JP3122762B2 - 2-deoxy-siro-inosose synthase, amino acid sequence, gene base sequence - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to glucose-6-
2-Deoxy-siro-inosose (2-De)
The present invention relates to a 2-deoxy-siro-inosose synthase that catalyzes a reaction for synthesizing oxy-scyllo-inosose, an amino acid sequence of the enzyme, and a gene base sequence encoding the amino acid sequence.

[0002]

2. Description of the Related Art 2-Deoxy-siro-inosose (DO)
I) Synthase is glucose-6-phosphate (G-6-
It is a cyclizing enzyme that catalyzes a reaction for synthesizing 2-deoxy-scyllo-inosose (DOI) using P) as a substrate (FIG. 1). DOI synthase is an enzyme involved in the biosynthesis of aminoglycoside antibiotics containing 2-deoxystreptamine as aglycone, and its product, D
OI is a substance useful as a pharmaceutical raw material or a chemical industrial resource. Until now, the DOI synthesis method has only a multi-step reaction and a method using toxic or expensive metals, and a short-step method has not been known so far.

[0003]

Considering the characteristics of the DOI synthase, the enzyme can be introduced into other microorganisms to efficiently use the abundant glucose as a raw material to efficiently use DOI as a useful resource. , It may be possible to produce in a short process. Heretofore, although the existence of DOI synthase has been expected, its actual state has not been clarified, and the properties of the enzyme have not been clarified. In addition, the isolation and purification of the enzyme were not successful, and the enzyme was not industrially used.

[0004]

Therefore, in order to obtain a means for industrially using DOI synthase, the present inventors have determined that Bacillus circulans SANK72 isolated from soil.
073, the DOI synthase was purified, and the characteristics of the enzyme were clarified. Furthermore, the amino acid sequence of the enzyme and the nucleotide sequence of the gene encoding the amino acid sequence were clarified for the first time. Furthermore, the DOI synthase is expressed in Escherichia coli,
The properties of the recombinant DOI synthase were determined.

[0005]

EXAMPLES (Culture of bacteria) Butyrosine-producing bacterium Bacillus circulans SANK72
The 073 suspension was cultured in nutrient glycerol medium (1% meat extract, 1% peptone, 0.5% NaCl, 1% glycerol). OD at 660 nm is 7.0-
Preculture was performed on a rotary shaker at 28 ° C. until 9.0. A part of the pre-culture was collected and further cultured until the OD reached 8.0-9.0. The cells were collected by centrifugation (6000 g, 30 minutes, 4 ° C.), and washed with a 50 mM Tris-HCl buffer solution (pH 7.7) (6000 g, 30 minutes, 4 minutes).
° C).

(Purification of DOI synthase) c
irculans cells (80 g wet weight) at 50 μM
phenylmethansulfonyl fl
suspended in 240 ml of Tris-HCl buffer containing uoride (PMSF) and 50 μM EDTA,
At 0 ° C. in an ice bath, Branson Sonifier 250
Sonication was performed with the mold. After sonication, 6
After centrifugation at 000 g for 30 minutes, ammonium sulfate (ammonium sulfate) precipitation was performed on the supernatant by a conventional method. 40-
For the precipitate obtained by 70% saturated ammonium sulfate precipitation,
50 mM T containing M PMSF and 50 μM EDTA
Dialysis was performed overnight at 4 ° C. in a ris-HCl buffer (pH 7.7). The dialysate was washed with 50 mM Tris-HCl.
The solution was poured into a DEAE Cellulofine A-800 column (Chisso) equilibrated with a buffer (pH 7.7), and the protein adsorbed on the column was removed from 0M (300 ml) to 0.
Elution was performed with a 4M (300 ml) NaCl gradient. DOI synthase activity was detected when the NaCl concentration was around 0.2M.

[0007] The active fraction was concentrated to 10 ml with Centriprep-10 (Amicon), and 50 mM Tris-H was added.
TSK- equilibrated with Cl buffer (pH 7.7)
gel AF-Blue Toyopearl 650
The concentrated sample was poured into ML (Tosoh). The adsorbed protein was eluted with a similar buffer containing 1 M NaCl. The active fraction was similarly concentrated to 2 ml,
50 mM Tris-HCl containing 0.1 M NaCl
HiLoad equilibrated with buffer (pH 7.7)
26/60 Superdex 200pg (FPL
The concentrated sample was poured into C). The active fraction was concentrated to 2 ml in the same manner, and concentrated to 50 ml containing 0.1 M NaCl.
Mono Q HR 10/10 (FPLC) equilibrated with M Tris-HCl buffer (pH 7.7)
The concentrated sample was poured. The adsorbed protein is N
Eluted at aCl concentrations of 0.13M to 0.16M. Enzyme activity was detected at a NaCl concentration of 0.14M. FIG. 2 shows SDS polyacrylamide gel electrophoresis (SDS-P) at each stage of purification of DOI synthase.
AGE). Lane 1 is the cell extract before purification, Lane 2 is after ammonium sulfate precipitation, Lane 3 is after DEAE purification, and Lane 4 is TSK-gel AF-Blue To.
After purification of yopearl 650 ML, lane 5 contains Sup
After purification of erdex200, lane 6 shows MonoQ purification, and lane 7 shows molecular weight markers. Table 1 shows D
The purification rate at each stage of OI purification is shown.

[0008]

[Table 1] Purification of DOI from C. circulans Step Total protein Total activity Specific activity ^b) Purification factor ^b) Recovery ^b) (mg) (U) (U / mg) (%) Extract 5516 nd ^a) nd ^a) nd ^a) nd ^a) 40-70% 2431 nd ^a) nd ^a) nd ^a) nd ^a) (NH ₄ ) ₂ SO ₄ DEAE 817 99.7 0.122 1 100 Blue ^C) 119 42.4 0.356 2.87 42.5 Sephadex 200 9.787 60.8 6.23 51.1 61.0 Mono Q 0.778 14.1 18.1 148 14.1 a) undetermined b) calculated after DEAE chromatography c) TSK-gel AF-Blue Toyopearl 650 ML

(Determination of molecular weight of DOI synthase) When the molecular weight of the enzyme obtained by the purification was analyzed by SDS-PAGE, two peptides of 42 kDa and 23 kDa were detected (FIG. 2, lane 7). On the other hand, native polyacrylamide gel electrophoresis (Native-PA
GE), a single band was detected (FIG. 3). Furthermore, gel filtration chromatography (TSK-g
el G3000SW), it was detected as a single band under native conditions, but 0.1% SDS
Under denaturing conditions containing, the two peptides are separated and detected, and isoelectric focusing (Mono PHR)
5/20), a single band was detected. The above results indicate that the obtained enzymes were 42 kDa and 23 kDa.
This indicates that the protein is a 65 kDa dimeric protein consisting of a peptide of Da.

(Measurement of DOI synthase activity) The activity of 2-deoxy-scyllo-inosose (DOI) synthase is determined by HP
Detected using LC method. A certain amount of enzyme solution (70μ
l) 5 mM G-6-P, 5 mM NAD ⁺ and 5 mM
50 mM Tris-HCl containing MCoCl ₂ (p
H7.7, final volume of 100 μl) and incubated at 46 ° C. for 1 hour. The reaction was stopped by adding 100 μl of methanol. Hydrochloric acid O-
(4-nitrobenzyl) hydroxylamine (NBH
20 μl of a pyridine solution (5 mg / ml) of A) was added to generate an oxime derivative. After heating the whole solution at 60 ° C. for one hour, air was blown in to remove the solvent.
The residue was dissolved in 2 ml of a mixture of chloroform and methanol (20: 1) and poured into Sep-Pak plus silica. The O- (4-nitrobenzyl) oxime derivative of DOI was eluted with a mixture of chloroform and methanol (5: 1). The eluate was evaporated and the residue was dissolved in 100 μl of methanol. A portion (5 μl) of the solution was TSK-gel ODS-80TM
It was injected into a CTR column (Tosoh) and the ultraviolet absorption at 262 nm was measured. The amount of O- (4-nitrobenzyl) oxime derivative of DOI was quantified by standard curve method. One unit of enzyme activity was defined as producing 1 nmol of DOI per minute.

(Properties of DOI synthase) The effect of pH on DOI synthase activity was examined.
To 8.5 was detected (Fig. 4). N
When the effect of temperature in the presence of AD ⁺ and cobalt ions was examined, the DOI synthase showed the maximum activity at 46 ° C. (FIG. 5). On the other hand, in the absence of NAD ⁺ and cobalt ions, the activity was lost at 46 ° C.
AD ⁺ and cobalt ions are thought to be used not only for the enzyme reaction but also for stabilizing the DOI synthase. Furthermore, the effects of various divalent metal ions on DOI synthase activity were examined (Table 2). Since no enzyme activity was observed in the presence of EDTA,
The presence of metal ions appears to be essential for the DOI synthase reaction. In addition, cobalt ion had the highest activity of enzymatic activity, but magnesium, calcium and manganese ions did not affect the enzyme activity. Meanwhile, copper,
Zinc ion reduced enzyme activity.

[0012]

[Table 2] Effect of divalent metal ions on DOI synthase activity Added ion Vmax Added ion Vmax None 0.353 Co ²⁺ 1 Mg ²⁺ 0.398 Ni ²⁺ 0.163 Ca ²⁺ 0.367 Cu ²⁺ 0 Mn ²⁺ 0.376 Zn ^{2 +} 0 Fe ²⁺ 0.099 EDTA 0

(Reaction Kinetics of DOI Synthase) Reaction Rate
Prior to analysis of the theory, the time-dependent changes in the DOI synthase reaction were detected.
After discussion, the reaction rate decreased 2 hours after the start of the reaction.
did. Therefore, in the reaction kinetic analysis, 1
The reaction was stopped at time. Enzyme reaction rate constant (K_catvalue
And K _mValue) from the initial rate of the DOI synthesis reaction
It was determined by performing a Weber-Bark plot. That
Results for G-6-P at 46 ° C., pH 7.7
K_mThe value is 9.0X10^-FourM, K for NAD_mvalue is
1.7X10^-FourM and K_catThe value is 7.3X10^-2s
^-1And K_cat/ K_mIs 82M^-1s^-1Met.

(Determination of N-terminal amino acid sequence of DOI synthase) The 42 kDa and 23 kDa subunits of DOI synthase purified from Circulans were
Separation was performed on 2.5% Tricine-SDS-PAGE. After electrophoresis, proteins were transferred from the gel to a PVDF membrane by electroblotting. Two bands were cut out by staining with Coomassie Brilliant Blue, and a peptide sequencer (Shimadzu, PP
The sequence was determined according to SQ-21).

(Cloning of DOI synthase gene)
Part of the determined N-terminal amino acid sequence (FNFAG
EHV) and 40 kA (5'-TTYGCNTTT)
YGGNGARCAYGT-3 ') and 40 kB (5'-
TNCCCRAANGCRAARTTRAA-3 ′) was designed and synthesized, and the 3 ′
Labeled with IG (digoxigenin-11-dUTP). B. The chromosomal DNA of C. circulans was cleaved with a restriction enzyme, the obtained hydrolyzate was separated by agarose gel electrophoresis, and the DNA fragment was transferred to a blotting membrane (Zeta Probe GT Genomic Tested Blotting membrane Biorad). Hybridization was performed according to the standard protocol of the kit. 50 for oligonucleotide (20mer)
Hybridization was performed at 65 ° C. for longer size DNA. Oligonucleotides were washed at 50 ° C. in 2 × SSC, and large size DNA was washed at 65 ° C. in 1 × SSC. The hybridized band was detected with a DIG fluorescence detection kit (Boehringer Mannheim).

[0016] DN hybridized with both probes
Among the A fragments, a 4.2 kbp fragment obtained by digesting chromosomal DNA with EcoRI was selected and ligated to pUC19. Using the obtained plasmid, E. coli JM10
Five transformations were performed. Transformants were transformed with ampicillin (50 μg / ml), isopropyl-β-D-thiogalactopyranoside (IPTG) (200 μg / ml) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyr. Luria containing noside (40 μg / ml)
-Selection was performed on Bertani (LB) agar medium. Transformants were transferred to Hybond-N + membranes (Amersham), solubilized, and the DNA fixed. Colony hybridization was performed using the same DIG probe for further screening.

(Determination of base sequence of DOI synthase gene) As a result, a positive clone pDS1 was obtained,
The nucleotide sequence was determined. . The nucleotide sequences of various restriction enzyme fragments of pDS1 were determined using Thermosequenase cycle sequencing kit (Amersham), M13 forward primer, M13 reverse primer (Amersham) and DNA sequencer (Li-cor4200 type). Determined by the dideoxynucleotide method. The total sequence of both strands was determined using DNA. The DNA sequence was analyzed by the GENETYX program (software development) and found to have three reading frames. EMBL, Genebank or SW using BLAST and FESTA software on the net
A homology search was performed on the ISSPROT data library. As a result, the deduced amino acid sequence encoded by pDS1 contained a portion completely matching the N-terminal amino acid sequence of the DOI synthase described above. But,
pDS1 contained only a portion of the gene encoding the 42 kDa subunit.

Therefore, the Ba of DIG-labeled pDS1 is
The mHI-EcoRI fragment was designed as a probe to pick up the entire gene. As a result of performing the same Southern hybridization using the probe, a 4.0 kbp BamHI-PstI fragment (pDS2) was identified.
The transformant obtained by binding to UC19 was screened by colony hybridization using the same probe. As a result, pDS2 was identified, and its nucleotide sequence was determined by the same method as for pDS1. Thus, it was estimated from the molecular size that pDS2 contained the entire 42 kDa subunit gene shown in SEQ ID NO: 1 of the DOI synthase. D
From the sequence of the OI synthase gene, it was found that the gene encodes a polypeptide (SEQ ID NO: 2) consisting of 368 amino acids and having a molecular weight of 40,746 Da.

(Expression of DOI synthase gene) In order to confirm the function of the DOI synthase gene, large-scale expression of the gene was attempted. Using pDS2 as a template, 40
kf (5'-GGTGGAGGGATCCATATGAC
GACTAA-3 ') and 40 kr (5'-AAAGC)
AAGCTTATCCTGGTTCATCC-3 ')
(Amersham Pharmacia) as a primer to perform a polymerase chain reaction (PCR) to amplify the DOI synthase gene. PCR is UIMA DN
Gene amplification PC using A polymerase (Roche)
R System 9700 (PerkinElmer, Upright Bioscience). 50 μg of pDS2
DNA, 60 pmol of both primers and 0.25 mM
Was added to the incubation buffer to perform the reaction (100 μl). PCR is 95
After initial denaturation at 1 ° C. for 1 minute, the enzyme is added, followed by 30 cycles of 95 ° C. for 30 seconds, 60 ° C. for 45 seconds, 72 ° C. for 30 seconds, and further reaction at 72 ° C. for 7 minutes. went. The product amplified by PCR is digested with BamHI and HindIII,
Cloning into the appropriate site of UC19, the resulting p
DS3 was isolated.

After determining the base sequence of pDS3, Nde
Degradation with I-HindIII resulted in the inserted DN
A is separated from the plasmid and the expression vector pET3
0b (+) bound to the NdeI-HindIII site. Escherichia coli BL21 (DE
3) was transformed. A transformant containing pDS4 was transformed with 6
The culture was pre-cultured at 37 ° C. until the OD at 00 nm reached 0.6 and a portion was inoculated into the culture medium. 600
Shake culture at 37 ° C. until the OD in nm reaches 0.6, add IPTG (final concentration 0.4 m
M), the culture was continued for another 2-3 hours. Centrifugation (400
0 g, 10 minutes, 4 ° C.)
The plate was washed with a 50 mM Tris-HCl buffer (pH 7.7) containing MCoCl ₂ . E. coli (pDS4)
The DOI synthase expressed in (recombinant DOI synthase) reached as much as 20% of the total soluble protein. FIG. 6 shows a pattern of SDS polyacrylamide gel electrophoresis, in which lane 1 is a molecular weight marker and lane 2 is pET.
E. coli extract containing 30b, lane 3 was E. coli extract containing pET30b after IPTG induction, lane 4 was pDS
E. coli extract containing E.4, lane 5 shows p.
E. coli extract containing DS4, lane 6 circu
lans-derived natural DOI synthase.

(Confirmation of activity of recombinant DOI synthase) p
Escherichia coli transformed with DS4 was treated with 0.2 mM CoC.
l_Two50 mM Tris-HCl buffer (pH
7.7) (5 g wet cells in 150 ml)
Under ice cooling (0 ° C) using Branson Sonifier 250
The cells were disrupted (2 times, 10 times). After cell disruption
Centrifugation (12,000 g, 30 minutes), and collect the supernatant.
Seeds were used for cell-free enzymatic reactions. 5ml cell-free
In the extract, glucose 6-phosphate or [6,
6-^TwoH_Two] -Glucose 6-phosphate, 5 mM NAD
⁺And 5 mM Co²⁺And incubate at 46 ° C for 2 hours.
The enzymatic reaction was performed by cubating. O- of the reaction product
Derivatize (4-nitrobenzyl) oxime to form a thin layer
Purified by chromatography (Merck, Kiesel
Gel F₂₅₄). 8.4mg glucose 6-phosphate
And about 3 mg of DOI O-
(4-Nitrobenzyl) oxime derivative was obtained. Raw
About adult¹H-NMR and^TwoH-NMR spectrum
Was confirmed.¹H-NMR (300 MHz, C
D_ThreeOD) δ: 1.90 (dd, J = 10.9, 13.
8 Hz, H-2ax), 3.19 (t, J = 8.8H)
z, H-4), 3.51 (dd, J = 4.9, 13.8)
Hz, H-2eq), 3.3-3.4 (H-3, H-
5), 4.03 (d, 9.0 Hz, H-6), 5.25
(S), 7.58 (d, 8.5 Hz), 8.21 (d,
8.5 Hz). [6,6-^TwoH _Two] -Glucose 6-li
Of similar derivatives of DOI products derived from acid^TwoH-NMR
(61.4 MHz, CH_ThreeOH) [delta]: 1.82, 3.4
6. From these results, the expected reaction product was confirmed,
Escherichia coli germination even in the absence of the 23 kDa subunit
The current product catalyzes the DOI synthase reaction and the resulting protein
The protein was proved to be a recombinant DOI synthase.

(Purification of Recombinant DOI Synthase) During purification, DOI synthase showed a tendency to aggregate under conditions of ammonium sulfate precipitation and dialysis. When stored in a 50 mM Tris-HCl buffer in the absence of cobalt ions, the enzyme activity was lost even during short-term storage. These results differ from recombinant DOI synthase in protein stability as compared to the native enzyme. Therefore, in the case of the recombinant 2-DOI synthase, the following purification method different from that of the natural enzyme was used. 0.2 mM recombinant 2-DOI synthase
Handling was performed in 50 mM Tris-HCl buffer (pH 7.7) containing CoCl ₂ . 4 ℃ or -75
Approximately 60% of the purified enzyme was inactivated after 10 days of storage, even when stored at <RTIgt;

The cell-free extraction solution was prepared by adding 0.2 mM CoC
50 mM Tris-HCl buffer containing l ₂ (p
H7.7) equilibrated with DEAE-Sepharose
Loaded on a Fast Flow column. The adsorbed protein was converted from 0 M NaCl (300 ml) to 0.4 M N
Elution was performed with a linear gradient of aCl (300 ml). DOI synthase activity was detected near the 0.2 M NaCl fraction. The active fraction was centriprep-10
(Amicon) and concentrated to 10 ml. Concentrate containing 5% 0.1M NaCl and 0.2mM CoCl ₂
HiLoad26 / 60Superdex20 equilibrated with 0 mM Tris-HCl buffer (pH 7.7)
Active fractions were similarly obtained by applying 0 pg (FPLC).
Lane 7 in FIG. 6 shows the pattern of the recombinant DOI synthase obtained by SDS polyacrylamide gel electrophoresis,
A single band of Da was shown. Table 3 shows the purification ratio at each stage of the recombinant DOI purification.

[0024]

Table 3 Purification of recombinant DOI synthase Step Total protein Total activity Specific activity Purification factor Recovery (mg) (U) (U / mg) (%) Extract 364 65.0 0.179--DEAE 84.7 57.4 0.678 3.8 88.3 Sephadex 200 45.9 49.1 1.07 6.0 75.5

(Properties of Recombinant DOI Synthase) pH,
Recombinant DOI for added metal ion or temperature
The activity characteristics of the synthase were studied. pH 6.5 to 8.
0 for 50 mM MOPS-KOH buffer,
pH 7.0-9.0 was examined using a Tris-HCl buffer. The activity of the recombinant DOI synthase was measured by modifying the HPLC method used for the natural DOI synthase. 1 μl of enzyme preparation was added to 10 μl of 50 m
M glucose 6-phosphate, 10 μl of 50 mM NAD
50 m with ⁺ and 79 μl of 0.2 mM CoCl ₂
It was mixed with an MTris-HCl buffer (pH 7.7, final reaction volume 100 μl, final concentration of glucose 6-phosphate and NAD ⁺ 5 mM) and incubated at 46 ° C. for 5 minutes. Thereafter, the enzyme activity was measured by the above-mentioned HPLC method. FIG. 7 shows the effect of pH on the enzymatic reaction of the recombinant DOI synthase. From FIG.
Maximum activity was observed between pH 7.5 and 8.5.
In FIG. 7, square points indicate the results of examination using the MOPS-KOH buffer, and circle points indicate the results of examination using the Tris-HCl buffer.

The temperature conditions and temperature stability of the enzyme reaction were studied. For optimal reaction temperature conditions, see 15
The study was carried out by performing an enzyme reaction at a temperature between 55 ° C and 55 ° C.
As shown by the square points in FIG. 8, in the enzyme reaction for 5 minutes,
Under the conditions containing NAD ⁺ and Co ²⁺ , the maximum activity was observed at around 55 ° C. When the temperature stability was examined by measuring the enzyme activity remaining after treatment at 20 ° C. to 60 ° C. for 5 minutes, the enzyme activity was reduced at 50 ° C. or more as shown by the circles in FIG. It is believed that the recombinant DOI synthase denatures at 50 ° C. or higher. Furthermore, the divalent metal ion requirement was analyzed in the presence of 1 mM of cobalt, magnesium, manganese, copper, iron, calcium, zinc and nickel ions (Table 4). The presence of cobalt ions is essential for the recombinant DOI synthase, and as described above, the recombinant DOI synthase could not be purified in the absence of cobalt ions. In addition, the enzyme activity disappeared in the presence of EDTA. Magnesium, calcium,
Manganese and iron did not promote or inhibit the enzyme activity, whereas copper and zinc strongly inhibited the enzyme activity.

[0027]

[Table 4] Effect of divalent metal ion on recombinant DOI synthase Additive ion Relative Vmax Additive ion Relative Vmax None 1.00 Co 2 ⁺ 1 Mg ²⁺ 1.06 Ni 0.79 Ca ²⁺ 0.87 Cu 0.43 Mn ²⁺ 0.93 Zn 0.05 Fe ²⁺ 0.93 EDTA 0

(Reaction Kinetics of Recombinant DOI Synthase)
Using 2 μM recombinant DOI synthase, the enzyme reaction was
The change with time was examined. As a result, 30 minutes after the start of the reaction
It showed a constant initial speed. Stop the reaction 5 minutes after the reaction starts
From the initial speed of the DOI synthesis reaction
By performing a peak plot, the rate constant (K
_catValue and K_mValue). As a result, 46 ° C, pH
K for glucose-6-phosphate at 7.7_mvalue
Is 2.1X10^-FourM, K_catThe value is 1.0s^-1And K
_cat/ K_mIs 4.8X10^ThreeM^-1s ^-1Met. Also,
K for NAD_mThe value is 2.3 × 10^-FiveM.

[0029]

According to the present invention, 2-deoxy-scyllo-inosose synthase, which catalyzes the reaction of synthesizing 2-deoxy-scyllo-inosose from glucose-6-phosphate, is purified, and the amino acid sequence of the enzyme is determined. And the gene sequence encoding the amino acid sequence was given.

[0030]

[Sequence List] <110> Applicant's name: Director of Tokyo Institute of Technology <120> Title of invention: 2-deoxy-scyllo-inosose synthase, amino acid sequence, gene base sequence <160> Number of sequences: 2 <210> SEQ ID NO: 1 <211> Sequence length: 1107 <212> Sequence type: nucleic acid <213> Origin: Bacillus circulans SANK72073 2-deoxy-scyllo-inosose synthase 42 kDa subunit <400> Sequence ATGACGACTA AACAAATTTG TTTTGCGGAC CGGTGTTTTA ACTTTGCATT CGGCGAACAT 60 GTTTTGGAAT CGGTTGAATC CTATATTCCC CGGGATGAAT TCGATCAATA TATCATGATT 120 TCGGACTCGG GGGTACCGGA CTCGATTGTT CATTATGCGG CCGAATACTT CGGCAAACTC 180 GCCCCTGTAC ATATTCTTCG CTTTCAGGGC GGAGAAGAAT ACAAAACACT TTCAACCGTG 240 ACAAATTTGC AAGAGCGGGC AATTGCTCTG GGAGCCAACC GAAGAACCGC TATCGTAGCG 300 GTTGGCGGAG GGTTAACCGG AAACGTTGCC GGAGTGGCGG CCGGCATGAT GTTTCGCGGG 360 ATTGCGCTTA TTCACGTTCC GACCACGTTT TTGGCGGCCT CCGATTCGGT TCTTTCGATT 420 AAGCAGGCTG TTAATTTAAC GAGCGGAAAG AACCTGGTCG GCTTTTATTA TCCGCCACGC 480 TTCGTGTTCG CCGATACCCG AATCTTGTCG GAGTCGCCGC CCCGTCAGGT GAAAGCGGGA 540 ATGTGCGAGC TGGTAAAAAA TATGCTGATT CTGGAAAACG ACAACAAGGA ATTTACAGAG 600 GATGATTTAA ATTCAGCCAA TGTGTATTCT CCGAAGCAGC TGGAGACGTT TATCAACTTC 660 TGCATATCGG CCAAAATGTC GGTATTAAGC GAAGATATTT ACGAGAAAAA GAAGGGCCTG 720 ATCTTTGAGT ACGGCCATAC GATCGGTCAT GCGATCGAGC TTGCCGAGCA GGGAGGGATC 780 ACGCACGGAG AAGCCATTGC AGTGGGCATG ATTTACGCCG CTAAAATAGC GAACCGGATG 840 AACCTGATGC CCGAACATGA CGTGTCCGCC CATTACTGGC TTTTAAATAA AATCGGGGCC 900 TTGCAGGATA TTCCGCTCAA ATCGGACCCG GATTCGATCT TCCATTATTT AATCCACGAT 960 AACAAGAGGG GCTACATTAA GCTGGATGAG GATAATTTGG GTATGATTTT ACTTAGCGGA1020 GTCGGTAAAC CGGCGATGTA TAACCAAACG CTGCTTACAC CGGTCAGAAA AACGCTCATA1080 AAAGAAGTGA TCCGGGAAGG GCTGTAA 1107 <210> SEQ ID NO: 2 <211> sequence length: 368 <212> Sequence type: amino acid <213> Origin: Bacillus circulans SANK72073 2- Deoxy - scyllo - Inoso over scan synthase 42kDa subunit <400> sequence MTTKQICFAD RCFNFAFGEH VLESVESYIP RDEFDQYIMI SDSGVPDSIV HYAAEYFGKL 60 APVHILRFQG GEEYKTLSTV TNLQERAIAL GANRRTAIVA VGGGLTGNVA GVAAGMMFRG 120 IALIHVPTTF LAASDSVLSI KQAVNLTSGK NLVGFYYPPR FVFADTRILS ESPPRQVKAG 180 MCELVKNMLI LENDNKEFTE DDLNSANVYS PKQLETFINF CISAKMSVLS EDIYEKKKGL 240 IFEYGHTIGH AIELAEQGGI THGEAIAVGM IYAAKIANRM NLMPEHDVSA HYWLLNKIGA 300 LQDIPLKSDP DSIFHYLIHD NKRGYIKLDE DNLGMILLSG VGKPAMYNQT LLTPVRKTLI 360 KEVIREGL 368

[Brief description of the drawings]

FIG. 1. Reaction catalyzed by 2-deoxy-scyllo-inosose synthase.

FIG. 2: SDS-PAGE pattern of purified 2-deoxy-scyllo-inosose synthase.

FIG. 3. Native PAGE pattern of purified 2-deoxy-scyllo-inosose synthase.

FIG. 4. Effect of pH on 2-deoxy-scyllo-inosose synthase activity.

FIG. 5: Effect of temperature on 2-deoxy-scyllo-inosose synthase activity.

FIG. 6: SDS-PAGE patterns of E. coli extracts containing pET30b and pDS4 before and after IPTG induction,
And purified, natural and recombinant 2-deoxy-silo-
SDS-PAGE pattern of inosose synthase.

FIG. 7: Effect of pH on recombinant 2-deoxy-scyllo-inosose synthase activity.

FIG. 8: Effect of temperature on recombinant 2-deoxy-scyllo-inosose synthase activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification symbol FI (C12N 9/54 C12R 1:19) (58) Investigated field (Int.Cl. ⁷ , DB name) WPI (DIALOG) BIOSIS ( DIALOG) CAS ONLINE (STN) DDBJ / EMBL / GenBank

Claims (9)

(57) [Claims] 1. A 2-deoxy-scyllo-inosose synthase having the following properties: (1) catalyzing a reaction for producing 2-deoxy-scyllo-inosose from glucose-6-phosphate; (3) Enzyme activity is promoted in the presence of cobalt ions; (4) Enzyme activity is suppressed in the presence of iron, zinc and copper ions; (5) NAD Perform the reaction using ⁺ as a coenzyme. 2. A gene comprising a base sequence represented by the following (a) or (b): (A) base numbers 1-110 shown in SEQ ID NO: 1 in the sequence listing
Genetic characterized by consisting of the nucleotide sequence represented by 7
Child . (B) 2-deoxy-scyllo-inosose synthase activity
A part of (a) is deleted,
A gene that has been replaced or added. 3. Bacillus circulans SANK720
73 2-deoxy-scyllo-inosose synthase 42k
3. The genetic of claim 2, which encodes a Da subunit.
Child . Characterized by comprising the amino acid sequence shown in 4. below (c) or (d), polypeptide. (C) amino acid numbers 1-3 shown in SEQ ID NO: 2 of the sequence listing
A polypeptide consisting of the amino acid sequence represented by 68. (D) 2-deoxy-scyllo-inosose synthase activity
A, a part of (c) deleted, substituted or added,
Polypeptide . 5. Bacillus circulans SANK720
73 2-deoxy-scyllo-inosose synthase 42k
The polypep according to claim 4, which is derived from a Da subunit.
Chid . 6. Glucose-6-phosphate is used as a raw material,
A method for producing 2-deoxy-scyllo-inosose by a catalytic reaction using the 2-deoxy-scyllo-inosose synthase according to claim 1. 7. An Escherichia coli transformant into which the gene according to claim 2 has been inserted. 8. A recombinant 2-deoxy-scyllo-inosose synthase obtained by expressing the gene according to claim 2 in Escherichia coli. 9. Glucose-6-phosphate is used as a raw material,
A method for producing 2-deoxy-scyllo-inosose by a catalytic reaction using the recombinant 2-deoxy-scyllo-inosose synthase according to claim 8.