JP3458791B2 - Thermostable lactate oxidase - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a lactate oxidase which catalyzes a reaction represented by L-lactic acid + oxygen → pyruvate + hydrogen peroxide, and more particularly to a lactate oxidase having high heat resistance.

[0002]

BACKGROUND OF THE INVENTION Lactate oxidase is an enzyme that catalyzes the production of pyruvic acid and hydrogen peroxide from L-lactic acid and oxygen, and is a very useful enzyme for measuring the concentration of lactic acid in body fluids such as blood. The enzyme has previously been used in Pediococcu (Pediococcu).
s) genus, Streptococcus genus, Aerococcus genus, Lactococcus (Lactoc)
It is known to exist in bacteria and the like belonging to the genus occus) (Japanese Patent Publication No. 58-4557 and Japanese Patent Publication No. 59-101).
No. 90, Japanese Patent Application Laid-Open No. 2-178886, Japanese Patent Application No. 9
-57808).

The inventors of the present invention have already shown that mutant lactate oxidase having thermostability (Japanese Patent No. 2624217).
Is inventing. This is intended to further improve thermostability by adding another mutation to the above enzyme.

[0004]

However, the heat resistance is insufficient in the above-mentioned prior art. That is, the lactate oxidase obtained from Streptococcus has low thermostability and is rapidly inactivated at a temperature higher than 34 ° C (Japanese Patent Publication Sho 58).
-4557), the heat stability of lactate oxidase obtained from Lactococcus is 50 ° C. or lower (Japanese Patent Application No. 9-5).
7808), the residual activity of lactate oxidase obtained from Aerococcus, which has the highest thermostability, decreases to 5% or less by heat treatment at 65 ° C. for 10 minutes (Japanese Patent Publication No. Sho 5).
9-10190). In addition, the patent number 26 of the present inventors
Also in the thermostable lactate oxidase of FIG.
The residual activity was reduced to 5% or less by the heat treatment at 0 ° C. for 20 minutes, and the heat resistance was not sufficient. By imparting heat resistance to lactate oxidase, it has the effects of prolonging its service life at room temperature, enabling long-term storage of the enzyme, and prolonging its service life when used in a lactate sensor and reducing the frequency of replacement.

The main object of the present invention is to develop a lactate oxidase having remarkable thermostability.

[0006]

In the present invention, a lactate oxidase having the amino acid sequence shown in FIG. 1, or
Lactic acid oxidase that catalyzes the reaction represented by L-lactic acid + oxygen → pyruvic acid + hydrogen peroxide, or one or more amino acids are deleted, substituted, or added in the lactate oxidase. Amino acid sequence
A technique relating to a lactate oxidase containing (wherein 160-position is glycine and 198-position isoleucine), and further, a DNA encoding the lactate oxidase, or a vector containing the DNA is provided.

As a result of extensive studies on the above problems, the present inventor has found that the lactate oxidase having the above-mentioned amino acid sequence has a remarkable heat resistance as compared with the conventional lactate oxidase, and solved the conventional problems.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The mutant lactate oxidase of the present invention is shown in FIG.
Insert the polynucleotide fragment described in 1. into a vector having an appropriate promoter (eg, lac promoter, tac promoter, etc.) and an antibiotic resistance marker (ampicillin resistance, kanamycin resistance, tetracycline resistance, chloramphenicol resistance, etc.), and Nasukudo (E. coli (E.
coli), B. subtilis, etc.), for example, Escherichia coli (Escherichia coli), and then the protein is expressed and then purified.

Embodiments of the invention will be described in detail in the section of Examples in accordance with Examples.

[0010]

[Example] Preparation of thermostable lactate oxidase gene The thermostable lactate oxidase gene of the present invention is a known technique based on the gene for the thermostable mutant lactate oxidase LOD1 shown in FIG. Law (site-directed mutagenes
is) was used (FIG. 6). More specifically, using a vector containing the LOD1 gene (pLOD1) as a template, a sense primer containing a mismatch at the 592nd base (Fig. 2)
And Quicksense using antisense primer (Fig. 3)
PCR with Pfu polymerase was performed according to ngeMutagenesis Kit (manufactured by STRATGENE, Funakoshi), and then 3 with DpnI.
The treatment was carried out at 7 ° C. for 1 hour. E. coli XL1-Blue was transformed with this DpnI-treated PCR product, and LB agar medium containing ampicillin (1% Tryptone, 0.5% YeastExtract, 0.5% NaC) was used.
1%, 1.5% Agar, 01% Ampicillin Sodium) and cultured overnight at 37 ° C. The obtained colonies were transferred to LB liquid medium containing ampicillin (1% Tryptone, 0.5% Yeast Extract, 0.5% NaCl, 0.01% Am
It was cultured overnight at 37 ° C. in a picillin), and the plasmid was extracted and purified using a Qiagen plasmid purification kit (QIAGEN). The base sequence of the enzyme portion of the obtained plasmid was confirmed by analyzing the base sequence using a DNA sequencer (ABI373: manufactured by Applied Biosystems).

The plasmid containing the mutant lactate oxidase gene obtained as described above was designated as pLOD12.

Expression of thermostable lactate oxidase and preparation of crude enzyme solution pLOD12, pLOD1, pLODwt (including the wild-type lactate oxidase gene obtained from Aerococcus, other parts are pLOD
12, the same plasmid as pLOD1) was used for each E.
The method of Dewar et al. [Dower et al., Nucleic Aci]
dsResearch, vol.16, No.13, pp6127-6145 (1988)], and electroporation was performed. Specifically, the plasmid and E. coli JM109 were mixed and allowed to stand in ice for 1 minute, and then 200 using Gene Pulser ™ (BioRad).
After introducing the gene by electroporation under the conditions of ohm, 25 μF, and 18 kV / cm, the cells were added to SOC medium (2% Try
ptone, 0.5% YeastExtract, 10mMNaCl, 2.5mMKCl, 10mMMgCl
It was cultured in 2,10 mM MgSO4,20 mM Glucose) at 37 ° C. for 1 hour.

After culturing, the bacterial cells were cultured in LAH medium (1% Tryptone, 0.5% Ye
astExtract, 0.5% NaCl, 1.5% Agar, 0.01% AmpicillinSodiu
m, 01% ABTS, 50mMLithium-L-Lactate, 1U / mlHorseradishPe
roxidase) and cultured overnight at 37 ° C. Pick up the resulting colony with a sterilized jig and use 4 ml of LA medium.
(1% Tryptone, 0.5% YeastExtract, 0.5% NaCl, 0.01% Ampicil
linSodium) and cultured overnight at 37 ° C, then 100mMIPT
4 μl of G (Isopropyl Thiogalactopyranoside) was added, and the mixture was further incubated at 37 ° C. for 3 hours. The bacterial cells were collected by centrifugation at 12000 rpm for 5 minutes, and 1 ml of HEPES buffer solution (10 mM HEPES, 1 mMEDT
The cells were washed with A, pH 7.3) and then suspended in 400 μl of the same buffer. PMSF (Phenylmethylsulfonylfluorid) was added to the cell suspension.
e) to a final concentration of 1 mM and sonicate in ice (V
P-60: manufactured by TAITEC). The cell debris was removed by centrifugation at 12000 rpm for 10 minutes, and the protein concentration of the upper layer solution was adjusted with the BCA protein assay kit.
(Pierce, Takara Shuzo). LOD12, LOD1, L
The protein concentration of ODwt was adjusted to be 50 μg / ml, and lactate oxidase activity was measured.

Lactic acid oxidase activity measurement Method of Ducan et al. [Ducan et al., BBRC, vol.164, N
o.2, pp919-926 (1989)].

HEPES buffer (1M, pH 7.3) was added to 2.7 ml of pure water.
120 μl, L-lactic acid lithium salt (96 mg / ml) 30 μl, 4-aminoantipyrine (30 mg / ml) 30 μl, phenol (3
1 mg / ml) 30 μl, HRP (Horseradish Peroxidase, 10
(0 U / ml) was added and mixed in a cuvette with an optical path length of 1 cm, 60 l of lactate oxidase solution was added, and immediately after stirring, the absorbance at 500 nm was measured with an absorptiometer (UV-365: Shimadzu). The change was measured. The crude enzyme solution was allowed to stand in a constant temperature bath at 70 ° C. for a certain period of time (5, 10, 10, 40 minutes) and then allowed to stand in ice to measure the enzyme activity (residual activity). Lactate oxidase LODwt obtained from Aerococcus (Japanese Patent Publication No. 59-10)
190), the residual activity became almost 0 after heating at 70 ° C. for 5 minutes. Fig. 8 shows LOD1 (heat-resistant mutant lactate oxidase described in Patent No. 2624217) and lactate oxidase of the present invention at 70 ° C.
The plot of the time-dependent change in the residual activity is shown.

[0016]

[Effect of the Invention] FIG. 7 shows the gene sequence and amino acid sequence of the lactate oxidase of the present invention. The underlined part is different from the lactate oxidase obtained from Aerococcus.

FIG. 8 shows the change over time in residual activity when the thermostable mutant lactate oxidase LOD1 shown in FIG. 4 and the lactate oxidase of the present invention were kept at 70 ° C. It can be seen that the lactate oxidase of the present invention has significantly improved heat resistance as compared with the thermostable mutant lactate oxidase shown in FIG.

[Brief description of drawings]

1] Amino acid sequence of mutant lactate oxidase LOD12

FIG. 2 is a gene sequence of a sense primer containing a mismatch at the 592nd base using a vector containing the LOD1 gene (pLOD1) as a template.

FIG. 3 is a gene sequence of an antisense primer containing a mismatch at the 592nd base using a vector containing the LOD1 gene (pLOD1) as a template.

[Fig. 4] Amino acid sequence of thermostable mutant lactate oxidase L0D1

FIG. 5 shows the sequence of the lactate oxidase gene of the present invention.

FIG. 6 shows the procedure of site-directed mutagenesis.

FIG. 7 shows the amino acid sequence of the lactate oxidase of the present invention and the gene sequence in association with each other.

FIG. 8 shows the difference in thermostability between the thermostable mutant lactate oxidase described in Patent No. 2624217 and the lactate oxidase of the present invention.

Continuation of front page (56) References Patent 2624217 (JP, B2) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/09 ZNA SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / G eneSeq BIOSIS / MEDLINE / WPID S (STN)

Claims (4)

(57) [Claims] 1. A lactate oxidase having the amino acid sequence shown in FIG. 2. The lactate oxidase according to claim 1, which has a catalytic action in a reaction represented by L-lactic acid + oxygen → pyruvic acid + hydrogen peroxide. 3. A DNA encoding lactate oxidase, wherein the DNA encodes the lactate oxidase according to claim 1 or 2 . 4. A vector comprising the DNA according to claim 3 .