Fermentation process for creatinine iminohydrolase

A fermentation process and improved aqueous nutrient medium are used for the production of urease-free creatinine iminohydrolase from an aerobic soil microorganism. In the process, an inoculum culture of the microorganism is transferred into a production medium to generate microorganisms in which creatinine iminohydrolase production has been induced, said nutrient medium containing a source of ammonia and after the ammonia is substantially exhausted, incrementally adding a solution of creatinine, controlling the pH by addition of .alpha.-ketoglutaric acid, and extracting urease-free creatinine iminohydrolase. An improved aqueous nutrient medium for use as the production medium is disclosed.

FIELD OF THE INVENTION 
The present invention relates to a process and an improved aqueous nutrient 
medium for growing an aerobic soil microorganism which produces 
urease-free creatinine iminohydrolase. 
BACKGROUND OF THE INVENTION 
Creatinine iminohydrolase is an enzyme which specifically hydrolyzes 
creatinine to ammonia. Accordingly, by contacting an aqueous liquid 
containing creatinine with this enzyme to generate ammonia, the presence 
and/or concentration of creatinine in the liquid can be determined by 
detecting the level of generated ammonia. This enzyme can therefore play 
an important role in the clinical laboratory where it can be used as a 
diagnostic test reagent for the determination of creatinine in biological 
liquids. 
Creatinine iminohydrolase, sometimes referred to as creatinine desimidase, 
has been obtained from various microorganisms. For example, J. Szulmajster 
in J. Bacteriolol, 75: 633 (1958) and Biochim Biophys Acta, 30: 154 (1958) 
describes a preparation of creatinine iminohydrolase obtained from the 
anaerobic, gram-positive microorganism Clostridium parapurtrificum. A 
method of growing the Clostridium parapurtrificum microorganism is also 
described in these Szulmajster publications. However, these publications 
relate specifically to an anaerobic, gram-positive microorganism, and the 
disclosed fermentation method for growing the microorganism requires a 
long time. Moreover, the amount of microbial cells grown and the yield of 
enzyme extracted therefrom is relatively small. 
U.S. Pat. Nos. 4,087,329 and 4,134,793 describe the production of the 
enzyme creatinine desimidase from one of several aerobic microbial sources 
including microorganisms of the genera Brevibacterium, Corynebacterium, 
Pseudomonas, and Arthrobacter. These patents further describe a nutrient 
medium which may be used for culturing microorganism of the aforementioned 
genera. These patents assert that the formulation of this nutrient medium 
can be widely varied and can contain any of a large number of specifically 
recited carbon and nitrogen sources, as well as other optional nutrients, 
including inorganic materials, a creatinine inducer, and the like. 
Goodhue, Esders, and Masurekar U.S. Pat. No. 4,276,377 describes and claims 
a creatinine iminohydrolase enzyme preparation free from urease activity 
obtained from an aerobic soil microorganism, preferably the aerobic soil 
microorganism ATCC 31546. Because this enzyme preparation is free of 
urease contamination and is highly specific for creatinine, creatinine 
assays can be performed with this enzyme without regard to interference by 
urea and other nitrogenous substances that are often present in biological 
aqueous liquids to be assayed for creatinine, e.g., serum. The urease-free 
creatinine iminohydrolase enzyme preparation described therein is 
therefore highly desirable. 
Masurekar's U.S. Pat. No. 4,275,164 describes a process for the production 
of creatinine iminohydrolase from an aerobic soil microorganism by 
transferring a growth microorganism to a production medium having a pH in 
the range 5 to 10 and extracting urease-free creatinine iminohydrolase. 
The production medium comprises a carbon source containing glucose or an 
amino acid precursor, a nitrogen source containing creatinine, trace 
nutrients and a buffer. Although the process produces excellent enzyme 
activity (as units per liter) and high specific activity (as units per 
gram dry cell mass), further improvement is susceptible to the toxic 
effects of fermentation medium components at high concentration and 
metabolites such as N-methylhydantoin which decrease productivity and 
yield. In addition, the use of complex nutrient sources such as yeast 
extract makes the fermentation susceptible to contamination by foreign 
microbes which decrease the fermentation productivity and enzyme activity, 
as well as themselves being a source of impurities that make subsequent 
enzyme recovery and purification more difficult. 
An improved process from that described in U.S. Pat. No. 4,275,164 for 
providing a creatinine iminohydrolase with increased yield would represent 
a clearly advantageous addition to the art. Such a process and nutrient 
medium used therein would be particularly desirable if useful with the 
aerobic soil microorganism ATCC 31546. 
SUMMARY OF THE INVENTION 
The present invention provides an improved fermentation process and 
improved aqueous nutrient medium for growing under aerobic conditions an 
aerobic soil microorganism from which a creatinine iminohydrolase enzyme 
preparation can be obtained. 
In one embodiment, the invention provides a fermentation process for 
production of creatinine iminohydrolase from an aerobic soil microorganism 
maintained on an aqueous maintenance medium containing creatinine under 
aerobic conditions. The fermentation process comprises the steps of 
1) transferring a seed culture of the microorganism to a production medium 
having a pH in the range of about 7 to 8 to generate, under aerobic 
conditions, a high concentration of the microorganism wherein the aqueous 
nutrient medium comprises: 
a) a carbon source comprising .alpha.-ketoglutaric acid and glucose, 
b) a nitrogen source comprising a source of ammonia, and 
c) salts which are sources of inorganic nutrients, and 
2) incrementally adding, when ammonia is substantially exhausted, a 
solution of creatinine at a rate of about 0.5 to 3 grams per liter of 
fermentation broth per hour to induce synthesis of creatinine 
iminohydrolase to a high level, and 
3) controlling the fermentation pH to about 7 to 8 by addition of an 
aqueous solution containing .alpha.-ketoglutaric acid, glucose, and 
inorganic salts, and 
4) extracting urease-free creatinine iminohydrolase from said medium. 
In a preferred embodiment, the improved fermentation process and improved 
aqueous nutrient medium of the invention have been found useful for 
production of urease-free creatinine iminohydrolase from an aerobic soil 
microorganism such as ATCC 31546. The term "urease-free" as defined herein 
refers to an enzyme preparation that in crude, unpurified form as 
extracted and separated from the microbial cells in which it was produced 
exhibits substantially no urease activity. A typical assay procedure for 
determining urease activity can be carried out using the method of 
Procedure 4 hereinafter. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The process of the present invention provides an improved aqueous nutrient 
medium for the growth of an aerobic soil microorganism, preferably the 
microorganism ATCC 31546, from which increased yields of creatinine 
iminohydrolase can be obtained. The microorganism identified as ATCC 31546 
has received this designation based on its deposit with the American Type 
Culture Collection, Rockville, Md. 20852 U.S.A. on July 26, 1979. The 
written description of ATCC 31546 which appears in commonly-owned U.S. 
Pat. No. 4,276,377 at column 3, lines 16-64, is hereby incorporated by 
reference. This microorganism has been tentatively assigned to the genus 
Flavobacterium and given the species name filamentosum. 
The fermentation process described hereinabove facilitates good growth of 
the microorganism and significantly improves yield of the enzyme. 
Unexpectedly, these highly advantageous results are achieved in the 
process by growing a seed culture of the microorganism and inducing enzyme 
production by a two phase process of 
1) transferring the seed culture of the microorganism to a production 
medium having a pH in the range of about 7 to 8 to generate, under aerobic 
conditions, a high concentration of the microorganism, said production 
medium representing an aqueous nutrient medium which comprises: 
a) a carbon source comprising .alpha.-ketoglutaric acid and glucose, 
b) a nitrogen source comprising a source of ammonia, and 
c) salts which are sources of inorganic nutrients, and 
2) after the first phase and when ammonia is substantially exhausted, 
incrementally adding a solution of creatinine at a rate of about 0.5 to 3 
grams of creatinine per liter of fermentation broth per hour to induce 
synthesis of creatinine iminohydrolase to a high level. 
The pH is controlled throughout the fermentation at about 7 to 8 by 
addition of an aqueous solution containing .alpha.-ketoglutaric acid, 
glucose and inorganic salts. High yields of creatinine iminohydrolase in 
excess of 11,400 units per liter of fermentation broth in 27.4 hours and a 
specific enzyme activity of 460 units per gram dry cell mass are obtained. 
Seed Medium 
The microorganism can be grown in any conventional medium such as that 
described in U.S. Pat. No. 4,275,164. Thus, a fresh sample of aerobic soil 
microorganism can be inoculated into a seed medium having a pH of from 
about 5 to about 10 to grow the microorganism. The seed medium can 
comprise nutrients including carbon sources such as glucose and 
.alpha.-ketoglutaric acid, nitrogen sources such as glutamic acid, 
creatinine, and ammonium salts, and inorganic salts which buffer the 
medium and supply trace inorganic nutrients. Other complex nutrients from 
natural sources such as vegetable or nonpeptic milk protein hydrolysates 
may be incorporated to supplement or replace the other medium components 
as described in U.S. Pat. No. 4,275,164. 
Production Medium 
The production medium useful herein comprises a carbon source, nitrogen 
source and inorganic salts. 
The carbon source is a mixture of .alpha.-ketoglutaric acid and glucose. 
The carbon source could also include other sources of carbon such as 
citric acid, fumaric acid, malic acid, lactic acid, and the like in place 
of or together with .alpha.-ketoglutaric acid and glucose. 
The amount of .alpha.-ketoglutaric acid in the initial medium can vary. 
Useful amounts have been found to be in the range of about 20 to 80 grams 
per liter, preferably about 20 to 30 grams per liter. The starting medium 
preferably contains about 30 to 100 grams per liter of total carbon 
source. 
The nitrogen source employed in the initial production medium comprises a 
source of ammonia such as ammonium sulfate, ammonium hydroxide, glutamic 
acid, and the like. The preferred nitrogen source is ammonium sulfate. 
The amount of ammonium sulfate in the initial medium can vary. Useful 
amounts have been found to be in the range of about 5 to 20 grams per 
liter, preferably about 10 to 15 grams per liter. 
Inorganic salts are employed in the production medium to satisfy inorganic 
nutrient requirements of the microorganism. Typically these trace 
inorganic salts are added in small quantities. Yeast extract and vitamins 
can also be present as optional sources of trace nutrients. 
Typical inorganic salts which can be present as trace nutrients include 
salts of phosphorus, sulfur, chlorine, potassium, sodium, magnesium, 
calcium, iron, zinc, manganese, molybdenum, and other salts. One inorganic 
salt mixture which has been found especially useful is an aqueous 0.1N HCl 
solution of the following composition, the concentration of each listed 
component based on the amount present in one liter of aqueous solution: 
______________________________________ 
MgSO.sub.4.7H.sub.2 O 12.2 g 
CaCl.sub.2.2H.sub.2 O 0.076 g 
FeSO.sub.4.7H.sub.2 O 2.8 g 
MnSO.sub.4.H.sub.2 O 1.7 g 
ZnSO.sub.4.7H.sub.2 O 0.06 g 
NaCl 0.6 g 
NaMoO.sub.4.2H.sub.2 O 
0.1 g 
______________________________________ 
The aqueous nutrient medium representing the production medium can also 
contain other salts, such as a phosphorous-containing salt, as well as 
salts also included in the salt mixture. A preferred phosphate salt is 
dipotassium hydrogen phosphate, K.sub.2 HPO.sub.4, typically present in an 
amount within the range of about 5 to 15 grams per liter, preferably about 
10 to 12 grams per liter. 
The pH of the aqueous nutrient medium representing the production medium is 
typically in a range of from about 7.0 to 8.0. The pH of the initial 
medium can readily be adjusted to a value in the aforementioned range by 
addition of a base such as KOH or NaOH, preferably NaOH. 
The production medium can also contain other optional components as will be 
appreciated by those skilled in the art. Because foaming is often 
encountered when growing microorganisms in a large-scale fermentation, 
foam control agents can be included in the medium. One such foam control 
agent which can be employed is a polyglycol such as Polyglycol P-2000, a 
tradename of Dow Chemical Company, Midland, Mich. Typically, when used, 
this foam control agent is employed in an amount up to about 0.5 gram per 
liter although lower amounts of 0.1 gram per liter have generally been 
found sufficient to control foaming. Other foam control agents can also be 
used; the main criterion for selection being minimal or no inhibition of 
microbial growth and enzyme synthesis at a concentration level that will 
control the foam. 
Fermentation Process For Enzyme Production 
The above-described production medium is advantageously employed in the 
first phase of the final step of a fermentation process suitable for 
large-scale production of creatinine iminohydrolase from an aerobic soil 
microorganism such as the microorganism ATCC 31546. 
This fermentation process employs a sample of the microorganism grown on a 
maintenance medium under aerobic conditions at pH conditions similar to 
those described above for the production medium. Thus, the pH is typically 
adjusted from about 5.0 to 10.0, preferably about 7.0 to 8.0 by addition 
of base, preferably NaOH or KOH, although other bases may also be 
employed. The temperature of the maintenance medium is typically within 
the range of from about 15.degree. to 42.degree. C., preferably about 
25.degree. to 30.degree. C. 
The maintenance medium typically comprises an aqueous medium including a 
carbon source preferably including one or more of the above-described 
amino acid precursors representing an organic acid free from amino groups 
together with glucose; a nitrogen source comprising creatinine; trace 
nutrients such as one or more inorganic salts and optionally yeast extract 
and vitamins; buffer; and agar. 
A maintenance medium which has been found especially useful has the 
following composition, the concentration of each listed component based on 
the amount present in one liter of the maintenance medium: 
______________________________________ 
Agar 20.0 g 
Fumaric acid (carbon source) 
10.0 g 
Creatinine (nitrogen source) 
5.0 g 
K.sub.2 HPO.sub.4 as buffer (anhydrous) 
5.0 g 
Salt solution 10.0 ml 
Distilled water 800.0 ml 
pH adjusted to 7.0 with KOH and volume made 
up to 1 liter with distilled water. 
______________________________________ 
The salt solution noted immediately hereinabove is a 0.1N HCl aqueous 
solution and has the following composition, the concentration of each 
listed component based on the amount present in one liter of salt 
solution: 
______________________________________ 
MgSO.sub.4.7H.sub.2 O 12.2 g 
CaCl.sub.2.2H.sub.2 O 0.076 g 
FeSO.sub.4.7H.sub.2 O 2.8 g 
MnSO.sub.4.H.sub.2 O 1.7 g 
ZnSO.sub.4.7H.sub.2 O 0.06 g 
NaCl 0.6 g 
NaMoO.sub.4.2H.sub.2 O 
0.1 g 
______________________________________ 
The fermentation process is initiated wherein a "fresh" sample of the 
microorganism is transferred to a microbial seed medium to grow the 
microorganism. The term "fresh" sample of microorganism refers to a sample 
of the microorganism which has been incubated and maintained in the 
maintenance medium at about 25.degree. C. for a relatively short duration, 
typically on the order of from about 24 to 72 hours, preferably about 48 
hours. 
To provide a ready supply of fresh sample of microorganism obtained from 
the maintenance medium, one can store the microorganism as a freeze-dried 
powder at temperatures in the range of from about 4.degree. to 25.degree. 
C., thereby suspending cellular growth processes of the microorganism. New 
cultures of the freeze-dried powder in the above-described maintenance 
medium can then be periodically initiated as desired. In this way, one can 
provide a continual supply of "fresh" sample of the microorganism for 
transfer to the microbial growth medium of the fermentation. 
Alternatively, one can also provide a supply of fresh sample of 
microorganism by storing it frozen in liquid nitrogen as described in the 
appended Procedures. 
The composition of the microbial seed medium in preparing the growing 
microorganism of the fermentation process, although important, can vary 
widely. In general, the microbial seed medium is selected to achieve 
maximal cell growth of the microorganism consistent with good yields of 
enzyme production in the process. A medium of the following composition is 
preferred: 
______________________________________ 
Na.sub.2 .alpha.-KG (Disodium 
12.5 g/l 
.alpha.-ketoglutarate) 
Creatinine 6.7 g/l 
Glucose 5.0 g/l 
MgSO.sub.4.7H.sub.2 O 0.8 g/l 
KH.sub.2 PO.sub.4 5.0 g/l 
Salt Solution 8.3 ml/l 
______________________________________ 
The temperature conditions for the microorganism in the seed medium can 
vary. Typically, good cell growth in the seed medium in the process occurs 
over a temperature range of from about 20.degree. to 37.degree. C., 
preferably about 25.degree. to 30.degree. C. 
The sample of microorganism which is transferred to the microbial seed 
medium is incubated in this seed medium for a period effective to obtain 
good cell growth of the microorganism. The effective time period of this 
can vary depending upon the composition of the medium as well as the 
number of cells transferred to the growth medium. In case of a preferred 
microbial seed medium, the effective incubation period for a sample of the 
microorganism obtained from the maintenance medium and inoculated into a 
flask containing 25 ml of the microbial seed medium is about 24 hours. 
Having grown the microorganism, the microbial seed medium containing the 
growing cells is transferred to the production medium. The microbial seed 
medium containing growing cells is typically transferred, in total, to the 
production medium. This microbial seed culture (i.e., the microbial seed 
medium and the growing cells contained therein) thus serves as an inoculum 
for the production medium. 
The production medium is contained in a fermentor. Such a fermentor has a 
capacity of at least 10 liters, typically 150 liters to 200,000 liters. 
Depending upon the size of the final production fermentor, the preparation 
of the microbial seed medium containing the growing cells can be carried 
out in stages to obtain a sufficient quantity of microbial seed culture to 
serve as the inoculum for the final production fermentor. 
For instance, in the case where the final production fermentor has a volume 
of approximately 150 liters, the growth is advantageously carried out in 
two stages. Typically, in each stage of the growth step the cell growth of 
the microorganism in the microbial seed medium is maximized and the 
resultant culture (containing both the medium and growing cells) is used 
as an inoculum for a succeeding stage in which the culture is introduced 
into a new, generally larger batch of microbial seed medium. 
The composition of the production medium employed in the present 
fermentation process is as described in the "Production Medium" section of 
this specification. Likewise, the pH conditions maintained during 
incubation of the microorganism in the production medium are identical to 
those described in the "Production Medium" section. Sufficient oxygen to 
maintain maximum enzyme production by the microorganism is also important. 
This can readily be determined by monitoring the dissolved oxygen 
concentration in the production medium. The air flow rate and the 
agitation rate of the medium are varied to prevent oxygen limitation. The 
incubation time for the microorganism in phase one of the fermentation 
process will vary depending on the specific composition of the production 
medium, the oxygen transport rate, temperature, and other conditions. 
Typical incubation times for a 14-liter production scale fermentor are 
within the range of from about 10 to 14 hours. As described above, if 
necessary or desirable, anti-foam agents can be added at an earlier stage 
of the fermentation multi-stage process to the microbial seed medium. 
An aerobic soil microorganism such as ATCC 31546 grown in the production 
medium of the present fermentation process can generally be grown over a 
reasonable range of temperatures to produce good yields of creatinine 
iminohydrolase enzyme. Good results can be obtained in a temperature range 
of from about 20.degree. to 37.degree. C. Best results have typically been 
achieved at a temperature of about 25.degree. to 30.degree. C. 
The fermentation process for production of creatinine iminohydrolase from 
the grown microorganism is carried out in two phases. In the first phase, 
the seed culture containing the microorganism is transferred to the 
production medium described above. The source of ammonia is supplied in 
excess to support rapid growth and only a small amount of creatinine 
iminohydrolase is actually produced. The end of the first phase 
corresponds with the exhaustion of ammonia and is marked by a sharp drop 
in the gas exchange rates as determined by a sudden decrease in the rates 
of oxygen consumption and CO.sub.2 production and a sudden increase in the 
dissolved oxygen concentration at fixed agitation and air flow rates. The 
second phase is initiated immediately upon observation of these changes. 
In the second phase of the production fermentation, a solution of 
creatinine is added incrementally by feeding at a fixed growth-limiting 
rate of from about 0.5 to 3 grams creatinine per liter per hour. The 
resulting nitrogen-limited condition in the presence of creatinine, the 
inducer of creatinine iminohydrolase, causes a high rate of enzyme 
synthesis. The preferred rate of addition of creatinine is from 1 to 1.5 
grams creatinine per liter per hour. 
During both phases, the pH is controlled at 7 to 8 by the addition of a 
solution containing .alpha.-ketoglutaric acid. We have found that in an 
uncontrolled fermentation, the pH tends to increase due to the depletion 
of the organic acid carbon source. Using the .alpha.-ketoglutaric acid 
addition controls the pH effectively and causes .alpha.-ketoglutaric acid 
to be replaced as it is consumed. Thus this method of pH control also 
allows lower starting concentrations of nutrients such as 
.alpha.-ketoglutaric acid, glucose, and salts which at high concentrations 
inhibit the growth of the microorganism and production of creatininase 
iminohydrolase. 
The solution of .alpha.-ketoglutaric acid advantageously contains at least 
300 grams per liter of .alpha.-ketoglutaric acid. The solution can 
comprise other materials as well, such as glucose and trace elements, such 
as Mg.sup.2+, Mn.sup.2+, Fe.sup.2+ and Zn.sup.2+ to insure that adequate 
amounts of these components are supplied at noninhibitory concentrations. 
Generally, the solution comprises from 
______________________________________ 
80 to 120 g/l Glucose 
5 to 10 g/l MgSO.sub.4.7H.sub.2 O 
0.1 to 1 g/l FeSO.sub.4.7H.sub.2 O 
0.1 to 1 g/l MnSO.sub.4 SO.sub.4.H.sub.2 O 
0.01 to 0.1 g/l ZnSO.sub.4.7H.sub.2 O. 
______________________________________ 
The rate of addition of .alpha.-ketoglutaric acid solution will vary 
according to the pH of the medium. The pH is monitored by a pH electrode, 
and additional solution is added automatically when the pH exceeds the 
desired value. Thus, the rate of addition is typically less than 1 ml per 
liter per hour at the beginning of the first phase and increases to 15 ml 
per liter per hour or more by the end of the second phase. 
The second phase of the production fermentation can generally run from 10 
to 15 hours. The length of time for this phase is determined by 
measurement of the creatinine iminohydrolase activity. When the activity 
level reaches a maximum, the fermentation is stopped. 
Following completion of the second phase, the desired urease-free 
creatinine iminohydrolase enzyme is recovered from the microbial cells in 
step 3. This can be accomplished by conventional means whereby the cells 
are disrupted by sonication, grinding, or the like; and the desired enzyme 
preparation is separated from the medium by organic solvent fractional 
precipitation or other conventional enzyme separation and purification 
techniques. An especially preferred method for recovering the desired 
enzyme and to obtain increased enzyme yield is described in McCollough, 
Esders and Lynn, U.S. Pat. No. 4,275,166.

The following nonlimiting example is provided to further illustrate the 
invention. In the example, the following materials were used: 
______________________________________ 
1. Microorganism - the aerobic soil microorganism 
ATCC 31546 
2. Medium No. 1 (microbial maintenance medium) 
Agar 20.0 g/l 
Fumaric acid (carbon source) 
10.0 g/l 
Creatinine (nitrogen source) 
5.0 g/l 
K.sub.2 HPO.sub.4 (anhydrous) 
5.0 g/l 
Modified Salt Solution C 10.0 ml 
Distilled Water 800.0 ml 
pH was adjusted to 6.7 with KOH, and made up 
to 1 liter with distilled water. 
Composition of Modified Salt Solution C: 
MgSO.sub.4.7H.sub.2 O 12.2 g/l 
CaCl.sub.2.2H.sub.2 O 0.076 g/l 
FeSO.sub.2.7H.sub.2 O 2.8 g/l 
MnSO.sub.4.H.sub.2 O 1.7 g/l 
ZnSO.sub.4.7H.sub.2 O 0.06 g/l 
NaCl 0.6 g/l 
NaMoO.sub.4.2H.sub.2 O 0.1 g/l 
Made up to 1 liter with 0.1 N HCl 
3. Medium No. 2 (seed medium) 
Glucose 5.0 g/l 
Na.sub.2 .alpha.-ketoglutarate 
12.5 g/l 
Creatinine 6.7 g/l 
K.sub.2 HPO.sub.4 5.0 g/l 
MgSO.sub.4.7H.sub.2 O 0.8 g/l 
Modified Salt Solution C 8.3 ml/l 
Initial pH 8.3 (requires no adjustment) 
4. Medium No. 3 (initial medium for production) 
.alpha.-Ketoglutaric acid 18.2 g/l 
Na.sub.2 .alpha.-ketoglutaric acid 
5.5 g/l 
Glucose 10.6 g/l 
(NH.sub.4).sub.2 SO.sub.4 10.9 g/l 
Creatinine 0.45 g/l 
K.sub.2 HPO.sub.4 10.0 g/l 
MgSO.sub.4.7H.sub.2 O 1.5 g/l 
KOH (brings medium to 17.0 g/l 
pH 7.05) 
Modified Salt Solution C 15.0 ml 
Polyglycol (2000) 0.15 ml 
The creatinine feed solution contains: 
Creatinine 74 g/l 
The .alpha.-ketoglutaric acid feed solution contains: 
.alpha.-Ketoglutaric acid 373.0 g/l 
Glucose 110.0 g/l 
MgSO.sub.4.7H.sub.2 O 6.7 g/l 
MnSO.sub.4.H.sub.2 O 0.2 g/l 
FeSO.sub.4.7H.sub.2 O 0.4 g/l 
ZnSO.sub.4.7H.sub.2 O 0.01 g/l 
______________________________________ 
In the example, the following procedures were used: 
Procedures 
1. Culture Preservation and Maintenance 
A culture of the microorganism ATCC 31546 was preserved by growing the 
culture for ten hours at 30.degree. C. in Tryp-Soy Broth, a "complex" 
medium composed of a vegetable protein hydrolysate sold by Scott 
Laboratories Inc. Fiskeville, R.I. The cells were then separated 
aseptically and resuspended in sterile 10% aqueous glycerol with Allen's 
salt solution (Allen, M. B., Archives of Microbiology, Vol. 32, p. 270-277 
(1959). A small volume, 0.5-2.0 ml of this culture was added to a sterile 
glass ampoule which was then sealed and stored in liquid nitrogen. To 
obtain a sample of the microorganism, the culture in the ampoule was 
thawed and the contents were aseptically transferred to Tryp-Soy Broth and 
grown for 10 hours at 30.degree. C. A loopfull of this culture was 
transferred aseptically to slants of Medium No. 1 incubated at 25.degree. 
C. 
2. Enzyme Production 
Enzyme production was achieved in a 14-liter fermentor as follows: 
First, a fresh sample of the microorganism ATCC 31546 culture grown for two 
days on Medium No. 1 slants as described in Procedure 1 above was 
obtained. From this slant, a loopfull of culture was inoculated into 25 ml 
of a microbial seed medium contained in each of four 250-ml Erlenmeyer 
flasks. The microbial seed medium employed consisted of Medium No. 2, 
referred to hereinabove. Following inoculation of the culture into the 
four Erlenmeyer flasks, the flasks were shaken at 200 rpm at 25.degree. C. 
for 24 hours to produce good cellular growth. Then the contents of the 
four flasks were transferred equally to two Fernbach flasks each 
containing 450 ml of Medium No. 2. The Fernbach flasks were shaken at 100 
rpm at 25.degree. C. for 16 hours. 
The first phase of the production fermentation was carried out by 
transferring the contents of the two Fernbach flasks into a 14-liter 
fermentor containing 6.6 liters of an initial production medium. The 
production medium employed consisted of Medium No. 3, referred to 
hereinabove. 
The temperature was maintained at 25.degree. C. The dissolved oxygen 
concentration was maintained at or above 20 percent of the air saturation 
concentration by agitation and aeration of the medium. The pH was 
controlled at 7.3 to 7.8 by automatic addition of the .alpha.-ketoglutaric 
acid feed solution described hereinabove as a part of Medium No. 3. 
The rates of oxygen consumption and carbon dioxide production were 
monitored with the aid of a mass spectrometer which provided measurements 
of the inlet and exhaust gas compositions. The rates were calculated by a 
material balance on the gas streams. Samples of the fermentation broth 
were taken periodically for off-line analysis of the cell dry weight 
concentration. In addition, 2.5 ml aliquots of the culture samples were 
centrifuted at 5000 rpm in a refrigerated centrifuge to separate the cells 
from the production medium. The supernatants containing the production 
medium were saved for later analysis of chemical component concentrations. 
The cells were disrupted and assayed for creatinine iminohydrolase 
activity as described in Procedures 4 and 5. 
Upon exhaustion of the ammonium supplied in the initial medium, the rates 
of oxygen consumption and carbon dioxide production were observed to 
decrease noticeably and suddenly. The dissolved oxygen concentration 
similarly increased. This marked the end of the first phase of production. 
The second phase of the production fermentation was carried out by 
initiating the addition of creatinine to the fermentor at a fixed rate of 
181 ml per hour immediately upon observing the changes in rates and 
dissolved oxygen noted above. The feed solution employed is described 
hereinabove as part of Medium No. 3. All other conditions were as 
described for the first phase. 
Due to the greatly increased microorganism concentration and rate of 
metabolism of carbon source, the rate of automatic addition of the 
.alpha.-ketoglutaric acid feed solution was much faster than at the 
beginning of the first phase of the production fermentation. The 
fermentation was halted after 27.4 hours of operation after the rate of 
increase in the creatinine iminohydrolase activity had slowed. 
3. Cell Disruption: Sonication 
A sample of well agitated fermentation mixture (5.0 ml) is centrifuged for 
five minutes at 17,000 rpm in a centrifuge tube, the supernate decanted 
and discarded, and the pellet resuspended in 4.9 ml of 0.1M tris phosphate 
buffer. The tris phosphate buffer is prepared by dissolving 12.1 grams of 
Sigma Chem. Co. T-1503 Trizma base in 800 ml of deionized water, adjusting 
the pH at 25.degree. C. to 7.5 with phosphoric acid and diluting with 
deionized water to a final volume of 1000 ml. 
Another 5.0 ml of 0.1M tris phosphate buffer is added and the sample is 
transferred to a Rosette cell for sonication. The Rosette cell is placed 
in an ice bath and sonicated at an output setting of 2 for five minutes 
using an Ultrasonic Inc. W-375A Sonifier having a 3/16 inch (9.5 mm) 
tapered microtip disrupter horn. The horn is placed carefully into the 
mixture to avoid touching the cell walls. A 5.0 ml sample of the sonicated 
material is transferred by pipette to a 10 ml volumetric flask and diluted 
to 10 ml with the 0.1M tris phosphate buffer. The total dilution factor 
from the original sample is 4X. 
4. Assay of Creatinine Iminohydrolase 
Creatininase converts creatinine to N-methylhydantoin and ammonia, thus it 
can be assayed by monitoring the rate of disappearance of NADPH 
(.beta.-nicotinamide adenine dinucleotide phosphate-reduced) during a 
second stage reaction in which .alpha.-ketoglutaric acid and ammonia are 
converted to glutamic acid in the presence of glutamate dehydrogenase. 
EQU .alpha.-Ketoglutarate+NH.sub.4.sup.+ 
+NADPH.revreaction.glutamate+NADP.sup.+ +H.sub.2 O 
The following stock solutions were prepared: 
A. 0.1M Bicine Solution 
Sigma B-3876 Bicine (16.3 g) is dissolved in 800 ml of deionized water, the 
pH adjusted to 7.6 with 1N KOH at 25.degree. C., and the solution diluted 
to 1000 ml with distilled water (store refrigerated). 
B. .alpha.-Ketoglutaric Acid/EDTA Solution 
Ethylenediaminetetracetic acid (EDTA) (0.4 g) and .alpha.-ketoglutaric acid 
(1.6 g) are dissolved in 80 ml of deionized water, the pH adjusted to 7.5 
at 25.degree. C. with 50 percent NaOH solution, and the mixture diluted to 
100 ml with deionized water (store frozen). 
C. 0.4M Creatinine Solution 
Sigma C-4255 creatinine (0.45 g) is dissolved in 10 ml of deionized water 
and stored frozen. 
D. 0.01M NADPH Solution 
Sigma N-1630 NADPH (0.080 g) is dissolved in 10 ml of deionized water and 
stored frozen. 
E. Stock Reaction Mixture 
The above stock solutions were used to prepare a stock reaction mixture 
having the following composition: 
______________________________________ 
0.1 M Bicine Solution A 7.4 ml 
.alpha.-Ketoglutaric Acid/EDTA Solution B 
1.0 ml 
0.4 M Creatinine Solution C 
1.0 ml 
Sigma G-2626 L-Glutamic Dehydrogenase 
300 U 
0.01 M NADPH Solution D 0.3 ml 
______________________________________ 
The fermentation samples were analyzed in a Caoz 219 Spectrophotometer by 
adding 10 ml of diluted sample prepared as in 3 above [Cell Disruption 
(Analytical Sample Preparation)] to 0.99 ml of the Stock Reaction Mixture 
E in a cuvette, covering the cuvette with parafilm, and placing in the 
sample compartment of the spectrophotometer. The decrease in NADPH is 
monitored by following the absorbence at 340 nm for 10 minutes and the 
calculated activity of the creatininase iminohydrolase is reported. 
EXAMPLE 1 
Results of Creatinine Iminohydrolase Production Using a Two-Phase 
Fermentation 
This example reports the increase in yield and productivity of creatinine 
iminohydrolase enzyme obtained from aerobic soil microorganisms grown by a 
two-phase fermentation process using an improved aqueous nutrient medium. 
The urease-free creatinine iminohydrolase-producing microorganism ATCC 
31546 described in Procedure 1 above was grown according to Procedure 2, 
also described above. The microbial cell pellets obtained as described in 
Procedure 2 were disrupted as in Procedure 3 and were assayed as described 
in Procedure 4 to determine their creatinine iminohydrolase activity and 
thus obtain a quantitative evaluation of the yield of creatinine 
iminohydrolase and the fermentation productivity. The results are set out 
below in Table I. The elapsed fermentation time was measured from the 
moment that the contents of the two Ferbach seed flasks were added to the 
production medium. 
The first phase of the fermentation extended from time 0 through 12.1 
hours. During this time interval, the cell mass concentration increased 
rapidly to 16 grams dry mass per liter and the oxygen consumption rate 
similarly increased to over 150 mmole per liter per hour, but relatively 
little increase in creatinine iminohydrolase was observed. The oxygen 
consumption rate was observed to decrease suddenly at 12 hours. This was 
taken to indicate that ammonia in the medium was exhausted, as confirmed 
by analysis of the ammonia concentration. Consequently, the feeding of the 
creatinine solution at a fixed rate of 181 ml per hour was begun at 12.1 
hours. This marked the beginning of the second phase. 
Shortly after the creatinine feed was begun, the creatinine iminohydrolase 
activity began to increase rapidly, indicative of the induction of its 
synthesis by creatinine. The increases in cell mass concentration and 
oxygen consumption rate in the second phase are indicative of the limiting 
effects of the creatinine feed on cell mass production and carbon source 
metabolism. 
The .alpha.-ketoglutaric acid solution, containing also glucose and 
inorganic salts, was fed automatically throughout the fermentation to 
control the medium pH between 7 and 8. In this way carbon limitation of 
the fermentation was automatically avoided without any danger of the 
carbon source concentration becoming too high and inhibiting cell mass and 
creatinine iminohydrolase production. Between 23 and 24 hours, however, it 
was necessary to add a small volume of aqueous solution containing 50 
grams of .alpha.-ketoglutaric acid that had been neutralized with KOH. 
This action was taken because a drop in the oxygen consumption rate was 
observed during this time interval, indicating a carbon source limitation 
despite the continuous addition of carbon to control the medium pH. It is 
evident, in a variation of this procedure, that the concentration of 
.alpha.-ketoglutaric acid in the initial production medium could be 
increased by about 10 grams per liter to avoid the occurrence of a carbon 
source limitation during the second phase. 
The fermentation was stopped at 27.43 hours when the rate of increase in 
creatinine iminohydrolase activity had slowed. At this point, the activity 
was over 11,400 units per liter and the specific activity was 460 units 
per gram dry cell mass. This represents a large and significant 
improvement over the results obtained using the method described in U.S. 
Pat. No. 4,275,164. 
TABLE I 
__________________________________________________________________________ 
Specific 
Elapsed 
Ammonia 
Cell Mass 
Creatinine 
Creatinine Imino- 
Oxygen Consump- 
Total Volume 
Fermentation 
Concentra- 
Concentration 
Imino- hydrolase activity 
tion rate 
.alpha.-ketoglutaric 
Time (Hours) 
tion (g/l) 
(g dry mass/L) 
hydrolase (units/l) 
(units/g dry mass) 
(mmole/l/hour) 
Acid Fed 
__________________________________________________________________________ 
(liters) 
0.0 2.0 0.51 69 135 3.8 0.012 
1.0 1.8 0.55 48 87 4.7 0.029 
2.0 2.3 0.66 66 100 6.6 0.032 
3.0 2.3 0.90 80 89 8.0 0.035 
4.0 2.2 1.0 72 71 15. 0.048 
5.0 1.6 1.5 71 46 16. 0.054 
6.0 2.3 2.2 125 58 16. 0.072 
7.0 1.8 2.9 123 43 32. 0.091 
8.0 1.6 4.1 108 26 46. 0.121 
9.0 1.8 5.6 132 24 65. 0.169 
10.1 0.7 8.2 141 17 97. 0.239 
11.1 0.4 13. 221 17 140 0.332 
12.1 0.0 16. 268 17 98 0.480 
13.0 0.0 17. 538 33 117. 0.539 
14.0 0.0 18. 1600 91 150 0.649 
15.0 0.0 18. 3020 168 150 0.788 
16.1 0.0 19. 4030 214 150 0.928 
17.0 0.0 20. 4560 226 150 1.04 
18.1 0.0 21. 5080 243 160 1.16 
19.1 0.0 21. 6590 309 160 1.29 
20.1 0.0 22. 7140 328 160 1.43 
21.1 0.0 23. 8380 371 160 1.58 
22.2 0.0 23. 8360 364 160 1.73 
23.2 0.0 24. 9000 381 150 1.89 
24.3 0.0 24. 9560 406 130 2.04 
25.2 0.0 24. 8960 368 150 2.41 
26.4 0.0 25. 10280 408 170 2.60 
27.7 0.0 25. 11440 457 160 2.80 
__________________________________________________________________________ 
The invention has been described in detail with particular reference to 
preferred embodiments thereof, but it will be understood that variations 
and modifications can be effected within the spirit and scope of the 
invention.