.beta.-galactoside-.alpha.-2, 6-sialyltransferase, and a process for producing from Photobacterium

BACKGROUND OF THE INVENTION
Recently, the biological activities of glycoconjugates such as glycoproteins, glycolipids etc. in animal cells have been revealed and the importance of sugar chains in glycoconjugates comes to be recognized. Sialic acid is mentioned as a saccharide present mainly at the nonreducing terminals of sugar chains in glycoconjugates, and sialic acid is considered to have particularly many functions where the physiological functions and biological activities of sugar chains are regarded as important. For this reason, the importance of sialyltransferase for transferring sialic acid to sugar chains is also recognized well.
For linking sialic acid to an oligosaccharide, a sugar chain in a glycoconjugate, etc., e.g. for linking sialic acid via an .alpha.-2,6-linkage to the 6-position of a galactose residue at the nonreducing terminal in a sugar chain, chemical synthesis and enzymatical synthesis methods are known. However, the chemical synthesis method is complicated in its procedures, and side reactions also occur, so it is extremely difficult to efficiently synthesize a product having sialic acid linked selectively to the 6-position of its galactose.
On the other hand, the enzymatical synthesis method is extremely simple as compared with the chemical synthesis method and no side reactions occur, so it is possible to synthesize the desired product in high yield. Enzymes used therein are obtained from internal organs such as the submaxillary gland, liver etc. in animals such as rats, pigs, humans etc. (Poulson et al., J. Biol. Chem. 252, 2356-2362 (1977), Weinstein et al., J. Biol.. Chem.257, 13835-13844 (1982), Miyagi et al., Eur. J. Biochem. 126, 253-261 (1982)). However, it is disadvantageous that these enzymes are expensive because they cannot be obtained in large amounts due to their difficult purification, and the stability of these enzymes are poor.
Every sialyltransferase purified so far is derived from animal cells and consists essentially of glycoprotein. A certain animal-derived sialyltransferase has already been cloned, and a gene for this sialyltransferase can be used in hosts such as yeast, COS-3 cells, COS-7 cells etc. to produce sialyltransferase as an enzymatically active protein.
However, the specific activity of the enzyme produced by these hosts is extremely low compared with the original sialyltransferase present in animal tissues or cells. For this phenomenon, it is estimated that although the original and recombinant enzymes have the same primary structure in their protein moiety, the sugar chain linked to the protein moiety in the sialyltransferase produced by hosts (yeast, COS-3 cells, COS-7 cells etc.) is different from the sugar chain in the original sialyltransferase present in animal cells, so that the specific activity of the recombinant is lower than that of the original enzyme.
SUMMARY OF THE INVENTION
The object of the present invention is to produce novel .beta.-galactoside-, .alpha.-2,6-sialyltransferase inexpensively in a large amount.
As a result of their eager research, the present inventors found that a microorganism belonging to the genus Photobacterium produces an enzyme for transferring sialic acid to a galactose residue etc. in a sugar chain of a glycoconjugate or in a free sugar chain.
Specifically, the present invention is .beta.-galactoside-.alpha.-2,6-sialyltransferase derived from a microorganism belonging to the genus Photobacterium, having the following physicochemical properties:
(1) action and specificity: transferring sialic acid from cytidine monophosphate-sialic acid to the 6-position of a galactose residue in a sugar chain of a glycoconjugate or in a free sugar chain, or to the 6-position of a monosaccharide having a hydroxyl group on carbon at the 6-position and being capable of forming a glycoconjugate;
(2) optimum pH: 5 to 6;
(3) optimum temperature: 30.degree. C.; and
(4) molecular weight: 64,000.+-.5,000 (determined by gel filtration).
Further, the present invention is .beta.-galactoside-.alpha.-2,6-sialyltransferase having the following physicochemical properties:
(1) action and specificity: transferring sialic acid from cytidine monophosphate-sialic acid to the 6-position of a galactose residue in a sugar chain of a glycoconjugate or in a free sugar chain, or to the 6-position of a monosaccharide having a hydroxyl group on carbon at the 6-position and being capable of forming a glycoconjugate;
(2) optimum pH: 5;
(3) optimum temperature: 30.degree. C.;
(4) pH stability: 4.5 to 6;
(5) thermal stability: keeping about 90% of the initial activity after heating at 35.degree. C. for 5 minutes and about 70% of the initial activity after heating at 45.degree. C. for 5 minutes;
(6) molecular weight: about 61,000 (determined by 12.5% SDS-polyacrylamide gel electrophoresis) and about 64,000 (determined by gel filtration); and
(7) isoelectric point: 4.6.
Further, the present invention is a process for producing .beta.-galactoside-.alpha.-2,6-sialyltransferase, which comprises culturing a microorganism belonging to the genus Photobacterium capable of producing .beta.-galactoside-.alpha.-2,6-sialyltransferase in a medium, and then recovering the enzyme from the microorganism.
Further, the present invention is a process for producing .beta.-galactoside-.alpha.-2,6-sialyltransferase, wherein the microorganism belonging to the genus Photobacterium capable of producing .beta.-galactoside-.alpha.-2,6-sialyltransferase is at least one member selected from the group consisting of Photobacterium damsela JTO160, ATCC 33539 and ATCC 35083.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the pH stability of the .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention.
FIG. 2 is a graph showing the thermal stability of the .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention.
FIG. 3 is a graph showing a .sup.1 H-NMR spectrum of .alpha.-2,6-sialylmethyl-.beta.-D-N-acetyllactosaminide (taken at 303 K) synthesized by use of the .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention.
FIG. 4 is a graph showing a .sup.13 C-NMR spectrum of .alpha.-2,6-sialylmethyl-.beta.-D-N-acetyllactosaminide (taken at 303 K) synthesized by use of the .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention.
FIG. 5 is a graph showing a mass spectrum of .alpha.-2,6-sialylmethyl-.beta.-D-N-acetyllactosaminide synthesized by use of the .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention.
FIG. 6 is a graph showing the result of analysis of pyridylaminated lactase in high performance liquid chromatography (HPLC).
FIG. 7 is a graph showing the result of HPLC analysis of pyridylaminated .alpha.-2,6-sialyllactose synthesized by use of the .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention derived from ATCC 33539.
FIG. 8 is a graph showing the result of HPLC analysis of pyridylaminated .alpha.-2,6-sialylactose synthesized by use of the .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention derived from ATCC 35083.
FIG. 9 is a graph showing the relative activity of .beta.-galactoside-.alpha.-2,6-sialyltransferase toward each monosaccharide where the .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention derived from Photobacterium damsela JTO160 was used to transfer sialic acid to each monosaccharide.

DESCRIPTION OF THE PREFERRED EMBODIMENT
Hereinafter, the present invention is described in detail.
The present invention relates to novel .beta.-galactoside-.alpha.-2,6-sialyltransferase which transfers sialic acid from cytidine monophosphate-sialic acid (CMP-sialic acid) to the 6 position of a galactose residue in a sugar chain of a glycoconjugate or in a free sugar chain or to the 6 position of a monosaccharide capable of forming a glycoconjugate, as well as to a process for producing the same.
The .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention can be obtained by culturing a microorganism belonging to the genus Photobacterium capable of producing .beta.-galactoside-.alpha.-2,6-sialyltransferase in a medium, and then recovering the resulting enzyme from the microorganism.
The microorganism used herein may be any microorganism belonging to the genus Photobacterium and having the ability to produce .beta.-galactoside-.alpha.-2,6-sialyltransferase. Preferable microorganisms are those belonging to the species Photobacterium damsela. Examples are Photobacterium damsela JTO160, ATCC 33539, and ATCC 35083. Every Photobacterium damsela is marine microorganisms, and Photobacterium damsela JTO160 was separated from seawater in Sagami Bay, Japan. By way of example, a method of screening Photobacterium damsela JTO160 is shown below.
As a source of the microorganism, seawater, sea sand or sea sludge is plated directly or after dilution with sterilized seawater onto a plate medium, Marine Broth 2216-agar medium (1.5% agar, manufactured by Difco laboratories) where the marine microorganisms are then grown. The microorganisms are subjected to single colony isolation in a usual manner and then each microorganism is cultured in a liquid medium, Marine Broth 2216 medium (Difco laboratories). After the microorganisms are grown sufficiently, they are harvested by centrifugation and then suspended in 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 (Kanto Kagaku K.K.) as surfactant. This suspension is treated with ultrasonication under cooling on ice whereby by the bacterial cells are disrupted. The solution containing the disrupted cells is examined in a usual manner for sialyltransferase activity, and a strain with sialyltransferase activity is obtained as the microorganism of the invention.
The bacteriological properties of Photobacterium damsela JTO 160 thus obtained are as follows:
______________________________________1) Morphological features(1) Form Bacillus, 1 .times. 0.5 .mu.m to 2 .times. 1 .mu.m in size(2) Motility present with flagella(3) Gram stainability negative(4) Spore formation absent2) Physiological properties(1) growth temperature 25 to 35.degree. C.(2) colony color not producing any characteristic colony color(3) O-F test F(4) catalase test positive(5) oxidase test positive(6) formation of gas from glucose positive(7) V-P reaction positive(8) gelatin degradability absent(9) starch degradability absent(10) nitrate reducibility present(11) fluorescence absent(12) attitude toward oxygen anaerobic(13) accumulation of glucose present(14) accumulation of .beta.-hydroxybutyric absent acid(15) utilization of .beta.-hydroxybutyric present acid(16) requirement for Na.sup.+ present(17) sensitivity to a pteridine derivative 10 .mu.g present 150 .mu.g present(18) quinone system Q-8, Q-7(19) GC content in bacterial DNA 42 (mol %)*(20) lipase activity present(21) argininedihyrase activity present(22) assimilability acetate absent maltose present L-proline present pyruvate absent D-xylose absent cellobiose absent D-galactose present D-galacturonic acid absent D-gluconate absent glucose present L-glutamate absent D-mannose present sucrose absent mannitol absent______________________________________ Note) *: Determined in HPLC.
From the above bacteriological properties, it was estimated that this microorganism is Photobacterium damsela, and finally this microorganism was identified as Photobacterium damsela due to its high homology (89%) in a DNA-DNA hybridization test with Photobacterium damsela GIFU 10450 (standard strain). This DNA--DNA hybridization test was carried out using a photobiotin label in a microplate.
Photobacterium damsela JTO160 was deposited as FERM BP-4900 under the conditions of the Budapest Treaty on Nov. 24, 1994 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan. Photobacterium damsela ATCC 33539 deposited as FERM BP-6135 and ATCC 35083 deposited as FERM BP-6136 both have been deposited with American Type Culture Collection (ATCC) and under the conditions of the Budapest Treaty and the bacteriological properties of ATCC 33539 are described in Int. J. Syst. Bact.32, 267 (1982) and by MacDonell et al. in Syst. Appl. Microbiol. 6, 171-182 (1985), and the bacteriological properties of ATCC 35083 are described by Grimes et al. in Microb. Ecol.10, 271-282 (1984).
The medium used for culture of the above microorganism is a medium containing a carbon source, a nitrogen source, inorganic matter etc. which can be utilized by the microorganism. The carbon source includes peptone, triptone, casein lysate, meat extract etc., preferably peptone. The nitrogen source is preferably yeast extract. The salt used is preferably a suitable combination of sodium chloride, iron citrate, magnesium chloride, sodium sulfate, calcium chloride, potassium chloride, sodium carbonate, sodium bicarbonate, potassium bromide, strontium chloride, sodium borate, sodium silicate, sodium fluoride, ammonium nitrate, hydrogen disodium phosphate, etc.
Marine Broth 2216 (Difco), that is, a medium containing all of the above ingredients, can also be used. Further, a medium prepared by adding peptone, yeast extract etc. to artificial seawater containing suitable salts may also be used.
The culture conditions are varied more or less depending on the composition of the medium. Usually, the culture temperature ranges from 15.degree. to 35.degree. C., preferably 20.degree. to 33.degree. C., the pH ranges from pH 6.8 to 8.8, preferably pH 7.3 to 8.2, and the culture period ranges from 8 to 48 hours, preferably 16 to 24 hours.
Before separation and isolation of the desired enzyme, the microorganism should be disrupted in a usual manner, for example by ultrasonication, French press, glass beads, dynomill, because the desired enzyme is present in cells. Ultrasonication is used in a preferred embodiment. After insolubles are removed from the disrupted microorganism, the enzyme can be purified completely from the supernatant, for example by column chromatography with a suitable combination of commercial cation and anion exchange columns, gel filtration column, hydroxyapatite column, CDP-hexanolamine agarose column, CMP-hexanolamine agarose column and hydrophobic column, as well as native-PAGE, etc.
.beta.-galactoside-.alpha.-2,6-sialyltransferase may be purified completely, but because partially purified .beta.-galactoside-.alpha.-2,6-sialyltransferase has enough activity, the .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention includes both the purified enzyme and the partially purified enzyme.
The .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention is characterized in that the enzyme is derived from microorganisms belonging to the genus Photobacterium, particularly microorganisms belonging to the species Photobacterium damsela and the action and substrate specificity of the enzyme are to transfer sialic acid from CMP-sialic acid to the 6-position of a galactose residue in a sugar chain of a glycoconjugate or in a free sugar chain, or to the 6-position of a monosaccharide having a hydroxyl group on carbon at the 6-position and being capable of forming a glycoconjugate. The optimum pH of the present enzyme is in the range of pH 5 to 6, the optimum temperature is 30.degree. C., and the molecular weight by gel filtration is 64,000.+-.5,000. The glycoconjugate, and the free sugar chain, carrying such a galactose residue may be any saccharides composed of galactose residues, regardless of whether the glycoconjugate and the free sugar chain are derived from animals or plants. Examples are disaccharides, free sugar chains (derived from glycoconjugates), glycoproteins, and glycolipids. The disaccharides include lactose, N-acetyllactosamine etc.; the free sugar chains from glycoconjugates (e.g. free sugar chains composed of galactose residues, derived from glycoconjugates) include an N-acetyllactosamine type of biantennary sugar chains etc. and N-acetyllactosamine type monosialylated of oligosaccharides etc.; the glycoproteins such as asialofetuin, asialo-.alpha..sub.1 -acid glycoproteins etc.; and the glycolipids include lactosylceramide and ganglioside saccharides. The monosaccharide having a hydroxyl group on carbon at the 6-position and being capable of forming a glycoconjugate may be any monosaccharide having a pyranose-type ring and containing a primary hydroxyl group at the nonreducing terminal. Examples are monosaccharides such as glucose, galactose, mannose, allose, talose etc. having a primary hydroxyl group at its reducing terminal, monosaccharides such as 3-O-methylglucopyranose, 3-O-methylgalactopyranose etc. having a substituent group at its alcoholic OH group, and monosaccharides such as methylgalactopyranoside, ethyl-glucopyranoside etc. having a substituent group at its glycosidic hydroxy group.
The enzyme of the present invention includes the enzyme derived from Photobacterium damsela JTO160, Photobacterium damsela ATCC 33539 and Photobacterium damsela ATCC 35083. By way of example, the enzyme derived from Photobacterium damsela JTO160 has the following enzyme properties and physicochemical properties:
(1) Action and substrate specificity
The enzyme transfers sialic acid from CMP-sialic acid to the 6 position of a galactose residue in a sugar chain of a glycoconjugate or in a free sugar chain, particularly the 6-position of galactose occurring in its nonreducing terminal, or to the 6 position of galactose occurring in an oligosaccharide such as lactose, N-acetyllactosamine etc., particularly the 6-position of galactose occurring in its nonreducing terminal, or to the 6-position of a monosaccharide having a hydroxyl group on carbon at the 6-position and being capable of forming a glycoconjugate such as galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine etc.
(2) Thermal stability
10 .mu.l solution of the enzyme was heated for 5 minutes in a water bath at 30.degree. C., 35.degree. C., 40.degree. C., 45.degree. C., 50.degree. C., 55.degree. C. and 60.degree. C., respectively, then its enzyme activity was examined, and its remaining activity was determined by comparison with the activity (as 100%) of a solution of the enzyme not heated. The result indicated that the enzyme had 100% of the initial activity at 30.degree. C., about 90% at 35.degree. C., and about 70% at 45.degree. C.
(3) pH stability
The enzyme was dissolved in acetate buffer (pH 3.7 to 5.0), cacodylate buffer (pH 5.0 to 7.3) and Tris-HCl buffer (pH 7.3 to 8.0), respectively, and 10 .beta.I of each enzyme solution was determined for enzyme activity. The result indicated that the enzyme was stable in the range of pH 4.5 to 6.
(4) Optimum temperature
The optimum temperature is in the range of 30.degree. C. to 35.degree. C. in which the enzyme is inactivated only at a minimum level and the enzyme reaction proceeds rapidly.
(5) Optimum pH
Acceptor substrate was dissolved in acetate buffer (pH 3.7 to 5.0), cacodylate buffer (pH 5.0 to 7.3) and Tris-HCl buffer (pH 7.3 to 8.0), respectively, and the enzyme was added thereto and examined for enzyme activity. The result indicated that the optimum pH of the enzyme was pH 5.
(6) Molecular weight
The molecular weight of the enzyme was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. In SDS-PAGE, the enzyme and a molecular marker (phosphorylase b with a molecular weight of 97,400; bovine serum albumin, 66,267; aldolase, 42,400; carbonic anhydrase, 30,000; trypsin inhibitor, 21,100; and lysozyme, 14,400) were electrophoresed in a usual manner on 12.5% polyacrylamide gel containing SDS, and the molecular weight was determined from its mobility. The result indicated that the molecular weight of the enzyme was about 61,000 (SDS-PAGE).
In gel filtration, a calibration curve was prepared by passing a molecular weight marker (aldolase with a molecular weight of 158,000; albumin, 68,000 and 45,000) through a gel filtration column AsahiPak GS-510 (Asahi Kagaku K.K.) in 20 mM phosphate buffer (pH 6.0) containing 0.2M NaCl. Then, the enzyme was passed through the column, and the molecular weight was determined on the basis of its elution position in the calibration curve. The result indicated that the molecular weight of the enzyme was about 64,000 (gel filtration).
(7) N-terminal amino acid sequence
The N-terminal amino acid sequence of the enzyme was determined by the Edman degradation method. 20 .mu.l solution of the enzyme (0.3 .mu.g/.mu.l) was subjected to SDS-PAGE (7.5% gel) in a usual manner, and the enzyme was transferred by Western blotting onto Biodaine A (Japan Paul K.K.) and its N-terminal amino acid sequence (12 amino acids from the N-terminal) was determined in Amino Acid Sequence Analyzer 477A and 120A Protein Sequencer (Applied Biosystems) The result indicated that the N-terminal amino acid sequence of the enzyme was X-Asn-Ser-Asp-Asn-Thr-Ser-Leu-Lys-Glu-Thr-Val (SEQ ID NO:1).
The above-described .beta.-galactoside-.alpha.-2,6-sialyltransferase of the invention can be used to transfer sialic acid to the 6-position of a monosaccharide or the 6-position of galactose occurring in oligosaccharides, glycoprotein sugar chains, etc. More specifically, monosaccharides capable of forming glycoconjugates galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine etc., oligosacchrides such as galactose-containing lactose, N-acetyllactosamine etc., glycoproteins such as asialofetuin, asialo-.alpha..sub.1 -acid glycoproteins etc., and glycolipids such as gangliosides etc., are used as acceptor substrates respectively, together with CMP-sialic acid i.e. a saccharide-donating substrate, and their corresponding sialo-derivatives can be produced by the action of the present enzyme.
The reaction conditions are not limited insofar as the enzyme is not inactivated. Specifically, the reaction temperature ranges from 15.degree. to 50.degree. C., preferably 25.degree. to 35.degree. C., and the reaction pH ranges from pH 4 to 7.5, preferably pH 5 to 6. Any buffer in this pH range can be used. The resulting sialo-derivatives (oligosaccharides, glycoproteins, glycolipids etc.) can be separated and purified by column chromatography etc.
EXAMPLES
The present invention is described in more detail by reference to the following examples, which however are not intended to limit the scope of the present invention.
Example 1
Production of .beta.-galactoside-.alpha.-2,6-sialyltransferase derived from Photobacterium damsela JTO160
A liquid medium Marine Broth 2216 was prepared in a usual manner, pipetted into test tubes (10 ml/tube) and sterilized in an autoclave. Photobacterium damsela JTO160 was harvested from colonies previously subcultured in Marine Broth 2216-agar plate (1.5% agar) and was then inoculated into 10 ml of Marine Broth 2216 liquid medium prepared above. The microorganism was pre-cultured at 30.degree. C. for 8 hours with stirring at 150 r.p.m.
The culture thus obtained was further cultured in the following manner. 500 ml Marine Broth 2216 prepared in a usual manner was introduced into 3000 ml flask and sterilized in an autoclave. 5 ml of the above culture was inoculated into the medium and cultured at 30.degree. C. with stirring at 150 r.p.m. for 16 hours. The resulting microorganism was recovered by centrifugation to give a pellet (wet weight: about 2 g).
The microorganism was suspended in 40 ml of 20 mM cacodylate buffer (pH 6.0) containing 1M NaCl and 0.2% Triton X-100 (surfactant available from Kanto Kagaku K.K.) and disrupted by ultrasonication under cooling on ice. The disruption was continued until the suspension showed 30% or less of the original absorbance. After the disruption, the bacterial solution was centrifuged at 100,500 g at 4.degree. C. for 1 hour to give a supernatant. The supernatant was introduced into a cellulose tube and dialyzed overnight at 4.degree. C. against 5000 ml of 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant, during which the buffer was exchanged 3 times with fresh one. After the dialysis, the precipitates occurring in the sample were removed by centrifugation at 100,500 g for 1 hour at 4.degree. C. . A crude enzyme solution was thus prepared from the solution of the disrupted microorganism.
The crude enzyme solution was adsorbed onto a strong anion exchange column Q-Sepharose HR 26/10 (Pharmacia) previously equilibrated with 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant. The enzyme was then eluted with a linear gradient of from 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant to the same buffer containing 1M NaCl (total elution volume: 1060 ml), and fractions with enzyme activity eluted with 0.25 to 0.35M NaCl were recovered and combined.
This enzyme solution was dialyzed overnight at 4.degree. C. against 5000 ml of 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant, during which the buffer was exchanged 3 times with fresh one. The dialyzate was adsorbed onto hydroxyapatite (Koken K.K.) previously equilibrated with 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant. The sample was eluted with a linear gradient of from 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant to 0.35M phosphate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant (total elution volume: 620 ml). Fractions with enzyme activity eluted with 0.08M to 0.16M phosphate buffer were recovered and combined. This enzyme solution was dialyzed overnight against at 4.degree. C. against 5000 ml of 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-200 as surfactant, during which the buffer was exchanged 3 times with fresh one.
The dialyzate was applied to Sephacryl S-200 (Pharmacia) i.e. a gel filtration column (2.6.times.60 cm) and passed through the column with 20 mM cacodylate buffer (pH 6.0) containing 0.1M NaCl and 0.2% Triton X-100 as surfactant, and fractions with enzyme activity, i.e. between 100 ml and 140 ml in elution volume, were recovered and combined. This enzyme solution was dialyzed at 4.degree. C. against 5000 ml of 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant.
The dialyzate was adsorbed onto a CDP-hexanolamine agarose column (0.7.times.1.2 cm) previously equilibrated with 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant, and the column was washed with 4 ml of the same buffer, and the sample was eluted with 8 ml of 2M NaCl. This fraction was dialyzed against 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 as surfactant. The SDS-PAGE of this fraction indicated one single band indicating a molecular weight of about 61,000. The specific activity of this fraction was 643-fold higher than that of the solution of the disrupted microorganism. The total activity was 257 U, and 19% of the original activity was recovered.
The activity of the present enzyme was determined by mixing a donor substrate (CMP-sialic acid) and an acceptor substrate (asialo-glycoprotein) with an enzyme solution and quantifying the sialic acid transferred during the enzyme reaction to the saccharide-accepting substrate. The detailed procedures for this measurement are as follows:
Asialofetuin was dissolved as the saccharide-accepting substrate at a concentration of 6.25 mg/ml in 20 mM cacodylate buffer (pH 6.0), and 80 .mu.l of the solution was used as the saccharide-accepting substrate solution. 10 .mu.l solution of CMP-sialic acid (6.7 nmol) where sialic acid was labeled with .sup.14 C (6,700 cpm/nmol) was added to the saccharide-accepting substrate solution, and the mixture was kept at 30.degree. C. 10 g .mu.l enzyme solution was added to it and reacted for 5 minutes at 30.degree. C. Then, the reaction solution was applied to a column (inner diameter, 10 mm; length, 160 mm) packed with Sephadex G-50 (Pharmacia) previously equilibrated with 0.1M NaCl. The eluent used was 0.1M NaCl. A void-volume fraction containing the saccharide-accepting substrate with sialic acid transferred to it by the enzyme reaction was collected and examined for radioactivity. The transferred sialic acid thus determined was used to determine enzyme activity where it was assumed that 1 U of the enzyme is the amount of the enzyme causing transfer of 1 nmol sialic acid per minute.
Example 2
Production of sialylmethyl-.beta.-D-N-acetyllactosamine
Sialylmethyl-.beta.-D-N-acetyllactosamine was produced using methyl-.beta.-D-N-acetyllactosamine as the acceptor substrate. 79.4 mg (200 .mu.mol) of methyl-.beta.-D-N-acetyllactosamine, 9.4 mg (16 .mu.mol) of CMP-sialic acid, and 270 U of the .beta.-galactoside-.alpha.-2,6-sialyltransferase obtained in Example 1 were dissolved in 1 ml of 20 mM cacodylate buffer (pH 6.0) and reacted at 30.degree. C. for 1 hour. The progress of the reaction was confirmed on a 1/10 scale under the same conditions by determining sialic acid transferred from CMP-sialic acid labeled with .sup.14 C as the donor substrate. After the reaction was finished, the reaction solution was adjusted to 10 ml with 1 ml distilled water. This solution was applied to a Dowex1-.times.2 (phosphate form) column (1.5.times.9 cm, 15.9 ml resin, manufactured by Muromachi Kagaku K.K.) previously equilibrated with distilled water.
The column was washed with 90 ml distilled water and the sample was then eluted with 60 ml of 5 mM sodium phosphate, pH 6.8 and collected (5 ml eluate/fraction). Each fraction was determined for glycoside-linked sialic acid by a periodate-resorcinol method. Fractions containing glycoside-linked sialic acid, eluted between 20 ml and 50 ml in elution volume, were collected and concentrated in a rotary evaporator. This concentrate of the reaction product was applied to an activated-carbon column (1.5.times.6.8 cm, 12 ml activated carbon, manufactured by Wako Pure Chemical Industries, Ltd.). Then, the column was washed with a 3-fold volume of distilled water relative to the column volume, and the sample was eluted with a 2-fold volume each of 10%, 20%, 50% and 100% ethanol relative to the column volume. Each fraction was determined for glycoside-linked sialic acid by the periodate-resorcinol method. Fractions containing glycoside-linked sialic acid, eluted with 10%, 20% and 50% ethanol, were combined and concentrated in a rotary evaporator.
The reaction product thus obtained was analyzed by .sup.1 H-NMR and .sup.13 C-NMR. The results are shown in FIGS. 3 and 4 respectively. From FIGS. 3 and 4, it became evident that the reaction product occurring in the enzyme reaction is .alpha.-2,6-sialylmethyl-.beta.-D-N-acetyllactosamine.
The reaction product was further analyzed by mass spectrometry. Its mass spectrum is shown in FIG. 5. The molecular weight of sialylmethyl-.beta.-D-N-acetyllactosamine is 688, and its corresponding peak (molecular weight: 689 (M+H.sup.+)) could be seen in the mass spectrum in FIG. 5.
From a control experiment using the donor substrate CMP-sialic acid where sialic acid was labeled with .sup.14 C, it was found that 16 .mu.mol of .alpha.-2,6-sialylmethyl-.beta.-D-N-acetyllactosamine could be produced under the present conditions.
Example 3
(1) Sialylation of pyridylaminated sugar chains
The enzyme reaction was carried out using the enzyme obtained in Example 1 and pyridylaminated sugar chains as the saccharide-accepting substrate. The pyridylaminated sugar chains used were N-acetyllactosamine type : biantennary sugar chain (Gal .beta.1-4GlcNAc .beta.1-2Man.alpha.1-6 (Gal.beta.1-4GlcNAc.beta.1-2Man.alpha.1-3) Man.beta.1-4GlcNAc.beta.1-4GlcNAc-PA, produced by Takara Shuzo Co., Ltd.) and 2 kinds of N-acetyllactosamine type monosialylated : biantennary sugar chain (NeuAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-2Man.alpha.1-6 (Gal.beta.1-4GlcNAc.beta.1-2Man.alpha.1-3) Man.beta.1-4GlcNAc.beta.1-4GlcNAc-PA, Gal.beta.1-4GlcNAc.beta.1-2Man.alpha.1-6 (NeuAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-2Man.alpha.1-3) Man.beta.1-4GlcNAc.beta.1-4GlcNAc-PA, produced by Takara Shuzo Co., Ltd.).
For any acceptor substrate, 2.0 .mu.M acceptor substrate, 5.7 .mu.M CMP-sialic acid, and 1 U enzyme were dissolved in 25 .mu.l of 20 mM cacodylate buffer (pH 6.0) and the mixture was reacted at 30.degree. C. for 20 hours. After the reaction was finished, the reaction solution was heated at 100.degree. C. for 2 minutes so that the enzyme was inactivated. Then, the reaction product was analyzed in HPLC.
Shimazu LC-10 (Shimazu) was used as HPLC system in which Takara PALAK Type R (Takara Shuzo Co., Ltd.) was used as the column. 10 .mu.l of the reaction solution containing the inactivated enzyme was injected into the column previously equilibrated with 100 mM acetate-triethylamine (pH 5.0) containing 0.15% N-butanol. Using eluent A (100 mM acetate-triethylamine, pH 5.0) and eluent B (100 mM acetate-triethylamine, pH 5.0, containing 0.5% n-butanol), the pyridylaminated sugar chain was eluted in a linear gradient (0 to 35 minutes) of from 30% to 100% eluent B and then with 100% eluent B (35 to 50 minutes) at a flow rate of 1 ml/min. at a column temperature of 40.degree. C. while monitoring the pyridylaminated sugar chain with fluorescence (Ex: 320 nm, Em: 400 nm).
As a result, there was detected a peak having the same retention time as in the elution position of N-acetyllactosamine type disialylated : biantennary sugar chain (NeuAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-2Man.alpha.1-6 (NeuAc.alpha.2-6Gal.beta.1-4GlcNAc.beta.1-2Man.alpha.1-3) Man.beta.1-4GlcNAc.beta.1-4GlcNAc-PA). It was thereby made evident that the present enzyme could be used to produce N-acetyllactosamine type disialylated:biantennary sugar chain from any of N-acetyllactosamine type:biantennary sugar chain and 2 kinds of N-acetyllactosamine type monosialylated:biantennary sugar chain.
Example 4
Production of .beta.-galactoside-.alpha.-2,6-sialyltransferase derived from Photobacterium damsela ATCC 33539 and ATCC 35083
ATCC 33539 and ATCC 35083 were cultured respectively in a liquid medium Marine Broth 2216 (Disco). After the microorganisms were sufficiently grown, they were harvested from the cultures by centrifugation. The microorganisms were suspended in 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100 (Kanto Kagaku K.K.) as surfactant. The cells were disrupted by ultrasonication of the suspension under cooling on ice to give crude enzyme solutions.
Example 5
(2) Sialylation of a pyridylaminated sugar chain
The enzyme reaction was carried out using each of the enzymes obtained in Example 4 and a pyridylaminated sugar chain as the saccharide-accepting substrate. Pyridylaminated lactose (Gal.beta.1-4Glc-PA, produced by Takara Shuzo Co., Ltd.) was used as the pyridylaminated sugar chain and analyzed in the same manner as in Example 3.
FIG. 6 shows the result of HPLC analysis of the acceptor substrate; FIG. 7 shows the result of HPLC analysis of the reaction product using the enzyme derived from ATCC 33539; and FIG. 8 shows the result of HPLC analysis of the reaction product using the enzyme derived from ATCC 35083.
In FIG. 7, the peak with a retention time of 5,500 indicates pyridylaminated a 2,6-sialyllactose, and in FIG. 8, the peak with a retention time of 5,499 indicates pyridylaminated a 2,6-sialyllactose. It was thereby made evident that the present enzyme could be used to produce pyridylaminateda 2,6-sialyllactose from pyridylaminated lactose.
Example 6
Sialylation of monosaccharides
The enzyme reaction was carried using the enzyme obtained in Example 1 and CMP-sialic acid as the donor substrate. In this reaction, methyl-.alpha.-D-galactose, methyl-.beta.-D-galactose, methyl-.alpha.-D-mannose, methyl-.beta.-D-mannose, methyl-.beta.-D-N-acetylglucosamine, and methyl-.alpha.-D-N-acetylglucosamine were used respectively as acceptor substrates. Specifically, 80 mM monosaccharide, 67 .mu.M CMP-sialic acid labeled with a radioisotope (6700 cpm/nmol) and 1 U enzyme were dissolved in 50 .mu.l of 20 mM cacodylate buffer (pH 6.0) and reacted at 30.degree. C. for 30 minutes.
After the reaction was terminated, the reaction solution was applied to a Dowex 1.times.2 (phosphate form) column (0.7.times.2 cm, 0.8 ml resin, manufactured Muromachi Kagaku K.K.) equilibrated with distilled water, and the reaction product was eluted with 5 mM phosphate buffer (pH 6.8). The determined radioactivity of the reaction product indicated that sialic acid was transferred to any of the monosaccharides. The activity of the enzyme towards each acceptor substrate was determined and expressed in percentage where the activity towards methyl- a -D-galactose was shown to be 100%. The result is shown in FIG. 9.
Example 7
Sialylation of asialofetuin and asialo-.alpha..sub.1 -acidic glycoprotein
Asialofetuin was dissolved as the acceptor substrate at a concentration of 6.25 mg/ml in 20 mM cacodylate buffer (pH 6.0), and 80 .mu.l of the solution was used as the saccharide-accepting substrate solution. 10 .mu.l 1 solution of CMP-sialic acid (6.7 nmol) where sialic acid was labeled with .sup.14 C (6,700 cpm/nmol) was added to the acceptor substrate solution, and the mixture was kept at 30.degree. C. 10 .beta.l of the crude enzyme solution obtained in Example 1 was added to it and reacted for 5 minutes at 30.degree. C. . Then, the reaction solution was applied to a column (inner diameter, 10 mm; length, 160 mm) packed with Sephadex G-50 (Pharmacia) previously equilibrated with 0.1M NaCl. The eluent used was 0.1M NaCl. A high-molecular, void-volume fraction containing asialofetuin with sialic acid transferred to it by the enzyme reaction was collected and examined for radioactivity. The result indicated that the radioactivity was 3125 cpm on average.
As the control, a solution was prepared by adding 10 .mu.l of 20 mM cacodylate buffer in place of the enzyme solution to a mixture of 80 .mu.l saccharide-accepting substrate solution and 10 .mu.l of CMP-(.sup.14 C-labeled) sialic acid (6.7 nmol, 6,7000 cpm/nmol). This control solution was incubated at 30.degree. C. for 5 minutes and then applied to the same gel filtration column as above, and the radioactivity of the void-volume fraction was determined in the same manner as above. The result indicated that the radioactivity was 30 cpm on average.
This radioactivity of the void-volume fraction as the control was subtracted from the above radioactivity of the void-volume fraction from the sample subjected to the enzyme reaction, whereby the sialic acid transferred by the enzyme was determined. The result indicated that 3 nmol sialic acid was transferred to the asialofetuin under the above conditions.
Separately, asialo-.alpha..sub.1 -acid glycoprotein was used as the acceptor substrate and the same enzyme reaction as above was carried out. The result indicated that the radioactivity of the void-volume fraction was 2985 cpm on average, while the radioactivity of the void-volume fraction from the control was 35 cpm on average. This result indicated that 2.9 nmol sialic acid was transferred to the asialo-.alpha..sub.1 -acid glycoprotein.
Effect of the Invention
According to the present invention, novel .beta.-galactoside-.alpha.-2,6-sialyltrantransferase derived from microorganisms can be provided inexpensively in a large amount.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 1(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: Not Relevant(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(v) FRAGMENT TYPE: N-terminal(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:XaaAsnSerAspAsnThrSerLeuLysGluThrVal1510__________________________________________________________________________

Number	Date	Country
8154673	Jun 1996	JPX
8173182	Jul 1996	JPX

.beta.-galactoside-.alpha.-2, 6-sialyltransferase, and a process for producing from Photobacterium

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