TW201529851A - Transformant of recombinant brazzein gene and method for producing soluble recombinant brazzein by using the same - Google Patents

Abstract

The invention relates to a transformant of recombinant brazzein gene. An inducible recombinant plasmid which includes an inducible promoter and a polynucleotide encoding brazzein and a constitutive plasmid are introduced into Bacillus licheniformis. Furthermore, the invention relates to a method for production of soluble recombinant brazzein. Incubating the transformant as described above, and adding inducer when the growth of bacteria reaches default cell concentration. The soluble recombinant brazzein is secreted to the culture medium by the transformant, and the soluble recombinant brazzein can be purified and concentrated.

Description

Transgenic strain with recombinant sweet protein gene and method for producing soluble recombinant sweet protein

The present invention relates to a Bacillus licheniformis transgenic strain, and more particularly to a Bacillus licheniformis transgenic strain for producing a soluble recombinant sweet protein and a method for producing the soluble recombinant sweet taste protein.

Sweeteners play an important role in people's daily diet. According to the source, it can be divided into natural and artificial sweeteners. Natural sweeteners include common sugars such as sugar, sucrose, glucose, etc., which have the advantages of high sweetness acceptance and safety, but relatively low sweetness and high calories. Excessive intake often leads to excessive The caloric intake is therefore easy to derive health problems such as obesity, diabetes and dental caries. In order to overcome these problems, more and more chemically synthesized artificial sweeteners such as Aspartame and Saccharin have been developed and replaced the role of the original natural sweetener, but some studies have pointed out that excessive Ingestion of these artificial sweeteners increases the risk of carcinogenesis.

Therefore, scholars have turned to research and isolate natural plant sweetness proteins, such as thaumatin, monellin and brazzein isolated from tropical African fruits. Their sweetness is thousands of times that of sucrose, with low calories, safety and availability. It is digested and decomposed into an amino acid. Among them, the aforementioned sweet protein brazzein is a sweet protein which is known to have a small molecular weight and is widely studied, and has a sweetness of 500 to 2000 times that of the same quality sucrose. In addition to the above advantages, the sweet protein brazzein has good thermal stability, pH stability, water solubility, similar flavor and sucrose, does not cause oral cavities, is not easily used by microorganisms, and can be eaten by diabetic patients. However, the extraction of materials from natural plants is limited by seasons, yields, tedious steps of increasing the cost, and the like. Therefore, in recent years, more and more scholars have tried to produce sweet protein brazzein using different expression systems.

Taking the prokaryotic expression system as an example, the prior art has used systems such as Escherichia coli and Bacillus subtilis as the expression system of the recombinant sweet protein brazzein. However, the above performance system has the following problems. First, when a transformant strain having a recombinant sweet protein brazzein gene is established, mutation of DNA is apt to occur, resulting in difficulty in embedding the correct DNA molecule. Furthermore, if an attempt is made to express the sweet protein brazzein in a continuous system, the amount of expression and the folding structure of the protein are not stable and it is difficult to secrete the bacteria outside the body.

One aspect of the present invention provides a transformant strain for producing a soluble recombinant sweet taste protein comprising: a host cell, wherein the host cell is Bacillus licheniformis . An inducible recombinant plastid, and a regulatory trait. Wherein the inducible recombinant plastid comprises one of the inducible expression element sequences arranged in sequence, one of the first nucleic acid fragments as shown in SEQ ID NO: 3, and the second nucleic acid fragment as shown in SEQ ID NO: 4, and the inducible expression The element sequence is selected from the group consisting of sequence identification number 1 and sequence identification number 2.

According to an embodiment of the invention, wherein the second nucleic acid fragment encodes a recombinant sweet taste protein as set forth in SEQ ID NO: 5.

According to another embodiment of the invention, the soluble recombinant sweet protein protein is secreted outside of the transformant strain.

According to still another embodiment of the present invention, the soluble recombinant sweet protein has a 1.6% alpha-helix secondary structure.

The transformed strain of the present invention can secrete the produced recombinant sweet taste protein extracellularly to obtain a soluble recombinant sweet taste protein. The soluble recombinant sweet taste protein produced has a 1.6% alpha-helix secondary structure, and has a disulfide bond to make the wrinkle configuration of the protein correct and active.

Another aspect of the present invention provides a method of producing a soluble recombinant sweet protein comprising: providing a liquid culture, wherein the liquid culture comprises the transformant of the present invention. The regulatory properties of the transformed strain continue to exhibit inhibitors to inhibit the expression of soluble recombinant sweet protein in the inducible recombinant plastid of the transformed strain. When the optical density of the cell concentration of the transformed strain reaches 0.5 to 0.6, isopropyl-β-D-thiogalactoside is added to induce the inducible recombinant plastid of the transformed strain to express the soluble recombinant sweet protein. And secreted outside the transgenic strain. Removal of the transformed strain to obtain soluble recombinant sweet taste protein Above the clear liquid. Finally, the soluble recombinant sweet protein was separated from the supernatant.

According to an embodiment of the invention, the inhibitor of the regulated property is LacI.

According to another embodiment of the present invention, the step of obtaining the supernatant comprises the step of treating the liquid culture at a temperature of from 70 ° C to 90 ° C for from 10 minutes to 50 minutes. The transformed strain is then removed by centrifugation to obtain a supernatant containing the soluble recombinant sweet protein.

According to still another embodiment of the present invention, the step of isolating the soluble recombinant sweet taste protein comprises separating the soluble recombinant sweet taste protein from the supernatant using a chromatography column.

According to still another embodiment of the present invention, the chromatography column comprises an anion chromatography column.

Thereby, the method of the present invention can induce the mass of the transgenic strain and then induce the large amount of the soluble recombinant sweet protein in a suitable time point (for example, a suitable cell density). Furthermore, the soluble recombinant sweet protein produced by the method of the present invention has a heat-resistant and protein secondary structure which is relatively loose compared with the conventional sweet protein, so that the separation method of the present invention can be used to obtain a high-purity soluble recombinant sweet taste. protein.

The Summary of the Invention is intended to provide a simplified summary of the present disclosure in order to provide a basic understanding of the disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

110‧‧‧UP element

120‧‧‧-35/-10 promoter region

130‧‧‧Lactose Control Group

140‧‧‧SD sequence

150‧‧‧UP element

160‧‧‧-35/-10 promoter region

170‧‧‧Lactose Control Group

180‧‧‧SD sequence

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

Figure 2 is a graph showing the growth of B. subtilis-induced expression systems at different time points.

Figure 3 is a graph showing the analysis of protein expression of the B. subtilis-induced expression system at different time points, both intracellularly and extracellularly.

Figure 4 is a graph showing the growth of a transformed strain of the present invention at different time points.

Figure 5 is a graph showing the analysis of protein expression at different time points in one aspect of the present invention.

Figure 6 is a graph showing the yield of soluble recombinant sweet protein of the transformed strain of the present invention at different time points.

Fig. 7 is a view showing the analysis of the fermentation process of the transformant strain (pSECS6- lacO _SACBAZ , pBL1) of the method for producing soluble sweet protein according to an embodiment of the present invention.

Fig. 8 is a graph showing the analysis of the fermentation process of the transformant strain (pSECS7- lacO _SACBAZ , pBL1) of the method for producing soluble sweet protein according to another embodiment of the present invention.

Fig. 9 is a view showing the protein expression analysis of each of the purification steps of the transformant strain (pSECS7- lacO _SACBAZ , pBL1) of the soluble sweet protein production method according to another embodiment of the present invention.

Figure 10 is a diagram showing the original film power of the soluble recombinant sweet protein of the present invention. Swimming analysis chart.

Figure 11 is a graph showing the molecular weight identification of the soluble recombinant sweet taste protein of the present invention.

Figure 12 is a graph showing the molecular weight identification of the soluble recombinant sweet protein of the present invention after DTT treatment.

Figure 13 is a graph showing the analysis of the secondary structure of the soluble recombinant sweet protein of the present invention.

The disclosure of the present specification provides a transformant strain for producing a soluble recombinant sweet protein, and a method for producing a soluble recombinant sweet protein. It is introduced into an inducible diplast in a host cell, wherein the inducible diplast comprises an inducible recombinant plastid and a regulatory trait, and the inducible recombinant plastid comprises a gene encoding a recombinant sweet protein to obtain a transformed strain and perform Liquid culture. When the liquid culture reaches a predetermined bacterial concentration, an inducer is added to the liquid culture to induce the aforementioned induced recombinant plastid to express a large amount of soluble recombinant sweet protein and secreted outside the transformed strain. The obtained soluble recombinant sweet protein has high yield and can be easily separated and purified, thereby being applied to mass production.

The term "host cell (or expression system)" as used in the present invention refers to a prokaryotic expression system which utilizes a large number of prokaryotic organisms to express a heterologous protein. In an exemplary embodiment of the invention, a suitable prokaryotic expression system can be, for example, Bacillus licheniformis . Bacillus licheniformis is a Gram-positive, facultative anaerobic spore-forming bacterium that has been approved by the US Food and Drug Administration (FDA) as a GRAS (generally recognized as safe) class.

The term "sweet protein" as used in the present invention refers to a sweet protein brazzein isolated from the wild plant Pentadiplandra brazzeana Baillon in western Africa, having a molecular weight of about 6.3 KDa and an amino acid sequence of 54 amino acids. The stereostructure has an alpha-helix and three antiparallel beta-strands containing eight cysteines and forming four pairs of disulfide bonds.

The term "soluble" as used in the present invention means that the recombinant protein expressed in the expression system can be secreted into the culture medium outside the cell and dissolved in water, and does not accumulate in the cells of the expression system to aggregate into water-insoluble inclusion bodies. Or other forms. In one embodiment of the invention, the recombinant sweet protein described above may have a message sequence added to its N-terminus to facilitate delivery of the expressed recombinant sweet protein to the outside of the cell.

The terms "inducible" or "sustainable" as used herein are referred to herein as plastids into a host cell, and the promoter is a sustained or inducible recombinant protein. A persistent expression system can continue to cause foreign genes to express foreign proteins, but if the foreign protein is harmful to the host, it can harm the host and even cause death. The inducible expression system needs to induce the expression of the foreign gene under specific induction conditions, and can avoid the damage caused by the continuous expression of the cytotoxic protein. In an exemplary embodiment of the invention, a transformant strain suitable for the present invention is an inducible expression system, and suitable specific induction conditions may include, but are not limited to, a predetermined bacterial concentration.

The term "α-helix" as used herein refers to the helical folding of a protein two structure. Due to the α-helix helical folding relationship, the C=O of an amino acid will hydrogen bond with the NH on the third amino acid downstream ( C=O...HN), the hydrogen bond of the maximum strength can be obtained, and becomes a solid cylindrical structure. Therefore, the higher the proportion of α-helix in the protein, the tighter the protein structure.

In the present invention, an inducible diplast is introduced into a host cell, wherein the inducible diplast comprises an inducible recombinant plastid and a regulatory trait, and the inducible recombinant plastid comprises a transgenic plant encoding a recombinant sweet protein gene. The liquid culture is carried out, and when the liquid culture reaches the preset bacterial concentration, the inducer is added to the liquid culture to induce the aforementioned induced recombinant plastid to express a large amount of soluble recombinant sweet protein and secreted outside the transformed strain. The soluble recombinant sweet taste protein expressed can be obtained by removing the transformed strain and removing the supernatant to obtain a large amount of high-purity soluble recombinant sweet protein.

The following examples are presented to illustrate some aspects of the invention, and are intended to be These examples are to be considered as limiting the scope of the invention, but the materials and methods for practicing the invention. All publications cited herein are hereby incorporated by reference in their entirety.

Test Example 1: Production of a transformant strain of soluble recombinant sweet protein (1) plastid construction

Please refer to Fig. 1, which is a structural diagram of an inductive expression element of the present invention. Part (A) is a structural diagram of SECS-6/ lacO . Part (B) is a structural diagram of SECS-7/ lacO .

An insert gene arrangement of the inducible recombinant plasmid pSECS6- lacO _SACBRZ according to an embodiment of the present invention comprises an inducible expression element SECS-6/ lacO , a first nucleic acid fragment and a second nucleic acid fragment. The inducible expression element SECS-6 /lacO sequence, as shown in SEQ ID NO: 1, sequentially comprises UP element 110, -35/-10 promoter region 120, which stimulates the transcription of the Bacillus subtilis sigma promoter. Lactose manipulation group 130 and SD sequence 140. The sequence of the first nucleic acid fragment, as shown in SEQ ID NO: 3, is the levansucrase message peptide SP _SACB of Bacillus subtilis. The second nucleic acid fragment sequence is a recombinant sweet protein as indicated by the sequence identification number code 4. The interposer fragment was first cut with EcoR I/ Sal I, and then the T4 ligase (T4 DNA ligase) was used in the appropriate molar number with the vector pHY300PLK (Takara Shuzo Co., Tokyo, Japan) cut through the same restriction enzyme. The ratios were joined to obtain the constructed pSECS6- lacO _SACBRZ .

An intervening gene arrangement of the inducible recombinant plasmid pSECS7- lacO _SACBRZ according to another embodiment of the present invention comprises an inducible expression element SECS-7/ lacO , a first nucleic acid fragment and a second nucleic acid fragment. The inducible expression element SECS-7/ lacO sequence, as shown in SEQ ID NO: 2, contains UP element 150, -35/-10, which stimulates the transcription of B. subtilis σ ^A -type promoter and σ ^D - type promoter. Promoter region 160, lactose manipulation group 170, and SD sequence 180. The sequence of the first nucleic acid fragment, as shown in SEQ ID NO: 3, is the fructan sucrase message peptide _PSACB of Bacillus subtilis. The second nucleic acid fragment sequence is a recombinant sweet protein as indicated by the sequence identification number code 4. The interposer fragment was first cut with EcoR I/ Sal I, and then the T4 ligase (T4 DNA ligase) was used in the appropriate molar number with the vector pHY300PLK (Takara Shuzo Co., Tokyo, Japan) cut through the same restriction enzyme. The ratios were joined to obtain the constructed pSECS7- lacO _SACBRZ .

The regulatory property of the present invention, pBL1, can be referred to "Regulated promoter for high-level expression of heterologous genes in Bacillus subtilis ." (Le Grice, SF, 1990), and sustainable expression of LacI as an inhibitor of the induced recombinant plastid lactose control group. In order to regulate the expression of the recombinant sweet protein of the target gene, the recombinant sweet protein is expressed when the inducer isopropyl-β-D-thiogalactoside is added.

(2) Transformation of Bacillus licheniformis

Transformation of the plastid into the B. licheniformis host can be accomplished, without limitation, by the following steps. First, Bacillus licheniformis electric competent cells were prepared, and a single colony was picked and inoculated into TSB culture solution (Tryptic soy broth), and shake culture was carried out at 37 ° C and 180 rpm. The bacterial culture was inoculated overnight to fresh TSB medium containing 1% glycine, and the initial OD ₆₀₀ was adjusted to 0.15. The mixture was shaken at 37 ° C and 180 rpm until the OD _{600 was} about 1.0, poured into a sterilized centrifuge bottle, and the cells were collected by centrifugation at 6000 x g for 15 minutes at 4 ° C for 30 minutes in an ice bath. After washing the cells twice with ice-cold sterilized deionized water, the cells were suspended in ice-cold sterilized SHMPYT buffer (0.25 M sucrose, 1 mM Hepes, 1 mM magnesium chloride, 1/250 to 1/500 of the volume of the culture solution). 20% (v / v) polyethylene glycol 6000 (PEG6000), 0.125% yeast extraction (yeast extract), 0.25% tryptose peptone (tryptone)] and dispensed into microcentrifuge tubes (100 μ L / tube), Stored at -80 ° C, which is Bacillus licheniformis electric competent cells.

During the electrotransformation experiment, Bacillus licheniformis competent cells were taken from a -80 ° C refrigerator and thawed on ice. Take 1 ~ 5 μ L was added plastid electrically competent Bacillus licheniformis cell, and the electrode tubes previously placed in the cold in an ice bath for 5 minutes, the electric field strength within the 10kV /, 200 [Omega] resistors, capacitors conditions of cm 25 μ F Electric transformation. The transformed cells were added to 1 mL of SB medium (3.5% tryptone, 2.0% yeast extract, 0.5% sodium chloride), shaken at 37 ° C, 120 rpm for 3 to 5 hours, and centrifuged to remove excess culture solution. Apply to LB solid medium (Luria-Bertani) containing appropriate antibiotics and incubate at 37 ° C for 16-18 hours. Observe the results. Firstly, the regulatory property pBL1 was electrotransformed into Bacillus licheniformis according to the above-mentioned electrorotation conditions, and after selecting small colonies for cultivation, the plastids were extracted by commercial kits or by conventional methods to limit the enzymatic cleavage and confirm the correct size of the plastids. . The inducible recombinant plasmids pSECS6- lacO _SACBRZ and pSECS7- lacO _SACBRZ were correctly _inserted into the B. licheniformis host containing the regulatory property pBL1. The transformed strain (pSECS6- lacO _SACBRZ , pBL1) and the transformed strain (pSECS7- lacO _SACBRZ , pBL1) were obtained.

(3) Transformation of Bacillus subtilis

In order to construct a transformant strain using Bacillus subtilis as a host cell as a comparative example of the present invention, the inducible recombinant plasmid and the regulatory property pBL1 are collectively transformed into a Bacillus subtilis host, and may be included, but not limited to, in the following steps. First, the B. subtilis electric competent cells were prepared, and a single colony was picked and inoculated into the LB culture solution, and shake culture was carried out at 37 ° C and 180 rpm. Inoculate overnight culture medium to fresh LB culture medium to adjust the initial OD ₆₀₀ to 0.1, shake culture at 37 ° C, 180 rpm to an OD _{600 of} about 1.0, and inoculate the bacterial solution to 1% at a ratio of 1:10. In a minimal medium of threonine, the cells were shaken at 37 ° C and 180 rpm to an OD _{600 of} about 1.0, and the cells were collected by centrifugation at 8000 × g for 10 minutes at 4 ° C. After ice-cooling to the sterile deionized water to wash the cells twice, the cells were suspended in ice cold sterile buffer SHMPYT culture volume of 1/250 to 1/500 of the in and dispensed into microcentrifuge tubes (100 μ L / Tube), stored at -80 ° C, that is Bacillus subtilis electric competent cells.

During the electrotransformation experiment, Bacillus subtilis electric competent cells were taken out from the -80 ° C refrigerator and thawed on ice. Take 1 ~ 5 μ L was added plastid electrically competent B. subtilis cells, and placed in advance of the electrode tube in ice, the ice bath for 5 minutes, the electric field intensity at 8.75kV /, 500 [Omega resistors, capacitors conditions of cm 25 μ F Electric transformation. The transformed cells were added to 1 mL of SB culture solution, shaken at 37 ° C, 120 rpm for 3 hours, centrifuged to remove excess culture solution, and then applied to LB solid medium containing appropriate amount of antibiotics, and cultured at 37 ° C for 16 to 18 hours. Observation results. First, the regulatory property pBL1 was electrotransformed into Bacillus subtilis according to the above-mentioned electrorotation conditions, and after the colony was selected for small-scale cultivation, the plastid was extracted by a commercial kit or by a known method to confirm the correct size of the plasmid by restriction enzyme digestion. The inducible recombinant plasmids pSECS6- lacO _SACBRZ and pSECS7- lacO _SACBRZ were correctly _inserted into the Bacillus subtilis host containing the regulatory property pBL1. Bacillus subtilis transformants (pSECS6- lacO _SACBRZ , pBL1) and B. subtilis transformants (pSECS7- lacO _SACBRZ , pBL1) were obtained.

Test Example 2: Performance of recombinant sweet protein (1) Performance in Bacillus subtilis host

Bacillus subtilis transformants (pSECS6- lacO _SACBRZ , pBL1) and Bacillus subtilis transformants (pSECS7- lacO _SACBRZ , pBL1) were cultured in LB medium containing appropriate antibiotics, and cultured overnight at 37 ° C, 180 rpm, and then The bacterial solution was inoculated into the LB medium containing appropriate antibiotics, the initial OD ₆₀₀ was adjusted to 0.1, and the culture was continued at 37 ° C, 180 rpm until the OD ₆₀₀ reached 0.5-0.6, at which time 1 mM isopropyl-β-D-sulfur was added. The galactosides induced recombinant sweet protein expression and no isopropyl-β-D-thiogalactoside as a control group. The cell density was measured at different induction time points and 1 mL of bacterial solution was taken out at 4 ° C. The cells were centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected for observation of extracellular protein expression by Tricine-SDS-PAGE protein electrophoresis and Western blotting. Part of the bacteria was dissolved in lysis buffer, and after ultrasonic disruption, the supernatant was collected by centrifugation and the intracellular protein expression was observed by Western blotting with Western blotting method and Tricine-SDS-PAGE protein electrophoresis.

Please refer to Fig. 2 and Fig. 3, Fig. 2 shows the growth of B. subtilis-induced expression system at different time points, and Fig. 3 shows the protein expression of B. subtilis-induced expression system in extracellular and intracellular at different time points. A) Part of the extracellular protein electropherogram and the Western blot method (B) are the intracellular protein electropherogram and the western blot method. Among them, "M" is a protein molecular weight marker, and rbrz is a sweet protein produced by the E. coli system as a positive control group.

The results showed that no extracellular recombinant sweet protein was detected in the culture. In the bacterial part, two B. subtilis transformants were able to detect intracellular recombinant sweet protein at 24 and 36 hours of culture. The recombinant sweet protein expression was highest in the intracellular expression of Bacillus subtilis (pSECS7- lacO _SACBRZ , pBL1) at 24 hours of culture, reaching 30.9 μg/ml; and in Bacillus subtilis (pSECS6- lacO _SACBRZ) , pBL1) yield is lower. It is speculated that the reason may be related to the rapid decline of the number of bacteria after the addition of the inducer, which may be caused by damage to the bacteria during protein synthesis or folding.

(2) Performance in Bacillus licheniformis host

A single colony of the transformed strain (pSECS6- lacO _SACEBRZ , pBL1) and the transformed strain (pSECS7- lacO _SACBRZ , pBL1) were cultured overnight in LB medium, then inoculated into TSB medium, and cultured at 37 ° C and 180 rpm. When the OD ₆₀₀ reached 0.5-0.6, 0.5mM isopropyl-β-D-thiogalactoside was added to induce recombinant sweet protein expression without isopropyl-β-D-thiogalactoside as a control group. The bacterial cell density was measured at different induction time points, and 1 mL of the bacterial liquid was taken out, centrifuged at 10,000 rpm for 10 minutes at 4 ° C, and the supernatant was collected for the performance of extracellular protein by Western blotting with Western blotting method and Tricine-SDS-PAGE protein electrophoresis.

Please refer to Fig. 4 to Fig. 6. Fig. 4 is a view showing the growth of the transformed strain of the present invention at different time points. Fig. 5 is a diagram showing the extracellular protein expression of the transgenic plants of the present invention at different time points, (A) is a protein electrophoresis map, and (B) is a Western blotting method. Figure 6 is a graph showing the yield of soluble recombinant sweet protein of the transformed strain of the present invention at different time points, (A) is the total yield, and (B) is the unit yield.

It can be seen from the growth curve that the B. licheniformis cell density is still steadily increased after the addition of the inducer. In terms of soluble recombinant sweet protein expression, the expression of transgenic strains (pSECS7- lacO _SACBRZ , pBL1) was higher than that of transgenic plants (pSECS6- lacO _SACBRZ , pBL1) at different time points. Both have maximum yields for 24 hours of culture. Transformation total strain _(pSECS6- lacO SACBRZ, pBL1) of 54 μ g / ml; Transformation strain _(pSECS7- lacO SACBRZ, pBL1) of 64 μ g / ml. After culturing conversion yields Transformation strain _(pSECS7- lacO SACBRZ, pBL1) 24 - hour Max Unit yield was _{18.74 μ g / ml / OD 600} .

(3) Production of soluble recombinant sweet protein by small yeast in Bacillus licheniformis host

A single colony of the transformed strain (pSECS6- lacO _SACBRZ , pBL1) and the transformed strain (pSECS7- lacO _SACBRZ , pBL1) was cultured overnight in TSB medium, and then an appropriate amount of the pre-culture solution was inoculated into 3L TSB medium to 37 Incubate at °C and 180 rpm, add 0.5 mM isopropyl-β-D-thiogalactoside at an OD ₆₀₀ of 0.5-0.6, and measure the fermentation parameters at 12, 24, 30, and 36 hours of culture. The supernatant was collected by centrifugation and the protein expression was observed by protein electrophoresis and Western blotting.

Please refer to Figures 7 to 8. Figure 7 is an analysis of the fermentation process of the transformed strain (pSECS6- lacO _SACBRZ , pBL1). Part (A) is a protein electropherogram, (B) is a Western _blot , and (C) is a growth. parameter. Figure 8 is an analysis of the fermentation process of the transformed strain (pSECS7- lacO _SACBRZ , pBL1). Part (A) is a protein electrophoresis pattern, (B) is a Western blotting method, and (C) is a growth. parameter.

The results showed that with the increase of fermentation time, the pH value decreased slightly and then increased gradually. The cell density and protein yield were almost the same. The transgenic plants (pSECS6- lacO _SACBRZ , pBL1) had the maximum yield of 36 hours after fermentation. μ g / mL; Transformation strain _(pSECS7- lacO SACBRZ, pBL1) 30 hours fermentation at Po maximum yield 28 μ g / mL.

Test Example 3: Purification of soluble recombinant sweet taste protein

The purification method comprises the following steps: cultivating the fermentation for 36 hours, separating the supernatant by high-speed centrifugation, concentrating the solution by 10 times, and heating it at 70 ° C to 90 ° C for 10 minutes to 50 minutes, and then centrifuging the supernatant to remove the heat. Stable protein, at which point the amount of hybrid protein is significantly reduced. The calculated isoelectric point of the soluble recombinant sweet protein was 6.7, and the pH of the supernatant was 8.2. Therefore, the pH of the supernatant was not adjusted, and the membrane was directly passed through a 0.45 μm filter membrane and purified by an anion chromatography column. However, the results showed that the soluble recombinant sweet protein was not bound to the anion chromatography column, but the heteroprotein was bound to it, thereby achieving the purification effect of separating the soluble recombinant sweet protein. The influent flowing through the anion chromatography column was then collected, and the concentrated centrifuge tube of the influent through 30 kDa was filtered through a membrane to remove a small amount of large molecular impurities.

Please refer to Figure 9. Figure 9 is a diagram showing the protein expression analysis of each purification step of the transformed strain (pSECS7- lacO _SACBRZ , pBL1). Part (A) is a protein electrophoresis pattern, and (B) is a Western blot method. Part C) is a final concentrated soluble recombinant sweet protein. "1" is the supernatant of 36 fermentation days, "2" is the supernatant concentrated by 10 times, "3" is the 10 times concentrated supernatant after heat treatment, and "4" is the passage through the anion chromatography tube. The influent of the column, "5" is the influent through the membrane filtration. "P" is a concentrated and purified sweet taste protein after concentration and purification.

Refer to Table 1 below for reference. Table 1 shows the protein content, yield and purity of the soluble recombinant sweet protein after the purification step.

The results show that through the above purification step, the soluble recombinant sweet protein can be successfully purified from two liters of the fermentation broth, the protein content can reach 10 mg, and the purity can reach 85%.

Test Example 4: Characterization of soluble recombinant sweet taste protein (1) Original gel electrophoresis and protein molecular weight identification

The purified soluble recombinant sweet protein was treated with the original state, treated with dithiothreitol (DTT) and treated with sodium dodecyl sulfate (SDS). Please refer to Figure 10. Figure 10 is an electrophoresis analysis of the original recombinant film of soluble recombinant sweet protein, wherein "1" is the original soluble recombinant sweet protein, "2" is the soluble recombinant sweet protein after DTT treatment, and "3" is the Soluble recombinant sweet protein after SDS treatment. The results show that the soluble recombinant sweet protein forms a structure of an oligomer upon addition of DTT, and the addition of SDS will aggregate it into a large group of macromolecules.

Since this is different from the previous literature that the sweet protein has only the structure of the monomer (Ming and Hellekant, 1994), further division is performed. The sub-quantity was identified to determine the actual molecular weight of the soluble recombinant sweet protein. Because SDS interferes with molecular weight identification, samples from the original and DTT treatments are analyzed. Please refer to Figure 11 and Figure 12. Figure 11 is a graph showing the molecular weight identification of soluble recombinant sweet protein. Figure 12 is a graph showing the molecular weight identification of soluble recombinant sweet protein after DTT treatment. It can be seen from the analysis results that the soluble recombinant sweet protein with or without DTT treatment has only a monomer structure and a molecular weight of 6366 Da.

(2) Analysis of secondary structure of soluble recombinant sweet protein

The purified soluble recombinant sweet protein was replaced with deionized water, diluted to an appropriate concentration, and protein secondary structure analysis was performed using circular dichroism spectra. When the polarized light passes through different protein samples, the different secondary structures of the protein will have different absorption degrees for different wavelengths and differently turned circular aurora, and the analyzed data (mdeg(θ)) will be converted into △ε used by the analysis software website. . The conversion formula is Δε=θ×[0.1×MRW/(P×Conc)×3298], wherein MRW (mean residure weight) is the average molecular weight of the protein/the number of amino acid residues; P is the optical path length (cm); Conc is the protein concentration (mg/ml). The Δε obtained at each wavelength was input to the prediction software K2D3 (Louis-Jeune et al., 2012) on the website to analyze the proportion of α-helix and β-strand in the secondary structure.

Please refer to Figure 13 and Table 2 below. Figure 13 is an analysis of the secondary structure of soluble recombinant sweet protein. Table 2 compares the secondary structure of soluble recombinant sweet protein and wild sweet protein.

The results showed that the secondary structural composition ratio of soluble recombinant sweet protein was 1.6% for α-helix and 22% for β-strand. In the previous literature (Caldwell et al ., 1998), the secondary structure of wild sweet protein from P. brazzeana Baillon was analyzed by NMR to be 16.6% for α-helix and 29.9% for β-strand. It can be seen from the analysis results that the secondary structure of soluble recombinant sweet protein and wild sweet protein is significantly different in the α-helix composition ratio, and the soluble recombinant sweet protein has a lower α-helix ratio and a looser structure.

(3) Analysis of pH and thermal stability of soluble recombinant sweet protein

a solution of pH 3 (citric acid), pH 4 (CH ₃ COOH), pH 5 (CH ₃ COOH), pH 6 (CH ₃ COOH), pH 7 (deionized water), pH 8 (NaHCO ₃ ), pH 9 (Na ₂ HPO ₄ ) The soluble recombinant sweet protein samples were diluted and heated at 98-100 ° C for 1 hour and at 80 ° C for 4 hours to observe the pH stability of the soluble recombinant sweet protein in food and the thermal stability after processing.

Please refer to Table 3 below. Table 3 compares the sweetness of soluble sweet protein to different pH values and heat treatment based on sucrose.

The results showed that heating at 98 ° C to 100 ° C for 1 hour, soluble recombinant sweet protein in the environment of pH 5 ~ 9, can taste the sweetness, At pH 3 and pH 4, the soluble recombinant sweet protein has a precipitate, and only tastes sour and has no sweet taste. When heated at 80 ° C for 4 hours, the soluble recombinant sweet protein can taste sweetness in the environment of pH 4-9. This result shows that the soluble recombinant sweet protein produced by Bacillus licheniformis has good thermal stability in a low acid to slightly alkaline environment, and has good thermal stability at a common pH value (pH 5-8) of processed foods.

(4) Sensory evaluation of soluble recombinant sweet protein

10 20- to 30-year-old product reviewers were selected for sensory evaluation of soluble recombinant sweet protein. (A) The product reviewer selects five different concentrations (1%, 5%, 10%, 20%, and 30%) of sucrose aqueous solution, and the evaluators are ranked according to the low to high concentration to observe the taster's sensitivity to sweetness. degree. (B) sweetness threshold determination test, was diluted with deionized water soluble recombinant protein sample sweetness of a range of concentrations (30 μ g / ml ~ 100 μ g / ml), please judge members from low concentration to high concentration sample sequentially tasting When the sweet taste is felt, the evaluation is stopped, and the first sample number that feels the sweetness is recorded. The lowest concentration average of the sweetness perceived by each taster is compared with the sucrose sweetness threshold (2%), which can be converted. The sweetness of soluble recombinant sweet protein relative to sucrose.

Please refer to Table 4 below. Table 4 shows the results of sweetness analysis of soluble recombinant sweet protein and sucrose by sensory evaluation.

The results showed that the soluble recombinant sweet protein was 266 times the sweetness of the same quality sucrose, and the sweetness per molecule was 4957 times that of sucrose.

According to the above test results, the transformed strain of the present invention can successfully produce soluble recombinant sweet protein, and the comparative example of the B. subtilis transformant can only secrete the recombinant sweet protein with the message peptide in the cell, but cannot be secreted. To the extracellular. The soluble recombinant sweet taste protein produced by the method for producing the soluble recombinant sweet protein of the present invention can obtain the high-purity soluble recombinant sweet protein by using the purification method of the present invention due to heat resistance and loose protein structure. The soy licheniformis was purified in 36 hours of fermentation to obtain a soluble recombinant sweet protein having a yield of about 10 mg and a purity of 85%. The purified soluble recombinant sweet protein was identified as brazzein by protein identification, and the disulfide bond was formed correctly in the protein structure. According to the sensory evaluation, the sweetness was 266 times that of the same concentration of sucrose, and the sweetness per molecule was 4957 times that of sucrose. Moreover, it has good thermal stability between pH 5 and 9, and has potential for application in the food industry.

The present invention has been disclosed in the above embodiments, but it is not intended to limit the invention, and the present invention can be modified and modified without departing from the spirit and scope of the invention. The scope is subject to the definition of the scope of the patent application.

<110> National Chung Hsing University

<120> A transformant strain having a recombinant sweet taste protein gene and a method thereof for producing a soluble recombinant sweet taste protein

<160> 5

<210> 1

<211> 116

<212> DNA

<213> Artificial sequence

<220> enhancer, promoter

<223> Inducible expression element SECS6-lacO

<400> 1

<210> 2

<211> 128

<212> DNA

<213> Artificial sequence

<220> enhancer, promoter

<223> Inducible expression element SECS7-lacO

<400> 2

<210> 3

<211> 87

<212> DNA

<213> Artificial sequence

<220> signal peptide

<223> nucleotide sequence of the message peptide

<400> 3

<210> 4

<211> 159

<212> DNA

<213>

<220> CDS

<223> Nucleotide sequence of recombinant sweet protein (brazzein)

<400> 4

<210> 5

<211> 53

<212> PRT

<213>

<220>

<223> Amino acid sequence of recombinant sweet protein (brazzein)

<400> 5

Claims (9)

A transformant strain for producing a soluble recombinant sweet taste protein, comprising: a host cell, wherein the host cell is Bacillus licheniformis ; an induced recombinant plastid, wherein the induced recombinant plastid comprises sequentially arranged An inducible expression element sequence, such as a first nucleic acid fragment represented by SEQ ID NO: 3 and a second nucleic acid fragment as shown in SEQ ID NO: 4, and the inducible expression element sequence is selected from the sequence identification number 1 and a group consisting of the sequences indicated by the sequence identification number 2; and a regulatory property. A transformant strain for producing a soluble recombinant sweet taste protein according to claim 1, wherein the second nucleic acid fragment encodes a recombinant sweet taste protein as shown in SEQ ID NO: 5. A transformant strain for producing a soluble recombinant sweet taste protein according to claim 2, wherein the soluble recombinant sweet taste protein is secreted outside the transformant strain. The transformant strain for producing a soluble recombinant sweet taste protein according to claim 2, wherein the soluble recombinant sweet taste protein has a 1.6% α-helix secondary structure. A method for producing a soluble recombinant sweet protein, comprising: A liquid culture is provided, wherein the liquid culture comprises the transformant according to any one of claims 1 to 4, wherein one of the transformants of the transformant continuously exhibits an inhibitor to inhibit the transformant One of the inducible recombinant plastids expresses the soluble recombinant sweet taste protein; when an optical density value of one of the transformed strains is 0.5 to 0.6, isopropyl-β-D-thiogalactoside is added Inducing the inducible recombinant plastid to express the soluble recombinant sweet taste protein and secreting it outside the transformed strain; removing the transformed strain to obtain a supernatant containing the soluble recombinant sweet taste protein; The soluble recombinant sweet taste protein was isolated from the supernatant. The method for producing a soluble recombinant sweet taste protein according to claim 5, wherein the inhibitory substance exhibits the inhibitor system as Lac I. The method for producing a soluble recombinant sweet taste protein according to claim 5, wherein the step of obtaining the supernatant further comprises: treating the liquid culture at a temperature of 70 ° C to 90 ° C for 10 minutes to 50 minutes; The transformed strain was removed by a centrifugation method to obtain the supernatant containing the soluble recombinant sweet protein. The method for producing a soluble recombinant sweet protein according to claim 7, wherein the step of isolating the soluble recombinant sweet protein further comprises The soluble recombinant sweet protein is isolated from the supernatant using a chromatography column. The method for producing a soluble recombinant sweet protein according to claim 8, wherein the chromatography column comprises an anion chromatography column.