KR101017717B1 - Bio-modification method of polyester fiber using lipolytic enzymes - Google Patents

Abstract

The present invention relates to a method for surface modification of polyester fiber, Sphingobium Chungbukkens ( Sphingobium chungbukense ) Biochemically modifying the surface of polyester fiber biochemically using a novel lipolytic enzyme derived from Cutinase family, which minimizes the mechanical properties of the fiber and improves the surface properties. Effects of increasing hydrophilicity, improving peeling properties, and improving antistatic properties can be obtained.

Enzyme, Sphingobium Chungbukkens, Polyester, Surface, Hydrophilic

Description

Bio-modification method of polyester fiber using lipolytic enzymes

The present invention relates to a method for surface modification of polyester fibers, and more particularly to the surface of polyester fibers biochemically using a novel lipolytic enzyme of cutinase-based Cutinase-derived Sphingobium Chungbuk-kens. It relates to a method of biochemical modification.

Polyester fiber, which is most used among synthetic fibers, is a hydrophobic fiber and has a high heat stability and strength by forming a dense structure. However, due to the rigidity, the wearability and appearance are significantly lower than those of natural fibers, and the hygroscopicity is low. have. In addition, this causes static electricity and fluffing, and in particular, the peeling generated due to the high toughness of the fiber is hardly eliminated, thereby reducing the aesthetic and emotional characteristics of the product, thereby limiting its application as a high quality garment material.

 In order to overcome these disadvantages, research and development are being made on methods for improving the peeling by improving the surface of the polyester fiber and improving soft touch and absorbency such as silk.

Among these surface modification methods, a processing method by alkali hydrolysis treatment, which is excellent in terms of economical aspects and ease of operation, is generally used. However, while the modification method by alkali hydrolysis has the advantages described above, there is no modification effect under weak alkali conditions, so the work must be performed under high temperature and strong alkali conditions, and thus the excellent strength of polyester fiber can be significantly reduced. However, there is a problem that it is difficult to control the process, there is a risk of overweight and can damage the fiber itself extensively. Therefore, by the alkali treatment method, it is difficult to obtain an appropriate level of surface modification treatment to obtain the improvement of the texture and the anti-pilling effect while maintaining the basic strength of the fabric itself.

In addition, the alkali processing requires a large amount of alkali, so it is difficult to apply the problem of environmental pollution processing with a large amount of waste water load and a composite material of natural fibers such as cotton and wool and composite fibers of regenerated fibers such as rayon and tencel and polyester. Problems are also pointed out.

On the other hand, in the processing of natural fiber and regenerated fiber material, processing is performed using enzymes such as pretreatment such as desizing or refining, or tactile improvement using an enzyme. In particular, enzymatic processing such as reducing the fine hairs of cotton fabrics by using cellulase has been introduced, for example, by treating the fabric with an enzyme to prevent peeling and to give flexibility.

However, the method of using enzymes in the surface modification process of polyester is still in the research stage, and the discovery of enzymes and optimization of the process for surface modification of polyester which have industrially meaningful results have not been made yet. Is not a reality.

The present invention has been made to solve the problem of the surface modification process of the polyester fiber described above, polyester fiber that can exhibit surface modification effects such as improved water absorption or peeling properties without a significant decrease in mechanical properties such as tensile strength An object of the present invention is to provide a reforming process.

In order to achieve the above object, the configuration of the present invention,

Sphingobium Chungbukkens KCTC 2955 BP ( Sphingobium) consisting of the amino acid sequence set forth in SEQ ID NO: 6 chungbukense KCTC 2955 BP) derived lipolytic enzyme; A recombinant lipolytic enzyme expressed from a transformant transformed with a vector comprising a polynucleotide encoding a sphingopium Chungbuk-kens derived lipolytic enzyme having the amino acid sequence set forth in SEQ ID NO: 6; And at least one selected from the group consisting of the transformant, the culture of the transformant, the cell-free culture of the transformant, the concentrate of the culture, and the concentrate of the cell-free culture. It is characterized in that the surface of the polyester fiber by hydrolyzing the surface of the polyester fiber by using a composition for modifying the polyester fiber comprising at least one lipolytic enzyme selected from the group consisting of a catalyst.

The polyester fiber modifying composition includes at least one enzymatic activity stabilizer selected from a stabilizer containing at least one type of cation selected from Ca ^{2 +,} Mg ^{2 +} , Fe ^{2 +} , Zn ^{2 +} or a nonionic surfactant It is preferable to include.

Hydrolysis of the polyester fiber by the lipolytic enzyme occurs in the active site of the enzyme, the active site is generally a three-dimensional space structure that can be optimally combined with the reactants and amino acid residues. In order to maintain the optimum catalysis, substrate specificity and stability of the enzyme, the three-dimensional structure of the enzyme active site must be maintained. Thus, the polyester fiber modification enzyme composition is selected from Ca ^{2 +} , Mg ^{2 +} , Fe ^{2 +} , Zn ²⁺ to help maintain the three-dimensional structure of the enzyme active site through the coordination bond with the amino acid of the enzyme The addition of stabilizers containing more than one type of cation leads to an improvement in the effect of the reforming treatment.

In addition, when using a water-insoluble substrate such as fiber, a surfactant may be used to enhance the interaction between the enzyme and the substrate. However, nonionic ionic surfactants, such as anionic surfactants or cationic surfactants, can act as substituents on amino acids charged on the protein surface of the enzyme, causing irreversible changes in the enzyme structure, causing inactivation of the enzyme. The use of surfactants is appropriate.

The temperature at the time of the said reforming process has a preferable range of 30-70 degreeC, and 40-60 degreeC is still more preferable. The pH is preferably in the weak alkali range of 6 to 9, and the neutral to weak alkali range of pH 7 to 8 is most preferred.

Under conditions outside the above ranges, it was found that the activity of the enzyme was inferior and the surface modification effect of the polyester fiber was not sufficient.

On the other hand, although the time required for the treatment varies depending on the treatment temperature, the treatment is preferably performed for 1 hour or more. At 60 ° C, the highest activity of the enzyme, the maximum modification effect can be obtained by treatment for 3 to 6 hours, and there is little improvement in surface modification effect even after further treatment.

The surface modification increases the hydrophilicity of the polyester fiber, thereby improving wettability and moisture content, thereby preventing static electricity. In addition, the improvement of the peeling properties will be shown.

The lipolytic enzyme used in the surface modification processing method of the polyester fiber of this invention is demonstrated in detail.

The lipolytic enzyme used in the surface modification processing method of the polyester fiber according to the present invention is a sphingodium Chungbukkens, for example, a lipolytic enzyme derived from Sphingodium Chungbukkens KCTC 2955 BP.

Sphingobium Chungbukkens ( Sphingobium) chungbukense ) Lipolytic enzyme is a kind of cutinase, a lipolytic enzyme that catalyzes the hydrolysis of polyester fiber, which is an insoluble substrate, even when the concentration of substrate is lower than that of other lipolytic enzymes. Since it exhibits a high initial reactivity, there is an advantage that it can effectively hydrolyze by acting on the ester bond of the polyester fiber.

The lipolytic enzyme was first cloned using a colony hybridization technique and a PCR technique in a phosphide library of Sphingobium chungbukense KCTC 2955 BP ( Sphingobium chungbukense KCTC 2955 BP). Encoded by bp polynucleotides, translated into 350 amino acids, has a common active region of lipolytic enzyme 'GXSX-G' in the 190th amino acid to 194th amino acid region, lipolytic activity and polymer plastic degradation Have activity.

The lipolytic enzyme is composed of the amino acid sequence of SEQ ID NO: 6, more preferably may have an amino acid sequence encoded by the polynucleotide of SEQ ID NO: 5, so long as the activity of the enzyme is maintained, A method known to those skilled in the art may be modified by deletion, substitution or insertion of amino acids.

The polynucleotide encoding the lipolytic enzyme may have a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 6, preferably may have a nucleotide sequence described in SEQ ID NO: 5, to maintain the activity of the enzyme As long as it is, a nucleotide may be modified by deletion, substitution or insertion by a conventionally known method.

In addition, the expression vector including the polynucleotide encoding the lipolytic enzyme may be appropriately selected according to the host used, and all conventional expression vectors capable of expressing a protein in a transformant are applicable. It may be an expression vector comprising a polynucleotide encoding a fusion partner (fusion-tag) capable of purifying the protein expressed by the transformant. Specifically, in the case of Escherichia coli, pET 21a (Novagen Co., Germany), pGEX 4T-1 (GE Healthcare, USA) or pHCE 19 (which can express the introduced foreign genes in a highly soluble form in a transformant with high efficiency) BioLeaders Co., South Korea), preferably may be pGEX 4T-1 or pHCE 19, more preferably pHCE 19, and in the case of yeast a promoter of GAL1, a gene encoding galactose, may be a foreign gene. PYES2 (Invitrogen Co., USA) which can be used for the expression of can be used.

The transformant was prepared using the expression vector, and the host used for the production of the transformant was a prokaryotic cell or a yeast such as E. coli, Bacillus sp. Strain and Pseudomonas sp. Strain. Eukaryotic cells, such as fungi, and the like, but are not limited thereto.

Preferably the expression vector is a polynucleotide having a gene sequence encoding a lipolytic enzyme of SEQ ID NO: 6, more preferably a polynucleotide of SEQ ID NO: 5 inserted into the pGEX 4T-1 vector, the host is preferably May be E. coli , for example E. coli BL21 (DE3).

Method for producing a lipolytic enzyme using the transformant is to culture the transformant using a culture medium, to obtain a lipolytic enzyme contained in the culture medium or a lipase present in the transformant It may be to include the step.

The culture medium, culture conditions and culture methods can be appropriately selected according to the type of transformant, for example, prepared by inserting a polynucleotide having a gene sequence encoding the lipolytic enzyme of SEQ ID NO: 5 into pHCE19 A transformant prepared by introducing a recombinant vector was cultured in a culture medium, or a recombinant vector prepared by inserting a polynucleotide having a gene sequence encoding the lipolytic enzyme of SEQ ID NO: 5 into pGEX4T-1 was introduced. After culturing the prepared transformant in a culture medium, IPTG (Isopropylthio-β-D-galactoside) can be added and cultured. The recombinant lipolytic enzyme prepared by the method expressed by the transformant may be purified by a conventionally known method, but may be used in the form of whole cells or cultures thereof or cell-free cultures thereof without purification.

The recombinant lipolytic enzyme prepared by expressing in the transformant transformed with the expression vector may be preferably a water-soluble lipolytic enzyme, the transformant may be preferably E. coli, for example E. coli BL21 (DE3). The expression vector may be a pGEX 4T-1 vector or pHCE 19 vector, preferably pHCE 19. For example, the recombinant lipase produced by the transformant prepared by introducing a vector of pHCE 19 into which the polynucleotide having the gene sequence encoding the lipase of SEQ ID NO: 5 is introduced may be His. It may be a fusion protein including a tag, and the size of the fusion protein may be 32 kDa.

On the other hand, the culture in the biocatalyst having a lipolytic activity used in the polyester fiber reforming process according to the present invention is a cell, preferably a microorganism, more preferably Sphingodium Chungbukkens, even more preferably The Sphingobi Chungbukkens KCTC 2955 BP ( Sphingobium) chungbukense KCTC 2955 BP) means a culture medium in which a transformant is transformed with a vector comprising a nucleotide sequence encoding a lipolytic enzyme derived from the culture medium, and the culture medium includes cultured microorganisms.

In the present invention, the cell-free culture means removing the cultured microorganisms in the culture medium in which the transformant is cultured.

The culture medium means a solid composition or a liquid composition containing nutrients necessary for culturing microorganisms including animal cells, plant cells or bacteria, and preferably, may be a liquid composition.

In the present invention, the concentrate means that the culture or the cell-free culture is concentrated.

When the surface treatment of the polyester fiber is modified by the polyester fiber reforming method using the lipolytic enzyme according to the present invention, the surface properties of the fiber, such as tensile strength, are hardly modified by modifying only surface properties. It is possible to obtain an effect of improving desirable properties as a garment material such as hydrophilicity such as water absorption, moisture content, and the like, and peeling property is improved.

In addition, the polyester fiber modified processing method according to the present invention does not require radical conditions such as the use of a large amount of alkali as in the prior art is preferable in terms of environmentally friendly with minimizing damage of the fiber.

In addition, since the lipolytic enzyme used in the polyester fiber modification processing method according to the present invention does not act on other materials, there is an advantage that it can be widely applied to materials such as products blended with fibers other than polyester fibers.

Production Example  One: Sphingobium Chungbuk Kens KCTC  2955 BP ( Sphingobium chungbukense KCTC 2955 BP Origin Phosmid  Production of the library

Production Example  1-1: Sphingobium Chungbuk Kens KCTC  Genome of 2955 Strain DNA ( Genomic DNA Extraction of

The phosphide library of Sphingobium Chungbukkens KCTC 2955 strain was produced using CopyControlTM Fosmid Library Production Kit (Epicentre, USA).

More specifically, in order to produce a phosphid library of sphingphodium Chungbukkens, after culturing the sphingobiium Chungbukkens KCTC 2955 strain using LB medium, the genomic DNA extraction kit (Solgent) for bacteria to the culture strain Co., South Korea) using genomic DNA was extracted to prepare the insert DNA.

The prepared insert DNA was diluted to a concentration of 0.5 ㎍ / ,, it was placed in an eppendorf tube and sheared to about 40 kb through vortexing. 20 μg of the sheared DNA, 8 μl of 10X End-repair buffer, 8 μl of 2.5 mM dNTP, 8 μl of 10 mM ATP, and 8 μl of End-repair enzyme mix were prepared to prepare a reaction solution with 80 μl of final volume. The reaction was carried out at room temperature for 45 minutes. 6X gel loading buffer was added to 1X in the reaction solution, and the reaction was carried out at 70 ° C. for 10 minutes to deactivate the end-repair enzyme mix.

Load the end-repaire DNA containing the end-repair enzyme in 1% low melting point agarose gel (LMP), perform electrophoresis, and cut a portion of the gel corresponding to 40 kb to the eppendorf tube. The gel corresponding to 40 kb was heated at 70 ° C. for 15 minutes to dissolve the gel. After the gel was removed by heating, the gel was transferred to a block of 45 ° C., and 50 × GELase buffer and GELase enzyme were pre-warm, and reacted for 1 hour or more. Thereafter, the GELase enzyme was inactivated at 70 ° C. for 10 minutes and at 4 ° C. for 5 minutes.

After performing the deactivation, it was centrifuged for 20 minutes at 13,000 rpm, the supernatant was separated and transferred to a new tube. 1/10 portion of 3 M sodium acetate and 2.5 times cold 100% ethanol were added to the supernatant and reacted for 10 minutes. After centrifugation at 13,000 rpm for 20 minutes to recover the DNA, the supernatant was removed and washed with 70% ethanol. Dissolve and recover DNA with an appropriate amount of TE buffer.

0.25 μg of recovered DNA, 1 μl of 10 × fast-link ligation buffer, 1 μl of 10 mM ATP, 1 μl of CopyControl pCC1FOS vector (0.5 μg / μl), and 1 μl of Fast-link DNA ligase were mixed and reacted at room temperature for 2 hours. After performing the reaction, the ligase was inactivated for 10 minutes under the condition of 70 ℃. After the ligation was completed, the ligater was mixed with 25 μl of MaxPlac Lambda Packaging Extracts and reacted at 30 ° C. for 90 minutes. 25 µl of MaxPlac Lambda Packaging Extracts was further added thereto and reacted at 30 ° C. for 90 minutes. Phage dilution buffer (10 mM Tris-HCl pH 8.3, 100 mM NaCl, 10 mM MgCl 2) was added to the final 1 μL, after which 25 μl of chloroform was added to the reaction to complete the reaction, and the supernatant was obtained. .

The supernatant, ie, the obtained packaged phage, was infected with EPI-300-T1R, which was prepared in advance, to prepare a phosphide library of Sphingobium Chungbukkens KCTC 2955 strain.

Production Example  1-2: Probe for lipolytic enzyme discovery DNA Made of

For the production of probe DNA for lipolytic enzyme discovery from the phosphid library prepared in Preparation Example 1-1, the conservative amino acid sequence of lipolytic enzymes of the strains of the genus Sphingomonas was confirmed.

Specifically, an amino acid sequence of a lipolytic enzyme is obtained from GenBank of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), and Clustal X (ftp: // ftp-igbmc. Multiple sequence comparisons were made using the u-strasbg.fr/pub/ClustalX/) program.

As a result of the multiple sequence comparison, the obtained lipolytic enzymes were identified as having a conserved amino acid sequence having the amino acid sequence 'M-H-G-G-G-M (SEQ ID NO: 1)' and the amino acid sequence 'S-A-G-G-N-L (SEQ ID NO: 2)'. Based on the two amino acid sequence was prepared / secured two DNA primer sequences of Table 1 below.

Primer Name Sequence SEQ ID NO: For probe atgcanggnggnggnatn 3 Rev probe agnttnccnccngcnga 4

Using the primers of Table 1 and using the phosphide library of the sphingobidium Chungbukkens strain of Preparation Example 1-1 as a template, PCR was performed under the conditions of Table 2 below. Probe DNA for southern blotting was performed to find lipolytic enzymes. The probe DNA fragment was about 260 bp in size as shown in FIG. 4.

Step  Temperature Reaction time First denatuation  95 ℃ 5 minutes Second denaturation  95 ℃ 30 seconds Annealing 55 ° C 30 seconds Extension 72 ℃ 20 seconds Cycle go to second denaturation 30 cycles Fnal extension 72 ℃ 5 minutes

Production Example  1-3: for the discovery of genes encoding new lipolytic enzymes Collini Hybridization

Colony hybridization for the discovery of genes encoding novel lipolytic enzymes was carried out using a phosphid library of Sphingodium Chungbukkens prepared in Preparation Example 1-1 and a Dig labeling and detection kit (Roche diagnostics, Germany). It was.

Specifically, the phosphide library was picked on a plate and incubated at 37 ° C. for at least 12 hours, and the cultured cells were adsorbed onto a nylon membrane (using a Hybond-N + filter). The adsorbed cells were lysed using a 10% SDS solution, and the DNA was denaturated using a denaturation solution (1.5 M NaCl, 0.5 M NaOH). Neutralization was performed using a neutralization solution (1.5 M NaCl, 0.5 M Tris-Cl pH 7.5), and 2X SSC (30 mM trisodium citrate, 300 mM NaCl) and UV-crosslinker were used to fix the DNA to the membrane. As a probe, about 260 bp of DIG-labeled product was used, and hybridization was performed at 57 ℃.

Washing solution I (2X SSC, 0.1% SDS), washing solution II (0.5X SSC, 0.1% SDS) and washing buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% (v / v) ) Washing was performed several times using Tween 20). After washing, the membrane is used for signal detection, blocking solution (maleic acid buffer, block reagent) and antibody solution (blocking solution, anti-digoxigenin-AP) and detection solution (0.1 M NaCl, 0.1 M Tris-Cl pH 9.5). In order to detect the signal, CDP-star (Roche diagnostics, Germany) was used. The result of performing the signal detection is shown in FIG.

The colony where signal detection of FIG. 1 is confirmed is named FL446.

Production Example  1-4: Cloning with Lipolytic Gene

As confirmed in FIG. 1, the sequence was analyzed using the method shown in FIG. 2 for the phosmid FL446 clone obtained with a signal through colony hybridization of Preparation Examples 1-3.

More specifically, the phosphine imide FL446 clones Bam H After cleavage using I restriction enzyme, DNAs of various sizes were eluted. DNA eluted in various sizes is Bam H After cloning into pBluescript II SK (-) (Stratagene Co., USA) vector cut with I, sequencing was performed. Sequence analysis revealed some sequences of the novel lipase gene in 2.3 kb of the clones of various sizes. In order to obtain the entire lyase enzyme gene sequence, pBluescript II SK (-), which was cloned with 2.3 kb of DNA, was cut with Sac II to obtain two DNAs of about 400 bp and 2000 bp, of which 400b was obtained. The bp DNA was cloned back into pBluescript II SK (-) to obtain a single clone, and the 2000 bp DNA containing the vector sequence was self-ligation to obtain a clone. The two clones obtained were subjected to sequencing, and through this process, a gene sequence assumed to be a gene of lipase was obtained. The gene suspected of the novel lipolytic gene was named EST1.

Production Example  1-5: Identification of New Lipolytic Enzyme Gene Sequences

As shown in FIG. 3 and SEQ ID NO: 5, EST1, a gene encoding the new lipolytic enzyme derived from the Sphingobium Chungbukkens KCTC 2955 BP strain, uses ATG as the start codon and TGA as the stop codon. A total of 1050 bp of open reading frames are included.

Production Example  1-6: Spingongum Chungbuk Kens KCTC  2955 BP  Derived New Lipolytic EST1 Gene Homology  inspection

Analysis of the similarity between the newly discovered EST1 lipase gene and other lipases previously reported in NCBI and similarity between lipases from other strains and uniqueness of the novel lipase EST1 gene of the present invention It was confirmed.

Specifically, similarity between the newly discovered EST1 lipase gene and amino acid sequence of other lipases previously reported in the NCBI was performed using NCBI's blast X program. This difference was confirmed, using the Clustal X program (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) and the newly discovered EST1 lipolytic gene and other previously reported lipolysis. Gene homology was analyzed between gene sequences of enzymes to predict gene homology between enzymes and active sites of newly discovered EST1, and the results are shown in Table 3 below.

As shown in Table 3, it was confirmed that the EST1 gene, a lipolytic enzyme of the present invention, had a similarity of 33.1 to 52.8% with the previously reported lipolytic enzyme. Among the other lipolytic enzymes, Marine Gamma proteobacterium HTCC 2143 showed the closest similarity with lipolytic enzyme (52.8%).

From the above results, it was confirmed that the EST1 gene, which is the gene of the lipase of the present invention, is a novel lipase gene not reported until now.

Production Example  1-7: New Lipolytic Enzymes EST1 Amino acid active site prediction

Genetic homology analysis between EST1 identified as a novel lipolytic enzyme in Preparation Example 1-6 and gene sequences of various conventional lipolytic enzymes was performed using Clustal X (ftp://ftp-igbmc.u-strasbg.fr / pub / ClustalX /) program, the gene sequence of the novel lipolytic enzyme EST1 of the present invention and the gene homology analysis results are shown in FIGS. 3 and 4.

As shown in FIG. 4, the amino acid sequence of GXSXG amino acid sequence, which is a common active site (conservation sequence) of another existing lipolytic enzyme, is 190- among the amino acid sequences of the novel lipolytic enzyme EST1 prepared in Preparation Example 1 of SEQ ID NO: 6. It was confirmed to exist in the 194 area. From the above-identified facts, EST1 indicates an active site common to lipolytic enzymes common in the amino acid sequences 190-194, and EST1 indicates an active site in the 190-194 amino acid sequence. It was classified as a new lipolytic enzyme with a site.

Production Example  2; New Lipolytic Enzyme EST1 Expression vector and transformant prepared by using the transformant EST1  Expression and Purification of Proteins

Production Example  2-1: Recombinant Novel Lipolytic Enzyme EST1 Expression vector and transformant preparation

In order to secure a large amount of the novel lipase EST1 as an active protein, that is, a water-soluble protein, a transformant capable of expressing the recombinant novel lipase EST1 was prepared.

Specifically, to prepare an EST1 expression vector comprising the novel lipolytic enzyme EST1 gene, three DNA primers containing restriction enzyme sites having the sequences shown in Table 4 and SEQ ID NOS: 7 to SEQ ID NO: 9 were prepared. Made to order (Bioneer, Korea).

Using the primers of Table 4 and PCR under the conditions of Table 5, the gene of the new lipolytic enzyme EST1 was amplified.

Step Temperature Reaction time First denaturation 94 ℃ 5 minutes Second denaturation 94 ℃ 60 seconds Annealing 68 ℃ 60 seconds Extension 72 ℃ 60 seconds Cycle go to second denaturation 30 cycles Final extension 72 ℃ 5 minutes

The expression vector was prepared by ligating the gene amplified by the PCR and the vector treated with the same restriction enzyme. In the case of the pET 21a vector and the pGEX 4T-1 vector, Eco R I and Xho I were treated, and the pHCE 19 vector was treated with Eco R I / Hind III. The electrophoresis of the gel is shown in Figure 5 (Fig. 5 (a)).

The produced expression vector was transformed into E. coli BL21 (DE3) (Studier, FA, and Moffatt, BA, 1986, 189, 113-130) using electrophoresis, and then ampicillin resistance ( Colonies with amphicillin-resistant were screened to obtain transformants that produce recombinant strains. A photo of electrophoresis on the agarose gel of the gene treated by the restriction enzyme used in each vector with the plasmid of the transformant according to each vector is shown in FIG. 5 (b).

As shown in FIG. 5, each strain had a gene at the same position near 1 kb, and each vector could be identified at a position near 4 kb, thus confirming that it had the novel lipase EST1 gene of the present invention.

Production Example  2-2: New Lipolytic Enzyme Through Recombinant Strains EST1 Expression and Purification

The monoclonals of the transformants obtained in Preparation Example 2-1 are cultured in LB medium (0.5% yeast extract, 1% bacto-peptone, 0.5% NaCl). After the inoculation, a portion of the spawn cell-free culture cultured all day at 37 ° C. at 200 rpm was inoculated (1%) in the secondary culture medium (LB medium + 100 mg / L ampicillin), followed by 200 at 37 ° C. Incubation at rpm induced expression of recombinant EST1 protein with the growth of the cells. In addition, the expression of recombinant genes from the transformants cloned using pET 21a and pGEX 4T-1 expression vectors was 0.5 mM of IPTG (isopropylthio-β-D-galactoside) when the OD600 of the culture reached 0.4. After inducing the gene expression, it was carried out by incubating for 4 hours under the same conditions.

Cell culture medium in which lipase was expressed by the above method was centrifuged at 13,000 rpm for 10 minutes, and only cells were separated. The cells obtained by performing the centrifugation were resuspended using 10 mM Tris-HCl, and then the cells were destroyed by using an ultrasonic grinder. In addition, the culture solution was centrifuged for 10 minutes under conditions of 13,000 rpm to separate the active protein and inactive protein.

The purified purified purified active protein was purified using Ni-NTA agarose resin for recombinant EST1 protein with His-tag, and glutathione agarose for recombinant EST1 protein with GST-tag. Purification was carried out.

SDS-PAGE (12% and 15%) analysis of purified EST1 protein and pre-purified protein samples obtained by the above purification process were performed to determine the expression level of the protein of the new lipolytic enzyme EST1, active protein, inactive type. Proteins and purified proteins were identified, and the results are shown in FIG. 6.

In the case of Figure 6a, lanes 1 and 2 are SDS-PAGE of the supernatant (1) and the culture cell pellet (2) of the culture medium of the transformant prepared by transducing the pHCE 19 expression vector into the BL21 (DE3) strain As a result of the analysis, lanes 3 and 4 show the supernatant (3) and the culture cell pellet (4) of the culture medium of the transformant prepared by transducing the pHCE 19 expression vector including the EST1 gene sequence into the BL21 (DE3) strain. As a result of SDS-PAGE analysis, lane 5 was isolated and purified from the supernatant of the culture medium of the transformant prepared by transducing the pHCE 19 expression vector including the EST1 gene sequence into BL21 (DE3) strain using Ni-NTA agarose resin. SDS-PAGE analysis of the purified EST1 recombinant protein obtained by the following procedure.

As shown in FIG. 6A, the recombinant gene cloned into the pHCE 19 expression vector produced a recombinant EST1 protein having a His-tag, which was confirmed to be expressed as 50% of the active protein. The protein was found to have a size of about 32 kDa.

In the case of Figure 6b, lanes 6 and 7 of the supernatant (6) and culture cell pellet (7) of the culture medium of the transformant prepared by transducing the pGEX 4T-1 expression vector to the BL21 (DE3) strain As a result of SDS-PAGE analysis, lanes 8 and 9 are supernatants (8) and cultured cells of the culture medium of the transformant prepared by transducing the pGEX 4T-1 expression vector including the EST1 gene sequence into the BL21 (DE3) strain. SDS-PAGE analysis of the pellet (9).

As shown in FIG. 6B, the EST1 recombinant gene cloned into the pGEX 4T-1 expression vector produced a recombinant EST1 protein having GST, which was confirmed to be expressed as 20% of the active protein. The recombinant protein was found to have a size of about 53 kDa.

From the above results, it was confirmed that the highest active protein is expressed when the recombinant protein is prepared using the pHCE 19 expression vector.

Production Example  2-3; New Lipolytic Enzyme EST1 Activity of lipolysis

To determine lipolytic activity, Kollattukudy, PE, RE Purdy, and IB Maiti. 1981. Cutinase from fungi and pollen, pp. 652-664. In JM Lowenstein (ed.), Methods in Enzymology , Vol. 71. A novel product prepared in Preparation Example 2-2 using para-nitrophenyl butylate (PNB, Sigma, USA), a substrate commonly used for lipolytic activity analysis in Academic Press, New York, USA). The lipolytic activity of the purified protein of EST1, a lipolytic enzyme, was measured.

The lipolytic activity was determined using 96-well microplates, each well containing 106.7 μl of phosphate buffer (0.1 M, pH 8.0), 13.3 μl of Triton X-100 solution (4 g / l), 13.3 μl. Filled with 200 μl of enzyme / substrate solution containing enzyme (added 1 mg of purified protein of EST1) and 66.7 μl of substrate (PNB 100 mg / l). Hydrolysis reaction in the wells was carried out for 5 minutes at 30 ℃. For the control, the reaction was carried out under the same conditions except that no enzyme was included. Experiments were carried out on 12 columns of a 96-well plate, and experiments were conducted under the same reaction conditions in each of the eight wells, and the averages were measured by measuring the absorbance over time (OD 405 nm) for each column. The result is shown in FIG. 7 (standard deviation is less than 5%). FIG. 7 shows the course of lipolysis over time of each EST1 purified protein enzyme against PNB substrate.

As shown in FIG. 7, the purified protein of the recombinant EST1 enzyme having His-tag prepared using pHCE 19 expression vector (FIG. 7A) and the recombinant having GST-tag prepared using pGEX 4T-1 expression vector. It was confirmed that all of the purified proteins of the EST1 enzyme (FIG. 7B) had superior substrate degrading activity compared to the control.

Example  One Sphingobium Chungbuk Kens KCTC  2955 BP  Of polyester fiber using strain-derived lipolytic enzyme Surface modification

Refining and washing

Charms fabric (75D / 36f × 75D / 72f, 187g / yd) of PET material is heated at 95 ° C for 1 hour under conditions of 3-30 g / l of refiner SS-30, 6 g / l of decanter SUNMOL PS, and bath ratio 1:35 After rinsing, cold water washing, 30 minutes washing at 100 ° C., hot water washing and cold water washing were performed three times in order to obtain a refined PET fabric from which the scouring agent was completely removed and used in subsequent surface modification tests.

Lipolytic enzyme Surface modification

The PET fabric after the scouring and washing with water was subjected to surface modification using a composition for modifying polyester fibers using the purified protein of EST1, a lipolytic enzyme prepared in Preparation Example 2-2.

In the treatment conditions, the bath ratio was 1:20, and the purified protein (Cutinase) of EST1 was used at a concentration of 3.5 g / l. The pH of the treatment solution was adjusted to 7.5, and treated at a temperature of 60 ° C. for 12 hours to hydrolyze the PET fiber surface by lipolytic enzymes.

Absorption test

After the refining and washing, the PET fabric sample and the PET fabric after the surface modification treatment with enzyme were measured by dropping 20 mg of water droplets onto the surface in accordance with AATCC test method 79-1992. .

After repeating the test 10 times, the average is shown in Table 6 below in comparison with the time required for the wet (wetting) after the untreated and the surface modification treatment by the enzyme.

In the case of untreated PET, it took more than 1,000 seconds for the fabric to wet, whereas in the case of surface modified PET fabric with enzymatic treatment, the fabric was completely wet in less than 10 seconds. It was confirmed that the hydrophilicity of the surface was greatly improved by modifying.

division Treatment condition Wetting time (sec.) Untreated PET Refining and washing 1010 Example 1 EST1 3.5g / ℓ 8.7 Comparative Example 1 NaOH 5% solution 215

Process water content ( Moisture regain ) Measure

With respect to the PET fabric subjected to the surface modification treatment by the enzyme obtained in Example 1 and the refined and washed PET fabric, the dry weight and the process moisture content weight of the sample were measured according to ASTM D 2654 method. The moisture content was calculated and shown in FIG. 8.

As shown in FIG. 8, it can be seen that the process moisture content of the PET fabric subjected to the surface modification treatment by enzyme in Example 1 was about 0.21%, indicating an improved effect compared to the untreated fabric. The absolute value of the process moisture content is not high because the hydrolysis by the enzyme is limited to the surface of the PET fiber.

Tensile Properties Measurement

Tensile strength was measured by KS K 0520 method by cutting the untreated PET fabric after the scouring and washing with water at room temperature for 24 hours and the fabric subjected to the surface modification treatment by the enzyme to a size of 100 mm in length and 25 mm in width. The tester used a universal testing machine (Zwick 1005, Load cell: 5KN), and measured the test 5 times for each sample at a test rate of 50㎜ / min and averaged the value.

The results of the tensile strength test are shown in FIG. 9. As shown in FIG. 9, the sample subjected to the surface modification treatment according to the present embodiment greatly increases the hydrophilicity of the surface as described above, improves the process water content, and decreases the tensile strength. It was confirmed that the excellent mechanical properties of the PET fibers do not significantly decrease.

Anti-pilling  exam

The anti-pilling test was carried out according to the ICT BOX method of KS K 0533 using the above-described refined and washed PET fabric and PET fabric having surface modification treatment by the enzyme obtained in Example 1. After photographing the surface state of each fabric is shown in Figure 10 and Figure 11, respectively.

As shown in the figures, in the case of untreated fabrics, a lot of fluffs were observed on the surface of the fabrics, and anti-pilling properties were shown in grades 3-4, whereas PET fabrics surface-treated with enzymes were almost observed with fluffs. It did not appear 4-5 grade.

It can be seen from the test results that the surface modification treatment according to the enzyme of the present invention greatly improves not only hydrophilicity but also anti-pilling property.

Fiber surface observation

On the other hand, the unfinished PET fabric after the refined and washed with water and the PET fabric subjected to the surface modification treatment by the enzyme obtained in Example 1 is shown by 3,000 times magnification using a scanning electron microscope shown in Figs. 12 and 13, respectively. It was.

As shown in the drawings, the untreated PET fiber of FIG. 12 maintains a smooth surface state without any irregularities or scratches, whereas the surface of the PET treated with the enzyme of FIG. It can be seen that a portion of the microscopically damaged to form irregular deformation, such as (scale).

Comparative example  1 of polyester fiber with alkali Surface modification

The other conditions were the same as in Example 1, and the surface modification treatment of the existing PET fiber by alkali was performed, and the absorption power, the process water content, and the tensile properties were tested to compare with the surface modification treatment results by the enzyme of Example 1. It was.

The alkali treatment was performed under conditions of 30 minutes treatment at a temperature of 95 ° C. using a NaOH 1% solution and a 5% solution.

Absorption and other tests were made under the same conditions as described in Example 1, and the results are also shown together in Tables 6, 8, and 9 showing the results for the above examples, respectively.

Through the above test results, the surface hydrotreatment of polyester fiber by alkali increased the hydrophilicity (wetting) of the surface or the improvement of the process water content while the surface modification by the enzyme treatment of Example 1 was significantly lower than the tensile treatment. The deterioration in properties is remarkable, indicating that the effect of alkali treatment is not limited to the surface.

Example  2 Sphingobium Chungbuk Kens KCTC  2955 BP  Of polyester fiber using strain-derived lipolytic enzyme Surface modification  In pH Absorption Change According to

The PET fabric was refined and washed under the same conditions as in Example 1, and surface modification treatment was performed on the PET fabric using the lipolytic enzyme EST1 purified protein. However, in order to consider the effect of surface modification treatment according to the pH change, the pH of the treatment solution was changed to 4.5, 5.5, 6.3, 7.3, 8.1, and 9.2, respectively, and the treatment time was 15 hours each.

For the fabric of the surface modification treatment was carried out in the same manner as in Example 1, the absorbency test was carried out, and the time taken for each sample to be wet was measured, and the results are shown graphically in FIG.

As shown in FIG. 14, the time required to wet the entire pH range was reduced to 1/3 or less than in the untreated fabric of Comparative Example 1, indicating that the surface modification treatment was performed. However, the treatment effect was found to be higher in the range of weakly acidic to weakly alkaline of pH 6.3-9.2, especially 6.3-8.1.

Example  3 Sphingobium Chungbuk Kens KCTC  2955 BP  Of polyester fiber using strain-derived lipolytic enzyme Surface modification  Change in absorbance according to treatment temperature

The PET fabric was refined and washed under the same conditions as in Example 1, and surface modification treatment was performed on the PET fabric using the lipolytic enzyme EST1 purified protein. However, the temperature of the treatment liquid was changed to 30 ° C., 40 ° C., 50 ° C., 60 ° C., 70 ° C., and 80 ° C., respectively, in order to consider the effect of surface modification treatment with the change of treatment temperature. It was set as 15 hours.

The fabric of the surface modification treatment was carried out in the same manner as in Example 1, the absorption test was carried out, and the time taken for each sample to get wet, the results are shown graphically in FIG.

As shown in FIG. 15, the time required for the wetting for the entire temperature range tested was reduced to 1/25 or less than that of the untreated fabric, indicating that the surface modification treatment was performed. However, the enzyme activity is lowered at a high temperature or a low temperature exceeding 70 ℃, the effect of the surface modification treatment was found to decrease slightly.

The highest treatment effect was found to be around 60 ° C.

Example  4 Sphingobium Chungbuk Kens KCTC  2955 BP  Of polyester fiber using strain-derived lipolytic enzyme Surface modification  Hydrophilicity with treatment temperature and time

The PET fabric was refined and washed under the same conditions as in Example 1, and surface modification treatment was performed on the PET fabric using the lipolytic enzyme EST1 purified protein.

However, in order to consider the change of surface modification treatment effect according to the change of treatment temperature and treatment time, two kinds of treatment liquids having a temperature of 30 ° C and 60 ° C were prepared, respectively, for 1 hour, 3 hours, The treatment was performed for 5 hours, 8 hours, 16 hours, and 21 hours to achieve surface modification.

The treated fabrics were subjected to an absorption test in the same manner as in Example 1, and the time taken to wet each sample was measured. The results are shown graphically in FIG. 16 and the process moisture content was measured. 17 is shown.

As shown in FIG. 16 and FIG. 17, it was confirmed that the treatment effect was slightly improved both in terms of the absorption rate and the improvement rate and the degree of improvement in the treatment at 60 ° C. rather than 30 ° C. FIG. However, in the treatment time, the improvement of hydrophilicity was observed until 8 hours at 30 ° C. and 5 hours at 60 ° C., but the improvement of the treatment effect was not significant even if the treatment was performed for longer time.

Example  5 Sphingobium Chungbuk Kens KCTC  2955 BP  Of polyester fiber using strain-derived lipolytic enzyme Surface modification  Changes in Absorption Capacity by Addition of Enzyme Stabilizer

The PET fabric was refined and washed under the same conditions as in Example 1, and surface modification treatment was performed on the PET fabric using the lipolytic enzyme EST1 purified protein.

However, in order to confirm the effect of the addition of the enzyme activity stabilizer to the treatment solution, the composition of the treatment solution was subjected to the surface modification treatment by differently as shown in Table 7 below. In addition, the temperature at the time of treatment was 61 degreeC, respectively, and the treatment time was 15 hours, respectively.

division Treatment liquid composition Sample 5-1 Untreated Sample 5-2 Purified Protein of EST1 Sample 5-3 Purified Protein of EST1 / CaCl ₂ 0.001M Sample 5-4 Purified Protein of EST1 / CaCl ₂ 0.005M Sample 5-5 EST1, Purified Protein / CaCl ₂ 0.01M Sample 5-6 Purified Protein of EST1 / CaCl ₂ 0.05M Sample 5-7 EST1 purified protein / Tween 80 1g / ℓ

The treated samples were each subjected to an absorption test in the same manner as in Example 1, and the time taken for each sample to wet was measured, and the results are shown graphically in FIG. 18.

As shown in FIG. 18, the time required for wetting is reduced by the addition of CaCl ₂ , a stabilizer, and Tween 80, a nonionic surfactant for enhancing the interaction between the enzyme and the substrate. It was confirmed that the addition of the enzyme activity stabilizer resulted in an improvement in the treatment effect.

Figure 1 is a phosphide clone of the phosphid library of Sphingobium Chungbukkens KCTC 2955 BP cut with Sac I, a phosphide clone FL446 clone using a conserved gene sequence region of conventional lipolytic enzymes. Photographs showing the results obtained using the prepared probe DNA and colony hybridization technique and X-film;

Fig. 2 shows a restriction fragment of 2.3 kb monoclonal Sac among clones cloned into pBluescript II SK (-) vector by cutting FL446 clone into Bam H I. Schematic diagram showing a process of discovering a new EST1 gene, a new lipolytic enzyme, by performing subcloning using II;

Figure 3 shows the gene of the novel lipolytic enzyme EST1 prepared in Preparation Example 1 and the amino acid sequence encoded by the gene;

4 is a lipolytic enzyme contained in the novel EST1 gene through multiple sequence alignment of the amino acid sequence encoded by the gene of the novel lipase EST1 prepared in Preparation Example 1 and the conventional lipase amino acid sequence. A conserved domain sequence of amino acids;

5 is a photograph showing the results of preparation and cloning of various recombinant EST1 expression vectors prepared for the production of recombinant proteins expressed by the gene of the novel lipolytic enzyme EST1;

6A and 6B are photographs showing the results of analyzing the EST1 protein produced therefrom by transducing a vector expressing recombinant EST1 into E. coli;

Figure 7 is a graph showing the results of measuring the lipid degradation ability over time on the para-nitro phenyl butyrate substrate of the recombinant protein EST1 recombinant protein purified using Ni-NTA agarose resin;

8 is a graph showing the process water content change of the untreated fabric and PET fabrics surface-treated in Example 1 and Comparative Example 1;

FIG. 9 is a graph showing changes in tensile strength of untreated fabrics and PET fabrics surface-treated in Example 1 and Comparative Example 1; FIG.

10 and 11 are photographs showing the anti-pilling test results of the untreated fabric and the surface-modified PET fabric in Example 1, respectively;

12 and 13 are photographs showing 3,000 times magnification of the fiber surface of the untreated fabric and the surface-modified PET fabric in Example 1, respectively, with a scanning electron microscope;

14 is a graph showing the change in absorbency of the surface-modified fabric according to the pH change of the treatment liquid in Example 2;

15 is a graph showing the change in absorbency of the surface-modified fabric with the change in treatment temperature in Example 3;

FIG. 16 and FIG. 17 are graphs showing changes in absorption capacity and process moisture content of surface-modified fabrics according to treatment temperature and time change in Example 4, respectively;

18 is a graph showing the results of changes in absorbency according to the addition of a stabilizer during surface modification in Example 5. FIG.

<110> KOREA DYEING TECHNOLOGY CENTER          KIM, Yang Hoon <120> Bio-modification method of polyester fiber using lipolytic          enzymes <130> DYETEC1 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 6 <212> PRT <213> Sphingobium chungbukense <400> 1 Met His Gly Gly Gly Met   1 5 <210> 2 <211> 6 <212> PRT <213> Sphingobium chungbukense <400> 2 Ser Ala Gly Gly Asn Leu   1 5 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DNA primer <400> 3 atgcanggng gnggnatn 18 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> DNA primer <400> 4 agnttnccnc cngcnga 17 <210> 5 <211> 1050 <212> DNA <213> Sphingobium chungbukense <400> 5 atggagaggc acagaatggc gaagctgttt caagacgtgc gggttgatcc acgaataaag 60 gcggttttcg gtgactatgg cgaaccagcc ggccccgaca tgtccagccg cgaggagctg 120 atcgagttca tgaacagtcc ggaggcacaa gaggccgtgg ccgcggacgc cgccttcttc 180 gcgtcggccg tatcggaaga atgcgccccg tcgcaaggca tcgtctacga aaccagggtc 240 tttacgtccc agcccgacgg caaccagatc aagatccaat atatccgtcc cgaaggggcc 300 ggcatcctgc cctgcgttta ttatatgcat ggcggcggaa tggcctatct gtctgccttc 360 gaccccaatt atcgcgcctg gggccgcatc atggcggcgc agggcgtggc ggtggcgatg 420 gtcgatttcc gcaatagcct gctcccctcg tcggcccccg aaatcgcccc cttcccagcc 480 ggtctgaacg attgcgtcgc cggactgaaa tgggtccatg cccatgcgga agaactccag 540 atcgacccgg acaagatcat cgtcgcgggc gaaagcggcg gcggcaacct ggcgctcgcc 600 accgcgctga agctgaaccg ggatggcgac atcgggctga tcaagggcgt ttatgcgctt 660 tgcccgttca tcgccggcca gtggccgcat cccgatcatc cgtcctcggc cgccaacgag 720 ggcattttca tcagcatcgg gaacaatcgc ctgaccctcg cctacggcat cgaagccttc 780 gagcggcagg acccactggc ctggccgatc ttcgcgtcgg aggctgatct caagggcctg 840 ccgcaaaccg tcatcagcgt gaacgaatgc gatccgctcc acgacgaggg cgtcgccttt 900 taccgcaaat tgctggcggc cggggttccc gcgcggtgcc gctcgctgat cggcacggtc 960 catgcggtcg aaatattgcc cagatgcgcg ccggatgtca gcgccaacac cgccaatgac 1020 atcgcctatc cggcgcggac tggaaagtga 1050 <210> 6 <211> 350 <212> PRT <213> Sphingobium chungbukense <400> 6 Met Glu Arg His Arg Met Ala Lys Leu Phe Gln Asp Val Arg Val Asp   1 5 10 15 Pro Arg Ile Lys Ala Val Phe Gly Asp Tyr Gly Glu Pro Ala Gly Pro              20 25 30 Asp Met Ser Ser Arg Glu Glu Leu Ile Glu Phe Met Asn Ser Pro Glu          35 40 45 Ala Gln Glu Ala Val Ala Ala Asp Ala Ala Phe Phe Ala Ser Ala Val      50 55 60 Ser Glu Glu Cys Ala Pro Ser Gln Gly Ile Val Tyr Glu Thr Arg Val  65 70 75 80 Phe Thr Ser Gln Pro Asp Gly Asn Gln Ile Lys Ile Gln Tyr Ile Arg                  85 90 95 Pro Glu Gly Ala Gly Ile Leu Pro Cys Val Tyr Tyr Met His Gly Gly             100 105 110 Gly Met Ala Tyr Leu Ser Ala Phe Asp Pro Asn Tyr Arg Ala Trp Gly         115 120 125 Arg Ile Met Ala Ala Gln Gly Val Ala Val Ala Met Val Asp Phe Arg     130 135 140 Asn Ser Leu Leu Pro Ser Ser Ala Pro Glu Ile Ala Pro Phe Pro Ala 145 150 155 160 Gly Leu Asn Asp Cys Val Ala Gly Leu Lys Trp Val His Ala His Ala                 165 170 175 Glu Glu Leu Gln Ile Asp Pro Asp Lys Ile Ile Val Ala Gly Glu Ser             180 185 190 Gly Gly Gly Asn Leu Ala Leu Ala Thr Ala Leu Lys Leu Asn Arg Asp         195 200 205 Gly Asp Ile Gly Leu Ile Lys Gly Val Tyr Ala Leu Cys Pro Phe Ile     210 215 220 Ala Gly Gln Trp Pro His Pro Asp His Pro Ser Ser Ala Ala Asn Glu 225 230 235 240 Gly Ile Phe Ile Ser Ile Gly Asn Asn Arg Leu Thr Leu Ala Tyr Gly                 245 250 255 Ile Glu Ala Phe Glu Arg Gln Asp Pro Leu Ala Trp Pro Ile Phe Ala             260 265 270 Ser Glu Ala Asp Leu Lys Gly Leu Pro Gln Thr Val Ile Ser Val Asn         275 280 285 Glu Cys Asp Pro Leu His Asp Glu Gly Val Ala Phe Tyr Arg Lys Leu     290 295 300 Leu Ala Ala Gly Val Pro Ala Arg Cys Arg Ser Leu Ile Gly Thr Val 305 310 315 320 His Ala Val Glu Ile Leu Pro Arg Cys Ala Pro Asp Val Ser Ala Asn                 325 330 335 Thr Ala Asn Asp Ile Ala Tyr Pro Ala Arg Thr Gly Lys ***             340 345 350 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> DNA primer <400> 7 cgcgaattca tggagaggca cagaatggc 29 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> DNA primer <400> 8 aatctcgagc tttccagtcc gcgccggat 29 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> DNA primer <400> 9 attaagcttt cagtggtggt ggtggtggtg 30  

Claims (3)

Sphingodium Chungbuk-kens derived lipolytic enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 6; A recombinant lipolytic enzyme expressed from a transformant transformed with a vector comprising a polynucleotide encoding a sphingopium Chungbuk-kens derived lipolytic enzyme having the amino acid sequence set forth in SEQ ID NO: 6; And at least one selected from the group consisting of the transformant, the culture of the transformant, the cell-free culture of the transformant, the concentrate of the culture, and the concentrate of the cell-free culture. Lipolytic enzyme, characterized in that to hydrolyze the surface of the polyester fiber using a composition for modifying the polyester fiber comprising at least one lipolytic enzyme selected from the group consisting of a catalyst to modify the surface of the polyester fiber Polyester fiber modification processing method used. The method of claim 1, wherein the polyester fiber-modifying composition comprises a stabilizer or a water-insoluble fiber substrate containing at least one type of cation selected from Ca ^{2 +,} Mg ^{2 +} , Fe ²⁺ , Zn ^{2 +} and an enzyme. A polyester fiber modified processing method using a lipolytic enzyme, characterized in that it comprises at least one enzyme activity stabilizer selected from nonionic surfactants to enhance the action. The polyester fiber modified processing method according to claim 1, which is treated for at least 1 hour at a temperature in the range of pH 6-9, 30-70 ° C.

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