3,806,416 United States Patent Office Patented Apr. 23, 1974 2 3,806,416 In addition, this invention provides a process for ob CREATINE AMDOHYDROLASE AND PROCESS taining creatinine amidohydrolase and creatine amidino FORTS PREPARATION Hans Mollering, Klaus Beaucamp, Michael Nelboeck hydrolase from microorganisms. The process essentially Hochstetter, and Hans Ulrich Bergmeyer, Tutzing, comprises culturing a microorganism in a creatinine Upper Bavaria, Germany, assignors to Boehringer containing medium, digesting same and obtaining the Mannheim GmbH, Mannheim-Waldhof, Germany creatinine amidohydrolase in pure form from the water No Drawing. Filed May 2, 1972, Ser. No. 249,589 soluble digestion fraction by known biochemical purifica Claims priority, application Germany, May 5, 1971, tion and fractionation methods carried out at at a pH P 21 22, 2980 value above 7.0, with the use of a test in which creatine Int, C. C07c 7/028, C12d 13/10 O formed from added creatinine is determined in known U.S. C. 195-62 2 Claims manner. Thereafter, if desired, any creatine amidinohy drolase present is separated by exchange chromatography ABSTRACT OF THE DISCLOSURE from the creatinine amidohydrolase obtained. creatinine amidohydrolase and creatine amidinohydro The following reactions are catalyzed by the two lase are prepared by a process comprising culturing a 5 enzymes provided by the present invention: microorganism in a creatinine-containing medium, digest (a) creatinine amidohydrolase ing same, obtaining the creatinine amidohydrolase in sub Creatinine - HO creatine stantially pure form from the water-soluble digestion (b) fraction and, optionally separating by ion exchange creatine amidinohydrolase chromatography any creatine amidinohydrolase present. 20 Creatine -- H0 sarcosine -- urea Since Equation a is that of an equilibrium reaction with the taking up or removal of water, the enzyme which The invention is concerned with two new enzymes, participates in this reaction is called creatinine amido creatinine amidohydrolase and cre tine amidinohydro hydrolase. lase, and with a novel process for the preparation thereof 25 from microorganisms. As starting material for obtaining the two new en In clinical chemistry, especially for the functional diag zymes, there can, in general, be used microorganisms in nosis of the kidney, the determination of the intermediate which the desired enzymes are adaptively enriched, this and end products of protein metabolism play an impor usually taking place by culturing the microorganisms in tant part. The products of this metabolism include 30 the presence of or with the addition of creatinine. creatinine and creatine. Methods for the determination A water-soluble fraction is obtained by digestion of of creatinine have already been frequently described. the microorganism so obtained. The usual digestion meth Most of the known methods are based on the nonenzy ods can be used, depending upon the strength or resist matic Jaffe reaction which, however, suffers from the dis ance of the cell membrane of the microorganism used. 35 High pressure dispersion and ultrasonic digestion have advantage of being non-specific. proved to be especially useful. Further examples of diges Furthermore, there has also been described a specific tion methods include the use of disintegration mills, for microbiological method of determination of creatinine example those of Balutini and Schlossmann, and diges with the use of isolated bacteria in which washed cell sus tion methods working on a similar basis. Chemical or pensions were used for the measurement of the creati 40 enzymatic digestion methods can also be used. nine and the formation of urea and ammonia was used as The creatinine amidohydrolase can be enriched from a measure of the enzyme action. However, it was not the so obtained water-soluble fraction by known bio possible to obtain soluble enzyme extracts capable of chemical purification methods by utilizing its ability to breaking down creatinine. A prerequisite for the provi convert creatinine into creatine for testing the result of sion of a specific creatinine determination with the help 45 any biochemical purification method used. The creatine of enzymes is, however, the discovery of appropriate formed can be determined by known methods, for exam soluble enzymes which can catalyze specific and measur ple, by the addition of adenosine triphosphate and meas able reactions of creatinine. With this object in view, urement in the usual way of the adenosine diphosphate experiments have been carried out with, for example, formed. It is, however, necessary to operate at pH values Corynebacterium, Pseudomonas aeroginosa, Pseudo 50 above 7 and to select a purification method which can monas ovalis, Pseudomonas eisenbergii and Clostridia, be used at these pH values. but all of these experiments were unsuccessful. According to a further method of following the progress Roche, Lacombe and Girard (BBA 6, 210/1950) char of the enrichment process, the creatine formed is split with acterized, in two types of Pseudomonas, creatinase, creatine amidinohydrolase, with the formation of sarco creatininase and a glycocyaminase as specific enzymes 55 sine and urea, and then to determine the urea in the usual which liberate urea from their substrates from the gua way, for example, by the use of urease. nidino group. Furthermore, Akamatsu et al. (Enzymol The extent to which the purification is carried out de ogia, 15, 122, 158, 173/1951) found, in soil bacteria, an pends upon the intended use of the enzyme or enzymes. enzyme which they called creatine-mutase and which If it is desired to obtain a preparation suitable for the brings about the equilibrium adjustment between creati 60 enzymatic determination of creatine, a single purification nine and creatine. The following course of breakdown of step suffices. Examples of such purification steps include the creatinine was assumed: a polyanion treatment, fractionation with organic solvents Creatinine2 creatine->urea and sarcosine->glycine->NH or chromatography. With the use of the above-mentioned methods of determination, it is possible, in a simple man We have now found and isolated, in a state of high 65 ner to ascertain the content of the desired enzyme and to purity, two enzymes in microorganisms, which enzymes enrich it to a considerable extent in a single step. appear to decisively participate in the breakdown of Since a comparatively long storage of the collected and creatinine, viz., a creatinine amidohydrolase and creatine frozen microorganisms could lead to a decrease of activity amidinohydrolase. of the desired enzymes, the cells are preferably further The present invention thus provides two new enzymes, 70 worked up as quickly as possible after collection. Two as well as a process for the isolation and purification microorganisms preferred for carrying out the process of thereof, the present invention are Alcaligenes spec, WS 51400 of 3 3,806,416 . 4. the family Achromo bactericeae and Penicillium WS conditions, the indicator changes color, in the case of the 90001. These two microorganisms are deposited, under Penicillium after 4 to 6 days and in the case of the Alcali the stated numbers, in the collection of the Bacteriologi genes in 2 to 3 days. The color change is clearly into cal Institute of the Technical University, Munich, Ger the alkaline region, brought about by the breakdown of many, at Weihenstephan. creatinine and creatine. Creatinine-utilizing microorga Alcaligenes spec. WS 51400 possesses the following nisms which do not bring about the breakdown according properties: strictly oxidative gram-negative, short rodlets to the above-mentioned metabolic scheme with the above with peritrichous flagellation. The microorganism is weak mentioned enzymes, do not bring about this alkalization. ly oxidase-positive, alkalizes litmus milk and is capable The microorganisms to be used according to the present of dentrification. Furthermore, the following properties O invention permit the economic production of creatinine have been ascertained: amidohydrolase and creatine amidinohydrolase from the Growth at 4 C. ------water-soluble protein fractions obtained from a digest of Growth at 41° C. ------the microorganisms. w Gelatine liquifaction ------Since creatinine amidohydrolase and also creatine ami Tributyrin fission ------5 dinohydrolase quickly lose their activity at pH values Egg yolk reaction ------of 6 and below and exhibit the greatest stability at pH Pigment formation ------values of about 8.0, the digestion is preferably carried Fermentation of: out with the use of an alkaline buffer, 0.1 M potassium Arabinose ------phosphate buffer (pH 8.0) being preferably used. The Glucose ------20 buffer and the buffer concentration used should preferably Maltose ------(--) be such that any further enrichment of the enzymes can Cellobiose ------(--) also be carried out in the buffer used for the digestion. Trehalose ------When carrying out the digestion by high pressure disper 2-ketogluconate ------sion, usually at about 700 to 800 ats., further purification Mannitol ------(--) 25 can, when using a polyanion treatment, be carried out m-Inositol ------aaa without previous separation of the cell residues. Examples Glycolate ------Om of polyanions which can be used include protamine sul Pelargonate ------few phate and water-soluble polyethyleneimines. It is preferred Adipate ------s to add a water-soluble polyethyleneimine, for example, p-Hydroxybenzoate ------30 in the form of a 10% solution of pH 8. When using a Phenylacetate ------raa Solution of this concentration, an about 5% addition Valine ------thereof, referred to the digest volume, is necessary to Arginine ------(-t-) achieve complete precipitation. The precipitate can then 3-Aminovalerate ------am be separated by physical means, for example, by filtering 35 or centrifuging, the desired enzymes remaining in the Tryptophane ------Supernatant, Betain ------Instead of a polyanion precipitation, a precipitation Hippurate ------with Organic solvents can also be advantageous, isopro Acetamide ------O panol preferably being used. In this case, the cell residues Benzylamine ------aaaa 40 are first removed after the digestion and then the organic According to present knowledge, this microorganism solvent is added at ambient temperature. The isopropanol belongs to the Achromobacteraceae family and is to be fractionation is preferably so carried out that at about assigned, with great probability, to the genus Alcaligenes. 25 C., 800 ml. of 90% isopropanol is added per litre of The other preferred mocroorganism, namely WS 90001, digest solution and the precipitate separated off. The is a fungus of the Penicillium genus. 45 Supernatant obtained is again mixed with 500 ml. isopro In order adaptively to enrich the desired enzymes in the panol per litre and the precipitate, which contains the microorganisms to be used according to the present in two desired enzymes, separated off. vention, these are preferably cultured with the addition of A combined use of the two above-mentioned methods, creatinine to the nutrient medium. The microorganisms namely polyanion precipitation and subsequent fractiona are advantageously allowed to grow on a nutrient sub 50 tion with organic solvents, is especially preferred. strate which contains glucose or glycerol, as a source of Not only the products obtained with a single enrichment carbon and creatinine, as well as salts and vitamins in the step but also those with the two combined steps are suffi amounts and compositions known in microbiology. An ciently pure for use in a specific creatinine determination. especially preferred nutrient medium has the following The enzymes creatinine amidohydrolase and creatine composition: 55 amidinohydrolase are hereby always obtained in a mix 0.5 wt. percent glucose or glycerol ture. If it is desired to separate the enzymes, the prepara 0.5 wt. percent creatinine tion so obtained is subsequently separated by exchange 0.08 wt. percent ammonium sulphate chromatography, a weakly basic ion eXchanger, such as 0.02 wt. percent magnesium sulphate hydrate diethylaminoethyl-Sephadex or diethylaminoethyl-cellu 0.05 wt. percent yeast extract 60 lose having proved to be especially useful. 1 mg, nicotinic acid The enzymes are adsorbed on the exchanger, for exam 1 mg. thiamine-p-aminobenzoic acid ple on diethylaminoethyl-"Sephadex,” at a low ion concen 1 mg. vitamin Bs tration, preferably below 0.1 M and more preferably of 0.1 mg. biotin about 0.01 to 0.05 M. Subsequently, the exchanger is 65 washed With an ion concentration of about 0.1 M, non together with traces of iron sulphate, calcium chloride active accompanying proteins thereby being removed. and manganese sulphate, dissolved in M/10 potassium Creatinine amidohydrolase can then be eluted with 0.2 phosphate buffer with a pH of 6 in the case of the Peni Mbuffer solution, whereas the creatine amidinohydrolase cillium or with a pH of 7 in the case of Alcaligenes. still remains on the exchanger. It can also be eluted by A strain of the microorganisms to be used according to 70 increasing the ion concentration to 0.5 M, for example, the present invention is obtained in the usual way on by using 0.2 M potassium phosphate buffer (pH 8.0) agar tilted tubes by the addition of 2% agar to a suitable With a content of 0.3 M sodium chloride or potassium nutrient substrate, preferably to the above-described nu chloride or a similar salt. trient Substrate, to which 5 ml./litre of a 0.05% bromo The above-described preferred embodiment of the proc thymol blue Solution is also added. Under optimum growth 75 ess according to the present invention, which combines 3,806,416 5 6 an isopropanol fractionation with an exchange chroma ture solution. The specific activity of creatinine amidohy tography, leads to an approximately 100 to 150 fold en drolase is 1600 IU/g. dry weight. richment of the enzymes and gives a creatinine amido hydrolase preparation with a specific activity of more than EXAMPLE 3 200 u./mg. The creatine amidinohydrolase is hereby en Alcaligenes spec. WS 51400 is cultured, as described in riched about 100 fold, a preparation with an activity of Example 2, in a working volume of 60 litres. As soon 3 u./mg. being obtained. as the desired stage of growth is reached, fresh nutrient According to another embodiment of the process ac substrate is added to the culture vessel and grown culture cording to the present invention, a very simple high puri solution is removed from the culture vessel at the same fication of the desired enzymes can be obtained without O rate. The dilution rate (flow velocity/working volume) separation when, in a batch process, sufficient exchanger is thereby 0.13 to 0.18 and preferably about 0.16, 500 is added to adsorb both enzymes. The exchanger is then litres of air are passed through the culture solution per separated and thereafter washed and subsequently eluted hour. There is thus obtained, by continuous culturing, in the manner described above. a yield of 3 to 5 g. dry bacteria mass per litre, with a A further possibility for the purification and enrich 5 specific activity such as is obtained by the batch process ment of the two enzymes is by means of salt precipitation described in Example 2. or salt fractionation, for example, with the use of ammo Alcaligenes spec. WS 51400 is thus well suited for con nium Sulphate. Thus, when using ammonium sulphate, tinuous culturing in the manner described above. Creatinine amidohydrolase precipitates at a concentration EXAMPLE 4 of 2.2 M and creatine amidinohydrolase or the mixture 20 of the two enzymes at a concentration of 2.7 M. Isolation of highly purified creatinine amidohydrolase Salt precipitation can also be used for obtaining the enzymes from solutions thereof, such as are obtained, for The cells (1 kg. dry weight) are separated from a example, by elution of the exchanger. By dialysis at about culture batch of Alcaligenes spec. WS 51400 cultured in 4. C., expediently against diluted buffer, for example 0.02 25 the presence of creatinine, then made up with 0.1 M po M diethanolamine buffer (pH 8.0), the enzymes can be tassium phosphate buffer (pH 8.0) to 20 litres and, with freed from Salts and other low molecular weight accom out cooling, digested by high pressure dispersion at a pres panying materials. sure of about 800 ats. The extract is mixed with 5 vol. The preparations so obtained can be stored for several percent of a 10% polyethyleneimine solution (mol. months in a frozen state, without loss of activity. 30 weight about 1800) of pH 8.0 (about 1 litre). After the If only creatinine amidohydrolase is desired, a further addition of potassium chloride (0.01 M end molarity) purification and enrichment step can also comprise a heat and ammonium chloride (0.1 M end molarity), there ing step at 60° C. Such heating for a period of 5 minutes are added, within the course of 10 minutes, 0.8 volumes does not lead to any noticeable reduction of the creatinine of 90% aqueous isopropanol per litre of extract, followed amidohydrolase activity. However, in contradistinction 35 by stirring for 30 minutes at ambient temperature. thereto, the creatine amidinohydrolase loses its activity The copious precipitate obtained is centrifuged off, under these conditions. whereafter 0.45 volumes of 90% isopropanol are added The new enzymes according to the present invention per litre of supernatant. can be used for scientific purposes, as well as for the spe The precipitate so formed, which contains creatinine 40 amidohydrolase and creatine amidinohydrolase, is sep cific determination of creatinine and creatine. arated off and taken up in 400 ml. 0.02 M potassium phos: The following examples are given for the purpose of phate buffer (pH 8.0). Undissolved residue is separated illustrating the present invention. off and the supernatant is applied to a diethylaminoethyl EXAMPLE 1. "Sephadex' column (3.5 cm. x 1 m.) which has been equil ibrated with the same buffer. Thereafter, the column is Enrichment of active fungal mycelia in a submersed washed with 1 vol. 0.1 M potassium buffer (pH 8.0) and shake culture then eluted with 0.2 M potassium phosphate buffer (pH 2 litres of a nutrient substrate containing, by weight, 8.0). The creatinine amidohydrolase-containing fractions 1% glucose, 0.5% creatinine, 0.08% ammonium sulphate, are combined and adjusted at pH 8.0 with ammonium Sul 0.02% magnesium sulphate heptahydrate, 0.05% yeast phate to 2.2 M. The precipitated enzyme is centrifuged extract, 1 mg. each of nicotinic acid, thiamine-p-amino off and dissolved in 0.02 M diethanol amine buffer (pH benzoic acid and vitamin B6, 0.1 mg. biotin and traces of 8.0) to give a volume of 100 ml. and then dialyzed for iron sulphate, calcium chloride and manganese sulphate, 4 hours at 4° C. against the same buffer. There is thus in 0.10 M potassium phosphate buffer (pH 6) are inocu obtained a total yield of 64% of a preparation with a lated in a shaking flask with 200 ml. of a 48 hours old specific activity of 303 u./mg. The following Table I sum pre-culture of Penicillium WS 90001 and cultured in a marizes the activities and yields obtained in the individual shaking apparatus for 4 days. The creatinine content drops steps of this process. in this time from 0.5% weight to scarcely 0.1% by weight and, after this time, the glucose is almost used up. Further TABLE more, the pH value increases to 7.5 to 8.0. There are thus 60 Protein Yield, obtained 3 to 4 g. Penicillium WS 90001 dry weight per U. (25° C.) ing. U.fmg. percent litre of culture solution with a content of 45 to 60 IU/g. Dispersion digestion.------2.8X10 98.6 2.8 00 dry weight of creatinine amidohydrolase. Polyethyleneimine super natant.------2.6X0 66.2 3.9 93 First isopropanol addition EXAMPLE 2. (supernatant).------2.6X05 32, 6 6.6 77 65 Second isopropanol addition In a culture flask containing 15 litres of the nutrient (precipitate).------197X105 6.9 28.5 71 Diethylaminoethyl medium described in Example 1 are cultured, with vigor "Sephadex' chromatog ous aeration, 1.5 litres of a well grown pre-culture of raphy (eluates)------18X105 0.59 303 64 Alcaligenes spec. WS 51400. During the whole culturing period, the pH is maintained constant at 7.0. The creati 70 nine and the glucose content are followed continuously. When carrying out the digestion ultrasonically, instead The creatine utilization takes place mainly in the second of by high pressure dipersion, the content of creatinine third of the log phase, whereas the glucose is broken down amidohydrolase, with about the same specific activity, first. The culture is maintained at 30° C. After 35 hours, can be increased 2.5 fold, referred to the dry weight of the 1.5 g. dry bacteria mass can be collected per litre of cul 75 microorganisms used. 3,806,416 7 8 The creatinine amidohydrolase obtained as described ultrasonically at pH 8.0. The suspension obtained was above has an equilibrium constant mixed, as described in Example 1, with 0.8 volume iso propanol, stirred for 30 minutes at ambient temperature CreatineE. . K=. . 1.27 and centrifuged. The supernatant is mixed with a further 0.45 volume isopropanol and again centrifuged. The pre (37° C.; pH 8.0). cipitate is taken up as described in Example 1 and chro The Michealis constant for creatinine as substrate matographed over a diethylaminoethyl-"Sephadex' col K=3.3X10-2M (37° C. pH 8.0). umn, only the creatinine amidohydrolase thereby being EXAMPLE S eluted. The following Table TV shows the details of this Separation of creatinine amidohydrolase and creatine O process. The preparation obtained was only suitable for amidinohydrolase the determination of creatinine. The procedure described in Example 4 is repeated. TABLE IW However, after elution of the creatinine amidohydrolase U. Protein Yield, from the exchanger column, the creatine amidonohydro 5 Step (25° C.) in g. U.fmg. percent lase is eluted with 0.2 M potassium phosphate buffer (pH UltraSonic digest------1.5X103 780 0.85 100 8.0), containing 0.3 M sodium chloride. The following Secondisopropanol addition (precipitate).------Table II shows the details. Diethylaminoethyl 1.2X10 0,119 10.1 80 'Sephadex' eluate-...------0.7x103 0.028 25 47 TABLE II 20 Separation of creatinine amidohydrolase and creatine amidinohydrolase on diethylaminoethyl-Sephadex' anion exchanger In the experiments described in the above examples the Creatinine Creatine amidohydrolase amidinohydrolase creatine formed from creatinine in the presence of cre Protein - - - atinine amidohydrolase was determined by the addition of Step in Ing. U. U.fmg. U. U.fmg. 25 adenosine triphosphate (ATP); in the presence of crea After isopropanol step...-- 75 860 4.9 44 0.25 Diethylaminoethyl tine phosphokinase (CPK), the creatine is converted into "Sephadex' wash creatine phosphate and adenosine diphosphate (ADP). Water------90 ------0 ------0.20 Mp3.0 (eluate).----- 29 787 27 0 ------ADP formed is converted into ATP with pyruvate kinase 0.50 MpH 8.0 (eluate).----- 13, 8 0.13 4. 3.0 (PK) and lactate dehydrogenase (LDH), as well as phos 30 phoenolpyruvate (PEP), with the consumption of NADH as measured optically. These known steps can be illus EXAMPLE 6 trated as follows: The procedure described in Example 4 is repeated. How ever, the diethylaminoethyl-"Sephadex' chromatography (1) CPK is replaced by a diethylaminoethyl-"Sephadex' batch proc 35 Creatine - APT - creatine phosphate - ADP CSS (2) PK Starting from 100 g. Alcaligenes spec. WS 51400, 100 ADP - PEP - pyruvate - RTP ml. of the second isopropanol precipitate are dissolved (3) LDE in 0.02 M potassium phosphate buffer (pH 8.0) and mixed Pyruvate -- NADH - Hit - lactate - NADt with such an amount of diethylaminoethyl-"Sephadex' 40 that in the supernatant there remain about 5% of each The creatine amidinohydrolase determination was car of the enzymes (about 20 g. of moist, pressed out ex ried out by measurement of the urea formed from creat changer). The exchanger is filtered off, washed with about inine, the urea thereby being measured by the urease 100 ml, 0.08 M potassium phosphate buffer (pH 8.0) process. and, for the joint elution of the two enzymes, stirred with 45 The Michealis constant for creatine was determined as 100 ml. 0.2 M potassium phosphate buffer (pH 8.0), follows: K=5x10-2M (25 C.; pH 7.6). containing 0.3 M potassium chloride, for 15 minutes at It will be understood that the specification and exam 4 C. and subsequently filtered. Both enzymes are present ples are illustrative but not limitative of the present in in the eluate. The following Table III shows that, by pre vention and that other embodiments within the spirit and cipitation with ammonium sulphate, there can be obtained 50 scope of the invention will suggest themselves to those a preparation containing 31.5 u./mg. creatinine amido skilled in the art. hydrolase and 1.1 u./mg. creatine amidinohydrolase. We claim: TABLE II 1. Soluble creatinine amidohydrolase having a specific Isolation of an enriched enzyme mixture of creatinine amidohydrolase 55 activity of more than 10.0 u./mg, and a pH optimum of and creatine amidinohydrolase from 00 g. (dry weight) Alcaligenes 8.0. spec. WS 51400 2. Soluble creatine amidinohydrolase having a specific Creatine activity of at least 2.0 u./mg. and a pH optimum of 7.6. Creatinine anidinos amidohydrolase hydrolase 60 References Cited Protein U.f U.f Step in g. U. mg. Ig. Kopper, Archives of Biochemistry, vol. 19, pp. 171 Digest------26.1 2.4X0 0.92 6X10. 0.023 172 (1949). Second isopropanol ad dition (precipitate)...... 43 198X10 13.9 3.8X0 0,265 Roche et al., Biochimica et Biophysica Acta, vol. 6, Diethylaminoethyl pp. 210-216 (1950). "Sephadex' eluate 0.5 M 65 pH 8-0------0.370 1,16X0 3.5 4, 1X02 1. i LIONEL M. SHAPIRO, Primary Examiner EXAMPLE 7 U.S. C. X.R. 7 g. (dry weight) Alcaligenes spec. WS 51400, grown 195-103.5 R in the presence of creatinine, were collected and digested 70