L-ribose isomerase, its preparation and uses

An L-ribose isomerase which isomerizes aldoses such as L-ribose, D-lyxose, D-talose, D-mannose, L-allose and L-gulose into their corresponding ketoses such as L-ribulose, D-xylulose, D-tagatose, D-fructose, L-psicose and L-sorbose. The enzymatic reaction is a reversible equilibrium reaction. The L-ribose isomerase can be obtained from microorganisms of the genus Acinetobacter.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to an L-ribose isomerase, its preparation and 
uses, more particularly, relates to an L-ribose isomerase which converts 
L-ribose into L-ribulose and vice versa, preparation thereof, 
microorganisms capable of producing the L-ribose isomerase, and a process 
for producing ketoses and aldoses using the L-ribose isomerase. 
2. Description of the Prior Art 
Biochemical industries have been developing in these days, and rare 
saccharides which had been put aside are in great demand in the field of 
saccharide chemistry. Thus, the establishment of these rare saccharides is 
strongly required. Although such rare saccharides can be produced by 
organic chemical methods, the production conditions are generally crucial 
and the yields of desired products are relatively low. Therefore, the 
organic chemical methods are not satisfactory as an industrial scale 
production. While enzymatic saccharide-conversion methods may be imagined 
as biochemical methods for producing rare saccharides but there was 
reported no isomerase, which acts on L-ribose or D-talose as a rare 
saccharide, and was not established the production method for such rare 
saccharides. 
SUMMARY OF THE INVENTION 
It has been strongly required an industrial-scale production method for 
rare saccharides such as L-ribose and D-talose. 
To attain the object the present inventors studied on an L-ribose isomerase 
and extensively screened microorganisms which produce such an enzyme. As a 
result, the inventors found that a newly isolated microorganism of the 
species Acinetobacter calcoaceticus LR7C strain, isolated from a soil in 
Miki-machi, Kita-gun, Kagawa, Japan, produces an L-ribose isomerase. The 
inventors also found that the L-ribose isomerase facilitates the 
production of rare saccharides when acts on aldoses or ketoses as 
substrates and established the present invention.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to an L-ribose isomerase, its preparation and 
uses, more particularly, relates to an L-ribose isomerase which converts 
L-ribose into L-ribulose and vice versa, preparation thereof, 
microorganisms capable of producing the L-ribose isomerase, and a process 
for producing ketoses and aldoses using the L-ribose isomerase. 
Followings are the identification results of the microorganism of the genus 
Acinetobacter, i.e., Acinetobacter calcoaceticus LR7C strain (FERM 
BP-5335). 
Identification Results of Acinetobacter calcoaceticus LR7C strain: 
A. Morphology 
Characteristics of cells when incubated at 27.degree. C. in nutrient broth 
agar 
Usually existing in a single rod form of 1.0-1.5.times.1.5-2.5 .mu.m; 
Motility: Positive (rotatory or vibratory motility); 
Asporogenicity: Positive; 
Flagellum: Positive; 
Gram stain: Negative; 
B. Cultural properties 
(1) Characteristics of colony formed when incubated at 27.degree. C. in 
nutrient agar plate with broth 
Shape: Circular colony having a diameter of about 0.1-1 mm after 2 days' 
incubation; 
Rim: Entire; 
Projection: Hemispherical shape; 
Gloss: Dull; 
Surface: Smooth; 
Color: Semitransparency, pale yellow; 
(2) Characteristics of colony formed when incubated at 27.degree. C. in 
slant nutrient agar with broth 
Growth: Satisfactory; 
Shape: Thread-like; 
(3) Characteristics of colony formed when incubated at 27.degree. C. in 
slant nutrient agar with trypton soya broth 
Growth: Satisfactory; 
Shape: Thread-like; and 
(4) Not liquefying gelatin when stab-cultured at 27.degree. C. in nutrient 
gelatin with broth. 
C. Physiological properties 
(1) Reduction of nitrate: Positive; 
(2) Accumulation of poly-.beta.-hydroxy butyrate: Negative; 
(3) Methyl red test: Negative; 
(4) VP-Test: Negative; 
(5) Formation of indole: Negative; 
(6) Formation of hydrogen sulfide Negative; 
(7) Hydrolysis of starch: Negative; 
(8) Utilization of citric acid: Positive; 
(9) Formation of pigment: Negative; 
(10) Oxidase: Negative; 
(11) Catalase: Positive; 
(12) Growth conditions: Growing at a temperature in the range of 
20.degree.-37.degree. C.; 
(13) Oxygen requirements: Aerobic; 
(14) Formation of acid from D-glucose: Positive; 
(15) Hemolysis: Negative; 
(16) .beta.-Xylosidase: Negative; 
(17) Utilization of carbon source Utilizing glutaric acid, malonic acid, 
phenyl lactic acid, azelaic acid, D-malic acid, ethanol, 2,3-butanediol, 
aconitic acid, D-ribose, D-xylose, L-arabinose and D-glucose; 
(18) Utilization of nitrogen source Utilizing L-phenyl alanine, 
L-histidine, L-aspartic acid, L-leucine, L-tyrosine, .beta.-alanine, 
L-arginine and L-ornithine but not utilizing histamine; 
(19) DNase: Positive; 
(20) Formation of 3-ketolactose: Negative; and 
(21) Mol % guanine (G) plus cytosine (C) of DNA: 42%. 
Based on these mycological properties, the microorganism was compared with 
those of known microorganisms with reference to Bergey's Manual of 
Determinative Bacteriology, Ninth Edition (1994). As a result, it was 
revealed that the microorganism was identified as a novel microorganism of 
the genus Acinetobacter. The present inventors identified this 
microorganism as the one of the species Acinetobacter calcoaceticus based 
on the data of not growing at 41.degree. C., negative hemolysis and 
gelatin hydrolysis, positive acid formation from glucose, and conditions 
of utilizing carbon and nitrogen sources. 
From these results, the present inventors named the microorganism 
"Acinetobacter calcoaceticus LR7C" and deposited it on Dec. 14, 1995, in 
National Institute of Bioscience and Human-Technology Agency of Industrial 
Science and Technology, Ibaraki, Japan. The deposition of the 
microorganism was accepted on the same day and has been maintained by the 
institute under the accession number of FERM BP-5335. 
In addition to the above-identified microorganism, other strains of the 
genus Acinetobacter and mutants thereof can be suitably used in the 
present invention as long as they produce the L-ribose isomerase according 
to the present invention. The above mutants can be obtained by physically 
treating microorganisms of the genus Acinetobacter with ultraviolet ray or 
.gamma.-ray, chemically treating the microorganisms with nitrosoguanidine, 
or by successively culturing the microorganisms in nutrient culture media 
containing D-lyxose to stably produce the present L-ribose isomerase and 
to increase the enzyme yield. Any other microorganism can be used in the 
present invention as long as it produces the present L-ribose isomerase. 
It is possible to produce the L-ribose isomerase by expressing the 
isomerase in transformants into which a gene encoding the isomerase is 
introduced. If necessary, protein engineering techniques can be used to 
increase the thermal stability of the present L-ribose isomerase or to 
widen the range of pH stability. Any nutrient culture medium such as 
synthetic- or natural-nutrient culture medium can be used in the invention 
as long as the above microorganisms can grow therein and produce the 
present L-ribose isomerase. One or more carbon-containing substances such 
as aldoses, ketoses and sugar alcohols can be used in the invention as 
carbon sources. Usually, D-lyxose can be advantageously used as carbon 
sources for the culture media. The nitrogen sources used in the present 
invention include inorganic nitrogen-containing compounds such as ammonium 
salts and nitrates and organic nitrogen-containing compounds such as urea, 
corn steep liquor, casein, peptone, yeast extract and beef extract. The 
inorganic ingredients used in the present invention include calcium salts, 
magnesium salts, potassium salts, sodium salts and phosphates. The 
microorganisms used in the invention can be cultured under aerobic 
conditions at a temperature of, usually, about 10.degree.-40.degree. C., 
preferably, about 20.degree.-35.degree. C., and at a pH of about 5-9, 
preferably, about 6-8.5. 
After completion of the culture of microorganisms, the present L-ribose 
isomerase is recovered from the culture. The cells in themselves can be 
used as an enzymatic agent because the enzyme mainly exists 
intracellularly. The intracellular enzyme can be extracted from the cells 
by a conventional technique, and the extracted enzyme can be used intact 
as a crude enzyme or used after purified by a conventional method. For 
example, extracts of homogenized cells can be purified by two or more 
techniques such as fractionations using polyethylene glycol, ion-exchange 
chromatographies, and gel filtration chromatographies to obtain enzyme 
preparations with an electrophoretically single protein band. 
The present L-ribose isomerase activity is assayed as follows: Mix 0.05 ml 
of 0.5M glycine-sodium hydroxide buffer (pH 9.0), 0.05 ml of 0.05M 
L-ribose, and an appropriate volume of an enzyme solution sufficient to 
give a total volume of 0.5 ml. Incubate the resulting solution at 
30.degree. C. for an enzymatic reaction and quantify the amount of formed 
L-ribose by the cysteine-carbazole method. One unit activity of the 
present L-ribose isomerase is defined as the amount of enzyme which forms 
one .mu.mole of L-ribose per minute. 
In addition to L-ribose, the present L-ribose isomerase acts on aldoses 
such as D-lyxose, D-talose, D-mannose, L-allose and L-gulose to isomerize 
them into their corresponding ketoses. 
The present L-ribose isomerase should not necessarily be purified to the 
highest possible level. For example, microorganisms, containing the 
L-ribose isomerase and being treated with toluene, can be suitably used 
intact in industrial-scale saccharide-transferring reactions. 
Microorganisms with the present L-ribose isomerase activity and partially 
purified preparations of the isomerase can be immobilized by conventional 
immobilization methods such as entrapping, adsorption and covalent bonding 
methods. The immobilized enzyme can be repeatedly used batchwise or 
continuously used after packed in columns. 
The reaction mixtures thus obtained usually contain both aldoses and 
ketoses. In general, the mixtures can be purified by two or more 
techniques of filtration using filter aids, filters and membrane filters, 
centrifugation to remove insoluble substances, decoloration with activated 
charcoals, and desalting using ion exchangers in H- and OH-form. The 
resulting mixtures can be concentrated to obtain syrupy products or dried 
into powdery solid products. If necessary, higher level crystallization 
steps can be employed: For example, fractionations using cation exchangers 
in alkaline metal and/or alkaline earth metal form or anion exchangers in 
bisulfite and/or boric acid form, and column chromatographies using silica 
gels readily produce high-purity saccharides. When the obtained 
saccharides are crystallizable, they can be arbitrarily prepared into 
crystalline products by conventional crystallization techniques. For 
example, the above reaction mixtures or saccharide solutions, either 
treated with or without appropriate purification methods, are admixed with 
one or more organic solvents in general such as lower alkyl alcohols 
including methanol, ethanol and isopropyl alcohol or concentrated and/or 
allowed to stand at relatively-low temperatures. These methods can be 
combined to obtain supersaturated solutions of the above saccharides, 
followed by crystallizing the solutions and separating the crystals to 
obtain solid products containing crystals. These saccharide products can 
be used as chemical reagents and used in food industries as sweeteners and 
quality-improving agents and in pharmaceutical and chemical industries as 
materials and intermediates. 
Among these saccharides, D-ribose, an isomer of L-ribose, is an essential 
component for DNA correlating deeply with cell growth. Therefore, L-ribose 
or derivatives thereof can be used as a replication inhibitory agent for 
nucleic acids: Examples of such an inhibitory agent include 
pharmaceuticals such as antiseptics, antiviral agents, anti-AIDS agents 
and antitumor agents. As is described above, L-ribose is readily prepared 
from L-ribulose by using the present L-ribose isomerase. The L-ribulose is 
not restricted to its origin: For example, the saccharide can be readily 
prepared by oxidation reactions using microorganisms of the genera 
Gluconobacter and Acetobacter, i.e., the species Gluconobacter frateurii 
and Acetobacter aceti. The following experiments explain the present 
invention in detail: 
EXPERIMENT 1 
Preparation of L-ribose isomerase from Acinetobacter calcoaceticus LR7C 
A liquid nutrient culture medium, consisting of 0.5 w/v % yeast extract, 
0.5 w/v % polypeptone, 0.5 w/v % salt and water, was adjusted to pH 7.0. 
Two liters of the medium was placed in a 2.5-L jar fermenter, sterilized 
by an autoclave at 120.degree. C. for 20 min, cooled and inoculated with a 
seed culture of Acinetobacter calcoaceticus LR7C (FERM BP-5335), which had 
been cultured for 4 days in a nutrient culture medium containing D-lyxose 
as a carbon source, which were then cultured at 30.degree. C. for 14 hours 
under aeration-agitation conditions. The culture medium was centrifuged to 
obtain cells in a yield of about 15 g wet; cells per one L of the culture 
medium. The wet cells were in a conventional manner disrupted with 
aluminum powder, admixed with 0.05M Tris-HCl buffer (pH 7.5) to extract 
the desired enzyme, and centrifuged to obtain a 200-ml supernatant as a 
crude enzyme solution, having a total enzyme activity of 2,870 units and a 
specific activity of 1.73 units/mg protein. 
EXPERIMENT 2 
Purification of L-ribose isomerase 
EXPERIMENT 2-1 
Polyethylene glycol fractionation 
The crude enzyme solution in Experiment 1 was ice-chilled, admixed with 
0.01M manganese chloride, allowed to stand for 30 min, and centrifuged to 
remove insoluble substances. To the resulting supernatant was added 
polyethylene glycol powder to give a final concentration of 15 w/v % and 
dissolved therein under stirring conditions, followed by centrifuging the 
mixture to remove the formed insoluble substances. The resulting 
supernatant was mixed with polyethylene glycol to give a final 
concentration of 25 w/v % and dissolved therein under stirring conditions, 
and the formed insoluble substances were centrifugally collected. 
EXPERIMENT 2-2 
Ion-exchange chromatography 
The sediments in Experiment 2-1 were dissolved in 0.05M Tris-HCl buffer (pH 
7.5), and the residual insoluble substances were removed by centrifugation 
to obtain a 13-ml supernatant which was then fed to a column packed with 
"DEAE-TOYOPEARL.RTM. 650M", a slightly alkaline anion-exchanger 
commercialized by Tosoh Corporation, Tokyo, Japan, to adsorb the desired 
enzyme on the exchanger, followed by eluting the enzyme from the column 
with a liner gradient solution of potassium chloride which increases from 
0M to 0.5M and collecting fractions with an L-ribose isomerase activity. 
EXPERIMENT 2-3 
Gel filtration chromatography 
The fractions with an L-ribose isomerase activity in Experiment 2-2 were 
concentrated, and the concentrate was fed to a column packed with 
"SEPHADEX.RTM. G-150", a bead-like dextran gel commercialized by Pharmacia 
LKB Biotechnology AB, Uppsala, Sweden, and eluted with 0.05M Tris-HCl 
buffer (pH 7.5) to obtain fractions with an L-ribose isomerase activity. 
Table 1 shows the protein amount, enzyme activity, enzyme yield, and 
enzyme purification degree in each purification step. 
TABLE 1 
__________________________________________________________________________ 
Protein 
Enzyme activity 
Yield 
Purification step 
(mg) (unit) (%) Purification degree 
__________________________________________________________________________ 
Crude enzyme 
1,660 
2,870 100 1.0 
PEG fraction 
450 2,570 90 3.3 
DEAE-TOYOPEARL .RTM. 
79.8 1,180 41 8.6 
SEPHADEX .RTM. 
32.9 796 28 14 
__________________________________________________________________________ 
Purity inspection by polyacrylamide gel disk electrophoresis for the 
finally purified enzyme, obtained as an eluate in the gel filtration using 
"SEPHADEX.RTM. G-150" in Table 1, revealed that the enzyme was purified up 
to show a single protein band, meaning that the enzyme was an 
electrophoretically highly-purified enzyme. 
EXPERIMENT 3 
Property of L-ribose isomerase 
Using a purified L-ribose isomerase, obtained by the method in Experiment 
2, the physicochemical properties were studied. 
EXPERIMENT 3-1 
Action 
When acting on L-ribose or L-ribulose in accordance with the method for 
assaying the present L-ribose isomerase activity, the purified L-ribose 
isomerase formed L-ribulose from L-ribose and vice versa. The enzymatic 
reaction is a reversible equilibrium reaction. 
EXPERIMENT 3-2 
Substrate specificity 
In accordance with the method for assaying the present L-ribose isomerase 
activity, the purified L-ribose isomerase activity for aldoses as 
substrates was assayed. Table 2 shows relative activities of the enzyme 
for the aldoses when the relative activity of the enzyme for L-ribose was 
regarded as 100. 
TABLE 2 
______________________________________ 
Substrate Relative activity (%) 
______________________________________ 
L-Ribose 100 
D-Lyxose 50 
D-Talose 44 
D-Mannose 3 
L-Allose 2 
L-Gulose 2 
______________________________________ 
As is evident from the results in Table 2, the L-ribose isomerase showed 
the highest activity on L-ribose. The L-ribose isomerase acted on other 
aldoses such as L-lyxose, D-talose, D-mannose, L-allose and L-gulose. 
These enzymatic reactions were reversible reactions, and the enzyme also 
acted on L-ribulose, D-xylulose, D-tagatose, D-fructose, L-psicose and 
L-sorbose as substrates corresponding to the above aldoses, respectively. 
Table 3 shows the data of ketoses and aldoses, produced from the above 
substrates, which were confirmed by ion-exchange chromatography, 
high-performance liquid chromatography, thin-layer chromatography, etc. 
TABLE 3 
______________________________________ 
Material Product 
______________________________________ 
L-Ribose L-Ribulose 
D-Lyxose D-Xylulose 
D-Talose D-Tagatose 
D-Mannose D-Fructose 
L-Allose L-Psicose 
L-Gulose L-Sorbose 
L-Ribulose L-Ribose 
D-Xylulose D-Lyxose 
D-Tagatose D-Talose 
D-Fructose D-Mannose 
L-Psicose L-Allose 
L-Sorbose L-Gulose 
______________________________________ 
As is evident from the results in Table 3, the present L-ribose isomerase 
acts on aldoses to convert them into their corresponding ketoses and vice 
versa. These enzymatic conversion reactions share a common enzymatic 
reaction mode. Chemical formula 1 is an example of the reaction mode. 
##STR1## 
The enzymatic reaction of the present L-ribose isomerase is a reversible 
reaction. FIG. 4 shows the results of the quantitative relationship of 
reversible equilibrium reactions between aldoses and ketoses under the 
conditions used for assaying the present L-ribose isomerase activity. 
TABLE 4 
______________________________________ 
L-Ribose:L-Ribulose = 70:30 
D-Lyxose:D-Xylulose = 70:30 
D-Talose:D-Tagatose = 12:88 
D-Mannose:D-Fructose = 30:70 
L-Allose:L-Psicose = 40:60 
L-Gulose:L-Sorbose = 25:75 
______________________________________ 
The Km, Michaelis constant, of the present L-ribose isomerase to L-ribose 
was 44 mM. 
EXPERIMENT 3-3 
Molecular weight 
(1) About 25,000-35,000 daltons on polyacrylamide gel electrophoresis 
(SDS-PAGE); 
(2) About 110,000-130,000 daltons on gel filtration method; 
The molecular weight on gel filtration, about 4 times higher than that on 
SDS-PAGE, indicates that the present L-ribose isomerase exists in 
tetramer. 
EXPERIMENT 3-4 
Isoelectric point (pI) 
The present L-ribose isomerase has a pI of about 4.0-5.5 on 
isoelectrophoresis using an agarose plate. 
Experiment 3-5 
Inhibition of activity 
The activity of the present L-ribose isomerase is slightly inhibited by 
L-arabitol and ribitol. 
EXPERIMENT 3-6 
Optimum temperature 
The optimum temperature of the present L-ribose isomerase was studied in 
accordance with the method for assaying the present L-ribose isomerase 
activity. As is shown in FIG. 1, the optimum temperature was about 
30.degree. C. when incubated at pH 9.0 for 10 min. 
EXPERIMENT 3-7 
Optimum pH 
The optimum pH of the present L-ribose isomerase was studied in accordance 
with the method for assaying the present L-ribose isomerase activity. As 
is shown in FIG. 2, the optimum pH was about 8-9 when incubated at 
30.degree. C. for 10 min. FIG. 2 shows the results of assay using citrate 
buffer, Veronal buffer, and glycine-sodium hydroxide buffer which are 
respectively indicated by the symbols ".largecircle.", "" and 
".increment.". 
EXPERIMENT 3-8 
Thermal stability 
The thermal stability of the present L-ribose isomerase was studied in 
accordance with the method for assaying the present L-ribose isomerase 
activity. As is shown in FIG. 3, the L-ribose isomerase was stable up to a 
temperature of about 30 C when incubated at pH 9.0 for 10 min. 
EXPERIMENT 3-9 
pH Stability 
The pH stability of the present L-ribose isomerase was studied in 
accordance with the method for assaying the L-ribose isomerase. As is 
shown in FIG. 4, the isomerase was stable at a pH of about 7-9 when 
incubated at 4.degree. C. for 24 hours. FIG. 4 shows the results of assay 
using citrate buffer, phosphate buffer, Tris-HCl buffer, and 
glycine-sodium hydroxide buffer which are respectively indicated by the 
symbols ".largecircle.", "", ".increment." and ".tangle-solidup.". 
Based on these data, the optimum pH and pH stability of the present 
L-ribose isomerase are substantially equal, meaning that the isomerase has 
an advantageous feature when used in an industrial-scale production. 
EXPERIMENT 3-10 
N-Terminal amino acid sequence 
A purified enzyme preparation, obtained by the method in Experiment 2-3, 
was dialyzed against distilled water, and an about 80 .mu.g of the enzyme 
with respect to a protein content was used as a sample for analyzing the 
N-terminal amino acid sequence. "PROTEIN SEQUENCER MODEL 473A", a protein 
sequencer commercialized by Applied Biosystems Inc., Foster City, USA, 
determined the amino acid sequence up to the fifth amino acid residue from 
the N-terminal to be the one in SEQ ID NO:1. More detail analysis of the 
above sample revealed that the enzyme contains the amino acid sequence of 
SEQ ID NO:2 as an N-terminal partial amino acid sequence. 
The followings are the preferred Examples according to the present 
invention: 
EXAMPLE 1 
Production of ketoses for aldoses 
Using a purified L-ribose isomerase obtained by the method in Experiment 2, 
ketoses were produced from aldoses. L-Ribose, D-lyxose, D-talose, 
D-mannose, L-allose and L-gulose were used as aldoses. While keeping 10 ml 
of 0.05M aldose solution at pH 9, 50 units of the purified L-ribose 
isomerase was added to the aldose solution, followed by the incubation at 
30.degree. C. for 10 hours. The reaction mixture was treated with 
activated charcoal and subjected to deionization and fractionation by 
column chromatography using a cation exchanger in Ca.sup.++ -form. The 
resulting fractions containing a desired product was concentrated in vacuo 
to obtain a purified product. Analyses on high-performance liquid 
chromatography and thin-layer chromatography revealed that the 
relationship between aldoses as materials and ketoses as products was 
substantially the same as that of Table 3. 
EXAMPLE 2 
Production of aldoses from ketoses 
Using a purified L-ribose isomerase obtained by the method in Experiment 2, 
aldoses were produced from ketoses. L-Ribulose, D-xylulose, D-tagatose, 
D-fructose, L-psicose and L-sorbose were used as ketoses. While keeping 10 
ml of 0.05M ketose solution at pH 9, 50 units of the purified L-ribose 
isomerase was added to the ketose solution, followed by the incubation at 
30.degree. C. for 10 hours. The reaction mixture was treated with 
activated charcoal and subjected to deionization and fractionation by 
column chromatography using a cation exchanger in Na.sup.+ -form. The 
resulting fractions containing a desired product was concentrated in vacuo 
to obtain a purified product. Analyses on high-performance liquid 
chromatography and thin-layer chromatography revealed that the 
relationship between aldoses as materials and ketoses as products was 
substantially the same as that of Table 3. 
EXAMPLE 3 
Production of L-ribose 
In accordance with the method in Example 2, 10 units of a purified L-ribose 
isomerase was added to 25 ml of 0.1M L-ribulose, followed by the 
incubation at 30.degree. C. for 15 hours to convert L-ribulose into 
L-ribose. The reaction mixture was in a conventional manner decolored with 
activated charcoal, desalted and purified with ion exchangers in H-- and 
CO.sub.3 -form, and fractionated by column chromatography using an anion 
exchanger in bisulfite-form heated to 40.degree. C. to obtain a purified 
L-ribose. The yield of the purified L-ribose to the material L-ribulose 
was about 60%, on a dry solid basis (d.s.b.). The product can be 
arbitrarily used in food products, cosmetics, pharmaceuticals, and their 
materials as a sweetener, quality-improving agent, humectant, and 
replication inhibitory agent for nucleic acids. 
EXAMPLE 4 
Production of D-talose 
In accordance with the method in Example 2, 10 units of a purified L-ribose 
isomerase was added to 50 ml of 0.1M D-tagatose, followed by the 
incubation at 30.degree. C. for 20 hours to convert D-tagatose into 
D-talose. The reaction mixture was in a conventional manner decolored with 
activated charcoal, desalted and purified with ion exchangers in H-- and 
CO.sub.3 -form, and fractionated by column chromatography using a cation 
exchanger in Ca.sup.++ -form, followed by concentrating the fractions 
containing a desired product to obtain a D-talose crystal. The yield of 
the obtained D-talose to the material D-tagatose was about 10%, d.s.b. The 
product can be arbitrarily used in food products, cosmetics, 
pharmaceuticals, and their materials as a sweetener, quality-improving 
agent and humectant. 
EXAMPLE 5 
Production of L-ribose from ribitol 
One hundred ml aliquots of a nutrient culture medium, consisting of 2 w/v % 
trypton soya broth, one w/v % glycerol and deionized water, were 
distributed to ten 500-ml shaking flasks, followed by autoclaving the 
flasks at 120.degree. C. for 20 min. Thereafter, the flasks were cooled 
and inoculated with a seed culture of Gluconobacter frateurii (IFO 3254) 
using a platinum loop, followed by incubation at 30.degree. C. for 2 days 
under shaking conditions. After completion of the culture, the cells were 
collected by centrifugation, and about 10 g wet alive cells was mixed with 
100 ml of 0.05M Tris-HCl buffer (pH 7.0) containing 5 w/v % ribitol. The 
mixture solution in a volume of 100 ml was placed in a 500-ml shaking 
flask, and incubated at 30.degree. C. for 20 hours under shaking 
conditions to convert ribitol into L-ribulose. Thereafter, the culture was 
centrifuged to remove cells, and the resulting supernatant was in a 
conventional manner decolored with activated charcoal, desalted with 
"DIAION SK1B (H-form)" and "DIAION WA30 (OH-form)", both of which are 
cation exchangers of Mitsubishi Chemical Corporation, Tokyo, Japan, and 
concentrated in vacuo to obtain a transparent syrup with a concentration 
of about 60 w/w %. The syrup was fractionated by column chromatography 
using "DOWEX 50W-X4", a cation exchanger in Ca.sup.++ -form commercialized 
by The Dow Chemical Co., Midland, Mich., USA, to obtain high L-ribulose 
content fractions which were then concentrated into an about 70 w/w % 
syrup. High-performance liquid chromatographic (HPLC) analysis using a 
column, 8.times.300 mm, packed with "MCIGEL CK-08EC", a gel in Ca.sup.++ 
-form commercialized by Mitsubishi Chemical Corporation, Tokyo, Japan, 
revealed that the product contained at least 97 w/w % L-ribulose, d.s.b. 
The yield of L-ribulose to the material ribitol was about 90%, d.s.b. 
Using the obtained L-ribulose, L-ribose was prepared as follows: According 
to the method in Experiment 1, about 50 g wet cells was obtained by 
centrifuging a culture of microorganisms and treated with toluene. The 
resulting cells were kneaded with 100 ml of 2.5 w/v sodium alginate. The 
slurry containing cells was dropped into 0.1M CaCl.sub.2 solution, which 
was being stirred by a magnetic stirrer, to form gels with a diameter of 
about 2 mm. The gels were filtered to obtain an immobilized enzyme with an 
L-ribose isomerase activity of about 5,000 units. An L-ribulose syrup 
obtained by the above method was diluted with water into an about 1.0M 
solution which was then mixed with about 50 units/g L-ribulose of the 
immobilized enzyme, and allowed to react at pH 8.5 and 10.degree. C. for 
15 hours to convert L-ribulose into L-ribose. The immobilized enzyme was 
collected by filtration, and the filtrate was similarly as in Example 3 
decolored, desalted, and fractionated by column chromatography to obtain 
high L-ribose content fractions while removing high L-ribulose content 
fractions. The high L-ribose content fractions were pooled, concentrated, 
crystallized by the addition of a seed crystal under stirring conditions, 
and separated to obtain high L-ribose content fractions while removing 
L-ribulose content fractions. The high L-ribose content fractions were 
pooled, concentrated, admixed with a seed crystal to crystallize L-ribose 
under stirring conditions, followed by separating the mixture to obtain a 
solid product containing L-ribose crystal. HPLC analysis as described in 
Example 5 revealed that the purity of the product was about 98 w/w %, and 
the yield of the L-ribose crystal to the material L-ribulose was about 
20%, d.s.b. The crystal can be arbitrarily used in foods, cosmetics, 
pharmaceuticals, and their materials as a sweetener, quality-improving 
agent, humectant and replication inhibitory agent for nucleic acids. The 
recovered immobilized enzyme can be repeatedly used in the present 
conversion reaction. Furthermore, the high L-ribulose content fractions, 
which were fractionated and removed from reaction mixtures, can be 
advantageously recycled as a material for L-ribose to increase the yield 
of L-ribose crystal. 
EXAMPLE 6 
Production of L-ribose from ribitol 
One hundred ml aliquots of a nutrient culture medium, consisting of 2 w/v % 
trypton soya broth, one w/v % glycerol and water, and distributed to two 
500-ml shaking flasks, followed by autoclaving the flasks at 120.degree. 
C. for 20 min. Thereafter, the flasks were cooled and inoculated with a 
seed of Acetobacter aceti (IFO 3281) using a platinum loop, followed by 
the incubation at 30.degree. C. for 2 days under shaking conditions to 
obtain a seed culture. 16.8 L of a nutrient culture medium, consisting of 
1.1 w/v % polypeptone, 0.2 w/v % "HINUTE SMP", a peptide solution of 
edible soy beans commercialized by Fuji Oil Co., Ltd., Tokyo, Japan, 1.68 
w/v % potassium dihydrogenphosphate, one w/v % glycerine and water, was 
placed in a 30-L jar fermenter, autoclaved at 120.degree. C. for 20 min, 
cooled to 30.degree. C., and adjusted to pH 7.2 by the addition of aqueous 
sodium hydroxide solution. To the nutrient culture medium was inoculated 
one v/v % of the above seed culture and incubated at 30.degree. C. for 22 
hours under aeration-agitation conditions, then mixed with 3.2 L aqueous 
ribitol solution containing 2 kg ribitol, which had been autoclaved at 
120.degree. C. for 20 min, and stirred for 27 hours under 
aeration-agitation conditions to convert ribitol into L-ribulose. The 
reaction mixture was membrane filtered to obtain a filtrate. HPLC analysis 
as described in Example 5 revealed that the filtrate contained at least 97 
w/w % L-ribulose, d.s.b. 
Using the obtained L-ribulose, L-ribose was prepared as follows: A seed of 
Acinetobacter calcoaceticus LR7C (FERM BP-5335) was inoculated into and 
incubated in a nutrient culture medium, containing D-lyxose as a carbon 
source, at 30.degree. C. for 4 days under shaking conditions. A portion of 
the culture was inoculated into and incubated in a fresh preparation of 
the same nutrient culture medium for 2 days, and this culturing step was 
repeated 5 times. After completion of these successive cultures, a portion 
of the final culture was incubated unto the surface of a nutrient agar 
plate containing D-lyxose as a carbon source and cultured to form 
homogenous colonies. One of the colonies as a seed was inoculated into a 
nutrient culture medium, consisting of 0.5 w/v % yeast extract, 0.5 w/v % 
polypeptone, 0.5 w/v % salt and water, which had been autoclaved at 
120.degree. C. for 20 min, and cultured at 30.degree. C. under shaking 
conditions to obtain a seed culture. Fifteen L of a fresh preparation of 
the same nutrient culture medium used in the above seed culture was placed 
in a 30-L jar fermenter, autoclaved at 120.degree. C. for 20 min, cooled 
to 30.degree. C., inoculated with one v/v % of the seed culture, and 
cultured at 30.degree. C. for 20 hours under aeration-agitation 
conditions. Thereafter, the culture was centrifuged to obtain wet cells, 
and about 50 g of which was treated with toluene, mixed with 50 mM glycine 
buffer (pH 9.0) in a volume of one ml per g wet cells to obtain a 110 ml 
enzyme solution containing 128 units/ml of L-ribose isomerase. The above 
filtrate containing L-ribulose obtained by the above method was adjusted 
to pH 9.0 by the addition of aqueous sodium hydroxide solution, mixed with 
manganese chloride to give a final concentration of 0.5 mM, mixed with 
about 5 units/g L-ribulose of the enzyme solution, and incubated at 
30.degree. C. for 24 hours to convert about 70% L-ribulose into L-ribose. 
The reaction mixture thus obtained was filtered with an ultrafilter, 
desalted and concentrated in vacuo to obtain 1.77 kg of an about 85 w/w % 
syrup. Ethanol was added to the syrup to crystallize L-ribose, and the 
crystal was separated, washed, dried in vacuo and pulverized to obtain an 
about 600 g high-purity L-ribose crystal. HPLC analysis as described in 
Example 5 confirmed that the purity of the L-ribose crystal was about 99.9 
w/w %, d.s.b. The yield of the L-ribose crystal to the material ribitol 
was about 30%. "GEIGERFLEX RAD-IIB", an x-ray diffraction analyzer using 
CuK.alpha. ray commercialized by Rigaku Corporation, Tokyo, Japan, 
revealed that the L-ribose crystal had diffraction angles (2.theta.) of 
16.3.degree., 20.1.degree., 21.3.degree., 21.4.degree. and 33.0.degree.. 
The L-ribose crystal can be arbitrarily used in food products, cosmetics, 
pharmaceuticals, and their materials as a sweetener, quality-improving 
agent, humectant or replication inhibitory agent for nucleic acids. 
As is evident from the above, the present L-ribose isomerase acts on 
L-ribose, D-lyxose, D-talose, D-mannose, L-allose and L-gulose to convert 
or isomerize them into their corresponding L-ribulose, D-xylulose, 
D-tagatose, D-fructose, L-psicose and L-sorbose, respectively. The 
enzymatic reaction is a reversible equilibrium reaction. Thus, the 
L-ribose isomerase can be used in the isomerization conversion reactions 
between the aldoses and ketoses. The L-ribose isomerase should not 
necessarily be allowed to act on its substrates after purified to a 
relatively-high level, and crude preparations of the L-ribose isomerase 
can be arbitrarily used to industrially produce rare saccharides. The 
present invention enables a readily production of rare saccharides which 
have not been readily available and will greatly influence on the fields 
of food, cosmetic, pharmaceutical and chemical industries. Therefore, the 
present invention has unfathomable industrial significance. 
While there has been described what is at present considered to be the 
preferred embodiments of the invention, it will be understood the various 
modifications may be made therein, and it is intended to cover in the 
appended claims all such modifications as fall within the true spirit and 
scope of the invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: N-terminal fragment 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ThrArgThrSerIle 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: N-terminal fragment 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ThrArgThrSerIleThrArgArgGluTyrAspGluTrpValArgGlu 
151015 
AlaAlaAlaLeuGlyLysAlaLeuArgTyr 
2025 
__________________________________________________________________________