KR20200098882A - Manufacturing Method of Pullulan Using recombinant glycosyltransferases - Google Patents

Abstract

The present invention relates to a process of producing sugar transferase, Dextransucrase and Neopullulanase, and synthesizing Panose, a type of triose, and Pullulan, a polymer of Panose, through the corresponding enzymatic reaction and, more specifically, to a process of constructing an expression vector of Dextransucrase and Neopullulanase, which can be expressed in E. coli in large quantities and has easy purification process, using molecular biology techniques, transforming it in E. coli to produce a large amount of recombinant Dextransucrase and Neopullulanas, producing Panose, a kind of triose, through the enzymatic reaction of recombinant Dextransucrase using sucrose and maltose as substrates, and synthesizing Pullulan, a sugar polymer, through the enzymatic reaction of recombinant Neopullulanase using the reaction product of Panose as a substrate.

Description

Manufacturing Method of Pullulan Using Recombinant Glucose Transferase {Manufacturing Method of Pullulan Using Recombinant Glycosyltransferases}

The present invention produces sugar transfer enzymes Dextransucrase and Neopullulanase, and through an enzymatic reaction, Panose, a type of tritanose, and Flulan, a polymer of pannose. ) To the synthesis process. In more detail, by using molecular biology techniques, a large amount of expression in E. coli is possible and an expression vector of neofluranase and dextranshukrase that can be easily purified is constructed, and it is transformed in E. coli to produce a large amount of recombinant dextranche. It is to provide a method for producing craze and neofluranase. In addition, pannose, a kind of samtangose, is produced through an enzymatic reaction using the recombinant dextran sucrose using sugar (Sucrose) and maltose as substrates, and the recombination using the pannose synthesis reaction product as a substrate. It relates to a process for synthesizing flurane, a sugar polymer, through an enzymatic reaction using neofluranase.

Flulan (Pullulan) is a high-molecular polymer of natural polysaccharides secreted from Aureobasidium Pullulans , a black yeast strain. Flurane is a type of carbohydrate derivative that has alpha-1,4 and alpha-1,6 bonds at the same time. It consists of maltriose units, and each maltriose monomer is in the form of alpha-1,6 glucoside bonds. Form a polymer body. Flurane is one of the substances that make up the cell wall of Aureobasidium Pullulans. A fluran layer is formed on the outside of the β-glucan layer, and can be obtained in large quantities through the fermentation process of black yeast. Due to the stepwise structural arrangement characteristics of flurane, it has the advantage of being superior in viscosity, no toxicity at all, and easy industrial application due to its strong durability compared to general polysaccharides such as starch. The mechanism of production of flurane is that when UDPG is converted to UDP in the phospholipids of Aureobasidium pullulans , the glucose molecules are attached to the phospholipids, and these biochemical reactions occur in a chain to form fluran precursors.

Fluran is a natural material that can replace hyaluronic acid, and if mass production is possible based on the development of an eco-friendly production process, it will be possible to supply it as an excellent moisturizing material with a relatively low production cost. In addition, fluran is widely used in various fields such as food additives, edible films, coatings, and medical materials as well as skin moisturizers. Especially, fluran is one of natural polymer materials and has excellent biodegradability, so it is the next generation biotechnology. It is in the limelight as a plastic material.

Currently, the production process of fluran is mainly a process of culturing a large amount of black yeast strain Aureobasidium pullulans and extracting fluran directly from microorganisms, but the cultivation of black yeast takes considerably longer than other microorganisms, as well as black. Due to the melanin pigment peculiar to the yeast strain, fluran, which is the main raw material for production, is colored, and as a result, the purity of fluran and the functionality of the product are poor. Therefore, it can be said that it is necessary to develop a new process that can efficiently produce high-purity flurane.

Korean Patent Registration No. 10-0739022 Korean Patent Registration No. 10-1882101 Korean Patent Registration No. 10-0414952

(Research Paper 0001) Barnett Christian. et al., Pullulan production by Aureobasidium pullulans growing on hydrolysed potato starch waste. CARBOHYDRATE POLYMERS, 1998, Vol. 38(3), pp. 203-209.

The production of fluran that has been studied so far is based on the method of culturing a large amount of black yeast strain Aureobasidium pullulan and extracting fluran directly from the recovered culture, focusing on shortening the production process or improving the purity of fluran. As of yet, there is no research and development in the process of artificially synthesizing flurane through methods other than black yeast strains. The process of producing fluran from black yeast strains not only increases the operating period of production equipment such as fermentors due to the long cultivation time of Aureobasidium pulllans , but also removes black pigment after production of fluran due to the coloring of melanin pigments that appear during black yeast cultivation. As a result, a new purification process is required, or the step of adding the pigment as an essential element to suppress the pigment during the cultivation step must be entered, and as a result, the production cost has a limitation. Accordingly, the present invention is to provide a manufacturing method capable of efficiently producing high-purity flurane.

In order to solve the above problem, the present invention provides a recombinant microorganism for producing dextran sucrase, transformed with a recombinant vector containing the dextran sucrase gene dsr S derived from Leuconostoc mesenteroides . do.

In one embodiment of the present invention, the recombinant vector may be possible when expressed in E. coli vector, whichever is less available and, pRDsu the present invention, one inserts the dextran sucrase gene dsr shoe S in pRSET_A vector.

In one embodiment of the present invention, (a) culturing a recombinant microorganism transformed with a pRDsu vector; (b) culturing the recombinant microorganism cultured in step (a) in a dextran sucrase-only expression medium; (c) extracting dextran sucrase from the culture medium obtained in step (b); And (d) purifying dextran sucrase into a single substance through affinity chromatography. It provides a method for mass-producing texturan sucrase containing.

In one embodiment of the present invention, the affinity chromatography is characterized in that the affinity chromatography using Ni-NTA.

In addition, the present invention provides a recombinant microorganism for producing neofluranase , transformed with a recombinant vector containing the neofluranase gene npl 38 derived from Geobacillus stearothermophilus TRS40.

In an embodiment of the present invention, the recombinant vector may be any vector that can be expressed in E. coli, and in the present invention, it may be a pRNPL vector in which the neofluranase gene npl 38 is inserted into pRSET_A.

In one embodiment of the present invention, (a) culturing a recombinant microorganism transformed with the pRNPL vector; (b) main culturing the recombinant microorganism cultured in step (a) in a neofluranase-only expression medium; (c) extracting neofluranase from the culture solution obtained in step (b); And (d) purifying neofluranase into a single substance through affinity chromatography. It provides a method for mass-producing neofluranase containing.

In one embodiment of the present invention, the affinity chromatography is characterized in that the affinity chromatography using Ni-NTA.

The present invention is characterized by a method for producing flurane from a matrix mixture of maltose and sugar through an enzymatic reaction using the mass-produced dextran sucrase and neofluranase.

In one embodiment of the present invention, (a) preparing a substrate mixture of maltose and sugar; (b) synthesizing pannose by enzymatic reaction by adding dextran sucrase produced from the recombinant microorganism to the substrate mixture of (a); (c) synthesizing flurane through an enzymatic reaction by adding neofluranase produced from the recombinant microorganism to the reaction product of step (b); And (d) extracting and purifying the synthesized fluran in step (c); containing, it provides a method for producing fluran.

The enzymatic reaction process of the present invention is relatively simple and easy to handle compared to the production process through microorganisms, and there is no concern of microbial contamination.

The process of producing fluran from Aureobasidium pullulans takes at least about 7 days because fluran is produced through a third culture process, but the fluran enzyme reaction process of the present invention can be synthesized within about 24 hours at an appropriate temperature. Is completed. This also affects the reduction of the production cost of fluran.

In the present invention, fluran synthesized through an enzymatic reaction process does not require a separate purification process because there is no coloration of the melanin pigment produced by microorganisms, and the yield is also increased by 20-30%.

1 is an analysis result of agarose gel electrophoresis of dextran sucrase gene amplification through a polymerase chain reaction (Polymerase Chain Recation) process.
2 is an analysis result of agarose gel electrophoresis of neofluranase gene amplification through a polymerase chain reaction (Polymerase Chain Recation) process.
3 is a schematic diagram showing the structural characteristics of the expression vector pRDsu into which the dextran sucrase gene is inserted.
4 is a schematic diagram showing the structural characteristics of the expression vector pRNPL into which the neofluranase gene is inserted.
5 is a TLC analysis result of the synthesis of pannose, a precursor of fluran, through an enzymatic reaction of a recombinant dextran sucrase.
6 is a result of SDS-PAGE analysis of large-scale expression of recombinant neofluranase from E. coli.

The present invention will be described in detail based on the following examples. The terms, examples, etc. used in the present invention are merely exemplified to describe the present invention in more detail and to aid understanding of those skilled in the art, and the scope of the present invention should not be interpreted as being limited thereto.

Technical terms and scientific terms used in the present invention represent the meanings commonly understood by those of ordinary skill in the art, unless otherwise defined.

Mass production of dextran sucrase

1. Construction of a recombinant expression vector with a gene of dextran sucrase inserted

A polymerase chain reaction using gDNA of Leuconostoc mesenteroides as a template DNA was carried out to obtain a dextran sucrase gene ( dsr S). Leukonostock mecenteroides was inoculated in Nutrient Broth (0.3% Beef Extract, 0.5% Peptone) and incubated at 27°C for 48 hours, and the cultured Leukonostock mecenteroides was spin down to harvest only the strain and diluted with TE buffer. Therefore, it was used as Template DNA without any special lysis process. The PCR process for amplification of dextran sucrase was performed using Taq polymearse, 0.5 µl of polymerase, 5 µl of 10x PCR buffer, 5 µl of 2.5 mM dNTP mixture, 10 nM/ul forward primer and 10 nM/ul Distilled water was added so that the PCR reaction solution containing 1 µl of each reverse primer and 2 µl of the template was 50 µl, and then PCR was performed.

At this time, pF primer (5'-ctcgagatgaggaaagaagccatcta-3') as a forward primer and pR primer (5'-aagctcctaccaatgttctattgcataaa-3') as a reverse primer were used, and PCR reaction conditions were predenaturation for 5 minutes at 94°C. , 94 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 3 minutes under the conditions of repeated reaction 30 times and induce an extension reaction for 7 minutes at 72 ℃. The resultant product was analyzed for size and concentration through Agarose gel electrophoresis, and as a result, it was confirmed that a dextran sucrase gene derived from leukonostock mecenteroides of about 3,000 bp was amplified (FIG. 1). The reaction product was purified with a DNA fragment purification kit for subsequent enzymatic reaction.

Afterwards, the purified dextransukrase gene ( dsr S) and the expression vector pRSET_A were cut by treatment with the same restriction enzyme and subjected to an enzymatic reaction of T4 DNA Ligase, and the expression vector pRDsu into which the dextransukrase gene was inserted. After constructing (Fig. 3), it was transformed in E. coli DH5a, and whether or not recombination was confirmed through colony PCR. The correctly recombined pRDsu vector was purified and isolated from E. coli and transformed into E. coli BL21 (DE3) for mass expression.

2. Mass expression of recombinant dextran sucrase from E. coli BL21 (DE3)

Expression of dextran sucrase was attempted using E. coli BL21 (DE3) transformed with pRDsu vector. Before attempting mass expression, variables such as medium composition, culture time and temperature were selected on a small-scale in order to explore the expression conditions of pRDsu optimized from E. coli, and compared and analyzed through SDS-PAGE, and then based on the optimized conditions. Dextran sucrase was produced in large quantities. BL21 (DE3) transformed with pRDsu was seeded in SOB broth containing the antibiotic ampicillin, and the culture was again carried out in the same SOB broth at a ratio of 1:50 (v/v), and the OD value in real time When the absorbance reached 0.7 by measuring, isopropyl 1-thio-β-D-galactoside (IPTG) was added so that the final concentration became 1 mM. After that, the expression was performed at 37°C for 5 hours, and the strain after expression was centrifuged at 6,000 rpm at 4°C for 10 minutes to harvest Cells.

The recovered pellets were washed with PBS buffer (137 mM NaCl, 27 mM KCl, 10 mM NaHPO ₄ , 1.8 mM KH ₂ PO ₄ ) and Cell Lysis buffer (10% sucrose, 0.1 M Tris-HCl, 0.05 M EDTA, 0.2 M After suspending the strain with NaCl, pH 7.9), the strain was completely crushed through ultrasound, and the strain lysate was centrifuged again at 16,000 rpm for 30 minutes to separate the water-soluble protein and the insoluble protein. Among them, the pellet section was washed with Cell wash buffer (2% Triton X-100, 1.0 M Urea), and then with Solubilization buffer (8 M Urea, 0.01 M Tris-HCl, 0.2 M Na ₂ HPO ₄ , pH 8.0). The inclusion body was completely dissolved. Each of the separated water-soluble and insoluble proteins was subjected to SDS-PAGE analysis to confirm the expression pattern of dextran sucrase. Take 40 ul of the protein to be analyzed, and mix 10 µl of 5x SDS-PAGE sample buffer (60 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue). The reaction was carried out in boiling water for 5 minutes, and the expression of dextran sucrase was confirmed by electrophoresis on 10% Acrylamide gel. As a result of the analysis, a dextran sucrase-expressing protein band was observed from the insoluble protein sample, indicating that the recombinant dextran sucrase was expressed in the form of an inclusion body protein.

3. Refolding Reaction of Recombinant Dextran Sucrase Protein

Recombinant proteins expressed in the form of inclusion bodies fail to form the correct protein structure, resulting in loss of intrinsic protein activity. Accordingly, there is a need to structurally restore the inclusion body of dextran sucrase produced through the protein refolding process.

The protein refolding reaction of dextran sucrase was carried out by a rapid dilution method, and a 20-fold dextran sucrase-specific refold buffer (50 mM Tris-HCl, 1 mM L-Glutathione oxidized, 1 mM L-Glutathione reduced, 1 mM Beta-merchaptoethanol, 1 mM DTT) to dilute the inclusion body of Dextransucrase and stir slowly at 160 rpm at 4°C.

4. Purification of recombinant dextran sucrase protein through affinity chromatography

Affinity chromatography using Ni-NTA was performed to purify the dextran sucrase protein in which six Histidines were fused to the N-terminus into a single substance. Equilibrated by flowing 10-fold binding buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH 8.0) to a column filled with Ni-NTA resin, and loading a protein sample containing dextran sucrase. And, impurities were completely removed through 10 times the washing buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole, pH 8.0). The target protein was eluted using an eight-fold elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole, pH 8.0), and the fraction showing the peak was recovered and subjected to SDS-PAGE analysis. The absorbance was measured at 280 nm, and the flow rate was 0.5 mL/min.

Mass production of neofluranase

1. Construction of a recombinant expression vector into which the gene of neofluranase was inserted

The process of constructing the recombinant expression vector of neofluranase was carried out almost the same as the dextran sucrase described above. In this project, a neofluranase gene ( npl 38) derived from Geobacillus stearothermophilus TRS40 was used as a target gene. Geobacillus stearothermophilus ( Geobacillus stearothermophilus ) TRS40 was completely purified on a spin column using a gene extraction kit, and then used as a template DNA for PCR. The amplification of npl 38 was 0.5 μl Taq DNA polymerase, 10x PCR buffer 5 μl, 2.5 mM dNTP mixture 5 μl, 10 nM/ul forward primer and 10 nM/ul reverse primer 1 μl, respectively, As the template, distilled water was added so that the PCR reaction solution containing 2 µl became 50 µl, and then PCR was performed.

At this time, pF primer (5'-ggatccatgtataaaatctgggaaaatgt-3') as a forward primer and pR primer (5'-ccatggttataaatctggtacccaatct-3') as a reverse primer were used, and PCR reaction conditions were predenaturation for 5 minutes at 94°C. , 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ under the conditions of 2 minutes repeated reaction 30 times and induce an extension reaction at 72 ℃ 7 minutes. The resulting product was analyzed for size and concentration through Agarose gel electrophoresis, and as a result, it was confirmed that the neofluranase gene of about 2,000 bp was amplified (FIG. 2). The reaction product was purified with a DNA fragment purification kit for subsequent enzymatic reaction.

After that, the purified neofluranase gene (npl38) and the expression vector pRSET_A were cut with the same restriction enzyme and digested, followed by enzymatic reaction of T4 DNA Ligase, and the expression vector into which the neofluranase gene was inserted, pRNPL (Fig. 4 ) Was constructed and transformed in E. coli DH5a, and the plasmid was isolated from each colony and subjected to restriction enzyme treatment to select correctly recombined pRNPL. Thereafter, pRNPL, which was confirmed for recombination, was purified and isolated from E. coli and transformed into E. coli BL21 (DE3) for mass expression.

2. Mass expression of recombinant neofluranase from E. coli BL21 (DE3)

The expression of neofluranase was attempted using pRNPL-transformed E. coli BL21 (DE3). In order to search for the expression conditions of pRNPL optimized from E. coli, conditions such as medium composition, culture time and temperature were compared in the small-scale in the same manner as in the above pRDsu and analyzed through SDS-PAGE.

As a result of the expression test, it was found that pRNPL was expressed in the form of a water-soluble protein in E. coli, and in order to utilize this point, it was decided to proceed with expression through an auto-induction media. In a sterilized Erlenmeyer flask, 200 ml of AIM broth (Triton 12 g/L, yeast extract 24 g/L, glucose 0.5 g/L, lactose 2.0 g/L, (NH4)2SO4 3.3 g/L, KH2PO4 6.8 g/L , Na2HPO4 7.1 g/L, MgSO4 0.15 g/L, and glycerol 4.0 ml/L are prepared and sterilized, and then ampicillin is added. BL21 (DE3) transformed with pRNPL is seed cultured in SOB broth and the culture is Main culture was performed in the above AIM broth at a ratio of 1:50 (v:v) and expressed for 8 hours at 37° C. Pellet was recovered by centrifugation, washed with PBS buffer, and the strain was suspended in Cell Lysis buffer. After that, the strain was completely crushed through ultrasound, and the strain lysate was centrifuged again at 16,000 rpm for 30 minutes to separate the soluble protein and the insoluble protein, and the Pellet part was suspended in Solubilization buffer to completely dissolve the inclusion body. The separated water-soluble protein and insoluble protein were analyzed by SDS-PAGE to confirm the expression pattern of neofluranase, 40 ul of the protein to be analyzed was mixed with 10 µl of 5x SDS-PAGE sample buffer and added to boiling water. The reaction was performed for 5 minutes and the expression of neofluranase was confirmed by electrophoresis on 10% Acrylamide gel (Fig. 6) As a result of the analysis, it was found that the recombinant neofluranase was expressed in the form of a water-soluble protein.

3. Purification of recombinant neofluranase protein through affinity chromatography

The purification process of neofluranase was also carried out through Ni-NTA affinity chromatography using 6x histamine end, which is the principle of purification of dextran sucrase protein, and all processes were performed in the same manner. Equilibrated by flowing 10-fold binding buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH 8.0) to a column filled with Ni-NTA resin, and loading a protein sample containing neofluranase. And, impurities were completely removed through 10 times the washing buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole, pH 8.0). The target protein was eluted using an eight-fold elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole, pH 8.0), and the fraction showing the peak was recovered and subjected to SDS-PAGE analysis. The absorbance was measured at 280 nm, and the flow rate was 0.5 mL/min.

Synthesis of flurane using recombinant sugar transferase

Dextran sucrase purified from a single substance was used as a catalyst for the synthesis of pannose enzyme. 50 mM Sodium acetate buffer (pH 5.5), 1 mM CaCl _2, 1% maltose and 5% sugar were mixed, and 10% of the final volume of recombinant dextran sucrase was added, followed by reaction at 25℃ for 24 hours. Thus, pannose was synthesized. The sample for which the reaction was completed was first analyzed through TLC (Thin Layer Chromatography). For TLC analysis, C ₁₈ silica gel was used as the stationary phase, and Acetic acid: Formic acid: Water = 7: 2: 1 was used as the mobile phase, and the developed plate was slightly immersed in a methanol solution containing 5% sulfuric acid and 5 at 121℃. The reaction was carried out for a minute to detect the spot, and the synthesis of pannose was confirmed through this (FIG. After that, for more precise qualitative and quantitative analysis, High Performance Liquid Chromatography (HPLC) analysis was performed. Analysis conditions are Column: SRT 30 cm x 7.8 mm ID, 5 μm, 300 Å, Solvent: 150 mM phosphate Buffer, pH 7.0, flow rate: 0.7 ml/min, Detector: RI Detector. Synthesis and yield were checked. After that, a large amount of fluran was synthesized using the purified neofluranase and pannose enzyme compound. The pH of the pannose enzyme compound was adjusted to 6.0, 10% recombinant fluranase was added thereto, and then reacted at 40° C. for 24 hours to synthesize fluran from the pannose substrate. After all enzymatic reactions were completed, the reaction was terminated using an ultrafiltration membrane and the contained enzymes were completely removed.To extract the synthesized flurane, ethanol equivalent to twice the total volume was added, and the sugar polymer was added for about 1 hour. Completely precipitated. Thereafter, the precipitated saccharide polymer was recovered and lyophilized to completely powder the flurane. After all identification was completed, HPLC was performed on the final reaction product to analyze the synthesis yield and purity of fluran.

Claims (5)

Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) derived from the dextran sucrase gene dsr S transformed with a recombinant vector containing a recombinant microorganism for the production of dextran sucrase.
Geobacillus stearothermophilus ( Geobacillus stearothermophilus ) Transformed with a recombinant vector containing the neofluranase gene npl 38 derived from TRS40 , a recombinant microorganism for the production of neofluranase .
(a) culturing the recombinant microorganism according to claim 1;
(b) culturing the recombinant microorganism cultured in step (a) in a dextran sucrase-only expression medium;
(c) extracting dextran sucrase from the culture medium obtained in step (b); And
(d) purifying dextran sucrase into a single substance through affinity chromatography;
Containing, a method for mass-producing textran sukrase.
(a) culturing the recombinant microorganism according to claim 2;
(b) main culturing the recombinant microorganism cultured in step (a) in a neofluranase-only expression medium;
(c) extracting neofluranase from the culture solution obtained in step (b); And
(d) purifying neofluranase into a single substance through affinity chromatography;
Containing, a method for mass-producing neofluranase.
(a) preparing a substrate mixture obtained by mixing maltose and sugar;
(b) synthesizing pannose by enzymatic reaction by adding the dextran sucrase of claim 3 to the substrate mixture of (a);
(c) synthesizing flurane through an enzymatic reaction by adding the neofluranase of claim 4 to the reaction product of step (b); And
(d) extracting and purifying the synthesized flurane of step (c);
Containing, a method for producing fluran.

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