Abstract:
This invention provides endo-α-N-acetylgalactosaminidase (endo-α-GalNAcase) from a microorganism belonging to the genus Alcarigenes. This endo-α-GalNAcase is very useful in the analysis of the structure and function of mucin-type sugar chains of glycoproteins, as it is an enzyme that cleaves O-glycosidic linkages of sugar chains of glycoproteins, releasing the sugar chain from said protein.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a method of producing endo-α-N-acetylgalactosaminidase from microorganisms. 
     2. Description of the Prior Art 
     In recent years, it has been found the important functions of the sugar-chain portions of complex carbohydrates for cell differentiation, cell growth, cell recognition, and the onset of many diseases that involve malignant tumors, in living organisms. 
     For complex carbohydrates such as glycoproteins the sugar chains of the glycoproteins or the like are bound to the peptide chain of the glycoproteins either via N-glycosidic linkages or O-glycosidic linkages. Of these two kinds of sugar chains, the sugar chains with 0-glycosidic linkages mainly exist in blood group substances and in glycoproteins involved in immunity. It has been found that such sugar chains with 0-glycosidic linkages have a variety of important physiological functions. In order to further identify these functions, the structural investigation of the sugar chains is indispensable. For this kind of analysis of the structure of the sugar chains, the use of various glycosidases that have high substrate specificity for the sugar chain structure in complex carbohydrates provides an important means. 
     Of such glycosidases, endo-α-N-acetylgalactosaminidase) (endo-α-GalNAcase) can be used as an enzyme that acts on sugar chains with a Gal β1→ 3GalNAc structure in which the GalNAc is bound the serine or threonine residues of proteins, to cleave the O-glycosidic linkages; thus, the enzyme releases the sugar chains from the proteins. This action of the enzyme is shown in the following formula: ##STR1## wherein Gal is galactose, GalNAc is N-acetylgalactosamine, Ser is serine, Thr is threonine, and X and Y are peptide chains. In this way, because endo-α-GalNAcase can release sugar chains with O-gylcosidic linkages from proteins, this enzyme is important for the structural analysis of the sugar chains of glycoproteins. 
     Previously, the activity of endo-α-GalNAcase has been found in the culture medium of Clostridium perfringens or Diplococcus pneumoniae only. Also, the purification and the characterization of this enzyme are not yet satisfactory, which decreases its practical value. 
     SUMMARY OF THE INVENTION 
     The method of producing endo-αN-acetylgalactosaminidase of this invention, which overcomes the above-discussed and numerous other disadvantages and deficiencies of the prior art, comprises the steps of growing a microorganism belonging to the genus Alcalicenes that produces enddo-αN-acetylgalactosaminidase, and isolating endo-α-N-acetylgalactosaminidase from the cultures of said microorganism. 
     In a preferred embodiment, the microorganism belonging to the genus Alcaligenes mentioned above is Alcaligenes sp. F-1906 (FERM BP-1857). 
     Accordingly, the invention described herein makes possible the objectives of (1) providing an endo-α-GalNAcase that is very useful in the structural and functional analysis of mucin-type sugar chains of glYcoproteins; and (2) providing a simple method for the production of endo-α-GalNAcase inexpensively on a large scale. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The inventors of this inventions searched widely in nature for microorganisms capable of producing endo-α-GalNAcase that acts on sugar chains with O-glycosidic linkages. As a result, this microorganism isolated from soil, that had properties suitable for the purposes mentioned above. 
     The bacteriological characteristics of this strain are shown in the following Table 1. 
     
                       TABLE 1______________________________________Bacteriological characteristics______________________________________(1) MorphologyShape of cells    Short rodsSize of cells     (0.04- 0.5) × (1.2- 1.8) μmMobility          NoneFlagellation      NoneSpores            NoneGram staining     Negative(2) Growth on different media(a) Meat-broth agar plates    Shape of colonies Small circles    Upward growth     Convex curve    Edge              Complete    Surface           Glossy(b) Meat-broth agar slanting    medium    Quality of growth Moderate    Surface           Glossy    Condition of growth                 Belt-like    Color             Yellowish-white when grown                 in a dark place, and yellow                 when grown in a light place    Gloss             Present    Transparency      Semi-transparent(c) Meat-broth liquid medium    Growth on surface None    Turbidity         Moderate    Precipitate       Moderate(d) Meat-broth gelatin    stab culture    Condition of growth                 Grows only on surface    Liquification of  Weakly positive    gelatin(e) Litmus milk    Coagulation       Negative    pH                Alkaline(3) Physiological characteristicsReduction of nitrate             +Denitrification reaction             -MR test           -VP test           -Indole production -Hydrogen sulfide production             -Starch hydrolysis +Utilization of citric acid             +Utilization of inorganic nitrogenSodium nitrate    +Ammonium nitrate  +Production of pigments             -Urease            -Oxidase           +Catalase          +Growth limitspH                5.5-9.4Temperature       20- 37° C.Oxygen requirements             Aerobic______________________________________Acid and gas production from sugars:Sugars            Acid   Gas______________________________________L-Arabinose       -      -D-Xylose          -      -D-Glucose         -      -D-Mannose         -      -D-Fructose        -      -D-Galactose       -      -Maltose           -      -Sucrose           -      -Lactose           -      -______________________________________Growth on medium containing 5% NaCl                    Weak3-Ketolactose production from lactose                    -Decomposition of protocatechuic acid                    -(Cleavage at ortho or meta position)Ability to oxidize gluconic acid                    -______________________________________ 
    
     This strain, with the above bacteriological characteristics, was classified and identified as a strain of the genus Alcaligenes by reference to Bergey&#39;s Manual of Systematic Determinative Bacteriology (8th edition). However, it was not possible to find any known species that had the same characteristics as those of this species. Thus, this strain was identified as a new species, and named Alcaligenes sp. F-1906 by the inventors. This strain has been deposited as FERM BP-1857 with the Fermentation Research Institute Agency of Industrial Science and Technology. 
     The endo-α-GalNAcase produced by this strain has the following enzymological and physiochemical characteristics. 
     (1) Action of the Enzyme 
     This enzyme, as shown below, acts on the sugar chains of glycoproteins and the like with O-glycosidic linkages to release Gal β1→3GalNAc: ##STR2## wherein X and Y are peptide chains. 
     (2) Substrate Specifitity 
     Glycopeptides or glycoproteins such as asialofetuin, asialocasein, and asialomucin, which has sugar chains with O-glycosidic linkages and Gal β1→ 3GalNAc α→Ser (or Thr) structures can be used as substrates. 
     (3) Measurement of the Enzyme Activity 
     The activity of this enzyme can be measured with asialofetuin as a substrate. The enzyme reaction is carried out in citrate buffer, pH 4.5, at 37° C., and then stopped by adding a borate buffer, pH 9.1, to this reaction mixture. The Gal β1→3GalNAc produced is measured by the method of Reissig. One unit of enzyme activity is defined as the amount of enzyme that releases 1 μmol of Gal β1,→3GalNAc in one minute. 
     (4) Optimum pH and Range of pH Stability 
     The optimum pH of the enzyme reaction is 4.5-5.0 The enzyme is stable in the pH range of 4.2-6.5. thin this pH range, the activity remains about 90% or more after the enzyme is left at 1 hour at 30° C. 
     (5) Optimum Temperature for the Enzyme Reaction and Range of Temperature for Stability 
     The optimum temperature for the enzyme reaction is 40-45° C. The temperature at which the enzyme is stable for 10 minutes when kept in a phosphate buffer, pH 6.0, is 30° C. or less. At 40° C., about 70% of the activity remains after such treatment. 
     (6) Effects of Inhibitors 
     The enzyme was tested for the effects of various kinds of substances. The results are shown in Table 2. The table shows that the enzyme is completely inhibited by mercury. Also, it is about 26-35% inhibited by copper ion or manganese ion. It is approximately 30% inhibited by cysteine, which is one of the SH reagents, while other SH reagents such as mercaptoethanol, N-ethylmaleimide, P-chloromercuribenzoic acid, and iodoacetic acid left the enzyme activity almost unaffected. Monosaccharides left the enzyme activity almost unaffected. 
     
                       TABLE 2______________________________________          Concentration                      Relative activityAdditives      (mM)        (%)______________________________________None                       100Mg             2.5         92Mn             2.5         65Zn             2.5         88Co             2.5         95Hg             2.5         0Ca             2.5         109Fe             2.5         104Cu             2.5         74Ethylenediamine-          2.5         97tetraacetic acidCysteine       2.4         68Mercaptoethanol          2.4         97N-Ethylmaleimide          2.4         105p-Chloromercuribenzoic          2.4         99acidIodoacetic acid          2.4         109Glucose        2.0         109Galactose      2.0         107Glucosamine    2.0         111Galactosamine  2.0         106Sialic acid    1.0         106______________________________________ 
    
     (7) Method for Purification 
     The purification of this enzyme can be done by the appropriate combination of salting-out and different kinds of chromatographic methods. 
     (8) Molecular Weight 
     The molecular weight of this enzyme was estimated to be approximately 160,000 by gel filtration on Sephadex® G-200, and approximately 160,000 by SDS-polyacrylamide gel electrophoresis. 
     (9) Polyacrylamide Gel Electrophoresis 
     The purified enzyme gave a single band on polyacrylamide gel electrophoresis. 
     As the microorganism belonging to the genus Alcaligenes used in this invention, any microorganism that produces endo-α-GalNAcase can be used. The Alcaligenes sp. F-1906 (FERM BP-1857) isolated by the inventors from soil is preferably used. 
     For the production of endo-α-GalNAcase by the microorganisms, the culture medium with mucin from pig gastric mucosa treated in 1 N sulfuric acid for one hour at 80° C. can be used. The concentration of mucin is 0.1-10%, and preferably 0.5-5%. The enzyme is produced by the aerobic culture of the microorganisms in such medium for about 48 hours at pH 6.5 and 28° C. 
     After the culture, the endo-α-GalNAcase can be collected and purified from the culture by a appropriate combination of known methods. Because this enzyme is secreted into the culture medium, the culture is centrifuged to remove the cells and the supernatant is fractionated with ammonium sulfate followed by ionexchange chromatography, gel filtration chromatography, affinity chromatography, etc., to purify the enzyme. The following example will illustrate the invention more precisely, but is not intended to limit the invention. 
     EXAMPLES 
     Example 1 
     Commercially available mucin from pig gastric mucosa was mixed with 1 N sulfuric acid and left for 1 hour at 80° C. for hydrolysis, which removed the sialic acid moiety of the mucin. The hydrolysate was added to the medium at the concentration of 1%. Five hundred milliliters of the medium was put into each of 40 flasks with a capacity of 2 liters, which were sterilized and were inoculated with 5 ml of pre-cultured Alcaligenes sp. F-1906. The flasks were cultured at 28° C. for 3 days. After the culture, the bacterial cells were removed by centrifugation, and the supernatant of the culture was obtained. Ammonium sulfate was added to this supernatant at 0-5° C. and the fractions that precipitated at the saturation of 40-80% ammonium sulfate were collected. This precipitate was dissolved in 0.01 M phosphate buffer, pH 6.0, and then dialyzed overnight against the same buffer. The dialysate was put on a DEAE-Sephadex® A-50 column (5.0×32.5 cm) equilibrated with 0.01 M phosphate buffer. After the column was washed with the same buffer containing 0.2 M NaCl, the enzyme was eluted with the same buffer containing 0.4 M NaCl. The fractions with enzyme activity were concentrated by precipitation with ammonium sulfate at the saturation of 80%, and dialyzed overnight against 0.01 M phosphate buffer, pH 6.0. The dialysate was then put on a hydroxyapatite column (2.0 x 20 cm) equilibrated with the same buffer. The column was washed with the same buffer and with 0.4 M phosphate buffer, pH 6.0, and then eluted with 0.5 M phosphate buffer, pH 6.0. The fractions with enzyme activity were collected, concentrated by ultra-filtration, and gel filtered on a Sephadex® G-200 column (1.0×110 cm) equilibrated with 0.01 M phosphate buffer, pH 6.0, containing 0.2 M NaCl. The fractions with enzyme activity were collected, concentrated, and put on a Con A Sepharose® column (0.5×3 cm) equilibrated with 0.05 M phosphate buffer, pH 6.0. The fractions with enzyme activity were eluted as the fractions that did not adsorb to the column with the same buffer, and 3 mg of endo-α-GalNAcase was obtained in purified form (relative activity, 2.2 units/mg; yield, 1.0%). 
     It is understood that various other modifications will be apparent to and can be readily made by those skilled in the art without departing from the scope and spirit of this invention. Accordingly, it is not intended that the scope of the claims appended hereto be limited to the description as set forth herein, but rather that the claims be construed as encompassing all the features of patentable novelty that reside in the present invention, including all features that would be treated as equivalents thereof by those skilled in the art to which this invention pertains.