WO1994001539A1

WO1994001539A1 - A highly specific sarcosine oxidase

Info

Publication number: WO1994001539A1
Application number: PCT/US1993/006620
Authority: WO
Inventors: Susan Long
Original assignee: Genzyme Corporation
Priority date: 1992-07-14
Filing date: 1993-07-14
Publication date: 1994-01-20
Also published as: AU4677793A

Abstract

A sarcosine oxidase isolated from a new Bacillus ATCC 55164 is described. This enzyme exhibits a high degree of specificity for sarcosine and a low degree of reactivity with other substrates such as N-ethyl glycine and is therefore useful for the determination of creatine in body fluids when used in combination with creatinine amidohydrolase and creatine amidinohydrolase.

Description

A highly specific sarcosine oxidase

Background

Sarcosine oxidase (E.C. 1.5.3.1) catalyses the oxidation of sarcosine (N-methyl glycine) to yield glycine, formaldehyde and peroxide. This reaction is useful for the enzymatic determination of sarcosine, creatinine or creatine. The enzymatic determination of creatinine in serum, plasma or urine which uses sarcosine oxidase together with creatinine amidohydrolase and creatine amidinohydrolase, as shown below, is of special importance in clinical diagnosis (e.g. as an indication of renal function disorder).

1. Creatinine + hLOl creatinine amidohydrolase •*. creatine 2. Creatine + H₂0 creatine amidinohydrolase , sarcosine + urea

3. Sarcosine + h O sarcosine oxidase ^ glycine + formaldehyde +H₂O

The stoichiometric relationship of each of the end products formed from creatinine in the final reaction is one, hence the quantity of any one of these compounds, glycine, formaldehyde or hydrogen peroxide is directly related to creatinine concentration and can be used to assay creatinine in a sample. Glycine and formaldehyde can be assayed, for example, using glycine dehydrogenase and formaldehyde dehydrogenase and hydrogen peroxide can be determined colormetrically in a simple way using peroxidase detection systems, e.g. Trinder type (Bergmeyer 'Methods in Enzymatic Analysis" Volume 1 (1983) p. 197).

The Kodak single-slide assay for creatinine determination is a two-point kinetic assay involving coupled reactions carried out within a dry, multilayered slide element. Peroxidase and a leuco dye react with hydrogen peroxide to form a colored dye, which is measured by reflectance spectroscopy. (Mauck, JC, et.al.. Clin. Chem. 22:1197-1198 (1986); Curme, HG.fita! ., a Chem.24: 1335-42 (1978)). The Kodak assay is easy to use and results are rapidly obtained. Therefore this method is widely used clinically.

Sarcosine oxidases have been isolated from a variety of microorganisms. The enzymes that have been characterized to date are of two general types. The first type is a relatively large enzyme with molecular weight 170-190,000 comprised of multiple subunrts. Sarcosine oxidases of this type have been isolated, for example, from species of Pseudomonas (Shimizu, S. fit al. Clinica Chimica Acta 185:241-252 (1989); Rikitaki fit -al_*. J- Biochem. £& 1109- 1117 (1979)); Arthrobαcter (Kaplan, A. fit. Q\„ MQL Cell Biochem. 2: 17-25 (1974) and Ogushi, S. fitfll. Chem* Et-aim..Bull 26:1445- 1450 (1988)); Alcαligenes (Kim, JM fit. t Agric. Q . Chem. 5_Q:281 1-2816 (1986)); and Corynebacterium (Suzuki, M. fit. at J. Biochem.22:599-607 (1981)).

The second type of sarcosine oxidase is smaller, and comprised of a single subunit. This second type of sarcosine oxidase has been isolated from species such as Streptomyces (Inouye, Y. fit at Chem. Pharm. βull- 25:4194-4202 (1987) and U.S. Patent No.4, 743549 issued May 10, 1988 to Mayr≤t al.); Cylindrocarpon (Mori, N. fit. at Agric. Biol. Chem. 44:1391-1397 (1980)); and Bacillus. For example, U.S. Patent No. 4,216,292 issued August 5, 1980 to Ikuta fit. al- describes sarcosine oxidase produced by Bacillus sp. 0618, which has a molecular weight of about 40,000 as measured by gel filtration and an isoelectric point of 4.7. The optimum pH for reaction is pH 9, however, it is stable in pHs ranging from 6-10. The optimum temperature for reaction is about 50, while the enzyme is completely inactivated after standing at 60'C for 10 minutes.

U.S. Patent No. 4,740,465 issued April 26, 1988 to Suzuki fit at describes a sarcosine oxidase, called "sarcosine oxidase N" produced by Bacillus sp. NS129. Sarcosine oxidase N has a molecular weight of about 49,000 as measured by gel filtration, and an isoelectric point of 5.30. The optimum pH for reaction is in the range of pH 6.7 to 10.0, and it is stable in the range of pH 6.5 to 11.5. The optimum temperature for reaction is in the range of 45 to 60^*C and the enzyme retains 75% activity when treated at 60'C for 10 minutes. Sarcosine oxidase N is reported to have a Michaelis constant (Km) value of 4.7 X 10^"3.

To be useful clinically in assays, a sarcosine oxidase must be highly pure and stable within the conditions used for clinical assays. Further, since the amount of sarcosine formed from the enzymatic degradation of creatine and creatinine is small, an appropriate sarcosine oxidase must react well with a low concentration of sarcosine (i.e. have a low Michaelis constant (Km) value). It must also exhibit a low reactivity with substrate analogs or derivatives, which may occur in clinical samples and interfere with the assay. For example, N-ethyl glycine (NEG), a metabolite of lidocaine, is known to react with the sarcosine oxidase used in the Kodak single-slide creatinine assay. Therefore, patients receiving lidocaine show false increases in serum creatinine. (Roberts, RT stat, £Ha Chem.24: 2569-2572 (1988)).

There is therefore a need for a highly pure sarcosine oxidase which is stable within the conditions commonly used for clinical assays and which has a high specificity for sarcosine and a reduced reactivity with other substrates, such as N- ethyl glycine.

Summary of the invention

The present invention relates to a newly isolated strain. Bacillus sp. ATCC 55164. The invention also relates to a novel sarcosine oxidase produced by the strain. The sαrcosine oxidase of the subject invention is highly specific for sarcosine and exhibits a low reactivity with other substrates, such as N-ethyl glycine. The optimum pH for activity is 7.5 to 8.0 when sarcosine is used as a substrate and the enzyme is stable over a pH range of 6 to 10. The optimum temperature for activity is in the range of 50- 60^*C and the present sarcosine oxidase retains 100% activity when treated at 65^*C for 10 minutes and 14% when treated at 70°C for 10 minutes. The molecular weight is estimated to be approximately 40kD by SDS PAGE.

The present invention further relates to processes for obtaining sarcosine oxidase from Bacillus sp. ATCC 55164 and to methods for using the enzyme so obtained in diagnostic assays to detect creatine, creatinine or sarcosine in clinical samples such as plasma, serum or urine.

Brief Description of the Drawings

Figure 1 is a graph showing the optimum pH of the sarcosine oxidase produced from Bacillus sp. ATCC 55164 .

Figure 2 is a graph showing the pH stability of the sarcosine oxidase produced from Bacillus sp. ATCC 55164.

Figure 3 is a graph showing the optimum temperature range of the sarcosine oxidase produced from Bacillus sp. ATCC 55164 .

Figure 4 is a graph showing the temperature stability of the sarcosine oxidase produced from Bacillus sp. ATCC 55164

Detailed description of the invention

Based on morphology, physiology and fatty acid profile, the microorganism described in the present invention is a strain belonging to the genus Bacillus. This Bacillus, designated Bacillus ATCC 55164, was isolated from soil and maintained in axenic culture as described in detail in Example 1. Bacillus ATCC 55164 can be maintained in pure culture in LB media or in any other media which supplies the Bacillus with the necessary vitamins and nutrients.

Bacillus ATCC 55164 has the following identifying characteristics.

1. Morphology (as detected by microscopic observation of cells cultured in Luria medium at 30^*C for 1 to 3 days): a) Size of ceils: rod of 0.5 x 2-3.5μm b) Polymorphism: swollen phase bright presporulation stationary phase ceils, 0.8 -1 x l-3μm size often found as clumps or aggregates of 2 to 10 or more cells. Polymorphism especially noticeable in cultures taken from Luria agar. c) Motility: vegetatively growing cells are motile d) Spores: form endospores in 1-2 days. Spores are ellipitical; size 0.5 x 0.5 - lμm; terminal or central position. e) Gram stain: positive f) Colony morphology: non-pigmented yellow, smooth surfaced colonies are formed after 20 hours at 30"C, diameter 1 mm.

2. Physiological properties

1. VP test: positive

2. Formation of indoie: negative 3. Formation of hydrogen sulfide: negative

4. Hydrolysis of starch: negative

5. Hydrolysis of casein: negative

6. Gelatin liquification: positive

7. Utilization of citrate (Simons medium) negative

8. Utilization of amino acids arginine. lysine and tryptophan: negative

9. Urease: positive

10. Catalase: positive 11. Oxidase: positive

12. Formation of acids from carbohydrates: negative when the following carbohydrates were used: glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose

13. Oxygen requirement: aerobic 14. Growth conditions: The pH range for growth is 6.0 - 7.5. The temperature range for growth is 25^* to 46^*C. 15. Resistance of spores to treatment at 85" for 10 minutes.

High yields of the enzyme sarcosine oxidase can be obtained by culturing Bacillus ATCC 55164 in a medium containing creatine or sarcosine. An example of a preferred culture medium is one which contains sarcosine (lOg/l). yeast extract (5g/l) tryptone (lOg/l) and sodium chloride (lOg/l). Culturing of the present strain is conducted ordinarily at 30'C for 16 to 24 hour. The initial pH of the medium is around ph 7.0 and conditions of shaking or aeration to mix the culture are necessary. The enzyme is normally found associated with the cells and can be recovered from harvested cells using any of a number of known techniques, for example, lysozyme treatment; ultrasonic disruption; or breakage using a French pressure cell, thereby releasing soluble enzyme into solution. Insoluble material is removed by centrifugation and enzyme is recovered by ordinary isolation and purification procedures. For example, sarcosine oxidase can first be recovered by precipitation with 65% - 85% ammonium sulphate; loaded directly onto a phenylsepharose column and eluted using an ammonium sulphate gradient of approximately 0.5M to 0.0M. The enzyme can be dialyzed against water and finally purified using an anion exchange (mono Q) column and a sodium chloride gradient of 0 to 0.3M.

The physical and chemical properties of the purified enzyme are described in detail below.

1. Enzyme action:

The enzyme, sarcosine oxidase, catalyses the oxidation of sarcosine to form glycine. formaldehyde and hydrogen peroxide. Enzyme activity was determined using a peroxidase linked colormetric assay performed as follows: enzyme solution (40mD was added to a reaction mixture (0.5ml, pH 7.75) consisting of 20mM Tris buffer, 1.5mM 4-aminoantipyrine; 2mM dichlorobenzenesulfonate, disodium salt; 200mM sarcosine and 5 units peroxidase and incubated at 37^*C. The reaction was followed by measuring the rate of color development at an absorbance of 510nm. One unit of enzyme activity is defined as the amount of enzyme which generates lμmol of hydrogen peroxide per minute under these conditions.

Substrate specificity

Specificity of sarcosine oxidase for different substrates was determined by adding enzyme (0.1 units) to a reaction mixture (0.5ml, pH 7.75) consisting of 20mM Tris buffer, 1.5mM 4 aminoantipyrine; 2mM dichlorobenzenesulfonate, disodium salt and 5 units peroxidase and lOOmM substrate. The mixture was incubated at 37^*C for 5 minutes. The absorbance of the reaction mixture at 510nm was monitored and compared with the absorbance of a control reaction performed without added enzyme. The relative activity for a substrate is expressed as a percentage of the activity when sarcosine is used as a substrate as shown below:

N-methyl D/L vαline 6.9

N-methyl D/L leucine 6.8

Glycine 1.5

Choline 0.8 Creatinine 0.7

Substrate specificity may also be determined by comparing relative rates of enzyme activity on different substrates and expressing these as activity ratios of one substrate for another. This type of comparison is done at very low substrate concentrations (e.g. as found under physiological conditions in serum samples). An activity ratio assay was performed as follows: enzyme (0.1 units) was added to reaction mixtures (0.5ml, pH 7.75) containing 20mM Tris buffer, 1.5mM 4- aminoantipyrine; 2mM phenol and ImM or 5mM substrate: either a) NEG or b) sarcosine. The mixture was incubated at 37°C and the absorbance monitored at 480nm for up to 60 minutes. The substrate activity ratio is calculated as the ratio of the change in OD/min NEG to change in OD/min sarcosine. Under these assay conditions, no reactivity was seen in the reaction with NEG.

3. Km Value The Km value (Michaelis constant) for sarcosine is 9mM at 37^*C at pH 7.75 (Tris buffer). The Km value for N-ethyl glycine is 108mM at 37^*C at pH 7.75 (Tris buffer).

4. Optimum PH ranae

Figure 1 is a graph showing the relative activity of sarcosine oxidase isolated from Bacillus ATCC 55164 assayed at various pHs with sarcosine as the substrate. The. figure shows that the optimum pH range of the present enzyme is 7.5 - 8.0. The buffers used were PIPES buffer solution (pH 6.0 - 7.0); HEPES buffer solution (pH 6.5 - 7.5); TRIS buffer solution (pH 7.0 - 8.0) and a phosphate buffer solution (pH 7.5 - 9.0).

5. pH stability

The present enzyme was incubated at 4^*C for 1 hr. in buffers of various pH values and then assayed to examine residue enzyme activity.

Figure 2 shows that the present enzyme is stable between pH 6.5 and 9.0.

6. Optimum reaction temperature

As shown in Figure 3, the optimum temperature range of the present enzyme is 50-60'C. 7. Heat stability

Figure 4 shows that the present enzyme lost no activity after treatment at 65'C and that even after incubation for 10 min. at that temperature, 14% residual activity was retained.

8. Molecular weight

An estimate of the size of the present enzyme was made by comparing the electrophoretic mobility of denatured protein with the electrophoretic mobility of proteins of known molecular weight using polyacrylamide gel electrophoresis. The present enzyme was found to be approximately 40kD.

The above described sarcosine oxidase is highly pure, stable and exhibits a high reactivity with sarcosine and a low reactivity with sarcosine analogs or derivatives such as N-ethyi glycine. Therefore, the enzyme according to the present invention is useful for a creatine/creatinine clinical assay.

Methods for detecting levels of creatine or creatinine in a fluid sample, such as serum, plasma or urine are well known in the art. However, in general, the sample is contacted with creatinine amidohydrolase and creatine amidinohydrolase and allowed to react. The sample is then contacted with sarcosine oxidase and allowed to react. Finally, the sample is assayed for the presence of a catalysis product such as glycine. formaldehyde and hydrogen peroxide. The presence of such a catalysis product indicates the original presence of creatine or creatinine in the sample.

The invention will now be illustrated by the following example, which is not intended to be limiting in any way.

Example 1: Isolation of sarcosine oxidase producers from natural environments

Sixteen different environments, including soil and water samples were screened for microorganisms, which were able to grow on sarcosine or the precursor creatine as the sole source of carbon or nitrogen. To this end, microorganisms were plated onto Minimal Enrichment Medium (8.2g Na₂HPO₄, 2.7g KH₂PO₄; 0.5g NaCI, O.lg MgSO . in lkL dH O) supplemented with Metal and Vitamin Stock (biotin, folic acid, riboflavin, Ca pantothenate, nicotinic acid, p-Amino Benzoic acid, pyridoxine HCL, thiamine HCL, boric acid, inositol. CuSO 4., KIO 4., FeCI, MnSO 4. and ZnSO 4 and lOmM

NH .SO, or 50mM sarcosine or 50mM creatine. Spore-formers were identified from

4 4 samples which had been subject to a heat step. Specifically, 0.1- 0.2g soil resuspended in 2ml sterile water or 2ml water samples from nature were heated to 70^*C for 10 minutes. The isolates which survived the heat step were recovered by culturing and purification on stringent media containing sarcosine or creatine as above.

Bacillus ATCC 55164 was identified in one soil sample of the many samples screened from sixteen different environments. Bacillus ATCC 55164 was initially cultured in BSM medium (lg yeast extract, 1.5g KCI, 0.5g K2HPO4, 0.25g Mg2SO4, 2.5g creatine or sarcosine in 1L H 0 at pH 7.0), but the cells grew poorly and produced low levels of activity. Of the standard growth medias tested, the strain was found to grow best (i.e. to give the best combination of biomass and enzyme activity) in LB medium (Gibco).

Under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, deposit of the above described strain of Bacillus was made with the American Type Culture Collection (ATCC) of Rockville, MD, USA on April 10, 1991 , where the deposit was given Accession Number ATCC 55164.

Applicants' assignee. Genzyme Corporation, represents that the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 11.14 and 35 USC 122. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least thirty years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicants' assignee acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.

Equivalents:

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A microorganism of the genus Bacillus and having the identifying characteristics of ATCC Deposit No. 55164 and mutants thereof.

2. A sarcosine oxidase enzyme produced by the microorganism of claim 1.

3. A sarcosine oxidase enzyme of Claim 2, which has a low reactivity with substrates other than sarcosine.

4. The enzyme of claim 3 which is stable over a temperature range of 50°C to 60'C and a pH range of 6.5 to 9.0.

5. The enzyme of claim 3 which has a molecular weight of about 40 kD.

6. A method for producing sarcosine oxidase comprising culturing a Bacillus microorganism having the identifying characteristics of ATCC 55164 under conditions permitting expression of sarcosine oxidase, harvesting cells of said microorganism from the culture medium, and recovering sarcosine oxidase from said harvested cells.

7. The method of claim 6 wherein sarcosine oxidase is recovered by the steps of breaking the cell membrane, allowing the sarcosine oxidase to be excreted into the recovery medium, and collecting the sarcosine oxidase from the recovery medium.

8. The method of claim 7 wherein the sarcosine oxidase is collected by precipitation from the recovery medium.

9. A method for the enzymatic determination of creatinine in a human fluid sample comprising the steps of:

a) contacting said sample with creatinine amidohydrolase and creatine amidinohydrolase; b) contacting the sample from step (a) with sarcosine oxidase obtained from a microorganism having the identifying characteristics of ATCC 55164, and c) assaying the sample from step (b) for the presence of a catalysis product selected from the group consisting of glycine, formaldehyde and hydrogen peroxide.

10. The method of claim 9 wherein the fluid sample also contains N-ethyl glycine.

11. A sarcosine oxidase enzyme having a Km value of about 9 mM for sarcosine

12. The sarcosine oxidase enzyme of Claim 1 1 exhibiting less than 10% relative activity with substrates chosen from the group consisting of the N-ethyl glycine,

N-methyl D/L vaiine, N-methyl D/L leucine, glycine, choline and creatine when sarcosine is used a s a substrate.

13. The sarcosine oxidase enzyme of Claim 11 which retains greater than 80% activity after incubation for 10 minutes at 60^*C.

14. The sarcosine oxidase enzyme of Claim 1 1 which has a molecular weight of about 40kD.

15. The sarcosine oxidase enzyme of Claim 1 1 which is produced by a microorganism.

16. The sarcosine oxidase enzyme of Claim 15, wherein the microorganism is from the genus Bacillus.

17. The sarcosine oxidase enzyme of Cliam 16, wherein the microorganism from genus Bacillus is ATCC Deposit No. 55164.

18. A sarcosine oxidase enzyme exhibiting less than 10% relative activity with subtrates chosen from the group consisting of the N-ethyl glycine, N-methyl D/L vaiine, N-methyl D/L leucine, glycine, choline and creatine when sarcosine is used as a substrate.

19. A sarcosine oxidase enzyme which retains greater than 80% activity after incubation for 10 minutes at 60^*C.

20. A sarcosine oxidase enzyme which has the physical and chemical properties of the sarcosine oxidase enzyme produced by the microorganism ATCC Deposit No.55164.