IL39363A - A process for obtaining creatinine amidohydrolase and optionally also creatine amidinohydrolase from microorganisms - Google Patents

Description

A FOR ALSO GfiSA The present invention is concerned with a process for obtaining the creatinine and possibly also the enzyme creatine from In clinical especially for the functional diagnosis of the the determination of the mediate and end products of protein metabolism plays an important metabolic products include creatinine and Methods for determination of creatinine already been frequently Most of the known methods are based upon the reaction suffers from the disadvantage of being a specific microbiological method of determination with the use of isolated bacteria has also been described in which washed cell suspensions used for the measurement of the creatinine and the formation of urea and ammonia was used as a measure of the enzyme it was not possible to obtain soluble enzyme extracts capable of breaking down A prerequisite for the provision of a specif c creatinine determination with the help of the discovery of appropriate soluble enzymes which can catalyse specific and reactions of Lacombe and Girard 21 0 1 950 in two types of and a as specific enzymes which liberate urea from their substrates from the Akamatsu et 1 351 in soil bacteria an enzyme which they called creatine mutase and which about the equilibrium adjustment between creatinine and The following course of breakdown hereby creatinine creatine urea and sarcosine glycine Israel In a simultaneously filed Patent Application there is described a process for separating and obtaining two new enzymes which participate decisively in the course of this These new enzymes were isolated from It is an object of the present invention to provide microorganisms which are especially suitable as starting materials for obtaining these new we call creatinine creatine amidinohydrolase Many attempts have already been made to break down creatinine enzymatically with the help of it has hitherto not been possible to obtain soluble extracts which can break down creatinine to a sufficient Experiments of this kind have been carried for with Co Pseudomonas Pseudomonas and the problem forming the basis of the presen invention is to provide microorgan a digest fraction of which may be used for obtaining the two new According to the present have now found that creatinine and creatine amidinohydrolase can be obtained from the WS or Alcaligenes possesses the following strictly oxidative short ro with peritricho s The is weakly alkalises litmus milk and is capable of the properties have growth at o growth at glycollate gelatine liqui action pelargonate tributyrin fission egg yolk reaction pigment formation phenylacetate fermentation valine arabinose arg ine glucose maltose tryptophane cellobiose betain trehalose hippurate acetamide mannitol According to the present this microorganism belongs to the family and is to be with great to the genus Alcaligenes The other preferred namely WS is a fungus of the Penicillium In order adaptatively to enrich the desired enzymes in the microorganisms to be used according to the present these are preferably cultured with the addition of creatinine to the growth The microorgan are advantageously allowed to grow on a nutrient substrate which contains glucose or glycerols as a source of and as well as salts and vitamins in the amounts and composition known in An especially preferred nutrient medium has the following glucose or glycerol creatinine ammonium sulphate magnesium sulphate hydrate yeast extract 1 nicotinic acid 1 acid 1 vitamin together with traces of iron calcium chloride and magnesium dissolved in potassium phosphate buffer with a of 6 in the case of the Penicillium or with a pH of 7 in the case of Alcaligenes A strain of the microorganisms to be used according to the present invention is obtained in the usual way on agar tilted tubes by the addition of agar to a suitable nutrient preferably to the nutrient to which 5 litre of a th blue solution is also Under optimum the indicator changes in the case of the Penicillium after to 6 days and in the case of the Alcaligenes in 2 to 3 The colour change is clearly into the alkaline brought about by the breakdown of creatinine and organisms which do not bring about the breakdown according to the oned metabolic scheme with the mentioned do not bring about this The microorganisms to be used according to the present invention permit the economic production creatinine amidohydrolase and creatine amidinohydrolase from the soluble protein fractions obtained from a digest of the microorganisms in the manner described in our mentioned simultaneously filed German Patent The following Examples are given for the purpose of illustrating the present Example Enrichment of active fungal mycelia in a submersed shake culture 2 litres of a nutrient substrate by ammonium magnesium sulphate heptahydrate yeast 1 each of acid vitamin biotin and traces of iron calcium chloride and manganese in OM potassium phosphate buffer are inoculated in a shaking flask of a hours old of 90001 and cultured in a shaking apparatus for The creatinine content drops in this time from by weight to scarcely by weight after this the glucose is almost used the pH value increases to to There are thus obtained 3 to Penicillium WS 90001 dry weight per litre of culture solution with a content of to dry weight of creatinine amidohydrolase Example In a culture flask containing litres of the nutrien medium described in Example 1 are with vigorous 1 litres of a well grown of Alcaligenes WS During the whole culturing the pH is maintained constant at The creatinine and the glucose content are followed The creatine utilisation takes place mainly in the second third of the log the glucose is broken down The culture is maintained at After 35 1 dry bacteria mass can be collected per litre of culture The specific activity of creatinine amidohydrclase is 1600 dry Example 3 Alcaligenes spec WS is as described in Example in a working volume of As soon as the stage of growth is fresh nutrient substrate is added to the culture vessel and grown culture solution is removed from the culture vessel at the same The dilution rate is thereby to and preferably about 500 litres of air are passed through the culture solution per There is thus by continuous a yield of to 5 dry bacteria mass per with a specific activity such as is obtained by the batch process described in Example Alcaligenes WS is thus well suited for continuous culturing in the manner described Both microorganisms are deposited in the collection of the Bacteriological Institute of the Technical at insufficientOCRQuality

Claims (6)

1. What we claim is :- 1. A process for obtaining creatinine amidohydrolase and optionally also creatine amidinohydrolase from a micro- ■ organism, wherein as microorganism there is used Alcaligenes sjoec . WS 5Ί 00 or Penicjill^ium WS 90001 .

2. A process according to claim 1 , wherein the microorganism has been cultured in a nutrient medium containing creatinine.

3. . A process according to claim 1 or 2 , wherein the microorganism has been cultured in a nutrient medium containing glucose or glycerol, as a source of carbon, as well as salts and vitamins.

4. . A process according to claim 1 for obtaining creatinin amidohydrolase and optionally creatine amidinohydrolase, substantially as hereinbefore described and exemplified.

5. » Creatinine amidohydrolase, whenever obtained by the process according to any of claims 1 to i|.

6. ." Creatine amidinohydrolase, whenever obtained by the process according to any of claims 1 ttorney for Applicants