KR20240083164A - Lipase having thermal stability and method for producing thereof - Google Patents

Abstract

The present invention relates to a new lipase isolated from Lactobacillus rhamnosus IDCC 3201 and a method for producing the same, which has excellent thermal and pH stability, maintains activity even when metal ions are added, and maintains organic solvent stability, making it a biocatalyst. It is highly utilized in various industrial fields such as:

Description

Lipase with heat stability and method for producing the same {LIPASE HAVING THERMAL STABILITY AND METHOD FOR PRODUCING THEREOF}

The present invention relates to a novel lipase with high heat stability and pH stability and a method for producing the same.

Lipase is a lipid hydrolyzing enzyme that decomposes lipids to produce fatty acids and glycerol. Depending on the reaction conditions, lipase can catalyze various chemical reactions such as hydrolysis of ester compounds as well as their synthesis reactions and transesterification reactions. Lipase has useful reaction characteristics such as high positional specificity, substrate specificity, and stereospecificity. It produces little industrial wastewater and by-products from the chemical catalyst process, and because the reaction occurs at a relatively low temperature, it has many advantages in terms of energy, making it an industrial It is being widely used.

In addition, lipase produced by organic solvent-resistant microorganisms is stable over a wide range of temperature and pH conditions, has excellent enzymatic activity, and is capable of performing aminolysis, esterification, and thiotransesterification (aminolysis), which are impossible in aqueous conditions. It can be used in the fine chemical industry because it has excellent reactivity in aqueous and hydrophobic systems, such as catalyzing reactions such as thiotransesterification and transesterification.

Republic of Korea Patent No. 10-1596435

Accordingly, the inventors of the present invention completed the present invention by discovering that a new lipase isolated from Lactobacillus rhamnosus IDCC 3201 has excellent heat stability and pH stability and maintains activity even when metal ions are added. .

Accordingly, the purpose of the present invention is to provide a novel lipase derived from Lactobacillus rhamnosus IDCC 3201, a method for producing the same, and a use thereof.

The present invention provides lipase represented by the amino acid sequence of SEQ ID NO: 1.

Additionally, the present invention provides a recombinant vector containing a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1.

Additionally, the present invention provides a transformant transformed with the recombinant vector according to the present invention.

Furthermore, the present invention includes the steps of transforming a host cell with a recombinant vector containing a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1; Culturing the transformed host cells; and isolating lipase from the culture medium.

Finally, the present invention provides a laundry additive or detergent composition containing a lipase containing the amino acid sequence represented by SEQ ID NO: 1.

The new lipase isolated from Lactobacillus rhamnosus IDCC 3201 of the present invention has excellent thermal and pH stability, maintains activity even when metal ions are added, and maintains stability even in organic solvents, so it can be used in various industrial fields such as biocatalysis. It is possible to utilize it.

Figure 1 is a schematic diagram showing the manufacturing process of lipase according to an embodiment of the present invention.
Figure 2 shows the results of SDS-PAGE analysis confirming the purification of lipase according to various concentrations of imidazole in an example of the present invention. Lane M: Protein marker, lane 1: pellet, lane 2: crude, lane 3: flow through, lane 4: 20mM imidazole, lane 5: 50mM imidazole, lane 6: 300mM imidazole, lane 7: 1M imidazole.
Figure 3 shows the results of confirming the lipid decomposition ability of lipase in an olive oil-containing plate in an example of the present invention (D: Dilution)
Figure 4 is a graph showing the optimal activity and stability of lipase according to temperature in an example of the present invention: (a) Optimal temperature of purified lipase in the temperature range of 10°C to 80°C. Activity assays were performed in 50 mM potassium phosphate buffer at pH 8.0. (b) Results of confirming the thermal stability of purified lipase at 40, 60, and 70 °C for up to 240 minutes.
Figure 5 is a graph showing the optimal activity and stability of lipase according to pH in an example of the present invention: (a) Optimal pH activity of lipase at 60 °C. (b) pH stability of purified lipase incubated in various buffers (pH 5 to 11) for 1 h at 60 °C.
Figure 6 shows the results of confirming whether purified lipase is affected by organic solvents of various concentrations (10% and 30%) in an example of the present invention. The activity of the purified lipase was confirmed through a lipase assay after preliminary incubation with an organic solvent for 1 hour at room temperature.
Figure 7 is a graph showing the results of measuring the enzyme activity of the lipase of the present invention added to a commercial laundry detergent in an example of the present invention.
Figure 8 is a photograph showing the results of evaluating the oil stain removal ability of the lipase of the present invention added to a commercial laundry detergent in an example of the present invention.

Hereinafter, the present invention will be described in more detail. However, the present invention may be implemented in various different forms, and the present invention is not limited to the embodiments described herein, and the present invention is only defined by the claims to be described later.

In addition, the terms used in the present invention are only used to describe specific embodiments and are not intended to limit the present invention. In the entire specification of the present invention, 'including' a certain element means that other elements may be further included rather than excluding other elements, unless specifically stated to the contrary.

The present invention relates to lipase represented by the amino acid sequence of SEQ ID NO: 1.

The lipase may be encoded by the nucleic acid sequence represented by SEQ ID NO: 2, but is not limited thereto.

The lipase may be derived from a Lactobacillus rhamnosus strain, for example, Lactobacillus rhamnosus IDCC 3201 ( Lactobacillus rhamnosus IDCC 3201), but is not limited thereto.

The lipase shows an activity of 60% or more at 50 to 90°C, and may, for example, show high activity at 60 to 70°C, but is not limited thereto. The lipase can maintain its activity for up to about 250 minutes even at a temperature ranging from 60 to 70° C., but is not limited thereto.

The lipase shows an activity of more than 60% at pH 5 to 9, and may show optimal activity at pH 8, but is not limited thereto. The lipase can maintain its activity for more than 1 hour even at pH 5 to 9, but is not limited thereto.

The lipase may have specificity for a substrate having 10 to 18 carbon atoms, for example, 12 to 16 carbon atoms, but is not limited thereto.

The lipase may maintain activity in the presence of metal ions, but is not limited thereto. The metal ion may be selected from the group consisting of Mg, Mn, Zn, Ca, Cu, Fe, and combinations thereof, for example, Mg or Cu, but is not limited thereto.

The lipase can maintain its activity in an organic solvent, for example, its activity can be better maintained in an organic solvent at a concentration of about 10%, but is not limited thereto. The organic solvent may be selected from the group consisting of acetone, glycerol, dimethyl sulfoxide, methanol, ethanol, acetonitrile, isopropyl alcohol, ethyl acetate, hexane, n-butyl acetate, and combinations thereof, for example, glycerol. , dimethyl sulfoxide, or acetone, but is not limited thereto.

Additionally, the present invention relates to a recombinant vector containing a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1.

As used herein, the term "vector" refers to a DNA preparation containing a polynucleotide of a gene operably linked to a suitable regulatory sequence to enable expression of a gene of interest in a suitable host, wherein the control sequence can initiate transcription. a promoter, optional operator sequences for controlling such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling termination of transcription and translation.

The vector of the present invention may include expression control elements such as a promoter, start codon, stop codon, polyadenylation signal and enhancer, secretion signal, etc., and may be manufactured in various ways depending on the purpose. The initiation codon and stop codon must be functional in the subject when the genetic construct is administered and must be in frame with the coding sequence.

The vector used in the present invention is not particularly limited as long as it can replicate in a host, and any vector known in the art can be used. For example, non-viral vectors or viral vectors can be used.

A representative example of the non-viral vector is a plasmid. Plasmid expression vectors are an FDA-approved gene delivery method that can be used in humans and are a method of delivering plasmid DNA directly to human cells. Unlike viral vectors, plasmid DNA has the advantage of being able to be purified to homogeneity. As a plasmid expression vector that can be used in the present invention, a mammalian expression plasmid known in the art can be used. For example, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91/08291), and pVL1392 (PharMingen) are representative examples, but are not limited thereto.

Additionally, a viral vector can be used as the expression vector of the present invention. Viral vectors include, for example, retrovirus, adenovirus, adenovirus-related virus, herpes virus, and avipoxvirus. The viral vector must meet the following criteria: (1) it must be able to infect the target cells and a viral vector with an appropriate host range must be selected accordingly, and (2) the delivered gene is preserved in the cell for an appropriate period of time. and can be expressed, and (3) the vector must be safe for the host.

The retroviral vector is designed so that all viral genes are removed or altered so that non-viral proteins are produced in cells infected by the viral vector. The main advantages of retroviral vectors for gene therapy are that they deliver large amounts of genes into replicating cells, accurately integrate the transferred genes into cellular DNA, and do not cause continuous infection after gene transfection. The retroviral vector certified by the FDA was manufactured using PA317 amphotropic retrovirus packaging cells (Miller, A.D. and Buttimore, C., Molec.Cell Biol., 6:2895-2902, 1986).

Non-retroviral vectors include adenovirus as mentioned above. The main advantage of adenovirus is that it carries large amounts of DNA fragments (36 kb genome) and has the ability to infect non-replicating cells at very high titers. Additionally, the herpes virus can also be usefully used for human gene therapy (Wolfe, J.H., et al., Nature Genetics, 1:379-384, 1992). In addition, known appropriate viral vectors can be used.

Other viral vectors that can be used for gene transfer into cells include murine leukemia virus (MLV), JC, SV40, polyoma, Epstein-Barr virus, papilloma virus, vaccinia, poliovirus, herpes virus, Sindbis virus, and lentivirus. May include viruses and other human and animal viruses.

The expression vector according to the present invention can be introduced into cells using methods known in the art. Examples include, but are not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun and other known methods for introducing nucleic acids into cells. It can be introduced into cells by the method (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988) .

Additionally, the vector of the present invention may additionally include a selection marker (selection market). A selection marker is used to select cells transformed with a vector, that is, to confirm whether a target gene has been inserted, and to impart a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. Markers that do so may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so transformed cells can be selected.

Additionally, the present invention relates to a transformant obtained by introducing the recombinant vector according to the present invention into a host cell.

The transformant of the present invention can be constructed by introducing a vector into a host cell in a manner in which a promoter can function.

In the present invention, “transformation” means introducing DNA into a host so that the DNA can be replicated as an extrachromosomal factor or through completion of chromosomal integration. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and can be performed by selecting an appropriate standard technique depending on the host cell, as known in the art. These methods include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and acetic acid. Lithium-DMSO method may be included, but is not limited thereto.

Since the expression level and modification of the protein varies depending on the host cell transformed with the expression vector, the host cell most suitable for the purpose can be selected and used. Host cells that can be used in the present invention include insect cell lines, yeast, fungi, bacteria, or algae.

The present invention includes the steps of transforming a host cell with a recombinant vector containing a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1; Culturing the transformed host cells; and a method for producing lipase comprising the step of isolating lipase from the culture medium.

The present invention relates to a laundry additive containing lipase represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the lipase represented by the amino acid sequence of SEQ ID NO: 1 may be added in an amount of 0.001 to 1 g, preferably 0.005 to 0.1 g, and more preferably 0.008 to 0.08 g per 100 ml of laundry detergent, but is limited thereto. That is not the case.

The present invention relates to a laundry detergent composition containing lipase represented by the amino acid sequence of SEQ ID NO: 1.

Additionally, the laundry detergent composition may include at least one type of surfactant selected from the group consisting of amphoteric surfactants, nonionic surfactants, and natural surfactants.

For example, anionic surfactants include sulfate surfactants, sulfonate surfactants, sulfoacetate surfactants, sulfosuccinate surfactants, phosphate surfactants, carboxylate surfactants, etc., and amphoteric surfactants can be used as anionic surfactants. Betaine surfactant can be used. Additionally, nonionic surfactants include ester surfactants, ether surfactants, acidic amide surfactants, and ester/ether surfactants.

Additionally, the laundry detergent composition may include carbonate. The carbonate can replenish alkali and prevent recontamination of the cleaning object, and is used to improve the cleaning effect by assisting a surfactant, and may include at least one of sodium sesquicarbonate and sodium bicarbonate.

In addition, the laundry detergent composition may include a pH adjuster, and the pH adjuster is at least one of citric acid, sodium citrate, malic acid, sodium malate, phmalic acid, sodium phmalate, succinic acid, sodium succinate, sodium hydroxide, and sodium monohydrogen phosphate. may contain one

In addition, the laundry detergent composition may include an anti-freeze agent, and the anti-freeze agent includes glycerin, polyethylene glycol, butylene glycol, propylene glycol, sorbitol, trehalose, collagen, salicylic acid, hyaluronic acid, chondroitin sulfate, sodium nitrate, It may be one or more selected from sodium PCA, amino acid, urea, electrolyte, pyrrolidone carboxylate, and collagen, and preferably may be propylene glycol. Additionally, the anti-freeze agent can also function as a moisturizer.

In addition, in the laundry detergent composition, the enzyme represented by the amino acid sequence of SEQ ID NO: 1 is used in addition to the lipase of the present invention to further increase the cleaning effect of the laundry detergent composition, and alkaline proteolytic enzymes can be mainly used. For example, the enzyme may be selected from the group consisting of protease, lipase, amylase, or mixtures thereof.

In addition, the laundry detergent composition includes, in addition to the above-mentioned components, additives commonly mixed in laundry detergents, such as fat ingredients, moisturizers, emollients, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, disinfectants, antioxidants, It may contain at least one of alcohol, coloring, fragrance, blood circulation stimulant, cooling agent, and antiperspirant. Since materials used as mixing additives can be known, detailed description thereof will be omitted.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example

Example 1. Isolation of lipase gene

Lactobacillus rhamnosus IDCC 3201 (Lactobacillus rhamnosus IDCC 3201) was obtained from Ildong Pharmaceutical and used. To extract the whole gene containing the target lipase gene , Lactobacillus rhamnosus IDCC 3201 was cultured overnight at 37°C in MRS broth, and the whole gene was extracted from 1 ml of the suspension using the protocol of Promega's Wizard Genomic DNA purification kit.

First, 1 ml of pre-culture fluid was centrifuged and the supernatant was removed. Then, 50mM EDTA and lysozyme were added, incubated at 37°C for 60 minutes, and the supernatant was removed through centrifugation. After adding the Nuclei lysis solution, incubating at 80°C for 5 minutes and cooling at room temperature, 3 μl of RNAse solution was added, and the culture was incubated at 37°C for 60 minutes and then cooled at room temperature. Afterwards, protein precipitation solution was added, left on ice, and the supernatant was obtained through centrifugation. After transferring the supernatant to isopropanol, remove the supernatant through centrifugation, add 70% ethanol, wash, remove ethanol through centrifugation, completely remove ethanol through evaporation, add rehydrate solution, and cool for 1 hour at 65°C. gDNA was extracted through rehydration for a period of time.

Example 2. Cloning of lipase gene

The lipase gene isolated in Example 1 was cloned using the primers shown in Table 1 below. Using the primers in Table 1, it was confirmed that the optimal annealing temperature was 54.7°C. After performing PCR at 54.7°C, only the target lipase gene was secured using the EZ-pure ^TM PCR purification kit Ver2. To secure a plasmid for cloning, plasmid was extracted from E. coli containing the pET-21a(+) plasmid using the Gene All Hybrid-Q ^TM plasmid rapidprep kit. After performing restriction enzyme cut on the target lipase gene and pET-21a(+) plasmid at 37°C for 2-4 hours using restriction enzymes ( The lipase gene was obtained using the EZ-pure ^TM PCR purification kit Ver2. A ligation reaction was performed on the sticky end plasmid and the lipase gene using T4 DNA ligase at 16°C overnight and at room temperature for 30 minutes. After the ligation reaction, transformation was performed and the sequence was confirmed through sequence alignment. Through this process, recombinant E. coli BL21 (DE3) containing the lipase gene (SEQ ID NO: 2) of Lactobacillus rhamnosus IDCC 3201 was obtained.

Example 3. Expression and purification of lipase

For lipase expression, recombinant E. coli BL21(DE3) was cultured in LA broth, and when the OD value at a wavelength of 600 nm reached 0.4 to 0.6, IPTG was added and cultured at 16°C for 16 hours. Afterwards, cells were collected through centrifugation and released into cell lysis buffer. Afterwards, the cell suspension was sonicated while remaining cold to break up the cells, and the supernatant was obtained through centrifugation. Then, the supernatant was flowed onto Ni-NTA agarose, and protein purification was performed by gradually flowing buffers with higher concentrations of imidazole in the following order: 20mM, 50mM, 100mM, 200mM, 300mM, 500mM, and 1M.

When separating His-tag recombinant proteins using affinity chromatography, when the protein sample is passed through Ni-NTA agarose gel to which Ni ²⁺ is bound, only Ni ²⁺ is attached to the hig-tag recombinant protein and the remaining proteins pass through the column. It comes out. At this time, the N-attached hig-tag recombinant protein is separated from the gel when a high concentration of imidazole is applied. However, since the extent to which the his-tag protein is bound to Ni-NTA agarose cannot be accurately determined, the separation conditions for the target protein were generally optimized by changing the imidazole concentration from low to high concentration. Figure 2 shows the results of SDS-PAGE analysis confirming the purification of lipase according to various concentrations of imidazole in an example of the present invention, and it was confirmed that the desired protein was well expressed. In addition, as a result of experiments under various imidazole concentrations, it was confirmed that the clearest expression was achieved at a concentration of 300 mM imidazole. The protein size of lipase (SEQ ID NO: 1) derived from Lactobacillus rhamnosus IDCC 3201 is about 23KD, and when the protein concentration was checked through BCA assay, it was found to be 442.4 ug/mL.

Example 4. Agar plate method

It was confirmed through the agar plate method that lipase produced using olive oil as a substrate could decompose olive oil.

As a result, as can be seen in Figure 3, it was found that the lipase produced was able to decompose lipids through the formation of a ring around it.

Example 5. Activity and stability of lipase according to temperature and pH

To determine the optimal temperature for the activity of lipase obtained through purification, para-nitrophenyl palmitate was used in 50mM potassium phosphate buffer (pH 8.0) at 10 to 80°C. For temperature stability, 50mM potassium phosphate buffer (pH 8.0), lipase was cultured at 40°C, 60°C, and 70°C, and the activity was measured at 30-minute intervals to determine the activity remaining up to 240 minutes. To check the optimal pH of lipase, use 50mM sodium acetate buffer for pH 5~6, potassium phosphate buffer for pH 6~7, 50mM Tris-HCl buffer for pH 7~9, and 50mM sodium carbonate buffer for pH 9~11. The reaction was performed at 60°C, which is the activation temperature. For pH stability, lipase was incubated for 1 hour in pH buffers identical to the optimal pH, and then activation was performed at 60°C.

As a result, as can be seen in Figure 4, the optimal temperature of the produced lipase was checked and it was found that it had the highest activity at 60°C (Figure 4a). In addition, to check the temperature stability, the activity was examined for up to 240 minutes, and the activity was maintained well even at high temperatures (60, 70 ° C), showing that lipase has strong stability even at high temperatures (Figure 4b).

In addition, as can be seen in Figure 5, the optimal pH of the produced lipase was confirmed to show high activity at pH 5-9 and the optimal pH was 8 (Figure 5a). Additionally, excellent activity retention ability was confirmed in the pH 5-6 and pH 8-9 ranges even after 1 hour of exposure to various pH ranges (Figure 5b).

Example 6. Confirmation of activity against metal ions

The activity of lipase was evaluated in the presence of metal ions. Lipase activity was confirmed in 50mM Tris-HCl buffer (pH 8.0) containing 1mM metal ion. The activity of the control group without metal ions was set at 100%.

As a result, as can be seen in Table 2, the activity of lipase was observed after adding various metal ions to the buffer. Compared to the control, the activity increased most significantly with magnesium and copper ions, and the activity was well maintained even in various ions. I was able to find out the truth.

Example 7. Substrate specificity

To confirm the substrate specificity of the produced lipase, the difference in enzyme activity according to the number of carbon chains (C10-18) in 50 mM Tris-HCl buffer (pH 8.0) was compared.

As a result of measuring the activity of the enzyme using para-nitrophenol with carbon numbers ranging from 10 to 18 as a substrate, it was confirmed that para-nitrophenyl laurate (C12) with carbon numbers of 12 showed the greatest activity (Table 3).

Example 8. Activity against organic solvents

It was confirmed whether the purified lipase was affected by organic solvents at various concentrations (10% (v/v) and 30% (v/v)). The activity of the purified lipase was confirmed through a lipase assay after preliminary incubation with an organic solvent for 1 hour at room temperature.

As a result, as can be seen in Figure 6, it was confirmed that enzyme activity was better maintained in 10% organic solvent than when the concentration was 30%. In particular, the activity of glycerol, dimethyl sulfoxide, and acetone increased more after adding organic solvents than when they were not added.

Example 9. Confirmation of usability as a laundry additive

In order to confirm the usability of the lipase produced through the processes of Examples 1 to 3 as a laundry additive, four types of commercial liquid detergents, Tamsa, Sugar bubble, Dawny, and Persil, which are easily available on the market, were purchased and used. 10 ml of each detergent was diluted with 100 ml of distilled water to obtain a final concentration of 10% (v/v). Additionally, in order to heat inactivate the enzymes inherent in existing commercial detergents, they were sterilized at 121°C for 15 minutes. And finally, 0.0004 g of lipase derived from L. rhamnosus IDCC 3201 was added to 100 ml of diluted detergent solution to a final concentration of 1% (v/v) and 5% (v/v) to determine the concentration of lipase in each diluted detergent solution. A detergent solution was prepared at 0.0004% (w/v), and after 2 hours at room temperature, enzyme activity was measured using para-nitrophenyl palmitate substrate in the same manner as the activity under the optimal temperature conditions above.

As a result, as shown in Figure 7, commercial detergents that inactivated the enzyme (Tamsa negative control, Dawny negative control, Sugar bubble nagative control, Persil negative control) had very low enzyme activity, while lipase produced from L. rhamnosus IDCC 3201 Added detergents (Tamsa, Dawny, Sugar bubble, Persil) have detergent concentrations of 1% (v/v) and 5% (v/v), which are conditions in which the detergent is diluted in water, similar to a washing environment, after 2 hours. All showed a residual activity of over 80%.

Through this, it can be confirmed that all enzymes present in the commercial detergent were inactivated, as the commercial detergent sterilized at high temperature did not show enzyme activity, and more than 80% of the remaining activity in the lipase-added group was produced by L. rhamnosus IDCC 3201. It can be seen that it is the activity of lipase.

In addition, lipase that can be used in the detergent industry must have durability under harsh conditions, including compatibility with various commercial detergents such as Tamsa, Sugar bubble, Dawny, and Persil, to be used as a detergent additive.

Therefore, these results indicate that lipase derived from L. rhamnosus IDCC 3201 has significant detergent compatibility and has the potential to be used as a detergent additive.

Next, in order to confirm the oil stain removal ability of lipase derived from L. rhamnosus IDCC 3201 as a laundry additive, the oil-decomposing activity of lipase in laundry detergent against chocolate, olive oil, and peanut oil stains was confirmed.

Specifically, a piece of cloth measuring 5 cm wide Then, chocolate, olive oil, and peanut oil were applied to the cloth to create oil stains. In the case of olive oil and peanut oil, oil-soluble dye (liquid candy color violet, Chefmaster) was added drop by drop to reveal color so that they could be easily distinguished with the naked eye, and then stains were made on the fabric. Afterwards, the stained cloth contained 100 ml of water, 100 ml of the heat-inactivated 1% (v/v) detergent solution prepared in Example 9, the heat-inactivated 1% (v/v) detergent solution, and 0.0008 g of lipase derived from L. rhamnosus IDCC 3201. was added to each different washing solution and then washed at 50°C and 150rpm. After 1 hour, the cloth was taken out, rinsed, and checked to see how much of the stain had been removed. The heat-inactivated detergent was manufactured by sterilizing Dawny, an existing commercial detergent, at 121°C for 15 minutes.

As a result, as shown in Figure 8, in the washing solution containing only water, the stain was almost intact on the piece of cloth, and in the washing solution containing water and a heat-inert detergent, the stain was removed compared to using only water, but was not completely removed. didn't However, the cleaning solution containing lipase derived from L. rhamnosus IDCC 3201 removed the most oil stains, and as a result, the white color of the natural fiber pieces was most clearly observed.

Today, laundry is mainly performed using detergents to remove oil contaminants in the home and industry, with most commercial detergents being hydrolytic enzyme-based detergents that are diluted with water before use. Therefore, lipase derived from L. rhamnosus IDCC 3201 is expected to show excellent stain removal ability even under high-intensity washing conditions such as washing machines. These results suggest that adding L. rhamnosus IDCC 3201-derived lipase to detergents in the future can provide ecological benefits by reducing the amount of detergent needed and can be used to develop new detergent products.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Sequence list electronic file attached

Claims (14)

Lipase represented by the amino acid sequence of SEQ ID NO: 1. According to claim 1,
The lipase is encoded by the nucleic acid sequence represented by SEQ ID NO: 2. According to claim 1,
The lipase is a lipase derived from Lactobacillus rhamnosus strain. According to clause 1.
The lipase has an activity of 60% or more at 50 to 90°C. According to clause 1.
The lipase has an activity of 60% or more at pH 5 to 9. According to clause 1.
The lipase has specificity for substrates having 10 to 18 carbon atoms. According to clause 1.
The lipase maintains activity in the presence of metal ions. According to clause 1.
The lipase maintains activity in an organic solvent. A recombinant vector containing a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1. A transformant obtained by introducing the recombinant vector of claim 9 into a host cell. Transforming a host cell with a recombinant vector containing a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1;
Culturing the transformed host cells; and
Separating lipase from the culture medium
A method for producing lipase comprising. A laundry additive containing lipase represented by the amino acid sequence of SEQ ID NO: 1. According to clause 12,
The lipase is a laundry additive, characterized in that it is added in an amount of 0.001 to 0.1 g per 100 ml of laundry detergent. A laundry detergent composition comprising lipase represented by the amino acid sequence of SEQ ID NO: 1.