Method for producing glucose isomerase

Abstract

To provide a method for producing glucose isomerase containing no nucleic acid by a simple operation.SOLUTION: The method for producing glucose isomerase comprises the steps of culturing a glucose isomerase-producing bacterium in a liquid medium, heat-treating the culture broth at 45-95°C for 1-60 min, and disrupting the bacterial cell wall.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing glucose isomerase. [Background technology]

[0002] Glucose isomerase is an enzyme that catalyzes the conversion of glucose to fructose and fructose to glucose. It is used as a food additive to produce isomerized sugar (fructose or glucose), to improve the sweetness of glucose-containing foods (such as plant-derived milk), and to improve the flavor of fermented foods and beverages.

[0003] A known method for mass-producing glucose isomerase involves liquid culture of natural or recombinant glucose isomerase-producing bacteria, followed by disruption of the cell walls of the bacteria to collect glucose isomerase liberated from the bacteria (Non-Patent Document 1 and Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2017 / 220551 [Non-patent literature]

[0005] [Non-Patent Document 1] Organic Synthetic Chemistry Vol. 38 No. 8 (1980) pp. 538-545 Summary of the Invention [Problem to be solved by the invention]

[0006] However, when the cell walls of a culture broth or a bacterial cell suspension are disrupted, a large amount of nucleic acids is contained in the disrupted solution, which increases the viscosity of the disrupted solution and places a burden on the purification of glucose isomerase. In other words, there are problems such as the complexity of the purification process in order to reduce the amount of leaked nucleic acid. Furthermore, if nucleic acid remains in the final product, when the product is immobilized on a column as an immobilized enzyme, the nucleic acid inhibits adsorption to the column, so it is preferable that the final product is free of nucleic acid. Therefore, an object of the present invention is to provide a method for producing nucleic acid-free glucose isomerase by a simple procedure. [Means for solving the problem]

[0007] Therefore, the present inventors have investigated means for preventing nucleic acids from being mixed into a glucose isomerase-containing solution during treatment in the early stages of production, and have found that by subjecting a glucose isomerase-producing bacterium to a specific heat treatment on the culture broth at a stage before the cell wall disruption treatment after liquid culture and before the cell wall disruption treatment, the amount of nucleic acids in the disrupted solution after the cell wall disruption treatment can be reduced or no nucleic acids can be mixed in. They have also found that by subsequently purifying glucose isomerase from the disrupted product in a conventional manner, high-purity glucose isomerase free from nucleic acids can be obtained, and have completed the present invention.

[0008] That is, the present invention provides the following [1] to [6]. [1] A method for producing glucose isomerase, comprising liquid culturing a glucose isomerase-producing bacterium, heating the culture broth at 45°C to 95°C for 1 minute to 60 minutes, and then disrupting the bacterial cell walls. [2] The method according to [1], wherein the cell wall disruption treatment is selected from the group consisting of enzyme treatment, osmotic shock, freeze-thawing, ultrasonic treatment, French press, disruption in a mortar, crushing with a homogenizer, and crushing with glass beads. [3] The method according to [1] or [2], wherein the glucose isomerase-producing bacterium is a glucose isomerase-producing bacterium belonging to the genus Streptomyces. [4] A production method according to any one of [1] to [3], wherein a disruption solution obtained after disruption of bacterial cell walls contains glucose isomerase-producing bacteria after disruption of bacterial cell walls, the disruption solution is subjected to solid-liquid separation in a state in which all or part of the nucleic acids are present in the cells of the producing bacteria, and glucose isomerase is obtained from the resulting supernatant. [5] A glucose isomerase obtained by the production method according to any one of [1] to [4]. [6] Pores are formed in the cell wall and / or cell membrane of the glucose isomerase-producing bacterium, and at least one selected from proteins, membrane proteins, and nucleic acids inside the producing bacterium is denatured; the amount of nucleic acid and / or sugar inside the producing bacteria is less than that of bacteria in which no pores are formed in the cell wall and / or cell membrane, the amount of nucleic acid and / or sugar inside the producing bacteria is higher than that of a producing bacteria in which pores are formed in the cell wall and / or cell membrane and in which proteins, membrane proteins and nucleic acids inside the producing bacteria are not denatured at all; Glucose isomerase producing bacteria. [Effects of the Invention]

[0009] According to the method of the present invention, nucleic acids are not mixed into the disrupted solution after disruption of the bacterial cell walls, and therefore, highly pure glucose isomerase can be produced by ordinary purification procedures without the need for complicated nucleic acid removal procedures. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the time course of glucose isomerase activity in the supernatant of the lysozyme-treated lysate with or without heat treatment (relative to the glucose isomerase activity (U / mL) in the supernatant of the lysate treated with lysozyme for 5 hours without heat treatment). [Figure 2] This figure shows the change over time in the amount of nucleic acid in the supernatant of the lysozyme-treated lysate with or without heat treatment (relative value to the amount of nucleic acid (mg / mL) in the supernatant of the lysate treated with lysozyme for 5 hours without heat treatment). [Figure 3]FIG. 1 shows the time course of the total sugar content in the supernatant of the lysozyme-treated lysate with or without heat treatment (relative to the total sugar content (mg / mL) in the supernatant of the lysozyme-treated lysate for 5 hours without heat treatment). DETAILED DESCRIPTION OF THE INVENTION

[0011] Terms used in this specification are used in the sense commonly used in the art unless otherwise specified.

[0012] As used herein, glucose isomerase refers to an isomerase enzyme that catalyzes the conversion of glucose to fructose and fructose to glucose. As described above, glucose isomerase is used as a food additive for producing isomerized sugar (fructose or glucose), improving the sweetness of glucose-containing foods (such as plant-derived milk), and improving the flavor of fermented foods and beverages. The glucose isomerase obtained in the present invention is preferably a thermostable glucose isomerase, and more preferably a thermostable glucose isomerase that maintains 80% or more of its activity after treatment at 80° C. for 30 minutes.

[0013] The optimum (optimum) temperature of the glucose isomerase obtained by the present invention is preferably in the range of 45°C or higher and 95°C or lower, more preferably in the range of 60°C or higher and 95°C or lower, even more preferably in the range of 70°C or higher and 95°C or lower, and particularly preferably in the range of 80°C or higher and 95°C or lower.

[0014] The temperature stability of the glucose isomerase obtained by the present invention is preferably 0°C or higher and 100°C or lower, more preferably 0°C or higher and 95°C or lower, even more preferably 0°C or higher and 90°C or lower, and particularly preferably 0°C or higher and 85°C or lower.

[0015] The optimum (optimum) pH of the glucose isomerase obtained by the present invention is not particularly limited, and is, for example, preferably 5 or more and 10 or less, more preferably 6 or more and 9 or less, and even more preferably 6 or more and 8 or less.

[0016] The pH stability of the glucose isomerase obtained by the present invention is not particularly limited, and is preferably 5 or more and 10 or less, more preferably 6 or more and 9 or less, and even more preferably 7 or more and 9 or less.

[0017] The metal ions that stabilize the activity of glucose isomerase obtained in the present invention are not particularly limited, and examples thereof include Mg 2+ , Co 2+ and other metal ions.

[0018] The activity of the glucose isomerase obtained by the present invention is not particularly limited and can be adjusted as appropriate. For example, it is preferably 1 to 1,000,000 GIU / mL, more preferably 100 to 500,000 GIU / mL, and even more preferably 1,000 to 200,000 GIU / mL. Here, the unit of glucose isomerase activity, "GIU," is an abbreviation for Glucose Isomerase Unit. In this specification, 1 GIU refers to the amount of enzyme that produces 1 mg of fructose in 60 minutes under the following conditions. (Reaction conditions) Substrate composition: 0.1M glucose (containing 10mM MgSO4) pH: pH 7.0 (0.1M K-Na phosphate buffer) Temperature: 70℃ Time: 30 minutes The activity of the glucose isomerase obtained in the present invention is measured by the following method: that is, by analyzing the reaction in which glucose isomerase isomerizes the substrate glucose to fructose. The reaction can be terminated by adding perchloric acid. The produced fructose can be quantified by the cysteine ​​sulfate method, and the glucose isomerase activity (GIU) can be calculated. The specific procedure is as described in the Examples.

[0019] One aspect of the present invention is a method for producing glucose isomerase, which comprises liquid culturing a glucose isomerase-producing bacterium, heating the culture broth at 45°C to 95°C for 1 minute to 60 minutes, and then disrupting the bacterial cell walls. Examples of glucose isomerase-producing bacteria include those belonging to the genera Streptomyces, Actinoplanes, Aspergillus, and Bacillus. However, glucose isomerase-producing bacteria belonging to the genus Streptomyces are preferred because they are heat-resistant glucose isomerase-producing bacteria. Examples of glucose isomerase-producing bacteria belonging to the genus Streptomyces include Streptomyces griseofuscus, Streptomyces murinus, Streptomyces phaeochromogenes, and Streptomyces rubiginosus. Furthermore, genetically modified forms of these glucose isomerase-producing bacteria belonging to the genus Streptomyces can also be used.

[0020] From the viewpoint of mass production, the glucose isomerase-producing bacteria are preferably cultured in liquid form. Liquid culture is preferably carried out by inoculating the glucose isomerase-producing bacteria into a liquid medium containing a carbon source such as glucose, xylose, or maltose, a nitrogen source such as ammonium phosphate, yeast extract, powdered soy protein, or corn steep liquor, and inorganic salts such as magnesium sulfate or cobalt chloride, at a pH of 6.5 to 7.6 and a temperature of 20°C to 35°C.

[0021] In the present invention, the culture broth after liquid culture is heat-treated. From the viewpoint of not inactivating glucose isomerase present in the bacteria and preventing the release of nucleic acids in the subsequent cell wall disruption treatment, this heat treatment is preferably carried out at 45°C to 95°C for 1 minute to 60 minutes, more preferably at 55°C to 85°C for 1 minute to 20 minutes, even more preferably at 60°C to 80°C for 1 minute to 10 minutes, and particularly preferably at 65°C to 80°C for 1 minute to 4 minutes. This heat treatment denatures the structures of proteins, nucleic acids, etc. in the culture medium, and further denatures membrane proteins that penetrate the cell membrane. Denaturing the membrane proteins in the cell membrane allows the bacterial cell shape to be maintained without losing its shape, even when holes are formed in the cell wall and / or cell membrane in the subsequent cell wall disruption treatment. As a result, glucose isomerase is released outside the bacterial cell, but nucleic acids, which have a larger molecular weight than glucose isomerase, remain inside the cell, presumably preventing leakage of nucleic acids. The heat treatment in the present invention is preferably performed on the culture broth after liquid culture, before solid-liquid separation, cell washing, buffer suspension, etc., from the viewpoint of preventing the release of nucleic acids in the subsequent bacterial cell wall disruption treatment. Heating of the culture broth is also preferred from the viewpoint of process simplification, since the heat treatment can be performed directly in the culture tank after the end of the culture.

[0022] After the heat treatment, the bacterial cell walls are disrupted to release the target glucose isomerase from the cells. Examples of the cell wall disruption treatment include enzyme treatment, osmotic shock, freeze-thawing, ultrasonic treatment, French press, mortar disruption, homogenizer grinding, and glass bead grinding. However, enzyme treatment is preferred from the viewpoint of preventing the release of nucleic acids and preventing the inactivation of glucose isomerase. Examples of enzyme treatment include lysozyme (also known as muramidase), glucosaminidase, amidase, and endopeptidase. Lysozyme and glucosaminidase hydrolyze glycosidic bonds in the glycan chains of the cell wall peptidoglycan layer, amidase hydrolyzes the amide bond between N-acetylmuramic acid and L-alanine, and peptidase hydrolyzes peptide bonds in the peptide chains of the cell wall peptidoglycan layer. Treatment with lysozyme or glucosaminidase, which degrades glycan chains, is preferred because of its high effectiveness in preventing the release of nucleic acids, and lysozyme treatment is more preferred. As lysozyme, commercially available products such as lysozyme derived from egg white can be used. The cell wall disruption treatment varies depending on the treatment method. For example, in the case of lysozyme treatment, lysozyme is added to a concentration of 0.001 to 0.1% and reacted at 45 to 55°C for several hours, preferably 1 to 5 hours, and more preferably 2 to 4 hours. Before the cell wall disruption treatment, it is preferable to separate the culture broth after the heat treatment into solids and liquids, and then wash the resulting cells and suspend them in a buffer solution. The solid-liquid separation method at this stage is preferably centrifugation.

[0023] The relative ratio of the amount of nucleic acid (mg / mL) released into the supernatant of the disrupted solution after cell wall disruption treatment (heat-treated cells / non-heat-treated cells) is preferably 0.8 or less, more preferably 0.7 or less, even more preferably 0.6 or less, and particularly preferably 0.5 or less.

[0024] In this specification, the amount of nucleic acid is measured by the orcin-HCl method, which is a known method.

[0025] The relative ratio of the amount of sugar (mg / mL) released into the supernatant of the disrupted solution after cell wall disruption (heat-treated cells / non-heat-treated cells) is preferably 0.8 or less, more preferably 0.7 or less, even more preferably 0.6 or less, and particularly preferably 0.5 or less.

[0026] In this specification, the sugar amount is measured by the known phenol-sulfuric acid method.

[0027] As described above, the heat treatment denatures the structures of proteins, nucleic acids, etc. in the culture medium, and further denatures membrane proteins that penetrate the cell membrane. Denaturing membrane proteins in the cell membrane allows the cell shape to be maintained without losing its shape, even if holes are formed in the cell wall and / or cell membrane during the cell wall disruption treatment performed after the heat treatment. As a result, glucose isomerase is released outside the cell, but nucleic acids, which have a larger molecular weight than glucose isomerase, remain inside the cell, presumably preventing leakage of nucleic acids. Therefore, another aspect of the present invention is a method for producing glucose isomerase, wherein pores are formed in the cell wall and / or cell membrane of a glucose isomerase-producing bacterium, and at least one selected from proteins, membrane proteins, and nucleic acids inside the producing bacterium is denatured, the amount of nucleic acid and / or sugar inside the producing bacteria is less than that of bacteria in which no pores are formed in the cell wall and / or cell membrane, the amount of nucleic acid and / or sugar inside the producing bacteria is higher than that of a producing bacteria in which pores are formed in the cell wall and / or cell membrane and in which proteins, membrane proteins and nucleic acids inside the producing bacteria are not denatured at all; It is a glucose isomerase-producing bacterium. Here, the amount of nucleic acids and / or sugars inside the glucose isomerase-producing bacteria of the present invention is preferably 30% or more less, more preferably 40% or more less, and even more preferably 50% or more less than that of bacteria having no pores at all in the cell wall and / or cell membrane, with the upper limit of the reduction being preferably 70% or less, more preferably 65% ​​or less. The amount of nucleic acids and / or sugars inside the glucose isomerase-producing bacteria of the present invention is preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more higher than that in bacteria in which pores have been formed in the cell wall and / or cell membrane and in which the proteins, membrane proteins, and nucleic acids inside the bacteria are not denatured at all. Here, the upper limit of the increase rate is preferably 70% or less, more preferably 65% ​​or less.

[0028] After disrupting the bacterial cell walls, the product is purified by a conventional method to obtain a highly purified glucose isomerase.

[0029] According to the method of the present invention, simply subjecting the culture broth after culture to the above-mentioned heat treatment allows all or part of the nucleic acids to be retained within the cells during the subsequent cell wall disruption treatment, suppressing the release of nucleic acids outside the cells and selectively releasing glucose isomerase. Therefore, the supernatant obtained by solid-liquid separation of the disrupted solution does not contain all or part of the nucleic acids, simplifying the subsequent purification procedure for glucose isomerase. Furthermore, according to the present invention, the release of not only nucleic acids but also sugars is suppressed, thereby reducing the burden on the purification process. [Example]

[0030] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0031] Example 1 (1) Cultivation of glucose isomerase-producing bacteria Streptomyces griseofuscus (ATCC 23916) was inoculated into a medium containing 5% by mass of corn steep liquor, 1% by mass of xylose, 1% by mass of glucose, 0.25% by mass of monoammonium phosphate, and 0.03% by mass of cobalt chloride (pH 7.0, medium sterilization was performed by sterilizing the xylose and glucose separately), and the medium was immersed and cultured at 30°C for 80 hours.

[0032] (2) Heat treatment of culture broth The resulting culture broth (enzyme activity: 521 U / ml, extracellular activity: 18 U / ml) was adjusted to pH 8.5 and heated at 80°C for 3 minutes. The cells were then collected by suction filtration. The cells were suspended in phosphate buffer (pH 7.4-7.69) to half the volume of the resulting culture broth, and lysozyme (product name: Lysozyme BIO, distributor: Nippon Biocon) was added to a concentration of 0.01%. The amounts of nucleic acids and total sugars contaminants and glucose isomerase activity of the disrupted cells treated with lysozyme at 50°C for 1, 1.5, 2, 3, and 5 hours were compared with those of unheated cells. The supernatant of the disrupted cells was used for various measurements. Glucose isomerase activity was measured as follows. 3.6 g of anhydrous crystalline glucose was mixed with 25 mL of 0.4 M K-Na phosphate buffer (pH 7.0) and 20 mL of 0.1 M magnesium sulfate solution, and the resulting mixture was diluted to 100 mL with distilled water. This was used as the substrate solution. The culture supernatant sample was appropriately diluted with 50 mM K-Na phosphate buffer (pH 7.0) to approximately 10 U / mL. 0.2 mL of the culture supernatant sample was dispensed into a test tube, and 0.8 mL of distilled water was added to make a 1.0 mL solution. 1.0 mL of the substrate solution was added while cooling in water, and the test tube was capped and incubated at 70°C for 30 minutes. After the reaction, 4.0 mL of a reaction stop solution (0.5 M perchloric acid solution) was added while cooling in water, and 4.0 mL of distilled water was added to make a 10 mL solution. Fructose was quantified using the resulting solution by the cysteine ​​sulfate method. Glucose isomerase activity was calculated using the following formula:

[0033]

number

[0034] Nucleic acids were measured according to the orcin-hydrochloride method, and total sugars were measured according to the phenol-sulfuric acid method.

[0035] The results are shown in Figure 1 (relative values ​​of supernatant activity), Figure 2 (relative values ​​of nucleic acid amount), and Figure 3 (relative values ​​of total sugar amount). Figure 1 shows relative values ​​when the activity (U / mL) of the supernatant of the homogenate after 5 hours of lysozyme treatment without heat treatment is taken as 100%. Figure 2 shows relative values ​​when the nucleic acid amount (mg / mL) of the supernatant of the homogenate after 5 hours of lysozyme treatment without heat treatment is taken as 100%. Figure 3 shows relative values ​​when the total sugar amount (mg / mL) of the supernatant of the homogenate after 5 hours of lysozyme treatment without heat treatment is taken as 100%. Even at the stage of the bacterial cell cake suspension before lysozyme treatment (no addition in the figure), the nucleic acid and total sugar impurities were at half the amount, and the viscosity of the disruption solution after 5 hours of lysozyme treatment was extremely low. Compared to the amount of nucleic acid in the supernatant of the disruption solution after 5 hours of lysozyme treatment without heat treatment, the amount of nucleic acid in the supernatant of the disruption solution after 5 hours of lysozyme treatment with heat treatment was approximately 48%, less than half. Also for total sugar, the amount of total sugar in the supernatant of the disruption solution after 5 hours of lysozyme treatment with heat treatment was approximately 67% of the amount of total sugar in the supernatant of the disruption solution after 5 hours of lysozyme treatment without heat treatment. In the disruption solution treated with lysozyme for 3 hours, the amount of nucleic acids in the supernatant of the disruption solution treated with lysozyme for 3 hours with heat treatment was approximately 43% of the amount of nucleic acids in the supernatant of the disruption solution treated with lysozyme for 5 hours without heat treatment. The total sugar amount in the supernatant of the disruption solution treated with lysozyme for 3 hours with heat treatment was approximately 56% of the total sugar amount in the supernatant of the disruption solution treated with lysozyme for 5 hours without heat treatment. Furthermore, the presence or absence of heat treatment did not affect enzyme activity, and no inactivation was observed. Furthermore, since the longer the lysozyme treatment time, the more contaminants tend to be eluted, the time was set to several hours, preferably around 3 hours.

[0036] (3) Purification of glucose isomerase after heat treatment The disrupted solution after the lysozyme treatment was purified by a conventional method and recovered as a glucose isomerase solution.

Claims

1. A method for producing glucose isomerase, comprising liquid culturing a glucose isomerase-producing bacterium, heating the culture broth at 45°C to 95°C for 1 minute to 60 minutes, and then disrupting the bacterial cell walls.

2. 2. The method according to claim 1, wherein the cell wall disruption treatment is selected from the group consisting of enzyme treatment, osmotic shock, freeze-thawing, ultrasonic treatment, French press, disruption in a mortar, crushing with a homogenizer, and crushing with glass beads.

3. 2. The method according to claim 1, wherein the glucose isomerase-producing bacterium is a glucose isomerase-producing bacterium belonging to the genus Streptomyces.

4. 2. The method according to claim 1, wherein the disruption solution after disruption of bacterial cell walls contains glucose isomerase-producing bacteria after disruption of bacterial cell walls, the disruption solution is subjected to solid-liquid separation in a state in which all or part of the nucleic acids are present in the cells of the producing bacteria, and glucose isomerase is obtained from the resulting supernatant.

5. A glucose isomerase obtained by the production method according to any one of claims 1 to 4.

6. Pores are formed in the cell wall and / or cell membrane of the glucose isomerase-producing bacterium, and at least one selected from proteins, membrane proteins, and nucleic acids inside the producing bacterium is denatured, the amount of nucleic acid and / or sugar inside the producing bacteria is less than that of bacteria in which no pores are formed in the cell wall and / or cell membrane; the amount of nucleic acid and / or sugar inside the producing bacteria is higher than that of a producing bacteria in which pores are formed in the cell wall and / or cell membrane and in which proteins, membrane proteins and nucleic acids inside the producing bacteria are not denatured at all; Glucose isomerase producing bacteria.

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