WO2002097077A1 - Procede de preparation de sucrose-phosphorylase - Google Patents

Procede de preparation de sucrose-phosphorylase Download PDF

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Publication number
WO2002097077A1
WO2002097077A1 PCT/JP2002/005088 JP0205088W WO02097077A1 WO 2002097077 A1 WO2002097077 A1 WO 2002097077A1 JP 0205088 W JP0205088 W JP 0205088W WO 02097077 A1 WO02097077 A1 WO 02097077A1
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sucrose
sucrose phosphorylase
phosphorylase
solution
activity
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PCT/JP2002/005088
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English (en)
Japanese (ja)
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Michiyo Yanase
Hiroki Takata
Takeshi Takaha
Takashi Kuriki
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Ezaki Glico Co., Ltd.
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Publication of WO2002097077A1 publication Critical patent/WO2002097077A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)

Definitions

  • the present invention relates to a method for obtaining a sucrose phosphorylase preparation and to a sucrose phosphorylase preparation.
  • Sucrose phosphorylase is an enzyme that acts on sucrose in the presence of inorganic phosphate to produce glucose-1-phosphate and fructose.
  • Sucrose hospholase can be applied to quantitative analysis of inorganic phosphate or sucrose in combination with, for example, phosphodalcomase and glucose-6-phosphate dehydrogenase. Therefore, sucrose phosphorylase is useful as a diagnostic enzyme and the like.
  • sucrose phosphorylase in combination with phosphorylase, a sugar unit can be linked to a darcan such as amylose, starch, glycogen, etc. by a 1,4,1-darcoside bond to extend the sugar chain.
  • sucrose phosphorylase In order to use sucrose phosphorylase for the above purpose, it is preferable to remove some contaminating enzymes and increase the purity of sucrose phosphorylase to some extent. For example, when sucrose phosphorylase is used for quantifying inorganic phosphate together with phosphodalcomase and glucose 16-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 a 1,4-dalcoside bond, it is preferable to remove amylase in addition to phosphatase. As microorganisms having sucrose phosphorylase, for example, the following are known.
  • sucrose phosphorylase Agrobacterium sp., Synecococcus sp., E. coli, Aspergillus niger, Moniia si tophi la> Sclerotinea escerotiorum, Chlamydomonas sp., Listeria monocytogenes and Streptococcus mitis.
  • conventional methods such as ion exchange chromatography have been used. Have been. So far, there has been no report that a heat treatment step was performed to prepare sucrose phosphorylase.
  • JP-A-2-23866 discloses a method for stabilizing sucrose phosphorylase. In this method, first, sucrose phosphorylase from Leuconostoc mesenteroides is roughly purified.
  • sucrose phosphorylase was rendered thermostable by the addition of sucrose. Furthermore, there was an optimal sucrose concentration in the preparation of sucrose phosphorylase, and it could not be expected at all whether the concentration was higher or lower than that of the sucrose phosphorylase.
  • sucrose phosphorylase is easily denatured 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.).
  • a conventionally known general preparation method for example, chromatography, dialysis, precipitation method, etc.
  • Another disadvantage is that a large amount of man-hour is required for pretreatment or washing of the column, or extremely expensive equipment is required for industrial mass production. For this reason, in the conventional technology, sucrose phosphorylase is used at a practical cost on an industrial mass production scale. There was no purification method. Disclosure of the invention
  • the present invention is intended to solve the above problems, and has as its object to provide a simple method for preparing sucrose phosphorylase.
  • 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 under which enzyme reaction by sucrose phosphorylase does not substantially occur. .
  • the sucrose phosphorylase preparation may have improved specific activity compared to the sucrose phosphorylase before heating.
  • the solution may be substantially free of inorganic phosphoric acid.
  • the concentration of sucrose in the solution may be between 1.5 and 50%.
  • the solution can be prepared by adding sucrose to a microbial extract containing sucrose phosphorylase or a crude product thereof.
  • the concentration of sucrose in the solution can be between 4 and 30%.
  • the concentration of sucrose in the solution can be between 8 and 30%.
  • the concentration of sucrose in the solution can be between 8 and 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 temperature may be between 40 ° C and 90 ° C.
  • the temperature may be 5 (TC to 80 ° C). In one embodiment, the temperature may be between 55X and 70C.
  • the sucrose phosphorylase may retain 50% or more of the activity of the sucrose phosphorylase before heating when heated at 55 for 30 minutes in the presence of 4% sucrose.
  • sucrose phosphorylase is derived from a Streptococcus bacterium.
  • sucrose phosphorylase is from Streptococcus mutans.
  • sucrose phosphorylase can be from Streptococcus thermophilus.
  • sucrose phosphorylase may be from Streptococcus piieu maraud iae.
  • sucrose phosphorylase may be from Streptococcus mitis.
  • the sucrose phosphorylase may be produced from a recombinant mesophile.
  • the sucrose phosphorylase can be produced from a recombinant E. coli or a recombinant B. subtilis.
  • sucrose phosphorylase preparation of the present invention is prepared by the above method, and has a specific activity of 3 OUZ mg or more.
  • 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.
  • Figure 3 shows the results of using sucrose phosphorylase heated in the presence of sucrose. It is a graph which shows the synthesis of mire.
  • 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 flux on sucrose phosphorylase stability.
  • FIG. 7 is a graph showing the effect of inorganic phosphate on sucrose phosphorylase stability.
  • sucrose phosphorylase Materials for preparation of sucrose phosphorylase
  • sucrose phosphorylase preparations of the present invention have good specific activity, and in a preferred embodiment have a specific activity of 30 units Zmg or more.
  • sucrose-phosphorylase preparation refers to liquids, semi-solids, and solids containing sucrose phosphorylase. Therefore, if necessary, enzymes, solvents, additives, etc. other than sucrose phosphorylase may be contained as long as the state of sucrose phosphorylase is not lost.
  • the sucrose phosphorylase preparation typically contains 0.0001% to 40% by weight of sucrose phosphorylase, preferably 0.001% to 100% by weight of the preparation. 20% by weight, more preferably 0.005% by weight to 10% by weight.
  • the term “specific activity” refers to the enzymatic activity per mg of protein in a sucrose phosphorylase solution. Therefore, when calculating the specific activity, the proteins that precipitate at the bottom of the container of the preparation are not taken into account. Therefore, for example, when insoluble protein precipitates in the preparation, sucrose phos The enzymatic activity per mg of protein contained in the holylase preparation is called the specific activity.
  • the enzyme unit of sucrose phosphorylase can be determined, for example, by the following method.
  • sucrose phosphorylase derived from Streptococcus mutans e.g., 50 ° C
  • sucrose phosphorylase derived from Leuconostoc e.g., 37 ° C
  • Glucose-1 monophosphate is quantified, for example, by the following method.
  • 300 1 measurement reagent 200 mM Tris—HC 1 (pH 7.0), 3 mM NADP, 15 mM magnesium chloride, 3 mM EDTA, 15 M glucose—1,6-diphosphate, 6 gZml Phosphodalcomase, 6 zgZm l Dulco-6-phosphate dehydrogenase
  • add an appropriately diluted solution of 600-1 containing glucose-1-phosphate and stir to obtain a reaction system. After keeping the reaction system at 30 ° C for 30 minutes, measure the absorbance at 340 nm using a spectrophotometer.
  • 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.
  • sucrose phosphorylase refers to sucrose whiskers. It refers to any enzyme that transfers phosphorylation by transferring a cosyl group to inorganic phosphate.
  • the reaction catalyzed by sucrose phosphorylase is represented by the following formula: Sucrose + inorganic phosphate ⁇ -D-glucose-1-phosphate + D-fructose
  • Sucrose phosphorylase is naturally contained in various microorganisms. Examples of microorganisms that produce sucrose phosphorylase include Leuconostoc mesenteroides, Streptococcus thermophi lus, Streptococcus mutans, Streptococcus
  • pneumoniae Pseudomonas sp., Clostridium sp., Pul lularia pul lulans, Acetobacter xylinum, Agrobacterium sp., Synecococcus sp., E. coli,
  • Chlamydomonas sp. Listeria monocytogenes and Streptococcus mitis, but are not limited thereto.
  • Sucrose phosphorylase can be from any microorganism that produces sucrose phosphorylase, such as a bacterium that produces sucrose phosphorylase.
  • Sucrose phosphorylase preferably has some degree of thermostability.
  • Sucrose phosphorylase is preferred as it has higher heat resistance when it is present alone.For example, when heated at 55 ° C for 30 minutes in the presence of 4% sucrose, sucrose phosphorylase before heating is preferred. Preferably, it retains 50% or more of its activity.
  • the sucrose phosphorylase is preferably derived from a bacterium belonging to the genus Streptococcus, more preferably from Streptococcus miitans, Streptococcus thermophi lusx Streptococcus pneumoniae or Streptococcus mitis.
  • the term "derived from" a microorganism does not only mean that the enzyme is directly isolated from the microorganism but also that the enzyme can be obtained by utilizing the microorganism in some way. That means. For example, when an enzyme gene of the microorganism is introduced into Escherichia coli and the enzyme is isolated from the Escherichia coli, the enzyme is isolated from the microorganism. It is called “origin”.
  • the sucrose phosphorylase used in the present invention can be isolated directly from a naturally occurring microorganism that produces sucrose phosphorylase 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.
  • 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.
  • the microorganism may be a microorganism that directly produces sucrose phosphorylase.
  • a gene encoding sucrose phosphorylase may be cloned, a microorganism that is advantageous for sucrose phosphorylase expression may be genetically recombined with the obtained gene to obtain a recombinant microorganism, and sucrose phosphorylase may be obtained from the obtained microorganism. .
  • Microorganisms used for genetic recombination with the sucrose phosphorylase gene should take into account various conditions such as easy expression of sucrose phosphorylase, easy cultivation, rapid growth and safety. Can be easily selected.
  • the microorganism used for genetic recombination is preferably a microorganism that does not substantially produce water-soluble proteins having higher thermostability than sucrose phosphorylase or produces them at low levels. 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 as a contaminant protein in the solution after heating. Therefore, it is preferable to use a mesophilic bacterium such as Escherichia coli or Bacillus subtilis for genetic recombination.
  • a mesophilic bacterium refers to a bacterium that grows in a medium temperature range, and generally 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 or expresses amylase and phosphatase at low levels for gene recombination. In recombinant E. coli or Bacillus subtilis Sucrose phosphorylase produced from warm bacteria is preferred for use in the method of the present invention because it is substantially free of thermostable water-soluble proteins.
  • Genetic recombination of the microorganism with the cloned gene 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 an inducible promoter.
  • “Operably linked” means that the promoter and the gene are linked so that expression of the gene is regulated by the promoter.
  • the culturing is preferably performed under inducing conditions.
  • Various inductive motors are known to those skilled in the art.
  • the cloned gene can also be linked to a signal peptide so that the sucrose phosphorylase produced is secreted extracellularly.
  • Signal peptides are known to those skilled in the art.
  • One skilled in the art can appropriately set the conditions for culturing the microorganism to produce sucrose phosphorylase.
  • Media suitable for culturing microorganisms, induction conditions suitable for each inducible promoter, and the like are known to those skilled in the art.
  • sucrose phosphorylase is recovered from the culture.
  • sucrose phosphorylase can be obtained in the supernatant by removing the cells of the microorganism by centrifugation. If the sucrose phosphorylase produced in the cells is not secreted out of the cells, the microorganisms are crushed by a treatment such as ultrasonic treatment, mechanical crushing, or chemical crushing to obtain a crushed microorganism solution. In the method of the present invention, the crushed microorganism solution may be used without purification. The microbial lysate may then be centrifuged to remove bacterial debris and the supernatant obtained.
  • the enzyme of the present invention was subjected to ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography.
  • T-chromatography, hydroxylapatite mouth chromatography and rectal chromatography It can be recovered by well-known methods including tin chromatography. The recovered product can be purified if necessary.
  • the concentration of sucrose phosphorylase in the solution is typically from 0.0001% to 40% by weight, preferably from 0.0001% to 20% by weight, based on the weight of the solution. Preferably it is 0.001% 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 is too small, the recovery of sucrose phosphorylase activity may be low.
  • Sucrose is a disaccharide with a molecular weight of about 342, represented by C H ⁇ Ou. Sucrose is present in all photosynthetic plants. Sucrose may be isolated from plants or chemically synthesized. From the viewpoint of cost, it is preferable to isolate sucrose from plants. Examples of plants containing a large amount of sucrose include sugarcane and sugar beet. Sugar cane contains about 20% sucrose in the juice. Sugar beet contains about 10-15% sucrose in the juice.
  • the sucrose used in the method of the present invention is pure.
  • any other contaminants may be contained as long as the effects of the sucrose of the present invention are not inhibited.
  • a raw sugar can be used in the present invention.
  • the concentration of sucrose in the solution is typically 4-30%, preferably 8-30%, more preferably 8-25, especially considering large volumes of sucrose phosphorylase solution. %.
  • the concentration of sucrose is Weight / Vo1 ume, that is,
  • the solvent that dissolves sucrose phosphorylase and sucrose can be any solvent that does not impair the enzymatic activity of sucrose phosphorylase.
  • a typical solvent is water.
  • the solvent may be water in a crushed microorganism obtained with sucrose phosphorylase when preparing the above sucrose phosphorylase.
  • the water may be any of soft water, intermediate water and hard water.
  • Soft water refers to water having a hardness of at least 20 °
  • intermediate water refers to water having a hardness of at least 10 ° and less than 20 °
  • 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.
  • the sucrose phosphorylase and the solution containing sucrose may contain any substance as long as it does not interfere with the interaction between sucrose hosphorylase and sucrose.
  • examples of such substances include buffers, components of microorganisms that produce sucrose phosphorylase, salts (such as NaCl), and media components.
  • the sucrose phosphorylase preparation is prepared by a method comprising heating a solution containing sucrose phosphorylase and sucrose under conditions under which substantially no enzymatic reaction by sucrose phosphorylase occurs.
  • the solution can be obtained, for example, by dissolving pure sucrose in a solution containing sucrose phosphorylase (eg, a supernatant containing sucrose phosphorylase). Alternatively, it can be obtained by mixing a solution containing sucrose phosphorylase and a solution containing sucrose (for example, sugar water, sugar cane juice concentrate, etc.).
  • the microorganism extract or Sucrose is added to the crude product.
  • the microorganism extract is a liquid extracted from a microorganism having a sucrose phosphorylase-producing ability and refers to a liquid containing sucrose phosphorylase. The crushed liquid and the culture supernatant described above are included in the microbial extract here.
  • the partially purified microorganism extract refers to a product obtained by partially or substantially removing any substance other than sucrose phosphorylase from the microorganism extract.
  • Crude purification of microorganisms can be performed by methods well known in the art. Examples of crude purification methods include concentration, removal of insolubles by membrane fractionation, and ammonium sulfate fractionation.
  • the crude product has a sucrose phosphorylase purity of typically about 0.001% to about 40%, preferably about 0.001% to about 20%, and more preferably about 0.001% to about 20%. 0.001% to about 10%.
  • the substance removed by the crude purification is, for example, water when the crude purification is performed by concentration, and when the insoluble matter is removed by the membrane fraction, for example, cell debris such as a cell wall, a cell membrane, nucleic acid, Proteins other than sucrose phosphorylase, and when roughly purified by ammonium sulfate fractionation, include, for example, cell debris such as cell walls and cell membranes, nucleic acids, and proteins other than sucrose phosphorylase.
  • Constructions under which substantially no enzymatic reaction by sucrose phosphorylase occurs are conditions under which substantially no phosphorylation reaction catalyzed by sucrose phosphorylase occurs. For example, it means that the phosphoric acid reaction does not occur at all or that it occurs very little. For example, a condition in which the concentration of inorganic phosphate does not exceed the Km value of sucrose phosphorylase for inorganic phosphate. Examples of such conditions include conditions in which only a small amount of inorganic phosphoric acid is present, and conditions in which the equilibrium of the reaction is greatly inclined in the opposite direction to the phosphoric acid reaction (for example, either glucose-11-phosphate or fructose). Conditions in which a large amount is present as compared to sucrose). Therefore, the method of the present invention is preferably performed without adding inorganic phosphoric acid.
  • the resulting solution is heated.
  • the temperature of the solution in this heating step is When heated for 30 minutes, the temperature is preferably a temperature at which 50% or more, more preferably 80% or more of the activity of sucrose phosphorylase contained in this solution before heating remains.
  • This temperature is selected according to the species of sucrose phosphorylase used and is generally preferably between 50 and 80, more preferably between 55 and 70 ° C.
  • the temperature is preferably 50 ° C to 60 ° C.
  • the heating time can be set at any time in consideration of the heating temperature, as long as the activity of sucrose phosphorylase is not significantly impaired.
  • the heating time is typically from 10 minutes to 90 minutes, more preferably from 30 minutes to 60 minutes.
  • Heating may be performed by any means, but it is preferable to heat while stirring so that heat is uniformly transmitted to the entire solution.
  • the solution is, for example, placed in a beaker and stirred.
  • An apparatus used for heating includes a water path.
  • sucrose phosphorylase increases thermostability by coexisting with sucrose. The mechanism is unknown.
  • sucrose reacts in some manner with sucrose phosphorylase in some manner, for example, when coexisting sucrose forms a complex with sucrose phosphorylase and has a more stable structure than sucrose phosphorylase alone and contaminating proteins. It is considered that sucrose phosphorylase is stabilized by exerting the action. Therefore, even when heated to a temperature at which single sucrose phosphorylase and contaminating proteins are denatured, sucrose phosphorylase in the presence of sucrose is still considered to be undenatured.
  • Precipitated contaminating proteins can be removed, if necessary, by methods known in the art. 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. If there is no problem in performing the step, subsequent steps may be performed without removing the precipitate.
  • 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 3 O UZmg or more, preferably 40 UZmg to 200 U / mg, more preferably 4 O UZmg to: I 0 O. UZm g.
  • the mechanism by which the sucrose phosphorylase preparations of the present invention have improved specific activity compared to the sucrose phosphorylase solution before heating is unknown. However, it is thought that this increase in specific activity may be mainly due to precipitation of contaminating proteins.
  • the increase in specific activity may be due to denaturation of a protein that inhibits phosphorylation by sucrose phosphorylase. In other words, it is thought that this may be caused by denaturation of a protein having an action of depriving sucrose phosphorylase of at least one of sucrose and inorganic phosphate, which are substrates of sucrose phosphorylase. Furthermore, it is conceivable that sucrose acts on sucrose phosphorylase in some manner to enhance the activity of sucrose phosphorylase.
  • the sucrose phosphorylase preparation obtained by the method of the present invention can be used for applications of conventionally known sucrose phosphorylase. For example, it can be used for quantitative analysis of inorganic phosphate or sucrose. It can be used as a diagnostic enzyme.
  • the sucrose phosphorylase preparation obtained by the method of the present invention can be used for higher temperature reactions as compared with untreated sucrose phosphorylase. it can.
  • the sucrose phosphorylase preparation of the present invention may be used as an enzyme solution without any operation after heat treatment, or may be used after removing precipitated contaminating proteins.
  • the sucrose phosphorylase preparation of the present invention can be subjected to a purification step such as column chromatography.
  • the present invention is advantageous because the sucrose phosphorylase preparation according to the method of the present invention has a small load on a purification step such as column chromatography.
  • the sucrose phosphorylase preparation of the present invention may be stored after being made into a powder by a treatment such as precipitation or lyophilization.
  • a treatment such as precipitation or lyophilization.
  • sucrose phosphorylase preparation of the present invention has less contaminants compared to the untreated state, when the sucrose phosphorylase preparation of the present invention is used for amylose synthesis, the effect that the maximum molecular weight of the obtained amylose is increased. Is obtained.
  • sucrose phosphorylase is heated in the presence of sucrose to prepare a sucrose phosphorylase preparation, and then sucrose, inorganic phosphoric acid, oligosaccharide and glucan phosphorylase are added to initiate the amylose synthesis reaction.
  • the precipitated contaminating protein may be removed if necessary before the start of the amylose synthesis reaction, but performing the amylose synthesis without removing the precipitate simplifies the entire process and reduces costs. It is preferable in terms of.
  • the method for preparing the sucrose phosphorylase preparation of the present invention has a smaller rate of denaturing sucrose phosphorylase than other general purification methods.
  • sucrose phosphorylase can be obtained efficiently at low cost. Therefore, there is an advantage that sucrose phosphorylase can be obtained at a lower cost as compared with other general purification methods.
  • 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 a pre-equilibrated anion exchange resin Q-Sepharose (Pharmacia), and washed with a buffer containing 200 mM sodium chloride. Subsequently, the mixture is eluted with a buffer containing 40 OmM sodium chloride, and the eluate is recovered.
  • glucan phosphorylase-containing solution that can be used in the present invention at this stage, but often requires further purification. If necessary,
  • Example 1 Preparation of a cell lysate of recombinant Escherichia coli containing S. mutans sucrose phosphorylase, and preparation of a sucrose phosphorylase enzyme solution from the cell lysate
  • Streptococcus mutans sucrose phosphorylase gene Frazier tti
  • sucrose phosphorylase gene was operably linked under the control of an isopropyl-j3-D-thiogalactobilanoside (IPTG) inducible promoter.
  • IPTG isopropyl-j3-D-thiogalactobilanoside
  • E. coli was plated on a plate containing LB medium containing the antibiotics ampicillin and IPTG and cultured at 37 ° C overnight. By selecting Escherichia coli grown on this plate, Escherichia coli into which the sucrose phosphorylase gene was introduced was obtained. It was confirmed that the obtained E. coli contains the sucrose phosphorylase gene by analyzing the sequence of the introduced gene. Ma In addition, it was confirmed by activity measurement that the obtained Escherichia coli expressed sucrose phosphorylase.
  • the Escherichia coli was inoculated into 1 liter of LB medium containing the antibiotics ampicillin and tetracycline, and cultured with shaking at 120 rpm at 37 ° C. for 6 to 7 hours. Thereafter, IPTG was added to this medium to a concentration of 0.04 mM, and the cells were cultured with shaking at 30 ° C. for an additional 18 hours. Next, this culture was centrifuged at 5,000 rpm for 5 minutes to collect E. coli cells. The obtained cells were suspended in 50 ml of 20 mM Tris-HCl buffer (PH 7.0) and then disrupted by sonication to obtain 50 ml of a cell disrupted solution. The crushed liquid contained about 15 UZmg of specific activity sucrose phosphorylase.
  • sucrose to the cell lysate obtained in 1.1 above and dissolve it.
  • the final concentration of sucrose is 4%, 8%, 12%, 16% and 20%, respectively, based on the solution after lysis. , 25% or 30% solutions were obtained.
  • a lysate containing no sucrose was used as a control (0% sucrose). These solutions were heated in a 55 ° C water bath. At the start of heating (0 minutes), samples were taken at 30, 60 and 90 minutes after the start of heating, and the activity of sucrose phosphorylase was measured according to the method described herein.
  • the residual activity was calculated based on the measured activity.
  • 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 (Brad ford, M., Anal. Biochem., 72, 248-254 (1976)).
  • the Bradford method is a colorimetric assay that binds a chromogenic substrate to all proteins contained in a solution.
  • a protein assay kit purchased from Nippon Bio-Rad Laboratories Co., Ltd., the measurement was carried out in accordance with the protocol, using siaglopurine as a standard.
  • sucrose phosphorylase specific activity of the extract before heating was about 15 UZmg protein, but when added with 4-20% sucrose and heated at 55 ° C for 30 minutes, it increased to 4 OUZmg protein or more. Thus, it was shown that the degree of purification was increased. On the other hand, when sucrose was 25% or more, the specific activity after the same treatment was only about 2 OUZmg protein (Fig. 2).
  • the bacterial cell lysate obtained in (1.1) above is mixed in the presence of 4%, 8%, 16%, 20%, 25%, or 30% sucrose as in (1.2) above. Heated at 55 ° C for 30 or 60 minutes.
  • the sucrose phosphorylase enzyme solution obtained after heating was mixed with sucrose, inorganic phosphoric acid, maltotetraose, sucrose phosphorylase and the potato-derived glucan phosphorylase produced in Production Example 1 above.
  • a mixed solution having a final concentration of 2% sucrose, 10 mM of inorganic phosphate, 10 M of maltotetraose, 1 UZm1 of sucrose phosphorylase, and 1 UZm1 of glucan phosphorylase derived from potato was obtained.
  • This mixture was subjected to calcination at 37 ° C. to synthesize amylose.
  • the reaction was stopped by heat treatment.
  • Amylose was similarly produced using a non-heated cell lysate as a control.
  • the amounts of glucose, fructose and glucose-1-phosphoric acid in the reaction product were measured.
  • Glucose was quantified using a measuring reagent (glucose AR_II) commercially available from Wako Pure Chemical Industries.
  • Fructose was quantified using a measurement kit (F—kit D—glucose ZD—fructose) commercially available from Roche.
  • Glucose-1 monophosphate was quantified by the following method.
  • 300 1 measurement reagents (20 OmM Tris-HCl (pH 7.0), 3 mM NADP, 15 mM magnesium chloride, 3 mM EDTA, 15 M glucose-1, 6-diphosphate, 6 gml Phosphodalco Mutase, 6 g / m 1 glucose-6-phosphate dehydrogenase)
  • a solution 600 1 containing sue-monophosphate was added and stirred to obtain a reaction system. After keeping the reaction system at 30 ° C for 30 minutes, the absorbance at 340 nm was measured using a spectrophotometer. The absorbance was similarly measured using glucose-11-sodium phosphate with a known concentration, and a standard curve was prepared. The absorbance obtained from the sample was applied to this standard curve to determine the concentration of glucose-1-monophosphate in the sample. The activity to produce 1 zmol glucose-1 monophosphate per minute was defined as 1 unit.
  • Amylose (DIM glucose equivalent)
  • Amylose yield (%) (Amylose (mM glucose equivalent)) ⁇ (Initial sucrose (mM)) X 100 Heating time 30 minutes, the yield of amylose compared to the case of 8-30% sucrose and the case of non-heating Was high.
  • the molecular weight of amylose was measured using the same reaction solution.
  • the molecular weight of glucan synthesized in the present invention was measured by the following method. First, after completely dissolving the glucan synthesized in the present invention with 1 N sodium hydroxide and neutralizing with an appropriate amount of hydrochloric acid, about 300 mg of the glucan was passed through a differential refractometer and a multi-angle light scattering detector. The weight-average molecular weight was determined by subjecting the gel filtration chromatography to the combined use.
  • Fig. 3 shows the results of the molecular weight of the product amylose in the cell lysate heated at 55 ° C for 30 minutes
  • Fig. 5 shows the results of the yield, and the cells heated at 55 ° C for 60 minutes.
  • the results of the molecular weight of the product amylose for the crushed liquid are shown in FIG. As a result, the following was found.
  • the molecular weight and yield of the product amylose tends to be lower due to impurities in the sucrose phosphorylase, especially the amylase activity. Therefore, this result indicates that a heat treatment in the presence of 4 to 30% sucrose, more preferably 8 to 30%, and even more preferably 8 to 25% sucrose can provide a sucrose phosphorylase enzyme solution with reduced impurities. Indicates that it was possible.
  • Amylose was produced in the same manner as in 3). As a result, when unheated cell lysate was used, As a result, high-molecular-weight amylose was obtained, which was about 1.5 times as large as that of the case.
  • Example 2 Preparation of a cell lysate of recombinant Bacillus subtilis containing S. mutans sucrose phosphorylase, and preparation of sucrose phosphorylase from the cell lysate
  • Bacillus subtilis was used instead of Escherichia coli used in Example 1.
  • PWH 1520 MoBiTech GmbH, Germany
  • a plasmid a plasmid
  • a tetracycline resistance gene as a selection marker in Bacillus subtilis
  • final xylose as an inducer
  • a lysate of recombinant Bacillus subtilis containing S. mutans-derived sucrose phosphorylase was prepared in the same manner as in Example 1.
  • the obtained lysate 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.
  • sucrose phosphorylase enzyme solution suitable for amylose synthesis could not be obtained.
  • sucrose phosphorylase Preparation of sucrose phosphorylase from cell lysate containing L. mesenteroides sucrose phosphorylase
  • sucrose phosphorylase activity was measured according to the method described herein. As a result, when heated in the presence of 4 to 30% of sucrose, a sucrose phosphorylase enzyme solution suitable for amylose synthesis was obtained.
  • sucrose phosphorylase enzyme solution suitable for amylose synthesis could not be obtained.
  • sucrose phosphorylase Preparation of sucrose phosphorylase from cell extract containing Escherichia coli mesenteroides sucrose phosphorylase (Escherichia coli extract model)
  • sucrose to this model cell lysate to obtain a final concentration of 4%, 8%, 12%, 16%, 20%, 25% or 30%, respectively, based on the lysed solution.
  • a solution was obtained.
  • a lysate containing no sucrose was used as a control (0% sucrose).
  • Sucrose phosphorylase activity was measured according to the method described herein. As a result, when heated in the presence of 4 to 30% of sucrose, a sucrose phosphorylase enzyme solution suitable for amylose synthesis was obtained.
  • sucrose phosphorylase enzyme solution suitable for amylose synthesis could not be obtained.
  • Substrates for sucrose phosphorylase include fructose, inorganic phosphate, and glucose-1-phosphate in addition to sucrose.
  • the effect of fructose on sucrose phos holase stability was examined as follows.
  • Fructose was added to and dissolved in the cell lysate containing sucrose phosphorylase derived from S. mutans prepared in (1.1) above so that the final concentration was 5% or 10%, respectively, to obtain a solution. .
  • the cell lysate to which no fructose was added was used as a control (0% sucrose).
  • As a control the case where the same concentration of sucrose was added was examined at the same time.
  • These solutions were heated in a 55 ° C water bath. At the start of heating (0 minutes), samples were taken at 30, 60 and 90 minutes after the start of heating, and the activity of sucrose phosphorylase was measured according to the method described herein. The residual activity and the specific activity were calculated based on the measured activities.
  • FIG. 6 shows the results regarding the residual activity. As a result, it was found that fructose did not have a sucrose phosphorylase stabilizing effect as in the case of sucrose.
  • the residual activity and the specific activity were calculated based on the measured activities.
  • FIG. 7 shows the results regarding the residual activity. As a result, it was found that inorganic phosphate did not have the sucrose phosphorylase stabilizing effect as in the case of sucrose. Industrial applicability
  • sucrose phosphorylase preparation and a method for preparing the same are provided.
  • 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, inorganic 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 hindered.
  • sucrose phosphorylase preparation of the present invention can reduce the load on column chromatography due to its high specific activity, and can be purified using a smaller column than when the specific activity is low. Also suitable for purification purposes.

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Abstract

Cette invention se rapporte à un procédé servant à produire une préparation de sucrose-phosphorylase. Ce procédé consiste à chauffer une solution contenant de la sucrose-phosphorylase et du sucrose dans des conditions propres à empêcher que toute réaction enzymatique favorisée par la sucrose-phosphorylase ne se produise. Cette invention concerne également la préparation de sucrose-phosphorylase produite par ce procédé, cette préparation ayant une activité spécifique de 30 U/mg ou plus.
PCT/JP2002/005088 2001-05-28 2002-05-24 Procede de preparation de sucrose-phosphorylase WO2002097077A1 (fr)

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CN107236696A (zh) * 2017-07-31 2017-10-10 江南大学 一种表达L. mesenteroides来源的蔗糖磷酸化酶重组枯草芽孢杆菌
CN109234220A (zh) * 2018-11-02 2019-01-18 南京工业大学 一株枯草芽孢杆菌基因重组菌及其构建方法与应用
CN109306357A (zh) * 2018-11-09 2019-02-05 沈阳农业大学 一种高效表达制备蔗糖磷酸化酶的方法
CN110343654A (zh) * 2019-08-15 2019-10-18 江南大学 一种产蔗糖磷酸化酶的基因工程菌
CN110656077A (zh) * 2019-11-07 2020-01-07 江南大学 一种生产蔗糖磷酸化酶的方法及其应用
WO2021142863A1 (fr) * 2020-01-17 2021-07-22 江南大学 Procédé de préparation de dextrine à chaîne droite
US11549133B2 (en) 2020-01-17 2023-01-10 Jiangnan University Preparation method of amylodextrin

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JP4604240B2 (ja) * 2004-10-04 2011-01-05 独立行政法人産業技術総合研究所 光阻害免疫能力回復剤及びその製造方法

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107236696A (zh) * 2017-07-31 2017-10-10 江南大学 一种表达L. mesenteroides来源的蔗糖磷酸化酶重组枯草芽孢杆菌
CN107236696B (zh) * 2017-07-31 2019-11-26 江南大学 一种表达L.mesenteroides来源的蔗糖磷酸化酶重组枯草芽孢杆菌
CN109234220A (zh) * 2018-11-02 2019-01-18 南京工业大学 一株枯草芽孢杆菌基因重组菌及其构建方法与应用
CN109234220B (zh) * 2018-11-02 2020-05-08 南京工业大学 保湿修复因子甘油葡萄糖苷的生物制备菌株及其构建方法与应用
CN109306357A (zh) * 2018-11-09 2019-02-05 沈阳农业大学 一种高效表达制备蔗糖磷酸化酶的方法
CN109306357B (zh) * 2018-11-09 2021-12-17 沈阳农业大学 一种表达制备蔗糖磷酸化酶的方法
CN110343654A (zh) * 2019-08-15 2019-10-18 江南大学 一种产蔗糖磷酸化酶的基因工程菌
CN110656077A (zh) * 2019-11-07 2020-01-07 江南大学 一种生产蔗糖磷酸化酶的方法及其应用
WO2021142863A1 (fr) * 2020-01-17 2021-07-22 江南大学 Procédé de préparation de dextrine à chaîne droite
US11549133B2 (en) 2020-01-17 2023-01-10 Jiangnan University Preparation method of amylodextrin

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