JP2000083674A - beta-PHOSPHOGLUCOMUTASE GENE, NEW RECOMBINANT DNA AND PRODUCTION OF beta-PHOSPHOGLUCOMUTASE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene encoding a protein having a specific amino acid sequence and β-phosphoglucomutase activity and usable e.g. for production of the above enzyme useful e.g. as a conjugate enzyme for a reagent for assay of α-and β-amylases. SOLUTION: This new β-phosphoglucomutase gene is such one as to encode a protein having an amino acid sequence shown by the formula or a protein having an amino acid sequence wherein one or plural amino acid(s) is (are) deleted, substituted or added in the amino acid sequence shown by the formula and consisting of a protein having β-phosphoglucomutase activity and to be useful e.g. for production of an enzyme catalyzing the reaction wherein β-D- glucose-1-phosphate is converted into β-D-glucose-6-phosphate and useful e.g. as a conjugate enzyme for a reagent for measurement of α-, and β-amylase activity and a component for an analytical reagent for inorganic phosphate. This gene is obtained from chromosomal DNA derived from Lactococcus lactis subsp. cremoris (IFO 3427) through cloning.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a β-phosphoglucomutase gene, a novel recombinant DNA, and a method for producing β-phosphoglucomutase.

[0002]

2. Description of the Related Art An enzyme that catalyzes a reaction for converting β-D-glucose-1-phosphate to β-D-glucose-6-phosphate is
It is called β-phosphoglucomutase. β-phosphoglucomutase (hereinafter abbreviated as β-PGM) is, for example, maltopentaose as a substrate, maltose phosphorylase, β-PGM and glucose-6-phosphate.
Reagent for measuring α-amylase activity using dehydrogenase as a coupling enzyme (Biological Sample Analysis, Vol. 9, 21-2
9, 1986), maltose phosphorylase, β
-PGM and glucose-6-phosphate dehydrogenase as main components for analysis of inorganic phosphate
No. 5676), and as one conjugate enzyme such as a reagent for measuring β-amylase. (U.S. Pat. No. 4,097,336) Conventionally, β-PGM has been used for Lactococcus lactis subsp. Cremoris (Lactococcus lactis subsp. Cremoris).
moris) (IFO 3427) in culture medium and β-PG
It is manufactured by collecting M. (Japanese Patent Application 9-17
No. 6622)

[0003]

However, when the conventional method for producing Lactococcus lactis subsp. Cremoris is used, the production amount is low and the purification method is complicated, so that the production cost is high. And the like.

[0004]

The present inventors have conducted various studies in view of the above-mentioned drawbacks. As a result, the present inventors have found that β-lactide originated from Lactococcus lactis subsp. Cremoris (IFO 3427).
Successful isolation and structure determination of the PGM gene, and furthermore, the gene encoding β-PGM was
A recombinant DNA inserted into A was obtained, and the recombinant was included in a strain belonging to the genus Escherichia, and a strain having β-PGM producing ability was cultured in a medium.
The present inventors have found that β-PGM is efficiently produced, and completed the present invention.

That is, the first invention is a β-PGM gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the sequence (a), and having β-PGM activity. The second invention is to insert the above-mentioned β-PGM gene into vector DNA. Novel recombinant D characterized by the following
NA, and the third invention is the recombinant DNA,
A fourth aspect of the present invention is a β-PGM, wherein the above-mentioned transformant or transductant is cultured in a medium, and β-PGM is collected from the culture. -It is a manufacturing method of PGM.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The β-PGM gene of the present invention can be obtained, for example, as follows. First, the original strain Lactococcus
Lactis subsp.cremoris (IFO3427), for example, Current Protocols in Molecular Biology (WILEY
Intersciense, 1989) and the like, and chromosomal DNA is extracted from the obtained strain.

[0007] A probe for hybridizing a gene can be obtained, for example, as follows. First, the original strain Lactococcus lactis subsp. Cremoris (IFO3427), for example,
Cultured by the method described in the specification of No. 76622, purified and β-P
Obtain GM protein. This is shown in Example 4 as it is.
It can be used for determination of the N-terminal amino acid sequence by 73A Protein Sequencer (manufactured by Applied Biosystems). Furthermore, this is digested with, for example, a protease such as lysyl endopeptidase, and C1
The peptide fragment was fractionated by HPLC using an 8 column (manufactured by Shiseido Co., Ltd.), and similarly, 473A Protein shown in Examples
The amino acid sequence inside the protein can be determined by the Sequencer. A synthetic DNA can be designed based on the amino acid sequence obtained in this way and used as a probe as it is, or a fragment amplified by PCR can be used as a probe.

The chromosomal DNA obtained above is, for example,
It is partially digested with a restriction enzyme such as Sau3AI or completely digested with SpeI or the like, and integrated into a vector DNA according to a conventional method. Examples of the vector DNA used include pUC119 (Takara Shuzo), pBR322 (Takara Shuzo), pBluescript II K
Plasmid DNAs such as S + (Stratagene) and pMAL-C2 (NEW England Labs); bacteriophage DNAs such as λENBL3 (Stratagene) and λDASH II (Funakoshi). Obtained recombinant DNA
Using, for example, E. coli K-12, preferably E. coli JM10
9. Transform or transduce DH5α (manufactured by Toyobo), XL1-Blue (manufactured by Funakoshi) or the like to obtain respective transformants or transductants.

[0009] Transformation can be performed, for example, by the method of DMMorrison.
(Method in Enzymology, 68, 326-331, 1979). Transduction can be performed, for example, by the method of B. Hohn (Method in Enzymology, 68, 299-309, 1979).

[0010] In order to obtain a novel recombinant DNA purified from the above-mentioned transformant or transductant, for example, the β-PGM gene can be obtained by Southern hybridization, colony hybridization or the like using the probe obtained above. Obtainable.

Further, using the DNA containing the above gene, the entire base sequence of the β-PGM gene was analyzed using the 370 DNA Sequencing System (manufactured by Applied Biosystems) shown in Examples (see SEQ ID NO: 2).
Next, the primary sequence of the amino acid of the polypeptide translated by the gene having the nucleotide sequence is determined. (See SEQ ID NO: 1)

In the amino acid sequence shown in SEQ ID NO: 1, one or more amino acids are deleted, substituted or added, and the β-PGM gene encoding the amino acid sequence that brings about β-PGM activity is all Included in the present invention.

In the amino acid sequence shown in SEQ ID NO: 1, one or more, preferably several amino acids are deleted, substituted or added, and β-
Β-P encoding an amino acid sequence conferring PGM activity
Any method can be used to obtain the GM gene, for example, a site-directed mutagenesis method, which is a well-known technique for generating a point mutation or a deletion mutation in a gene; selectively cleaving the gene; A method of removing or adding nucleotides and linking genes; an oligonucleotide mutagenesis method, and the like.

To produce β-PGM using the transformant or transductant having the ability to produce β-PGM obtained as described above, for example, a strain belonging to the genus Escherichia, Can do it. In order to culture the above microorganism, it may be cultured by a usual solid culture method, but it is preferable to employ a liquid culture method as much as possible.

The medium for culturing the microorganism may be, for example, potassium dihydrogen phosphate in one or more nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor or soybean or wheat koji leachate. One or more inorganic salts such as dipotassium hydrogen phosphate, magnesium sulfate, ferric chloride, ferric sulfate or manganese sulfate are added, and if necessary, saccharide raw materials, vitamins and the like are appropriately added. Can be

The initial pH of the medium is suitably adjusted to 7-9. The culture is performed at 25 to 42 ° C, preferably 37
6 to 24 hours at around ℃, aeration and stirring deep culture, shaking culture,
It is preferably carried out by static culture or the like. After cultivation,
To collect β-PGM from the culture, ordinary enzyme collecting means can be used.

The cells are separated from the culture by, for example, filtration, centrifugation, etc., and washed. Then, it is preferable to collect β-PGM from the cells. in this case,
Although the cells can be used as they are, freezing and thawing, ultrasonic crusher, French press, a method of destroying the cells using various destruction means such as dynamyl, cells using a cell wall lysing enzyme such as lysozyme, etc. It is preferable to collect β-PGM from cells by a method of lysing cell walls, a method of extracting enzymes from cells using a surfactant such as Triton X-100, or the like.

From the crude enzyme solution thus obtained, β-
In order to isolate PGM, a method used for ordinary enzyme purification can be used. For example, it is preferable to carry out an appropriate combination of ammonium sulfate salting out, organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, adsorption chromatography, electrophoresis and the like.

Β-PGM obtained by the present invention has the following physicochemical properties. (a) Action: converts β-D-glucose-1-phosphate to β-D-glucose-6-phosphate. (b) Substrate specificity: acts on β-D-glucose-1-phosphate and has no effect on α-D-glucose-1-phosphate. (c) Optimum pH and stable pH range: Optimum pH is 6.8 to 7.3
The stable pH range is 5.0 to 9.5 after treatment at 30 ° C. for 30 minutes. (d) Range of suitable working temperature: The range of suitable working temperature is 35 to 42 ° C. (e) Molecular weight: about 25,000 (SDS-PAGE).

(F) Method of measuring titer: The titer of β-PGM was measured by the following method. 1 μmol of β-D- per minute
The amount of the enzyme that converts glucose-1-phosphate into β-D-glucose-6-phosphate is defined as 1 unit (U).

1. Preparation of reagents: 20 mM KCl, 2.0 mM MgCl ₂ , 0.8% (W /
V) Triton X-100, 0.3 mM β-D-glucose-
20 mM phosphate buffer (pH 7.0) containing 1,6-phosphate, 2.2 mM β-D-glucose-1-phosphate, 1.2 mM NADP and glucose-6-phosphate dehydrogenase (12 U / ml) ) Is prepared. 2. Activity measurement 3.0 ml of the above reagent was preliminarily heated at 37 ° C. for 5 minutes, and then 0.03 ml of an appropriately diluted enzyme was added to start the reaction. The change in absorbance at 340 nm in one minute is measured. Incidentally, the control was 0.03 of the enzyme during the above operation.
All measurements were performed by the same operation except that purified water was added instead of ml. 3: Calculation of titer From each value of the sample absorbance and blank absorbance obtained by the above-mentioned measurement method, the titer was obtained by the following formula. U / ml = (sample absorbance−blank absorbance) /min.×
16.24 x dilution ratio

(G) Conditions for deactivation by pH, temperature, etc.
This enzyme has a pH of 5.0 at 30 ° C. for 30 minutes.
It is stable at 9.5, and gradually deactivates on the acidic or alkaline side. In addition, the thermostability of the present enzyme when treated at each temperature for 15 minutes using 50 mM phosphate buffer (pH 7.0) containing 2 mM EDTA was examined. The enzyme is stable up to about 50 ° C,
% Or more of the activity remains.

(H) Influence of metal ion and coenzyme: The same substrate / enzyme mixture as used in the above titration method,
When metal ions were removed, Mg ²⁺ ions, Mn ²⁺ ions, Co ²⁺ ions or Ni ²⁺ ions were added, and D-glucose-1,6-diphosphate was removed or added. In each case, the activity of the present enzyme was measured. As a result, when the metal ion or D-glucose-1,6-diphosphate is removed, the activity of the present enzyme is hardly expressed. This enzyme reaction is activated when Mg ²⁺ ion, Mn ²⁺ ion, Co ²⁺ ion or Ni ²⁺ ion is added, and when D-glucose-1,6-diphosphate is added. (I) Inhibition and stabilization: Various metal salts shown in Table 1 (each at a concentration of 1 mM) were added to the same substrate / enzyme mixture as used in the above titration assay, and the enzyme activity was measured. The effects were investigated. The results are as shown in Table 1. The enzyme showed Zn ²⁺ ion, Cu ²⁺ ion and Hg ²⁺
Strongly inhibited by ions, Ca ²⁺ ions and Fe
^Although inhibited by ²⁺ ions, no metal salt contributing to stabilization is found.

[0024]

[Table 1] (J) Purification method: Isolation and purification of the enzyme can be carried out according to a conventional method, for example, ammonium sulfate precipitation, organic solvent precipitation, adsorption treatment using an ion exchanger, ion exchange chromatography, hydrophobic chromatography. , Gel filtration chromatography, adsorption chromatography, affinity chromatography, electrophoresis and the like are used alone or in appropriate combination. (K) Km value: From the Lineweaver-Burk plot, the Km value for β-D-glucose-1-phosphate is about 0.23 mM. (L) SDS-polyacrylamide gel electrophoresis: SDS-polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE) using a polyacrylamide gel having a concentration gradient of acrylamide 4 to 20% (W / V) by a conventional method. As a result, a single band of the present enzyme was observed. The relative mobility (Rf) after energizing at 40 mA for 60 minutes is 0.65.

[0025]

EXAMPLES The present invention will be described more specifically with reference to the following examples. (Example)

(1) Preparation of chromosome of the original strain Lactococcus lactis subsp. Cremoris
(IFO 3427) in M17 medium (0.5% bactotryptone, 0.5%
% Bactosoyton, 0.5% meat extract, 2.5% yeast extract, 0.05% ascorbic acid, 0.025% magnesium sulfate, 1.9% dipotassium phosphate, pH 7.0) 100 ml, shake culture at 30 ° C for 20 hours and culture I got The culture was collected by centrifugation at 7,000 rpm for 5 minutes to obtain cells. Current Protoc
ols in Molecular Biology (WILEY Intersciense, 1989)
According to the method described, 0.1 mg of chromosomal DNA was obtained.

(2) Determination of Internal Amino Acid Sequence of Purified Enzyme β prepared by the method described in Japanese Patent Application No. 9-176622.
-PGM enzyme sample on a C18 column (Shiseido)
The protein was purified to a single band by staining with DS-PAGE using Coomassie blue, and the N-terminal amino acid sequence was determined to have 48 residues as shown in SEQ ID NO: 3 using a 473A Protein Sequencer (manufactured by Applied Biosystems).

(3) Design of Probe and Southern Hybridization Among the partial amino acid sequences obtained above, a site where codon degeneracy was low was selected, and primers for preparing a probe used for hybridization were designed. When an attempt was made to amplify a gene fragment from a chromosome by PCR using the designed primer, a fragment of about 150 bp was amplified. It was confirmed by the 373A DNA Sequencer (manufactured by Applied Biosystems) that this approximately 150 bp fragment encodes the amino acid sequence obtained above. Therefore, this gene fragment was DIG-labeled using a PCR-DIG-Probe-synthesys kit (manufactured by Boehringer) to obtain a probe. The Southern hybridization membrane converts chromosomal DNA to BamH
I, EcoRI, HindIII, PvuII, SpeI and the like were completely digested and separated by 0.8% agarose gel electrophoresis.
It was prepared by transferring to bond-N + (manufactured by Amersham) using an ordinary method. Southern hybridization is DIG-Lumine
This was performed using a scent Detection kit (Boehringer). As a result, a band of about 5.0 Kbp was detected in the chromosomal DNA treated with SpeI.

A DNA fragment of about 5 kbp was recovered from an agarose gel in which a SpeI digest of chromosomal DNA was electrophoresed, and pBl
960 strains which were incorporated into the SpeI site of uescriptII SK + (Stratagene) were prepared, colonies were transcribed to Hybond-N +, and colony hybridization was performed in the same manner as Southern hybridization described above.
The strain was positive. Plasmids were recovered from these and the nucleotide sequence was determined using the 370A DNA Sequencing System (manufactured by Applied Biosystems). Included. Also, in the base sequence of the obtained chromosome fragment, a stop codon was confirmed on the same frame as the frame encoding the N-terminal amino acid sequence, and it was confirmed that the β-PGM gene was completely contained. . In addition, β-
The PGM gene encoded a total length of 663 bp and 221 amino acids. (SEQ ID NOS: 1 and 2)

(4) Confirmation of Activity Based on the nucleotide sequence information confirmed above, N-terminal and C-terminal primers described in SEQ ID NOs: 4 and 5 to which an NdeI site was added were prepared. Only the structural gene was amplified by the PCR method using the primers prepared above, and connected to pUTE500K '(described in JP-A-8-205861) NdeI site using T4Ligase (Takara Shuzo). This plasmid was named pUG1. E. coli (E. coli) using the obtained plasmid pUG1
DH5α (manufactured by Toyobo Co., Ltd.) was transformed by the method of DMMorrison to obtain a transformant, E. coli DH5α (pUG1).
Escherichia coli (E. coli) DH5α (pUG1) has been deposited as FERM BP-6502 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology. Escherichia coli DH5α (pUG1) in TY medium containing 50 μg / ml ampicillin (1% bactotryptone, 0.5%
After culturing with 10 ml of Bacto yeast extract, 0.5% sodium chloride, pH 7.0) at 37 ° C for 20 hours, the culture solution is disrupted by sonication, centrifuged, and the supernatant is subjected to the enzyme described above. The β-PGM activity measured by the activity measurement method was about 1.2 U / ml.

[0031]

According to the present invention, β-PGM can be produced efficiently at low cost.

[0032]

[Sequence list] SEQUENCE LISTING <110> KIKKOMAN CORAPORATION <120> A β-phosphoglucomutase gene, a novel recombinant DNA and a process for the production of β-phosphoglucomutase <130> P1997 <160> 5 <210> 1 <211> 221 <212> PRT <213> Lactococcus lactis subsp.cremoris (IFO 3427) <400> 1 Met Phe Lys Gly Val Leu Phe Asp Leu Asp Gly Val Ile Thr Asp 5 10 15 Thr Ala Glu Phe His Tyr Arg Ala Trp Gln Lys Leu Gly Gln Glu 20 25 30 Ile Gly Ile Thr Ile Asp Arg Thr Phe Asn Glu Gln Leu Lys Gly 35 40 45 Val Ser Arg Glu Asp Ser Leu Ser Leu Leu Leu Ala Tyr Gly Gly 50 55 60 Lys Glu His Ser Phe Ser Lys Glu Glu Phe Ala Glu Leu Ala Lys 65 70 75 Arg Lys Asn Asp Tyr Tyr Leu Glu Met Ile Gln Thr Ile Glu Pro 80 85 90 Lys Asp Val Phe Pro Gly Val Val Pro Leu Leu Asp Ser Leu Lys 95 100 105 Glu Glu Lys Ile Lys Ile Ala Leu Ala Ser Ala Ser Lys Asn Gly
110 115 120 Pro Phe Leu Leu Glu Lys Met Gly Leu Thr Pro Tyr Phe Asp Ala 125 130 135 Ile Ala Asp Pro Ala Glu Ala Ala Ser Gly Lys Pro Ala Pro Asp 140 145 150 Ile Phe Leu Leu Ala Ala Lys Ala Val Gly Leu Lys Ala Glu Glu 155 160 165 Cys Ile Gly Ile Glu Asp Ala Gln Ala Gly Ile Gln Ala Ile Leu 170 175 180 Ser Ser Gly Ala Gln Pro Ile Gly Val Gly Arg Ala Glu Asp Leu 185 190 195 Gly Gl
u Glu Ile Pro Leu Val Pro Asp Thr Th
r Phe Leu Thr Ile 200 205 210 Asp Tyr Leu Lys Glu Lys Trp His Asp His Gly 215 221

<210> 2 <211> 663 <212> DNA <213> Lactococcus lactis subsp. Cremoris (IFO 3427) <400> 2 atg ttt aaa ggg gta ttg ttc gat tta gac ggc gtc att acc gat act 48 gcc gaa ttc cat tat cgt gct tgg caa aag ctt ggt cag gaa atc ggc 96 att acg atc gac cgc aca ttc aac gaa cag cta aaa ggc gtc agc cga 144 gaa gac tct ctc tct ttg ctt tta gct tt gg ga t tca aag gaa gaa ttt gca gaa tta gca aaa aga aaa aat gat tat 240 tat tta gag atg atc cag acg atc gaa cca aaa gat gtt ttc cct ggg 288 gtc gtc cct tta cta gat tct tta ata gaa gaa ata gaa gaa ata ctt gct tct gca agt aaa aat ggg cct ttc cta ctt gaa aaa atg ggt 384 ctc aca cct tac ttt gac gct att gcc gac ccg gca gaa gca gca agc 432 gga aaa cct gcg cca gat atc ttc ctc gtt 480 cta aaa gca gaa gaa tgt atc ggg atc gag gac gcg caa gct ggt atc 528 caa gcc atc ctc tca agt ggt gct caa cca att ggc gtg gga cgt gct 576 gaa gac ctt gga gag gag atc ccg ctt gt cc gtt ctt 6 24 acg att gat tac tta aaa gaa aag tgg cat gac cat gga 663

<210> 3 <211> 48 <212> PRT <213> Lactococcus lactis subsp. Cremoris (IFO 3427) <400> 3 Met Phe Lys Gly Val Leu Asp Leu Asp Val Ile Thr Asp Thr Ala 5 10 15 Glu Phe His Tyr Arg Ala Trp Gln Lys Leu Gly Gln Glu Ile Gly 20 25 30 Ile Thr Asn Asp Arg Thr Phe Asn Asp Gln Leu Lys Gly Val Ser 35 40 45 Arg Ala Asp 48

<210> 4 <211> 32 <212> DNA <213> Artifical Sequence <400> 4 cga cgg aga ttc ata tgt tta aag ggg tat tg 32

<210> 5 <211> 34 <212> DNA <213> Artifical Sequence <400> 5 cga cgg aga ttc ata tgt tat cca tgg tca tgc c 34

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 1/21 C12R 1:19) (C12N 9/90 C12R 1:01) (C12N 9/90 C12R 1 : 19) F term (reference) 4B024 AA03 AA05 BA07 CA04 CA06 DA06 EA04 HA01 HA11 4B050 CC03 DD02 LL02 LL05 4B065 AA01X AB01 BA02 CA27

Claims (4)

[Claims] 1. A β-phosphoglucomutase gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (b) an amino acid sequence (a) consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and having β-phosphoglucomutase activity Protein 2. A novel recombinant DNA comprising the β-phosphoglucomutase gene according to claim 1 inserted into a vector DNA. 3. A transformant or transductant containing the recombinant DNA according to claim 2. 4. A method for producing β-phosphoglucomutase, which comprises culturing the transformant or transductant according to claim 3 in a medium, and collecting β-phosphoglucomutase from the culture.