DD280333A1 - PROCESS FOR OBTAINING MILK FAELLING ENZYMES - Google Patents

Abstract

The invention relates to a process for the production of dairy enzymes from extracts of mammalian males or from culture broths of - optionally also recombinant - microorganisms or animal cell lines which form such enzymes. It aims to effectively provide dairy enzyme from initial solutions of the above labeling for the food industry. The object associated with this objective is achieved in its step I in such a way that in the weakly acidic, weakly ionic starting solution with the active dairy enzyme (preferably native or recombinant chymosin) as adsorbent, a solid, macroporous, fast-sedimenting Traeger, as Enzyme-linked ligand histidine is introduced. Adsorbents with porosic glass, with silica gel or with an organic copolymer as carriers are preferably used. The isolation of the dairy enzyme is successful in the overall process, d. H. including washing and desorption, in high yield (70% to 80%) and purity, the respective adsorbent can be easily regenerated and thus cyclically reused.

Description

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Field of application of the invention

The invention relates to a method for obtaining milk-precipitating enzymes from extracts of mammalian stomachs or from culture broths of such - optionally recombinant - bacterial or fungal microorganisms or animal cell lines, which enzymes of this specificity such. B. able to synthesize chymosin.

The field of application of the invention lies primarily in that area of the food industry in which microbiological and biotechnological process solutions are used. Furthermore, the invention can also be used in the manufacture of biofin chemicals.

Characteristic of the known state of the art

Milk-precipitating enzyme preparations have hitherto been obtained from calf stomach extracts (livestock enzymes) or from culture broths of corresponding microbial producers by means of process steps such as ultrafiltration, rotary evaporation and / or spray drying. For example, in the concentration of chymosin from such magnextractants to spray-dried rennet powder other non-specific for milk clotting proteases, especially pepsin, enriched with. For the separation of pepsin from such chymosin solutions are

a) Fractionation on DEAE-cellulose (Dairy Board Federation Standard, 110, 1982), as well as

b) the affinity chromatographic separation

to pepstatin-aminohexyl agarose (Kabayashi & Murakami, Agric. Biol. Chem., 42, 1978, 2227-2231),

on the synthetic peptide Val-D-Leu-Pro-Phe-Val-D-Leu, immobilized on Sepharose (Pohl, et al., Anal. Biochem., 139, 1984, 265-271),

- histidine-Sepharose (Amourache & Vijayalakshmi, J. Chromatogr., 303, 1984, 285-290) as well as

cibacrown blue agarose (S.Subramanian, Prepar. Biochem., 17,1987,297-312).

For the specific concentration of chymosin from culture broths of recombinant E. coli strains, there is a valid publication (Strop et al., In: Abstract, The 18 ^lh Linderstrdm-Lang Conference, Aspartic Proteinases, July 1988, Elsinore, Danmark), in turn the use of the above-mentioned synthetic peptide, also coupled to a carbohydrate carrier such as Sepharose, has been reported.

For the mentioned technical solutions, the following disadvantages exist: On the one hand, each of the methods mentioned requires the recourse to hard-to-reach starting material; On the other hand, column chromatography of large quantities of liquid can only be carried out in long periods. In addition, in these processes, the starting solutions must be freed from opacifying suspended matter before the column chromatographic separation steps.

Goal of the Itrfindunu

The invention aims to effectively provide milk-fattening enzyme for the food industry.

Explanation of the essence of the invention

The invention has for its object to describe an improved method, containing from liquids, r.ie milk-precipitating enzyme, the target product can be obtained in one step.

According to the invention, this object is achieved in principle as follows: In an active, lactic acid-containing, aqueous, weakly acidic starting solution, which is characterized by its conductivity as weakly ionic, is as a adsorbent, a solid, macroporous, fast sedimontierender carrier to which as enzyme-binding Ligand the amino acid histidine is coupled introduced. After enzyme binding and sedimentation, the loaded adsorbent is washed intensively with water and with weakly acidic buffer solutions, which are likewise characterized as being weakly ionic by virtue of their conductivity. After washing, the enzyme activity

- either with weakly acidic buffers at elevated conductivity values (variant a)

- or with strongly acidic aqueous solutions with subsequent renewed adjustment of the eluate to pH values in the weakly acidic range (variant b)

replaced.

A starting solution of the above labeling containing an active milk-precipitating enzyme can according to the method herein

from mammalian gastric extracts,

- from culture broths of fermented bacterial or fungal microorganisms,

- from culture broths of fermented stable L-form strains of bacterial or fungal origin or

- be prepared from culture broths of fermented animal cell lines.

In the further embodiment of the invention, for the adsorption step of the process, in each case an active starting solution containing milk-precipitating enzyme having a pH between pH 5.2 and pH 6.5 and having a conductivity of less than 15 mS cm ^{-1 is} prepared Adsorbent, which is to be introduced into a starting solution of this nature, preferably stir, as the carrier porous glass, silica gel or in each case an organic copolymer, eg macroporous copolymer based on acrylonitrile vinyl acetate, for this purpose such a carrier material of this marking with pore sizes or After introducing an adsorbent of this kind, the solution is stirred for a period of 2 hours After sedimentation of the loaded adsorbent, the supernatant is decanted off The sedimented loaded adsorbent is then thoroughly placed in a small vessel, preferably in a small vessel Frit or a column, first with water and then with 0.1 M acetate buffer, pH 5.5, which is added to a conductivity of 15mS cm " ¹ NaCl added, washed (see. this also the findings shown in Fig.2).

The separation of the milk-precipitating enzyme from the loaded, washed adsorbent (with histidine as ligand) takes place in variant a) with buffer solution whose pH is in the range vpt pH 5.2 to pH 6.5 and up to one conductivity above 15 m "cm" ¹ chantrope lo: ien, preferably Na * - and Cl "ions, have been added. As a release buffer, sodium acetate buffer (0.1 M) is preferably used in this variant (see also Fig. 2).

In variant b), the milk-precipitating enzyme activity is removed from the loaded, washed adsorbent at pH values below pH 3.5 with 5% acetic acid or with hydrochloric acid (0.1 N to 0.2 N); Subsequently, in the eluate - preferably by adding sodium hydroxide solution - the pH is readjusted to pH 5.5.

It has also proved to be favorable in variant a) of the process-appropriate desorption step to add to the release buffers, if appropriate, monohydric or polyhydric alcohols (cf., example 5) or a 3 M urea solution (cf., example 3). After the desorption step, the enzyme solution is ready for use; this can be stored in lyophilized, spray-dried or liquid state. With the aid of the method described above, it is possible in one step to concentrate the milk-precipitating enzyme activity to 20 to 100 times at yields of 70% to 80%. The separation of residual proteins is more than 95%.

The enzyme activity is determined for laboratory strengths> 200 with the aid of the following modification of the TGL-compliant milk clotting test (TGL 29273/24 of the Milchinduurie der DDR); For laboratory strengths <200, a milk agar plate test is used (see below).

1. Modified milk coagulation test

10% of skimmed milk powder in 0,004M CaCl ₂ reconstructed milk is filled to each 1 ml portions into tubes and preincubated at 35 ^C C. Then 10μΙ enzyme solution are added and the clotting time determined by coagulation at the edge of the tube. The Labstärke (L) is calculated as follows:

L = -χ Lab starch P / D t ₂

t, = coagulation time of the milk with standard lab

t ₂ = coagulation time of the milk with enzyme of unknown rennet strength

Lab strength P- Lab strength of the standard lab, based on 1 part fixed lab at X parts of ingested milk within 40 minutes D - dilution factor of the dissolved standard lab in the standard solution (usually 0.25 g / 50 ml H ₂ O; D = 200)

2. Miichagar test

to 15ml of 1.5% (r) agar (agarose), dissolved in 0.2 M sodium acetate, 0.01 M CaCl _2, pH 6.5, are added at 5O ⁰ C 1.5 ml of reconstituted skim milk (see above TGL) , The mixture is poured on a glass plate of 8cm χ 15cm. In the agar layer are punched at a distance of 1.5 cm holes with a diameter of 0.3 mm, in which then 7μΙ of the corresponding sample solution and their dilutions are filled.

In addition, a standard enzyme solution is carried in 5 different (1: 5) diluted concentrations. The plate is incubated in a moist chamber at 37 ° C. for 18 hours and the activity read from the prepared calibration curve after measuring the diameter of the coagulation halos (see Fig. 1).

The advantages of the invention are seen in that

- the specific binding of the enzyme to the carrier immediately from the solution succeeds,

- Concentration to at least one-third of the starting solution is possible in one step,

- succeed in a step, a substantial purification of residual proteins,

- when using the fast sedimentation according to the invention immobilized carrier (porous glass, silica gel or macroporous organic copolymer above labeling) the tedious column chromatography stage can be bypassed,

the binding of the enzyme in the method-specific use of specific heavy carriers is also possible without problems, even from cloudy solutions or culture broths, and furthermore that turbidity through washing and repeated sedimentation can be quickly removed from the carrier,

- The carrier can be cleaned quickly and inexpensively, for example, with ethanolic HCl solution and after equilibration with buffer solution, so with 0.1 M sodium acetate (pH 5.6) or citrate-phosphate buffer according to Mcllvain (pH 5.5 ), again with full binding capacity is usable as well

- Recombinant DNA residues are not bound unt 'thus not contaminating the enzyme end product.

embodiments

Example 1:

100 ml of 0.1 M sodium acetate buffer pH 5.5 are added to 1 g Hansen Labpuiver and stirred overnight. After centrifugation an ionic strength of 8 mS cm ^-1 and a lab starch of 1: 670 were present, ie 100 ml of the solution thickens 67 l milk The solution is placed in a polyethylene bottle and 20 ml histidine silica gel XWP 500 (particle size about ΙΟΟμητι; nm) after derivatization with 3-aminopiopyltriethoxysilane according to the instructions of RA Laursen (In: HMWeetall, Immobilized Enzymes, Antigens, Antibodies and Peptides, Marcel Dekker Inc., New York, 1975) with 1 g of histidine according to the glutaraldehyde method of Cambiasso et al., Immunochem., 12, 273, 1975) was added, and no milk clotting activity was observed in the supernatant after 2 hours of stirring and sedimentation of the histidine silica gel The loaded carrier was washed 3 times with 50 ml of 0.1 M sodium acetate buffer Desorption is carried out with 2 × 20 ml of the above sodium acetate buffer, pH 5.5, which additionally contains 1.5M NaCl The rennet strength of the eluate obtained (40 ml) was 1: 1450, ie 5 81 milk can be thickened, which corresponds to a yield of 86%.

Example 2:

The binding of chymosin from Hansen-Labpulver to histidine silica gel is carried out as in Example 1. After washing the support (see Example 1) is desorbed with 3x 10ml of 5% acetic acid. After adjusting the pH to pH 5.5 with 2N NaOH, the rennet starch was 1: 2300, d. H. the 30 ml eluate contains the clotting activity for 69I milk, which corresponds to a yield of 100%.

Example 3:

Eight liters of a culture filtrate of the prochymosin-forming, recombinant strain Proteus mlrabllls L99 (pKM 1336) are added after submerged cultivation with 2 N hydrochloric acid until the acidity has reached the value of pH 2.5. After incubation for 4 hours at room temperature, the mixture is centrifuged, the pH of the supernatant is adjusted to pH 5.5 with 4N sodium hydroxide solution, and this solution is allowed to stand at 4 ° C. for 18 hours. In this supernatant both n => ch agarose gel electrophoresis and after transformation experiments no DNA could be detected.

After adjusting the conductivity of the thus treated culture filtrate to 1OmS cm " ¹ by dilution with water (final volume

101), a lab strength of 1:45 was determined. 50 ml of histidine silica gel are introduced into the solution prepared in this way, and stirring is continued (see Example 1) for 2 hours. Upon completion of the stirring, the silica gel segments within 15 minutes and the supernatant is then decanted off. In the latter, no enzyme activity was detectable with the TGL coagulation test.

The loaded histidine silica gel is washed as described in Example 1. For desorption of the bound chymosin 2x 100 ml of 0.1 M sodium acetate, 1.5M NaCl; pH 5.5; used. The enzyme activity of the eluate was 1635 at a concentration of 50: 1, which corresponds to a yield of 28%. Repeated treatment of the carrier with 200 ml of a 3 M urea solution in 0.1 M sodium acetate; pH 5.5; the eluate contained 1: 1065 laboratory starches, corresponding to 47% yield. The total yield of the combined eluates was thus 75,000.

Example 4:

Recombinant strain L99 (pKM 636), which is called 10 l culture culture, is subjected to the procedure described in Example 3 until the chymosin-laden histidine silica gel has been washed. The initial bar thickness was 1:32, i. 101 filtrate put 3201 milk thick. The desorption of the chymosin is carried out by twice Einrühron of 100ml of 5% acetic acid, the pH of the first time with acetic acid is readjusted to pH 3.0. In the eluate, the pH is readjusted to pH 5.5 with 2 N NaOH. The lab strength of the eluate was 1: 1050 at a concentration of 50: 1, which corresponds to a yield of 66%.

Example 5:

Eight liters of one after acid activation to the conductivity of 1OmS cm ' ¹ according to Example 3 set culture filtrate of the recombinant strain L99 (pKM 636), the initial Labstärke 1:45, corresponding to 3601 thickness capacity of the total solution was 50ml histidine silica gel added and stirred for 2 hours at pH 5.5. After settling the silica gel and removing the supernatant, the silica gel is washed as in Example 3. Then the absorbent is divided into four equal parts of 12 ml each and stirred with 2x25ml each of the following eluent:

a) 5% acetic acid

b) 5% acetic acid, 10% glycerin

c) 0.2NHCl

d) 0.05 N citric acid; pH 3.0.

The use of a total of 50ml eluent per serving means a volume reduction to 1:40. The results of this trial are summarized in the table.

Example 6:

200 ml of the culture filtrate (L = 1:45) used in Example 5 are mixed with 5 ml of porous glass (particle size about 100 pm, pore size about 50 nm), to which histidine has been coupled as described in Example 1, and Stirred for 2 hours. The elution is carried out with 2x 10 ml of 5% acetic acid.

After neutralization, the Labstärke was 1: 290, which corresponds to a yield of 64% at tenfold concentration.

Table 1

Desorption of recombinant chymosin from histidine silica gel with various eluents

cleaning volume protein rennet % l / mg n times step ml salary Strength activity protein cleaning (L) the back Total- won lösur.g (I milk) culture 2,000 7,8-10 ³ 90 100 12 _ S) 0.5MCH ₁ COOH 50 55 55 61 1000 83 b) 0.5MCH ₃ COOH 10% glycerin 50 90 70 78 778 65 O 0,2NHCI 50 95 66 73 695 58 d) 0.05 M lemons acid, pH 3.0 50 102 76 84 745 62 e) Rechrom, from a) anHistidin- Sepharoso 60 4.8 53 59 11040 920

Claims (9)

1. A method for obtaining milk-precipitating enzymes, in particular of native and recombinant chymosin, from aqueous solutions using a carrier-ligand adsorbent, characterized dciJu'ch that - In a weakly acidic, based on their conductivity as weakly ionic characterized, active milk-precipitating enzyme-containing starting solution as adsorbent a solid, macroporous, rapidly sedimenting carrier to which is coupled as the enzyme-ligand histidine, brings, - Washing the loaded adsorbent after binding d9S enzyme and sedimentation with water and with weakly acid, characterized by their conductivity as weakly ionic buffer solutions and then - Of each washed, adeny adsorbent, the enzyme activity a) with weakly acidic buffers at elevated conductivity values or b) with strongly acidic solutions with subsequent readjustment of the eluate to pH values in the weakly acidic range replaces. 2. The method according to claim 1, characterized in that one provides an active milk-containing enzyme-containing starting solution having a pH between pH 5.2 and pH 6.5 and having a conductivity of less than 15mS cm " ¹ provides. Process according to claims 1 and 2, characterized in that a starting solution containing the active milk precipitating enzyme has the characterization above - mammalian stomach extracts, Culture broths of fermented bacterial or beneficial microorganisms, - Culture broths of fermented stable L-form strains of bacterial or fungal origin or - Culture broths of fermented animal cell lines
provides. 4. Vr rfahren according to claim 1 to 3, characterized in that one - In the adsorbent to be introduced as a carrier porous glass, silica gel or an organic polymer uses and - After 2 hours of stirring, the loaded adsorbent sediment and the supernatant decanted. 5. The method according to claim 4, characterized in that one uses in the introduced adsorbent a! S carrier porous glass, silica gel or an organic copolymer having pore sizes or mesh sizes over 50 nm. 6. The method according to claim 1 to 5, characterized in that the sedimented, loaded adsorbent intensively with water and then with 0.1 M acetate buffer, pH 5.5, which has been added up to a conductivity of 15mS cm " ¹ NaCl , washes. 7. The method according to claim 1 to 6, characterized in that the einadenen, washed adsorbent, the milk-precipitating enzyme with buffer solution whose pH is in the range of pH 5.2 to pH 6.5 and that up to a conductivity above 15 mS cm " ¹ chaotropic ions have been added, peel. 8. The method according to claim 1 to 6, characterized in that one dissolves the einadenen, washed adsorbent, the milk-precipitating enzyme at pH values below pH 3.5 with 5% acetic acid or with 0.1 N to 0.2 N hydrochloric acid and then adjust the pH in the eluate back to pH 5.5. 9. The method according to claim 7 and 8, characterized in that the Ablösepuffern optionally monohydric or polyhydric alcohols or a 3M urea solution added.