KR101700346B1 - Method of improving cytopermeability of substrate of endoenzyme and method of manufacturing products of endoenzyme reaction from substrate - Google Patents
Method of improving cytopermeability of substrate of endoenzyme and method of manufacturing products of endoenzyme reaction from substrate Download PDFInfo
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Abstract
Description
The present invention relates to a method for enhancing the cell permeability of a substrate to an intracellular enzyme, and more particularly, to a method for enhancing the cell permeability of a substrate to an intracellular enzyme by treating a Gram positive bacterium producing an intracellular enzyme with a predetermined substance It's about how you can. The present invention also relates to a method for effectively producing an intracellular enzyme reaction product from a substrate using a method of improving the cell permeability of the substrate to the intracellular enzyme.
D-allulose is an epimer of
Because of these characteristics, alulose is a good source to replace sugar, but it belongs to a rare sugar, a monosaccharide that rarely exists in the natural world. Therefore, a method of efficiently producing alulose is needed to be applied to the food industry. Conventional alululose production methods are mainly chemical processes. Bilik et al. Proposed a method of converting fructose to alulose using the catalytic action of molybdic acid ions. McDonald has reported that 1,2: 4,5-di-δ-isopropylidene-beta-D-fructopyranose (1,2: 4,5- To produce aluloses in three chemical treatment steps. Doner also produced aluloses by heating fructose with ethanol and trimethylamine. However, these chemical production methods have a disadvantage in that they are expensive, while their efficiency is low and by-products are generated in large quantities.
Representative biological methods for producing aluloses include a method in which fructose is directly reacted with D-alulose 3-epimerase to convert to alulose, and a method in which a strain producing D-alulose 3-epimerase as an intracellular enzyme And reacting fructose with fructose to convert it to alulose. Among them, the method of producing aluloses by using a direct reaction with an enzyme has a limitation in its application because enzyme activity may be greatly lowered due to changes in temperature and pH, and enzyme immobilization is not easy. In addition, D-alulose 3-epimerase is an intracellular enzyme (endoenzyme), which is expensive and time consuming for purification. On the other hand, the method of producing aluloses using the reaction with cells is simple, and the cell wall of the cells protects the intracellular enzyme D-alulose 3-epimerase, which is advantageous in producing alulose for a long time . However, when the cell is a Gram-positive bacterium, intracellular inflow of fructose, which is a substrate, due to a thick cell wall, and extracellular leakage of the enzyme reaction product, alulose, are not smooth, and the enzyme reaction rate is lowered, Problems such as a decrease in productivity occur. To solve this problem, Korean Patent Laid-Open Publication No. 10-2014-0122043 discloses a method of increasing the permeability of a cell membrane by treating a strain of Ensifer adhaerens, which has an activity of converting fructose into a cytosine , with a surfactant, Triton X-100 have. In addition, the registration method of Republic of Korea Patent No. 10-0832339 discloses a treated toluene in the Sino separation tank emptying in (Sinorhizobium) YB-58 KCTC 10983BP strain having the activity to convert fructose into psicose cells increase the productivity of the psicose Lt; / RTI > However, the method disclosed in the above-mentioned prior art is not satisfactory in terms of the productivity level of psychosis from a commercial point of view and needs to be improved.
It is an object of the present invention to provide a method for improving the cell permeability of a substrate to an intracellular enzyme by treating a Gram-positive bacterium producing an intracellular enzyme with a predetermined substance . It is also an object of the present invention to provide a method for producing an intracellular enzyme reaction product from a substrate using a method for enhancing the cell permeability of the substrate to an intracellular enzyme.
Since endogenous enzymes in Gram positive bacteria are protected by thick cell walls composed of 80 ~ 90% of peptidoglycan and cell membranes composed of proteins and lipids, intracellular enzymes the intracellular inflow of the substrate must be performed smoothly for an effective reaction between the endoenzyme and the corresponding substrate. The inventors of the present invention have found that when Gram-positive bacteria are treated with a specific substance or a combination of specific substances, the cell wall or cell membrane structure of Gram-positive bacteria is changed and the intracellular inflow of the substrate is smoothly performed, The reaction activity was remarkably increased, and the present invention was completed.
In order to solve the above-mentioned object, one example of the present invention is a method for treating Gram-positive bacteria producing an endo-enzyme, which comprises treating a Gram-positive bacterium producing an endozyme with an antibiotic alone or with a surfactant alone or with an antibiotic or a surfactant, And a step of modifying the cell wall or cell membrane structure of Gram-positive bacteria by treating the cells with an antibiotic, a surfactant, and an organic solvent to improve cell permeability of the substrate to an intracellular enzyme. In an embodiment of the present invention, the antibiotic is penicillin, the surfactant is sorbitan monolaurate, and the organic solvent is toluene. In the method according to an example of the present invention, the change of cell wall or cell membrane structure of Gram-positive bacteria means that a part of components constituting the cell wall or cell membrane is removed or the cell wall or cell membrane structure is loosely changed. For example, treatment of Gram-positive bacteria with penicillin can break down a portion of the peptidoglycan layer that constitutes the cell wall to facilitate the influx of the substrate, and can inhibit Gram-positive bacteria from Sorbitan monolaurate, Or toluene, part of the lipid layer constituting the cell membrane may be removed to facilitate the inflow of the substrate.
In the method according to an example of the present invention, the type of the intracellular enzyme is not limited to an isomerizing enzyme or an epimerizing enzyme, and it is preferably a ketose 3-epimerizing enzyme in consideration of the availability of the enzyme reaction product. In the present invention, the ketose 3-epimerase has an activity of forming a corresponding D- or L-ketohexose by epimerizing D- or L-ketohexose at three positions, and has a D- or L- Mutual conversion between Tos and D- or L-alulose, mutual conversion between D- or L-tagatose and D- or L-sorbos. In addition, in the present invention, the ketose 3-epimerase has an activity of forming the corresponding D- or L-ketopentose by epimerizing D- or L-ketopentose at three positions, D- or L - catalyze the interconversion between xylose and D- or L-libulose. The ketohexose is a hexose having a ketose structure, specifically, fructose, alulose, tagatose and sorbose. D- or L-ketohexose is a D-form or an L-form . The ketopentose is a pentose having a ketose structure, and specifically refers to xylose and ribulose, D- or L-ketopentose refers to D-form thereof and L - means the sieve. In the method according to an example of the present invention, the ketose 3-epimerase is preferably an allylose 3-epimerase or a tagatose 3-epimerase, more preferably an allylose 3-epimerase. The above-mentioned alulose 3-epimerase catalyzes the mutual conversion between fructose and alulose, and may have an amino acid sequence of SEQ ID NO: 1 as a specific example.
In the method according to an example of the present invention, the Gram-positive bacteria are transformed by introduction of a gene encoding a ketose 3-epimerase or introduction of a recombinant vector containing a gene encoding a ketose 3-epimerase A recombinant strain is preferable. In addition, the host strain of the recombinant strain is preferably a food-stable Gram-positive strain. The food-safe Gram-positive strains generally mean GRAS (generally accepted as safe) strains which are considered to be safe. Examples include strains of Bacillus sp., Corynebacterium sp. ), And the like. The strains are industrial microorganisms that produce chemicals with diverse uses in fields such as feed, medicine, and food. These strains are easy to genetically manipulate and mass culture or have high stability under various process conditions. In addition, since these strains have a relatively rigid cell membrane structure as compared with other bacteria, they exhibit biological characteristics that the cells remain in a stable state even under the influence of osmotic pressure due to high sugar concentration and the like. Specific examples of the GRAS (generally accepted as safe) strain include bacillus subtilis , Corynebacterium glutamicum , and the like.
In the method according to an embodiment of the present invention, the treatment concentration of the penicillin is preferably 2 to 30 mg / L, more preferably 10 to 30 mg / L. The treatment concentration of the sorbitan monolaurate is preferably 1 to 5% (w / v), more preferably 1.5 to 5% (w / v). The treatment concentration of the toluene is preferably 2.5 to 30% (v / v), more preferably 5 to 30% (v / v).
In order to solve the above-mentioned object, another example of the present invention is a method for treating a Gram-positive bacterium producing an intracellular enzyme with an antibiotic alone, a surfactant alone, an antibiotic or a surfactant, an antibiotic or an organic solvent, Enhancing the cell permeability of the substrate to the intracellular enzyme by treating with an activator and an organic solvent; And converting the substrate into an intracellular enzyme reaction product by reacting a substrate for an intracellular enzyme with Gram-positive bacteria having improved cell permeability of the substrate to the intracellular enzyme, and a method for producing an intracellular enzyme reaction product from the substrate . In a method according to another embodiment of the present invention, the antibiotic is penicillin, the surfactant is sorbitan monolaurate, and the organic solvent is toluene. Technical characteristics such as intracellular enzymes, Gram-positive bacteria, penicillin treatment concentration, sorbitan monolaurate treatment concentration, and toluene treatment concentration constituting the method according to another example of the present invention are not particularly limited, The description of the method for enhancing the cell permeability of the substrate to the enzyme is the same as that described above, and thus the description thereof is omitted.
In the method according to another embodiment of the present invention, the substrate and the intracellular enzyme reaction product can be selected in various ways according to the kind of the intracellular enzyme. In consideration of the availability of the intracellular enzyme reaction product, the substrate is fructose, And the intracellular enzyme reaction product is preferably alulose.
The method of improving the cell permeability of a substrate to an intracellular enzyme according to an example of the present invention can be used to change the cell wall or cell membrane structure of a Gram-positive bacterium producing an endoenzyme, do. Therefore, the productivity of the reaction product can be greatly increased by reacting the substrate for Gram-positive bacteria and the intracellular enzyme with improved cell permeability to the intracellular enzyme by the method of the present invention. In addition, by using the Gram-positive bacteria having improved cell permeability to the intracellular enzyme by the method of the present invention, the cost and time for purification of the enzyme can be reduced, the immobilization can be facilitated, and the intracellular enzymes A reaction efficiency similar to that of a direct reaction can be expected.
FIG. 1 shows the result of measuring enzyme activity of a recombinant strain according to the kind of antibiotics when Corynebacterium glutamicum DS00001, a recombinant strain, was treated with various antibiotics.
FIG. 2 shows the result of measuring enzyme activity of the recombinant strain according to the treatment concentration of penicillin when the recombinant strain Corynebacterium glutamicum DS00001 was treated with penicillin.
FIG. 3 shows the result of measuring enzyme activity of a recombinant strain according to the type of surfactant when the cells of Corynebacterium glutamicum DS00001, a recombinant strain, were treated with various surfactants.
FIG. 4 shows the result of measuring enzyme activity of the recombinant strain according to the treatment concentration of
FIG. 5 shows the result of measuring enzyme activity of a recombinant strain according to the kind of an organic solvent when a cell of Corynebacterium glutamicum DS00001, which is a recombinant strain, was treated with various organic solvents.
FIG. 6 shows the result of measuring enzyme activity of the recombinant strain according to the treatment concentration of toluene when the cells of the recombinant strain Corynebacterium glutamicum DS00001 were treated with toluene.
FIG. 7 shows the result of measuring the enzyme reaction activity of a recombinant strain after treating the recombinant strain Corynebacterium glutamicum DS00001 with various combinations of antibiotics, surfactants and organic solvents.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.
1. Preparation of a recombinant strain producing D-alulose 3-epimerase
Genomic DNA was extracted from Flavonifractor plautii KCTC 5970, which was distributed at the Korea Microorganism Resource Center, and used as a template, and a gene encoding D-allose 3-epimerase Polymerase chain reaction (PCR) was carried out using a primer for cloning the polynucleotide of SEQ ID NO: 2 and Ex-Taq (TAKARA) polymerase. Table 1 below shows Flavonifractor < RTI ID = 0.0 > alulose 3-epimerase from the genomic DNA of E. coli , E. coli , E. coli , C. plautii , and the like. The primers shown in Table 1 were manufactured by Bioneer co., KR.
Then, the target DNA was isolated from the PCR product using the gel extraction kit (Qiagen), bound to Easy T-vector (promega), and the base sequence analysis of the isolated DNA was performed by Bioneer co., KR Respectively. As a result, it was confirmed that the target DNA amplified by PCR corresponds to the polynucleotide of SEQ ID NO: 2. Then, the target DNA amplified by the PCR reaction was inserted into the same restriction enzyme recognition site of the Corynebacterium shuttle vector PDS vector using the restriction enzymes Pst I and BamH I to construct a recombinant plasmid pDSFDPE Respectively. Thereafter, a recombinant plasmid pDSFDPE was inserted into the cells of Corynebacterium glutamicum ATCC 13032 strain by electroporation to prepare a recombinant strain. The Corynebacterium glutamicum ATCC 13032 strain, in which the recombinant plasmid pDSFDPE was inserted, was named Corynebacterium glutamicum DS00001, and the microorganism preservation center of Korea Institute of Bioscience and Biotechnology And received grant number KCCM 80101.
Then, a single colony of the recombinant strain was inoculated in 15 ml of LB-ampicilline medium (Difco) and pre-cultured for 6 hours at 37 ° C and 200 rpm. Then, the preculture was inoculated into 500 ml of LB-ampicilline medium and cultured under shaking conditions at 37 DEG C and 200 rpm. Then, when the absorbance (at 600 nm) of the culture was 0.5, IPTG was added at a concentration of 0.1 mM to induce the overexpression of the target enzyme. At the induction of overexpression, the culture was switched to 16 ° C and 150 rpm and maintained for about 16 hours. Then, the culture of the recombinant strain was centrifuged at 13000 rpm for 2 minutes to remove the supernatant, and the cells of the recombinant strain were recovered.
Cells of the recovered recombinant strains were suspended in lysis buffer (50 mM Tris-
2. Improvement of cell permeability of substrate by antibiotic treatment of recombinant strain
Example 1: Antibiotic treatment of recombinant strains and analysis of enzyme activity according to antibiotics
In a culture container containing a BHI medium (Brain heart infusion broth), a recombinant strain Corynebacterium ( Corynebacterium glutamicum ) DS00001 was added in an amount of 2 vol% (vol%) based on the total volume of the medium, the culture container was transferred into a shaking incubator, and cultured under shaking conditions at 30 ° C and 200 rpm. When the absorbance (at 600 nm) of the culture solution reached 0.6 to 0.8, the antibiotics were added to the culture container to a predetermined concentration, cultured at 30 ° C and 200 rpm for 20 hours with shaking, and centrifuged to obtain a recombinant strain Was recovered. In addition, as a control, cells of the recombinant strain recovered by centrifuging the culture without using antibiotics were used. The types of antibiotics used to treat the microbial cells of the recombinant strains and the antibiotic treatment concentrations based on 1 liter of the culture are as follows.
Penicillin: 2 mg / l, 5 mg / l
* Ethambutol: 50 mg / l, 100 mg / l
Ethionamide: 50 mg / l, 100 mg / l
Isoniazid: 50 mg / l, 100 mg / l
Then, the fructose solution and the buffer solution of PIPES [piperazine-N, N'-bis (2-ethanesulfonic acid)] were mixed and the recovered recombinant strain was added thereto to obtain a recombinant strain having a fructose concentration of 50 mM, , A pH of about 7.0, a PIPES concentration of 50 mM, and reacted at 65 DEG C for 10 minutes. Then, the amount of the reaction product, alulose, was analyzed by high performance liquid chromatography (HPLC), and the enzyme reaction activity of the recombinant strain was evaluated by the amount of the produced alulose. The enzymatic reaction of the recombinant strain means a reaction in which fructose is converted to alulose by D-alulose 3-epimerase present in the cells of the recombinant strain. The HPLC analysis conditions are as follows.
* Column type: Aminex HPX-87C carbohydrate column
* Column temperature: 80 ° C
* Sample volume (inject volume: 10 μl
* Sample flow rate: 0.6 ml / min
* Run time: 25 min
FIG. 1 shows the result of measuring enzyme activity of a recombinant strain according to the kind of antibiotics when Corynebacterium glutamicum DS00001, a recombinant strain, was treated with various antibiotics. In FIG. 1, the enzyme reaction activity of the recombinant strain is shown as 100 relative to the enzyme reaction activity of the recombinant strain used as a control (indicated as Wild in FIG. 1). The recombinant strains, as shown in Figure 1 from Corynebacterium glutamicum (Corynebacterium glutamicum ) DS00001 was treated with penicillin, the enzyme activity of the recombinant strain was the highest.
Example 2: Penicillin treatment of recombinant strains and enzyme reaction activity according to penicillin concentration
The recombinant strains were treated with the same conditions and methods as in Example 1 except that the recombinant strains were treated with penicillin at various concentrations and the enzyme reaction activity of the recombinant strains was evaluated. The treatment concentration of penicillin based on 1 liter of the culture in Example 2 is as follows.
* Penicillin: 1 mg / L, 2 mg / L, 5 mg / L, 7 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L and 30 mg / L
FIG. 2 shows the result of measuring enzyme activity of the recombinant strain according to the treatment concentration of penicillin when the recombinant strain Corynebacterium glutamicum DS00001 was treated with penicillin. In FIG. 2, the enzyme reaction activity of the recombinant strain is shown as 100 relative to the enzyme reaction activity of the recombinant strain used as a control (the penicillin treatment concentration is 0 in FIG. 1). As shown in FIG. 2, when the recombinant strain Corynebacterium glutamicum DS00001 was treated with penicillin at a concentration of 20 mg / L, the enzyme reaction activity of the recombinant strain was the highest, and the recombinant strain Was increased about 2.5 times as compared to the control.
3. Improvement of cell permeability of substrate by surfactant treatment of recombinant strain
Example 3: Analysis of Enzyme Reaction Activity by Surfactant Treatment and Surfactant Type in Recombinant Strain
In a culture container containing a BHI medium (Brain heart infusion broth), a recombinant strain Corynebacterium ( Corynebacterium glutamicum ) DS00001 was added in an amount of 2 vol% (vol%) based on the total volume of the medium, the culture container was transferred into a shaking incubator and cultured with shaking at 30 캜 and 200 rpm for 48 hours. Thereafter, the culture solution was centrifuged to recover the cells of the recombinant strain. Thereafter, cells of the recovered recombinant strain were added to 50 ml of a surfactant dilution solution (the surfactant was diluted to a predetermined concentration using water) and left on ice for 15 minutes to treat the cells of the recombinant strain. Then, the cells were washed with the reaction buffer and centrifuged twice to collect the cells of the recombinant strain. In addition, as a control, cells of a recombinant strain recovered by centrifuging the culture without using a surfactant were used. The type and concentration of the surfactant used to treat the cells of the recombinant strain are as follows. The treatment concentration of the surfactant is expressed as a percentage of the weight of the surfactant based on the total volume of the surfactant diluent solution. For example, if the treatment concentration of the surfactant is 1%, the amount of surfactant contained in 100 ml of the surfactant diluent solution is 1 g.
* Triton X-100 (Polyoxyethylene octyl phenyl ether): 0.2%, 0.5%
* CTAB (Cetyl trimethylammonium bromide): 0.2%, 0.5%
Tween 20 (Polyoxyethylene (20) sorbitan monolaurate): 2%, 5%
Tween 40 (Polyoxyethylene (20) sorbitan monopalmitate): 2%, 5%
Tween 80 (Polyoxyethylene (20) sorbitan monooleate): 2%, 5%
* Span 20 (Sorbitan monolaurate): 2%, 5%
Span 80 (Sorbitan monooleate): 2%, 5%
Then, the fructose solution and the buffer solution of PIPES [piperazine-N, N'-bis (2-ethanesulfonic acid)] were mixed and the recovered recombinant strain was added thereto to obtain a recombinant strain having a fructose concentration of 50 mM, 4 g / l, a concentration of PIPES of 50 mM and a pH of about 7.0 was prepared, and then reacted at 65 DEG C for 10 minutes. Then, the amount of the reaction product, alulose, was analyzed by high performance liquid chromatography (HPLC), and the enzyme reaction activity of the recombinant strain was evaluated by the amount of the produced alulose. The enzymatic reaction of the recombinant strain means a reaction in which fructose is converted to alulose by D-alulose 3-epimerase present in the cells of the recombinant strain. The HPLC analysis conditions are as follows.
* Column type: Aminex HPX-87C carbohydrate column
* Column temperature: 80 ° C
* Sample volume (inject volume: 10 μl
* Sample flow rate: 0.6 ml / min
* Run time: 25 min
FIG. 3 shows the result of measuring enzyme activity of a recombinant strain according to the type of surfactant when the cells of Corynebacterium glutamicum DS00001, a recombinant strain, were treated with various surfactants. In FIG. 3, the enzyme reaction activity of the recombinant strain is shown as 100 relative to the enzyme reaction activity of the recombinant strain used as a control (indicated by Control in FIG. 3). As shown in FIG. 3, the recombinant strain had the highest enzyme activity when the recombinant strain Corynebacterium glutamicum DS00001 was treated with
Example
4: Analysis of Enzyme Reaction Activity by
The recombinant strain was treated with the same conditions and method as in Example 3 except that the recombinant strain was treated with various concentrations of
Span 20: 0.5%, 1%, 2%, 3%, 4%, 5%
FIG. 4 shows the result of measuring enzyme activity of the recombinant strain according to the treatment concentration of
4. Improvement of cell permeability of substrate by organic solvent treatment of recombinant strain
Example 5: Treatment of recombinant strains with organic solvent and enzyme reaction activity according to organic solvent type
In a culture container containing a BHI medium (Brain heart infusion broth), a recombinant strain Corynebacterium ( Corynebacterium glutamicum ) DS00001 was added in an amount of 2 vol% (vol%) based on the total volume of the medium, the culture container was transferred into a shaking incubator and cultured with shaking at 30 캜 and 200 rpm for 48 hours. Thereafter, the culture solution was centrifuged to recover the cells of the recombinant strain. Thereafter, cells of the recovered recombinant strain were added to 50 ml of an organic solvent dilution solution (the organic solvent was diluted to a predetermined concentration using water), and the cells were allowed to stand on ice for 15 minutes to treat the cells of the recombinant strain. Then, the cells were washed with the reaction buffer and centrifuged twice to collect the cells of the recombinant strain. As a control, cells of the recombinant strain recovered by centrifuging the culture without using an organic solvent were used. The type and concentration of the organic solvent used for treating the cells of the recombinant strain are as follows. The treatment concentration of the organic solvent is expressed as a percentage of the volume of the organic solvent based on the total volume of the organic solvent dilution solution. For example, if the treatment concentration of the organic solvent is 1%, the amount of the organic solvent contained in 100 ml of the diluted organic solvent solution is 1 ml.
1-propanol: 10%, 20%
Iso-propanol: 10%, 20%
Ethanol: 10%, 20%
Methanol: 10%, 20%
* Acetone: 10%, 20%
DMSO (Dimethyl sulfoxide): 10%, 20%
Toluene: 10%, 20%
* Hexane: 10%, 20%
Then, the fructose solution and the buffer solution of PIPES [piperazine-N, N'-bis (2-ethanesulfonic acid)] were mixed and the recovered recombinant strain was added thereto to obtain a recombinant strain having a fructose concentration of 50 mM, 4 g / l, a concentration of PIPES of 50 mM and a pH of about 7.0 was prepared, and then reacted at 65 DEG C for 10 minutes. Then, the amount of the reaction product, alulose, was analyzed by high performance liquid chromatography (HPLC), and the enzyme reaction activity of the recombinant strain was evaluated by the amount of the produced alulose. The enzymatic reaction of the recombinant strain means a reaction in which fructose is converted to alulose by D-alulose 3-epimerase present in the cells of the recombinant strain. The HPLC analysis conditions are as follows.
* Column type: Aminex HPX-87C carbohydrate column
* Column temperature: 80 ° C
* Sample volume (inject volume: 10 μl
* Sample flow rate: 0.6 ml / min
* Run time: 25 min
FIG. 5 shows the result of measuring enzyme activity of a recombinant strain according to the kind of an organic solvent when a cell of Corynebacterium glutamicum DS00001, which is a recombinant strain, was treated with various organic solvents. In FIG. 5, the enzyme reaction activity of the recombinant strain is shown as 100 relative to the enzyme reaction activity of the recombinant strain used as a control (indicated by Control in FIG. 5). The recombinant strains, as also seen in five of Corynebacterium glutamicum (Corynebacterium glutamicum ) DS00001 was treated with toluene (Toluene), the enzyme reaction activity of the recombinant strain was the highest.
Example 6: Analysis of Enzyme Reaction Activity by Toluene Treatment and Toluene Treatment Concentration of Recombinant Strain
The recombinant strains were treated with the same conditions and methods as in Example 5 except that the recombinant strains were treated with various concentrations of toluene diluting solution to evaluate the enzyme reaction activity of the recombinant strains. The treatment concentration of toluene in Example 6 is as follows. The treatment concentration of toluene is a percentage of the volume of toluene based on the total volume of the toluene dilution solution. For example, if the treatment concentration of toluene is 1%, the amount of toluene in 100 ml of diluted toluene solution is 1 ml.
Toluene: 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%
FIG. 6 shows the result of measuring enzyme activity of the recombinant strain according to the treatment concentration of toluene when the cells of the recombinant strain Corynebacterium glutamicum DS00001 were treated with toluene. In FIG. 6, the enzyme reaction activity of the recombinant strain was relatively represented by the enzyme activity of the recombinant strain used as a control (when the toluene treatment concentration was 0 in FIG. 6) as 100. As shown in FIG. 6, when the recombinant strain Corynebacterium glutamicum DS00001 was treated with 20% (v / v) toluene, the enzyme reaction activity of the recombinant strain was the highest, and the recombinant strain Was increased about 1.8 times as compared with the control group.
5. Improvement of cell permeability of the substrate by various combinations of antibiotics, surfactants and organic solvents in the recombinant strains
From the results of Examples 1 to 6, it can be seen that the type of treatment agent of Corynebacterium glutamicum DS00001 strain for improving the cell permeability of fructose, which is a substrate for D-alulose 3-epimerase, The optimum concentrations of these were selected as follows.
Penicillin: 20 mg / l
Span 20 (Sorbitan monolaurate): 3% (w / v)
Toluene: 20% (v / v)
Then, the cells of Corynebacterium glutamicum strain DS00001 were treated with various combinations of the selected treatment agents, and the enzyme reaction activity of the treated recombinant strains was analyzed.
In the penicillin-treated group, cells of the recombinant strain Corynebacterium glutamicum DS00001 were treated in the following manner. In a culture container containing a BHI medium (Brain heart infusion broth), a recombinant strain Corynebacterium ( Corynebacterium glutamicum ) DS00001 was added in an amount of 2 vol% (vol%) based on the total volume of the medium, the culture container was transferred into a shaking incubator, and cultured under shaking conditions at 30 ° C and 200 rpm. Penicillin (Penicillin) was added at a concentration of 20 ㎎ / ℓ to the culture container when the absorbance (at 600 nm) of the culture reached 0.6 ~ 0.8 and cultured at 30 ° C and 200 rpm for 20 hours with shaking. And the cells of the recombinant strain were recovered.
(Penicillin + Span) treated group, the cells of the recombinant strain recovered in the penicillin treated group were treated with a 3% concentration of
(Penicillin + Toluene) treated group, the cells of the recombinant strain recovered in the Penicillin-treated group were treated with a 20% toluene dilution solution using the method described in Example 5 or Example 6. [
(Penicillin + Span + Toluene) treated group, the cells of the recombinant strain recovered in the penicillin treated group were treated with a
(Span + Toluene) treated group was transformed with the recombinant strain Corynebacterium glutamicum ) DS00001 were treated. In a culture container containing a BHI medium (Brain heart infusion broth), a recombinant strain Corynebacterium ( Corynebacterium glutamicum ) DS00001 was added in an amount of 2 vol% (vol%) based on the total volume of the medium, the culture container was transferred into a shaking incubator and cultured with shaking at 30 캜 and 200 rpm for 48 hours. Thereafter, the culture solution was centrifuged to recover the cells of the recombinant strain. Then, the cells of the recovered recombinant strain were added to 50 ml of the dilution solution of
In addition, the cells of the recombinant strain recovered by centrifuging the culture without any treatment as a control were used.
Then, the fructose solution and the buffer solution of PIPES [piperazine-N, N'-bis (2-ethanesulfonic acid)] were mixed and the cells of the recombinant strain recovered in each treatment group were added thereto. A bacterial suspension having a bacterial cell concentration of 4 g / l, a PIPES concentration of 50 mM and a pH of about 7.0 was prepared and reacted at 65 ° C for 10 minutes. Then, the amount of the reaction product, alulose, was analyzed by high performance liquid chromatography (HPLC), and the enzyme reaction activity of the recombinant strain was evaluated by the amount of the produced alulose. The enzymatic reaction of the recombinant strain means a reaction in which fructose is converted to alulose by D-alulose 3-epimerase present in the cells of the recombinant strain. The HPLC analysis conditions are as follows.
* Column type: Aminex HPX-87C carbohydrate column
* Column temperature: 80 ° C
* Sample volume (inject volume: 10 μl
* Sample flow rate: 0.6 ml / min
* Run time: 25 min
FIG. 7 shows the result of measuring the enzyme reaction activity of a recombinant strain after treating the recombinant strain Corynebacterium glutamicum DS00001 with various combinations of antibiotics, surfactants and organic solvents. In FIG. 7, the enzyme reaction activity of the recombinant strain was relatively represented by 100 as the enzyme reaction activity of the recombinant strain used as a control (indicated as Not-treated in FIG. 7). As shown in FIG. 7, when the cells of the recombinant strain Corynebacterium glutamicum DS00001 were sequentially treated with the
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Therefore, the scope of the present invention should be construed as including all embodiments falling within the scope of the appended claims.
<110> DAESANG CORPORATION <120> Method of improving cytoperability of substrate of endoenzyme and method of manufacturing products of endoenzyme reaction from 기판 <130> DP-15-1060 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 294 <212> PRT <213> Artificial Sequence <220> ≪ 223 > D-allulose 3-epimerase derived from Flavonifractor plautii <400> 1 Met Asn Pro Ile Gly Met His Tyr Gly Phe Trp Ser His Asn Trp Asp 1 5 10 15 Glu Ile Ala Tyr Ile Pro Leu Met Glu Lys Leu Ala Trp Leu Gly Phe 20 25 30 Asp Ile Cys Glu Val Ala Ser Ala Glu Trp Gly Tyr Tyr Asp Asp Ala 35 40 45 Arg Leu Arg Glu Leu Lys Ala Cys Ala Asp His Asn Gly Leu Gly Ile 50 55 60 Thr Tyr Ser Ile Gly Leu Glu Ala Lys Tyr Asp Leu Ala Ser Asp Asp 65 70 75 80 Pro Ala Val Arg Glu Asn Gly Ile Arg His Val Thr Arg Ile Leu Glu 85 90 95 Ser Met Pro Lys Val Gly Ala Ile Leu Asn Gly Val Ser Tyr Ala 100 105 110 Gly Trp Gln Ala Leu Pro Asp His Gly Ile Thr Leu Asp Glu Lys Arg 115 120 125 Arg Lys Glu Glu Leu Ala Leu Glu Ser Met Ser Arg Leu Met Lys Val 130 135 140 Ala Glu Asp Cys Gly Val Leu Tyr Cys Cys Glu Val Val Asn Arg Phe 145 150 155 160 Glu Gln Tyr Leu Leu Asn Thr Ala Lys Glu Gly Val Glu Phe Val Lys 165 170 175 Arg Leu Gly Ser Pro Asn Ala Arg Val Leu Leu Asp Thr Phe His Met 180 185 190 Asn Ile Glu Glu Asp Ser Met Val Asp Ala Ile Leu Glu Ala Gly Pro 195 200 205 Trp Leu Gly His Phe His Val Gly Glu Asn Asn Arg Arg Pro Ala Gly 210 215 220 Ser Thr Asn Arg Leu Pro Trp Lys Asp Met Ala Ala Ala Leu Lys Gln 225 230 235 240 Val Asn Tyr Gln Gly Ala Ile Val Met Glu Pro Phe Val Leu Met Gly 245 250 255 Gly Thr Ile Pro Tyr Asp Ile Lys Val Trp Arg Asp Leu Ser Gly Gly 260 265 270 Ala Gly Glu Ala Gly Leu Asp Glu Met Ala Gly Arg Ala Cys Arg Phe 275 280 285 Leu Lys Glu Leu Thr Ala 290 <210> 2 <211> 885 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding D-allulose 3-epimerase derived from Flavonifractor plautii <400> 2 atgaacccga ttggaatgca ctacggcttc tggagccaca actgggacga gattgcatac 60 atacccctga tggagaagct ggcctggctg ggctttgaca tctgcgaggt ggcctccgcc 120 gagtggggct attacgacga cgccaggctg cgggagctga aggcctgcgc cgatcacaac 180 ggcctgggca tcacctattc catcggcctg gaggccaaat acgacctggc cagcgacgat 240 ccggcggtgc gggagaacgg catccgccat gtcacccgca tcctggagag catgcccaag 300 gtgggggcgg ccatcctcaa cggcgtgtcc tacgccgggt ggcaggccct gcccgaccac 360 ggaatcaccc tggacgagaa gcgccgcaag gaggagcttg ccctggagtc catgtcccgg 420 ctcatgaagg tggcggagga ctgcggcgtg ctctactgct gcgaggtggt caaccgcttc 480 gagcagtacc tgctcaacac cgccaaagag ggcgtggagt ttgtcaagcg cctgggcagt 540 cccaacgccc gggtgctgct ggataccttc cacatgaaca tcgaggagga cagcatggtg 600 gacgccattc tggaggcggg cccctggctg gggcatttcc acgtggggga gaacaaccgc 660 cgccccgccg gctccaccaa ccgcctgccc tggaaggaca tggccgccgc cctcaagcag 720 gtgaactacc agggggccat tgtgatggag cccttcgtgc tcatgggggg taccattccc 780 tatgatatca aggtctggcg ggatctcagc ggcggggccg gggaggccgg gctggacgag 840 atggcgggcc gggcctgccg gttcctcaag gagctgaccg cgtaa 885 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer for cloning D-allulose 3-epimerase gene <400> 3 agtcactgca gaccctactt agctgccaa 29 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for cloning D-allulose 3-epimerase gene <400> 4 aattcggatc cttacgcggt cagctccttg 30
Claims (13)
Wherein the antibiotic is penicillin, the surfactant is sorbitan monolaurate, the organic solvent is toluene, the endoenzyme is a ketose 3-epimerase, the gram- A method for improving the cell permeability of a substrate to an intracellular enzyme, characterized in that it is Corynebacterium glutamicum
And converting the substrate into an intracellular enzyme reaction product by reacting a substrate for an intracellular enzyme with Gram-positive bacteria having improved cell permeability of the substrate to the intracellular enzyme,
Wherein the antibiotic is penicillin, the surfactant is sorbitan monolaurate, the organic solvent is toluene, the endoenzyme is a ketose 3-epimerase, the gram- A method for producing an intracellular enzyme reaction product from a substrate, characterized in that it is Corynebacterium glutamicum
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| KR100832339B1 (en) * | 2006-12-11 | 2008-05-26 | 솔젠트 (주) | Novel sinorhizobium sp. yb-58 strain converting fructose into psicose, and method for producing psicose using the same strain |
| KR20140122043A (en) * | 2013-04-09 | 2014-10-17 | 주식회사 삼양제넥스 | Strain of Ensifer genus and method for producing psicose using the same |
| KR101577147B1 (en) * | 2014-10-01 | 2015-12-11 | 경상대학교산학협력단 | Preparing method for psicose |
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| KR100832339B1 (en) * | 2006-12-11 | 2008-05-26 | 솔젠트 (주) | Novel sinorhizobium sp. yb-58 strain converting fructose into psicose, and method for producing psicose using the same strain |
| KR20140122043A (en) * | 2013-04-09 | 2014-10-17 | 주식회사 삼양제넥스 | Strain of Ensifer genus and method for producing psicose using the same |
| KR101577147B1 (en) * | 2014-10-01 | 2015-12-11 | 경상대학교산학협력단 | Preparing method for psicose |
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| CN109666620A (en) * | 2018-05-29 | 2019-04-23 | 中国科学院天津工业生物技术研究所 | A kind of engineered strain producing Tagatose, construction method and application |
| CN109666620B (en) * | 2018-05-29 | 2022-05-10 | 中国科学院天津工业生物技术研究所 | Engineering strain for producing tagatose, construction method and application thereof |
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