DE2659915C3 - Process for the recovery of 5 'nucleotides from microorganisms - Google Patents

Description

It is known that microorganisms with a high RNA content grow faster than those with a low one RNA content. Such microorganisms would therefore make an excellent source of 5 'nucleotides represent, if it were possible, in a simple way, the RNA in the microorganisms in 5 'nucleotides / u run over and win the latter / u.

It is known that the nucleotides / u from isolated RNA to win. It is also known. RNA from microorga Extract nisms / ti:

a) alkaline extraction

German Offenlegungsschnft Ib 70 110 J

b) s.iiirc extraction

c) Treatment with high sal / concentration
(N ₁ ICLKCLNH ₄ (I)

(I) Treatment with detergents (Dodecylsiilf.il etc.)

All chest processes have the disadvantage that the microorganisms are sometimes mimicked in a very complicated manner. In the alkaline extraction of microorganisms, the extracted RNA is partially denatured so that it can no longer be quantitatively cleaved enzymatically into soluble mononucleotides.

The invention is therefore the object / tigriiiulc. To create a method for obtaining nucleotides from microorganisms which does not have the disadvantages of the known methods described above . In particular, it is an aim of the invention to create a simple method which can be carried out on an industrial scale and does not require expensive apparatus , can be carried out without the addition of foreign substances which produce RNAse and does not require any previous treatment to kill and digest the microorganisms and is environmentally friendly.

This particular object is achieved according to the invention by the method according to claims 1 to 3. The further object is achieved by the method according to claims 1 to 6

In practicing the method of the invention, the nucleic acid is converted into 5 'nucleotides only which appear in the medium, with no 2 'and / or 3' nucleotides being formed. Depending on The nucleic acid content of the microorganism used can therefore easily be determined in advance and which one 5 'nucleotide content in the medium, the heating treatment at 63 to 67 ° C can be ended. It is there It is crucial that the exact temperature limits between 63 and 67 ° C, preferably 65 ° C, are adhered to and the pH value always remains in the specified range. Since then the becoming free the nucleotides the pH value drops in the course of the heat treatment by adding a base, preferably caustic soda, potassium hydroxide or ammonia, the specified pH value range is observed.

Endogenous nucleases are activated by the method according to the invention and this leads to Cleavage of the nucleic acid to form the desired 5'-nucleotides adenosine monophosphate (AMP), guanosine monophosphate (GMP), cyttidine monophosphate (CMP) and uridine monophosphate (UMP). The required heating time depends on the one used Microorganism, especially its content of endogenous nucleases. Since the nuclease content is very can be different, the heating time is also very different and is generally between about 20 minutes and about 20 hours.

In the course of the heating treatment according to the invention, the protein is already largely converted into the insoluble state. The precipitation of the protein is then completed by the brief boiling, that is to say by heating between about 90 and 100 ^° C. at normal pressure or higher temperatures at overpressure. The insoluble crude protein is then separated from the suspension, for example by removal or filtration, and can then be used as food, and can be further processed in any way. The filtrate contains the desired Icolide

All are suitable for the process according to the invention Types of microorganisms or protozoa such as yeast. H.iktcricn. Mushrooms and the like Examples of suitable microorganisms are Sauna romvces cereviswe. S.n ι h.iroinyccs carlsbergensis. Candida boidinii as ran. Nocardia erylropolis as Mycobacterium. ll.killiis iiieus and ll.u illus snhtilis (ur Bacillaceae. Strcpto'.osuis l.itt .ilis for streptococci. PseiiihmuHiiis fliitirescens fur I'svuiltinuiiiiitlcn. Aspurgilliis nigcr for .mold. As already mentioned. the microorganisms are preferably used in freshly harvested, viable l'orm, the Heil.ll so how they are obtained from the / üchtungsmi'diiim. However, it can also be treated by the known methods of preserving living microorganisms Some things have been used, e.g. And gentle dried or lyophilized protozoa

The heating time can be reduced by adding a material having ribonuclease activity will.

These include purified ribonuclease preparations as well as pre-purified or raw biological ones Understood preparations containing ribonuclease. Seedlings of grasses and cereals are preferred as material with ribonuclease activity, in particular malt turkey germ or extracts therefrom, preferably malt germ extract. In this embodiment, the method according to the invention is also available RNase-free microorganisms such as E. coli MRE 600 mutant applicable.

A particular advantage of the method is that the ribonucleic acid practically quantitatively in the 5 'nucleotides is cleaved. It is therefore possible to get out to obtain the nucleotides preparattv the filtrate after separation of the crude protein. The procedure is too environmentally friendly, as the cell breakdown can be burned without difficulty, which is the case with one with salt, Acid or alkali carried out digestion material is not possible

The following examples explain the invention further.

Examples 1-7

Each 2001 an aqueous slurry of the microorganisms listed in Table 1 with one

ίο Dry matter content of approx. 15 to 20% will be increased Heated to 65 ° C. and kept at this temperature for 15 hours with gentle stirring. The pH value is thereby always kept between 7 and 8.5 by adding sodium hydroxide solution. The sowing is then briefly opened boils and the crude protein is separated using separators and dried The yield is between 25 and 32 kg of crude protein. Table 1 shows the RNA content of the Starting material and the resulting cleavage of the RNA in the protein filtrate

Tabel 1 Biomass genus RNA content *) RNA cleavage Tabel 2 RNA splitting with example example Malt germ extract Saccharomyces yeast 0.5-1.5% 100% 100% 1 cerevisiae 8th Saccharomyces yeast 1.8% 87% 90% 2 carlsbergensis 9 Candida ί Jidinii yeast 1.6% 11.3% 26% 3 (ta. 14 mte old) 10 Nocardia erytrnpolis Mycobact 1.6% 77% 100% 4th Bacillus cersus Bac. iaceae 2.2% 16% 11th 30% 5 (at least 1 year old) 12th Streptococcus Streptococci 2.3% 100% 100% 6th faecal is 13th Pseudomonas Pseudomonads 5.1% 55% •> C7. 7th fluorescein 14th

*) The RNA content was isolated according to L. W. Waltschewa et al (Arch. Biochem. Biophys. 176,630 (1976)), and by measuring the Light absorption at 260 nm according to II. C. Sugo (Arch. Biochem. Uiophys. 125,924 (1968)).

As Table I shows, the Be process according to the invention runs very well in fresh microorganisms good yield of 5 'nucleotides. For a long time stored microorganisms the yield is lower, which is attributed to the reduced RNase content will.

Examples 8-14

a) J kg males / germs are ground with JO I water added and held at 65'C for 40 min. The suspension obtained is directly or after Centrifuge the insoluble malt fragments in the following procedure.

b) 200 l each of a slurry of the same microorganisms as in Examples I to 7 the same Tmikcnmasschalt, as described there, are heated to approx. 65 "C, with the after a) obtained malt mixture or the extract offset and then between 2 and 1j hours at bVC further hit / i. The work-up then takes place as described in Examples I to 7. The yield was 2 "> and 35 kg of crude protein, the average content of pure white at 70%.

Table 2 shows the cleavage of the RNA that was achieved. the The yield of 5 'nucleotides was approx. 7 in each case with approx. 10% RNA content in the dry substance up to 800 g on each of the four different 5 'nucleotides, with a lower RNA content dem Starting material correspondingly less.

Example 15

As described in the previous examples, a slurry of 100 g dry substance Saccharomyces cerevisiae with a content of approx. 45% protein and 1.5 to 3% RNA in the dry matter without the addition of a material with Ribonuclea.sewirektiven heated at 65 ° C, boiled and that Crude protein separated. The composition of the filtrate is given in Table 3 below.

Tabel J Kiltr.it
lot T. S. salary protein 5'- Nuclcnl idc example • 10 g 167 ,,
drs 5-
(I. ■ 10 ",.
IS. 15th

Claims (6)

Patent claims: 1. A method for obtaining 5'-NucIeotiden from microorganisms, characterized in that an aqueous slurry of Microorganisms are heated to 63 to 67 ° C at a pH value between 7 and 8.5 until the 5'-nucleotide content in the medium has reached the desired level, then briefly boiled the insoluble Crude protein is separated from the suspension and the 5'-nucleotides are obtained from the solution. 2. The method according to claim 1, characterized in that the pH value by adding a aqueous base is kept in the specified range. 3. The method according to claim 2, characterized in that sodium hydroxide solution as the aqueous base, Potash lye or ammonia is used 4. A method for obtaining 5'-nucleotides from microorganisms, characterized in that an aqueous suspension of the microorganisms with the addition of a material with ribonuclease activity at a pH between 7 and 8.5 63 to 67 ° C is heated until the 5'-nucleotide content in the medium has reached the desired level, then briefly boiled the insoluble crude protein is separated from the suspension and the 5 'nucleotides can be recovered from the solution. 5. The method according to claim 4, characterized in that the material with ribonuclease activity is cereal seedlings or an extract thereof be used. 6. The method according to claim 4, characterized in that the pH value by adding a π aqueous base, such as sodium hydroxide solution, potassium hydroxide solution or ammonia, is kept in the specified range.