L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase...

Details L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase... L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase...

1998-07-17

2001-03-06

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S189000

Reexamination Certificate

active

06197562

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a novel L-sorbose dehydrogenase (hereinafter referred to as SDH) and a novel L-sorbosone dehydrogenase (hereinafter referred to as SNDH) both derived from
Gluconobacter Oxydans
T-100. More particularly, the present invention relates to a novel SDH, a novel SNDH, a DNA encoding same, an expression vector containing said DNA, a host cell transformed (transfected) with said expression vector, and the production of the SDH and SNDH by culturing the host cell.
The SDH and SNDH of the present invention are enzymes useful for producing 2-keto-L-gulonic acid.
BACKGROUND ART
2-Keto-L-gulonic acid (hereinafter referred to as 2KLGA) is a key intermediate in the synthesis of L-ascorbic acid. For industrial production, 2KLGA is chemically synthesized from L-sorbose by oxidation according to the Reichstein's method.
On the other hand, it is well known that many microorganisms have an ability to convert L-sorbose to 2KLGA through a two-step enzymatic oxidation by SDH and SNDH. Namely, SDH catalyzes the. oxidation of L-sorbose to L-sorbosone, and SNDH catalyzes the oxidation of L-sorbosone to 2KLGA. However, because of the low productivity of 2KLGA obtained by using these microorganisms, they have not been applied to the industrial production yet.
It is desirable to provide efficient and simplified methods for the production of 2KLGA.
DISCLOSURE OF THE INVENTION
In an attempt to accomplish the above-mentioned objects, the inventors of this invention conducted intensive studies to find an SDH and an SNDH having preferable properties, and succeeded in producing a novel SDH and a novel SNDH having desirable properties for producing 2KLGA and developed the studies, which resulted in the completion of the present invention.
Accordingly, the present invention relates to a novel SDH derived from
Gluconobacter oxydans
T-100 (FERM BP-4188), which is characterized by:
(1) an ability to catalyze the conversion of L-sorbose into L-sorbosone,
(2) a molecular weight of 58,000 dalton (SDS-PAGE), and
(3) an N-terminal amino acid sequence of Thr—Ser—Gly—Phe—Asp—Tyr—Ile—Val—Val—Gly—Gly—Gly—Ser—Ala(SEQ ID NO: 5). Further, the present invention relates to an SDH having an amino acid sequence shown in the Sequence Listing, Sequence No. 1 to be mentioned later.
The present invention also relates to a novel SNDH derived from
Gluconobacter oxydans
T-100, which is characterized by:
(1) an ability to catalyze the conversion of L-sorbosone into 2KLGA,
(2) a molecular weight of 50,000 dalton (SDS-PAGE), and
(3) an N-terminal amino acid sequence of Asn—Val—Val—Ser—Lys—Thr—Val—Xaa—Leu (SEQ ID NO: 6, Xaa being an unidentified amino acid). The present invention further relates to an SNDH having an amino acid sequence shown in the Sequence Listing, Sequence No. 2.
The present invention also relates to a DNA which encodes the above-mentioned SDH and/or SNDH, an expression vector which contains said DNA, a host cell transformed (transfected) by said expression vector and a process for producing the SDH and/or SNDH, which comprises culturing said host cell (transformant) in a medium and recovering the SDH and/or SNDH from the resulting culture.
The
Gluconobacter oxydans
T-100 to be used in the present invention is a 2KLGA-high-producing mutant derived from
Gluconobacter oxydans
G716 (wild strain) by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutagenesis in a conventional manner.
The
Gluconobacter oxydans
G716 was isolated from a persimmon, and has the following morphological and physiological properties. The method described in Bergey's Manual of Systematic Bacteriology Vol. 1 (1984) and the method described in Manual for Identification to Medical Bacteria (S. T. Cowan, 2nd. Edition, 1985) were principally employed for the taxonomic study.
1. Morphological properties
The
Gluconobacter oxydans
G716 is a Gram-negative, motile bacterium. The cell shapes are rod, occurring both singly and in pairs, and rarely in chains. Morphological characteristics of
Gluconobacter oxydans
G716
Gram stain
negative
color of colony
pale
spore
negative
cell shape
rod
motility
positive
flagella
3-8 polar flagella
2. Physiological characteristics
Physiological characteristics of the
Gluconobacter oxydans
G716 are summarized in the following table.
Physiological characteristics of
Gluconobacter oxydans
G716
Conditions
Characteristics
growth in air
+
at 4° C.
−
at 22° C.
+
at 30° C.
+
at 40° C.
−
catalase
+
oxidase
−
gelatin liquefaction
−
nitrate reduction
−
aesuclin hydrolysis
−
acid formation
L-arabinose
+
D-cellobiose
+
Dulcitol
+
D-galactose
+
D-glucose
+
glycerol
+
D-mannitol
+
D-mannose
+
D-xylose
+
D-lactose
−
maltose
−
D-raffinose
−
rhamnose
−
D-sorbitol
−
sucrose
−
D-trehalose
−
mol% G + C of the DNA
60.0
Ubiquinone
Q10
The organism is aerobic, showing no growth under anaerobic conditions. Optimum temperature is 22 to 30° C., showing no growth at 40° C. and 4° C. The best medium for growth is SY medium that is composed of 2.5% sorbitol and 0.5% yeast extract (pH 6.4). Strong ketogenesis occurs from glucose and glycerol.
The
Gluconobacter oxydans
T-100 to be used in the present invention has the morphological and physiological properties identical to those of
Gluconobacter oxydans
G716.
The new SDH and new SNDH of the present invention can be prepared by recombinant DNA technology, polypeptide synthesis and the like.
In case where recombinant DNA technology is employed, the new SDH and/or new SNDH can be prepared by culturing a host cell transformed (transfected) with an expression vector containing a DNA encoding the amino acid sequence of the new SDH and/or new SNDH in a nutrient medium and recovering the same from the obtained culture.
Particulars of this process are explained in more detail in the following.
The host cell includes, for example, microorganisms such as bacteria (e.g.
Escherichia coli, Gluconobacter oxydans
and
Bacillus subtilis
), yeast (e.g.
Saccharomyces cerevisiae
), animal cell lines and cultured plant cells. Preferred examples of the microorganisms include bacteria, especially strains belonging to the genus Escherichia (e.g.
E. coli
JM109 ATCC 53323,
E. coli
NM538 ATCC 35638,
E. coli
HB101 ATCC 33694,
E. coli
HB101-16 FERM BP-1872 and
E. coli
294 ATCC 31446) and the genus Bacillus (e.g.
Bacillus subtilis
ISW1214), yeast, especially strains belonging to the genus Saccharomyces (e.g.
Saccharomyces cerevisiae
AH22), and animal cell lines [e.g. mouse L929 cell and Chinese hamster ovary (CHO) cell].
When a bacterium, especially
E. coli
or
Bacillus subtilis
is used as a host cell, expression vector is usually composed of at least promoter, initiation codon, DNA encoding amino acid sequence(s) of the new SDH and/or new SNDH, termination codon, terminator region, and replicatable unit.
When a yeast or an animal cell is used as a host cell, the expression vector is preferably composed of at least promoter, initiation codon, DNA encoding amino acid sequences of signal peptide and the new SDH and/or new SNDH and termination codon, and it is possible that enhancer sequence, 5′- and 3′-noncoding region of the new SDH, 5′- and 3′-noncoding region of the new SNDH, splicing junctions, polyadenylation site and replicatable unit are also inserted.
The promoter for expressing the new SDH and/or new SNDH in bacteria comprises, for example, promoter and Shine-Dalgarno (SD) sequence (e.g. AAGG). Preferable promoters include, for example, conventionally employed promoters (e.g. PL-promoter and trp-promoter for
E. coli
) and promoter of the SNDH chromosomal gene.
The promoters for expressing the new SDH and/or new SNDH in yeast include, for example, the promoter of the TRP1 gene, the ADHI or ADHII gene, and acid phosphatase (PH05) gene for
S. cerevisiae.
The promoters for expressing the new SDH and/or new SNDH in mammalian cells include, for example, SV4

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