JPS62208281A - Production of creatinase - Google Patents

Abstract

PURPOSE:To efficiently produce creatinase without adding creatine, creatinine, etc., to a culture medium, by cultivating a strain of the genus Escherichia transformed by a gene coding creatinase. CONSTITUTION:A DNA derived from a DNA containing a gene coding creatinase, e.g. Flavobacterium U-188 (FERM-P No.2922) strain, is inserted into a vector DNA, e.g. plasmid pBR322DNA to prepare a recombinant DNA, which is used to transform or transduce a strain of the genus Escherichia, e.g. Escherichia coli HB101 (ATCC33694), etc., having the ability to produce creatinase. The resultant strain is then cultivated under aerobic condition to collect the aimed creatinase from the obtained culture.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for producing creatinase.

creatinase (creatine amidinohydrolase)
Also called idinohydrolase)
acts on creatine to produce urea and sarcosine (5arc
It is an enzyme that hydrolyzes osine), and its reaction formula is as follows.

Creatinase creatine + H20 urea + sarcosine and this creatinase is extremely useful as an enzyme for quantifying creatine in creatine-containing substances such as serum and urine.

[Conventional technology]

Traditionally, creatinase has been used, for example, in Frapopacterium A
It is produced by culturing a strain belonging to the genus Flavobacterium and having the ability to produce creatinase in the presence of creatinine, creatine, or a mixture thereof, and collecting creatinase from the culture. -8395 publication).

[Problem that the invention seeks to solve]

However, when using the above creatinase production method, it is essential to add expensive reatinine, creatine, etc. to the culture medium, so the above creatinase production method is economically disadvantageous and the production operation is complicated. There were problems such as.

[Means for solving problems]

Therefore, as a result of various studies in order to solve the above-mentioned problems, the present inventors have developed a recombinant D in which a DNA containing a gene that feeds creatinase is inserted into a vector DNA.
NA was obtained, and this recombinant was transformed into Escherichia (Esch
When a strain having the ability to produce creatinase, which is included in a strain belonging to the genus Erichia, is cultured in a medium, creatinase is efficiently produced without adding any creatinine, creatine, or a mixture thereof to the medium. Based on these findings, the present invention was completed.

That is, the present invention provides a recombinant D in which a DNA containing a gene encoding creatinase is inserted into a vector DNA.
This is a method for producing creatinase, which is characterized by culturing a strain belonging to the genus Escherichia that contains NA and has the ability to produce creatinase in a medium, and collecting creatinase from the culture.

The present invention will be explained in detail below.

First, D containing the gene encoding creatinase
The preparation of NA will be described.

Flavobacterium U-188
No. (FERM P-NL2922) Co.
A culture is obtained by culturing in exactly the same manner as the method described in Publication No. 2-8395. This culture was incubated for example at 3000 r,
p, m, or more, preferably 8000 to 10000 r,
Centrifugation is performed for 5 minutes or more, preferably 10 to 15 minutes, at p, m, to obtain Flavobacterium U-188 strain +7) cells.

From this bacterial body, for example, the method of Saito and Miura [Biochem,
Biophys, Acta, (Biochem, Biop
hys.

ACta, vol. 72, p. 619 (1963) K. S. Kirby's method Biochem, J.
), Vol. 64, p. 405 (1956). Chromosomal DNA can be obtained by the method of Ko et al.

Next, a restriction enzyme that produces overhanging ends, such as 5au3AI (manufactured by Toyobo Co., Ltd.), is added to this chromosomal DNA at a temperature of 30"C or higher, preferably at 37°C, and at an enzyme concentration of 1 to 10.
Units)/d for 20 minutes or more, preferably 1 to 2 hours, to obtain a mixture of various chromosomal DNA fragments.

From the DNA fragment mixture thus obtained, a DNA fragment mixture is obtained, for example, by ordinary 7-galose gel electrophoresis, further purified by a purification means such as phenol extraction, and further concentrated by a concentration means such as ethanol precipitation. Then, a purified DNA fragment mixture (which includes a DNA fragment containing the gene encoding creatinase) is obtained.

On the other hand, vector DN that can be used in the present invention
A may be of any kind, such as plasmid vector DNA, bacteriophage vector DNA, etc., but specifically, for example, plasmid pBR3.
22DNA [Bethesda Research Laboratories (
Bethesda Research Laborato
[manufactured by Ries] and the like are preferred.

A restriction enzyme that produces overhanging ends, such as Bam Hl (manufactured by Takara Shuzo Co., Ltd.), is added to the vector DNA at a temperature of 3.
0°C or higher, preferably 37°C, enzyme concentration 10-100
0 units)/at for 1 hour or more, preferably 2 to 3
Digestion is performed over a period of time to obtain cleaved vector DNA.

Then, Flavobacterium U- obtained as above
188-derived DNA fragment mixture containing the gene encoding creatinase and the cut vector DNA
For example, E. coli DNA ligase (manufactured by Niney England Bio-Lapse), T4 DNA, etc.
! + gauze (manufactured by Boehringer Mannheim), preferably T4 DNA ligase, at a temperature of 4 to 37°C, preferably 4 to 16°C, and an enzyme concentration of 1 to 100 units for 1 hour or more, preferably 6 to 24 hours. Recombinant DNA is obtained.

Using this recombinant DNA, for example, -12
, preferably E. coli HBIOI (ATCC33694
), Escherichia coli DHI (ATCC 33849), Escherichia coli χ-1776 (ATCC 31244), etc. are transformed or transduced to obtain the respective strains. This transformation was carried out using D.M. Mauriso7 (D, M, Mo
Methods in Enzymology
, Vol. 68, pp. 326-331 (1979). Transduction is also carried out by the method of B. Hohn (1979).
You can do one thing by

By screening the above-mentioned strains for strains that have creatinase-producing ability, we found that the strain contains a recombinant DNA in which DNA containing a gene encoding creatinase was inserted into vector DNA, and belongs to the genus Thruetsu'7zlisia that has creatinase-producing ability. strains can be obtained.

In order to obtain a novel recombinant DNA purified from the strain obtained in this way, for example, B. gooley (P.
Guerry et al. [Jay, Bacteriology]
(J, Bacteriology) Volume 116, No. 1
064-1066 (1973)], the method of D. B. Clewe 11 [J. Bacteriology Vol. 110, pp. 667-676 (1972)], etc. can.

In order to produce creatinase using a strain belonging to the genus Escherichia which contains a recombinant DNA obtained by inserting the DNA containing the gene encoding creatinase obtained as described above into the vector DNA and has the ability to produce creatinase, this method is used. Although the bacterial strain may be cultured using a conventional solid culture method, it is preferable to use a liquid culture method.

In addition, as a medium for culturing the above-mentioned bacterial strain, for example,
J extract, hefton, meat extract, cornstap liquor or soybean or wheat mill infusion, with one or more nitrogen sources such as monopotassium phosphate, dipotassium phosphate,
One or more inorganic salts such as magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate are added, and if necessary, carbohydrate raw materials, vitamins, etc. are added as appropriate.

In addition, it is appropriate to adjust the initial pH of the culture medium to 7-9. In addition, the culture is carried out at 30-42°C, preferably at 37°C.
It is preferable to carry out the culture for 4 to 24 hours, preferably for 6 to 8 hours, by aeration and agitation deep culture, shaking culture, stationary culture, etc.

After completion of the culture, creatinase can be collected from the culture using a conventional enzyme collection method.

For example, the bacterial cells may be destroyed by ultrasonication or grinding using conventional methods, the enzyme may be extracted using a lytic enzyme such as lysozyme, or the enzyme may be shaken or left in the presence of toluene, etc. This enzyme is excreted from the bacterial body by autolysis. This solution is filtered, centrifuged, etc. to remove solid portions, and if necessary, nucleic acids are removed using streptomycin sulfate, protamine sulfate, or manganese sulfate, and then ammonium sulfate, alcohol, acetone, etc. are added to the solution for fractionation. The precipitate is collected, dialyzed against water, and then vacuum dried to obtain a crude enzyme preparation.

Furthermore, in order to obtain a purified product of creatinase, for example, DEAE-cellulose (di-ethyl amino ethyl cellulose, manufactured by Braun, USA), DEAE-Sephadex (di-ethyl amino ethyl・Sephadex, Sweden) ・Manufactured by Almasia), QA
Purification is performed by adsorption/elution using an ion exchange material such as E-Sephadex (manufactured by Pharmacia, Sweden), or Sephadex G-200 (manufactured by Pharmacia, Sweden), or Sephadex G-200 (manufactured by Pharmacia, Sweden), or Sepharose 6B (manufactured by Pharmacia, Sweden). By appropriately selecting and combining gel filtration methods using hytroxylapatite (Bio-Gel HT, manufactured by Bio-Rad, USA), electrophoresis using polyacrylamide gel, etc. , a highly purified creatinase preparation can be obtained.

The physicochemical properties of purified creatinase obtained by the above purification method are exactly the same as those of creatinase described in Japanese Patent Publication No. 52-8395.

〔Effect of the invention〕

As is clear from the above, according to the present invention, creatine, creatinine, etc. are not added to the culture medium,
Since creatinase can be obtained efficiently, the present invention is extremely useful industrially.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example (1) Chromosomal DNA of Flavobacterium U-188 strain
Preparation of Flavobacterium U-188 (FERM P-Han2
922) strain in TY medium (1% (W/V) Bacto-trypton [manufactured by Dirco, 0.5% (W/V) Bacto yeast extract]. (Bacto-yeast
extract) [manufactured by Difco,
0.5% (W/V) NaCl (p) (7,2) l 1
00 d, and cultured with shaking at a temperature of 30° C. for 8 hours to obtain a culture.

This culture was centrifuged for 10 minutes at 1000 o r, p, m in a conventional manner to obtain 0.5 y of wet bacterial cells.
Saito and Miura's method [Biochem, Biophys, A.
cta, ), vol. 72, p. 619 (1963)
Chromosomal DNA was obtained by

Next, 0.2 mg of this chromosomal DNA and 3A of restriction enzymes! (manufactured by Toyobo Co., Ltd.) 1.45 units in 10mM) lithium-hydrochloric acid buffer (50mM NaC 1%)
(containing lomMMgs04 and lmM dithiothreitol) (pH 7.4), and heated to 37°C.
The reaction was carried out at C for 1 hour. The reaction-completed solution was reduced to 0.7 by a conventional method.
% (W/V) agarose gel (manufactured by Takara Shuzo Co., Ltd.) After electrophoresis, DNA fragments with a size of 4 to 8 Kb (kilobase pair) were collected using 7-LCA Young (R,
C, A, Yang) etc. [Methods in Enzymol
ogy), Vol. 68, pp. 176-182 (1979
2000) to obtain the eluate, which was subjected to phenol extraction treatment using a conventional method, and then ethanol precipitation treatment.
Flavobacterium U-18 digested with au3A+
10 μy of chromosomal DNA fragments of 8 strains were obtained.

(2) Novel recombinant plasmid pKLS511DN
Preparation of A Plasmid pBR322DNA [Bethesda Research Laboratories (Bethesda Re5ear
chLaboratories)] 10μ, S+
and restriction enzyme BamHI (manufactured by Takara Shuzo Co., Ltd.) 140 units at 50 mM)! JS hydrochloric acid buffer (100mM NaC
1, and 10 mM Mg5O4) (pH 7,
4) and reacted at a temperature of 37°C for 2 hours to obtain a digestive fluid, and the vaginal fluid was subjected to phenol extraction and ethanol precipitation using a conventional method to obtain plasmid pBR digested with BamHI.
322 DNA was obtained. Next, this BamHI-digested plasmid pBR
322DNALoμ11 5au3 obtained in (1) above
．． Chromosomal DNA fragment lOμy of Flavobacterium U-188 strain digested with AI and 2 units of T4 DNA
Ligase [Boehringer Mannheim
ger Mannheim)] at 6.6mMMg.
C12, 10mM dithiothreitol and 10mM
66mM) Liss-HCl buffer (pH 7 containing TP)
, 5) and reacted at a temperature of 16°C for 10 hours.
A was connected.

Next, D.M. Morrison (D.M.

Morrison's method [Methods in Enzymolog]
y), Vol. 68, pp. 326-331 (1979)
Escherichia coli H8101 strain (A
TCC33694) was ligated as above.
3200 ampicillin-resistant and tetracycline-sensitive transformants were obtained.

Whether or not the thus obtained transformed strain had creatinase activity was tested as follows.

Each strain was inoculated with SuT-containing 0.2% (W/V) creatine (Sigma) and 50 μl/me ampicillin.
It was inoculated into Y medium 51Rt and cultured at a temperature of 30°C for 24 hours to obtain a culture solution. This is 350Or, p, m, 10
Centrifugation treatment was performed for 1 minute to obtain wet bacterial cells, and the bacterial cells were 1% (W
/V) 10mM phosphate buffer containing creatine (p
H7,0) was suspended in 1 ml, 20 μl of toluene was added thereto, and after reacting at a temperature of 37°C for 1 hour,
A reaction stop solution was obtained by heat treatment at a temperature of 100°C for 5 minutes.

Next, this solution was centrifuged for 10 minutes at 3500 r, p, m in a conventional manner to obtain a reaction solution, and this reaction solution was
．． 3 units/me sarcosine oxy-yase (manufactured by Seiun Pharmaceutical Co., Ltd.), 0.07% (W/V) 4-7 minoantibiline, 0.2% (W/V) 2.4-dichlorophenol sulfonation solution, and 70 units/me peroxidase (manufactured by Toyobo Co., Ltd.) was added at 0 and Lmt, respectively, and the reaction solution turned red is a transformed strain having creatinase activity.

Escherichia coli (E.coli) HB101, a transformed strain with creatinase activity thus obtained.
(pKLs511) was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 992 (FERM BP-99).
It has been deposited as 2).

(3) Isolation of recombinant plasmid pKLs511 DNA Tryptone 1% (W/v), yeast extract 0.5% (W/v)
V), and a medium consisting of NaC1O, 5% (W/V)
E. coli HB 101 (p
KLs511) was inoculated with 20 ml of culture solution, and the temperature was 37.
After 3 hours of shaking culture at °C, 0.21 g of chloramphenicol was added to the culture solution, and the culture was further cultured at the same temperature for 20 hours to obtain a culture solution.

Next, this culture solution was heated at 10,000 r, p by a conventional method.
, m.

Centrifugation was performed for 10 minutes to obtain wet bacterial cells, which were then centrifuged for 20 minutes.
ml containing 25% (W/V) sucrose6
After suspending in 50mM Tris-HCl buffer (pH 8,0), 10μ of lysozyme, 0.25M
8 ml of EDTA solution (pH 8,0) and 20% (W
/V) Eight doses of sodium dodecyl sulfate solution were added to each, and kept at a temperature of 60° C. for 30 minutes to lyse the bacteria to obtain a lysate.

To this lysate, 5M NaCl solution L3d was added and treated at a temperature of 4°C for 16 hours.
Centrifuge at 0 r, p, m for 30 minutes to obtain the extract.
After phenol extraction by a conventional method, ethanol precipitation was performed by a conventional method to obtain a precipitate.

Next, this precipitate was dried under normal vacuum, and then
It was dissolved in 6 ml of 10 mM) lithium-hydrochloric acid buffer (pH 7,5) containing 1 mM EDTA, to which was added 0.2 scraps of cesium 6I chloride and 10 mg/d ethidium bromide, and then dissolved in a conventional manner. 390
Equilibrium density gradient centrifugation was performed using an ultracentrifuge at 00 r,p,m for 42 hours to obtain the recombinant plasmid pK.
After isolating Ls511 DNA and removing ethidium bromide using n-butanol, 950 μy of purified recombinant plasmid pKLS511 DNA was obtained by dialysis against 10 mM Lis-HCl buffer (pH 7.5) containing 1 mM EDTA. I got it.

(4) E. coli HBIOI (pKLs)
511) and isolation of the enzyme, purified tryptone 1% (W/V), yeast extract 0.5% (W/V)
/V), and 2 g of medium consisting of 0.5% (W/V) salt.
was dispensed into the culture tank of a stirring-type small culture device (manufactured by Iwashiya Co., Ltd.) and sterilized under high pressure using a conventional method.Escherichia coli ( E. coli) HB 101 (pKLs51
Inoculating 20 wg of the culture solution in 1) and culturing with aeration for 8 hours at a temperature of 37°C was repeated 5 times to obtain 30 wg of moist bacterial cells.
I got g.

100 t of 0.05M phosphate buffer (pH 8.0) containing 1mM 2-mercaptoethanol containing these bacterial cells.
After suspending in
Centrifuge for 30 minutes at 5000 r, p, m, and add 120 tIIl (0
, 8 units/me).

The thus obtained crude enzyme solution was subjected to nucleic acid removal treatment using streptomycin sulfate, and then applied to a DEAE-cellulose column (in advance with 0.05 M!J acid buffer containing 1 mM 2-mercaptoethanol). (2×30 cm) to obtain the non-adsorbed section. This was prepared using DEAE-
Sephadex A-50 column (1,4X40 cm)
After adsorption to the solution, eluate containing creatinase was obtained at a salt concentration range of 0.1 to 0.3 mol by elution using a gradient elution method with a salt concentration of 0 to 1.5 mol.

This creatinase-containing eluate was transferred to a Sepharace 6B column (2X 108 c
m) to collect the creatinase activity fraction,
After further concentration using a conventional method, 43 units of purified creatinase powder was obtained by freeze-drying. In addition,
The yield was 45%.

Claims (3)

[Claims] (1) D containing the gene encoding creatinase
A method for producing creatinase, which comprises culturing in a medium a strain belonging to the genus Escherichia that contains recombinant DNA in which NA is inserted into vector DNA and has the ability to produce creatinase, and collects creatinase from the culture. (2) D containing the gene encoding creatinase
NA is DNA derived from Flavobacterium U-188 strain
A method for producing creatinase according to claim 1. (3) Vector DNA is plasmid pBR322DN
A method for producing creatinase according to claim 1.