DE2245342A1 - Stable crystalline pullulanase prodn. - using cholate treatment for selective denaturing of protease - Google Patents

Abstract

Stable, protease-free crystalline pullulanase (I) is produced by (a) culturing Aerobacter aerogenes ATCC 15050 continuously or batchwise in a liq. medium contg. maltose, soluble starch or pullulan as C source at conventional temp. and pH under aerobic conditions; (b) sepg. the cell mass from the medium; (c) extracting the cell mass with an aq. buffer soln. in the presence of a cholate; (d) dialysing the extract; and (e) isolating (I) by fractional pptn. with acetone in the presence of dodecyl sulphate.

Description

Process for the production of crystallized pullulanase The enzyme pullulanase of the bacterium Aerobacter aerogenes is important as an auxiliary enzyme for structure elucidation of amylopectins and glycogens as well as in the starch processing industry, for example to break down the starch of unmalted grain in beer production.

A process for the production of pullulanase is according to DOS 1 517 752 known. It is also known that pullulanase nests the external membrane of the cells is associated (cf. H. Bender, Arch. Mikrobiol. 71, 331 (1970)), it can be derived from replace this with detergents. According to K.Wallenfels, H.Bender and I.R. Rasched Biochem. biophys. Res. Commun. 22, 254, (1966) and K.Wallenfels and I.R. Rasched, Biochem.Z. 344, 524 (1966) can be used to remove sodium dodecyl sulfate. The one made in this way However, the enzyme is not pure. Rather, it contains even after repeated chromatography on columns traces of a proteolytic enzyme, which is slow but steady Decrease in pullulanase activity causes (see B.Eisele, I.R. Rasched and K. Wallenfels, European Journal of Biochemistry Vol. 26, 62 (1972).

The invention relates to a method. Pullulanase from the cells of To extract, free from protease and crystallize Aerobacter aerogenes.

The invention relates to a process for the production of crystalline, stable pullulanase by continuous or discontinuous cultivation of Aerobacter aerogenes ATOG 15050 in a liquid nutrient medium, the maltose, Contains soluble starch or pullulan as a carbon source, with the usual ones Temperature and pH conditions under aerobic conditions by isolating the pullulanase from the cells, which is characterized in that the separated from the nutrient medium cell mass extracted in an aqueous buffer solution in the presence of cholate, the Dialyzed extract and from it the pullulanase by fractional precipitation separated with acetone in the presence of dodecyl sulfate.

Aerobacter aerogenes is generally used to carry out the process grown in a nutrient medium that contains between 0.4 and 1.2 percent by weight of Naitose, Contains soluble starch or pullulan.

A nutrient medium is preferred for continuous culture, which contains 0.4 to 1.0 percent by weight of maltose, in particular 0.5 to 0.7 percent by weight Maltose.

For the discontinuous culture are also used as a carbon source 0.4 to 1.0 percent by weight maltose is preferred. This is particularly advantageous also a mixture of maltose and glucose, in particular 0.2 to 0.6 percent by weight Maltose and just as much glucose are used.

For discontinuous culture it can also be used as a carbon source soluble starch can be used, preferably 0.6 to 1.2 percent by weight, in particular 0.7 to 0.9 percent by weight soluble starch.

The aim is to cultivate the Bacillus Aerobacter aerogenes in discontinuous culture made. The thus obtained Culture is then continuously bred further, with an advantage according to Czapek-Dox modified minimal medium (as indicated in Example 1) and as a carbon source Maltose can be used.

Those obtained in the steady state of continuous culture Cells are separated from the nutrient medium, expediently by centrifuging. she are then in a weak al.

Kalischen buffer solution - expediently in 0.1 to 0.01 molar tris (hydroxymethyl) aminomethane mineral acid buffer solution from a pH value between 7.5 and 8.7 - suspendiort, which also has a soluble Cholate, especially an alkali cholate such as sodium cholate, and advantageously also a contains soluble magnesium salt such as magnesium chloride; good results have been achieved, when both salts are each in a molarity between 1-10² and 5.10-2 in the buffer solution are included.

The weight ratio of the separated cell mags to the buffer solution is between 1: 1 and 1: 3. The temperature is between 20 and 40, preferably 25 37 ° C The extraction of the cells of Aerobacter aerogenes thereby the buffer solution in the presence of chloat and magnesium salt, it is best done by stirring or shaking for 3 to 6 hours The extract obtained after separating the cells contains generally between 75 and 80 percent by weight of the original pullulanase activity.

The extract is then dialyzed. It is advantageous to dialyze initially for 24 to 48 hours against water and then for 24 to 36 hours long against a 0.03 to 0.06 molar citrate buffer solution with a pH between 4.8 and 5.5.

This will remove salts like sodium cholate and a larger part inactive material precipitated. The dialyzed extract is expediently durcll Centrifugation clarified.

The cell-free, dialyzed, clear extract obtained in this way is treated with alkali dodecyl sulfate - expediently with the sodium salt - added; one of these uses 1,2 with advantage up to 1.8 percent by weight. The mixture is then subjected to fractional precipitation with acetone subject. In the first fraction, which is expediently up to about 41 percent by volume Contains acetone, inalctive accompanying substances fall out.

The pullulanase is then, preferably after Kochsa: Lzzusatz, precipitated at an acetone content of about 48 - 53 percent by volume. The precipitation will in a buffer solution - expediently a neutral phosphate buffer solution - added and mixed with streptomycin sulfate to precipitate small amounts of nucleic acids; with It is advantageous to use between 1.2 and 1.8 percent by weight of streptomycin sulfate.

After separating the nucleic acids, a clear solution is obtained which generally about 70 percent by weight of that originally present in the culture Contains pullulanase activity. The enzyme is expediently carried out from this solution Addition of ammonium sulfate obtained crystallized.

The crystalline enzyme contains 65% of the total activity with a specific Activity of 38 units per milligram of protein. It can be done with the well-known Metlloden ' the protein purification, for example by column chromatography, completely purified gt be; it then has a specific activity of 60 units per milligram Protein. For many purposes, however, the degree of purity of the ioiikristaliisats is sufficient, since other glycosidases cannot be detected in it.

According to the method according to the invention, pullulanase is obtained from the cells Obtained from Aerobacter aerogenes protease-free and stable in crystalline state. This only requires a few steps of extraction and purification. The new The method can thus be carried out much more easily than the known methods and is preferred for production on an industrial scale. In addition are the yields and in particular the stability of the according to the new process produced pullulanase superior to the prior art. Besides, it is surprising that the addition of cholate necessary to achieve stability denatures the protease but not the pullulanase.

Example 1 a) First, Aerobacter aerogenes ATCC 15050 becomes discontinuous grown in a nutrient medium containing 0.4% glucose and 0.4% maltose. the The culture obtained in this way is placed in a minimal medium modified according to Czapek-Dox (NaNO3 0.3%; KH2PO4 0.1%; MgSO4. 7 H2O, 0.05%; KC1 0.05%; Sodium citrate 0.05%; FeSO4.7 H2O, 0.0025%) below. the following conditions in a fermenter continuously targeted: temperature 28 ° C, stirrer speed 400 revolutions per minute, Air supply one volume of air per volume of culture liquid and minute, dilution rate 0.2 per hour. A 0.6% maltose syrup is used as the carbon source or a syrup made from roughly equal parts of maltose and glucose (Mor Sweet der Maizena Cmbll, Hamburg).

In the state of equilibrium of this continuous culture are the Enzyme concentrations at 1.5 to 1.8 pullulanase units per milliliter of culture fluid with a cell density of 2.0 to 2.5 milligrams dry rolls per milliliter.

b) One kilogram of the freshly centrifuged Aerobacter cells aerogenes from the equilibrium state of continuous culture with a content of 1.72 pullulanase units per milliliter of the original suspension in 1.2 liters m / 30 tris- (hydroxymethyl) -aminomethane-H2SO4 -buffer of pH 8.0 below Addition of 2.5. 10-² mol. Per liter of magnesium chloride and 3. 1-2 moles per liter Suspended sodium cholate and stirred vigorously for four hours at 300C.

The mass is then rotated for 30 minutes at 20,000 revolutions per minute Centrifuged minute.

The total activity of the suspension, which has a volume of two liters was 100 units per milliliter before centrifugation, correspondingly a total of 200,000 units of pullulanase.

In the 1.3 liters of the cell-free obtained by centrifugation There are 120 units of 1 pululanase per milliliter of the supernatant, accordingly 156,000 units in total; that is 78 / o of the original activity.

The specific activity of the pullulanase thus obtained became 4.5 units per milligram of protein determined.

c) The cell-free, clear supernatant is obtained by adding tris (hydroxymethyl) aminomethane brought to pTI 8.0 and initially against running tap water for 48 hours and then dialyzed against 0.5 molar phosphate-citrate buffer of pII 5.0. The emerging Precipitate is centrifuged off at 20,000 revolutions per minute and discarded. The supernatant is adjusted to pH 7.2. After adding 1.5% sodium dodecyl sulfate acetone is added dropwise in the cold under Huhren.

The precipitate formed at an acetone concentration of 41% by volume is centrifuged at 20,000 revolutions per minute and discarded.

To promote precipitation, 2 table salt are added to the supernatant and further acetone was added dropwise up to a concentration of 50% by volume. The emerging Precipitate is centrifuged off at 12,000 revolutions per minute and in 14-0 milliliters 0.01 molar phosphate buffer solution of pH 7.0 was added. After adding 1.5% Streptomycin sulfate, the solution is left in the cold for ten hours and then centrifuged at 20,000 revolutions per minute.

Before the addition of acetone, the activity of the dialyzed and supernatant clarified by centrifugation 108 pullulanase lines per milliliter, At 1.4 liters, that's a total of 1 51 200 pullulanase units.

The activity of the obtained at an acetone concentration of 50% Precipitate is 860 pullulanase units per milliliter, that's one Amount of 160 milliliters 139,200 pullulanase units in total, accordingly 69.5% of the total initial activity.

The activity of the clear supernatant after separation with streptomycin sulfate precipitated nucleic acids is 920 I'ullulanase units per milliliter., das with a volume of 150 milliliters obtained, 138,000 pullulanase units are correspondingly 69% of the total initial activity.

The specific activity is 28 units per milligram of protein.

d) The enzyme solution obtained as described above according to c) is with stirring at 0 ° C., saturated ammonium sulfate solution was added dropwise to a level of 7.0) possible precipitate at an ammonium sulphate concentration of 18% is centrifuged off, taken up in 15 milliliters 0.01 molar phosphate buffer at p 7.0, centrifuged and the clear supernatant returned to the enzyme solution. When an arumonium sulfate concentration of 2 (3 9 is reached, the crystallization of pullulanase begins. The ammonium sulfate concentration is slowly increased to 30%. To complete the crystallization one leaves stand at 4 ° C for two days.

The activity of the centrifuged and in 0.01 molar phosphate buffer solution crystals dissolved by pH 7.0 is after clarification of the solution by centrifugation at 20,000 revolutions per minute 1,300 pullulanase units per milliliter, that is a total of 130,000 pullulanase units in total, corresponding to 65% of the initial one Total activity.

The specific activity is 38 units per milligram of protein.

e) From the enzyme solution obtained as described under d), the Pullulanase by adding dropwise ammonium sulfate solution saturated in the cold be obtained crystallized at 0 ° C.

The activity of the 0.01 molar phosphate buffer solution in 50 milliliters dissolved crystals is 2,270 pullulanase units per milliliter, that is with a volume of 55 milliliters, a total of 125,000 pullulanase units.

The specific activity is 48.5 units per milligram of protein.

Example 2 The enzyme solution obtained according to 1 d) is counteracted for 24 hours 0.01 molar phosphate buffer solution of pH 7.0 dialyzed and then lyophilized. 3.7 g of dry substance with a salt content of 1.5 are obtained. The activity is 40 units per milligram of organic dry matter, corresponding to 129,500 units all in all.

Example 3 A sample of the pullulanase solution obtained according to 1 d) is 6 days at room temperature, avoiding microorganism growth kept. There was no loss of activity during this period.

Example 4 Aerobacter aerogenes ATCC 15050 becomes discontinuous with a supply of air grown in a nutrient medium containing 0.8 percent by weight soluble starch as a carbon source in the nutrient medium modified according to Czapek-Dox (as indicated in Example 1 a) contains. The temperature is 280C. The breeding will be at the end of the logarithmic Cells stopped growing. You get 2.3 to 2.6 pullulanase units per milliliter of culture fluid with a cell density of 3.0 to 3.2 dry cells per milliliter. The pullulanase is isolated as described in Example 1 b-e.

Claims (7)

P a t e n t a n s p r ü c h e 1) Process for the production of crystalline, stable pulliilanase by continuous or discontinuous cultivation of Aerobacter aerogenes ATCC 15050 in a liquid nutrient medium containing maltose, soluble starch. or pullulan as a carbon source, with the usual temperature and pH conditions under aerobic conditions, by isolating the pullulanase from the cells, thereby characterized in that the cell mass separated from the nutrient medium is in an aqueous Buffer solution extracted in the presence of cholate, the extract dialyzed and extracted from it pullulanase by fractional precipitation with acetone in the presence of dodecyl sulfate separates. 2) Method according to claim 1, characterized in that sodium cholate is used used at a concentration of 1.10-² to 5.10-² moles per liter. 3) Process according to claims 1 and 2, characterized in that 0.10 to 0.01 molar solution of tris (hydroxySmethyl) aminomethane mineral acid used with a pH between 7.5 and 8.7. 4) Method according to claim 3, characterized in that the weight ratio the separated cell mass to the Tris buffer solution is between 1: 1 and 1: 3. 5) Method according to claim 3, characterized in that one of the Buffer solution magnesium ions at a concentration of 1.10-2 to 5.10-² mol per Liters added. 6) Process according to claims 1 and 2, characterized in that the extraction is carried out at a temperature between 25 and 37 oc. 7) Process according to claims 1 and 2, characterized in that the extraction is carried out with stirring for 3 to 6 hours.