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    • 专利摘要:
    IMMOBILIZATION OF PSYCHOSIS-EPIMERASE AND A METHOD FOR THE PRODUCTION OF D-PSYCHOSIS USING IT. The present invention relates to a method for successively producing D-psychosis from D-fructose or D-glucose using a psychose-epimerase derived from Agrobacterium tumefaciens, which is expressed in food safety form.
    公开号:BR112012007215B1
    申请号:R112012007215-3
    申请日:2010-09-01
    公开日:2021-08-31
    发明作者:Yong Ho Hong；Jin Ha Kim；Sung Bo Kim；Jung Hoon Kim；Young Mi Lee；Seung Won Park
    申请人:Cj Cheiljedang Corporation；
    IPC主号:
    专利说明:

    technical field
    [001] The present invention relates to a method for successively producing D-psychose from D-fructose or D-glucose using a psychose-epimerase derived from Agrobacterium tumefaciens, which is expressed in food safety form. State of the Art
    [002] D-psychose has been widely used as a functional sweetener that has a sweet taste similar to sugar, but is a very low-calorie monosaccharide. In particular, D-psychosis is known as a "rare sugar" because it is rarely found in nature, and even when found, it only occurs in small amounts.
    [003] D-psychose is an epimer of D-fructose and the intensity of its sweetness and its types are quite similar to those of D-fructose. However, unlike D-fructose, when absorbed by the body, D-psychose is rarely metabolized and has approximately zero calories. Furthermore, thanks to its action in relieving abdominal obesity by suppressing enzymatic activities related to lipid synthesis, D-psychose can be used as an effective ingredient in the formulation of dietetic foods. Furthermore, while sugar alcohols, which are widely used as sugar substitutes, have the disadvantage of causing side effects such as diarrhea when ingested in excess, D-psychosis has almost no side effects (Matsue, t., y. Baba, M. Hashiguchi, K. Takeshita, K. Izumori and H. Suzuki 2001. Dietary D-psychosis, a C-3 epimer of D-fructose, suppresses hepatic lipogenic enzyme activity in rats.Asia PAC j Clin. Nutr. 10:233-237.;Matsuo, T. and K. Izumori, 2004. D-psychosis, a rare sugar that provides no calories other than beneficial effects for clinical nutrition. Asia PAC j Clin. Nutr. 13 :S127).
    [004] For these reasons, D-psychosis is gaining prominence in the field of the food industry as a dietary sweetener and therefore there is a growing need to develop an efficient production method of D-psychosis.
    [005] Conventional methods of producing D-psychosis using a psychose-epimerase have been focused on developing a process of epimerization of D-psychosis from D-fructose, using a massively expressed recombinant enzyme in a recombinant e. coli or a host cell comprising the same. However, the biotechnological production of D-psychosis using such a recombinant e. colie inadequate for the production of D-psychosis as a food product in terms of food safety. To produce D-psychosis properly for use as food, it is necessary to develop an industrial-scale production method using a psychosis-epimerase expressed in a host cell, which is a strain considered GRAS (generally recognized as safe), as well. as a species capable of being produced on an industrial scale or using a host comprising the same.
    [006] The GRAS condition is assigned to substances that are generally recognized as safe when specialists qualified by their scientific training and experience assess their safety through scientific procedures under the conditions of intended use. GRAS is a system implemented in the USA but its importance and necessity is recognized internationally as well as in the USA. That's why people are paying attention to the development of the GRAS concept.
    [007] However, for the industrial production of D-psychosis it is necessary to develop a method of production of D-psychosis through a cheaper substrate. Currently, due to the high substrate specificity of a psychose-epimerase, only expensive D-fructose is used in the production of D-psychosis. Disclosure Technical problem
    [008] The present invention has been actively studied in the production of D-psychose on an industrial scale as food, reaching the conclusion that the successive production of D-psychose from D-fructose or D-glucose can be obtained using immobilization in order to provide a stable environment for enzymatic activation aimed at the production of D-psychose on an industrial scale and using a D-glucose isomerase together with a psychose-epimerase.
    [009] One of the objectives of the present invention is to provide a food-safe psychosis-epimerase for the preparation of D-psychosis usable as a food product.
    [0010] Another objective of the present invention is to provide a method of successive production of D-psychose from D-glucose or D-fructose, using a psychose-epimerase and a D-glucose isomerase with immobilized food safety. technical solution
    The present invention provides a method of preparing D-psychose through D-fructose, using a psychose-epimerase derived from Agrobacterium tumefaciens that is expressed in a GRAS lineage.
    [0012] The method of the present invention comprises the following steps: expressing a psychose-epimerase derived from Agrobacterium tumefaciens in a GRAS lineage; and preparing D-psychosis from D-fructose using expressed psychose-epimerase.
    [0013] The method of the present invention may further comprise the following steps: separating a psychose-epimerase from the culture of a GRAS strain capable of expressing a psychose-epimerase derived from Agrobacterium tumefaciens; and preparing D-psychose from D-fructose using the separate psychose-epimerase.
    The GRAS strain expressing a psychose-epimerase derived from Agrobacterium tumefaciens may be a strain transformed with a recombinant expression vector including a gene encoding the aforementioned enzyme.
    [0015] The psychose-epimerase used in the preparation of D-psychose according to the present invention can be presented in the form contained in a GRAS lineage culture expressing the psychose-epimerase derived from Agrobacterium tumefaciens or it can be isolated from the culture. The GRAS lineage can be a lineage transformed with a recombinant expression vector including a gene encoding a psychose-epimerase. The GRAS lineage may be Corynebacterium sp., preferably Corynebacterium glutamicum KCCM 11046.
    It is preferable that the Agrobacterium tumefaciens-derived psychose-epimerase used in the method of the present invention has the amino acid sequence SEQ ID NO: 1.
    [0017] The method of preparing D-psychose according to the present invention may further include the step of immobilizing the psychose-epimerase in a vehicle. Suitable vehicle for the present invention may include alginate, preferably sodium alginate, but is not limited thereto.
    [0018] The method of preparation of D-psychosis according to the present invention may further comprise steps consisting in filling a column with the vehicle in which the psychosis-epimerase is immobilized, and supplying the filling column (packed-bed column ) with a D-fructose solution.
    [0019] The present invention also provides a method of preparing D-psychose from D-glucose, comprising the step of immobilizing a psychose-epimerase and a D-glucose isomerase towards the vehicles. Suitable vehicle for the present invention may include alginate, preferably sodium alginate, but is not limited thereto.
    In the method described above, the psychose-epimerase can be contained in a GRAS lineage culture capable of expressing the psychose-epimerase derived from Agrobacterium tumefaciens or it can be isolated from the culture. The GRAS lineage can be a lineage transformed with a recombinant expression vector including a gene encoding psychose-epimerase. Furthermore, the GRAS lineage can be Corynebacterium SP., preferably Corynebacterium glutamicum KCCM 11046.
    It is preferable that the Agrobacterium tumefaciens-derived psychose-epimerase used in the method of the present invention has the amino acid sequence SEQ ID NO: 1.
    [0022] In addition, the present invention further provides a method of preparing D-psychose from D-glucose, comprising the following steps: immobilizing a psychose-epimerase and a D-glucose isomerase in vehicles; filling a column with vehicles so that psychose-epimerase and D-glucose isomerase are immobilized; and supplying a column with a D-glucose solution. The vehicles in which the psychose-epimerase and D-glucose isomerase are immobilized can be filled in the same column or respectively in two separate columns. However, it is preferable that the vehicle is filled in a separate column, in which case the two separate columns are connected to each other.
    The present invention provides a Corynebacterium glutamicum KCCM 11046 strain useful for the preparation of D-psychose from D-fructose or D-glucose.
    [0024] The term "food safety" in the scope of this descriptive report means that a particular product is safe enough to be used as food. Therefore, the term "expressed in food safety form" in this specification means that a target gene has been expressed in a strain known to be safe and without harmful effects even though it was used as food, for example, a GRAS strain.
    [0025] In one embodiment of the present invention, a method of transforming the GRAS lineage with a recombinant vector may include an electroperforation, but is not limited thereto and may be carried out using any transformation method well known in the art by those skilled in the art. dedicated to the field of the present invention.
    In one embodiment of the present invention, the recombinant vector including the gene encoding the psychose-epimerase derived from Agrobacterium tumefaciens may include the gene encoding the psychose-epimerase operatively linked to a promoter active in the bacterium Corynebacterium sp. The promoter may have one of the sequences SEQ ID NOs: 2 to 8 that have been confirmed to express a target gene more efficiently than a tac promoter in Corynebacterium sp. (Korean Patent, publication NO 20060068505).
    [0027] The term "psychose-epimerase" used in this specification means a D-psychose-3-epimerase having a converting activity from D-fructose to D-psychose.
    In an embodiment of the present invention, the psychose-epimerase derived from Agrobacterium tumefaciens may have the amino acid sequence SEQ ID NO: 1 or may be constituted by functional fragments thereof. Functional fragment means a fragment that includes mutations by substitution, insertion, deletion or similar processes of some amino acids in the amino acid sequence SEQ ID NO: 1, when it has a converting activity from D-fructose to D-psychose.
    [0029] In an embodiment of the present invention, the GRAS strain may be a Corynebacterium sp.
    [0030] The Corynebacterium sp. is an industrial microorganism that produces chemical compounds that have various applications in animal feed, medicine and food fields, etc., including L-lysine, L-threonine and various nucleic acids. Such a Corynebacterium sp. it is a GRAS strain (generally recognized as safe) and has properties that make it easy to genetically manipulate and grow on a large scale. In addition, the Corynebacterium sp. it has high stability in various process conditions, it has a relatively rigid cell wall structure when compared to other bacteria, thus having the biological property of existing in a cell mass in a stable state even under high osmotic pressure due to the high concentration of sugar, etc.
    [0031] In an embodiment of the present invention, the Corynebacterium sp. it could be Corynebacterium glutamicum.
    [0032] In an embodiment of the present invention, the GRAS strain expressing the psychose-epimerase derived from Agrobacterium tumefaciens may be a recombinant strain of Corynebacterium glutamicum KCCM 11046.
    Psychosis-epimerase can be easily modified by mutagenesis conventionally known to those skilled in the art, such as directed evolution and directed mutagenesis, etc. Therefore, we can conclude that recombinant enzymes with a certain range of sequence homology, such as sequence homology of 70% or greater, preferably 80% or greater, and more preferably 90% or greater for the psychose-epimerase amino acid sequence represented by SEQ ID NO: 1 and being expressed as an active form in the GRAS lineage; and a host cell comprising the same are within the scope of the present invention.
    [0034] The culture of the GRAS lineage expressing the psychose-epimerase derived from Agrobacterium tumefaciens can be obtained by culturing the bacterium transformed in medium and low culture media that can be easily selected by those skilled in the art, depending on the properties of the lineage. The culture method can include any culture method known to those skilled in the art, such as batch culture, continuous culture and fed-batch culture, but is not limited thereto.
    The method of preparing D-psychose according to an embodiment of the present invention may comprise a step of separating the psychose-epimerase from the GRAS lineage culture expressing the psychose-epimerase derived from Agrobacterium tumefaciens.
    [0036] In an embodiment of the present invention, cells separated from the cultured lineage expressing the Agrobacterium tumefaciens-derived psychose-epimerase by centrifugation, filtration, etc.; or the supernatant obtained by homogenization and centrifugation of the separated cells; or psychose-epimerase which is separated and purified from the supernatant by fractionation, chromatography, etc. can be used as an enzyme to convert D-fructose to D-psychose.
    In one embodiment of the present invention, the psychose-epimerase may be in the form of cell mass in culture or as a psychose-epimerase separated from the cell mass.
    [0038] In an embodiment of the present invention, the step of preparing the D-psychosis may further include the step of immobilizing the psychose-epimerase or the mass of cells expressing the same in a vehicle.
    [0039] As in the case of immobilization of the enzyme or cell mass in a vehicle an environment can occur for the maintenance of activity for a long period of time, immobilization has been used in the industrial production method using an enzyme or a microorganism. The vehicle applicable in the immobilization may include alginates such as sodium alginate, but is not limited thereto.
    [0040] In an embodiment of the present invention, the immobilization may be carried out using sodium alginate as a vehicle. Sodium alginate which is a naturally occurring colloidal polysaccharide abundantly present in the algae cell wall consists of β-D-mannuronic acid and aL-guluronic acid and is formed by random β-1,4 bonds in the content, so it is good that the cell mass or enzymes can be stably immobilized therein.
    [0041] In an embodiment of the present invention, immobilization can be done using 1.5% to 4.0% of sodium alginate as a vehicle.
    [0042] In a configuration of the present invention, immobilization can be done using 2% sodium alginate as a vehicle.
    [0043] In an embodiment of the present invention, if sodium alginate is used as a vehicle for immobilization, immobilization can be done by adding 1 to 2 times the volume of an aqueous solution of sodium alginate to a sample containing the enzyme or cell mass, reducing the resulting mixture to a 0.1 M calcium ion solution to create the enzyme or alginate cell mass complex into granules using a syringe pump and a vacuum pump.
    [0044] In an embodiment of the present invention, the step of immobilizing the psychose-epimerase in the vehicle may further include the steps that consist of filling a column with the immobilized enzyme and supplying the filled column with a solution of D-fructose.
    [0045] The column to be filled with the vehicle in which the enzyme or cell mass is immobilized and the method of filling the column with it can be easily selected by those skilled in the art, depending on the enzyme or cell mass, or the vehicle used in immobilization.
    [0046] In an embodiment of the present invention, it is possible to prepare a filling column by filling the column with the immobilized enzyme. The enzymatic reaction, that is, the conversion of D-fructose into D-psychose, can be done by supplying the filling column with a solution of D-fructose which is its substrate.
    [0047] In an embodiment of the present invention, when the filling column that is filled with the cell mass including the psychose-epimerase is supplied with the D-fructose solution at a constant concentration, the epimerization reaction occurs through the resulting immobilized cell mass. in the conversion of D-fructose to D-psychose. Converted D-psychosis is subject to separation and purification using separation columns and is then available as pure D-psychosis.
    [0048] The term "immobilization reactor" used in this descriptive report means a reactor whose reaction for the production of D-psychosis occurs by the cell mass or enzyme immobilized on the vehicle, or the column filled with the cell mass or with the enzyme immobilized on the vehicle. That is, immobilization means that a substance providing biological activities, in this case a psychose-epimerase or a D-glucose-epimerase or a mass of cells including the same, is immobilized in the vehicle.
    [0049] The term "operational stability" used in this descriptive report means that a biological reactor for the successive production of a target product, such as D-psychosis, can be operated maintaining an adequate level of productivity when compared to the initial activity and , in general, is represented by an operational period.
    [0050] In an embodiment of the present invention, the column filled with the vehicle in which the psychose-epimerase expressing cells are immobilized is supplied with 300 g/L of D-fructose at an influx rate of 0.1 min/ml to 50°C, which makes it possible to guarantee 25 days or more of operational stability.
    [0051] The present invention further provides a method of preparing D-psychose from D-glucose, comprising the step of immobilizing a psychose-epimerase and a D-glucose isomerase in vehicles.
    [0052] As described above, as D-fructose used as a substrate for psychosis-epimerase is expensive, it is necessary to produce D-psychose from a cheaper substrate like D-glucose for the production of D-psychose in industrial scale. D-glucose isomerase is an enzyme capable of converting D-glucose into D-fructose, and thus, it can provide a substrate for a psychose-epimerase, converting D-glucose into D-fructose. Therefore, it turns out that it is possible to produce D-psychose from D-glucose using these two enzymes.
    In an embodiment of the present invention, the psychose-epimerase may be the culture of the GRAS lineage expressing the psychose-epimerase derived from Agrobacterium tumefaciens, or the cell mass or the psychose-epimerase separated from the culture.
    [0054] In an embodiment of the present invention, the D-glucose isomerase can be the culture of the GRAS lineage expressing the D-glucose isomerase, or the cell mass or the D-glucose isomerase separated from the culture.
    In one embodiment of the present invention, the psychose-epimerase may have the amino acid sequence SEQ ID NO: 1.
    [0056] In an embodiment of the present invention, D-glucose isomerase may be commercially available. For example, D-glucose isomerase or immobilized D-glucose isomerase commercially available from Novozymes A/S (Bagsvaerd, Denmark) can be used.
    [0057] D-glucose isomerase is a representative enzyme for the conversion of D-glucose into D-fructose and currently has been widely used for the production of fructooligosaccharides. Among the enzymes currently used for industrial production, D-glucose isomerase is one of the enzymes whose activities and mechanisms have been clearly identified.
    [0058] In one embodiment of the present invention, the GRAS lineage may be a lineage transformed with a recombinant vector including a gene encoding psychose-epimerase and a gene encoding D-glucose isomerase or a lineage co-transformed with recombinant vectors including each of the genes described above.
    [0059] In a configuration of the present invention, the GRAS lineage may be the bacterium Corynebacterium sp.
    [0060] In an embodiment of the present invention, the GRAS lineage expressing the psychose-epimerase derived from Agrobacterium tumefaciens may be the recombinant lineage of Corynebacterium glutamicum KCCM 11046.
    [0061] In an embodiment of the present invention, the vehicle for the immobilization may be sodium alginate.
    [0062] In an embodiment of the present invention, immobilization can be performed using 1.5% to 4.0% of sodium alginate as a vehicle.
    [0063] In a configuration of the present invention, immobilization can be performed using 2% sodium alginate as a vehicle.
    [0064] In an embodiment of the present invention, if sodium alginate is used as a vehicle for immobilization, said immobilization can be carried out by adding 1 to 2 times the volume of an aqueous solution of sodium alginate to a sample containing the enzyme or cell mass, reducing the resulting mixture to a 0.1 M calcium ion solution to create the enzyme or alginate cell mass complex into granules using a syringe pump and a vacuum pump.
    [0065] In an embodiment of the present invention, the gene encoding psychose-epimerase and the gene encoding D-glucose isomerase can be expressed in the same GRAS lineage or in two different GRAS lineages, respectively, and the immobilization step can be made by immobilizing the cells of the cultured GRAS strains or a mixture of two enzymes separated from the cells in the vehicles.
    In an embodiment of the present invention, the culture of the GRAS lineage expressing the psychose-epimerase or cell mass or the psychose-epimerase separated from the culture; and D-glucose isomerase can be immobilized in vehicles, respectively.
    [0067] In an embodiment of the present invention, the method of the invention may further comprise the step of filling the column with the vehicle in which the cells or enzymes are immobilized.
    [0068] The method of preparing D-psychose from D-glucose according to the present invention may further comprise the steps of filling the column with immobilized psychose-epimerase and D-glucose isomerase and supplying the filled column with a D-glucose solution.
    [0069] In an embodiment of the present invention, the column filling can be prepared by filling the column with the immobilized enzymes. The enzymatic reaction, that is, the conversion of D-glucose into D-psychose, can be done by supplying the filling column with the D-glucose solution, which is its substrate.
    In one embodiment of the present invention, the immobilized psychose-epimerase and the D-glucose isomerase can be filled in separate columns, respectively.
    [0071] The column to be filled with the vehicle in which the enzymes or cells are immobilized and the method of filling the column with it can be easily selected by those skilled in the art, depending on the enzymes or cell mass, or the vehicle used for immobilization. In one embodiment of the present invention, it is possible to fill two separate columns with the immobilized vehicle of the psychose-epimerase and the immobilized vehicle of the D-glucose isomerase, respectively, connecting these two columns together to transport the D-fructose as a product of isomerization of the column filled with D-glucose isomerase to column filled with psychose-epimerase, and induce the reaction of conversion of D-fructose to D-psychose. When the solution of D-glucose as a substrate is supplied to the immobilization reactor composed of these two communicated columns, the D-glucose is converted to D-fructose in the column filled with D-glucose isomerase, and the D-fructose transferred to the column filled with psychosis-epimerase is converted to D-psychosis. Thus, it is possible to obtain D-psychose as an end-product of D-glucose. beneficial effects
    [0072] The method of preparation of D-psychose according to the present invention can massively and successively produce D-psychose in terms of food safety that can be used as a food product resulting from D-glucose or D-fructose. Brief description of the drawings
    Fig. 1 schematically illustrates a process of construction of a recombinant expression vector pCJ-1-ATPE including a gene encoding a psychose-epimerase derived from Agrobacterium tumefaciens.
    [0074] Fig. 2 illustrates the results of measuring the activity of psychose-epimerase derived from Agrobacterium tumefaciens by HPLC for the method according to an embodiment of the present invention, where Fig. 2a shows a standard reference for each sugar, and Fig. 2b shows conversion by reactions.
    [0075] Fig. 3 illustrates % conversion of D-fructose to D-psychose using the recombinant strain for the method according to an embodiment of the present invention under combined conditions of 100 g/L (3a), 300 g/L (3b) or 500 g/L (3c) of D-fructose, and 40°C, 45°C or 50°C of a reaction temperature.
    [0076] Fig. 4 illustrates the productivity of D-psychose depending on an influx rate of D-fructose as substrate for the method according to an embodiment of the present invention.
    [0077] Fig. 5 illustrates the operational stability of an immobilization reactor at a reaction temperature of 50°C for the method according to an embodiment of the present invention. A relative activity (%) means an activity relative to the activity at the time the operation is started.
    [0078] Fig. 6 illustrates the conversion of % D-glucose to D-psychose through the use of the immobilized recombinant strain expressing the psychose-epimerase and the immobilized D-glucose isomerase under combined conditions of 100 g/L (6a) , 300 g/L (6b) or 500 g/L (6c) of D-glucose, and 40 °C, 45 °C or 50 °C reaction temperature for the method according to an embodiment of the present invention.
    [0079] Fig. 7 illustrates the productivity of D-psychose depending on an influx rate of D-glucose for the method according to an embodiment of the present invention. Best Mode
    [0080] In the following, the present invention is illustrated in greater detail by the following specific examples. However, these examples are for illustrative purposes only and do not limit the scope of the present invention. Measurement of psychose-epimerase and D-glucose isomerase activities
    [0081] The activities of a psychose-epimerase and a D-glucose isomerase were measured using D-fructose and D-glucose as a substrate, respectively. The enzymes or samples containing them were added to 50 mM PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) buffer (pH 8.0) containing 100 g/L of substrates, followed by the reaction at 50C for one hour and the reaction was then stopped by heating the reaction to 100°C for 5 minutes. Concentrations of D-fructose, D-glucose and D-psychose were measured by HPLC equipped with an RI detector (index of refraction). HPLC analysis was performed by injecting a sample into the Supelcogel Pb column at 80°C and then passing distilled water as a mobile phase through the column at a rate of 0.5 ml/min. D-psychose was separated at a retention time of about 32 minutes, and its % conversion and productivity were calculated based on the amount of D-psychose as a reference standard. For the comparative analysis of enzyme activities, one unit of an enzyme was defined as the amount of enzyme to produce 1 μmole of D-psychose per minute at pH 8.0 and 50°C. Example 1: Recombinant strain expressing a psychosis-epimerase (1) Preparation of a recombinant strain
    [0082] The polymerase chain reaction (PCR) was performed to amplify a gene encoding a psychose-epimerase using a genomic DNA from Agrobacterium tumefaciens ATCC 33970 as a template and oligonucleotides of SEQ ID NOs: 9 and 10 with recognition sites restriction enzyme introduced Pstl and Xbal, respectively, as primers. PCR conditions were as follows: after denaturation at 95C for 30 seconds, 26 cycles of 30 seconds at 95C, 30 seconds at 55°C and 1 minute at 68C, and then extension at 68C for 10 minutes. For the mass expression of the psychose-epimerase encoded by the amplified gene, the amplified PCR product was digested with the restriction enzymes Pstl and Xbal and inserted into a pCJ-1 shuttle vector derived from the bacterium Corynebacterium sp (deposited at the Korean Culture Center of Microorganisms (KCCM), an international depository institution, on November 6, 2004, under deposit number KCCM-10611), to construct a recombinant expression vector pCJ-1-ATPE. Fig. 1 schematically shows the method of preparing the recombinant expression vector pCJ-1-ATPE including the gene encoding the psychose-epimerase derived from Agrobacterium tumefaciens.
    [0083] The recombinant expression vector pCJ-1-ATPE was introduced into a Corynebacterium glutamicum ATCC 13032 by transformation using electroperforation to prepare the recombinant strain expressing the gene encoding the psychose-epimerase derived from Agrobacterium tumefaciens. The recombinant strain was designated as Corynebacterium glutamicum ATPE and deposited at the Korean Microorganism Culture Center (KCCM, Hongje-1-dong, Seodaemum-gu, Seoul, Korea) under number KCCM 11046 on October 26, 2009 under the Treaty of Budapest. (2) Expression of a psychosis-epimerase
    [0084] MB (Bacto-tryptone 10 g/L, Bacto yeast extract 5 g/L, NaCl 5 g/L, Soytona 5 g/L) including 10μg/ml of kanamycin were inoculated with the recombinant strains obtained above (1) , respectively, at the initial concentration of OD600 = 0.1, followed by incubation at 30°C for 24 hours to induce the expression of recombinant psychose-epimerase. A 5 L-jar fermenter containing 3 L of a modification medium (D-glucose 80 g/L, Soytona 20 g/L, (NH4)2SO4 10 g/LKH2PO4 1.2 g/L, MgSO4 1.4 g/ L) including 10 μg/ml of kanamycin was inoculated with the culture obtained above at OD600 = 0.6, followed by incubation at 30°C for 20 hours. In order to measure the activity of the expressed psychose-epimerase, the culture was centrifuged at 8000x g for 10 minutes for the collected cells. Harvested cells were then resuspended in a 50 mM EPPS buffer (pH 8.0) (Sigma-Aldrich) and the resulting suspension was subjected to ultrasound resulting in cell lysis. The cell lysate was subjected to centrifugation to obtain a supernatant containing the psychose-epimerase, and the epimerization reaction of D-fructose was performed using the supernatant as an enzyme. Measurement of psychosis-epimerase activity as described above was used in the epimerization reaction. Fig. 2b shows the results of the HPLC analysis of the epimerization reaction. Example 2: Immobilization of a recombinant strain and preparation of D-psychosis (1) Immobilization of a recombinant strain
    For the production of D-psychose on an industrial scale, Corynebacterium glutamicum ATPE (KCCM 11046) expressing the psychose-epimerase derived from Agrobacterium tumefaciens and prepared in the above example 1 was immobilized on the vehicle in this example. For the immobilization of the recombinant strain, the Corynebacterium modification medium used in example 1 was first inoculated with the recombinant strain with an initial concentration of OD600 = 0.6 followed by incubation at 30°C for 20 hours. Upon completion of the incubation, the resulting culture was subjected to centrifugation to recover the cells, which were resuspended in the 50 mM EPPS buffer (pH 8.0) to 20%. The resuspended cells of the recombinant strain were added in 2% (v/v) of an aqueous solution of sodium alginate. The resulting mixture was reduced to a solution of 100 mM CaCl 2 by using a syringe pump and a vacuum pump to create a cellular alginate complex in which cells were trapped in sodium alginate granules. (2) Preparation of D-psychosis using immobilized recombinant strain
    [0086] A packed-bed XK16 column (16 mm X 20 mm, Amersham pharmacia) was filled with the recombinant strain immobilized in sodium alginate above (1) followed by measurement of the % conversion package of D-fructose to D- psychosis. In order to determine an optimal reaction temperature of an immobilization reactor and a substrate concentration of D-fructose, the reaction of conversion of D-fructose to D-psychose was carried out under combined conditions of 40°C, 45°C and 50°C of reaction temperatures, and 100 g/L, 300 g/L and 500 g/L of D-fructose concentration. According to Figs. 3a to 3C, it was found that the best conversion is obtained at a temperature of 50C and 100 g/L of D-fructose concentration, and that 300 g/L and 500 g/L of D-fructose concentration also promotes high conversion. Furthermore, unlike other D-glucose conversion reactions, the epimerization reaction for D-psychose showed low temperature dependence. (2 - 1) Productivity as a function of a D-fructose influx rate
    [0087] The packed-bed XK16 column was filled with the immobilized recombinant strain as described above by measuring the productivity of D-psychose depending on an influx rate of D-fructose. At this time, based on the results above (2), the D-fructose concentration and the reaction temperature were fixed at 300 g/L and 50°C, respectively. Productivity was measured by adjusting the influx rate of D-fructose, a substrate with 0.4, 1, 2, 4 and 8 h-1 space velocity (1/h). The results are illustrated in Table 1 below and in Fig. 4.Table 1. Productivity and % conversion depending on a D-fructose influx rate
    (2-2) Operational stability
    [0088] The operation was performed at 50C using the immobilized recombinant strains that were filled in the XK16 filling column until their specific activities reached 50%, respectively, in order to measure the operational stability of the immobilized recombinant strains that were filled in the column. At this time, the concentration of D-fructose substrate was 300 g/L and its influx rate was 0.1 min/ml (spatial velocity 0.4 h-1). It was found that 25 days or more of operational stability could be maintained under these reaction conditions.
    [0089] Fig. 5 shows the operational stability for a recombinant strain immobilized at a reaction temperature of 50°C. Example 3: Preparation of D-psychosis from D-glucose using immobilized recombinant strain/enzyme (1) Productivity as a function of a reaction temperature and a concentration of D-glucose
    [0090] XK16 fill columns (16 mm X 20 mm, Amersham pharmacia) were filled with the recombinant strain immobilized in sodium alginate in example 2 above, and in 10 g of the immobilized D-glucose isomerase (Novozymes, Denmark), respectively, followed by measuring the % conversion of D-glucose to D-psychose. In order to determine the ideal reaction condition for the two mixed enzymes, the reaction of converting D-glucose to D-psychose was carried out under combined conditions of 40C, 45C or 50C reaction temperature and 100 g/L, 300 g/L or 500 g/L of D-glucose concentration. Fig. 6 shows the conversion of % D-glucose to D-psychose depending on changes in reaction temperature and substrate concentration. According to Figs. 6a to 6c, unlike the epimerization reaction for D-psychosis, the D-glucose isomerization reaction was a temperature-dependent reaction. That is, the higher the reaction temperature, the faster the conversion rate of D-glucose to D-psychose. This was found to have an effect on the productivity of D-psychosis. The best conversion of D-glucose to D-fructose was achieved at a temperature of 50°C and there was no significant difference in reaction rate as substrate concentration increased. These results demonstrate that it is possible to operate the immobilization reactor under the conditions of a higher substrate concentration and to increase the productivity of D-psychose from D-glucose. (2) Productivity as a function of a D-glucose influx rate
    [0091] The two fill columns were filled with the immobilized recombinant strain, including an immobilized psychose-epimerase and an immobilized D-glucose isomerase, respectively, and the productivity of D-psychose was measured as a function of an influx rate of D-glucose . Based on the results of the above examples, the D-glucose concentration and the reaction temperature were fixed at 300 g/L and 50°C, respectively. Productivity was measured by adjusting the influx rate of D-fructose, a substrate with 0.4, 1, 2, 4 and 8 h-1 space velocity (1/h). The results are illustrated below in Table 2 and Fig. 7. Table 2. Productivity and % conversion depending on a D-glucose influx rate
    权利要求:
    Claims (12)
    [0001]
    1. Method to prepare D-psychose from D-fructose characterized by the fact that it uses a psychose-epimerase derived from Agrobacterium tumefaciens, and expressed in a GRAS lineage (generally recognized as safe), where the GRAS lineage is Corynebacterium glutamicum KCCM 11046, comprising the steps of: expressing the psychose-epimerase derived from Agrobacterium tumefaciens in the GRAS lineage; eprepare D-psychosis from D-fructose using the expressed psychosis-epimerase that is contained in a GRAS strain culture, immobilized in a vehicle.
    [0002]
    2. Method according to claim 1, characterized by the fact that the psychose-epimerase derived from Agrobacterium tumefaciens has the amino acid sequence of SEQ ID NO: 1.
    [0003]
    3. Method according to claim 1, characterized by the fact that the step of preparing D-psychosis comprises the step of immobilization of the GRAS lineage expressing the psychose-epimerase in a vehicle.
    [0004]
    4. Method according to claim 3, characterized in that the vehicle is sodium alginate.
    [0005]
    5. Method according to claim 3, characterized in that it further comprises the steps of filling a column with the vehicle in which the GRAS lineage expressing psychosis-epimerase is immobilized, and providing a D-fructose solution to the filled column .
    [0006]
    6. Method for preparing D-psychose from D-glucose characterized by the fact that it comprises the step of immobilizing a psychose-epimerase and a D-glucose isomerase in vehicles, in which the psychose-epimerase is derived from Agrobacterium tumefaciens, and expressed in a GRAS lineage (generally recognized as safe), where the GRAS lineage is Corynebacterium glutamicum KCCM 11046, and where the psychose-epimerase is contained in a culture of the GRAS lineage, immobilized in a vehicle.
    [0007]
    7. Method according to claim 6, characterized by the fact that the GRAS strain is a strain transformed with a recombinant vector including a gene encoding a psychosis-epimerase.
    [0008]
    8. Method according to any one of claims 6 and 7, characterized by the fact that the psychose-epimerase derived from Agrobacterium tumefaciens has the amino acid sequence of SEQ ID NO: 1.
    [0009]
    9. Method according to claim 6, characterized in that the vehicle is sodium alginate.
    [0010]
    10. Method according to claim 6 or 7, characterized in that it further comprises the steps of filling a column with vehicles in which the GRAS lineage expressing psychose-epimerase and D-glucose isomerase are immobilized and providing a solution of D-fructose to column filled by vehicle.
    [0011]
    11. Method according to claim 10, characterized in that each of the vehicles in which the GRAS lineage expressing psychose-epimerase and D-glucose isomerase are immobilized, respectively, is filled in a separate column, and the two columns are connected in communication with each other.
    [0012]
    12. Corynebacterium glutamicum strain KCCM 11046 characterized by the fact that it is useful for the preparation of D-psychose from D-fructose or D-glucose.
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    同族专利:
    公开号 | 公开日
    CN102869783A|2013-01-09|
    PT2470668T|2016-12-20|
    EP2470668A2|2012-07-04|
    BR112012007215A2|2021-02-17|
    KR20110035805A|2011-04-06|
    WO2011040708A2|2011-04-07|
    HRP20161658T1|2017-01-13|
    US8735106B2|2014-05-27|
    CN102869783B|2016-03-09|
    HK1175207A1|2013-06-28|
    JP2013501519A|2013-01-17|
    PL2470668T3|2017-03-31|
    WO2011040708A3|2011-07-07|
    HUE031275T2|2017-07-28|
    EP2470668A4|2013-04-10|
    ES2614891T3|2017-06-02|
    EP2470668B1|2016-09-14|
    US20120244580A1|2012-09-27|
    SI2470668T1|2017-01-31|
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    法律状态:
    2021-03-02| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2021-03-09| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2021-06-15| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-08-31| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/09/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |
    优先权:
    申请号 | 申请日 | 专利标题
    KR20090092784|2009-09-30|
    KR10-2009-0092784|2009-09-30|
    KR1020090118465A|KR20110035805A|2009-09-30|2009-12-02|Method of producing d-psicose using immobilized d-psicose 3-epimerase|
    KR10-2009-0118465|2009-12-02|
    PCT/KR2010/005902|WO2011040708A2|2009-09-30|2010-09-01|Immobilization of psicose-epimerase and a method of producing d-psicose using the same|
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