JP2005013228A - Method for producing phosphorylase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a phosphorylase from a phosphorylase-producing bacterium with a high purity in a simple manner. <P>SOLUTION: This method for producing the phosphorylase comprises culturing the phosphorylase-producing bacterium in a culture medium having a concentration of phosphoric acids or their salt of ≥50 mM, and collecting the phosphorylase formed in the culture medium. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing phosphorylase from a phosphorylase-producing bacterium.

  Phosphorylase is an enzyme that catalyzes the phosphorolysis of substrate sugars in the presence of inorganic phosphate. Maltodextrin phosphorylase, sucrose phosphorylase, maltose phosphorylase, cellobiose phosphorylase, trehalose phosphorylase and the like are known depending on the type of sugar of the substrate. Various phosphorylases are useful as enzyme reagents for quantification of inorganic phosphate and saccharides as reaction substrates, or as enzymes that can be used for the synthesis of various glycosides using phosphorylated saccharide as a substrate by utilizing the reversibility of the reaction. is there.

It is known that phosphorylase is possessed by many microorganisms such as Leuconostoc as intracellular or intracellular enzymes.
In order to use phosphorylase for the above-mentioned purposes, it is desirable to increase the purity of the enzyme preparation to some extent. In order to obtain such an enzyme preparation, after cultivating the phosphorylase-producing bacteria, Complicated processes such as body washing, cell disruption, enzyme fraction collection, and purification are required (see, for example, Patent Documents 1 to 5). At this time, in addition to the need to add a lytic enzyme, chelating agent, etc. for disrupting the cells, most of the cell components are eluted, increasing the load of the purification process to increase the phosphorylase purity. Cannot be avoided.

As a method for reducing such a load, a method for increasing the phosphorylase level in the microbial cells by, for example, optimizing the composition of the medium (see, for example, Patent Documents 6 to 7), or using the gene recombinant to increase the target phosphorylase level. A method for producing a pure substance (for example, see Non-Patent Document 1 and Patent Documents 3 to 5) and a simple purification method (for example, see Patent Document 8) are known, but the process itself cannot be changed. It is not always satisfactory.
Japanese Unexamined Patent Publication No. 1-91778 JP-A-8-280382 Japanese Patent Laid-Open No. 10-14580 Japanese Patent Laid-Open No. 10-276785 JP-A-10-327887 Japanese Patent Laid-Open No. 2-154686 Japanese Patent Laid-Open No. 3-4785 JP 2002-345458 A Kitao et al., J. Ferment. Bioeng., 73, 179-184 (1992)

  The present invention relates to providing a method for producing phosphorylase efficiently and simply from a phosphorylase-producing bacterium.

  The present inventors examined a method for efficiently preparing a phosphorylase fraction from a phosphorylase-producing bacterium. When a phosphorylase-producing bacterium was cultured on a high-phosphate medium, phosphorylase was produced in a high yield in the culture supernatant. The present inventors have found that phosphorylase can be produced efficiently without performing an operation such as collecting the enzyme from the microbial cells.

  That is, the present invention provides a method for producing phosphorylase, comprising culturing a phosphorylase-producing bacterium in a medium having a concentration of phosphoric acid or a salt thereof of 50 mM or more and collecting the phosphorylase produced in the medium. It is.

  Moreover, this invention provides the culture medium for phosphorylase production whose density | concentration of phosphoric acid or its salt is 50 mM or more.

  According to the present invention, phosphorylase can be efficiently and easily produced by an extremely simple process of culturing a phosphorylase-producing bacterium and then collecting (purifying cells) and purifying the culture supernatant.

The phosphorylase-producing bacterium used in the present invention is a bacterium having phosphorylase in the microbial cells or cells, and any bacterium that produces phosphorylase in the culture supernatant in the presence of a certain concentration of phosphates or salts thereof. Is, for example, Escherichia, Bacillus, Klebsiella, Streptococcus, Corynebacterium, Thermus, Thermococcus, Thermotoga, Leuconostoc, Pseudomonas, Clostridium, Acetobacter, Pullularia, Agrobacterium, Synecoccus, , Monilia, Sclerotinea, Chlamydomonas, Lactobacillus, Neiserria, Enterococcus, Lactococcus, Plesiomonas, Catellatospora, Kineosporia, Micrococcus, Arthrobacter, Brevibacterium, Flamobacterium, Anthro Genus, Thermoanaerobium, Rhizopus, Chaetomium, Acreomonium, Byssochilamys, Cercospora, Glomerella, Examples include bacteria belonging to the genus Humicola, Myceliophthora, Rhizomucor, Rosellinia, Sclerotinia, Sporidiobolus, Sterigmatomyces, Thermoascus, Thielavia, Tyromyces, Erwinia, Ruminococcus, and Cellvibrio.
In addition, such phosphorylase-producing bacteria can be obtained by natural or UV irradiation, chemical mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and ethylmethane sulfonate (EMS) (C. Guhrie & GR Fink, Methods in Enzymology). vol. 194, pp 273-281 Academic Press Inc.) and the like, and may be a mutant having improved phosphorylase production ability.

Among, Leuconostoc genus, bacteria are preferred genus Corynebacterium, Leuconostoc is Leuconostoc genus mesenteroides, Leuconostoc lactis, Leuconostoc fallax, Leuconostoc citreum, Leuconostoc carnosum, Leuconostoc argetinum, Leuconostoc pseudomesenteroides, the genus Corynebacterium Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium hoagii , Corynebacterium Vitaeruminis and Corynebacterium pilosum are more preferable. Among them, Leuconostoc mesenteroides , Leuconostoc mesenteroides KSM-SP1 (FERM P-19737) and Leuconostoc mesenteroides KSM-SP78 (FERM AP-20037 (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, 2004) Corynebacterium vitaeruminis , Corynebacterium glutamicum KSM-MP669 (FERM AP-20036 (Incorporated Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, received on April 28, 2004))) is particularly preferable.

  The phosphorylase is produced by culturing the phosphorylase-producing bacterium in a medium (phosphorylase production medium) in which the concentration of phosphoric acid or a salt thereof is 50 mM or more.

  Examples of the phosphoric acids or salts thereof added to the medium include phosphoric acid, metaphosphoric acid, tripolyphosphoric acid, polyphosphoric acid, diphosphoric acid, polymetaphosphoric acid, and salts thereof, and the salt is preferably a sodium salt or a potassium salt. . Examples of particularly preferable salts of phosphoric acids include monopotassium phosphate, dipotassium phosphate, monosodium phosphate, disodium phosphate, and the like. A mixture of phosphoric acid and its salt or several phosphoric acid salts is mixed. And more preferably used.

  The concentration of phosphoric acid or a salt thereof in the medium is generally required to be 50 mM or more from the viewpoint of effect, but is preferably 50 mM to 1.5 M, more preferably 50 mM to 1 M, particularly 100 mM to 1 M. The range of is preferable. Moreover, 400 mM to 1.2 M is preferable for bacteria of the genus Leuconostoc, and 100 mM to 600 mM is preferable for the genus Corynebacterium.

  The medium used in the present invention may be any medium as long as it can grow a phosphorylase-producing bacterium, and in addition to the phosphoric acid and its salt, a liquid medium containing a carbon source, a nitrogen source, metal minerals, vitamins and the like Can be used.

  Examples of the sugar added to the medium include monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Two or more of these may be used in combination.

  Examples of carbon sources other than carbohydrates include organic acid salts such as acetates, and examples of nitrogen sources include inorganic and organic substances such as ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium phosphate, and ammonium acetate. Ammonium salts, urea, peptone, meat extract, yeast extract, nitrogen-containing organic substances such as casein hydrolyzate, amino acids such as glycine, glutamic acid, alanine, methionine, etc., and metal minerals include, for example, sodium chloride, sulfuric acid Examples thereof include ferrous iron, magnesium sulfate, manganese sulfate, zinc sulfate, calcium carbonate, and the like. These may be used alone or in combination as necessary.

  The culture method is performed by appropriately adjusting the pH and temperature so that the microorganisms can sufficiently grow. In addition to shaking culture, anaerobic culture, stationary culture, and culture using a fermentor, the culture means can also use resting cell reaction and immobilized cell reaction.

  Thus, when a phosphorylase-producing bacterium is cultured in a high-phosphate medium, the phosphorylase is produced and accumulated in a high yield in the culture supernatant, and the target phosphorylase can be easily obtained by collecting this.

  The method for collecting phosphorylase from the medium may be performed according to a known method, for example, by separating and removing cells and combining ultrafiltration, salting out, ion exchange, hydrophobic chromatography, gel filtration, drying, and the like. Can be done by. Even when the immobilized enzyme is produced, it can be produced outside the cells without complicated operations.

In addition, the activity measurement of the obtained phosphorylase can use the general phosphorylase activity measuring method. An example is the method of Weinhausel (Enzyme Microb. Technol., 17 , 140-146 (1995)).

  According to the present invention, phosphorylase can be produced directly outside the bacterial cells, and the enzyme can be obtained without complicated operations such as disrupting the bacterial cells. Furthermore, since phosphoric acids, which are low-molecular compounds, are only added to the medium, the load on the purification process is greatly reduced, and the preparation of high-purity phosphorylase is easy.

  In addition, by adding a substrate such as disaccharide, oligosaccharide, polysaccharide and phosphoric acid or a salt thereof directly to the culture solution, sugar phosphate can be produced without any special operation. Furthermore, it is also possible to obtain a glycoside without any special operation by allowing a compound that becomes an aglycone and sugar phosphate to directly act on the culture solution.

Example 1 Production of phosphorylase by bacteria belonging to the genus Leuconostoc

Leuconostoc mesenteroides JCM9693, Leuconostoc carnosum JCM9695, Leuconostoc argentinum JCM11052, Leuconostoc pseudomesenteroides JCM11045, and Leuconostoc mesenteroides JCM9693 strains are treated with MNNG (C. Guhrie & GR Fink, Methods in Enzymology Inc. 281) the resulting phosphorylase highly productive mutants Leuconostoc mesenteroides KSM-SP1 (FERM P -19737) and Leuconostoc mesenteroides KSM-SP78 (FERM AP -20037), and Nurimatsu the MR agar medium (Oxoid Ltd.), 30 in anaerobic conditions Incubated at 0 ° C. One platinum loop of this bacterium in liquid medium (1% yeast extract (Difco), 1% polypeptone (Nihon Pharmaceutical), 0.04% magnesium sulfate heptahydrate, 0.02% manganese chloride tetrahydrate, 0.0002% sulfuric acid Ferric tetrahydrate, 400, 800, 1000 mM phosphate buffer, 5%, 10%, 15% sucrose) and static culture at 30 ° C. for 2 days. The sucrose phosphorylase activity of the culture supernatant after removing the cells by centrifugation was measured.

The sucrose phosphorylase activity in the supernatant was partially modified by the method of Weinhausel (Enzyme Microb. Technol., 17 , 140-146 (1995)). Enzyme reaction solution (200 mM potassium phosphate buffer (pH 7.0), 90 mM sucrose, 100 mM Tris-acetate buffer (pH 6.8), 20 μL of culture supernatant sample diluted appropriately on 96-well microplate, 2 mM EDTA, 10 mM magnesium sulfate, 2 mM NAD, 10 μM glucose-1,6-diphosphate, 1.2 unit / ml phosphoglucomutase (from rabbit muscle, manufactured by Roche Diagnostics), 1.2 unit / ml glucose 180 μL of 6-phosphate dehydrogenase (from Leuconostoc mesenteroides , manufactured by Roche Diagnostics) was added, and the increase in absorbance due to G1P generation at 340 nm was measured at 37 ° C. One unit (U) of the enzyme unit was an amount that produced 1 μmol of glucose-1-phosphate (G1P) per minute. The results are shown in Table 1.

  From Table 1, it was confirmed that the amount of phosphorylase produced outside the cells increased by increasing the phosphate concentration in the medium.

Example 2 Production of phosphorylase by Corynebacterium
Corynebacterium vitaeruminis JCM1323, Corynebacterium callunae IFO15359, Corynebacterium glutamicum JCM1321 and Corynebacterium phosphorylase high-producing mutant strains obtained by the glutamicum JCM1321 to MNNG treatment Corynebacterium glutamicum KSM-MP669 the (FERM AP-20036), SCD agar medium (Nippon Pharmaceutical Co., Ltd. ) And cultured at 30 ° C. One platinum loop of this bacterium is inoculated into a liquid medium (0.67% Yeast Nitrogen Base (Difco), 50, 100, 200 mM phosphate buffer (pH 7), 15% dextrin (derived from sigma potato)), 30 Shaking culture was performed at 0 ° C. for 6 days. The maltodextrin phosphorylase activity of the culture supernatant after removing the cells by centrifugation was measured.

The maltodextrin phosphorylase activity in the supernatant was obtained by partially modifying the method of Weinhausel (Enzyme Microb. Technol., 17 , 140-146 (1995)). Enzyme reaction solution (200 mM potassium phosphate buffer (pH 7.0), 2% dextrin, 100 mM Tris-acetate buffer (pH 6.8), 2 mM EDTA, 10 mM magnesium sulfate, 2 mM NAD, 10 μM glucose-1,6-diphosphate, 1.2 unit / ml phosphoglucomutase (from rabbit muscle, manufactured by Roche Diagnostics), 1.2 unit / ml glucose-6 -Phosphate dehydrogenase (from Leuconostoc mesenteroides , manufactured by Roche Diagnostics) was added in an amount of 180 μL, and the increase in absorbance due to G1P production at 340 nm was measured at 37 ° C. One unit (U) of the enzyme unit was an amount that produced 1 μmol of glucose-1-phosphate (G1P) per minute. The results are shown in Table 2.

  From Table 2, it was confirmed that the amount of phosphorylase produced outside the cells increased by increasing the phosphate concentration in the medium.

Claims (3)

  A method for producing phosphorylase, comprising culturing a phosphorylase-producing bacterium in a medium having a concentration of phosphoric acid or a salt thereof of 50 mM or more and collecting the phosphorylase produced in the medium.   The method for producing phosphorylase according to claim 1, wherein the phosphorylase-producing bacterium is a bacterium of the genus Leuconostoc or Corynebacterium.   A medium for producing phosphorylase, wherein the concentration of phosphoric acid or a salt thereof is 50 mM or more.