KR101171434B1 - Mining and improvement of isatin-generating enzyme, and their combined use with ß-glucosidase for the production of natural indirubin - Google Patents

Abstract

The present invention relates to a method for producing indirubin, which is used as a dye, a pharmaceutical raw material, and a physiologically active substance by acting on an indican contained in a natural plant, beta-glucosidase (β- The present invention provides a method for producing a large amount of indirubin by transforming microorganisms including genes encoding glucosidase and isatin generating enzyme.

More specifically, the present invention relates to the discovery and improvement of the oxygenase that exhibits selective oxidative activity at the C-2 position of the indoksil during the preparation of indirubin, registered in GenBank by bioinformatics The functional oxygena selected by searching for and extracting the relevant gene from the sequence and inserting the transformed recombinant strain into the LB solid medium containing ampicillin and indican to obtain a reddish clone. It is to provide an agent and a preparation method thereof.

In addition, after transforming the recombinant gene produced by random mutagenesis to increase the activity of the discovered oxygenase to a recombinant strain containing glucosidase, a clone containing a gene with an increased indyrubin-producing ability compared to the control was obtained. It is to provide a method for producing indirubin using the improved genetic resources as a process.

 Oxygenase, Glucosidase, Indican, Indoksil, Isatan, Indirubin

Description

Mining and improvement of isatin-generating enzyme, and their combined use with β-glucosidase for the production of natural indirubin}

The present invention relates to a method for producing indirubin, which is used as a raw material for dyes and pharmaceuticals by acting on indicans contained in the side plants, and β-glucosidase in the side plants containing indicans. The present invention relates to a method for producing indirubin by adding a new enzyme together.

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 produced 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. In addition to its function as a dyeing material, the range of application as a food additive or a physiologically active material as an eco-friendly material is gradually increasing. Indirubin, which is a kind of natural products, is known to be produced from indican, which is a plant material, as a natural substance with red color. Indican is an aromatic compound known to have high amounts in Polygonum tinctorium plants, a precursor to indigo that has been used in cosmetics and paints since ancient Roman times, and is currently the main plant material used for dyeing clothing and materials. Indigo-based dyes have been used mainly in the form of natural extracts depending on traditional methods, but there are problems such as non-uniformity in the production process, differences in the dye composition of the plant material, and non-standardization of the fermentation-maturation process. Is mainly used. 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 actual dyeing. Recently, many studies are being actively carried out to produce indigo by the standardized biotechnological method.

Indirubin is a reddish compound of the indigoid compound family, which has been reported to be used as a drug with various effects in ancient Chinese pharmacopoeia, and is a unique plant-derived natural pigment that can be used as a substitute dye. Or dye. In addition to cheonsa dye materials, alternative drugs are used to treat leukemia, have an anti-inflammatory effect, and are very useful as psoriasis and skin rash treatments. However, there is little known information about a clear synthetic pathway or related genes or proteins in vivo, and now only a by-product amount is available. Incidentally, there have been studies on the content of indigo, the indigo precursor and isatitin necessary for the production of indirubin, and indigo produced by the plant's own enzyme. There are no studies on mass-producible enzymes that are commonly used for biotransformation.

As a result, there is no known technique for producing selective indirubin, and indigo production process using biotechnological methods, ie, indole is produced from tryptophanase, an amino acid, followed by addition of dioxygenase. The production route of indirubin produced at low concentrations during the production of sea indigo is known (see FIG. 1). Indirubin produced as a by-product besides the desired indigo is made by istanane, which is synthesized by the oxidation reaction of C-2 and C-3 simultaneously by deoxygenase, but the oxidation specificity of the enzyme is It was reported to be high and mass production was very difficult because it was rarely reacted in two places at the same time. As a means of overcoming this, several oxidase-based enzyme modifications have been attempted, but no significant increase in production capacity has been reported.

The present invention has been found from previous studies on indican-containing plants compared to the existing process of converting indole into indoxyl or isatin to produce indirubin with high industrial value. By adding β-glucosidase to generate indokyl from indicane and introducing oxygenase with selective oxidation position of indylyl to form isatin, consequently the specific mass production of indirubin can be achieved. The invention was completed.

Accordingly, it is an object of the present invention to provide a method and enzyme for the discovery and improvement of an oxygenase that can selectively induce oxidation in a specific part of the indoctrin, thereby enabling the specific mass production of indirubin.

The present invention also provides a new enzyme process that can specifically produce large quantities of natural indirubin from a plant or extract thereof, ie, a method of combining betaglucosidase and oxygenase.

For the specific mass production of indirubin of the present invention, the reaction characteristics of existing oxidase or oxidoreductase are analyzed to target oxygenases that are selective at the C-2 position of indole or oxidatively induced at the C-3 position. Discovering or improving enzymes capable of producing isatin in the reading room. Therefore, if the C-3 site is oxidized in the reading room or the enzyme that can induce additional oxidation only in the C-2 position was determined that the isatin was able to induce the desired reaction. Eventually, a beta-glucosidase is formed from the indyl acid from the indica, and the selected or produced enzyme issatin is produced from the indose room, thereby enabling the production of indirubin (indirubin) in large quantities. It is possible.

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.

After the production of competent cells with glucosidase using the principle of use as a sole carbon source of D-glucose desorbed from indican as a result of beta-glucosidase activity for the implementation of the present invention, In order to confirm the ability to produce indirubin, a cloned oxygenase gene source was transformed, cultured in a minimal medium, and a strategy of selecting red clones with growth was used [see FIG. 2].

First of all, we attempted a systematic discovery of oxygenase, which can induce oxidation in a specific part of the reading room. The evolutionary tree was analyzed by collecting all oxygenase gene sequences and amino acid sequences in the GeneBank and PDB databases. 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. Particular considerations included sub-aguasses as the main search resources, including those previously reported to be active on indole or structurally similar substrates. As a result, it was confirmed that the enzyme source encoded by a gene derived from Rhodococcus spp. Can be selectively oxidized to the C-2 position of the reading room. All the amino acid sequences of sub-Aguas classified by this process were identified using the Clustal X program and then searched using the BLAST-P program in GenBank. (putative enzyme) but could be obtained a new group of enzymes that have not been reported activity in the laboratory.

In the present invention, when analyzing the characteristics of the oxygenase identified through the systematic excavation process, most of the excavated phosphorylated oxygenase was found in soil microorganisms (Rhodococcus and streptomyces) known to have a general aromatic compound resolution, and enzymes There is little known information about the characteristics. The inventors found that the selected oxygenases evolved from a common ancestor and showed a general characteristic of being active against the same substrate, with similar sized amino acids (393-404) and more than 35% high sequence similarity. 1 to 8 (SEQ ID NOS: 1 to 4 are nucleotide sequences, and SEQ ID NOs: 5 to 8 are amino acid sequences).

The present invention is a plant extract as a method to solve the problem of substrate specificity and the simultaneous reaction of the double oxidation method to induce oxidation of indole C-2 and C-3, which is a problem in the existing indirubin production pathway Decomposition products of indican glucosidase, that is, microorganisms capable of selective oxidation of a part of the existing enzyme or the C-2 position which is relatively easy to oxidize by using an indoksil that has already been oxidized to C-3 as an intermediate. Oxygenase subfamily was used. By incorporating glucosidase patented in our laboratory, we have completed the discovery of an enzyme with excellent activity capable of producing indirubin from indican in a single or continuous process.

In the case of the oxygenase discovered by the present inventors, the production of istane directly in the reading room can lead to the effective production of indrubin, which is a combination of the reading room and the istane, in a single process. However, since the substrate stability of the reaction chamber, which is a reaction substrate, is low and is spontaneously oxidized upon contact with air, it is difficult to apply it directly to the production process even if the organic synthesis of the economic chamber is possible. Therefore, in order to develop an economical and environmentally friendly process, a method for converting the glucosidase and the oxygenase of the present invention into a single or continuous process, which has been previously extracted in the indican obtained in the form of a plant or fractionated components and extracts It was estimated. In order to confirm this possibility, it was confirmed whether the production of indirubin was possible by adding two enzymes to the powder extracted with acetone and the plant itself as a natural hazard material. As a result, indirubin was found to be difficult or insignificant in the physiologically crushed or dried side, but the red product was easily identified in the solvent-extracted powder.

The process of preparing the two enzyme sources of the above-described process where reactivity was detected and indirubin was detected by using an enzyme expressed by leaking in E. coli without artificial manipulation or by cloning each of them in a constant expression vector or single The vector was prepared by co-expressing two enzymes. In the latter case, it was found that both the process of attaching a promoter to each of the two enzyme sources or cloning in series behind one promoter, such as the polycistronic expression principle, was possible. In this case, it is very important to use a transcriptional coupling to place the ribosome binding site (RBS) in front of each ORF so that translation can proceed separately. In another process, by inserting genes of two enzyme sources into a non-essential for cell viability, it was found that eliminating the use of vectors or antibiotics was advantageous for the production of pharmaceutical products.

In order to facilitate the purification and purification of pure water for the characterization of enzymes, and to mass production advantageously for field application, a stable expression system using a general fusion protein (MBP; glutathione-S-transferase; His-tag, etc.) Tried to build. However, unlike glucosidase, which is an effective expression induced in the fusion of MBP or his-tag, it was confirmed that the effect of oxygenase was insignificant or not increased. This was judged to be due to the properties of known similar oxygenases being inserted or interacted with the cell membrane. Since the production capacity of indirubin is dependent on oxygenase, which has a relatively low activity compared to glucosidase, which produces indokyl in indica, it is a process to express the two enzyme sources in their own sequence without fusion protein or tagging. It was finally confirmed as favorable to the phase.

As a result of inducing random mutations to improve the properties of oxygenase, which is a rate-limiting step of overall production capacity, and confirming whether the production capacity of indirubin was increased, the mutant enzyme source having one or two amino acid sequences changed was better. It was investigated. On the basis of the easy creation of a mutant enzyme prepared by a relatively simple process, the enzyme source selected in the present invention can be improved to an enzyme having better characteristics using protein engineering techniques or a cellulase which can be used for pretreatment of plants. If additional enzyme sources are considered, it is possible to adjust the substrate pretreatment time, enzyme titer, and simplify the process, thus enabling efficient biological production of natural pigments with a wide range of applications in the pharmaceutical field. It is thought that the health problems can be minimized.

The present invention, as described above, by using beta-glucosidase and oxygenase subfamily to confirm that the production of indirubin from indicans by using the selected enzymes in the production of indirubin in the plant itself, powder or extract Show that it is possible. Therefore, it is expected to be a new manufacturing process that can be used in various fields such as the production of natural pigments that can be used as medicines or cosmetics, paints, eco-friendly wallpaper or clothing by using plants. In addition, the manufacturing process is eco-friendly and natural materials can be used to develop new functional medicines that can be directly applied to the human body. Incidentally, the advantages of pure recombination enzyme process can easily overcome the problems in terms of cost, reproducibility and uniformity, thereby improving or replacing the complex production process and unstable product quality using the existing indole.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

Example 1 Screening for Oxygenase Gene Sources Presumed to Have Activity in the Indoctrin

Generally, it is known that indigo is mainly produced in indican-containing plants and there is no indirubin production or only a very small level by nonspecific oxidation process. Therefore, although the characteristics of plant-derived glucosidase related to indigo production are described in part, there are no reported results regarding enzyme sources related to the site-specific oxidation of the indole chamber. Therefore, we attempted to search for highly probable enzyme sources that would have activity in the doxo room by analyzing oxygenase, substrate specificity, and reaction degree mentioned in the existing indole process.

To date, many methods have been reported to produce indigoid pigments using deoxygenase enzymes as indole substrates. Oxygenase is known to be able to oxidize various substrates according to the change in substrate specificity induced by mutations, although the substrates that react with enzymes that oxidize the substrates specifically using oxygen are known. Previous studies have shown that wild-type enzymes and enzymes produced through mutation-inducing processes can produce indole, isatin, oxyindole, and deoxyindole by oxidizing C-2 and C-3 positions using indole as a substrate. It is known that the production of various indigoid pigments, including indigo and indirubin, is possible in principle. As such enzymes, toluene ortho-monooxygenases of Burkholderia cepacia G4 and Pseudomonas mendocina KR1 have been reported. In addition, enzyme sources called chloroperoxidase and indole oxygenase have been reported to be active. In addition, attempts are being made to produce indirubin through cytochrome P450 (CYP2W1, CYP2A6) derived from human cells. All of the above mentioned enzymes have the potential to be used for the production of indirubin in that they can oxidize indole and change the oxidation site of indole through mutation induction (Table 1).

Table 1. Genotypes Investigated to Enable Indirubin Production

Strain gene Gene ID Protein ID Burkholderia cepacia G4 toluene-4-monooxygenase   AF349675 AAL50370.1
AAL50371.1
AAL50372.1
AAL50373.1
AAL50374.1
AAL50375.1 Pseudomonas mendocina KR1 toluene-4-monooxygenase M65106.1 AAA25999.1
AAA26000.1
AAA26001.1
AAA26002.1
AAA26003.1 Homo sapiens cytochrome P450 (CYP2W1) NM_017781 (54905) NP_060251 Homo sapiens
cytochrome P450 (CYP2A6)  AK312964 BAG35803.1 Leptoxyphium fumago Chloroperoxidase X04486.1 CAA28172.1 Rhodococcus jostii RHA1 Indole oxygenase NC_008271.1 YP_709118 Bacillus megaterium Cytochrome p450 BM-3 P14779.2 Rhodococcus sp. Pigment production hydroxylase M55641 AAA26187.1

In Table 1, the characteristics of the oxidative or oxidation-reduction enzyme family proteins, which are estimated to be oxidizable, are as follows. The gene encoding the known toluene ortho-monooxygenase ( Burkholderia cepacia G4) enzyme consists of six cistrons of tomA012345. It is a pair of 211-kDa hydroxylase (tomA1A3A4), 40-kDa NADH oxidoreductase (tomA5), and 10.4-kDa cofactor-less regulatory protein (tomA2). Structurally, there is an active site for the substrate binding site and hydroxylation reaction and requires NADH. It is also known that TOM mutant enzyme (α-subunit of TomA3) can produce indigoide-based compounds based on indole. Toluene-4-monooxygenase (T4MO, Pseudomonas mendocina KR1) is an enzyme that converts toluene to p-cresol and consists of tmoABCDEF operon. It is known that expression is induced only when toluene, phenol or structurally similar substances are present in the medium.

As a type of cytochrome P450, CYP2W1 and CYP2A6 catalyze the monooxygenase reaction on individual substrates. Oxygen molecules and NADPH (NADH) are required to oxidize the substrate. However, they are known to be hardly expressed in E. coli. Known properties have been reported to produce indigo when expressed with NADPH-P450 reductase, which can be converted to oxindole or isatin with monooxygenase activity against indole. Chloroperoxidase derived from leptoxyphium fumago produces nitroso-compounds by oxidizing the nitrogen of arylamine. When hydrogen peroxide is added to the reaction solution, oxidized indole produces more than 99% oxindole. It shows the maximum activity in acid (pH3 ~ 4). Unlike peroxidase, the active site is linked through cysteine residues, so it has similar characteristics to that of P450 series enzymes.

Rhodococcus family of indole oxygenases are known to produce indigo by oxidizing indole and to produce some indirubin in side reactions. However, because the enzyme is associated with the cell membrane (association) function, it is difficult to overexpress, the activity is reduced during purification, the exact characteristics are not known. Bacillus megaterium p450 BM-3 has two domains, 54 kDa cytochrome P450 and 64 kDa NADPH-P450 reductase. The enzyme oxidizes saturated fatty acids and unsaturated fatty acids with 12 to 20 carbons sequentially from the end. NADPH is required as a coenzyme, and it is known to be expressed in the cytoplasm of E. coli. It is expressed in a soluble state and is studied on various substrate specificities. A type of mutant enzyme is known to produce indirubin. However, the low conversion rate of the substrate has the disadvantage that a large amount of protein is required. Pigment production hydroxylase of Rhodococcus sp . (ATCC 21145) is a small 42 kDa protein that has the function of Acyl-CoA dehydrogenase. E. coli is known to produce indigo and red pigments, but little research has been done on the properties of proteins. These materials are very similar to the absorbances of indigo and indirubin (552 nm, 603 nm) (560 nm, 610 nm), and are known to be insoluble in water, ethanol, and methanol, and in low proportions in chloroform.

Strains and cell lines containing the aforementioned preselected enzyme source were distributed in various distribution organs (KCTC, KCCM, ATCC, etc.), and the activity was measured by culturing the cells in the optimal medium specified in each organ. To this end, recombinant Escherichia coli containing pre-explored beta-glucosidase was cultured to disrupt cells, and then mixed with the disruption solution of the cell line with oxygenase at neutral pH and indican was added as a substrate. The activity was measured under various reaction conditions and reducing power, resulting in oxidative induction of T4MO, CYP2W1, CYP2A6, Rhodococcus- derived indole oxygenase and BM-3 family enzymes, and the specific position of the doxoroom in Pigment production hydroxylase. It was investigated.

Example 2 Cloning and Activity Comparison of Candidate Genes with Activity in the Reading Room

Each strain used in the experiment to amplify oxidative or redox-related enzyme genes is shown in Table 1. Toluene ortho-monooxygenase, CYP2W1, CYP2A6, 2E1, 2C19, indole oxygenase, cytochrome P450 BM-3, chloroperoxidase, non-heme chloroperoxidase, and pigment production hydroxylase were used as genes for cloning and activity measurement. Strains were used in the laboratory-owned strains and pre-institutions (KCTC, KACC, ATCC). E. coli XL1-Blue (recA1 endA1 gyrA46 thi-1 hsdR17 supE44 relA1 lac F '[proAB laclqZM15 Tn10 (Tetr) c]) was used as a cloning and expression host for the genes to be selected. Protein expression for activity measurement was used pTrc99A (Pharmacia) plasmid from which the lacI gene was removed to induce overexpression without an inducer. Isolation of chromosomes, which are the template for PCR amplification, was performed using whole cells secured using the medium specified in the LB medium and strain incubator, and transgenic strains were 25-37 ° C and LB added with ampicillin (100 μg / mL). The culture was carried out using the medium.

Genes cloned into pTrc99A plasmid from each strain were amplified by PCR using primers prepared on the basis of sequences registered in GenBank. Pfu polymerase was used for the PCR reaction, and the total amount was adjusted to 25 μl by adding 20 ng of template DNA, and then typical conditions were used. The prepared gene and pTrc99A plasmid were digested with restriction enzymes Nco I and Hind III and electrophoresed on 0.8% agarose gel and recovered using Wizard SV gel & PCR clean-up system (Promega). Ligation was performed at 4 ° C. for 12 hours after mixing the plasmid and the amplified gene.

E. coli XL1-Blue competent cells used for transformation were prepared by inoculating SOB containing 20 mM MgCl ₂ and incubating at 37 ° C. for 3 hours, followed by treatment with TB solution containing 20 mM hexaminecobalt chloride. The prepared cells were used while stored at -80 ℃. Transformation was performed by mixing each recombinant DNA with competent cells according to the general thermal shock method, and then standing on ice for 30 minutes, then thermal shock at 42 ° C. for 90 seconds, and leaving it on ice for 2 minutes. After transformation, the cells were plated on LB solid medium to induce growth within 15-20 hours, and colony cracking was used to obtain clones into which the desired gene was inserted.

All the genes prepared by the above process were transformed into E. coli competent cells prepared with beta-glucosidase, and then observed on red medium indirubin was plated on minimal medium containing indican. In parallel, incubation of cells with beta-glucosidase and hosts with respective oxygenases in order to confirm the ability of indirubin to produce higher sensitivity, followed by reaction solution (1-2% triton X-100 and 2- Buffer solution containing 5 mM indican) was pretreated at room temperature for 15 minutes to induce a reaction. In the case of liquid reactions, the products and substrates in the reaction solution were analyzed by absorbance or by high performance liquid chromatography (HPLC). As a result, similar to the results of wild-type whole cell strains, the concentrations of T4MO and CYP2W1, indole oxygenase and BM-3 enzymes, and pigment production hydroxylase showed a clear but distinct indierubin production. However, after the experiment to the expression level, the variation of the activity of the protein and significant oxidative present in all but the Rhodococcus origin indole oxygenase was performed on the indole oxygenase.

Example 3 Discovery of Gene Group with High Activity in Human Reading Room

On the basis of the oxygenase sequence derived from Rhodococcus described above, an additional discovery of oxygenase, which can induce oxidation in a specific part of the human dorsal chamber, was systematically attempted. After collecting resources with oxygenase-like sequences found for all oxygenase gene sequences and amino acid sequences in the GeneBank and PDB databases, paraloges in strains such as those with very high amino acid sequences ), But the orthologue, but the evolutionary relationship is very close, and the schematic was reconstructed. Afterwards, the conservative sequence of the finally selected genes was analyzed in consideration of the evolutionary distance of each gene sequence, the substrate specificity of the known enzyme, and the substrate spectrum. At this time, Clustal X program which is generally used was used for analysis of conservation sequence. A hypothetical ORF sequence consisting of the inferred conserved sequence and the preferred amino acid sequence was searched using the BLAST-P program in GenBank. The new enzyme group was not able to be secured [FIG. 3].

In the present invention, when analyzing the characteristics of the oxygenases identified through the systematic excavation process, it is confirmed that most of the excavated phosphorylated oxygenases are present in soil microorganisms ( Rhodococcus and streptomyces ) known to have general aromatic compound degradability. There is no known information on the enzyme properties. As a result of confirming the selective oxidation at the C2 position of the indoctrin by the enzyme source encoded by the gene for these resources, it was confirmed that there was a distinct activity in all cases. We show that the selected oxygenases evolved from a common ancestor and showed 35% or higher sequence similarity with amino acids (393 to 404) of similar size to show that it is a common feature to be active against the same substrate. It is shown by the nucleotide sequence (SEQ ID NO: 1-4) and amino acid sequence (SEQ ID NO: 5-8) represented by 1 to 8 [Fig. 3].

Example 4 Preparation and Selection of Mutagen Library for Improvement of Indole Oxygenase

In the case of the selected indole oxygenase (GenBank ID; NC_008271.1), the conversion yield of the initially added indican was found to be less than <10%, despite showing distinct activity. Most of the enzymes found in the subfamily showed similar or low yields, suggesting the need for increased yields due to process improvements or the improvement of the protein itself. To confirm this possibility, the library was constructed by inducing random mutations in wild-type or mixed genes to be excavated. In general, tag polymerase has a low calibration function and can easily increase mutagenesis rate by adding different amounts of dNTP and changing concentrations of MnCl ₂ and MgCl ₂ . The PCR reaction was performed by adjusting the total amount to 25 μl using 1-5 mM MnCl ₂ , 5-20 mM MgCl _2, 0.2-1 mM dNTP using tag polymerase. Random mutant PCR was performed by 5 minutes denaturation at 95 ° C, 1 minute denaturation at 95 ° C, annealing at 55 ° C for 1 minute, and extension / 35cycle at 1-3 ° C for 1-3 minutes. The amplified genes were electrophoresed on 0.8% agarose gel and recovered using Wizard SV Gel and PCR Clean-up system. Ligation was performed at 4 ° C. for 12 hours after mixing the vector and the amplified mutant gene pool. Each recombinant DNA was transformed into competent cells and plated in LB solid medium containing 1 mM indole (proprietary substrate of wild type enzyme).

After 16 hours of incubation, colonies of different color from wild-type enzyme were selected first. When colonies were cultured in an indole-containing medium, they were found to have a smaller size than the normal medium, which could affect the growth of bacteria. In the results of other researchers, the use of indole as a substrate is known to be limited by the toxicity of the substance. In addition, indole does not dissolve well in water, so there is a disadvantage in that it must be dissolved using an organic solvent, and there is a limit in the amount of added substrate. As a result, it was found that the previous study had an effect on the production of the desired indirubin or the discovery of protein, and as a result, it may be detrimental to the discovery or production of an excellent enzyme source.

It was predicted that the primary source of enzyme was a change in reactivity to indole, resulting in a different color than the wild-type enzyme (which may contain indirubin). We predicted that clones with different beta-glucosidase activity in the medium containing indican could be selected for clones with different production levels of indirubin due to increased activity in the doxoroom or differences in protein expression. . On the basis of this, LB medium containing indican was transformed into a host having a glucosidase cloned with a tac promoter in the pACYC184 vector, and the clones having a different color from the control group (glucosidase + wild type oxidase) were secondarily selected. Finally, the resultant was treated with a buffer reaction solution containing indican to finally screen for red light-like strains such as indirubin. In this case, when only beta-glucosidase was added to the reaction solution containing indican as a control, only indigo blue of indigo blue was produced. When indican was treated only by mutagens, no change in color was observed. As a result of confirming the amino acid sequence (SEQ ID NOS: 9 and 10) of the two selected mutants, it was confirmed that one (OM1) and two (OM2) mutations were induced, respectively [FIG. 4]. They have superior activity compared to wild type enzymes, and it was confirmed that about 20% of the initially added indican was converted to indirubin.

Specific clones identified during screening (OM1), wild-type enzymes, even when reacted with 1 mM indole, in addition to the production of red indirubin spectra with beta-glucosidase in the reaction solution containing indicans Interesting results have been obtained, showing a darker red color. Therefore, it was confirmed that in the case of the mutant enzyme, it is possible to oxidize the indole or the doxyl chamber at one or more positions. In the case of the newly selected enzyme source as described above, it was confirmed that an increase in substrate specificity or inactivation for indole or doxyl may be easier than that of other oxygenase subgroups. This suggests that the mutation space of a protein is large, and it is expected to be widely used in similar substrates or processes in the future.

Example 5 Indirubin Production Confirmation Using Selected Enzyme Sources

The enzyme source finally selected by the above-described process was inoculated in LB liquid medium containing ampicillin (100 µg / ml) for 12 hours incubation with ampicillin (100 µg / ml) for indirubin production and comparison with the standard sample. 1% of the culture was re-inoculated with 10 ml of LB containing ampicillin (100 µg / ml) and incubated at 37 ° C. until OD ₆₀₀ = 2. The culture solution was centrifuged at 12000 rpm for 5 minutes at 1.6 ml, and the supernatant was discarded. The remaining cells were dispersed by adding 200 µl of reaction solution (50 mM Tri-HCl buffer containing 1% Triton X-100, pH7.0). After that, the substrate was treated with the substrate as shown in Table 2 to induce the reaction.

Table 2. Composition of reaction solution for confirmation of indirubin production

No. Triton x-100 Indican Isatin Indole Glucosidase Indole oxygenase Final volume One One% 1 mM 0.1 ml 0.5 ml 2 One% 1 mM 1 mM 0.1 ml 0.5 ml 3 One% 1 mM 0.1 ml 0.1 ml 0.5 ml 4 One% 1 mM 0.1 ml 0.5 ml 5 One% 1 mM 1 mM 0.1 ml 0.5 ml 6 One% 0.1 ml 0.5 ml

As a result, when glucosidase and mutant source (OM1) were added to indican as a substrate as shown in Fig. 5a, it was confirmed that the indirubin estimator produced a distinct reddish color compared to the control group. This result was similar to the color of the reaction solution in which indicane and glucosidase, which were used as indirubin production control, were directly added to the product (Isatin) instead of the mutant enzyme. It was found that isatin was produced in the human chamber by the mutant enzyme efficiently. The development of the reactants was attempted to compare this with the standard sample on thin layer chromatography. After drying 0.5 ml of the reaction solution, 0.2 ml of DMSO was added to dissolve it, and 0.5 µl of the solution was added dropwise to the TLC plate (silica gel) five times. After spotting and repeatedly drying, the diffusion of the material was suppressed as much as possible, and then developed in a developing solvent for a predetermined time, and the results were confirmed. The developing solvent was used in a mixture of toluene: acetone: chloroform = 2: 1: 1. As shown in Fig. 5b, it is clearly distinguished from indigo, indican, and indole as a control group, and the Rf values of all products of the reactants having different concentrations at the same position as the indirubin standard (sample 1-5) are consistent. Confirmed.

This result was separated by high performance liquid chromatography (HPLC) and verified by mass spectrometer (Mass spectrophotometer), and showed the Rt and molecular weight that exactly matched the standard sample, thereby finally confirming that the reaction product of the present invention was indirubin. 6].

Example 6 Fusion of Beta-glucosidase Gene and Oxygenase Gene

The discovered oxygenase and the mutant enzyme source produced were found to be very large (data not shown) with molecular weight <200 kDa and have multimer properties. Therefore, overexpression is difficult and the effect on cell physiology was confirmed. In addition, in the case of glucosidase which should be co-expressed, it was confirmed in the previous study that the amount of independent expression was not high. In order to overcome some of these problems and reduce the metabolic burden associated with each independent expression, glucosidase-oxygenase fusion proteins have been attempted [Figure 7]. As a result of preparing the fusion protein by general end-to-end method, it was found to be difficult to express as a functional protein due to intracellular stress, absence of chaperone, and burden caused by giant fusion protein. However, since the activity is measured and the cytotoxicity is partially reduced, the bi-functional fusion protein may be produced by more advanced fusion methods such as selective fusion or grafting of specific domains. It could be confirmed.

Example 7 Indirubin Production Capacity Evaluation by Control of Expression Method

The process was evaluated by cloning each gene (glucosidase and oxygenase) using two vectors, pACYC184 and pTrc99A, which are stable in Escherichia coli hosts and have low metabolic physiological effects. . At this time, in order to increase the expression level of oxygenase, which is a rate-limiting step of the overall reaction rate, it was cloned into pTrc99A, and glucosidase having a relatively high activity and expression level was cloned into pACYC184. Another expression strategy was tested for cloning the oxygenase and glucosidase in the operon form after inserting the promoter into the pACYC184 plasmid [FIG. 8]. As a result, it was confirmed that the co-expression is weak, but the relative amount of indirubin production is favorable, and also advantageous to the stability of the plasmid. Industrially, in order to utilize this enzyme expression system, the addition of antibiotics to keep the plasmid stable in the cell must be performed in parallel. As a means of overcoming this, recombinant cells will be prepared by inserting improved enzymes and glucosidase with increased indirubin production into the chromosome of the host. In this case, a method of inserting an appropriate promoter to increase the protein production will be used as a means of preventing the degradation of production capacity due to the limited number of copies.

In addition, as a factor to be considered in the process, since the present invention generates a dodecyl room in indica using glucosidase, and then generates isatin by oxidase, the dodecyl room and isatin are dimerized in the presence of oxygen to indyrubin. In this case, two molecules of the reading chamber may be first dimerized to generate indigo. Therefore, the production amount of indirubin is greatly affected by the oxygen concentration. Indeed, as evidence of the above prediction results, it was confirmed that after reacting indican with both enzymes, the red color became darker with time when the initial reaction solution with weak red color was left. This provides an advantageous condition for the excess oxygen in the initial reaction to form indigo, a toxin chamber dimer before the toxin chamber is formed into isatin by the oxygenase, and after the concentration is lowered, the highly selective enzyme reactant (Isatin). It was confirmed that the increase of indirubin amount is increased by). In addition, it was further confirmed that an increase in indirubin production at 37 ° C than at 30 ° C was associated with decreased oxygen solubility with an increase in enzyme inactivation. Therefore, it is thought that the reaction temperature and the amount of oxygen should be appropriately controlled in the process of producing indirubin using indican as a substrate.

1 is a diagram illustrating the indie-rubin production process by the new process compared with the existing process.

2 is a diagram illustrating a screening strategy for the discovery of oxygenase with indirubin production capacity.

Figure 3 is a gene and amino acid sequence of the oxygenase subfamily confirmed indirubin production capacity by a systematic discovery process.

Figure 4 is an amino acid sequence of the mutated enzyme discovered after the production by random mutagenesis to increase the production capacity of indirubin.

5 is a result of analyzing the reaction solution (a) and the reaction solution observed in the process of confirming the production of indirubin using the enzyme source excavated by TLC.

6 is a mass spectrophotometer result of finally confirming the indirubin compound converted to glucosidase and oxygenase in indica.

Figure 7 is a schematic diagram showing the manufacturing process of the fusion protein as a kind of indirubin production strategy using two enzyme sources.

8 is a schematic diagram of the expression strategy of the two enzyme sources by individual expression (a), co-expression (b), chromosomal integration process.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> MINING AND IMPROVEMENT OF ISATIN-GENERATING ENZYME, AND THEIR          COMBINED USE WITH BETA-GLUCOSIDASE FOR THE PRODUCTION OF NATURAL          INDIRUBIN <140> 10-2009-0095227 <141> 2009-10-07 <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 1224 <212> DNA <213> Rhodococcus jostii RHA1 plasmid pRHL3 <400> 1 ctatacatcc atcgtgagtg cgtcgtccgc gccgatcaac aggcgcccgt agacctcttt 60 gccgacctcg ggggtgacgt aggcgtgccg ccccgcgatc tctgcatcgc gccagatctg 120 gttgaggacg ttcgcgtcgg cgaacgagcc ggccccgttg gtggtcatca ggatgtcgat 180 cccgtccttg acgagctccg cgatgatccc ggtgttgttc cggatccgcc cgcgggtgat 240 caggtcgggc aactcaccac gtgcggcggc gttgtcgatg tcggcggtgg cctgctgcat 300 caggagctcg gccatgttga acttcgttgc ggccgccgcc acgccgagct gatgcgtcgg 360 cgagttcttc gccgccgtgt acctggtgta ggccacccgc ttgttcggca gtttctccag 420 tgtcagctcc atcgcatgcc gagccagacc gagctgcgcg ccgacgagaa tcagcgcggc 480 gaccgggatg aacgccatgt tggagttgcg ttcggtgctc ttgtacgggg tggcgaagtc 540 cgcctcgcgc attgcgacga accgctgaat tcggtggtcc gggacgaagt ggtcttccac 600 gacgatggtg tcactgcccg atcccttcat ccccgccacg aaccaggtcg gcttaatcgt 660 ccacgcatcc ttggggatga gcgcgagcgc acgcgggtcc tcgccttcct cgacgtcgag 720 ggcgataccc agggtgcccc agtccgcggc gaacgatccg gacgaatacg gccactggcc 780 actgacgatg taaccgcctt cgacccgctc ggccttgcta ccgggcgtga agatgccgca 840 gcatttggcg tcgggattcg cgccgaacac gtcgtcttgc gcctgctgcg agaacgtggt 900 gccgaaccag gtgcacacgt tgagcagtgc gaccgcccat gccgtggaac cgtcgccctt 960 cgcgacctcg gcgaccgtct ccatcgcggt ccgaaggttt tcctcgtagc cgccgtagcg 1020 gcggggcacg aacagcttga acaggccctc ggcctccagc gcggccatca cctccggcgc 1080 ggcacgccgg tcagcggacc cctggtcggc gtactggcgg atcagcggga tgagcgcggt 1140 cgccttgccg atgacctccc ggttcggacg cgaggcgtcg acgtcggcgg tctccgtgct 1200 ggtgggttcg agcgtctgag tcat 1224 <210> 2 <211> 1206 <212> DNA <213> Rhodococcus opacus B4 <400> 2 tcagatgagg ggcgtgatct ggtcgtcgcg gccgaggagg gccttgccgt acacctcgta 60 gttgactgcc ggcaaggtca ccgcatgacg tgcagcgaca ttcgagtcac gccagatgcg 120 ttgcagtgga ttgttttccc cgaagctgcc ggcgccgtgt gcggagacga ggatgttgat 180 ggccttggtg atgtggtcga ggacccagcc cgtgtcggcg cggacacggg aacgggcgat 240 cacgtcgggg tagacgccgc gtgccgcagc ggcgtcgatg tcgtcggcgg cgcggtaggc 300 gtgcaggtgg gcggtgtcga tgagcatcgc ggcctcggcg atctggagct ggaacgccac 360 cgagtcggac tgggcggtgt agaaggtgta ggaaatggcc ttcgtgtccg ctttggcggt 420 gaccagttcg agggccttgc gtcccaggcc gagctgcggg ccggcaagca cgagcgagag 480 cagcggaacg aacgccgacc ggtagaggat ctcgtcggtg tgctcggtgg cgtagtcgcc 540 gccgatcgcg gcggggacgg gcatgatgcg gtgctcgggg acgaatacgt cggtggcgat 600 caggcagttc gacccggagg acttcatgcc ggcgacgaac caggtttcct cgaaaccgag 660 gtccgtgcgg gggatcaggg ccagtccctg gtcgatcacc tcgccgtccg cgttggtgat 720 cgggatgccc aggaccgccc agctcgcgtg ccaggagcca gagttgtagt accagcggcc 780 ggtcaccttg aagccgccct cgaccttgac tgcctccgac gtcggcgaga gcactccgga 840 caccttcgca tccggcgtgt cggcccagac gtcgtcctgt gcctgttcgg ggaacaggcc 900 ggtcagccag gcgcagacgt tgcagagggt gacgacccag gcggtgccgc cgtcggcctc 960 gccgaccgcg gcggacacgt cgagcatggt gcgcatcgag gtttcgtagc cgccgtagcg 1020 gcggggctgg gcgatcttga aggcgccggc gtcggtgagg gcggtgatcg actcctcgac 1080 gacgcggcgg tccgcttctc cttgcgcgga gttcttcgcc agcatcggct gcagatcgcg 1140 gactcggccg acgatctcgt cggcactggg gacggtggtg agcggggtgt cggtgacagt 1200 ggtcat 1206 <210> 3 <211> 1215 <212> DNA <213> Rhodococcus opacus B4 <400> 3 tcagacgaag ggggatacct gctcgtcgat gccgagcagg gcgcgtccgt agatctcctg 60 gttgatgttc aggttgatga aggcgtgccg ggagctggtt tccagatcac gccagatccg 120 ctgcagcggg ttgacctgcg cgaagctgcc ggcgccgctg acgttgagca gcagttcgac 180 cgcctcgcgg cacaggttcg cggcctgcgc ggcgtccatc cgcgcccgcg cccgctcgac 240 gaccggcatc tgcctgccgg cggcggccgc ggcgtcgatc tcgtctgcgc cgcggaacgc 300 gaggaggcgg gcctgatcga tcagggagcg ggcggcggcc atgttcagtt gggtggacgg 360 tgcgtcgatc gaccgctggt agaacgagta cgcgatcggc ttgtccttct gcagcgtcgc 420 caggaccgcg tcgtaggcgc cctccgccat gcccagcacg ggaccgacga gcaccagcgc 480 caggacgggg acgagcgagg agtgatacag ggtttcgtcc tggaactccg aggcggtgcg 540 cccctccatc atgtcggtga ccgacaggat ccgggcgtcg gggacgaaga cgtcgtcggc 600 gacgagggtg tcgctgccgg aaccggacat gcccgcgacg taccaggtgt tgtcgatcga 660 cagatcggtc atcgggatcc acgccgacgc cgccccgacg gcgtcaccgt cggcgttggt 720 cagggggata ccgacgttgg cccagctcgc gtggtgcgat gccgaggcga atccccaccg 780 tccggtgacc cggtacccgc cgtcgacccg caccgaggtc gaggtcgggg tgaacacgcc 840 gcaggtggcg atgcgcggat cggcgcccag cacgtcccgc tgcaccgact cggggtaggc 900 gccgacgaag aacgacgtca cgttgctcag cgtggtgacc cacgcggtgg acccgcacga 960 ccggccgagt tccagcgaca cctccaggaa ggtgcggaaa tcctcctccg ctccgccgaa 1020 gcgtgcgggt ttggtgatgt cgaacaggcc ggcctcgcgc agggcgtcga tgttctcctc 1080 gggcacgcgg cggtccgcct cggcgcgcgc ggcgttctcg cggagcagtg ggccgagcgc 1140 ccgcgcggcg ctgaccaggc gttcccgctc ggaaaccggc gtcgtcgtgt gcggggccgt 1200 gtcggtgatc gtcat 1215 <210> 4 <211> 1182 <212> DNA <213> Streptomyces ghanaensis ATCC 14672 <400> 4 atgaagacca tcgaggcgcc gacgcgcgaa gaactcgtcc gccgtgcatc ggacctggtt 60 ccgcttctgc ggaggaacgc gctcgccggt gaggagaacc gccgactgcc gcaggagacg 120 gtcgacgccc tgaccgaagc cggaattctc aagatgcgcg tcccgcaccg ctacggcggc 180 tacgagtccg acatgcgcac ggtcgtcgac accatcgccg agctcgcccg cggtgacggc 240 tccgccgcct ggaccgtctc cgtgtgggcc atcagctcct ggatggtcgg cctcttcccc 300 gacgaggtcc aggacgaggt cttctcgacc cccgacgtac gcatcagcgg catcctcagc 360 ccgggcgcca tggccacccc ggtccccggc ggtgtggtgc tcaacgggaa gtggtcgttc 420 aactccggtg tccagcacag ccagtggaac accaacgccg ccatcctcat cggcgaggac 480 ggcgagccgc agcccatcat ggtggccatc cccgtctccg acctgacgat cgtggacgac 540 tggcacaccg ccggcctgcg cggcaccggc agcgtcacca cgatcgccga gaacctgttc 600 gtcccgcagg aacgcgtact gccgatgatc ccggtgctcc agggccagca ccgctcggtg 660 ctcaacgccg gttccccgct ctacagcgcg ccgttcatgc ccaccgcctg cgccaccatc 720 tgcgccccgg ccctgggcct cgcccgcgcg gccaaggagg ccttcctgga gcgcctgccc 780 ggccggaaga tcacgtacac ggcgtacgag aaccaggcgg aggccccgct gacgcacctc 840 caggtcgccg aggccacggt gaagatcgac gaggcggagt tccacgcgca ccgcaccgcg 900 cggatggtcg acgccaaggg cgccgccggc gaggagtgga cgctggagga gcgggcccgg 960 gtccgcctgg acctcggtgc cgtcacccag cgcgccaagg aggccgtgga cctgctcaac 1020 acggccagcg gcggctcgtc catctactcc tccgtgccga tccagcgcat cgagcgggac 1080 gtgcagaccg tcaacctgca cgcgatcctc cacccgaaca ccaacctcga gctgtacgga 1140 cggatcgtct gcggccagga gcccaacacc gtctacctgt ag 1182 <210> 5 <211> 407 <212> PRT <213> Rhodococcus jostii RHA1 <400> 5 Met Thr Gln Thr Leu Glu Pro Thr Ser Thr Glu Thr Ala Asp Val Asp   1 5 10 15 Ala Ser Arg Pro Asn Arg Glu Val Ile Gly Lys Ala Thr Ala Leu Ile              20 25 30 Pro Leu Ile Arg Gln Tyr Ala Asp Gln Gly Ser Ala Asp Arg Arg Ala          35 40 45 Ala Pro Glu Val Met Ala Ala Leu Glu Ala Glu Gly Leu Phe Lys Leu      50 55 60 Phe Val Pro Arg Arg Tyr Gly Gly Tyr Glu Glu Asn Leu Arg Thr Ala  65 70 75 80 Met Glu Thr Val Ala Glu Val Ala Lys Gly Asp Gly Ser Thr Ala Trp                  85 90 95 Ala Val Ala Leu Leu Asn Val Cys Thr Trp Phe Gly Thr Thr Phe Ser             100 105 110 Gln Gln Ala Gln Asp Asp Val Phe Gly Ala Asn Pro Asp Ala Lys Cys         115 120 125 Cys Gly Ile Phe Thr Pro Gly Ser Lys Ala Glu Arg Val Glu Gly Gly     130 135 140 Tyr Ile Val Ser Gly Gln Trp Pro Tyr Ser Ser Gly Ser Phe Ala Ala 145 150 155 160 Asp Trp Gly Thr Leu Gly Ile Ala Leu Asp Val Glu Glu Gly Glu Asp                 165 170 175 Pro Arg Ala Leu Ala Leu Ile Pro Lys Asp Ala Trp Thr Ile Lys Pro             180 185 190 Thr Trp Phe Val Ala Gly Met Lys Gly Ser Gly Ser Asp Thr Ile Val         195 200 205 Val Glu Asp His Phe Val Pro Asp His Arg Ile Gln Arg Phe Val Ala     210 215 220 Met Arg Glu Ala Asp Phe Ala Thr Pro Tyr Lys Ser Thr Glu Arg Asn 225 230 235 240 Ser Asn Met Ala Phe Ile Pro Val Ala Ala Leu Ile Leu Val Gly Ala                 245 250 255 Gln Leu Gly Leu Ala Arg His Ala Met Glu Leu Thr Leu Glu Lys Leu             260 265 270 Pro Asn Lys Arg Val Ala Tyr Thr Arg Tyr Thr Ala Ala Lys Asn Ser         275 280 285 Pro Thr His Gln Leu Gly Val Ala Ala Ala Ala Thr Lys Phe Asn Met     290 295 300 Ala Glu Leu Leu Met Gln Gln Ala Thr Ala Asp Ile Asp Asn Ala Ala 305 310 315 320 Ala Arg Gly Glu Leu Pro Asp Leu Ile Thr Arg Gly Arg Ile Arg Asn                 325 330 335 Asn Thr Gly Ile Ile Ala Glu Leu Val Lys Asp Gly Ile Asp Ile Leu             340 345 350 Met Thr Thr Asn Gly Ala Gly Ser Phe Ala Asp Ala Asn Val Leu Asn         355 360 365 Gln Ile Trp Arg Asp Ala Glu Ile Ala Gly Arg His Ala Tyr Val Thr     370 375 380 Pro Glu Val Gly Lys Glu Val Tyr Gly Arg Leu Leu Ile Gly Ala Asp 385 390 395 400 Asp Ala Leu Thr Met Asp Val                 405 <210> 6 <211> 401 <212> PRT <213> Rhodococcus opacus B4 <400> 6 Met Thr Thr Val Thr Asp Thr Pro Leu Thr Thr Val Pro Ser Ala Asp   1 5 10 15 Glu Ile Val Gly Arg Val Arg Asp Leu Gln Pro Met Leu Ala Lys Asn              20 25 30 Ser Ala Gln Gly Glu Ala Asp Arg Arg Val Val Glu Glu Ser Ile Thr          35 40 45 Ala Leu Thr Asp Ala Gly Ala Phe Lys Ile Ala Gln Pro Arg Arg Tyr      50 55 60 Gly Gly Tyr Glu Thr Ser Met Arg Thr Met Leu Asp Val Ser Ala Ala  65 70 75 80 Val Gly Glu Ala Asp Gly Gly Thr Ala Trp Val Val Thr Leu Cys Asn                  85 90 95 Val Cys Ala Trp Leu Thr Gly Leu Phe Pro Glu Gln Ala Gln Asp Asp             100 105 110 Val Trp Ala Asp Thr Pro Asp Ala Lys Val Ser Gly Val Leu Ser Pro         115 120 125 Thr Ser Glu Ala Val Lys Val Glu Gly Gly Phe Lys Val Thr Gly Arg     130 135 140 Trp Tyr Tyr Asn Ser Gly Ser Trp His Ala Ser Trp Ala Val Leu Gly 145 150 155 160 Ile Pro Ile Thr Asn Ala Asp Gly Glu Val Ile Asp Gln Gly Leu Ala                 165 170 175 Leu Ile Pro Arg Thr Asp Leu Gly Phe Glu Glu Thr Trp Phe Val Ala             180 185 190 Gly Met Lys Ser Ser Gly Ser Asn Cys Leu Ile Ala Thr Asp Val Phe         195 200 205 Val Pro Glu His Arg Ile Met Pro Val Pro Ala Ala Ile Gly Gly Asp     210 215 220 Tyr Ala Thr Glu His Thr Asp Glu Ile Leu Tyr Arg Ser Ala Phe Val 225 230 235 240 Pro Leu Leu Ser Leu Val Leu Ala Gly Pro Gln Leu Gly Leu Gly Arg                 245 250 255 Lys Ala Leu Glu Leu Val Thr Ala Lys Ala Asp Thr Lys Ala Ile Ser             260 265 270 Tyr Thr Phe Tyr Thr Ala Gln Ser Asp Ser Val Ala Phe Gln Leu Gln         275 280 285 Ile Ala Glu Ala Ala Met Leu Ile Asp Thr Ala His Leu His Ala Tyr     290 295 300 Arg Ala Ala Asp Asp Ile Asp Ala Ala Ala Ala Arg Gly Val Tyr Pro 305 310 315 320 Asp Val Ile Ala Arg Ser Arg Val Arg Ala Asp Thr Gly Trp Val Leu                 325 330 335 Asp His Ile Thr Lys Ala Ile Asn Ile Leu Val Ser Ala His Gly Ala             340 345 350 Gly Ser Phe Gly Glu Asn Asn Pro Leu Gln Arg Ile Trp Arg Asp Ser         355 360 365 Asn Val Ala Ala Arg His Ala Val Thr Leu Pro Ala Val Asn Tyr Glu     370 375 380 Val Tyr Gly Lys Ala Leu Leu Gly Arg Asp Asp Gln Ile Thr Pro Leu 385 390 395 400 Ile     <210> 7 <211> 404 <212> PRT <213> Rhodococcus opacus B4 <400> 7 Met Thr Ile Thr Asp Thr Ala Pro His Thr Thr Thr Thr Pro Val Ser Glu   1 5 10 15 Arg Glu Arg Leu Val Ser Ala Ala Arg Ala Leu Gly Pro Leu Leu Arg              20 25 30 Glu Asn Ala Ala Arg Ala Glu Ala Asp Arg Arg Val Pro Glu Glu Asn          35 40 45 Ile Asp Ala Leu Arg Glu Ala Gly Leu Phe Asp Ile Thr Lys Pro Ala      50 55 60 Arg Phe Gly Gly Ala Glu Glu Asp Phe Arg Thr Phe Leu Glu Val Ser  65 70 75 80 Leu Glu Leu Gly Arg Ser Cys Gly Ser Thr Ala Trp Val Thr Thr Leu                  85 90 95 Ser Asn Val Thr Ser Phe Phe Val Gly Ala Tyr Pro Glu Ser Val Gln             100 105 110 Arg Asp Val Leu Gly Ala Asp Pro Arg Ile Ala Thr Cys Gly Val Phe         115 120 125 Thr Pro Thr Ser Thr Ser Val Arg Val Asp Gly Gly Tyr Arg Val Thr     130 135 140 Gly Arg Trp Gly Phe Ala Ser Ala Ser His His Ala Ser Trp Ala Asn 145 150 155 160 Val Gly Ile Pro Leu Thr Asn Ala Asp Gly Asp Ala Val Gly Ala Ala                 165 170 175 Ser Ala Trp Ile Pro Met Thr Asp Leu Ser Ile Asp Asn Thr Trp Tyr             180 185 190 Val Ala Gly Met Ser Gly Ser Gly Ser Asp Thr Leu Val Ala Asp Asp         195 200 205 Val Phe Val Pro Asp Ala Arg Ile Leu Ser Val Thr Asp Met Met Glu     210 215 220 Gly Arg Thr Ala Ser Glu Phe Gln Asp Glu Thr Leu Tyr His Ser Ser 225 230 235 240 Leu Val Pro Val Leu Ala Leu Val Leu Val Gly Pro Val Leu Gly Met                 245 250 255 Ala Glu Gly Ala Tyr Asp Ala Val Leu Ala Thr Leu Gln Lys Asp Lys             260 265 270 Pro Ile Ala Tyr Ser Phe Tyr Gln Arg Ser Ile Asp Ala Pro Ser Thr         275 280 285 Gln Leu Asn Met Ala Ala Ala Arg Ser Leu Ile Asp Gln Ala Arg Leu     290 295 300 Leu Ala Phe Arg Gly Ala Asp Glu Ile Asp Ala Ala Ala Ala Ala Gly 305 310 315 320 Arg Gln Met Pro Val Val Glu Arg Ala Arg Ala Arg Met Asp Ala Ala                 325 330 335 Gln Ala Ala Asn Leu Cys Arg Glu Ala Val Glu Leu Leu Leu Asn Val             340 345 350 Ser Gly Ala Gly Ser Phe Ala Gln Val Asn Pro Leu Gln Arg Ile Trp         355 360 365 Arg Asp Leu Glu Thr Ser Ser Arg His Ala Phe Ile Asn Leu Asn Ile     370 375 380 Asn Gln Glu Ile Tyr Gly Arg Ala Leu Leu Gly Ile Asp Glu Gln Val 385 390 395 400 Ser Pro Phe Val                 <210> 8 <211> 393 <212> PRT <213> Streptomyces ghanaensis ATCC 14672 <400> 8 Met Lys Thr Ile Glu Ala Pro Thr Arg Glu Glu Leu Val Arg Arg Ala   1 5 10 15 Ser Asp Leu Val Pro Leu Leu Arg Arg Asn Ala Leu Ala Gly Glu Glu              20 25 30 Asn Arg Arg Leu Pro Gln Glu Thr Val Asp Ala Leu Thr Glu Ala Gly          35 40 45 Ile Leu Lys Met Arg Val Pro His Arg Tyr Gly Gly Tyr Glu Ser Asp      50 55 60 Met Arg Thr Val Val Asp Thr Ile Ala Glu Leu Ala Arg Gly Asp Gly  65 70 75 80 Ser Ala Ala Trp Thr Val Ser Val Trp Ala Ile Ser Ser Trp Met Val                  85 90 95 Gly Leu Phe Pro Asp Glu Val Gln Asp Glu Val Phe Ser Thr Pro Asp             100 105 110 Val Arg Ile Ser Gly Ile Leu Ser Pro Gly Ala Met Ala Thr Pro Val         115 120 125 Pro Gly Gly Val Val Leu Asn Gly Lys Trp Ser Phe Asn Ser Gly Val     130 135 140 Gln His Ser Gln Trp Asn Thr Asn Ala Ala Ile Leu Ile Gly Glu Asp 145 150 155 160 Gly Glu Pro Gln Pro Ile Met Val Ala Ile Pro Val Ser Asp Leu Thr                 165 170 175 Ile Val Asp Asp Trp His Thr Ala Gly Leu Arg Gly Thr Gly Ser Val             180 185 190 Thr Thr Ile Ala Glu Asn Leu Phe Val Pro Gln Glu Arg Val Leu Pro         195 200 205 Met Ile Pro Val Leu Gln Gly Gln His Arg Ser Val Leu Asn Ala Gly     210 215 220 Ser Pro Leu Tyr Ser Ala Pro Phe Met Pro Thr Ala Cys Ala Thr Ile 225 230 235 240 Cys Ala Pro Ala Leu Gly Leu Ala Arg Ala Ala Lys Glu Ala Phe Leu                 245 250 255 Glu Arg Leu Pro Gly Arg Lys Ile Thr Tyr Thr Ala Tyr Glu Asn Gln             260 265 270 Ala Glu Ala Pro Leu Thr His Leu Gln Val Ala Glu Ala Thr Val Lys         275 280 285 Ile Asp Glu Ala Glu Phe His Ala His Arg Thr Ala Arg Met Val Asp     290 295 300 Ala Lys Gly Ala Ala Gly Glu Glu Trp Thr Leu Glu Glu Arg Ala Arg 305 310 315 320 Val Arg Leu Asp Leu Gly Ala Val Thr Gln Arg Ala Lys Glu Ala Val                 325 330 335 Asp Leu Leu Asn Thr Ala Ser Gly Gly Ser Ser Ile Tyr Ser Ser Val             340 345 350 Pro Ile Gln Arg Ile Glu Arg Asp Val Gln Thr Val Asn Leu His Ala         355 360 365 Ile Leu His Pro Asn Thr Asn Leu Glu Leu Tyr Gly Arg Ile Val Cys     370 375 380 Gly Gln Glu Pro Asn Thr Val Tyr Leu 385 390 <210> 9 <211> 407 <212> PRT <213> Artificial Sequence <220> <223> Indole oxygenase mutant 1 K89R, A138T <400> 9 Met Thr Gln Thr Leu Glu Pro Thr Ser Thr Glu Thr Ala Asp Val Asp   1 5 10 15 Ala Ser Arg Pro Asn Arg Glu Val Ile Gly Lys Ala Thr Ala Leu Ile              20 25 30 Pro Leu Ile Arg Gln Tyr Ala Asp Gln Gly Ser Ala Asp Arg Arg Ala          35 40 45 Ala Pro Glu Val Met Ala Ala Leu Glu Ala Glu Gly Leu Phe Lys Leu      50 55 60 Phe Val Pro Arg Arg Tyr Gly Gly Tyr Glu Glu Asn Leu Arg Thr Ala  65 70 75 80 Met Glu Thr Val Ala Glu Val Ala Arg Gly Asp Gly Ser Thr Ala Trp                  85 90 95 Ala Val Ala Leu Leu Asn Val Cys Thr Trp Phe Gly Thr Thr Phe Ser             100 105 110 Gln Gln Ala Gln Asp Asp Val Phe Gly Ala Asn Pro Asp Ala Lys Cys         115 120 125 Cys Gly Ile Phe Thr Pro Gly Ser Lys Thr Glu Arg Val Glu Gly Gly     130 135 140 Tyr Ile Val Ser Gly Gln Trp Pro Tyr Ser Ser Gly Ser Phe Ala Ala 145 150 155 160 Asp Trp Gly Thr Leu Gly Ile Ala Leu Asp Val Glu Glu Gly Glu Asp                 165 170 175 Pro Arg Ala Leu Ala Leu Ile Pro Lys Asp Ala Trp Thr Ile Lys Pro             180 185 190 Thr Trp Phe Val Ala Gly Met Lys Gly Ser Gly Ser Asp Thr Ile Val         195 200 205 Val Glu Asp His Phe Val Pro Asp His Arg Ile Gln Arg Phe Val Ala     210 215 220 Met Arg Glu Ala Asp Phe Ala Thr Pro Tyr Lys Ser Thr Glu Arg Asn 225 230 235 240 Ser Asn Met Ala Phe Ile Pro Val Ala Ala Leu Ile Leu Val Gly Ala                 245 250 255 Gln Leu Gly Leu Ala Arg His Ala Met Glu Leu Thr Leu Glu Lys Leu             260 265 270 Pro Asn Lys Arg Val Ala Tyr Thr Arg Tyr Thr Ala Ala Lys Asn Ser         275 280 285 Pro Thr His Gln Leu Gly Val Ala Ala Ala Ala Thr Lys Phe Asn Met     290 295 300 Ala Glu Leu Leu Met Gln Gln Ala Thr Ala Asp Ile Asp Asn Ala Ala 305 310 315 320 Ala Arg Gly Glu Leu Pro Asp Leu Ile Thr Arg Gly Arg Ile Arg Asn                 325 330 335 Asn Thr Gly Ile Ile Ala Glu Leu Val Lys Asp Gly Ile Asp Ile Leu             340 345 350 Met Thr Thr Asn Gly Ala Gly Ser Phe Ala Asp Ala Asn Val Leu Asn         355 360 365 Gln Ile Trp Arg Asp Ala Glu Ile Ala Gly Arg His Ala Tyr Val Thr     370 375 380 Pro Glu Val Gly Lys Glu Val Tyr Gly Arg Leu Leu Ile Gly Ala Asp 385 390 395 400 Asp Ala Leu Thr Met Asp Val                 405 <210> 10 <211> 407 <212> PRT <213> Artificial Sequence <220> <223> Indole oxygenase mutant 2 V248I <400> 10 Met Thr Gln Thr Leu Glu Pro Thr Ser Thr Glu Thr Ala Asp Val Asp   1 5 10 15 Ala Ser Arg Pro Asn Arg Glu Val Ile Gly Lys Ala Thr Ala Leu Ile              20 25 30 Pro Leu Ile Arg Gln Tyr Ala Asp Gln Gly Ser Ala Asp Arg Arg Ala          35 40 45 Ala Pro Glu Val Met Ala Ala Leu Glu Ala Glu Gly Leu Phe Lys Leu      50 55 60 Phe Val Pro Arg Arg Tyr Gly Gly Tyr Glu Glu Asn Leu Arg Thr Ala  65 70 75 80 Met Glu Thr Val Ala Glu Val Ala Lys Gly Asp Gly Ser Thr Ala Trp                  85 90 95 Ala Val Ala Leu Leu Asn Val Cys Thr Trp Phe Gly Thr Thr Phe Ser             100 105 110 Gln Gln Ala Gln Asp Asp Val Phe Gly Ala Asn Pro Asp Ala Lys Cys         115 120 125 Cys Gly Ile Phe Thr Pro Gly Ser Lys Ala Glu Arg Val Glu Gly Gly     130 135 140 Tyr Ile Val Ser Gly Gln Trp Pro Tyr Ser Ser Gly Ser Phe Ala Ala 145 150 155 160 Asp Trp Gly Thr Leu Gly Ile Ala Leu Asp Val Glu Glu Gly Glu Asp                 165 170 175 Pro Arg Ala Leu Ala Leu Ile Pro Lys Asp Ala Trp Thr Ile Lys Pro             180 185 190 Thr Trp Phe Val Ala Gly Met Lys Gly Ser Gly Ser Asp Thr Ile Val         195 200 205 Val Glu Asp His Phe Val Pro Asp His Arg Ile Gln Arg Phe Val Ala     210 215 220 Met Arg Glu Ala Asp Phe Ala Thr Pro Tyr Lys Ser Thr Glu Arg Asn 225 230 235 240 Ser Asn Met Ala Phe Ile Pro Ile Ala Ala Leu Ile Leu Val Gly Ala                 245 250 255 Gln Leu Gly Leu Ala Arg His Ala Met Glu Leu Thr Leu Glu Lys Leu             260 265 270 Pro Asn Lys Arg Val Ala Tyr Thr Arg Tyr Thr Ala Ala Lys Asn Ser         275 280 285 Pro Thr His Gln Leu Gly Val Ala Ala Ala Ala Thr Lys Phe Asn Met     290 295 300 Ala Glu Leu Leu Met Gln Gln Ala Thr Ala Asp Ile Asp Asn Ala Ala 305 310 315 320 Ala Arg Gly Glu Leu Pro Asp Leu Ile Thr Arg Gly Arg Ile Arg Asn                 325 330 335 Asn Thr Gly Ile Ile Ala Glu Leu Val Lys Asp Gly Ile Asp Ile Leu             340 345 350 Met Thr Thr Asn Gly Ala Gly Ser Phe Ala Asp Ala Asn Val Leu Asn         355 360 365 Gln Ile Trp Arg Asp Ala Glu Ile Ala Gly Arg His Ala Tyr Val Thr     370 375 380 Pro Glu Val Gly Lys Glu Val Tyr Gly Arg Leu Leu Ile Gly Ala Asp 385 390 395 400 Asp Ala Leu Thr Met Asp Val                 405  

Claims (11)

After isolation and amplification of the oxygenase gene that additionally oxidizes the C-2 position of the indole compartment in which the C-3 position of the indole is oxidized, the recombinant E. coli transformed into the vector and transformed into E. coli 1 to 100 mM indican, Incubated in a medium containing 0.5-1.5 wt.% NaCl and 0.2-0.8 wt.% Yeast extract, wherein the indoksil is formed from indicane by beta-glucosidase and oxidizes the C-2 position of the indoksil. A method for producing indirubin, characterized in that istane is formed in a doxoroom by an oxygenase enzyme represented by any one of SEQ ID NO: 1 to SEQ ID NO: 10. delete delete The method of claim 1, The beta-glucosidase is a microorganism transformed with a gene derived from a Shinorhizobium meliloti strain ( E. coli XL1-BLUE [pTrc99A-glu (SM)] (Accession No. KCTC 11236BP). Method for producing indirubin to be. delete The method of claim 1, The culturing is a method for producing indirubin, characterized in that the culture by injecting oxygen with aeration amount 0.1 ~ 2.0 vvm. delete delete The method of claim 1, The vector is a method for producing indirubin, characterized in that the modulator is expressed as a pTrc99A expression vector removed. The method of claim 1, A method for producing indirubin using recombinant E. coli, wherein the recombinant plasmids including the genes of the beta-glucosidase and the oxygenase are simultaneously transformed or two genes are cloned into one plasmid. The method of claim 10, A method for producing indirubin using recombinant E. coli, which introduces a gene in the recombinant plasmid into a host chromosome.

Images