JP2001245670A - Method for producing water-soluble pqq glucose dehydrogenase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a glucose dehydrogenase(GDH) using pyrrolo-quinoline quinone(PQQ) as a coenzyme using yeast in view of the fact that a method for culturing yeast in a high population density is established particularly in the food industry, that it is known that yeast has a high ability of producing a recombinant DNA product, and that selecting an appropriate host and signal peptide permits the extracellular secretion of a recombinant DNA product. SOLUTION: A method is provided that permits intracellular or extracellular accumulation and production of PQQGDH using recombinant yeast cell having a transformed PQQGDH structural gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a glucose dehydrogenase (G) having pyrroloquinoline quinone (PQQ) as a coenzyme.
DH).

[0002]

2. Description of the Related Art PQQGDH has been known as an Acinetobacter calcoace which produces the enzyme.
A method for culturing Ticus and extracting PQQGDH from the culture, and a method for further extracting Acinetobacter
The structural gene encoding P. alcoaceticus-derived PQQGDH is inserted into a commonly used expression vector of Escherichia coli, transformed into Escherichia coli, and the transformed Escherichia coli is cultured.
A method of extracting GDH has been used.

[0003]

In order to produce PQQGDH in large quantities, PQ based on recombinant DNA technology is required.
The method using transformed E. coli containing QGDH was excellent. On the other hand, yeast is known to have a high-density culture technology established mainly in the food industry, and to have a high ability to produce recombinant DNA products. Furthermore, in yeast, the recombinant DNA product can be secreted and produced extracellularly by selecting an appropriate host and signal peptide.

[0004]

Means for Solving the Problems As a result of searching for microorganisms that have been used in recombinant DNA experiments, the present inventors have paid attention to yeast as a host. That is, it has been found that yeast can express and produce PQQGDH in cells by transforming the yeast with a yeast expression vector containing the structural gene of PQQGDH. Alternatively, it has been found that yeast also expresses and produces PQQGDH in cells by inserting a structural gene of PQQGDH into the yeast genome by homologous recombination. Further, by transforming a yeast expression vector into which a structural gene of PQQGDH has been inserted downstream of the secretory signal of yeast, yeast can express extracellular PQQGDH.
It has been found that QGDH is secretory expressed and produced. It has also been found that yeast can secrete and express PQQGDH extracellularly by inserting a gene in which a structural gene of PQQGDH is inserted downstream of the yeast secretion signal into the yeast genome by homologous recombination.

That is, the present invention has been industrially advantageously achieved by the present invention having the following constitution.

[1] A yeast cell transformed with a structural gene for water-soluble glucose dehydrogenase (PQQGDH) having pyrroloquinoline quinone as a coenzyme.

[2] The yeast cell according to [1], wherein the yeast cell is Sac
Charomyces genus Hansenula, Klu
A microorganism selected from the group consisting of the genus yveromyces and the genus Pichia.

[3] In [1], the yeast cell is Pic
hia pastoris.

[4] PQ for culturing the transformant of [1]-[3] and collecting PQQGDH from the obtained cells
Method for producing QGDH.

[5] A method for producing PQQGDH by culturing the transformant of [1]-[3] and recovering PQQGDH secreted and produced in the medium.

[6] The transformant according to [4] or [5] is a Saccharomyces genus Hansenul
a genus, Kluyveromyces genus or Pich
PQQG using a microorganism selected from the group consisting of the genus ia
Method for producing DH.

[7] PQQG wherein the transformant according to [4] or [5] is Pichiapastoris
Method for producing DH.

[8] PQQGDH produced by the method according to [4]-[7].

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION A water-soluble glucose dehydrogenase (PQQGD) using the pyrroloquinoline quinone of the present invention as a coenzyme
The yeast cell transformed with the structural gene of H) is, for example, A
cineobacter calcoaceticu
This is a transformed yeast in which the structural gene of water-soluble PQQGDH derived from s is inserted into an expression vector that replicates in yeast.

Alternatively, the yeast cell into which the structural gene of PQQGDH has been transformed is, for example, Acinetobacter
Water soluble PQQGD derived from r calcoaceticus
The transformed yeast is a transformed yeast in which the structural gene of H is inserted by homologous recombination on the main chromosome of the yeast.

In the present invention, the PQQGDH expressed and produced in yeast is the previously reported Acine
tobacter calcoaceticus LM
D79.41 or a protein having an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence of the enzyme, and having glucose dehydrogenase activity. In the present invention, yeast cells transformed with PQQGDH used for expression production of PQQGDH include Saccharomyc.
genus Hansenula, Kluyveromyc
A group consisting of the genus es or the genus Pichia can be mentioned. Among them, yeast cells used as hosts are Pic
hia pastoris is preferred.

PQQG transformed using yeast cells
PQQGDH expressed by the DH gene can be recovered. For example, PQQGDH expressed in cells is obtained by disrupting yeast cells according to a conventional method such as French press, ultrasonic disruption, or zymolyase treatment, and then subjecting the resulting cell disrupted solution to ultracentrifugation, desalting, Furthermore, PQQGDH purified by anion exchange chromatography, hydrophobic biochromatography and the like can be obtained.

Alternatively, PQQ transformed with yeast cells
When PQQGDH expressed by the GDH gene is secreted and produced extracellularly, a culture solution of the yeast cells is collected, and a supernatant after removing the cells by centrifugation is prepared.
PQQG purified by desalting and further anion exchange chromatography and hydrophobic biochromatography
DH can be obtained.

Preferably, PQ transformed with yeast cells
In order to secrete and produce extracellular PQQGDH expressed by the QGDH gene, a gene was constructed in which the structural gene of PQQGDH was inserted downstream of the signal sequence of a secretory peptide of yeast, and this was inserted into a yeast expression vector or yeast main chromosome. Inserted into the vector for recombinant PQQ
A transformed yeast secreting and producing GDH can be obtained.

More preferably, in order to secrete and produce extracellular PQQGDH expressed by the transformed PQQGDH gene in yeast cells, the secretory signal sequence of the α-factor, which is a secretory peptide of yeast, is used for the structural gene of PQQGDH. Is constructed and inserted into a yeast expression vector or a yeast main chromosome integration vector to obtain a transformed yeast that secretes and produces recombinant PQQGDH.

Also, the present invention provides a PQQGD
PQQGDH produced by transformed yeast into which the structural gene of H has been integrated.

[0023]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Pichia pastori
Construction of Secretion Expression Vector pPIC9GB2 in A. cineobacter calcoacetic
and a plasmid pGB into which the structural gene (about 1.45 kbp) of the water-soluble PQQGDH-us (PQQGDH-B) is inserted as a template, and about 1.38 kbp of a region not containing the signal sequence of PQQGDH-B.
(Xho I-EcoRI fragment) gene fragment shown below with forward primer and reverse primer P
Amplified by CR. Forward primer 5'-GGCTCGAGAAAAGAGATGTTCC
TCTAACTCCATCTCAA-3 'reverse primer 5'-GGGAATTCTTACTTAGGCCTATAT
AGGTGAACTTAATGAGAG-3 '

The plasmid pGB contains the vector pTrc9
9A (manufactured by Pharmacia) at the multicloning site of Acinetobacter calcoacet.
It is the one into which the structural gene encoding PQQGDH derived from icus was inserted (FIG. 1). Using this plasmid as a template, the PCR reaction was carried out according to a standard method using TaqDN.
A polymerase, template DNA, forward primer, reverse primer, dGTP, dATP,
Using a reaction solution containing dCTP and dTTP, using a thermal cycler, 30 cycles of 94 ° C. for 3 minutes, then 94 ° C. for 3 minutes, 50 ° C. for 2 minutes, and 72 ° C. for 2 minutes, and finally for 10 minutes at 72 ° C. Was performed under the following conditions.

The obtained DNA fragment was cloned into Xho in the multiple cloning site of pPIC9, a vector for secretion expression in Pichia containing the secretion signal of α-factor.
Inserted into each of the I-EcoRI sites, pPIC9G
B2 was constructed (FIG. 2).

Example 2 Recombinant Pichia pas
Preparation of toris The constructed pPIC9GB2 was electroporated (applied potential 1500 V, charge capacity 25 μF, resistance 200
Ω) by Pichia pastoris KM71
(His-MutS), and Pichia pastoris in which the gene was integrated into the genome was selected on a minimal medium plate containing no histidine.

Example 3 Pichia pastori
secretion production of PQQGDH-B in the recombinant Pichia pastoris KM obtained
From 71 strains (His-MutS), Buffer
d PMS-DCI was applied to the culture supernatant obtained by culturing using the Minimal Methanol (BMM) medium.
The GDH activity was visually observed by the P system, and strains in which the GDH activity was confirmed were selected. This Recombinant Pichi
Growth and secreted PQQG of a pastoris
The change over time in the production of DH-B was examined as follows. 150ml x 8 Buffered Gl as preculture
ycerol-complex Medium (BMG
Using Y), the culture broth that had grown to an OD600 of 5.2 was collected (3000 × g, 5 min., Room item).
p. ), This is 100ml x 4 Buffered
Methanol-complex Medium (B
MMY). The OD600 at this time was 22. The final concentration is set to 0.4 every 24 hours for inducing expression.
Methanol was added to a concentration of 5%, and the time course of the cell concentration of the recombinant Pichia pastoris and the production amount of PQQGDH secreted into the culture solution when culturing was performed at 30 ° C. using a rotary shaker was examined.

After 24 hours, the OD600 reaches 72,
Subsequent cell concentrations were in a steady state. The PQQGDH production reached 180,000 U / l after 24 hours,
After 4 hours, it was 210,000 U / l.

[Brief description of the drawings]

FIG. 1 shows a restriction map of pGB.

FIG. 2 shows a restriction map of pPIC9GB2.

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[Procedure amendment]

[Submission date] November 2, 2000 (200.11.
2)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0029

[Correction method] Change

[Correction contents]

After 24 hours, the OD600 reaches 72,
Subsequent cell concentrations were in a steady state. The PQQGDH production reached 180,000 U / l after 24 hours,
After 4 hours, it was 210,000 U / l.

[Sequence list]

Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) (C12N 1/19 C12R 1:85) C12R 1:85) (C12N 1/19 (C12N 1/19 C12R 1:78) (C12R 1:78) (C12N 9/04 D (C12N 9/04 C12R 1:84) C12R 1:84) (C12N 9/04 D (C12N 9/04 C12R 1:85) C12R 1:85) (C12N 9 / 04 (C12N 9/04 C12R 1:78) C12R 1:78) C12N 15/00 ZNAA

Claims (8)

[Claims] 1. A yeast cell transformed with a structural gene of water-soluble glucose dehydrogenase (PQQGDH) having pyrroloquinoline quinone as a coenzyme. 2. The method according to claim 1, wherein the yeast cell is Sacch.
genus aromyces Hansenula, Kluyv
A microorganism selected from the group consisting of the genus eromyces or the genus Pichia. 3. The method according to claim 1, wherein the yeast cell is Pichia.
pastoris. 4. culturing the transformant of claim 1-3,
PQQGDH for collecting PQQGDH from the obtained cells
Manufacturing method. 5. The method according to claim 1, wherein the transformant is cultured.
PQQ for recovering PQQGDH secreted and produced in a medium
A method for producing GDH. 6. The transformant according to claim 4 or 5, wherein the transformant is Saccharomyces genus Hansenula,
A method for producing PQQGDH using a microorganism selected from the group consisting of the genus Kluyveromyces or the genus Pichia. 7. The transformant according to claim 4 or 5, wherein the transformant is P
A method for producing PQQGDH which is Ichia pastoris. 8. A PQQGDH produced using the method of claim 4-7.