KR100984480B1 - Recombinant microorganisms harboring a ?-glucosidase and their use for the production of indigo dyes - Google Patents

Abstract

The present invention is a β-glycosidase (β-glucosidase) which is a key enzyme useful in the production of indigo blue and indirubidin used as a raw material for dyes and pharmaceuticals by acting on indicans or derivatives thereof contained in natural plant material The present invention relates to a transformed microorganism comprising a gene encoding and a method for producing an indigo dye using the same. According to the present invention is expected to be able to use in various fields, such as the production of natural dyes using plants, cosmetics and paints. In addition, it is expected that the manufacturing process will be advantageous for the development of medicines related to immunity and antibacterial activity using eco-friendly and natural materials. As a side note, the advantages of a single enzymatic process are expected to overcome the shortcomings of traditional indigo dyes, namely, high cost, reproducibility, and homogenization, and can replace traditional production processes using indole or tryptophan. There will be.

Glucosidase, Indican, Indigo

Description

Recombinant microorganisms harboring a β-glucosidase and their use for the production of indigo dyes}

The present invention is a gene encoding β-glucosidase, which is a key enzyme useful in the production of indigo blue and indirubividin used as a raw material for dyes and pharmaceuticals by acting on indicans or derivatives thereof contained in plant plants. It relates to a method for producing a transformed microorganism comprising the and indigo dye using the same.

With the history of mankind, natural dyes have been used for a long time before the synthetic dye preparation method was established, and most of them are made from natural materials, that is, plant extract-oriented materials. Unlike synthetic dyes, natural dyes are harmless to the human body and do not cause any environmental pollution problems derived from synthetic or extraction processes. When used as a dye solution, which is a major application field of dyes, it is known to have a natural and harmonious color and texture, so that colors can not be expressed by synthetic dyes. Representative indigo blue and indirubidin as a natural product is a dye material of blue and red color is produced from indican, a material material contained in plants. Indikanes are known to have high amounts in Polygonum tinctorium plants . Taking advantage of these characteristics, Indigo has been used as a material for cosmetics and paints since Roman times, and it is still the main material used for dyeing eco-friendly cloth. As mentioned above, indigo-based dyes have been mainly used in the form of natural extracts depending on the traditional method, but due to the non-uniformity of the production process, the difference in the dye composition of the material plants, and the non-standardization of the fermentation-maturation process. As shown in FIG. 1A, chemical synthesis of indigo is enabled, and synthetic raw materials are used as most dyes in dyeing solutions. However, with the recent reports of dyeing of clothing, food or cosmetic ingredients, the possibility of remaining disease-causing substances including cancer and allergies has emerged, and the environmental pollution problem of organic synthesis has emerged, and according to health, environmental pollution and consumer preferences, There is a growing interest in dyes. There is an increasing interest and research on natural dyes in Korea, which has been applied to dyeing a lot, and many researches are actively underway to replace indigo production by biotechnology. Indigo can be used in the food industry in the form of cosmetic raw materials or indigotin derivatives, and is being used as an antimicrobial agent for the treatment of skin diseases and promoting the immune response.

Biological methods for producing industrially valuable indigo blue are generally divided into fermentation method and enzyme conversion method. The first method that requires biological activity is the conventional method used for dyeing cloth. It uses sediment-type indigo blue produced after immersing the spines in water for a certain period of time to induce fermentation and ripening. In other words, the dye is produced using the enzymatic activity of the plant itself or naturally contained microorganisms in the environment, such as the time and temperature of the immersion. The resulting indigo blue is recovered as a precipitate by adding lime and used as a dye. When dyes are prepared in this way, it is difficult to standardize because the time required is long, the particles are not uniform, and the quality varies depending on the type and amount of lime used or other immersion compounds. Therefore, the dyeing time is also not constant, which generally takes a long time and is not uniform due to uneven particles. In addition to these problems, due to the dyeing fastness, reproducibility, production limitations and storage problems of natural dyes, their use range is limited and they are being traded at high prices, and thus they are not popularized. The second method of biological production is to produce indigo blue via indole (indoxyl) after the conversion of aromatic compounds such as tryptophan, naphthalene, toluene from bacteria into indole (indole) through the enzymatic reaction as shown in [1b] will be. E. coli, the host used in this method, is not capable of metabolism of aromatic compounds, as well as enzymes that convert to the human labyrinth, which leads to highly complex recombinant gene production or alternatively, the use of Pseudomonas , which contains most of the related active enzymes. Can be reviewed. However, in the case of organic solvents such as naphthalene and toluene, it is difficult to mix with an aqueous solution because of the hydrophobicity and has a very strong toxicity, there is a problem that the residue of the raw aromatic compound from the host cell as well as the dye produced. As an alternative there is a fermentation method produced by the process of indole in tryptophan produced during glucose metabolism as shown in [1c] but these also can not be put to practical use due to low production yield, difficulty in culturing high concentration of cells, growth inhibition by metabolites, etc. have.

Contrary to this, there have been reports that Novozyme 188 and novagram G enzymes can convert indicans into indigo, but their production has been cut off due to their high cost and low demand. In addition, there have been many studies on indigo produced by the activity of indigo and istanan B, which are indigo precursors in plants, and the activity of the plant's own enzymes. There are no studies on the mass production enzymes that can be used in general.

The present inventors have focused on the fact that indican is converted to indigo by β-glucosidase in a plant, and the subdifamily of sub-family of β-glucosidase family discovered by a systematic process. After confirming the activity of the activity to find a superior enzyme source through the process of comparison and analysis, the present invention was completed.

It is therefore an object of the present invention to provide a transformed microorganism which produces β-glucosidase having the activity of producing indigo-based dyes and pharmaceuticals from a plant or extract thereof.

Another object of the present invention is to provide a composition for producing an indigo dye from a plant or extract thereof, comprising β-glucosidase derived from a Shinorhizobium meliloti strain. .

In addition, another object of the present invention includes adding a β-glucosidase derived from a Sinoraizolium melorirot strain, or a microorganism producing the same, to a plant or an extract thereof to convert indican to an indigo dye. It is to provide a method for producing a dye.

The first aspect of the present invention relates to a gene encoding β-glucosidase having a nucleotide sequence of SEQ ID NOs: 1-8. The gene is a gene encoding β-glucosidase having an activity of producing indigo dyes from a plant or an extract thereof.

The second aspect of the present invention relates to a recombinant plasmid comprising the gene. The recombinant plasmid is a recombinant plasmid characterized by being expressed as a maltose binding protein (MBP) fusion protein.

The third aspect of the present invention provides a transforming microorganism E. coli XL1-BLUE [pTrc99A-glu (SM)], which produces β-glucosidase having activity to produce indigo dyes from a plant or extract thereof (Accession No. KCTC 11236BP). The β-glucosidase is a transforming microorganism derived from the Shinorhizobium meliloti strain.

A fourth aspect of the present invention relates to a composition for producing indigo dyes from a plant or extract thereof, comprising β-glucosidase derived from a Shinorhizobium meliloti strain. . The β-glucosidase is a composition having a nucleotide sequence of SEQ ID NO: 8, and is characterized by having activity on X-gal (5-bromo-4-chloro-3-indolyl-bD-galactopyranoside). This means that X-gal can be used as a reporting system for determining whether or not to clone a gene using the enzyme of the present invention as a substrate.

The fifth aspect of the present invention comprises adding a β-glucosidase derived from a cynorazobi meliloti strain, or a microorganism producing the same, to a plant or an extract thereof to convert the indican to an indigo dye. It is about how to produce. Converting the indican to indigo dye,

a) inserting the β-glucosidase gene into the vector;

b) transforming the vector into a host cell and then culturing the cell to produce a transformant; And

c) adding the plant or extract to the medium in which the transformant is cultured and reacting:

It provides a method for producing an indigo dye comprising a.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

For the implementation of the present invention the desired substrate specificity is achieved using the availability of D-glucose desorbed from indicans as a sole carbon source as a result of β-glucosidase activity and a color or fluorescent substrate that can be introduced relatively easily. I searched for a child's family and tried systematic excavation. First, all glucosidase gene sequences and amino acid sequences in GeneBank and PDB databases were collected to analyze the evolutionary tree. Of these members, if the amino acid sequence is a paralogue in a strain, such as when the similarity is very high (> 35%), the phylogenetic tree was reconstructed except for orthologous but extremely close evolutionary relationships. . Subsequently, each subfamily is reclassified into genetic evolutionary distance, substrate specificity, and substrate spectrum, and the strains representing each sub- subfamily are obtained from environmental samples, strain banks, and laboratory holding strains. Measured. As a result, it was confirmed that one sub-agua had high activity against the target substrate, indican. Putative glucosidase after all the amino acid sequences of sub-Aguas classified by this process were identified using the Clustal X program and then scanned using the BLAST-P program in GenBank. A new group of enzymes classified as a family of enzymes but whose activity was not reported for indicans could be obtained.

When analyzing the properties of β-glucosidase a-agua identified through a systematic excavation process in the present invention, most of the indican decomposed β-glucosidase is found in general soil microorganisms (Rizobium, cynorazobibium, Agrobacterium) And Flobobacterium) and include Swanella and Thermus, which are frequent in fields, farmland, and aquatic environment. The present inventors show that the similar β-glucosidases evolved from a common ancestor and showed a similar sequence of amino acids (431-459) and more than 35% high sequence similarity to show that it is a common feature to be active against the same substrate [ Table 1], the base sequence of each strain represented by SEQ ID NO: 1 to 8 is as follows.

TABLE 1

1) Thermus caldophilus )

(SEQ ID NO 1)

2) Thermus thermiphilus HB8

(SEQ ID NO: 2)

3) RY Summers sheath (Thermus sp .)

(SEQ ID NO 3)

4) Agrobacterium tumefaciens

(SEQ ID NO: 4)

5) Bradyrhizobium japonicum

(SEQ ID NO: 5)

6) Shewanella baltica )

(SEQ ID NO: 6)

7) Flavobacterium johnsoniae

(SEQ ID NO: 7)

Shinorhizobium meliloti )

(SEQ ID NO: 8)

The present invention is a microorganism selected through a systematic excavation process in order to compensate for the time required for indigo production, a complicated process, a production non-uniformity, and a reproducibility problem during dyeing, or a drawback of poor fastness. Derived β-glucosidase subfamily was used to find enzymes with high activity in indican and its derivatives, which are precursors of indigo production.

In the case of the discovered β-glucosidase family of enzymes, the present inventors have demonstrated that production of indigo dyes in a single process is possible if the indoksil and D-glucose can degrade the bonds of polymerized indicans in the form of glycoside bonds. It was thought that this would be possible. However, it was confirmed that the discovered enzymes have activity on indicans, but when pure indicans are used in the actual indigo production process, it is difficult to apply them directly to the production process because they have to be purified or used organically synthesized substrates. Therefore, in order to develop an economical and environmentally friendly process, the plant itself or fractionated components, extracts should be used. To confirm this possibility, it was confirmed whether the enzyme could act on the natural plant itself. As a result, it was difficult to detect distinct enzyme reactions in the powders that were simply broken by physical methods. This is consistent with the existing results, and it is known that the enzyme is difficult to access to indicans mainly distributed in vacuoles or nearby tissues. As a solution to this problem, an attempt was made to detect enzyme activity in an indican-containing solution extracted from plant powder.

When comparing the characteristics of the enzyme expressed by the leaking (leaking) in E. coli without artificial manipulation, the β-glucosidase derived from the relatively high expression and high activity of cynorazobi melilotiti strains was high. Therefore, based on this, the mass production for the identification and application of the enzyme was attempted. In this case, for mass production of the protein is produced using a fusion label or a fusion protein, in the present invention to build a more stable expression system using a maltose binding protein (MBP) fusion protein.

For the purpose of inducing overexpression of proteins and increasing their solubility, they are generally overexpressed using a fusion protein such as glutathione-S-transferase (GST) or MBP. Available on site. In addition, the use of amylose (amylose) resin has the advantage that it is easy to pure separation of proteins and continuous operation by immobilization.

According to the present invention, if indigo dye is produced in a plant containing a large amount of indican, a natural material, after mass production of the selected enzyme source using recombinant genetic techniques, it is possible to adjust the processing time, enzyme titer, and simplify the process. Efficient biological production of natural dyes will be possible, and environmental pollution and health problems will be minimized.

As described above, the present invention shows that it is possible to produce indigo dyes from the side plants themselves, powders or extracts by using enzymes selected by confirming the activity of the β-glucosidase family of subfamily indica. Therefore, it is expected to be used in various fields such as production of natural dyes using plant, cosmetics and paints. In addition, it is expected that the manufacturing process will be advantageous for the development of medicines related to immunity and antibacterial activity using eco-friendly and natural materials. Incidentally, the single enzymatic process is expected to overcome the shortcomings of the traditional indigo dye method, i.e., high cost, reproducibility and homogenization. The complex production process using indole or tryptophan Could be replaced.

Hereinafter, the present invention will be described in detail with reference to Examples. The present invention is not limited to the following examples, and any person having ordinary skill in the art to which the present invention pertains may make various modifications without departing from the gist of the present invention as claimed in the claims. Such changes are intended to fall within the scope of the claims.

Example 1 Excavation of Indican Degrading Strains by a Systematic Process

After confirming β-glucosidase-related enzyme resources, which are the targets for the activity of indicans, from the gene and protein information banks, the overall sequencing and amino acid sequences were analyzed and the orthologs and similar paralogs in the genes with high similarity were analyzed. After the exclusion, the phylogenetic tree was reconstructed based on 141 genes derived from a total of 61 strains. Subsequent ecologically related strains and known substrate specificities were then simplified to sub- subfamily. Eleven strains with genes representing each subfamily were selected from strains known for the whole genome sequence, and then the activity and relative titers of the target substrate indican were determined using a glucose assay kit (glucose). assay kit (Sigma) and high performance liquid chromatography (HPLC) equipped with uBondapak C18 column were compared and analyzed. As a result, strains of the genus Riizobial, one of the sub- subfamily in the phylogenetic tree, were found to have high activity against indicans.

Based on the conserved sequence of glucosidase from the strains of the genus Rizovial to verify the confirmed results and to secure an extended source of enzymes, the BLAST-P program was used to enhance the activity of indicans. Strains with eight predictable enzyme sources were identified and shown in the following [Table 1], and the strains of each strain are shown in the following [Fig. 2].

The activity of the selected strains on indican was confirmed both in the whole cell utilization process through the above reactions and in the nutrient medium containing indican (LB or NB) and at least medium fed with indican as a carbon source. In each case, solid medium containing 1 ~ 5 mM indican was incubated for 3 to 5 days at the optimum growth temperature of each strain.

[ Example  2] of the gene encoding the related enzyme from the identified strain Cloning

The strains of [Table 1] selected for the acquisition of genes related to indican resolution were used by preservation in laboratory-owned strains and published institutions (KACC, MICRO BANK). Cultivation of the strain for genomic DNA extraction was carried out at 25 ~ 50 ℃ using the medium specified by the LB medium and the distribution institution, the genomic DNA extraction from the cultured cells using a promega genomic DNA prep kit. Primers used for amplification of genes to be cloned into pTrc99A (Pharmacia), which is a genomic DNA as a template, were prepared based on sequences registered in GenBank. Polymerase chain reaction (PCR) reaction was performed using Pfu polymerase and template DNA was added by adding 20ng each. PCR conditions were repeated 30 times for 5 minutes denaturation at 95 ° C, 1 minute denaturation at 95 ° C, 1 minute annealing at 55 to 60 ° C, and 2 minutes extension at 72 ° C. It was. In this example, only four strains were first tested in consideration of glucosidase sequence similarity within 8 selected strains and favorable thermal stability in practical application. Each cloning condition is as follows.

(1) Thermus caldophilus

The pTrc99A plasmid was digested with EcoR I and Hind III restriction enzymes, and then a recombinant plasmid was prepared by amplifying the β-glucosidase region of the Summer Caldophilus strain by PCR. To this end, the Thermos Caldophilus chromosome was used as a template, and a forward primer (5'-TAGAATTCAACGCCGAAAAGTTT-3 ') and a reverse primer (5'-TAAAGCTTTCACTCTGGCTGGGG)

-3 ') was used.

Forward primer: 5'-TAGAATTCAACGCCGAAAAGTTT-3 '(SEQ ID NO: 9)

Reverse primer: 5'-TAAAGCTTTCACTCTGGCTGGGG-3 '(SEQ ID NO: 10)

(2) Thermus thermophilus HB8

The pTrc99A plasmid was digested with EcoR I and Hind III restriction enzymes, and then the recombinant plasmid was prepared by amplifying the β-glucosidase region of the Summers thermophilus HB8 strain by PCR. For this purpose, the Thermos Thermophilus HB8 chromosome was used as a template, and a forward primer (5'-TAGAATTCAACGCCGAAAAATTC-3 ') and a reverse primer (5'-TAAAGCTTTTAGGTCTGGGCCC

G-3 ').

Forward primer: 5'-TAGAATTCAACGCCGAAAAATTC-3 '(SEQ ID NO: 11)

Reverse primer: 5'-TAAAGCTTTTAGGTCTGGGCCCG-3 '(SEQ ID NO: 12)

(3) Flavobacterium johnsoniae

The pTrc99A plasmid was digested with Nco I and Hind III restriction enzymes, followed by flavobacterium johnsonie strains. The recombinant plasmid was prepared by amplifying the β-glucosidase region by PCR. For this purpose, the forward primer (5'-GCCCATGGGTAAAATTGAAAACTCATTT-3 ') and the reverse primer (5'-GCAAGCTTTTAAGA) were used as the template for the Flavobacterium johnsonie chromosome.

TAAAAAATCTTTAAA-3 ') was used.

Forward primer: 5'-GCCCATGGGTAAAATTGAAAACTCATTT-3 '(SEQ ID NO: 13)

Reverse primer: 5'-GCAAGCTTTTAAGATAAAAAATCTTTAAA-3 '(SEQ ID NO: 14)

(4) Sinorhizobium meliloti

The pTrc99A plasmid was digested with Nco I and Hind III restriction enzymes, followed by cloning of the β-glycosidase region of the cynorazobilium melorirot strain to produce a recombinant plasmid. To this end, the genome of the cyno- razobiium melorirot strain was used as a template, and a forward primer (5'-ATCCATGGTGATCGAAGCCAAGA-3 ') and a reverse primer (5'-ATAAGCTTTCATCCC) were used.

PCR was performed using GGCTTGT-3 ').

Forward primer: 5'-ATCCATGGTGATCGAAGCCAAGA-3 '(SEQ ID NO: 15)

Reverse primer: 5'-ATAAGCTTTCATCCCGGCTTGT-3 '(SEQ ID NO: 16)

Genes amplified under the above conditions were electrophoresed on agarose gel (0.8%) to recover only fragments of a desired size and then inserted into expression vectors cut with the above-described restriction enzymes to prepare recombinant plasmids. The recombinant plasmid prepared was introduced into E. coli XL1-Blue, transformed, and recovered by amplification in large quantities through whole cell culture. As a result of confirming the nucleotide sequence and amino acid sequence of the gene inserted into each of the recovered recombinant plasmids, it was confirmed that it was identical with the sequence of the discovered β-glycosidase gene. Among the recombinant plasmids recovered above, E. coli XL1-Blue [pTrc99A-glu (SM)] transformed with the β-glycosidase gene of the cynorazobi memelirot strain was named E. coli XL. The deposit was made on November 23, 2007, with the accession number KCTC 11236BP, to the Gene Bank, an international depository institution located at 52, Eun-dong Biotechnology Research Institute.

Example 3 Expression and Activity of β-Glycosidase in Transgenic Strains

In order to confirm the expression level and activity of the recombinant β-glycosidase prepared in Example 2, the recombinant cells activated in a solid medium were inoculated into LB liquid medium containing ampicillin (50 µg / ml) at 37 ° C. Incubated for 10 hours. 1% of the culture solution was reinoculated into LB liquid medium containing 10ml 50µg ampicillin, and then cultured at 37 ° C and 180rpm to have an absorbance of 0.5 at OD ₆₀₀ . Thereafter, 1 mM IPTG (isopropyl-thiogalatoside) was added, and then protein expression was induced at 37 ° C. for 100 minutes. Expression-induced cells were recovered using a centrifugal separator and the cells were lysed and the expression levels of enzymes were measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A host transformed with an empty vector without inserts was used as a control and no activity of β-glycosidase enzyme was detected. As a result, all recombinant β-glycosidase, as shown in the following [Fig. 3] is expressed in the most insoluble state when expressed as a single protein (sole protein) without using a fusion tag (fusion tag) or protein Confirmed. There was no clear expression of the Summer Caldophyllase enzyme, and the expression levels of Summers Thermophilus HB8 and Flavobacterium johnsonie were overexpressed in about 30% of the total protein, but over 90% were insoluble form. It became. The enzyme of cynorazobi memeliotti was overexpressed to account for more than 45% of the total host protein, but only 20% was soluble. This expression pattern was found to be maintained even when the temperature was lowered or the host was expressed, indicating that the expression in E. coli was unstable. Therefore, when inducing expression in E. coli, it would be more advantageous to measure the activity of the protein expressed in small amounts in the absence of derivatives. Therefore, under the same conditions as above, the presence or absence and activity of the protein expressed using the fluorescent substrate was confirmed. To this end, MUG (4-methylumbelliferyl- ㅯ -D-glucopyranoside, Sigma-Aldrich), a fluorescent substrate, was cultured by smearing transformants onto LA plates containing 2 mM [Figure 4a], and 5 mM MUG on the cultured cells. 1% agarose gel (50 mM sodium-acetate) containing the solution was poured and reacted for 5 minutes [right of FIG. 4a], and then it was confirmed whether fluorescence appeared at 350 to 375 nm. As a result, as shown in [FIG. 4A], all of the enzymes except for the enzymes derived from Flavobacterium johnsonie strains were found to have distinct fluorescence by activity. Among them, the transformant having an enzyme derived from the cynorazobi melloriti strain showed the highest fluorescence intensity, indicating a relatively high activity.

In general, in the case of the E. coli host used in the experiment, it is known that various kinds of glucosidase family enzymes exist. Therefore, an enzyme diagram (zymogram) was performed on a native gel to confirm whether the activation of the fluorescent substrate was caused by an enzyme of the host or by an excavated enzyme. Each recombinant cell was incubated and disrupted in the above-described conditions to collect the supernatant and electrophoresed on 8% undenatured gel. The developed gel was washed with distilled water or a buffer solution, a 1% agarose gel (50mM Na-acetate) containing 5mM MUG was overlapped, and then reacted at 37 ° C for 5 minutes to identify a fluorescence protein band. As a result, as shown in the following [4b] to show the fluorescence at different positions, it was confirmed that the fluorescent substrate is decomposed by each of the discovered enzymes, rather than the degradation of the fluorescent substrate by the same enzyme in the host. On the enzyme diagram as in the solid medium, it was observed that the enzyme derived from cynorazobimelellioteti was high in expression and high in activity. Β-glycosidase activity was not observed for hosts transformed with the empty vector with no insert as a control.

In addition, β-glucosidase of cynorazobimelelliote showed distinct activity against X-gal (5-bromo-4-chloro-3-indolyl-D-galalactopyranoside) having similar characteristics to that of indican or MUG. ].

Example 4 Indican Degradation Activity of Excavated β-Glucosidase

The activity of the indican of the enzymes whose activity was demonstrated by fluorescence substrate was confirmed by the following method. LA cells containing 1mM pure indican and M9 minimal medium were plated with recombinants containing the genes whose activity was confirmed, and then cultured at 37 ° C., respectively, and the indigo blue was observed. First, the growth of recombinant microorganisms with β-glucosidase gene discovered in M9 minimal medium containing 1 mM indican (FIG. 6A) was confirmed. 2 to 3 days after incubation as shown in [FIG. 6], except for transformants with flavobacterium johnsonie enzymes using indican as a carbon source, cynorazobib melloritti and Somers Caldophilus, Somers Summer Filler Escherichia coli with β-glucosidase of HB8 strain was grown and produced indigo blue of indigo blue. In LA medium (FIG. 6b), the same results as in M9 minimal medium were produced indigo blue of β-glucosidase of cynorazobi memeliotti, Summers caldophyllus, and Summers thermophilus HB8 strains. In addition, as shown in the fluorescent substrate, it was confirmed that the activity of the transformant having the cyno- razobiium melilloti enzyme was the strongest, indicating that the activity was relatively high. In the case of β-glucosidase of the Flavobacterium johnsonie strain, it is unclear in the following [FIG. 6], but it was confirmed to produce a small amount of indigo after 5-6 days of culture. It is assumed that this is caused by low enzymatic activity and is expected to have only a slight degree of activity necessary for minimal cell growth. No growth or activity was observed in the host transformed with an empty vector with no insert as a control.

Example 5 Indican Degradation in the Extracted Extract of β-Glucosidase

In order to detect the enzyme activity in the indican-containing powder extracted from the plant, the transformant was plated and cultured in a LA solid medium, and the extract extracted in dissolving in methanol was spread out to confirm that indigo of blue color was produced. To prepare the extract, the leaf was lyophilized and the ground powder was suspended in acetone (10 ml / g) for 1 hour, centrifuged and the supernatant was recovered and dried at 70 ° C. As a result, when the side extract was treated to the cultured cells as shown in [Fig. 7], it was confirmed that the indigo was produced even in the extract of the form in which several substances were mixed, as the color of indigo blue increased. As shown in the results of FIGS. 4 and 6, it was confirmed that the enzyme derived from the Sino-Rizobium melloriti strain had the highest activity. Confirmation of the reaction result of the indigo blue color was confirmed by comparing the absorbance and characteristic peak of the commercial indigo, retention time on HPLC.

Example 6 Selection of Stable Expression System of Selected Enzymes

As described above, in the case of an enzyme derived from cynorazobimeliiloti, when expressed as a single protein, 80% or more of the enzyme is generated in an insoluble state. Therefore, a stable expression system capable of solving this problem and sufficient amount of protein required for production are secured. In order to perform the following experiment. The increase in the solubility of the expressed protein in E. coli is generally dependent on the fusion label or protein used, and therefore, the histidine-tagging and MBP fusion systems are used to increase the solubility. Cloning into pQE30 (Qiagen) and pMAL-c2X expression vector (Newland Biolabs) that can be utilized was attempted. The forward nucleotide sequence of the PCR primers prepared for insertion into the pQE30 and pMALc2X plasmids was 5'-ATGGATCCATGATCGAAGCCAAGA-3 '( BamH I), and the reverse nucleotide sequence was 5'-ATAAGCTTTCATCCCGGCTTGT-3' ( Hind III).

Forward primer: 5'-ATGGATCCATGATCGAAGCCAAGA-3 '(SEQ ID NO: 17)

Reverse primer: 5'-ATAAGCTTTCATCCCGGCTTGT-3 '(SEQ ID NO: 18)

In order to confirm the expression level and solubility of the expression-induced β-glucosidase, the cells activated in LA medium were inoculated in LB medium containing ampicillin (50 µg / ml) and incubated at 37 ° C for 10 hours. 1% of the culture was re-inoculated into 10 ml (50 μg ampicillin) LB liquid medium and incubated at 37 ° C. and 180 rpm to have an absorbance of 0.5 at OD ₆₀₀ . After addition of 1 mM IPTG as an expression inducer, the expression of protein was induced at 37 ° C. for 100 minutes. The expression-induced cells were recovered using a centrifuge, washed three times with a phosphate-buffered saline (PBS) solution, and centrifuged. The recovered cells were suspended in 4 ml buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.2) and sonicated (5 sec pulse on, 10 sec pulse off, amplify by 37%). It was crushed using. After centrifugation at 12,000rpm for 30 minutes, the supernatant and the precipitate were separated, and a 2 ㅧ sample loading buffer was added to induce denaturation at 100 ° C. for 10 minutes. The prepared sample was slowly cooled to room temperature and then loaded onto a 10% polyacrylamide gel, followed by electrophoresis (20 mA, 1 hour). The developed gel was stained with a staining solution (Brillient blue R-250) for 2 hours and then detained. As a result, when expressed with a histidine-labeled as shown in [Fig. 8], the solubility was low similar to the case of expressing with a single protein. These properties were observed to be maintained even in the experiments with varying expression and culture conditions. On the contrary, in the case of the MBP fusion protein, more than 85% was confirmed to be expressed as a soluble and active protein, confirming that it is stably expressed. In the case of the amount of protein expression, it was confirmed that overexpression of about 30% of the whole host protein.

1 is a diagram illustrating a chemical synthesis method and a bioconversion method of tryptophan into a precursor and a bioconversion method of an aromatic compound into a precursor using a conventional indigo dye production method.

Figure 2 is a diagram showing the phylogenetic tree of the discovered β-glucosidase gene.

Figure 3 is an SDS-PAGE showing the expression of β-glucosidase in E. coli XL1-blue transformed with a gene having activity against indican.

(1: control, 2: Flavobacterium johnsonie, 3: Summers Thermophilus HB8, 4: cynorazobilium melototti, 5: Summers Caldophyllus; P: insoluble fraction, S: soluble fraction)

Figure 4 shows the results of the enzyme β-glucosidase enzymes are shown in solid medium and unmodified gel.

(a-1: control, 2: Flavobacterium johnsonie, 3: Summers Thermophilus HB8, 4: cynorazobilium melototti, 5: Summers Caldophilus;

b-1: Summers Caldophilus, 2: Sinorazobib melloritti, 3: Summers Thermophilus HB8, 4: Flavobacterium Johnsonier, 5: Control)

Figure 5 is a figure confirming the activity of X-gal of β-glucosidase enzyme derived from the Sinorazobi meliloti strain in LA medium.

(1: recombinant strain containing β-glucosidase enzyme, 2: control with an empty vector, 3: control with an enzyme active on X-gal)

Figure 6 is a figure confirming the activity of the β-glucosidase enzymes on indikanes in M9 minimal medium (a) and LA medium (b).

(1: Control, 2: Flavobacterium Johnsonie, 3: Summers Thermophilus HB8, 4: Sinoraizolium melototti, 5: Summers Caldophyllus)

Figure 7 is a diagram showing the results of the activity confirmed by the extract extract for the practical use of the selected β-glucosidase.

(1: Control, 2: Flavobacterium Johnsonie, 3: Summers Thermophilus HB8, 4: Sinoraizolium melototti, 5: Summers Caldophyllus)

8 is a diagram showing the amount of expression of the fusion protein soluble induced overexpression of the selected β-glucosidase.

(1: His tag fusion protein, 2: MBP fusion protein; P: insoluble fraction, S: soluble fraction)

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Recombinant microorganisms harboring a B-glucosidase and their          use for the production of indigo dyes <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 1272 <212> DNA <213> Thermus caldophilus <400> 1 atgaccgaga acgccgaaaa attcctttgg ggagtggcca ccagcgccta ccagattgag 60 ggggccaccc aggaggacgg ccgggggcct tccatctggg acgccttcgc ccagcgcccg 120 ggggccatcc gggacgggag cacaggggag cccgcctgcg accactaccg ccgctacgag 180 gaggacatcg ccctgatgca atccctcggg gtgggggcct accgcttctc cgtggcctgg 240 ccccggatcc tccccgaggg ccgggggcgg atcaacccca agggcctcgc cttctacgac 300 cgcctggtgg accggcttct cgcttccggg atcacgccct ttctcaccct ctaccactgg 360 gacctgcctt tggcccagga ggagcgggga ggctggcgga gccgggagac cgccttcgcc 420 ttcgccgagt acgccgaggc ggtggcccgg gccctcgccg accggttgcc cttcttcgcc 480 accctgaacg agccctggtg ctcggccttc ctcgggcact ggacggggga acacgccccc 540 ggcctcagga acctggaagc ggccctccgc gccgcccacc acctcctcct gggccacggc 600 ctcgccgtgg aggccttgag ggccgcgggg gcgaagcggg tggggatcgt cctcaacttc 660 gccccgcgct acggcgagga ccccgaggcg gtggacgtgg ccgaccgcta ccacaaccgc 720 ttcttcctgg accccatcct gggcaagggg tatcccgaaa gccccttccg agaccccccg 780 cccgtcccca tcctctcccg cgacctggag ctcgtggcaa ggcccctgga cttcctgggg 840 gtgaactact acgcccccgt ccgcgtggcc ccggggacgg ggaccttgcc cgtgcgctac 900 cttcccccgg aagggccggc cacggccatg gggtgggagg tctaccccga ggggctttac 960 cacctcttga agcgcctcgg ccgggaggtg ccctggcccc tttacgtcac ggaaaacggg 1020 gccgcctacc ccgacctctg gacgggagag gccgtggtgg aggaccccga gcgggtggcc 1080 tacctcgagg cccacgtgga gggccgcctc cgggcccggg aagaaggggt ggacctccgg 1140 ggctacttcg tctggagcct catggacaac tttgagtggg cgttcggcta cacccggcgc 1200 ttcggcctct actacgtgga cttccccagc cagaggcgca tccccaaaag gagcgccctc 1260 tggtaccaat ga 1272 <210> 2 <211> 1296 <212> DNA <213> Thermus thermophilus <400> 2 atgaccgaga acgccgaaaa attcctttgg ggagtggcca ccagcgccta ccagattgag 60 ggggccaccc aggaggacgg ccgggggcct tccatctggg acgccttcgc ccagcgcccc 120 ggggccatcc gggacgggag cacaggggag cccgcctgcg accactaccg ccgctacgag 180 gaggacatcg ccctgatgca atccctcggg gtgcgggcct accgcttctc cgtggcctgg 240 ccccggatcc tccccgaggg ccgggggcgg atcaacccca agggcctcgc cttctacgac 300 cgcctggtgg accggcttct cgcttccggg atcacgccct ttctcaccct ctaccactgg 360 gacctgcctt tggccctgga ggagcgggga ggctggcgga gccgggagac cgccttcgcc 420 ttcgccgagt acgccgaggc ggtggcccgg gccctcgccg accgggtgcc cttcttcgcc 480 accctgaacg agccctggtg ctcggccttc ctcgggcact ggacggggga acacgccccc 540 ggcctcagga acctggaagc ggccctccgc gccgcccacc acctcctcct gggccacggc 600 ctcgccgtgg aggccttgag ggccgcgggg gcgaggcggg tggggatcgt cctcaacttc 660 gccccggcct acggcgagga ccccgaggcg gtggacgtgg ccgaccgcta ccacaaccgc 720 ttcttcctgg accccatcct gggcaagggg tatcccgaaa gccccttccg agaccccccg 780 cccgtcccca tcctctcccg cgacctggag ctcgtggcaa ggcccctgga cttcctgggg 840 gtgaactact acgcccccgt ccgcgtggcc ccggggacgg ggaccttgcc cgtgcgctac 900 cttcccccgg aagggccggc cacggccatg gggtgggagg tctaccccga ggggctttac 960 cacctcttga agcgcctcgg ccgggaggtg ccctggcccc tttacgtcac ggaaaacggg 1020 gccgcctacc ccgatctctg gacgggagag gccgtggtgg aggaccccga gcgggtggcc 1080 tacctcgagg cccacgtgga ggccgccctc cgggcccggg aagaaggggt ggacctccgg 1140 ggctacttcg tctggagcct catggacaac tttgagtggg ccttcggcta cacccggcgc 1200 ttcggcctct actacgtgga cttccccagc cagaggcgca tccccaaaag gagcgccctc 1260 tggtaccggg agcggatcgc gcgggcccag acctaa 1296 <210> 3 <211> 1296 <212> DNA <213> Thermus sp. <400> 3 atgaccgaga acgccgaaaa gtttctgtgg ggggtagcca ccagcgccta ccagattgag 60 ggggccaccc aggaggacgg gcgggggcct tccatctggg acaccttcgc ccgccgcccc 120 ggggccatcc gggacggaag cacaggggag cccgcctgcg accactacca ccgctacgag 180 gaggacatcg cccttatgca atccctcggg gtgggggtct atcgcttctc cgtggcctgg 240 ccccggatcc tccccgaggg ccgggggcgg atcaacccca agggcctcgc cttttacgac 300 cgcctggtgg accggcttct cgcggcgggg atcacgccct tcctcaccct ctaccactgg 360 gacctgcccc aggccctcga ggaccggggc ggctggcgga gccgggagac cgccttcgcc 420 ttcgccgagt acgccgaggc ggtggcccgg accctcgccg accgggtgcc cttcttcgcc 480 accctgaacg agccctggtg ctcggccttc ctcgggcact ggacggggga acacgccccc 540 ggcctcagga acctggaggc ggcccttcgc gccgcccacc acctcctcct ggggcacggc 600 ctcgccgtgg aggccttgag ggccgcgggg gcgaagcggg tggggatcgt cctcaacttc 660 gccccggcct acggcgagga ccccgaggcg gtggacgtgg ccgaccgcta ccacaaccgc 720 tacttcctgg accccatcct gggcaggggg tatcccgaaa gcccctttca agaccccccg 780 cccactccca tcctctcccg tgacctggag ctcgtcgcaa ggcccctgga cttcctagga 840 gtgaactact acgcccccgt ccgcgtggcc ccggggacgg ggcctttgcc cgtgcgctac 900 cttcccccgg aggggccggt cacggccatg gggtgggagg tctaccccga ggggctttac 960 cacctcttga agcgcctcgg ccgggaggtg ccctggcccc tttacatcac ggaaaacggg 1020 gccgcctacc ccgacctctg gacgggagag gccgtcgtgg aggaccccga gcgggtggcc 1080 tacctcgagg cccacgtgga ggccgccctc cgggcccggg aagaaggggt ggacctcagg 1140 ggctacttcg tctggagcct catggacaac tttgagtggg ccttcggcta cacccggcgc 1200 ttcggcctct actacgtgga cttccccagc cagaggcgca tccccaaaag gagcgccctc 1260 tggtaccggg agcggatcgc gcgggcccag ctctga 1296 <210> 4 <211> 1380 <212> DNA <213> Agrobacterium tumefaciens <400> 4 atgaccgatc cccaaacgct tgcagcccgt ttccccggtg atttcctgtt cggcgtcgcg 60 accgcctcgt tccagattga gggtgcaacc aaggcagatg gccgcaaacc ctccatctgg 120 gatgccttct gcaacatgcc aggccatgtc ttcgggcgtc acaatggcga tatcgcctgc 180 gatcattaca atcgctggga agaggacctc gatctcatca aggagatggg tgtcgaggcc 240 tatcgtttct ccatcgcctg gccgcgcatc attcccgacg gtttcggacc gatcaacgaa 300 aagggtctgg atttttacga ccgactggtt gatggctgca aggcgcgcgg tatcaagacc 360 tatgcgacgc tttaccattg ggatctgccg ctgaccctga tgggcgacgg cggctgggcc 420 tcgcgctcca ccgcccatgc tttccagcgt tacgccaaaa ccgtcatggc ccgtctgggc 480 gaccggctgg atgcggtggc gaccttcaac gaaccctggt gcgcggtgtg gctcagccat 540 ctctacggca tccatgcgcc gggcgagcgc aacatggagg cggccttagc ggccatgcac 600 cacatcaatc tcgcccatgg tttcggcgtc gaggcgtctc gccatgtagc gccaaaagtg 660 ccggtggggc tggtgctgaa cgcccattcc gtcattccgg cttctaacag cgaggccgat 720 ctcaaggctg ccgaacgggc gttccagttc cacaatggcg cgtttttcga tccggtcttc 780 aagggcgaat atccggccga gatgatggaa gcgcttggtg atcgcatgcc agtcgtggag 840 gcggaagacc tcgccatcat cagccagaag ctcgactggt ggggcctgaa ttattatacg 900 ccgatgcgcg ttgccgacga cgcgacgccg ggtgctgaat tccccgccac gacgccggct 960 ccggcggtca gcgaggtgaa gaccgatatc ggctgggagg tctacgcccc ggcgctcaag 1020 tcgctggtgg agacactcta caagcgttac gacctgccgg aatgctacat taccgaaaac 1080 ggcgcctgct ataatatggg catcgaaaac ggcgaggtga atgaccagcc gcggctcgat 1140 tattacgccg aacatctcgg catcgtcgcc gatctcatcc gcgacggtta cccgatgcgc 1200 ggatatttcg cctggagcct gatggataat ttcgaatggg cagagggcta tcgcatgcgc 1260 ttcggcctcg tgcatgtgga ttatgacacg caggtgcgta cgctgaagaa cagcgggaag 1320 tggtatagtg ctttggcttc gcgttttccg aaggggaacc atggtgtggt gaaggggtga 1380                                                                         1380 <210> 5 <211> 1353 <212> DNA <213> Bradyrhizobium japonicum <400> 5 atgaataaaa ttgaaaactc atttttgaac aaaaaccaat ttggggaaga tttcttgtgg 60 ggtgtttcta ccgctgcttt ccaaattgaa ggagcacatg attctgatgg aaaaggttct 120 tctatttggg acgtttttac ttctcaaaaa ggaaaaataa aaaacggaca tcatgccctg 180 actgcctgcg atttctataa ctcgtaccaa aatgatatag acttgattcg ggaattgaat 240 attccaaatt ttaggttttc tataagctgg ccccgaatta tgccaactgg agttcatccg 300 gtaaatcagg caggaattga ttattacaat aaaattatag actctctgct ggcatctgga 360 atcgaaccct ggataacgct ttaccactgg gatttaccgc acgctctaga agtaaaagga 420 ggctggacta accgtgaatc ggttaactgg ttttcagaat atgtagaagt ttgtgcgcag 480 tattttggtg atcgtgtcaa aaactggatg gtaatcaatg aaccatctgt ttttaccgga 540 gctggttatt tcttaggaat tcacgctcct ggaaaaaaag gaatcacaaa ttatctaaaa 600 gccatgcatc acgttacttt agcgacagct gccggcgcca ggatattacg aaacaaggtt 660 ccagaagcaa acataggaac gactttttca tgtacgcata ttgaagcggc aacagaaagc 720 gcgaaagatg ttgaagccgc aaaacgtgtc gatactttac tcaacagaac atttatcgaa 780 cctattttag gattaggata tccgcaaaaa gatcttccgg tacttaaaaa actcaacaat 840 tatattttag aagacgattt aaacaatctc gatttcgact ttgattttat tggattacag 900 tgttataccc gcgaagttgt aaaatcttct atccttactc cctacattgg tgccgaatta 960 gtgagtgccg aaaaaaggaa tgtaatttct accgaaatgg gctgggaagt atatcctccg 1020 gcattgtacc atgttctgga gaaattcaat aaatacgata aaatcaaaaa aatcattatt 1080 accgaaaacg gcgcggcttt tccagatacg gttacaaacg gaaaagtatt cgacattaaa 1140 cgaacacatt atattcagga ccatttagaa caaatcttaa aggccaaaaa aaatggtcta 1200 aatgtagaag ggtattttgt gtggagttta actgataact tcgaatgggc cgaaggctac 1260 aacgctcgct tcggactaat tcatgtcgat ttcgaaactc aaaaaagaac cattaaaaac 1320 tcaggattat ggtttaaaga ttttttatct taa 1353 <210> 6 <211> 1356 <212> DNA <213> Shewanella baltica <400> 6 atgaaaatat ctttaccaaa gaactcgaga ctccaaagcg aagcgtttac ttttggtgtt 60 gcaaccgctt cctttcaaat cgaaggtggc gtggactctc gccaaacctg tatttgggat 120 accttttgtg caacaccaga taaaatccgt gatgcctcca atggcgatgt cgcctgcaac 180 cacctgaatc tatggcaaga agatatcgcc ttaatcgcgt cactcggggt ggatgcctat 240 cgtttttcca tcgcatgggg acgcgtctta aatcaagatg gcagcattaa tcagcaggga 300 gttgatttct acattggcat tctagacgaa ctaaaacgta gaaatatcaa agcatttgtc 360 acgctttacc attgggatct tcctcaacat attgaggacc aaggcggctg gttaaaccga 420 gataccgctt atcttttcaa agactatgct gacaaaataa gccaagcctt cggcgaccga 480 gtgtattcct acgccacttt aaacgaaccc ttttgcagct catatttagg ctatgagtca 540 ggcattcacg cccccggctt aatgaaaaaa gcctatggcc gccaatcggc tcaccaccta 600 ttactcgccc acggtttagc gatgcaagtg ctgcaaaaga acagccctaa cagcatgaat 660 ggcatagtgc ttaacttcac gccttgctac gcattaaccc aaagtgctgc cgatattcaa 720 gccgcaaaac aagccgatga ttactttaac cagtggtata tcaagcccat tttcgatgcg 780 gcatacccag agcttatcgc ggcattagcg ccagaagata gaccggaaat tcacgacggc 840 gaccttgagc ttatcagtca acccattgac tttttagggg tcaactttta tacccgcgcc 900 gtatatcagg ccgatgccga acatggattt gtgcaagttg atttaccagg ggtaaccaaa 960 accgacatag gctgggagat ctatcctcag gcatttaccg atttattggt ttctttagat 1020 cacatctatg atttaccgcc tattttcatc acagaaaatg gcgccgccat ggacgataaa 1080 tgtattgatg gacgtgtcga tgacttcgat aggctcagct attaccagca ccatttaacc 1140 gcagtagaca atgccatagt acaaggtgtt aacattcagg gttactttgc ctggagcttg 1200 atggataatt ttgagtgggc ggaaggctac ttaaaacgtt ttggcattgt ctatgtggat 1260 tatgccagcc aaacccgaac gataaaggcc agtggtcacg cctacagtga cttgattcgc 1320 tcaagggccc acttaaccaa taacaataat aaataa 1356 <210> 7 <211> 1335 <212> DNA <213> Flavobacterium johnsoniae <400> 7 atgccgacaa cgcaactgcc aaacaccgcc agcctgcgcc ccggtttcat ctggggcgtt 60 tccacctcca gcttccagat cgaaggggca acgaaggagg acgggcgcgg actcagcatc 120 tgggacatct attgtcggtc cggtgaaatc aaaaatcacg ataccggcga cgtcgcgtgt 180 gaccattatc atcgctaccg cgaagatgtc ggcctcatga agacgttggg cgtacaggcg 240 taccggttct cggttgcctg gccgcgcgtc ctgccgctgg gactcggatc cgccaacgag 300 gccggcgttt cattctatga ccggctgatc gacgagctcg tggccgccgg tatcgaaccg 360 tggctgtgcc tgtatcattg ggacctgccg caggccctgg aggagcgcgg cggctggctg 420 aaccgtgagt cggcggcgtg gttcgccgac tatgtgacgt tgatcgccgc gcgatttggc 480 gaccgggtca agcgctttgc gacattcaac gagccgtcga tcttcagcct gttcagccgt 540 tcgctcggca agcgggagca tagcagcgag gacaagttcc accgcatgat ccacaacgtc 600 aatctcgccc acggggcggc ggtggatgtg cttcgcgcca acgtgatcgg cgcctcgatc 660 ggctgtatcc ataatcgaca gccatgccgg ccttccagtg caagcgaggc cgatgaagcg 720 gctgcggcac ggctggatgt gtactggaac gcggcctttc cggatccgca atgccgcggc 780 gaatatccgc catcgatgcg cgcggcaatc gaaccgcata tgcagccagg cgacctgacg 840 cggatttgcc gtccgctgga ttggttcggg ctgaaccact atagcccggt ctatgtgaag 900 gcacgggcgg attcgatgct gggctacgac ttcggcgaca agcccgccag cgtccccttg 960 accccgatcg gatggccgat cgatcccgag gccttcagcg agacgttgca ggcggtccgc 1020 acacggtacg gcctgccgat ttacgtgctc gagaatggct acggcgattc cggccagccc 1080 gaccagaccg gtgcggtgat cgacccgggc cggatagagt tcctgaaggc ctatatcaac 1140 gcgatgaata acgccgcggc ccacggcgtc gatgttcgtg gctatttcgt ctggtctctg 1200 ctcgacaatt tcgagtgggc ctccggttac agcatcaggt tcggcctgac ctatgtcgat 1260 tatgcttcgc tgcggcgaat cccgaaatcg tctttcggct ggtatagccg ggctgatcag 1320 ggcggtgcag ccatg 1335 <210> 8 <211> 1377 <212> DNA <213> Shinorhizobium meliloti <400> 8 atgatgatcg aagccaagaa actcgcagcg cgctttcccg gcgactttgt cttcggcgtt 60 gcaaccgcat ccttccagat cgagggagcc agcaaggcgg atggtcgcaa agcctccatc 120 tgggacgcct tctccaatat gccggggcgc gtttacgggc gccacaacgg cgacgtcgcc 180 tgcgaccatt acaaccggct ggagcaggac ctggacctca tcaagagcct cggcgtcgag 240 gcctaccgct tctcgatcgc ctggccgcgg atcgtgccgg agggcacggg gccgatcaac 300 gagaaggggc tcgactttta cgaccgcctt gtcgacggac tgaaggcgcg cggcatcaag 360 gcctttgcga ccctctatca ctgggacctg ccgctggcgc tgatgggcga cggcggctgg 420 acggcacgca cgaccgccta tgcataccag cgttacgcca agaccgtgat cgcgcgtctg 480 ggggatcgtc tcgacgcggt cgcgaccttc aacgaaccgt ggtgttccgt ctggctcggc 540 catctctacg gtgtccatgc accgggcgag cgcaacatgg atgcagcact tgccgcactg 600 cacttcacca atctcgccca cgggttgggg gtcgcggcga tccgctcgga acggccggaa 660 ctgccggtcg gcatcgtcat caatgcccat tcggtctatc ccggcagcaa cagcgccgag 720 gacaaggccg cggcggagcg cgccttcgat ttccacaacg gcgtcttctt cgatccgatc 780 ttcaagggcg agtatccgga ggatttcctc tccgcgctcg gcgaacgcat gccggcgatc 840 gaggacggcg acatggcaac gatcgcccag ccgctcgatt ggtgggggct caactattat 900 acgccgatgc gggtttcggc agaccccgcg aaaggcgccg aatatccggc gaccgtcaat 960 gcgaaacctg tcagcaacgt gaagaccgat attggctggg aagtctatgc gccggctttg 1020 ggcagcctgg tggagacgct caatgcgcgc tacaggctcc ccgactgcta catcaccgag 1080 aatggcgcct gttacaacat gggcgtcgag aacggtaccg tcgacgatca gcctcggctc 1140 gactacatct cagaccacct cgccgtcacc gccgatctca tcgccaaggg ttatccgatg 1200 cggggctatt tcgcctggag cctgatggat aacttcgaat gggccgaggg ctaccggatg 1260 cgcttcggca tcgttcacgt cgattacgag acacaggtcc gcacgatcaa gaaaagcggt 1320 cgctggtata aggacctggc ggaacggttt ccaagcggca accacaagcc gggatga 1377 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tagaattcaa cgccgaaaag ttt 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 taaagctttc actctggctg ggg 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tagaattcaa cgccgaaaaa ttc 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 taaagctttt aggtctgggc ccg 23 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gcccatgggt aaaattgaaa actcattt 28 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gcaagctttt aagataaaaa atctttaaa 29 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 atccatggtg atcgaagcca aga 23 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ataagctttc atcccggctt gt 22 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 atggatccat gatcgaagcc aaga 24 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ataagctttc atcccggctt gt 22  

Claims (11)

A gene encoding β-glucosidase consisting of the nucleotide sequence of SEQ ID NO: 8. delete Recombinant plasmid comprising the gene of claim 1. The method of claim 3, wherein The recombinant plasmid is a recombinant plasmid characterized in that it is expressed as a Maltose binding protein (MBP) fusion protein. Transgenic microorganism E. coli XL1-BLUE [pTrc99A-glu (SM)], which produces β-glucosidase having the activity of producing indigo dyes from a plant or extract thereof (Accession No. KCTC 11236BP) ). delete A composition for producing an indigo dye from a plant or extract thereof comprising β-glucosidase encoded by a gene consisting of the nucleotide sequence of SEQ ID NO: 8. delete The method of claim 7, wherein The composition is a composition having activity on X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside). Β-glucosidase encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 8, or transformed microorganism E. coli XL1-BLUE [pTrc99A-glu (SM)] (Accession No. KCTC 11236BP) A method of producing an indigo dye, comprising converting indican to an indigo dye in addition to a plant or extract thereof. delete

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