L-&agr;-glycerophosphate oxidase gene, recombinant DNA, and...

Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase

Reexamination Certificate

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C435S071200, C435S252300, C435S440000, C536S023200

Reexamination Certificate

active

06303357

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to an L-&agr;-glycerophosphate oxidase (to be referred to as GPO hereinafter) gene, a novel recombinant DNA, a modified GPO and a method for the production of GPO.
BACKGROUND ART
GPO is known as an enzyme which catalyses a reaction in which dihydroxyacetone phosphate and one molecule of hydrogen peroxide are formed from L-&agr;-glycerophosphate and one molecule of oxygen.
So far, bacteria belonging to the genus Streptococcus, the genus Lactobacillus, the genus Pediococcus (JP-A-58-165789 and JP-A-57-198085; the term “JP-A” as used herein means an “unexamined published Japanese patent application”), the genus Leuconostoc and the genus Aerococcus (JP-A-55-15746) are known as microorganisms which produce GPO, and the GPO produced by these bacteria can be used as an enzyme for clinical inspection and reagent use, e.g., for the measurement of lipase activity and determination of triglyceride, glycerol, ATP and the like.
Production of GPO by these conventional GPO-producing bacteria has several problems. For example, when a GPO-producing bacterium belonging to the genus Streptococcus is used, it entails problems such as considerably high cost. Thus, in recent years, a method for the production of GPO by means of recombinant DNA techniques has been reported using a bacterium belonging to the genus Streptococcus (JP-A-2-454), and it is known that the GPO produced by this method can be used as an enzyme for the clinical inspection reagent.
However, in order to use GPO in the aforementioned field, great concern has been directed toward the development of GPO having more superior stability and excellent properties such as heat resistance and reactivity and the production of GPO with a low cost. That is, since shift over to liquid reagents is in progress in the recent field of clinical inspection, development of more stable GPO having higher reactivity and higher reliability is expected.
SUMMARY OF THE INVENTION
In view of the above, it therefore becomes an object of the invention to provide a novel GPO-producing bacterium and also to provide a GPO having an amino acid sequence which is different from that of the naturally existing GPO but still having the GPO activity, in which physicochemical properties of the enzyme are modified by genetic engineering techniques.
Accordingly, the invention provides a GPO-producing bacterium newly screened from a natural source and also provides a recombinant DNA which can be replicated, in which a recombinant DNA obtained by replacing an amino acid of a specified position of the amino acid sequence deduced from DNA coding for the GPO by other amino acid is integrated into a vector, as well as a transformant containing the recombinant DNA and a method for the production of GPO having modified properties, which uses the same.
DETAILED DESCRIPTION OF THE INVENTION
With the aim of solving the aforementioned problems, the inventors of this invention have conducted intensive studies and revealed nucleotide sequence of a DNA fragment coding for GPO using a newly screened GOP producing bacterium and subsequently succeeded in obtaining GPO having further improved properties by modifying an amino acid sequence deduced from the nucleotide sequence.
That is, the inventors have carried out screening of GPO-producing bacteria from a broad range of natural sources and found that a bacterial strain isolated from a soil sample produces a novel GPO.
Bacteriological properties of the strain isolated by the inventors were identified with reference to Bergey's Manual of Systematic Bacteriology, vol. 2 (1986).
(1) Morphology
Gram positive, sub-spherical to ovoid, single coccus or 2 to 8 linked cocci, no spore formation, no motility.
(2) Cultural Properties (Culturing on Trypto-Soy Agar Plate at 37° C. for 24 Hours)
Shape of colony: circular
Surface of colony: smooth
Periphery of colony: entire
Rise of colony: convex
Gloss of colony: opaque, glistening
Color of colony: white
(3) Physiological Properties
Growth at 10° C.: yes
Growth at 45° C.: yes
Growth at 6.5% NaCl: yes
Growth at pH 9.6: yes
Growth with 40% bile medium: yes
Temperature resistance (60° C., 30 min.): yes
Hydrolysis of;
arginine: positive
hippurate: negative
esclin: positive
gelatin: negative
starch: negative
Reduction of;
methylene blue: positive
TTC [2,3,5-triphenyltetrazolium chloride (in the presence of 0.5% glucose)]: negative
tellurite: positive
Formation of acid from; glycerol:
weakly positive
cellobiose: positive
L-arabinose: positive
maltose: positive
ribose: positive
lactose: positive
adonitol: negative
melibiose: positive
glucose: positive
sucrose: positive
sorbose: negative
trehalose: positive
rhamnose: positive
inulin: negative
mannitol: positive
melezitose: negative
sorbitol: negative
raffinose: weakly positive
arbutin: positive
soluble starch: weakly positive
Assimilation of;
pyruvic acid: negative
citric acid: negative
malic acid: negative
arginine: negative
serine: negative
Formation of yellow pigment (insoluble): no Sensitivity for; bacitracin: resistant
optochin: resistant
Catalase: negative
Cytochrome oxidase: negative
Litmus milk: acidic, no coagulation, reduces litmus
Formation of gas from glucose: negative
VP test: positive
Hemolysis: negative to weakly &agr;-hemolytic
Behavior on oxygen: facultative anaerobe
Urease: negative
SF medium: growth
Bile solubility: negative
Reduction of nitrate: negative
Gas formation from malic acid (in the presence of glucose): positive
As shown in Table 1, this strain belongs to the genus Enterococcus, because it is a Gram positive facultative anaerobic coccus which forms a chain and does not form gas from glucose and, as is evident also from Table 2, these properties coincide with the definition of the genus Enterococcus.
TABLE 1
Genus name
Arrangements
Gas (glucose)
Staphylococcus
irregular clusters
Stomatococcus
irregular clusters
Enterococcus
chains, pairs

Leuconostoc
pairs, chains
+
Pediococcus
tetrads
Aerococcus
tetrads
This strain
chains, pairs

TABLE 2
Genus Enterococcus
GPO producer
Shape of cell
ovoid
sub-spherical-
ovoid
Linkage
single, 2 or short
single, 2 to 8
linkage
Gram staining
positive
positive
Spore
no formation
no formation
Motility
positive or
negative
negative
Oxygen
facultative
facultative
anaerobe
anaerobe
Optimum growth
about 35° C.
32 to 45° C.
temperature
Growth at 10° C.
positive
positive
and 45° C.
Temp. resistance
positive
positive
(60° C., 30 min.)
Growth at 6.5%
positive
positive
NaCl or pH 9.6
Hydrolysis of
positive
positive
pyridonyl-&bgr;-
naphthylamide
Final main
L-lactic acid
not tested
metabolite of
glucose
Also, as shown in Tables 3 and 4, this strain can grow at 10° C., 45° C., 6.5% sodium chloride and pH 9.6 and with 40% bile, so that it belongs to the group of enterococci.
TABLE 3
Pyogenic
Oral
streptococci
streptococci
This
1
2
3
4
5
6
7
8
9
10
11
12
13
14
strain
Growth at 10° C.

d

+










+
Growth at 45° C.





d
d
d
d
d
d
d
d
d
+
Growth at 6.5%

d









d
d

+
NaCl
Growth at pH 9.6














+
Growth with 40%

d



d
d

d
d
d
d
d
NT
+
bile
&agr;-Hemolysis



+
+
d
+
+






−wk
&bgr;-Hemolysis
+
d
+
+











Arginine
+
+
+
NT
+

+

d

+



+
hydrolysis
Hippurate

+













hydrolysis
Esculin hydrolysis
d

d
+
d
+
d

d
+
+
d
d
+
+
TABLE 4
Lactic
Other
This
Ente

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