JP4656620B2 - Preparation of sucrose phosphorylase - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for obtaining a sucrose phosphorylase preparation and to a sucrose phosphorylase preparation.
[0002]
[Prior art]
Sucrose phosphorylase is an enzyme that acts on sucrose in the presence of inorganic phosphate to produce glucose-1-phosphate and fructose. Sucrose phosphorylase can be applied to quantitative analysis of inorganic phosphate or sucrose in combination with, for example, phosphoglucomutase and glucose-6-phosphate dehydrogenase. Therefore, sucrose phosphorylase is useful as a diagnostic enzyme.
[0003]
Further, by using sucrose phosphorylase in combination with phosphorylase, sugar chains can be linked to glucans such as amylose, starch, glycogen, etc. by α-1,4-glucoside bonds to extend sugar chains.
[0004]
In order to use sucrose phosphorylase for the above-mentioned purpose, it is preferable to remove some contaminating enzymes and increase the purity of sucrose phosphorylase to some extent. For example, when quantifying inorganic phosphate using sucrose phosphorylase together with phosphoglucomutase and glucose-6-phosphate dehydrogenase, it is preferable to remove phosphatase in advance. When sucrose phosphorylase is used in an extension reaction for adding a sugar unit to glucan by an α-1,4-glucoside bond, it is preferable to remove amylase in addition to phosphatase.
[0005]
For example, the following is known as a microorganism having sucrose phosphorylase. Leuconostok Mecenteroides ( Leuconostoc mesenteroides ) , Streptococcus mutans ( Streptococcus mutans ) , Pseudomonas species ( Pseudomonas sp. ) , Clostridium species ( Clostridium sp. ) , Pullulia Pullance ( Pullularia pullulans ) , Acetobacter xylinum ( Acetobacter xylum ) , Agrobacterium species ( Agrobacterium sp. ) , Synecococcus species ( Synecoccus sp. ) , E. coli ( E. coli ) , Aspergillus niger ( Aspergillus niger ) , Moniria Sitophila ( Monilia sitophila ) , Sclerotinea Esserothiolum ( Sclerotinea escerioorum ) , Chlamydomonas species ( Chlamydomonas sp. ) . In the case of preparing sucrose phosphorylase from an extract of cells of these microorganisms containing sucrose phosphorylase or a culture supernatant of these microorganisms, a conventional technique such as normal ion exchange chromatography has been used. So far, there has been no report that a heat treatment step has been performed to prepare sucrose phosphorylase. In the purification using conventional ion exchange chromatography, the load on the column is heavy in order to allow the extract of bacterial cells rich in contaminating proteins, culture supernatant, etc. to flow through the column, so it is necessary to perform purification on a large column. was there. There was a problem of cost and time. Moreover, it is difficult to remove contaminant enzymes such as amylase by a simple purification method other than ion exchange chromatography (for example, ammonium sulfate precipitation method). Therefore, there has been a problem that the molecular weight of amylose synthesized using sucrose phosphorylase is small. Further, when phosphatase is mixed, there is a problem that amylose synthesis is inhibited and the yield of amylose is lowered. Furthermore, when a sucrose degrading enzyme other than sucrose phosphorylase is mixed, it cannot be used for quantitative analysis of sucrose and phosphate. Thus, in order to utilize sucrose phosphorylase for industrial applications, a simple preparation method is required.
[0006]
JP-A-2-23866 (Title of Invention: Method for Stabilizing Sucrose Phosphorylase) shows a method for stabilizing sucrose phosphorylase. In this method, first, sucrose phosphorylase derived from Leuconostoc mesenteroides is roughly purified. Next, saccharides and organic acids are added to the crude sucrose phosphorylase at 1% (w / v) or more, or alkali metal salts, alkaline earth metal salts, or chelating agents are added at about 5 mM, An amino acid is contained about 10 mM or more. This solution is lyophilized to obtain a powdered enzyme. The powdered enzyme was stored at 30 ° C. for 30 days, and as a result, the residual activity was high (83 to 94%) compared to no addition (residual activity 68%). Furthermore, this enzyme powder is stable for more than 6 months when stored at 4 ° C. However, JP-A-2-23866 does not disclose a method for preparing or purifying sucrose phosphorylase by heating in a sucrose solution.
[0007]
Other enzyme preparation methods include a method in which a heat treatment step is added to the preparation of a heat-stable enzyme. Many examples of this method have been reported. In addition, there are some published examples in which the thermal stability of an enzyme increases when a substrate is added. For example, amylase has high heat resistance in the presence of starch. Phosphorylase has high heat resistance in the presence of phosphoric acid, glucose-1-phosphate, and the like. In these cases, heat resistance generally increases as the substrate concentration increases (although there is a saturation point).
[0008]
However, enzyme stabilization by such a substrate is not a general one. For example, even when fructose or phosphoric acid as a substrate is added to sucrose phosphorylase, no heat stabilization effect is observed.
[0009]
Therefore, it has not been easily guessed that sucrose phosphorylase is heat-resistant by the addition of sucrose. In addition, there is an optimum sucrose concentration in the preparation of sucrose phosphorylase, and it was completely impossible to predict that the preparation efficiency would be lowered at higher or lower concentrations.
[0010]
Furthermore, when the specific activity is increased by increasing the purity of sucrose phosphorylase by a conventionally known general preparation method (for example, chromatography, dialysis, precipitation method, etc.), there is a disadvantage that sucrose phosphorylase is easily denatured. Alternatively, there are drawbacks that a great amount of man-hours are required for column pretreatment or washing, or that extremely expensive equipment is required for industrial mass production. For this reason, in the prior art, there was no method for purifying sucrose phosphorylase at a practical cost on an industrial mass production scale.
[0011]
[Problems to be solved by the invention]
The present invention is intended to solve the above-described problems and aims to provide a simple method for preparing sucrose phosphorylase.
[0012]
[Means for Solving the Problems]
The method for preparing a sucrose phosphorylase preparation of the present invention includes a step of heating a solution containing sucrose phosphorylase and sucrose under conditions where an enzyme reaction by sucrose phosphorylase does not substantially occur.
[0013]
In one embodiment, the sucrose phosphorylase preparation may have an improved specific activity compared to the sucrose phosphorylase prior to heating.
[0014]
In one embodiment, the solution may be substantially free of inorganic phosphoric acid.
[0015]
In one embodiment, the concentration of sucrose in the solution can be 1.5-50%.
[0016]
In one embodiment, the solution can be prepared by adding sucrose to a microbial extract containing sucrose phosphorylase or a crude product thereof.
[0017]
In one embodiment, the concentration of sucrose in the solution can be 4-30%.
[0018]
In one embodiment, the concentration of sucrose in the solution can be 8-30%.
[0019]
In one embodiment, the concentration of sucrose in the solution can be 8-25%.
[0020]
In one embodiment, the temperature of the solution in the heating step may be a temperature at which 50% or more of the activity of the sucrose phosphorylase contained in the solution before heating remains when the solution is heated for 30 minutes. .
[0021]
In one embodiment, the temperature can be between 40 ° C and 90 ° C.
[0022]
In one embodiment, the temperature can be between 50 ° C and 80 ° C.
[0023]
In one embodiment, the temperature can be between 55 ° C and 70 ° C.
[0024]
In one embodiment, the sucrose phosphorylase can retain 50% or more of the activity of the sucrose phosphorylase prior to heating when heated at 55 ° C. for 30 minutes in the presence of 4% sucrose.
[0025]
In one embodiment, the sucrose phosphorylase is Streptococcus ( Streptococcus ) It is derived from the genus bacteria.
[0026]
In one embodiment, the sucrose phosphorylase is Streptococcus mutans ( Streptococcus mutans ) Is from.
[0027]
In one embodiment, the sucrose phosphorylase is Streptococcus thermophilus ( Streptococcus thermophilus ) It can be derived from.
[0028]
In one embodiment, the sucrose phosphorylase is Streptococcus pneumoniae ( Streptococcus pneumoniae ) It can be derived from.
[0029]
In one embodiment, the sucrose phosphorylase can be produced from a recombinant mesophilic bacterium. In a preferred embodiment, the sucrose phosphorylase can be produced from recombinant E. coli or recombinant Bacillus subtilis.
[0030]
The sucrose phosphorylase preparation of the present invention is prepared by the above method and has a specific activity of 30 U / mg or more.
[0031]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0032]
<Material of sucrose phosphorylase preparation>
The sucrose phosphorylase preparation of the present invention has good specific activity, and in a preferred embodiment, the specific activity is 30 units / mg or more. As used herein, “sucrose phosphorylase preparation” refers to liquid, semi-solid and solid containing sucrose phosphorylase. Therefore, an enzyme other than sucrose phosphorylase, a solvent, an additive, and the like may be included as long as the state of sucrose phosphorylase is not lost. The sucrose phosphorylase preparation is typically from 0.0001% to 40% by weight, preferably from 0.001% to 20% by weight, more preferably from 0 to 40% by weight, where the weight of the preparation is 100. 0.005% by weight to 10% by weight.
[0033]
As used herein, “specific activity” refers to enzyme activity per mg of protein in a sucrose phosphorylase solution. Therefore, when calculating the specific activity, the proteins that settle to the bottom of the preparation container are not taken into account. For this reason, for example, when insoluble protein precipitates in the preparation, the enzyme activity per 1 mg of protein contained in the sucrose phosphorylase preparation excluding the insoluble protein is referred to as specific activity.
[0034]
The enzyme unit of sucrose phosphorylase is obtained, for example, by the following method.
[0035]
Mix 25 μl of 10% sucrose and 20 μl of 500 mM phosphate buffer (pH 7.0). To this mixed solution, 5 μl of an appropriately diluted enzyme solution from which insoluble protein has been removed is added and stirred to obtain a reaction system. The reaction system is brought to an appropriate temperature (for example, Streptococcus mutans ( Streptococcus mutans ) In the case of sucrose phosphorylase derived from, for example, 50 ° C., Leuconostok ( Leuconostoc ) In the case of sucrose phosphorylase derived from, for example, it is maintained at 37 ° C. for 20 minutes, and then heated at 100 ° C. for 5 minutes to stop the reaction. Thereafter, glucose-1-phosphate in the reaction product is quantified.
[0036]
Glucose-1-phosphate is quantified, for example, by the following method. 300 μl of measurement reagent (200 mM Tris-HCl (pH 7.0), 3 mM NADP, 15 mM magnesium chloride, 3 mM EDTA, 15 μM glucose-1,6-diphosphate, 6 μg / ml phosphoglucomutase, 6 μg / ml glucose-6 To the phosphate dehydrogenase), 600 μl of a solution containing appropriately diluted glucose-1-phosphate is added and stirred to obtain a reaction system. After holding this reaction system at 30 ° C. for 30 minutes, the absorbance at 340 nm is measured using a spectrophotometer. Absorbance is measured in the same way using sodium glucose-1-phosphate having a known concentration, and a standard curve is prepared. The absorbance obtained in the sample is applied to this standard curve to determine the glucose-1-phosphate concentration in the sample. Usually, the activity to produce 1 μmol of glucose-1-phosphate per minute is defined as 1 unit (U).
[0037]
The main raw materials of the sucrose phosphorylase preparation of the present invention are, for example, sucrose phosphorylase, sucrose, and a solvent in which it is dissolved.
[0038]
(1) Sucrose phosphorylase:
As used herein, “sucrose phosphorylase” refers to any enzyme that undergoes phosphorolysis by transferring the α-glucosyl group of sucrose to phosphoric acid. The reaction catalyzed by sucrose phosphorylase is shown by the following formula:
[0039]
[Chemical 1]
Sucrose phosphorylase is naturally contained in various microorganisms. Examples of microorganisms that produce sucrose phosphorylase include Leuconostok Mecenteroides ( Leuconostoc mesenteroides ) , Streptococcus thermophilus ( Streptococcus thermophilus ) , Streptococcus mutans ( Streptococcus mutans ) , Streptococcus pneumoniae ( Streptococcus pneumoniae), Pseudomonas species ( Pseudomonas sp. ) , Clostridium species ( Clostridium sp. ) , Pullulia Pullance ( Pullularia pullulans ) , Acetobacter xylinum ( Acetobacter xylum ) , Agrobacterium species ( Agrobacterium sp. ) , Synecococcus species ( Synecoccus sp. ) , E. coli ( E. coli ) , Aspergillus niger ( Aspergillus niger ) , Moniria Sitophila ( Monilia sitophila ) , Sclerotinea Esserothiolum ( Sclerotinea escerioorum ) ,and Chlamydomonas species ( Chlamydomonas sp. ) However, it is not limited to these.
[0040]
The sucrose phosphorylase can be derived from any microorganism that produces sucrose phosphorylase (eg, a bacterium that produces sucrose phosphorylase). Sucrose phosphorylase preferably has a certain degree of heat resistance. Sucrose phosphorylase is preferred as it has higher heat resistance when present alone. For example, when heated at 55 ° C. for 30 minutes in the presence of 4% sucrose, it preferably retains 50% or more of the activity of sucrose phosphorylase before heating. Sucrose phosphorylase is preferably Streptococcus ( Streptococcus ) Derived from bacteria of the genus, more preferably Streptococcus mutans ( Streptococcus mutans ) , Streptococcus thermophilus ( Streptococcus thermophilus ) Or Streptococcus pneumoniae ( Streptococcus pneumoniae ) Is from.
[0041]
In this specification, the phrase “derived from” a microorganism does not mean that the enzyme is isolated directly from the microorganism, but that the enzyme is obtained by utilizing the microorganism in some form. Say. For example, when an enzyme gene of the microorganism is introduced into E. coli and the enzyme is isolated from the E. coli, the enzyme is said to be “derived” from the microorganism.
[0042]
The sucrose phosphorylase used in the present invention can be directly isolated from the microorganisms that produce sucrose phosphorylase existing in nature as described above. The sucrose phosphorylase used in the present invention may be isolated from a microorganism that has been genetically modified using a gene encoding sucrose phosphorylase isolated from these microorganisms.
[0043]
The sucrose phosphorylase used in the method of the present invention can be prepared, for example, as follows. First, a microorganism that produces sucrose phosphorylase is cultured. This microorganism may be a microorganism that directly produces sucrose phosphorylase. Alternatively, a gene encoding sucrose phosphorylase may be cloned, a microorganism advantageous for sucrose phosphorylase expression may be recombined with the obtained gene to obtain a recombinant microorganism, and sucrose phosphorylase may be obtained from the obtained microorganism. .
[0044]
A microorganism used for gene recombination with a sucrose phosphorylase gene can be easily selected in consideration of various conditions such as ease of expression of sucrose phosphorylase, ease of culture, speed of growth, and safety. The microorganism used for gene recombination is preferably a microorganism that does not substantially produce a water-soluble protein having higher heat resistance than sucrose phosphorylase, or produces only at a low level. This is because such a heat-resistant water-soluble protein is unlikely to be denatured in the heating step of the present invention and is likely to be present in the solution as a contaminating protein after heating. Therefore, for gene recombination, it is preferable to use mesophilic bacteria such as Escherichia coli or Bacillus subtilis. A mesophilic bacterium refers to a bacterium that grows in a medium temperature range, and generally refers to a bacterium having an optimum growth temperature of about 20 ° C to about 40 ° C. Since sucrose phosphorylase preferably does not contain amylase and phosphatase as contaminants, it is more preferable to use a microorganism that does not produce amylase and phosphatase or expresses it at a low level for genetic recombination. Sucrose phosphorylase produced from mesophilic bacteria such as recombinant Escherichia coli or Bacillus subtilis is substantially free of thermostable water-soluble proteins and is therefore preferred for use in the method of the present invention.
[0045]
Genetic recombination of microorganisms with cloned genes can be performed according to methods well known to those skilled in the art. When using a cloned gene, it is preferred that this gene be operably linked to a constitutive or inducible promoter. “Operably linked” means that a promoter and a gene are linked so that expression of the gene is regulated by the promoter. When using an inducible promoter, culturing is preferably performed under inducing conditions. Various inducible promoters are known to those skilled in the art.
[0046]
The cloned gene can also be linked to a signal peptide so that the sucrose phosphorylase produced is secreted outside the cell. Signal peptides are known to those skilled in the art.
[0047]
A person skilled in the art can appropriately set conditions for culturing microorganisms to produce sucrose phosphorylase. A medium suitable for culturing microorganisms, conditions for induction suitable for each inducible promoter, and the like are known to those skilled in the art.
[0048]
After incubation for an appropriate time, sucrose phosphorylase is recovered from the culture medium. When the produced sucrose phosphorylase is secreted outside the cells, sucrose phosphorylase can be obtained in the supernatant by removing the cells of the microorganisms by centrifugation. When the sucrose phosphorylase produced in the microbial cells is not secreted outside the microbial cells, the microorganisms are crushed by a treatment such as ultrasonic treatment, mechanical crushing, and chemical crushing to obtain a microbial crushing solution. In the method of the present invention, the microorganism disrupting solution may be used without being purified. The microbial disruption solution can then be centrifuged to remove bacterial debris and the supernatant obtained. From these supernatants obtained, the enzyme of the present invention was converted to ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl chromatography, It can be recovered by well known methods including apatite chromatography and lectin chromatography. The recovered product can be purified as necessary.
[0049]
The concentration of sucrose phosphorylase contained in the solution is typically 0.00001 to 40% by weight, preferably 0.0001 to 20% by weight, more preferably 0.001 based on the weight of the solution. Weight to 10% by weight. If the weight of sucrose phosphorylase is too large, it may be difficult to increase the specific activity by the method of the present invention. If the amount used is too small, the recovery of sucrose phosphorylase activity may be low.
[0050]
(2) Sucrose:
Sucrose is C ₁₂ H _{twenty two} O ₁₁ And a disaccharide having a molecular weight of about 342. Sucrose is present in every plant that has photosynthetic ability. Sucrose may be isolated from plants or chemically synthesized. In view of cost, it is preferable to isolate sucrose from plants. Examples of plants containing a large amount of sucrose include sugar cane and sugar beet. Sugar cane contains about 20% sucrose in the juice. Sugar beet contains about 10-15% sucrose in the juice.
[0051]
The sucrose used in the method of the present invention is preferably pure. However, any other impurities may be included as long as the effect of the sucrose of the present invention is not inhibited. For example, in the present invention, raw sugar can be used.
[0052]
The concentration of sucrose contained in the solution is typically 4-30%, preferably 8-30%, more preferably 8-25%, especially considering the processing of large amounts of sucrose phosphorylase solution. . In the present specification, the concentration of sucrose is Weight / Volume, that is,
(Weight of sucrose (g)) × 100 / (volume of solution (ml))
Calculate with If the sucrose is too heavy, it may be difficult to increase the specific activity by the method of the present invention. If the amount used is too small, the recovery rate of sucrose phosphorylase activity may be low, and the specific activity may be difficult to increase.
[0053]
(3) Solvent:
The solvent for dissolving sucrose phosphorylase and sucrose can be any solvent as long as it does not impair the enzyme activity of sucrose phosphorylase. A typical solvent is water. The solvent may be water in the microorganism disruption liquid obtained accompanying the sucrose phosphorylase when preparing the sucrose phosphorylase.
[0054]
The water may be any of soft water, intermediate water and hard water. Soft water refers to water having a hardness of 20 ° or more, intermediate water refers to water having a hardness of 10 ° to less than 20 °, and hard water refers to water having a hardness of less than 10 °. The water is preferably soft water or intermediate water, more preferably soft water.
[0055]
(4) Other ingredients:
The sucrose phosphorylase and the solution containing sucrose may contain any substance as long as it does not interfere with the interaction between sucrose phosphorylase and sucrose. Examples of such substances include buffers, components of microorganisms that produce sucrose phosphorylase, salts (such as NaCl) and media components.
[0056]
<Preparation of sucrose phosphorylase preparation>
The sucrose phosphorylase preparation is prepared by a method comprising heating a solution containing sucrose phosphorylase and sucrose under conditions in which an enzymatic reaction by sucrose phosphorylase does not substantially occur.
[0057]
The solution can be obtained, for example, by dissolving pure sucrose in a solution containing sucrose phosphorylase (for example, a supernatant containing sucrose phosphorylase). Alternatively, it can be obtained by mixing a solution containing sucrose phosphorylase and a solution containing sucrose (eg, sugar water, sugarcane juice concentrate, etc.). In one preferred embodiment, sucrose is added to the microbial extract or a crude product thereof. Here, the microorganism extract refers to a liquid extracted from a microorganism having the ability to produce sucrose phosphorylase and containing sucrose phosphorylase. The crushed liquid and the culture supernatant described above are included in the microorganism extract herein. As the extraction method, any known method can be used. Here, the crudely purified product of the microbial extract refers to a product obtained by partially or substantially removing any substance other than sucrose phosphorylase from the microbial extract. The crude purification of microorganisms can be performed by methods well known in the art. Examples of the crude purification method include concentration, insoluble matter removal by membrane fractionation, and ammonium sulfate fractionation.
[0058]
The crude product typically has a sucrose phosphorylase purity of about 0.00001% to about 40%, preferably about 0.0001% to about 20%, more preferably about 0.001% to about 10%. is there.
[0059]
The substance to be removed by rough purification is, for example, water when roughly purified by concentration, and when insoluble matter is removed by the membrane fraction, for example, cell debris such as cell wall and cell membrane, nucleic acid, When the protein is other than sucrose phosphorylase and is roughly purified by ammonium sulfate fractionation, for example, cell debris such as cell wall and cell membrane, nucleic acid, protein other than sucrose phosphorylase, and the like.
[0060]
The “conditions in which an enzyme reaction by sucrose phosphorylase does not substantially occur” are conditions in which a phosphorylation reaction catalyzed by sucrose phosphorylase does not substantially occur. For example, it refers to a condition in which a phosphoric acid reaction does not occur at all or very little. For example, the conditions are such that the concentration of phosphoric acid does not exceed the Km value for phosphoric acid of sucrose phosphorylase. Examples of such conditions include conditions in which there is only a small amount of inorganic phosphoric acid, conditions in which the reaction equilibrium is greatly inclined in the opposite direction to the phosphoric acid reaction (for example, either glucose-1-phosphate or fructose). And the like are present in large amounts compared to sucrose). Therefore, it is preferable to carry out the method of the present invention without adding phosphoric acid.
[0061]
The resulting solution is heated. The temperature of the solution in this heating step is such that when this solution is heated for 30 minutes, 50% or more, more preferably 80% or more of the activity of sucrose phosphorylase contained in this solution before heating remains. Is preferred. This temperature is selected according to the species of sucrose phosphorylase used, and is generally preferably 50 ° C to 80 ° C, more preferably 55 ° C to 70 ° C. For example, Streptococcus mutans ( S. mutans ) In the case of sucrose phosphorylase, this temperature is preferably 50 ° C to 60 ° C.
[0062]
The heating time can be set at an arbitrary time in consideration of the heating temperature as long as the activity of sucrose phosphorylase is not significantly impaired. The heating time is typically 10 minutes to 90 minutes, more preferably 30 minutes to 60 minutes.
[0063]
The heating may be performed using any means, but it is preferable to perform the heating while stirring so that the heat is uniformly transferred to the entire solution. The solution is stirred, for example, in a beaker. A water bath is mentioned as an apparatus used for a heating.
[0064]
Many contaminant proteins other than sucrose phosphorylase present in the solution can denature and precipitate during heating. When sucrose phosphorylase coexists with sucrose, thermal stability is increased. This mechanism is unknown. However, sucrose has some effect on sucrose phosphorylase in some way, for example, the coexisting sucrose forms a complex with sucrose phosphorylase and has a more stable structure than single sucrose phosphorylase and contaminating proteins This is considered to stabilize sucrose phosphorylase. Therefore, even when heated to a temperature at which single sucrose phosphorylase and contaminating protein denature, sucrose phosphorylase in the presence of sucrose is still considered to be undenatured. Of course, the increase in thermal stability is not necessarily due to such a mechanism, but may be due to other mechanisms. As long as a stabilizing effect is obtained by the method described in the claims of the present application, the method is included in the present invention regardless of the mechanism of the stabilization.
[0065]
The precipitated contaminating protein can be removed by a method known in the art, if necessary. For example, precipitated contaminating proteins can be removed by centrifuging the solution to remove the precipitate or by filtering the solution through a suitable membrane. However, even if precipitated contaminating proteins are present, if there is no problem in performing the subsequent steps, the subsequent steps may be performed without removing the precipitate.
[0066]
In this way, the sucrose phosphorylase preparation of the present invention is obtained. The sucrose phosphorylase preparation of the present invention preferably has an improved specific activity compared to the sucrose phosphorylase solution before heating. The specific activity of the sucrose phosphorylase preparation of the present invention is typically 30 U / mg or more, preferably 40 U / mg to 200 U / mg, more preferably 40 U / mg to 100 U / mg.
[0067]
The mechanism by which the sucrose phosphorylase preparation of the present invention has an improved specific activity compared to the sucrose phosphorylase solution before heating is not known. However, it is thought that this increase in specific activity can be mainly attributed to the precipitation of contaminating proteins. In addition, it is considered that the increase in specific activity may be due to protein denaturation that inhibits the phosphorylation reaction by sucrose phosphorylase. That is, it is considered that it may be caused by denaturation of a protein having an action of depriving sucrose phosphorylase from at least one of sucrose and phosphoric acid, which are substrates of sucrose phosphorylase. Furthermore, it is considered that the activity of sucrose phosphorylase is improved by the action of sucrose on sucrose phosphorylase in some manner.
[0068]
The sucrose phosphorylase preparation obtained by the method of the present invention can be used for conventionally known sucrose phosphorylase. For example, it can also be used for quantitative analysis of inorganic phosphate or sucrose. It can also be a diagnostic enzyme.
[0069]
The sucrose phosphorylase preparation obtained by the method of the present invention can be used for a reaction at a higher temperature than sucrose phosphorylase in an untreated state. The sucrose phosphorylase preparation of the present invention may be used as an enzyme solution after heat treatment without any operation, or may be used after removing contaminating proteins that have precipitated. In addition, when a sucrose phosphorylase preparation with higher purity is required, the sucrose phosphorylase preparation of the present invention can be subjected to a purification step such as column chromatography. In this case, the sucrose phosphorylase preparation according to the method of the present invention is advantageous because the load on the purification step such as column chromatography is small. The sucrose phosphorylase preparation of the present invention may be stored after being powdered by a process such as precipitation or lyophilization. When the sucrose phosphorylase preparation of the present invention is industrially used, it is preferable from the viewpoint of cost to use it without performing any operation.
[0070]
Since the sucrose phosphorylase preparation of the present invention is less contaminated than the untreated state, when the sucrose phosphorylase preparation of the present invention is used for amylose synthesis, the maximum molecular weight of the resulting amylose is increased. Is obtained.
[0071]
In one preferred embodiment, a sucrose phosphorylase preparation is prepared by heating sucrose phosphorylase in the presence of sucrose, and then sucrose, phosphate, oligosaccharide and glucan phosphorylase are added to initiate the amylose synthesis reaction. Here, before the start of the amylose synthesis reaction, the precipitated contaminating protein may be removed if necessary, but performing amylose synthesis without removing the precipitate can simplify the entire process and reduce costs. It is preferable in terms.
[0072]
The preparation method of the sucrose phosphorylase preparation of the present invention has a lower ratio of denaturing sucrose phosphorylase than other general purification methods. In addition, since the necessary processing procedure is very simple and expensive equipment is not required, sucrose phosphorylase can be obtained efficiently at low cost. Therefore, it has an advantage that sucrose phosphorylase can be obtained at a low cost as compared with other general purification methods.
[0073]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited only to the following examples.
[0074]
(Production Example 1 Preparation Method of Potato Tuber-Derived Glucan Phosphorylase)
Peel 1.4 kg of commercially available potato tubers. Grind the peeled tubers with a juicer to obtain a ground liquid. Subsequently, this ground liquid is filtered through gauze to obtain a filtrate. Tris buffer (pH 7.0) is added to the filtrate to a final concentration of 100 mM to obtain an enzyme solution. This enzyme solution is heated in a 55 ° C. water bath for another 10 minutes after the solution temperature reaches 50 ° C. After the heating, the enzyme solution is centrifuged at 8,500 rpm for 20 minutes using a centrifuge (manufactured by Beckman, AVANTI J-25I) to remove insoluble proteins and the like to obtain a supernatant.
[0075]
To the obtained centrifugal supernatant, ammonium sulfate is added so as to be 100 g / L, and then left at 4 ° C. for 2 hours to precipitate proteins. Next, using a centrifuge (AVANTI J-25I, manufactured by Beckman Co., Ltd.), centrifugation is performed at 8,500 rpm for 20 minutes to remove insoluble proteins and the like, and a supernatant is obtained. Further, ammonium sulfate is added to the obtained supernatant to a final concentration of 250 g / L, and then left at 4 ° C. for 2 hours to precipitate proteins. Subsequently, it is centrifuged at 8,500 rpm for 20 minutes using a centrifuge (manufactured by Beckman, AVANTI J-25I) to recover insoluble protein.
[0076]
The recovered insoluble protein is suspended in 150 ml of 25 mM Tris buffer (pH 7.0). Dialyze the suspended enzyme solution against the same buffer overnight. The dialyzed sample is adsorbed on an anion exchange exchange resin Q-Sepharose (Pharmacia) previously equilibrated and washed with a buffer containing 200 mM sodium chloride. Subsequently, elution is carried out with a buffer containing 400 mM sodium chloride, and the eluate is collected to obtain a partially purified potato tuber-derived glucan phosphorylase-containing solution.
[0077]
Depending on the potato purchased, it becomes a glucan phosphorylase-containing solution that can be used in the present invention at this stage, but often requires further purification. If necessary, fractionation by gel filtration chromatography using Sephacryl S-200HR (Pharmacia), etc., or fractionation by hydrophobic chromatography using Phenyl-TOYOPARL 650M (Tosoh Corporation), etc. Thus, a purified potato glucan phosphorylase-containing solution can be obtained.
[0078]
(Example 1: Streptococcus mutans ( S. mutans ) Preparation of recombinant E. coli cell disruption solution containing sucrose phosphorylase and preparation of sucrose phosphorylase enzyme solution from the cell disruption solution)
(1.1 Recombination Streptococcus mutans ( Streptococcus mutans ) (Method for preparing cell disruption solution containing sucrose phosphorylase)
Streptococcus mutans ( Streptococcus mutans ) The sucrose phosphorylase gene (Ferretti, JJ et al., Ingbritt. Infect. Immun. 56: 1585-88) was selected from the selectable marker gene Amp. ^r , Tet ^r And incorporated into pKK388-1 to obtain a plasmid pKK388-SMSP. In this plasmid, the sucrose phosphorylase gene was operably linked under the control of an isopropyl-β-D-thiogalactopyranoside (IPTG) inducible promoter. This plasmid was introduced into E. coli TG-1 (manufactured by STRATAGENE) by the competent cell method. The Escherichia coli was plated on a plate containing LB medium containing antibiotics ampicillin and IPTG and cultured overnight at 37 ° C. By selecting E. coli grown on this plate, E. coli into which the sucrose phosphorylase gene was introduced was obtained. It was confirmed by analyzing the sequence of the introduced gene that the obtained Escherichia coli contained the sucrose phosphorylase gene. In addition, it was confirmed by activity measurement that the obtained Escherichia coli expressed sucrose phosphorylase.
[0079]
This Escherichia coli was inoculated into 1 liter of LB medium containing antibiotics ampicillin and tetracycline, and cultured with shaking at 37 ° C. for 6 to 7 hours while shaking at 120 rpm. Then, IPTG was added to this medium so that it might become 0.04 mM, and it culture | cultivated by shaking at 30 degreeC for further 18 hours. Next, this culture solution was centrifuged at 5,000 rpm for 5 minutes to collect E. coli cells. The obtained bacterial cells were suspended in 50 ml of 20 mM Tris-HCl buffer (pH 7.0), and then disrupted by ultrasonic treatment to obtain 50 ml of bacterial cell disruption solution. This crushed liquid contained sucrose phosphorylase having a specific activity of about 15 U / mg.
[0080]
(1.2 Preparation of sucrose phosphorylase by heating at 55 ° C. in the presence of sucrose)
The microbial cell disruption solution obtained in 1.1 above is dissolved by adding sucrose, and the final concentration of sucrose is 4%, 8%, 12%, 16%, 20% based on the solution after dissolution, respectively. 25% or 30% solutions were obtained. A cell disruption solution to which sucrose was not added was used as a control (0% sucrose). These solutions were heated in a 55 ° C. water bath. Sampling was performed at the start of heating (0 minutes), 30 minutes, 60 minutes and 90 minutes after the start of heating, and the activity of sucrose phosphorylase was measured according to the method described herein.
[0081]
Based on the measured activity, the residual activity was calculated.
[0082]
Residual activity was calculated as follows:
[0083]
[Expression 1]
Each sample was centrifuged at 12000 rpm for 5 minutes to obtain a supernatant. The amount of protein contained in the supernatant was measured using the Bradford method (Bradford, M., Anal. Biochem., 72, pages 248-254 (1976)). The Bradford method is a colorimetric assay in which a chromogenic substrate is bound to all proteins contained in a solution. Here, a protein assay kit purchased from Nippon Bio-Rad Laboratories was used, and bovine γ globulin was measured as a standard according to the protocol.
[0084]
Specific activity was calculated as follows:
[0085]
[Expression 2]
The results for residual activity are shown in FIG. 1, and the results for specific activity are shown in FIG. As a result, the following was found. When sucrose was not included (0%), the activity decreased to about 10% after heating at 55 ° C. for 30 minutes.
[0086]
In the case of containing 4% or more of sucrose, 50% or more of the activity remained even after heating for 30 minutes. In particular, when 8% or more of sucrose was contained, 80% or more of the activity remained even after heating for 30 minutes.
[0087]
The sucrose phosphorylase specific activity of the extract before heating was about 15 U / mg protein, but when 4 to 20% sucrose was added and heated at 55 ° C. for 30 minutes, it was over 40 U / mg protein. It was shown that the degree of purification was improved. On the other hand, when sucrose was 25% or more, the specific activity after the same treatment remained at about 20 U / mg protein (FIG. 2).
[0088]
In the case of heating at 55 ° C. for 60 minutes, the specific activity was remarkably improved in the case of the extract added with 8 to 25% sucrose, and reached about 60 U / mg protein. On the other hand, when sucrose was 4% or less, a significant decrease in activity was observed, resulting in a decrease in specific activity. Further, when sucrose was 30% or more, no significant specific activity was improved (FIG. 2).
[0089]
In the case of heating at 55 ° C. for 90 minutes, the specific activity was remarkably improved in the case of the extract added with 12 to 25% sucrose, and reached about 70 U / mg protein (FIG. 2).
[0090]
(1.3 Amylose production (molecular weight and yield) using sucrose phosphorylase enzyme solution obtained after heating at 55 ° C. in the presence of sucrose)
In the presence of 4%, 8%, 16%, 20%, 25%, or 30% sucrose, the cell disruption solution obtained in (1.1) is the same as (1.2) above. And heated at 55 ° C. for 30 or 60 minutes. The sucrose phosphorylase enzyme solution obtained after heating is mixed with sucrose, inorganic phosphate, maltotetraose, sucrose phosphorylase, and potato-derived glucan phosphorylase produced in Production Example 1, so that the final concentration is 2% sucrose, A mixed solution of 10 mM inorganic phosphate, 10 μM maltotetraose, 1 U / ml sucrose phosphorylase, and 1 U / ml glucan phosphorylase derived from potato was obtained. This mixture was incubated at 37 ° C. overnight to synthesize amylose. The reaction was stopped by heat treatment. Amylose was produced in the same manner using a non-heated cell disruption solution as a control.
[0091]
Next, the amounts of glucose, fructose and glucose-1-phosphate in the reaction product were measured. Glucose was quantified using a measurement reagent (glucose AR-II) commercially available from Wako Pure Chemical Industries. Fructose was quantified using a measurement kit (F-kit D-glucose / D-fructose) commercially available from Roche. Glucose-1-phosphate was quantified by the following method. First, 300 μl of a measurement reagent (200 mM Tris-HCl (pH 7.0), 3 mM NADP, 15 mM magnesium chloride, 3 mM EDTA, 15 μM glucose-1,6-diphosphate, 6 μg / ml phosphoglucomutase, 6 μg / ml glucose- 6-phosphate dehydrogenase), 600 μl of a solution containing appropriately diluted glucose-1-phosphate was added and stirred to obtain a reaction system. The reaction system was kept at 30 ° C. for 30 minutes, and then the absorbance at 340 nm was measured using a spectrophotometer. Absorbance was measured in the same manner using sodium glucose-1-phosphate having a known concentration, and a standard curve was prepared. The absorbance obtained in the sample was applied to this standard curve, and the glucose-1-phosphate concentration in the sample was determined. The activity for producing 1 μmol of glucose-1-phosphate per minute was defined as 1 unit.
[0092]
The amount of amylose produced was calculated based on the following formula:
[0093]
[Equation 3]
Furthermore, the yield of amylose was calculated based on the following formula:
[0094]
[Expression 4]
In the heating time of 30 minutes, when 8-30% of sucrose was contained, the yield of amylose was high compared to the case of non-heating.
[0095]
The molecular weight of amylose was measured using the same reaction solution. The molecular weight of the glucan synthesized in the present invention was measured by the following method. First, the glucan synthesized in the present invention is completely dissolved with 1N sodium hydroxide, neutralized with an appropriate amount of hydrochloric acid, and then about 300 μg of glucan is combined with a differential refractometer and a multi-angle light scattering detector. The weight average molecular weight was determined by subjecting it to filtration chromatography.
[0096]
Specifically, Shodex SB806M-HQ (manufactured by Showa Denko) is used as a column, and a multi-angle light scattering detector (DAWN-DSP, manufactured by Wyatt Technology) and a differential refractometer (Shodex RI-71, manufactured by Showa Denko) are used as detectors. ) Were used in this order. The column was kept at 40 ° C., and 0.1 M sodium nitrate solution was used as an eluent at a flow rate of 1 mL / min. The signals obtained were collected using data analysis software (trade name ASTRA, manufactured by Wyatt Technology), and analyzed using the same software to determine the weight average molecular weight.
[0097]
The product amylose molecular weight results for the cell disruption solution heated at 55 ° C. for 30 minutes are shown in FIG. 3, the yield result in FIG. 5, and the cell disruption solution heated at 55 ° C. for 60 minutes. The results of the molecular weight of the product amylose are shown in FIG. As a result, the following was found.
[0098]
In the heating time of 30 minutes, when 4 to 25% of sucrose was contained, high molecular weight amylose could be synthesized as compared with the case of non-heating. When sucrose was contained in an amount of 8 to 25%, particularly high molecular weight amylose could be synthesized. The amylose yield was high when 8-30% sucrose was included.
[0099]
In the heating time of 60 minutes, when sucrose was contained in an amount of 8 to 30%, high molecular weight amylose could be synthesized as compared with the case of non-heating. When sucrose was contained in an amount of 16 to 25%, particularly high molecular weight amylose could be synthesized.
[0100]
The molecular weight and yield of the product amylose tends to be smaller due to impurities in sucrose phosphorylase, especially amylase activity. Therefore, this result shows that sucrose phosphorylase enzyme solution with reduced impurities could be obtained by heat treatment in the presence of sucrose 4-30%, more preferably 8-30%, and still more preferably 8-25%. Is shown.
[0101]
(1.4 Specific activity improvement effect by heating at 60 ° C. in the presence of sucrose)
The cell disruption solution obtained in (1.1) was heated at 60 ° C. for 30 or 60 minutes in the presence of 20% or 30% sucrose in the same manner as in (1.2) above. Amylose was produced in the same manner as in (1.3) above using the sucrose phosphorylase enzyme solution obtained after heating. As a result, about 1.5 times higher molecular weight amylose was obtained compared to the case of using a non-heated cell disruption solution.
[0102]
(Example 2: Streptococcus mutans ( S. mutans ) Preparation of cell disruption solution of recombinant Bacillus subtilis containing sucrose phosphorylase and preparation of sucrose phosphorylase from the cell disruption solution)
B. subtilis was used instead of E. coli used in Example 1, pWH1520 (MoBiTec GmbH, Germany) capable of replicating in both B. subtilis and E. coli was used as a plasmid, and a tetracycline resistance gene was used as a selectable marker in B. subtilis. And similar to Example 1 except that xylose is used as the inducer at a final concentration of 0.5% by weight, Streptococcus mutans ( S. mutans ) A cell disruption solution of recombinant Bacillus subtilis containing the derived sucrose phosphorylase was prepared.
[0103]
The obtained bacterial cell disruption solution was heated at 55 ° C. for 30 minutes or 60 minutes in the presence of 4 to 30% sucrose in the same manner as in (1.2) above to obtain a preparation. The resulting preparation was tested for amylose production according to (1.3) and (1.4) above. As a result, it was found that a sucrose phosphorylase enzyme solution suitable for amylose synthesis was obtained.
[0104]
On the other hand, as a result of heating the cell disruption solution of Bacillus subtilis without adding sucrose at 55 ° C. for 30 minutes or 60 minutes, the activity of the obtained solution was lowered. That is, a sucrose phosphorylase enzyme solution suitable for amylose synthesis could not be obtained.
[0105]
( Leuconostok Mecenteroides ( L. mesenteroides ) Preparation of sucrose phosphorylase from cell disruption solution containing sucrose phosphorylase)
Leuconostok Mecenteroides ( L. mesenteroides ) NCIMB8699 strain was cultured in 1 liter of LM medium (2% sucrose, 2% yeast extract, 1% peptone, 2% dipotassium hydrogen phosphate, 0.02% NaCl) at 25 ° C. for 24 hours. Subsequently, centrifugation was performed at 8000 rpm, and the bacterial cells were collected. The obtained bacterial cells were treated with lysozyme and further treated with ultrasound to obtain a disrupted bacterial cell solution containing sucrose phosphorylase.
[0106]
In this cell disruption solution, sucrose is added and dissolved so that the final concentration is 4%, 8%, 12%, 16%, 20%, 25% or 30%, respectively, based on the solution after dissolution, A solution was obtained. A cell disruption solution to which sucrose was not added was used as a control (0% sucrose). These solutions were heated in a 50 ° C. water bath for 30 or 60 minutes. The activity of sucrose phosphorylase was measured according to the method described herein. As a result, a sucrose phosphorylase enzyme solution suitable for amylose synthesis was obtained when heated in the presence of 4-30% sucrose.
[0107]
On the other hand, when sucrose was not added, the obtained solution did not have sufficient activity. That is, a sucrose phosphorylase enzyme solution suitable for amylose synthesis could not be obtained.
[0108]
( Leuconostok Mecenteroides ( L. mesenteroides ) Preparation of sucrose phosphorylase from bacterial cell extract containing sucrose phosphorylase (E. coli extract model)
From recombinant E. coli Leuconostok Mecenteroides ( L. mesenteroides ) Assuming that sucrose phosphorylase was prepared, a model cell disruption solution was prepared by adding the cell disruption solution of Escherichia coli TG-1 to high-purity sucrose phosphorylase manufactured by Oriental Yeast.
[0109]
In this model cell disruption solution, sucrose is added and dissolved so that the final concentration is 4%, 8%, 12%, 16%, 20%, 25% or 30% based on the solution after dissolution, A solution was obtained. A cell disruption solution to which sucrose was not added was used as a control (0% sucrose). These solutions were heated in a 50 ° C. water bath for 30 or 60 minutes. The activity of sucrose phosphorylase was measured according to the method described herein. As a result, a sucrose phosphorylase enzyme solution suitable for amylose synthesis was obtained when heated in the presence of 4-30% sucrose.
[0110]
On the other hand, when sucrose was not added, the obtained solution did not have sufficient activity. That is, a sucrose phosphorylase enzyme solution suitable for amylose synthesis could not be obtained.
[0111]
(Comparative Example 1: Effect of fructose on stability of sucrose phosphorylase)
In addition to sucrose, sucrose phosphorylase substrates include fructose, phosphate, and glucose-1-phosphate. The effect of fructose on sucrose phosphorylase stability was examined as follows.
[0112]
Prepared in (1.1) above Streptococcus mutans ( S. mutans ) Fructose was added to and dissolved in a cell disruption solution containing sucrose phosphorylase derived so that the final concentration was 5% or 10%, respectively, to obtain a solution. A cell disruption solution without addition of fructose was used as a control (0% sucrose). As a control, the same concentration of sucrose was also examined. These solutions were heated in a 55 ° C. water bath. Sampling was performed at the start of heating (0 minutes), 30 minutes, 60 minutes and 90 minutes after the start of heating, and the activity of sucrose phosphorylase was measured according to the method described herein.
[0113]
Based on the measured activity, residual activity and specific activity were calculated.
[0114]
The results for residual activity are shown in FIG. As a result, it was found that fructose does not have the effect of stabilizing sucrose phosphorylase like sucrose.
[0115]
(Comparative Example 2: Effect of phosphoric acid on stability of sucrose phosphorylase)
The effect of phosphate on sucrose phosphorylase stability was examined as follows.
[0116]
Prepared in (1.1) above Streptococcus mutans ( S. mutans ) Sodium phosphate was added to the cell disruption solution containing sucrose phosphorylase derived from sodium phosphate so that the final concentrations would be 40 mM, 100 mM, and 400 mM based on the solution after dissolution to obtain a solution. A bacterial cell disruption solution to which sodium phosphate was not added was used as a control (no addition). As a control, the case where 10% sucrose was added was also examined. These solutions were heated in a 55 ° C. water bath. Sampling was performed at the start of heating (0 minutes), 30 minutes, 60 minutes and 90 minutes after the start of heating, and the activity of sucrose phosphorylase was measured according to the method described herein.
[0117]
Based on the measured activity, residual activity and specific activity were calculated.
[0118]
The results for residual activity are shown in FIG. As a result, it was found that sucrose phosphorylase stabilizing effect was not recognized in phosphoric acid as in the case of sucrose.
[0119]
【The invention's effect】
According to the present invention, sucrose phosphorylase preparations and methods for preparing them are provided. According to the method of the present invention, a highly purified sucrose phosphorylase preparation can be easily obtained by removing contaminating proteins and increasing the specific activity. The sucrose phosphorylase preparation of the present invention is suitable for uses such as amylose synthesis, phosphate quantification, and fructose quantification. Since the sucrose phosphorylase preparation of the present invention has a relatively low content of contaminating proteins, the reaction using sucrose phosphorylase is not easily disturbed. In particular, when used for amylose synthesis, there is an advantage that the yield of amylose can be increased and / or the maximum molecular weight of the obtained amylose is increased. Furthermore, since the sucrose phosphorylase preparation of the present invention has a high specific activity, it can reduce the load on column chromatography and can be purified with a smaller column than when the specific activity is low, so that the sucrose phosphorylase can be further purified. Is also suitable.
[Brief description of the drawings]
FIG. 1 is a graph showing the stability of sucrose phosphorylase in the presence of sucrose.
FIG. 2 is a graph showing the stability of sucrose phosphorylase in the presence of sucrose.
FIG. 3 is a graph showing amylose synthesis using sucrose phosphorylase heated in the presence of sucrose.
FIG. 4 is a graph showing amylose synthesis using sucrose phosphorylase heated in the presence of sucrose.
FIG. 5 is a graph showing amylose synthesis using sucrose phosphorylase heated in the presence of sucrose.
FIG. 6 is a graph showing the effect of sucrose or fructose on sucrose phosphorylase stability.
FIG. 7 is a graph showing the effect of phosphoric acid on sucrose phosphorylase stability.

Claims (20)

A method for preparing a sucrose phosphorylase preparation comprising:
Heating a solution comprising sucrose phosphorylase and sucrose under conditions such that the phosphate concentration of the solution does not exceed the Km value for the phosphate of the sucrose phosphorylase .   The method of claim 1, wherein the sucrose phosphorylase preparation has an improved specific activity compared to the sucrose phosphorylase prior to heating. Said solution of an inorganic phosphoric acid that does not contain A method according to claim 1.   The method according to claim 1, wherein the solution is prepared by adding sucrose to a microorganism extract containing sucrose phosphorylase or a crude product thereof.   The method according to claim 1, wherein the concentration of sucrose in the solution is 1.5 to 50%.   The method of claim 1, wherein the concentration of sucrose in the solution is 4-30%.   The method according to claim 1, wherein the concentration of sucrose in the solution is 8-30%.   The method of claim 1, wherein the concentration of sucrose in the solution is 8-25%.   The temperature of the solution in the heating step is a temperature at which 50% or more of the activity of the sucrose phosphorylase contained in the solution before heating remains when the solution is heated for 30 minutes. The method of any one of these.   The method according to claim 9, wherein the temperature is 50 ° C. to 80 ° C.   The method according to claim 9, wherein the temperature is 55 ° C. to 70 ° C.   The sucrose phosphorylase retains at least 50% of the activity of the sucrose phosphorylase before heating when heated at 55 ° C for 30 minutes in the presence of 4% sucrose. The method described in 1. The sucrose phosphorylase is derived from Streptococcus (Streptococcus) bacteria, the method according to any one of claims 1 to 11. The sucrose phosphorylase is derived from Streptococcus mutans (Streptococcus mutans), the method according to any one of claims 1 to 11. The sucrose phosphorylase is derived from Streptococcus thermophilus (Streptococcus thermophilus), The method according to any one of claims 1 to 11. The sucrose phosphorylase is derived from Streptococcus pneumoniae (Streptococcus pneumoniae), The method according to any one of claims 1 to 11.   The method according to any one of claims 1 to 16, wherein the sucrose phosphorylase is produced from a genetically modified mesophilic bacterium.   The method according to any one of claims 1 to 16, wherein the sucrose phosphorylase is produced from genetically modified E. coli.   The method according to any one of claims 1 to 16, wherein the sucrose phosphorylase is produced from genetically modified Bacillus subtilis. A sucrose phosphorylase preparation with a specific activity of 40 U / mg or more, prepared by the method of claim 1.