soluble saccharides in detergents and detergents.