KR101664814B1 - A recombinant vector, a recombinant yeast containing vector, and a producing method of aldehyde dehydrogenase 6 using the yeast - Google Patents
A recombinant vector, a recombinant yeast containing vector, and a producing method of aldehyde dehydrogenase 6 using the yeast Download PDFInfo
- Publication number
- KR101664814B1 KR101664814B1 KR1020150152500A KR20150152500A KR101664814B1 KR 101664814 B1 KR101664814 B1 KR 101664814B1 KR 1020150152500 A KR1020150152500 A KR 1020150152500A KR 20150152500 A KR20150152500 A KR 20150152500A KR 101664814 B1 KR101664814 B1 KR 101664814B1
- Authority
- KR
- South Korea
- Prior art keywords
- recombinant
- aldehyde
- yeast
- aldehyde dehydrogenase
- vector
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/66—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L9/00—Disinfection, sterilisation or deodorisation of air
- A61L9/01—Deodorant compositions
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Birds (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
The present invention relates to a recombinant vector, a recombinant yeast comprising the recombinant vector, and a method for producing aldehyde dihydrogenase 6 using the recombinant yeast. More specifically, the present invention relates to a recombinant vector comprising an aldehyde dehydrogenase 6 gene A vector, a recombinant yeast transformed with the recombinant vector, and a method for producing aldehyde dihydrogenase 6 using the recombinant yeast. The aldehyde dehydrogenase 6 prepared above can be used as an environmentally friendly odor reducing agent having high stability and high efficiency due to its improved function and can be mass-produced because it uses an enzyme in yeast, so that it can be used economically It is expected that the application range of deodorant, medicines, cosmetic additives, etc. will be expanded.
Formaldehyde is a typical cause of aldehyde odor. Formaldehyde has a structure containing an aldehyde group with one carbon and has strong irritating odor at room temperature. Formaldehyde is detected in factory wastes, tobacco smoke, furniture, industrial adhesives, varnishes, etc., and if it leaks for a long time, it causes headaches and vomiting and mutations. It is harmful to the human body due to its high reactivity with proteins, nucleic acids and lipids, and is designated as a first-level carcinogen by the International Agency for Research on Cancer (IARC) and the World Health Organization (WHO).
In addition, with the recent aging of our society, the aging ratio in 2000 was 7.2%, and it is expected to enter the aging society with an aging ratio of over 14% by 2020. It is expected that it will take a shorter time to enter a super-aged society with an aging ratio of 20% or more. As a result, the proportion of elderly people is increasing, and worries and anxieties about the odor of the elderly are increasing.
It is a unique smell that can be called "evidence of aging", which is different from bad odor and sweat odor to all ages. The problem of this smell is becoming a big problem not only at home but also at work. This odor, which is a product and evidence of aging, can lead to the loss of self-confidence and lethargy of the elderly because of the emotional problems such as embarrassment and shame in the interpersonal relationship of the elderly, The main source of this aging odor is 2-nonenal, a type of unsaturated aldehyde. This aldehyde is colorless and is a component of old beer or buckwheat odor and is composed of 9 carbon chains and is insoluble in water. 2-nonenal is rarely produced in childhood or adolescence, but as the aging of the body progresses due to the excess of active oxygen in the body since the age of 40, the metabolism ability decreases and decomposition and release of waste products are not active. .
As the industry develops, the aldehyde odor is regarded as pollution, and it is being actively studied as it is expanded to the object of odor. The smell related to Sick House Syndrome is caused by formaldehyde. In order to remove this odor, there are many companies that utilize materials showing excellent efficacy for eliminating malodorous aldehydes such as phytoncide, which are now used in woodland in forests. In addition, research on the development of deodorants that directly treat odorous areas in the case of old-age odors has been underway, and deodorization filters for deodorizing deodorizing filters that can be installed in places where odorous odors are present are under development.
In addition, deodorized antibacterial products, ie odor absorbing cloths and odor absorbing materials, which absorb and neutralize odor causing substances, have been developed, and products such as clothing and bedding have been marketed. Deodorants and deodorants for various aldehyde odors, such as aerosol type spray products, have also been developed.
However, since these methods are not a method for eliminating the root causes of aldehyde odor, they are only temporary effects that are not persistent, and they are still in the early stage of development, and thus they are expensive.
Accordingly, the present inventors have found that, while studying a substance that can fundamentally remove an aldehyde-based odor, an aldehyde dihydrogenase 6 (ALD6), which is one of enzymes for oxidizing and decomposing aldehyde, And the possibility of its use as an aldehyde-based odor reducing agent was confirmed, and the present invention was completed by providing a method for improving the function and mass production of ALD6.
An object of the present invention is to provide a recombinant vector comprising yeast-derived aldehyde dehydrogenase (ALD 6) gene represented by SEQ ID NO: 1.
Another object of the present invention is to provide a recombinant yeast overexpressing aldehyde dihydrogenase 6 transformed with the above recombinant vector.
It is still another object of the present invention to provide a process for producing an aldehyde dihydrogenase 6 comprising the step of culturing the recombinant yeast.
It is another object of the present invention to provide an aldehyde dihydrogenase 6-produced composition for alleviating odor of an aldehyde-based compound prepared by the above-described method.
In order to solve the above-mentioned problems, the present invention provides a recombinant vector comprising the yeast-derived aldehyde dehydrogenase 6 gene represented by SEQ ID NO: 1.
According to a preferred embodiment of the present invention, the yeast may be Saccharomyces cerevisiae s2805.
According to another preferred embodiment of the present invention, the recombinant vector may further comprise a GFP (green fluorescent protein) gene represented by SEQ ID NO: 2.
According to another preferred embodiment of the present invention, the recombination vector may have a cleavage map of FIG.
The present invention also provides a recombinant yeast overexpressing aldehyde dehydrogenase 6 transformed with the above-mentioned recombinant vector.
According to a preferred embodiment of the present invention, the recombinant yeast MY access to three saccharide Levy jiae (Saccharomyces cerevisiae s2805.
The present invention also provides a method for producing an aldehyde dihydrogenase 6 comprising the step of culturing the above-mentioned recombinant yeast.
The present invention also provides an aldehyde dihydrogenase 6-produced composition for alleviating the odor of the aldehyde-based compound prepared by the above process.
According to a preferred embodiment of the present invention, the aldehyde-based compound may be formaldehyde.
According to another preferred embodiment of the present invention, the aldehyde-based compound may be at least one selected from the group consisting of nonenal, octanal, nonanal, and decanal.
The recombinant yeast overexpressing the aldehyde dihydrogenase 6 transformed with the recombinant vector of the present invention is suitable for mass production of the aldehyde dihydrogenase 6, and the aldehyde dihydrogenase 6, which is overexpressed in the recombinant yeast, Is superior to the chemical synthetic material or the substance using the microorganism itself and is safe and environment friendly and harmless to the human body, it is possible to overcome the side effects of the chemical deodorant and the rejection against the microbial preparation.
Further, since the aldehyde dihydrogenase 6 can be mass-produced by the process for producing aldehyde dihydrogenase 6 of the present invention, the economic burden can be reduced, and the application range of the aldehyde dihydrogenase 6 can be broadened as a deodorant, a medicine, and a cosmetic additive.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a protein expression vector containing an ALD6 gene. FIG.
2 is a diagram showing a process for producing a recombinant vector pYES2 :: ALD6 :: GFP.
3 is a fluorescence microscope photograph showing overexpression of ALD6 protein in MBTL-SYL-1 as a result of fluorescence microscopy analysis.
FIG. 4 is a graph showing the expression levels of aldehyde dehydrogenase 6 by measuring NADH after culturing MBTL-SYL-0 and MBTL-SYL-1 and extracting the enzyme.
FIG. 5 is a graph showing the effect of reducing formaldehyde according to the concentration of enzyme through the emission of Vibrio fischeri by culturing MBTL-SYL-1, reacting with formaldehyde after extracting the enzyme.
FIG. 6 is a graph showing reduction effect of formaldehyde according to enzyme treatment time through emission of Vibrio fischeri by reacting MBTL-SYL-1 with formaldehyde after enzyme is extracted.
As described above, conventional deodorization antibacterial processing products for deodorizing odor removing and various types of deodorizing and deodorizing agents have been developed. However, since they are not a method for eliminating the root cause of aldehyde odor, It is in the early stage of development and has a disadvantage that it is expensive.
Accordingly, the present inventors have proposed an aldehyde-based odor reduction composition comprising aldehyde dihydrodisin 6 (ALD6) which can fundamentally remove aldehyde odor as a material that can be used economically and economically, Respectively.
Further, the present invention provides a production method capable of enhancing the function and mass production of ALD6, and the aldehyde dihydrogenase 6 produced by the method improves the function, is more safe than the substance using the chemical synthetic substance or the microorganism itself, is environmentally friendly, Therefore, it can be applied variously to pharmaceuticals and cosmetics as well as deodorant.
Hereinafter, the present invention will be described in more detail.
A "recombinant vector" as used herein includes any cloning or expression vector containing the desired cloned gene (s).
&Quot; Expression "as used herein refers to the process by which a polypeptide is produced from a structural gene. This process involves transcription of the gene into mRNA and translation of such mRNA into the polypeptide (s).
All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention.
The present invention provides a recombinant vector comprising yeast-derived aldehyde dehydrogenase (ALD6) gene represented by SEQ ID NO: 1.
The aldehyde dihydrogenase 6 is one of the enzymes that oxidize and decompose aldehyde. The aldehyde dihydrogenase 6 is an enzyme that plays a role in resistance to aldehyde stimulation in and out of a cell in an organism.
The yeast MY access to three saccharide Levy jiae (Saccharomyces cerevisiae s2805. Furthermore, the yeast-derived aldehyde dehydrogenase (ALD6) gene represented by SEQ ID NO: 1 can obtain the DNA sequence of the ALD6 gene from the genomic data of S. cerevisiae from the NCBI genbank.
Also, a pair of nucleotide primers capable of specifically amplifying the yeast-derived aldehyde dehydrogenase (ALD6) gene represented by SEQ ID NO: 1 was prepared, and the sequence thereof was as follows:
ALD6-F: 5'-GCC GGA TCC ATG ACT AAG CTA CAC TTT-3 '(SEQ ID NO: 3); And
ALD6-R: 5'-GGT GCG GCC GCC AAC TTA ATT CTG ACA-3 '(SEQ ID NO: 4)
(SEQ ID NO: 1) through PCR (polymerase chain reaction) using an oligonucleotide primer pair having the nucleotide sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4, using the ALD6 gene represented by SEQ ID NO: .
The amplified ALD6 gene (SEQ ID NO: 1) was inserted into pYES2 vector digested with restriction enzymes BamHI and NotI and cut at the same restriction sites to obtain yeast-derived aldehyde dehydrogenase (SEQ ID NO: 1) , ALD6) gene can be produced.
The recombinant vector prepared as described above may further include a GFP (green fluorescent protein) gene represented by SEQ ID NO: 2.
The green fluorescent protein (GFP) is a green fluorescent protein, which is a protein capable of observing how the green light-emitting proteins function in vivo in vivo. Thus, the GFP gene can be inserted into the recombinant vector to confirm the expression of ALD6.
The GFP gene may be obtained, for example, from a vector such as, but not limited to, pEGFP-C1 (BD Bioscience Clontech, USA).
Also, a pair of nucleotide primers capable of specifically amplifying the GFP gene represented by SEQ ID NO: 2 was prepared, and the sequence thereof was as follows:
GFP-F: 5'-ATT GCG GCC GCA GTG AGC AAG GGC GAG-3 '(SEQ ID NO: 5); And
GFP-R: 5'-GGT GCA TGC TCT AGA TCC GGT GGA TCC -3 '(SEQ ID NO: 6).
(GFP gene) (SEQ ID NO: 2) through PCR (polymerase chain reaction) using oligonucleotide primer pairs having the nucleotide sequences shown in SEQ ID NOS: 5 and 6 with the GFP gene shown in SEQ ID NO: 2 as a template, .
The amplified GFP gene (SEQ ID NO: 2) was digested with restriction enzymes NotI and SphI, and the same restriction site of the recombinant vector into which the ALD6 gene represented by SEQ ID NO: 1 was introduced was cut to add the GFP gene (SEQ ID NO: 2) A recombinant vector comprising the ALD6 gene of SEQ ID NO: 1 of the present invention and the GFP gene of SEQ ID NO: 2 can be prepared.
The recombinant vector comprising the ALD6 gene represented by SEQ ID NO: 1 and the GFP gene represented by SEQ ID NO: 2 may have the cleavage map shown in FIG. 1, but the present invention is not limited thereto.
The recombinant vector of the present invention can be used as a basic vector, which is inserted into chromosomal DNA of yeast and stably maintained during transformation. For example, pYES2 can be used, but is not limited thereto.
The present invention also provides a recombinant yeast overexpressing aldehyde dehydrogenase 6 transformed with the above-mentioned recombinant vector.
The recombinant yeast is three Levy jiae My process as Saccharomyces (Saccharomyces cerevisiae s2805.
The recombinant yeast having the recombinant vector may be used in various gene delivery methods, including, but not limited to, transformation, transfection, conjugal mating or electroporation, Lt; / RTI >
The recombinant yeast (MBTL-SYL-1) transformed with the recombinant vector of the present invention containing the ALD6 gene represented by SEQ ID NO: 1 and the GFP gene represented by SEQ ID NO: 2 was cultured to induce protein overexpression, As a result of observing the expression level of ALD6 protein by LysoTracker reacting with LysoTracker by fluorescence microscope, it was confirmed that ALD6 protein was overexpressed in cytoplasm as shown in Fig.
The present invention also provides a method for producing an aldehyde dihydrogenase 6 comprising the step of culturing the above-mentioned recombinant yeast.
The medium and culture conditions can be suitably selected depending on the host cell. The conditions such as the temperature, the pH of the medium and the incubation time can be appropriately adjusted so as to be suitable for cell growth and mass production of the protein at the time of culturing.
FIG. 4 shows that the NADH of the aldehyde-stressed recombinant yeast and the aldehyde-unreacted recombinant yeast were measured using the fact that the enzyme reaction of the aldehyde dehydrogenase 6 was dependent on the coenzyme NAD and produced NADH as a product thereof. Aldehyde dehydrogenase 6 expression level, indicating that the expression level of ALD6 was increased in MBTL-SYL-1.
Thus, it was confirmed that the method for producing ALD6 using the recombinant yeast of the present invention is suitable for mass production of ALD6.
The present invention also provides an aldehyde dihydrogenase 6 produced by the above-described method for reducing odor of an aldehyde compound.
In the composition for reducing odor of the aldehyde-based compound, the aldehyde-based compound may be formaldehyde.
Formaldehyde is an aldehyde-based compound associated with odor associated with sick house syndrome. The composition for reducing odor of formaldehyde is not limited thereto. For example, it may be used in the form of a deodorant or deodorant for removing odor related to sick house syndrome. It can be used in various forms such as liquid, aerosol, gel, etc., and thus can be applied to interior goods, medical equipment, furniture, interior and exterior walls, living room flooring, building materials and walls.
MBTL-SYL-1 was cultured to extract the enzyme, and the extracted enzyme was reacted with formaldehyde to form Vibrio As a result of observing the reduction effect of formaldehyde according to the concentration of the enzyme and the reaction time through the emission of fischeri , as shown in FIGS. 5 and 6, when the amount of the enzyme was 1 to 2 mg and the treatment time was 1 to 24 hours The effect of reducing formaldehyde was good.
Thus, it can be confirmed that the aldehyde dihydrogenase 6 prepared by the production method of the present invention can be used as a composition for reducing odor of formaldehyde associated with odor associated with sick house syndrome.
In addition, in the composition for aldehyde-based compound odor reduction, the aldehyde-based compound may be at least one selected from the group consisting of nonenal, octanal, nonanal, and decanal.
An aldehyde-based compound associated with nonane, odnanal, octanal, noranal, and decanal is an aldehyde-based compound associated with senile odor. The composition for reducing odor of at least one aldehyde compound selected from the group consisting of nonenal, octanal, For example, it may be used in the form of a deodorant or deodorant for removing old odors, and further may be used as a solution, suspension, emulsion, gel, cream, lotion, powder, soap, body shade, ≪ / RTI > or other commonly available formulations.
Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
To yeast ALD6 Production of a recombinant vector containing the gene
In this embodiment, we used the three Levy jiae my process in the eukaryotic microorganism Saccharomyces (Saccharomyces cerevisiae s2805, American Type Culture Collection, ATCC ® 4002805 TM), gene using pYES2 (Invitogen, Carlsbad, CA, USA) as a carrier vector Recombination was carried out. S. cerevisiae From the genomic data, the DNA sequence of the ALD6 gene was obtained from NCBI's genbank (www.ncbi.nlm.nih.gov) and the GFP gene was obtained from the vector pEGFP-C1 (BD Bioscience Clontech, USA) A nucleotide primer having SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 capable of specifically amplifying the corresponding
Next, genomic DNA was extracted from S. cerevisiae , and PCR was carried out using a pair of oligonucleotide primers having a nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4 as a template, The nucleotide sequence of
All of these genes were used after purification with GeneAll PCR DNA purification kit.
Preparation of recombinant vector (pYES2 :: ALD6 :: GFP)
In this Example, pYES2 :: ALD6 :: GFP, which is a vector for expression of ALD6, was prepared using yeast-protein expression vector pYES2 (Invitogen, Carlsbad, CA, USA) as a basic vector.
The DNA fragment of the ALD6 gene amplified in Example 1 was digested with BamHI and NotI, and inserted into the pYES2 vector in which the same restriction sites were cleaved, which was named pYES2 :: ALD6 (see FIG. 2). Here, the GFP gene gene was digested with NotI and SphI and then introduced into a truncated vector of the same restriction site, which was named pYES2 :: ALD6 :: GFP (see FIG. 1).
The prepared vector pYES2 :: ALD6 :: GFP was transformed into S. cereviae s2805, a yeast strain, by electroporation, and the colonies obtained from the SD solid plate culture medium were transferred to a fresh medium to obtain a single colony. The DNA sequence of the ALD6 gene and the GFP gene inserted into pYES2 :: ALD6 :: GFP was confirmed by DNA sequencing method. The recombinant microorganism containing only the pYES2 vector was named MBTL-SYL-0, and the pYES2: : Recombinant microorganism containing ALD6 :: GFP was named MBTL-SYL-1.
Overexpression of ALD6 protein in recombinant yeast MBTL-SYL-1
In this Example, the recombinant microorganism MBTL-SYL-1 prepared in Example 2 was inoculated in 15 ml of SD liquid medium (6.7 g / L yeast nitrogen base, 5 g / L amino acid and 20 g / L glucose) , And cultured with shaking at 160 rpm until the OD value became 3.0.
After removing the microorganism from the obtained culture solution, add 15 ml of SG liquid medium (6.7 g / L yeast nitrogen base, 5 g / L amino acid and 20 g / L galactose). Protein overexpression was induced by culturing the resulting culture solution (6.67 mL, OD value = 0.4) in 50 mL of SG liquid medium at 30 DEG C and 160 rpm for 10 hours.
To confirm the expression site of ALD6, LysoTracker, which specifically reacts only with lysosomes, was treated with inducible cells and the degree of ALD6 expression was observed by fluorescence microscopy.
Specifically, the first fluorescence microscopic photograph of MBTL-SYL-0 (control group) and MBTL-SYL-1 of FIG. 3 showed that the GFP gene was expressed, indicating that the MBTL-SYL- , And the second photograph is the observation of the lysosomes in the yeast through the dyed portion (red) in MBTL-SYL-0 and MBTL-SYL-1. The third picture shows the combination of the green fluorescence moiety and the red moiety stained with a lycoperside. When ALD6 is expressed in the lysosomes, it shows yellow fluorescence.
Therefore, as shown in FIG. 3, the green fluorescence and the red fluorescence partially overlap but are not perfectly combined, so that the protein is overexpressed in the cytoplasm.
Comparison of activity of aldehyde dihydrogenase 6 extracted from recombinant yeast
In this Example, the NADH of the recombinant yeast that gave aldehyde stress and the recombinant yeast that did not induce aldehyde stress were measured using the fact that the enzyme reaction of aldehyde dehydrogenase 6 was dependent on coenzyme NAD and produced NADH as a product thereof. The level of expression of aldehyde dehydrogenase 6 was determined.
The recombinant yeasts MBTL-SYL-0 and MBTL-SYL-1 prepared in Example 2 were treated with 0.2 mM formaldehyde under the same culture conditions as in Example 3, and cultured with shaking until the OD value reached 0.8 . 100 [mu] l of 0.4 N HCl / 0.5 M trizma base was treated with galactose induction to break the cell wall. NADH of the extract was measured for fluorescence using NADH assay kit (Promega), and the expression level thereof was compared. As a result, a higher amount of ALD6 was detected in the MBTL-SYL-1 recombinant yeast as shown in FIG.
Extracted from recombinant yeast Aldehyde Dihydrogenase Check the effect of 6
In this Example, the enzyme was extracted from the recombinant yeast prepared in Example 2, and the extracted enzyme was treated with formaldehyde to confirm the reduction. The procedure is as follows.
The recombinant yeast MBTL-SYL-1 prepared in Example 2 was shake-cultured under the same culture conditions as in Example 3. Subsequently, the cells were washed with 1 ml of distilled water and then centrifuged at 13000 rpm for 1 minute to remove the supernatant. After adding 10 mM of PIC (Protein inhibitor cocktail) and PMSF (lysis buffer) to make the cells and the volume 1: 1, add the glass beads to the same volume as the cells After 30 seconds of vortexing for 30 seconds, the reaction was repeated 5 times at 4 ° C and centrifuged at 13000 rpm at 4 ° C for 10 minutes to collect the supernatant (enzyme).
The bioluminescence, Vibrio fischeri were cultured in LB medium at 30 ° C. for 12 hours and main cultured for 3 hours. Then, 200 μl of the fischeri was treated with only the enzyme at OD 0.4, and the enzyme was reacted with formaldehyde (0, 10 min, 30 min, 1 hr, 3 hr, 12 hr, 24 hr) and the amount of enzyme (0 mg, 0.1 mg, 0.5 mg, 1 mg 1.5 mg, The results are shown in FIGS. 5 and 6, respectively, and the decrease in formaldehyde was confirmed.
As described above, since ALD6 produced using the recombinant yeast (MBTL-SYL-1) of the present invention effectively oxidizes and decomposes formaldehyde, it can be applied to a composition for reducing odor of formaldehyde which is a cause of odor related to sick house syndrome there was.
<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> A RECOMBINANT VECTOR, A RECOMBINANT YEAST CONTAINING VECTOR, AND A PRODUCING METHOD OF ALDEHYDE DEHYDROGENASE 6 USING THE YEAST <130> 1042263 <160> 7 <170> KoPatentin 3.0 <210> 1 <211> 1503 <212> DNA <213> Artificial Sequence <220> <223> ALD 6 gene <400> 1 atgactaagc tacactttga cactgctgaa ccagtcaaga tcacacttcc aaatggtttg 60 acatacgagc aaccaaccgg tctattcatt aacaacaagt ttatgaaagc tcaagacggt 120 aagacctatc ccgtcgaaga tccttccact gaaaacaccg tttgtgaggt ctcttctgcc 180 accactgaag atgttgaata tgctatcgaa tgtgccgacc gtgctttcca cgacactgaa 240 tgggctaccc aagacccaag agaaagaggc cgtctactaa gtaagttggc tgacgaattg 300 gaaagccaaa ttgacttggt ttcttccatt gaagctttgg acaatggtaa aactttggcc 360 ttagcccgtg gggatgttac cattgcaatc aactgtctaa gagatgctgc tgcctatgcc 420 gacaaagtca acggtagaac aatcaacacc ggtgacggct acatgaactt caccacctta 480 gagccaatcg gtgtctgtgg tcaaattatt ccatggaact ttccaataat gatgttggct 540 tggaagatcg ccccagcatt ggccatgggt aacgtctgta tcttgaaacc cgctgctgtc 600 acacctttaa atgccctata ctttgcttct ttatgtaaga aggttggtat tccagctggt 660 gtcgtcaaca tcgttccagg tcctggtaga actgttggtg ctgctttgac caacgaccca 720 agaatcagaa agctggcttt taccggttct acagaagtcg gtaagagtgt tgctgtcgac 780 tcttctgaat ctaacttgaa gaaaatcact ttggaactag gtggtaagtc cgcccatttg 840 gtctttgacg atgctaacat taagaagact ttaccaaatc tagtaaacgg tattttcaag 900 aacgctggtc aaatttgttc ctctggttct agaatttacg ttcaagaagg tatttacgac 960 gaactattgg ctgctttcaa ggcttacttg gaaaccgaaa tcaaagttgg taatccattt 1020 gacaaggcta acttccaagg tgctatcact aaccgtcaac aattcgacac aattatgaac 1080 tacatcgata tcggtaagaa agaaggcgcc aagatcttaa ctggtggcga aaaagttggt 1140 gacaagggtt acttcatcag accaaccgtt ttctacgatg ttaatgaaga catgagaatt 1200 gttaaggaag aaatttttgg accagttgtc actgtcgcaa agttcaagac tttagaagaa 1260 ggtgtcgaaa tggctaacag ctctgaattc ggtctaggtt ctggtatcga aacagaatct 1320 ttgagcacag gtttgaaggt ggccaagatg ttgaaggccg gtaccgtctg gatcaacaca 1380 tacaacgatt ttgactccag agttccattc ggtggtgtta agcaatctgg ttacggtaga 1440 gaaatgggtg aagaagtcta ccatgcatac actgaagtaa aagctgtcag aattaagttg 1500 taa 1503 <210> 2 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> gfp gene <400> 2 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga 720 ctcagatctc gagctcaagc ttcgaattct gcagtcgacg gtaccgcggg cccgggatcc 780 accggatcta gataa 795 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> ALD6 forward primer <400> 3 gccggatcca tgactaagct acacttt 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> ALD6 reverse primer <400> 4 ggtgcggccg ccaacttaat tctgaca 27 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GFP forward primer <400> 5 attgcggccg cagtgagcaa gggcgag 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GFP reverse primer <400> 6 ggtgcatgct ctagatccgg tggatcc 27 <210> 7 <211> 2298 <212> DNA <213> Artificial Sequence <220> <223> sequence for ALD6 :: GFP <400> 7 atgactaagc tacactttga cactgctgaa ccagtcaaga tcacacttcc aaatggtttg 60 acatacgagc aaccaaccgg tctattcatt aacaacaagt ttatgaaagc tcaagacggt 120 aagacctatc ccgtcgaaga tccttccact gaaaacaccg tttgtgaggt ctcttctgcc 180 accactgaag atgttgaata tgctatcgaa tgtgccgacc gtgctttcca cgacactgaa 240 tgggctaccc aagacccaag agaaagaggc cgtctactaa gtaagttggc tgacgaattg 300 gaaagccaaa ttgacttggt ttcttccatt gaagctttgg acaatggtaa aactttggcc 360 ttagcccgtg gggatgttac cattgcaatc aactgtctaa gagatgctgc tgcctatgcc 420 gacaaagtca acggtagaac aatcaacacc ggtgacggct acatgaactt caccacctta 480 gagccaatcg gtgtctgtgg tcaaattatt ccatggaact ttccaataat gatgttggct 540 tggaagatcg ccccagcatt ggccatgggt aacgtctgta tcttgaaacc cgctgctgtc 600 acacctttaa atgccctata ctttgcttct ttatgtaaga aggttggtat tccagctggt 660 gtcgtcaaca tcgttccagg tcctggtaga actgttggtg ctgctttgac caacgaccca 720 agaatcagaa agctggcttt taccggttct acagaagtcg gtaagagtgt tgctgtcgac 780 tcttctgaat ctaacttgaa gaaaatcact ttggaactag gtggtaagtc cgcccatttg 840 gtctttgacg atgctaacat taagaagact ttaccaaatc tagtaaacgg tattttcaag 900 aacgctggtc aaatttgttc ctctggttct agaatttacg ttcaagaagg tatttacgac 960 gaactattgg ctgctttcaa ggcttacttg gaaaccgaaa tcaaagttgg taatccattt 1020 gacaaggcta acttccaagg tgctatcact aaccgtcaac aattcgacac aattatgaac 1080 tacatcgata tcggtaagaa agaaggcgcc aagatcttaa ctggtggcga aaaagttggt 1140 gacaagggtt acttcatcag accaaccgtt ttctacgatg ttaatgaaga catgagaatt 1200 gttaaggaag aaatttttgg accagttgtc actgtcgcaa agttcaagac tttagaagaa 1260 ggtgtcgaaa tggctaacag ctctgaattc ggtctaggtt ctggtatcga aacagaatct 1320 ttgagcacag gtttgaaggt ggccaagatg ttgaaggccg gtaccgtctg gatcaacaca 1380 tacaacgatt ttgactccag agttccattc ggtggtgtta agcaatctgg ttacggtaga 1440 gaaatgggtg aagaagtcta ccatgcatac actgaagtaa aagctgtcag aattaagttg 1500 taagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 1560 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 1620 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 1680 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 1740 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 1800 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 1860 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 1920 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 1980 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 2040 gccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 2100 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 2160 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 2220 ggactcagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga 2280 tccaccggat ctagataa 2298
Claims (9)
(b) extracting aldehyde dihydrogenase (6) from the recombinant yeast. The composition for alleviation of malodor of formaldehyde according to any one of
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150152500A KR101664814B1 (en) | 2015-10-30 | 2015-10-30 | A recombinant vector, a recombinant yeast containing vector, and a producing method of aldehyde dehydrogenase 6 using the yeast |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150152500A KR101664814B1 (en) | 2015-10-30 | 2015-10-30 | A recombinant vector, a recombinant yeast containing vector, and a producing method of aldehyde dehydrogenase 6 using the yeast |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR101664814B1 true KR101664814B1 (en) | 2016-10-11 |
Family
ID=57161705
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020150152500A KR101664814B1 (en) | 2015-10-30 | 2015-10-30 | A recombinant vector, a recombinant yeast containing vector, and a producing method of aldehyde dehydrogenase 6 using the yeast |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101664814B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220117742A (en) | 2021-02-17 | 2022-08-24 | 주식회사 피코엔텍 | Hangover Treatment Composition Which Comprises Aldehyde Dehydrogenase and Glutathione |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015506681A (en) * | 2012-01-06 | 2015-03-05 | フイルメニツヒ ソシエテ アノニムＦｉｒｍｅｎｉｃｈ Ｓａ | Genetically modified yeast cells |
-
2015
- 2015-10-30 KR KR1020150152500A patent/KR101664814B1/en active IP Right Grant
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015506681A (en) * | 2012-01-06 | 2015-03-05 | フイルメニツヒ ソシエテ アノニムＦｉｒｍｅｎｉｃｈ Ｓａ | Genetically modified yeast cells |
Non-Patent Citations (4)
| Title |
|---|
| 1. Haze S, Nakashiyuki Y, Nakashiyuki S, Kohno Y, Sawano K, Ohta H, Yamazaki K, 2-Nonenal Newly Found in Human Body Odor Tends to Increase with Aging, Journal of Investigative Dermatology(2001), 116, 520-524; |
| 2. William B.Rizzo, Fatty Aldehyde and Fatty Alcohol Metabolism: Review and Importance for Epidermal Structure and Function, Biochimica et Biophysica Acta 1841(2014), 377-389 |
| 3. Markus B, Jan G, Fenja S, Melanie P, Jens S, Oxidation of Fatty Aldehydes to Fatty Acids by Escherichia coli Cells Expressing The Vibrio harveyi Fatty Aldehyde Dehydrogenase(FALDH), World J Microbiol Biotechnol(2013), 29:569-575. |
| GenBank Accession Number NM_001183875 (2014.08.22.)* * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220117742A (en) | 2021-02-17 | 2022-08-24 | 주식회사 피코엔텍 | Hangover Treatment Composition Which Comprises Aldehyde Dehydrogenase and Glutathione |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10870841B1 (en) | Nucleic acid-guided nucleases | |
| EP1572993B1 (en) | Choline oxidases | |
| ES2320531T3 (en) | PROCESS TO PRODUCE BIOPTERINS. | |
| WO2003008602A3 (en) | Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an attenuated ugpb gene | |
| JP2002507111A (en) | Haloperoxidase from Curbularia verruclosa and nucleic acids encoding the same | |
| KR20080028902A (en) | Genetically modified yeast of the species issatchenkia orientalis and closely related species and fermentation processes using same | |
| CN108138121A (en) | Long chain dicarboxylic acid is produced with microorganism high level | |
| RU2642290C2 (en) | Application of endogenic dnaase activity to reduce dna content | |
| KR101664814B1 (en) | A recombinant vector, a recombinant yeast containing vector, and a producing method of aldehyde dehydrogenase 6 using the yeast | |
| KR101876172B1 (en) | Designer cells for enantioselective reduction of ketones and use thereof in efficient production of enantioenriched alcohols | |
| CA3172787A1 (en) | Methods for cell free protein expression of mature polypeptides derived from zymogens and proproteins | |
| WO2006085535A1 (en) | Method for producing biopterin using tetrahydrobiopterin biosynthesis enzyme | |
| US20110262412A1 (en) | Bacteriocin based methods to control lactic acid bacterial growth | |
| Young et al. | Bacteriophage T4 gene 55.9 encodes an activity required for anaerobic ribonucleotide reduction. | |
| JP2021514679A (en) | Recombinant oxalate decarboxylase expressed by filamentous fungal host cells | |
| EP1437401B1 (en) | Oxidoreductase | |
| JP2002506638A (en) | Haloperoxidase with modified pH characteristics | |
| JP3330941B2 (en) | Method for producing heme protein | |
| JPH0353879A (en) | Improved enzyme oxidation of cephalosporin to glutaryl-7- amino-cephalosporanic acid | |
| KR101646007B1 (en) | Composition for removing aldehyde-based odor with lysosome or lysosomal enzyme and method using the same | |
| US20210261986A1 (en) | Biological devices for producing oxidized zinc and applications thereof | |
| CN110358753B (en) | Fusion protein based on CjCas9 and VPR core structural domain, corresponding DNA targeting activation system and application thereof | |
| Vigetti et al. | Human allantoicase gene: cDNA cloning, genomic organization and chromosome localization | |
| WO2021046364A1 (en) | Genelight cultures and extracts and applications thereof | |
| EP1847599A1 (en) | Polypeptides with perhydrolase activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AMND | Amendment | ||
| AMND | Amendment | ||
| AMND | Amendment | ||
| X701 | Decision to grant (after re-examination) | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20191001 Year of fee payment: 4 |