# Patent

## Title
Method and apparatus for the manufacture of a product having a substance

## Abstract

The disclosure is made on a polydeoxyribonucleic acid having a base sequence encoding an amino acid sequence of a polypeptide which is a creatinase derived from a creatinase-producing microorganism belonging to the genus of Bacillus. The bsae sequence of the creatinase gene DNA and amino acid sequence of the creatinase have been clarified. The transformant having this polydeoxyribonucleic acid as well as a process for preparing the creatinase at a high productivity with the application of genetic engineering technique are disclosed.

## Background
1. Field of the Invention

The present invention relates to a novel polydeoxyribonucleic acid having a genetic information of creatinase.

The invention also relates to a transformant having said polydeoxyribonucleic acid, and to the creatinase and a process for preparing the same through expression by the transformant of the genetic information of said polydeoxyribonucleic acid.

2. Description of the Background

Creatinase is a enzyme which catalyzes the reaction of creatine and water to produce urea and sarcosine. This substance has been known to exist in microorganisms belonging to genera of Pseudomonas Journal of General Microbiology, 14, 351,(1956)], Artherobactor [Molecular & Cellular Biochemistry, 3, 9, (1974)], Alcaligenes, . Penicilium (Japanese Patent Publication No. 7674/1981), Flavobacterium, Micrococcus, Colinebacterium (Japanese Patent Publication No. 8395/1977), Bacillus (Japanese Patent Publication No. 17465/1986), Actinobacillus (Japanese Patent Laid-open No. 67484/1981), and Acinetobactor (Japanese Patent Laid-open No. 67485/1981). Also, creatinase derived from Pseudomonas cutida by the use of genetic engineering technology has been reported.

Since creatinase is a hydrogenase having creatine as its substrate, it can be used for quantitation of creatine existing in a body fluid such as serum. It can also be utilized, through measurement of the quantity of creatine produced in the creatinine-creatinase creatine-producing system, for quantitation of various substances which are the substrates of enzymes affecting the creatine producing system as well as for the determination of enzymatic activities involved in the creatine-producing system. Creatinase is thus a very important reagent for use not only in laboratory experiment but also in clinical diagonosis.

Creatinase-producing microorganisms reported heretofore which are utilized without recourse to genetic engineering technique yield only a poor creatinase-producing efficiency so that the use of an enzyme-inducing substance such as creatine or the like which can help producing creatinase has been indispensable. This renders the cost of the creatinase production expensive. In addition, according to this method removal of other types of enzymes, such as creatininase or the like, which may be present together with creatinase, is very difficult. The cost of purification to obtain a high purity creatinase makes the overall production cost even higher. Thus, the use of these creatinase-producing microorganisms have not always been effective and convenient route for providing creatinase as reagents for abundant use in laboratory experiment or clinical diagonosis.

The creatinase derived from creatinase-producing microorganisms belonging to the genus of Bacillus also requires the addition of an expensive enzyme-inducing substance in the course of its preparation. In addition, there has been no report on the detailed chemical structure of the polypeptide constituting this enzyme.

The present inventors have undertaken extensive studies in order to improve the productivity of this creatinase, and have succeeded in obtaining the gene of this creatinase and further in clarifying its primary structural analysis. Further, the inventors have established a method for preparing the creatinase at a high productivity with the application of genetic engineering technique.

In addition, through analysis of the DNA sequence of the creatinase gene induced from the creatinase-producing microorganisms belonging to the genus of Bacillus the inventors have found that the enzyme possessed amino acid sequence and base sequence structures which are quite different from those of creatinase produced by conventionally known genetic engineering technique. The inventors also found that proteins obtained exhibited unexpectedly excellent creatinase activities. These findings have led to the completion of this invention.

The novel process for preparing creatinase according to this invention does not require the use of an expensive enzyme-inducing substance such as creatine in the course of creatinase production, and is thus a commercially advantageous process.

Accordingly, an object of this invention is to provide a polydeoxyribonucleic acid having a base sequence encoding an amino acid sequence of a polypeptide which is the creatinase derived from a creatinase-producing microorganism belonging to the genus of Bacillus or of a polypeptide having said creatinase as its constituent.

Another object of this invention is to provide a transformant having this polydeoxyribonucleic acid.

A further object is to provide a polypeptide comprising a creatinase having an amino acid sequence starting from the N-terminal of the following formula, or a polypeptide having such creatinase as its constituent: ##STR1## wherein A represents an amino acid residue, an acetyl group, or a hydrogen atom and B represents an amino acid residue or --OH or NH.sub.2.

A still further object of this invention is to provide a process for preparing creatinase comprising:

inserting a polydeoxyribonucleic acid which is a creatinase gene derived from a creatinase-producing microorganism belonging to the genus of Bacillus or a polydeoxyribonucleic acid having said creatinase gene as its constituent into a expression vector thus producing a recombinant DNA,

producing a transformant by introducing said recombinant DNA into a host microorganism and culturing said transformant to cause the transformant to manifest the genetic information of said polydeoxyribonucleic acid, and

collecting the polypeptide of said creatinase or the polypeptide having said creatinase as it constituent.

Other objects, features and advantages of the invention will hereinafter become more readily apparent from the following description.

FIG. 1 is a schematic drawing showing the construction of pCR1 vector, indicating sites of incision of the pCR1 vector with the use of restriction endonucleases EcoR I, Hind III, Ban II, Bgl II, Pst I, Sph I, and CIa I.

FIGS. 2-1 and 2-2 show the base sequence of the creatinase gene DNA (from 5' through 3'), and FIGS. 3-1 and 3-2 show the amino acid sequence of the translational product produced therefrom.

In the polypeptide of formula (I), the amino acid residue represented by A may be one or more amino acid residues. Preferred examples of A are a hydrogen atom, a methionine, or a signal polypeptide. The group represented by B ma be an acid amide or one or more amino acid residues.

A polydeoxyribonucleic acid which codes for a creatinase gene acting as a catalyst in the reaction of creatine and water to produce urea and sarcosine, or a polydeoxyribonucleic acid having said gene as its constituent, may be any polydeoxyribonucleic acid so long as it contains said creatinase gene itself. Given as examples of said creatinase gen itself are creatine represented by the following formula (II) starting from the N-terminal: ##STR2## as well as polydeoxyribonucleic acids having a base sequence encoding an amino acid sequence of a polypeptide comprising creatinase which catalyzes the reaction of creatine and water to produce urea and saccosine. The polydeoxyribonucleic acid can be any polydeoxyribonucleic acid so long as the same possesses any codon among several codons corresponding to each of the amino acids constituting the amino acid sequence of (II) of the creatinase polypeptide. It may also be a polydeoxyribonucleic acid having at its 5'-end one or more codons other than a nonsense codon and/or at its 3'-end one of more codons. A typical example of such polydeoxyribonucleic acid is the one having a base sequence represented starting from the 5'-end by the formula (III): ##STR3## in which X represents a codon other than TAA, TAG and TGA, or a hydrogen atom, and Y stands for a codon or a hydrogen atom.

Concerning the base sequence of formula (III), the codon represented by X may be any codon so long as the same codes for an amino acid. In addition, X may possess at its 5'-end one or more codons encoding amino acids. Preferable examples of X are ATG or a polydeoxyribonuclei acid corresponding to a signal peptide.

The codon represented by Y can be any codon selected from translation termination codons and codons encoding an amino acid. Y may possess at its 3'-end one or more codons encoding amino acids, provided that in this case it is desirable that a translation termination codon be provided at the 3'-end of these codons.

A polydeoxyribonucleic acid having a creatinase gene as its constituent, a polydeoxyribonucleic acid which is a gene having a base sequence encoding the amino acid sequence of formula (II), or a polydeoxyribonucleic acid represented by formula (III), can be prepared by the process comprising the following steps. A DNA of a creatinase-producing microorganism which is a donor of a creatinase gene is first separated and purified, followed by treatment with ultrasonic wave or with a restriction endoneuclease. This DNA and a digested linear expression vector DNA are joined with a DNA ligase blunt or cohesive ends of the two DNAs to form a closed circle. The recombinant DNA vector thus obtained is introduced into a reproducible host microorganism. Microorganisms having said recombinant DNA vector collected by means of a screening using the vector marker and the creatinase activity as indicators ar cultured. Said recombinant DNA vector is then separated from the cultured microorganisms and purified, from which the creatinase gene polydeoxyribonucleic acid is collected.

Any creatinase-producing microorganisms can be used as a creatinase gene-donating microorganism for the purpose of this invention. An example is Bacillus SP B-0618 strain (FERM BP-0750) disclosed in Japanese Patent Publication No. 17465/1986.

Transformed microorganisms derived from the genus of Bacillus, to which creatinase-producing capability was provided by the use of genetic engineering technique, can also be employed as a creatinase gene-donating microorganism.

The method of collecting a DNA induced by the gene-donating microorganism is now exemplarily illustrated. Any one of the above-mentioned gene-donating microorganisms is first cultured in a liquid culture medium under aeration for 1 to 3 days. The broth thus cultured is subjected to centrifugation to collect the microorganism, which is then lysed to produce a bacteriolysis containing a creatinase gene. Treatment with a cell wall lysing enzyme such as lysozyme or .beta.-glucanase is used for the bacteriolysis, in combination, as required, with other enzyme such as protease or a surface active agent such as sodium laurylsulfate. In addition, physical digestion of cell walls by means of freeze-thawing or French press, for example, may be employed together with the bacteriolysis.

Conventional methods of purification, including, for example, phenol extraction, protease treatment, ribonuclease treatment, precipitation from alcohol, and centrifugation, can be applied either independently or in combination to the separation and purification of DNA from the bacteriolysis.

Digestion of the DNA of the microorganism thus separated and purified can be carried out by means of treatment with ultrasonic wave or a restriction endoneuclease, for instance. In order to ensure easy joining of the DNA fragments and the vector DNA, however, the use of a restriction endoneuclease is preferable, especially those having an activity to a specific nucleotide sequences such as EcoR I, Hind III, BamH I, or BamH II.

Desirable vectors employed are those reconstructed for use as a genetic recombinant DNA through an artificial treatment of a phage or a plasmid DNA which is capable of growing autonomically in host bacterial cells.

In case where Escherichia coli is used as a host microorganism, for example, .lambda.gt..lambda.C, .lambda.gt..lambda.B, or the like is used as a phage.

As a plasmid, pBR322, pBR325, pACYC184, pUC12, pUC13, pUC18, pUC19, or the like is used when Escherichia coli is the host microorganism, pUB110, pC194, or the like is used when Bacillus subtillis is the host microorganism, and YRp7, pYC1, YEp13, pJDB, YIp1, or the like is used when Saccharomyces cerevisiae is the host microorganism. In addition, suttlevectors may be employed, which can autonomically grow in host bacterial cells of either or both of Gram-positive and Gram-negative microorganism, for instance, in either or both of Escherichia coli and Saccharomyces cerevisiae. These vectors are desirably digested into vector fragments by the use of the same restriction endoneuclease as that used in breaking the above-mentioned creatinase gene-donating microorganism DNA.

Conventional method of using DNA ligase can be employed for joining the bacterial DNA and the vector fragment. For instance, the cohesive end of the bacterial DNA and that of the vector fragment are first annealed, and then a recombinant DNA of the bacterial DNA and the vector fragment can be prepared by the action of a suitable DNA ligase. If required, the annealed bacterial DNA--vector fragment is introduced into the host microorganism to produce the recombinant DNA with the aid of in vivo DNA ligase.

Any microorganisms can be used as host bacteria, which allow autonomic and stable growth of the recombinant DNA and are capable of manifesting the character of the extraneous DNA. Examples of such microorganisms include Escherichia coli DH1, Escherichia coli HB101, Escherichia coli W3110, Escherichia coli C600, and the like when Escherichia coli is used as a host bacterium, Bacillus subtillis 207-25 [Gene, 34, 1-8, (1985)], Bacillus subtillis 207-21 [Journal of Biochemistry, 95, 87-93, (1984)], Bacillus subtillis BD170 [Nature, 293, 481-483, (1981)], Bacillus subtillis M (ATCC6051), and the like when Bacillus subtillis is used as a host bacteria, Saccharomyces cerevisiae AH-22 [Gene, 39, 117-120, (1985)], Saccharomyces cerevisiae BWG1-7A [Molecular & Cellular Biology, 6, 355-367, (1986)], and the like when Saccharomyces cerevisiae is used as a host bacteria.

Introducing the recombinant DNA into the host microorganism may be performed in the presence of calcium ion when the host microorganism is a bacterium belonging to the genus of Escherichia. When a bacterium belonging to the genus of Bacillus is used as a host microorganism, either the competent cell method, protoplast method, or micro-injection method can be used. For the host bacterium belonging to the genus of Saccharomyces the protoplast method or lithium acetate method may be employed.

The transformant bacteria thus obtained, when cultured in a nutrient medium, were found to stably produce a large amount of creatinase. Introducing purposive DNA into the host microorganism can be retrieved by means of detection of the microorganism which can manifest a drug resistance marker of the vector on which the purposive recombinant DNA is held as well as creatinase at the same time. For instance, those bacteria which grow in a selective culture medium of the drug resistance marker and which produce creatinase can be selected.

The recombinant DNA possessing creatinase gene once selected in this manner may be easily extracted from the transformant microorganism for introducing into another host bacterium. Alternatively, a creatinase gene DNA can be digested using a restriction endoneuclease or the like from a recombinant DNA possessing a creatinase gene, and is joined with a vector fragment obtained in a similar manner. The joined DNA--vector fragment is then introduced into the host microorganism.

A creatinase mutein which is a mutant produced by genetic engineering technique from a creatinase gene of this invention possessing substantial creatinase activity can be prepared by amplifying this mutant by the use of various methods, producing the recombinant DNA by inserting the mutant into a vector, and introducing this recombinant DNA into the host microorganism.

The base sequence of the creatinase gene prepared by the method described above was sequenced by the deoxy method [Science, 214, 1205-1210 (1981)]. The amino acid sequence of creatinase was determined based on the base sequence.

The method employed in the determination of the amino acid sequence of the portion constituting the N-terminal of the creatinase peptide is now discussed. The creatinase gene-donating microorganism capable of producing creatinase were first cultured in a nutrient medium to produce and accumulate creatinase in the bacteria. The cultured bacteria were collected from the broth by filtration, centrifugation, or the like means. The collected bacteria were then digested either by mechanical means or enzymatic means using lysozyme or the like, and to the digested bacteria were added EDTA and/or a suitable surface active agent, as required, to solubilize creatinase, which was then separated as an aqueous solution. This aqueous solution of creatinase was condensed or subjected to ammonium sulfate fractionation, gel filtration, adsorption chromatography, or ion exchange chromatography to obtain high purity creatinase. The amino acid sequence of the portion constituting the N-terminal of the creatinase peptide was determined on this high purity creatinase using a liquid phase protein sequencer (Beckman System 890ME, manufactured by Beckman, Inc.). In this manner, it was confirmed that the amino acid sequence of said portion is identical to the N-terminal amino acid sequence of the creatinase obtained by genetic engineering technique.

Culturing the transformant host microorganism is conducted under the conditions determined taking the nutrient--physiological characteristics of the host microorganism into consideration. In most of the cases a liquid culturing is employed. In an industrial scale production, however, culturing under deep aerobic stirring is more advantageous. A wide variety of nutrients conventionally used for culturing bacteria can be used for culturing the host microorganism. Specifically, any nutritious carbon compounds can be used as carbon sources, including, for example, glucose, sucrose, lactose, maltose, fructose, molasses, and the like. As nitrogen sources, any available nitrogen compounds can be employed, inclusive of peptones, meat extracts, yeast extracts, casein hydrolysates, and the like. Other ingredients, including salts such as phosphates, carbonates, and sulfates, as well as salts of magnesium, calcium, potassium, iron, manganese, zinc, and the like, and certain types of amino acids or vitamins, may be used as appropriate.

The culturing temperature can be varied in a range in which the bacteria can grow and produce creatinase. The preferable temperature range is 20.degree.-42.degree. C. for the Escherichia coli, 30.degree.-37.degree. C. for Bacillus subtillis, and 25.degree.-35.degree. C. for Saccharomyces cerevisiae. The culturing time may be varied to some degree depending on the culturing conditions. Basically, the culturing is terminated at the time when the yield of creatinase reaches maximum. In usual practice, it takes about 12-48 hours when the host bacterium is Escherichia coli, 18-42 hours when the host bacterium is Bacillus subtillis, and 24-48 hours when the host bacterium is Saccharomyces cerevisiae. It is possible to change the pH of the culture media within the range in which the bacteria can grow and produce creatinase. The especially preferable pH range is 6-8 for Escherichia coli, about 7 for Bacillus subtillis, and 5-7 for Saccharomyces cerevisiae.

Creatinase may be served for use in the form of culture broth as it contains bacteria. The creatinase contained in the culture broth, however, is generally used after separation of the bacteria therefrom by filtration, centrifugation, or the like means. In case creatinase is contained in the bacterial body, the bacteria is first separated by means of filtration or centrifugation. The collected bacteria are then digested either by mechanical means or enzymatic means using lysozyme or the like, and to the digested bacteria are added a chelating agent such as EDTA and/or a suitable surface active agent, as required, to solubilize creatinase, thus collecting creatinase as an aqueous solution.

The solutions containing creatinase thus obtained are then condensed by evaporation in vacuo or by the use of a filter, and subjected to salting-out treatment with ammonium sulfate, sodium sulfate, or the like, or to fractional precipitation using a hydrophilic organic solvent such as methanol, ethanol, acetone, or the like. The precipitate is dissolved into water, and the solution is dialyzed through a semipermeable membrane to eliminate low molecular weight impurities. Alternatively, the precipitate is refined by means of gel filtration, adsorption chromatography, ionexchange chromatography, or the like using an adsorbent or a gel filtration agent. Purified creatinase is produced from the creatinase-containing solution obtained by the use of these various means through vacuum evaporation, lyophilization, or the like.

In the description of this specification, amino acids, peptides, nucleic acids, and nucleic acids-related compounds are abbreviated according to the standard prevailing in the art. Some of the examples of the abbreviation are listed below. Also, all designation of amino acids denote the L-isomers.

Other features of the invention will become apparent in the course of the following description of the exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

A chromosome DNA was prepared from strain of Bacillus sp B-0618 (FERM BP-0750) by the following method. The strain was cultured with shaking in 150 ml of a normal bouillon medium containing 0.5% of sodium thiosulfate at 37.degree. C. overnight. The cultured broth was centrifuged at 3,000 rpm for 10 minutes to collect bacterial cells, which was suspended into 5 ml of a solution containing 10% sucrose, 50 mM Tris hydrochloric acid (pH 8.0), and 50 mM EDTA. To the suspension, 1 ml of lysozyme solution (10 mg/ml) was added, and the mixture was incubated at 37.degree. C. for 15 minutes, followed by addition of 1 ml of 10% SDS (sodium dodecylsulfate). To the suspension thus obtained the equal volume of a mixed solvent of chloroform and phenol (1:1) was added, and the mixture was stirred and centrifuged at 10,000 rpm for 3 minutes to separate water and solvent layers. To the separated water layer, 2-fold volume of ethanol was moderately added, and the mixture was stirred slowly with a glass rod so as to cause the DNA wound around the rod. The DNA separated in this manner was dissolved into 10 ml of a solution containing 10 mM Tris hydrochloric acid (pH 8.0) and 1 mM EDTA (such a solution is hereinafter referred to as "TE"). This solution was treated with the equal volume of chloroform--phenol mixed solvent, and was again centrifuged to separate the water layer. To this solution, 2-fold volume of ethanol was further added, and the DNA was again separated from the mixture in the same manner as described above. This finally obtained DNA was dissolved into 2 ml of TE.

Escherichia coli pM191 carring pACYC 184 therein [J. Bacteriol, 134, 1141 (1981); ATCC37033]was cultured with shaking in 1 liter BHI medium (produced by Difco Co.). When the turbidity of the broth reached OD.sub.660 =1.0, spectinomycin was added to it at a final concentration of 300 .mu.g/ml. Shaking of the broth at 37.degree. C. was continued for at least 16 hours. Upon termination of the shake culturing the broth was centrifuged at 3,000 rpm for 10 minutes to collect bacteria, from which the plasmid DNA was prepared according to the lysozyme--SDS method and the cesium chloride--ethidium bromide method [Maniatis et al, Molecular Cloning, 86-94, Cold Spring Harbor (1982)].

(1) Two (2) .mu.1 (about 0.5 .mu.g) of Bacillus sp chromosome DNA prepared in Example 1, 1 .mu.l of 10-fold concentration of Hind III digestion buffer [100 mM Tris hydrochloric acid (pH 7.5), 70 mM MgCl.sub.2, 600 mM NaCl, 70 mM mercapto ethanol], 1 .mu.l of Hind III (10 unit/.mu.l; produced by Takara Shuzo Co., Ltd.), and 6 .mu.l of water was mixed and incubated at 37.degree. C. for 1 hour for digestion. Plasmid pACYC 184 DNA (about 0.3 .mu.g) was separately digested using Hind III according to the similar manner. To this was added 0.6 unit of alkaline phosphatase (produced by Takara Shuzo Co., Ltd.; hereinafter referred to as "BAP") and the mixture was incubated at 65.degree. C. for 1 hour. The two DNA solutions thus prepared were mixed together, and to this mixed DNA solution, 0.1 volume of  3M sodium acetate was added. Subsequently, the solution was treated with the equal volume of a chloroform-phenol mixed solvent and centrifuged to separate the water layer. To this the twice amount of ethanol was added, and DNA was precipitated by means of centrifugation and dried in vacuo. The dried DNA was dissolved into 89 .mu.l of water, and to this 10 .mu.l of 10-fold concentration of Leigation buffer [0.5 M Tris hydrochloric acid (pH 7.6), 0.1 M MgCl.sub.2, 0.1 M dithiothreitol, 10 mM spermidine, 10 mM ATP) and 1 .mu.l of T4 DNA ligase (175 unit; produced by Takara Shuzo Co., Ltd.) were added and mixed, and the mixture was allowed to stand at 4.degree. C. overnight. This DNA solution was treated with a chloroform-phenol mixture, and the DNA was precipitated by ethanol, dried in vacuo, and dissolved into 10 .mu.l of TE.

(2) Escherichia coli DH 1 (Stock No. ME8569; provided by National Gene Research Institute) was cultured in 100 ml of BHI medium (Brain Heart Infusion, produced by Difco Co.), collected at the logarithmic growth phase by centrifugation (10,000 rpm, 2 minutes), and suspended into 40 ml of ice-cooled solution containing 30 mM potassium acetate, 100 mM RbCl, 10 mM CaCl.sub.2, 50 mM MnCl.sub.2, and 15% glycerin (pH 5.8). After having been allowed to stand at 0.degree. C. for 5 minutes, the suspension was centrifuged to remove the supernatant. The cells were suspended into 4 ml of an ice-cooled solution containing 10 mM MOPS buffer (produced by Dotite Co.), 75 mM CaCl.sub.2, 10 mM RbCl, and 15% glycerin (pH 6.5), and the suspension was left at 0.degree. C. for 5 minutes to obtain competent cells.

(3) To 200 .mu.l of the cell suspension 10 .mu.l of the DNA solution prepared in (1) above was added. After the mixture was allowed to stand still at 0.degree. C. for 30 minutes, 1 ml of BHI medium was added to it, and was kept at 37.degree. C. for 90 minutes. An aliquote of the mixture (0.1 ml) was spread on a BHI agar plate containing 25.mu.g/ml of chloramphenicol, and cultured overnight at 37.degree. C. to produce a transformant. This transformant was replicated on a creatinase medium plate (composition: peptone: 5 g, meat extract: 2 g, yeast extract: 5 g, NaCl: 1 g, K.sub.2 HPO.sub.4 : 1 g, MgSO.sub.4 : 0.5 g, peroxidase: 500 IU, sarcosine oxidase: 500 IU, dianisidine: 0.1 g, creatine: 11.5 g, agar: 15 g, distilled water: 1 liter; pH 7.0), and was further cultured overnight at 37.degree. C..

The periphery of 3 colonies among about 3,000 transformants was colored into charcoal. One of the three strains was named Escherichia coli DHI pCR1 (Deposited with Fermentation Research Institute, Agency of Industrial Science and Technology; Deposition No. 9495, FERM P-9495). This strain was cultured after purification in a BHI medium overnight at 37.degree. C. to determine its creatinase productivity according to the creatinase activity measurement method described below. As a result, the creatinase activity was found to be a 0.5 u/ml.

The plasmid contained in the Escherichia coli DHI pCR1 strain was separated according to the method described in Example 2, and the plasmid containing a creatinase gene and pACYC 184 gene was named pCR1.

The creatinase activity was determined by the following method in this invention.

Three solutions were first prepared: First Solution: 0.2M phosphate buffer (pH 7.5) 0.5 ml 50mM creatine aqueous solution 4.5 ml Second Solution: 0.2M Tris hydrochloric acid buffer 0.5 ml (pH 8.0) 15mM 4-aminoantipyrine aqueous solution 0.5 ml 0.2% phenol              0.5 ml peroxidase (50 u/ml)     0.5 ml sarcosine oxidase (30 u/ml) 1.0 ml 0.5mM p-chloromercury benzene 2.0 ml Third Solution: 0.5mM p-chloromercury benzene ______________________________________

A half ml of the first solution was pipetted into a test tube and equilibrated at 37.degree. C. for 3 minutes. To this 10 .mu.l of an enzyme solution was added and maintained at 37.degree. C. for precisely 5 minutes. To the mixture were added 0.5 ml of the third solution and succeedingly 0.5 ml of the second solution. After maintaining this mixture at 37.degree. C. for further 20 minutes, 1.5 ml of distilled water was added and absorbance was measured at a wave length of 500 nm. One unit of the enzyme was defined as the amount to produce 1.mu. mol of hydrogen peroxide per minute.

The pCR1 plasmid DNA was prepared from Escherichia coli DH1 pCR1 strain in the same manner as described for the preparation of pACYC 184. A pCR1 DNA creavage map was prepared using restriction endoneucleases CIa I, EcoR I, Hind III, Bgl II, Pst I, Sph I (all produced by Takara Shuzo Co., Ltd.), and Ban II (produced by Toyobo Co., Ltd.). The results are shown in FIG. 1. The base sequence of DNA containing creatinase gene was determined according to Dideoxy Method using M13 phage [Science, 214, 1205-1210 (1981)]. FIG. 2 shows the base sequence and amino acid sequence of the creatinase gene.

Escherichia coli DH1 pCR1 was cultured in 20 liters of BHI medium (produced by Difco Co.) through aeration-stirring at 37.degree. C. for 16 hours using a 30-liter jar fermenter. The cultured bacteria were collected by centrifugation at 5,000 rpm for 10 minutes. The creatinase productivity achieved was 5.0 u/ml. The bacteria were washed with 2 liters of physiological saline and suspended into 2 litters of 10 mM phosphate buffer (pH 7.0)

To the suspension thus prepared were added 1 mg/ml of lysozyme chloride and EDTA-2Na (to provide 2 mM concentration). After incubation at 37.degree. C. for 60 minutes with stirring, the mixture was centrifuged at 15,000 rpm for 10 minutes and 1.7 liters of the resulting supernatant was collected.

To the supernatant was slowly added 680 g of ammonium sulfate to salt out a protein precipitate. The precipitate was dissolved into 200 ml of 10 mM phosphate buffer (pH 7.0) and was then subjected to a Sephadex G-25 column for desalting. Subsequently, the desalted solution was subjected to DEAE-Cephallose CL-6B ion exchange chromatography to collect the active fraction. This fraction was desalted and freeze dried to give 4.08 g of a powdery product. The yield achieved was 42%, with the specific activity of the product being 10.3 u/mg.

This invention has clarified the creatinase gene derived from creatinase-producing microorganisms belonging to the genus of Bacillus and the amino acid sequence of the creatinase. In addition, the invention has provided a novel and efficient process for preparing creatinase using the creatinase gene and based on various genetic engineering technique.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

## Claims

1. An isolated and purified DNA sequence which encodes the amino acid sequence of a Bacillus creatinase, ##STR4## wherein A represents an amino acid residue or a hydrogen atom and B represents an amino acid residue or --OH.

2. The DNA sequence of claim 1, which starting from the 5'-end has the formula: ##STR5## wherein X represents a codon other than TAA, TAG or TGA, or a hydrogen atom, and Y represents a codon or a hydrogen atom.

3. A transformed microorganism, which comprises the DNA sequence of claim 1.

4. A transformed microorganism which comprises the DNA sequence of claim 2.

5. The transformed microorganism of claim 3, which is a microorganism belonging to the genus of Escherichia.

6. The transformed microorganism of claim 5, which is Escherichia coli.

7. A process for preparing a creatinase, comprising: claim 1.

8. The process of claim 7, wherein said transformed microorganism belongs to the genus Escherichia.

9. The process of claim 7, wherein said transformed microorganism is Escherichia coli.