KR20130071660A - Novel cinnamoyl esterase from lactobacillus acidophillus - Google Patents

Abstract

PURPOSE: Cinnamoyl esterase derived from Lactobacillus Acidophillus is provided to hydrolyze sugar-esterase bond linked to hydrocinnamic acid, and to prevent and treat diabetes. CONSTITUTION: Cinnamoyl esterase derived from Lactobacillus Acidophillus has an amino acid sequence of sequence number 2. A gene encodes cyannamoyl esterase and is denoted by sequence number 1. A recombinant vector contains the gene. A method for preparing cynnamoyl esterase comprises: a step of preparing a recombinant cynnamoyl esterase expression vector; and a step of transducing the expression vector to a microorganism and transforming.

Description

Novel Cinnamoyl Esterase from Lactobacillus Acidophillus from Lactobacillus ashdophyllus

The present invention is a Cinnamoyl esterase protein derived from Lactobacillus acidophillus, a gene encoding the protein, a recombinant vector comprising the gene, and a transformed vector using the recombinant vector. It relates to a microorganism and probiotics containing the microorganism.

It is estimated that there are more than 200 million people with diabetes worldwide, which is almost a disaster called AIDS in the 21st century based on complications and economic losses. This trend is expected to reach 300 million patients by 2020. In the United States, on average, over 170 billion dollars are spent on diabetes each year. It is known that Koreans are more likely to develop diabetes. Furthermore, the number of diabetics has increased rapidly since the 1980s due to the side effects of rapid westernization of lifestyle. At present, complications are severe, with half of adults with vision loss and 40% of kidney dialysis patients being diabetic. As a result, the mortality rate has risen, rising from seventh place in Korea in 1993 to fourth place in 2003. According to the current trend, it is reported that the domestic diabetic population can reach 7.2 million people by 2030, which is equivalent to 1 person for every 7 Korean population. Diabetes mellitus hyperglycemia itself is a problem, but the complications associated with chronicization is more severe, hyperlipidemia is accompanied by more than 70% of adult diabetic patients, more than 90% of adult diabetic patients are susceptible to infectious diseases. There is also a high risk of hypertension, arteriosclerosis, cerebral infarction, myocardial infarction and obstructive atherosclerosis due to cardiovascular vascular disorders. In particular, as hyperglycemia persists, three major complications such as retinopathy, nephropathy, and hand and foot appear, and diabetic neuropathy, peripheral neuropathy and autonomic neuropathy.

Diabetes is largely classified into type 1 diabetes and type 2 diabetes. Among these, type 2 diabetes is divided into insulin resistant diabetes, which plays a major role in insulin resistance, and insulin deficiency diabetes, which plays a major role in insulin secretion. Currently, more than 90% of Korean diabetics are known to have type 2 diabetes, and the number of patients is steadily increasing due to aging and lifestyle changes. Type 2 diabetes is characterized by: 1) low sensitivity to insulin in peripheral tissues, 2) decreased insulin secretion in the pancreas, 3) high endogenous glucose production in the liver, and 4) high food intake and absorption in the intestine. It is known to work and develop.

Since the causes of diabetes are very diverse and complex, none of the currently developed diabetes treatments shows effective treatment effects with monotherapy alone. Currently used therapeutic agents include insulin, sulfonylurea, and biguanide. Among these, insulin is known to be the most effective and effective, but its duration is long. It is short and does not show oral action, so there is a disadvantage to be used as an injection. Oral hypoglycemic agents include sulfonylurea and biguanide drugs. Sulfonic acid-based drugs promote the secretion of insulin produced by the beta cells of the pancreas, thereby lowering blood glucose levels, but causing liver toxicity and hypoglycemia. Biguanide-based drugs have side effects such as nausea, anorexia and diarrhea. Therefore, the development of oral diabetes therapeutics of other mechanisms that can reduce these side effects have been required, but the development is still insufficient.

Recently, research on the relationship between diabetes and environmental factors has attracted much attention. Especially, it has been announced that the composition of gut microbial environment system is very different in diabetic patients. This is increasing and research in this area is underway.

Among the studies on the role of these intestinal bacteria, Roesch et al. Suggested an important link between diabetes and intestinal microbial species. They found that their backs were predominant compared to the intestinal microbial distributions of rats that were susceptible to diabetes. These results are consistent with previous studies showing that the direct oral administration of lactic acid bacteria induces changes in the autonomic neurotransmission of the autonomic nervous system, thereby promoting insulin secretion and lowering sugar levels.

Polysaccharides on plant cell walls, on the other hand, are the most abundant organic compounds that occur naturally. It can be divided into three major groups: cellulose, hemicelluloses and pectin. These compounds consist of hydroxycinnamic acid, which is widely distributed in nature, such as ferulic, p-coumaric acid, caffeic, and sinapic acid. . These compounds generally have sugar residues ester bonds and are known to play an important role in the structure of plant cell wall. Among them, the most abundant hydrocinnamic acid is chlorogenic acid (5-caffeoylquinic acid), in which caffeic acid is ester-bonded with quinic acid.

These hydrocinnamic acids belonging to phenols are present in high concentrations in the form of fibers in plants such as coffee, tea, apples, pears, potato tubers, tomatoes, spinach, broccoli, and peaches. Most of the fiber is excreted through bowel movements, but in part, hydrocinnamic acid is produced from the fibrin in these foods through enzymatic degradation. These enzymes are detected in the epithelial cells of the small intestine, but most of them are detected in the colonic epithelial cells, and the enzymes secreted by the intestinal microorganisms are considered to be important factors for producing these useful hydrosinamic acids. Therefore, lactic acid bacteria that secrete large amounts of these enzymes are expected to be deeply involved in mechanisms such as promoting insulin secretion.

Chlorogenic acid and caffeic acid, which are two types of hydrocinnamic acid, are known to induce blood circulation in the human body. In particular, oxidation of low-density lipoprotein (LDL) in vitro It has been found that by inhibiting it can have a protective action against cardiovascular disease. It has also been demonstrated to have high antioxidant activity, such as free radical scavenging and metal ion chelating, in vitro and to inhibit specific enzymes involved in the risk of hydroperoxide formation associated with multiple mechanisms involving free radicals. lost.

Caffeic acid has the effect of preserving α-tocopherol in the human body, not only inhibits oxidative rancidity of foods in situ, but also inhibits mutagenic and carcinogenic N-nitroso compounds and prevents DNA damage in vitro It has been proven that it can be inhibited. In addition, it is known to prevent oxidative damage associated with cancer in vivo, attracting attention as an anti-cancer functional natural antioxidant. Recent studies have also shown that sustained intake of caffeic acid is also effective in preventing brain cancer.

Cinnamoyl esterase (EC), on the other hand, is an enzyme that hydrolyzes several sugar-ester bonds linked to hydroxynamic acid. Cinamoyl esterase to date is Streptomyces bacteria such as olivochromogenes or Pseudomonas fluorescens or Penicillium The enzyme has been isolated and purified from fungi such as pinophilum and Aspergillus species. It is divided into type A (FAE-III, feruloyl esterase) with specificity and type B (CinnAE, cinnamoyl esterase) with substrate specificity for methylester binding of caffeic acid and para-coumaric acid ( p- coumaric acid).

Therefore, the conversion of aromatic compounds into the form of high absorption rate through the enzymatic action from natural materials can be applied to not only the provision of novel functional probiotics foods but also to the antibiotic supplement feed additives having a high absorption rate using them. Industrial practicality is very high. However, to date, under the premise of preventing and treating diabetes, “specific lactobacillus and its enzymes” are involved in the production of hydrocinnamic acid by the action of enzymes in various dietary fiber components in the large intestine, thereby promoting insulin secretion It has not been found to provide a means.

Accordingly, the present inventors have isolated human intestinal derived cinnamoyl esterase activity excellent strains that are unique to non-diabetic patients compared to the diabetic group, from which the novel genes of cinnamoyl esterase are identified and their functions identified. The present invention has been completed.

Domestic patent application number: 10-2010-0040003

It is an object of the present invention to provide novel enzymes and genes thereof which hydrolyze sugar ester bonds of hydrosinamic acids.

Another object of the present invention to provide a recombinant vector comprising the gene.

Another object of the present invention to provide a microorganism transformed using the recombinant vector.

It is still another object of the present invention to provide a method for producing a novel enzyme that hydrolyzes a sugar ester bond of hydrocinnamic acid.

Another object of the present invention to provide a probiotic containing the transforming microorganism.

In order to solve the above problems, the present invention provides a Cinnamoyl esterase protein derived from Lactobacillus acidophillus.

The present invention also provides a gene encoding the protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a microorganism transformed using the recombinant vector.

In another aspect, the present invention comprises the steps of preparing the recombinant vector; And it provides a method for producing cinnamoyl esterase comprising the step of transforming by introducing the prepared vector into the microorganism.

In another aspect, the present invention provides a probiotic containing the transforming microorganism.

Cinnamoyl esterase protein derived from Lactobacillus acidophillus of the present invention hydrolyzes sugar-ester bonds linked to hydrocinnamic acid, thereby preventing and treating diabetes. It is useful for.

Figure 1 shows the ethyl ferulate esterase activity in the medium containing ethyl ferulate (ethyl ferulate).
Figure 2 confirms the chlorogenic acid and caffeic acid detected by HPLC / ELSD.
Figure 3 shows the result of confirming the cinnamoyl esterase gene through PCR.
4 is a recombinant vector comprising a cinnamoyl esterase gene.
5 shows the results of HPLC analysis of recombinant E. coli BL21 (DE3) and control E. coli BL21 (DE3).
6 is a result comparing the expression level of cinnamoyl esterase (cinnamoyl esterase) according to the IPTG concentration.
Figure 7 is a result of comparing the expression amount of cinnamoyl esterase (cinnamoyl esterase) with temperature.
8 shows the result of confirming the purified cinnamoyl esterase through SDS-PAGE.
9 is a graph showing the optimal active pH of cinnamoyl esterase.
10 is a graph showing the optimal activity temperature of cinnamoyl esterase.
11 is a result of confirming the pH stability of cinnamoyl esterase.
12 shows the results of confirming the thermal stability of cinnamoyl esterase.

In one embodiment, the present invention provides a Cinnamoyl esterase protein from Lactobacillus Acidophillus.

The range of Cinnamoyl esterase according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from Lactobacillus acidophillus and a functional equivalent of the protein. . Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2.

According to the present invention, the Cinnamoyl esterase protein derived from Lactobacillus acidophillus is not only a protein having its native amino acid sequence, but also an amino acid sequence variant thereof is also included in the scope of the present invention. The invention is a variant of the Cinnamoyl esterase protein from Lactobacillus acidophillus, which is a natural amino acid sequence and one or more amino acid residues deleted, inserted, non-conservative or conservative substitutions thereof or By combination is meant a protein having a different sequence. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In some cases, it may be modified by phosphorylation, sulfation, acetylation, glycosylation, methylation, farnesylation, or the like.

The present invention relates to a Cinnamoyl esterase protein derived from Lactobacillus Acidophillus, or a variant thereof, from natural or synthetic (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156). , 1963) or by DNA recombination methods based on DNA sequences (Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989).

The gene of the present invention includes both genomic DNA and cDNA encoding the Cinnamoyl esterase protein derived from Lactobacillus Acidophillus, genomic DNA and cDNA are known in the art It can manufacture according to a method.

Genomic DNA, for example, the invention extracts genomic DNA from a cell having a Cinnamoyl esterase gene derived from Lactobacillus Acidophillus and the genomic library (vector is for example plasmid, Phage, cosmid, BAC, PAC, etc. may be used) and colonies or plaques using probes prepared based on DNA encoding the protein of the present invention (eg, SEQ ID NO: 1). It may also be prepared by hybridization, or by preparing a primer specific for DNA encoding the protein of the present invention (eg, SEQ ID NO: 1) and performing a polymerase chain reaction (PCR) using the same. have.

cDNA, for example, the invention synthesizes cDNA based on mRNA extracted from a cell having a Cinnamoyl esterase gene derived from Lactobacillus acidophillus (Lactobacillus Acidophillus), and synthesized cDNA λZAP CDNA library may be prepared by inserting into a vector, and the cDNA library may be developed to be prepared by colony or plaque hybridization or PCR.

Preferably, the Cinnamoyl esterase gene derived from Lactobacillus Acidophillus of the present invention may include a nucleotide sequence represented by SEQ ID NO: 1. In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Cinnamoester esters derived from Lactobacillus acidophillus which can be used as genes encoding the Cinnamoyl esterase protein derived from Lactobacillus acidophillus of the present invention. (Cinnamoyl esterase) nucleic acid molecule is a functional equivalent of the nucleic acid molecule constituting it, for example, Although some sequences of the Cinnamoyl esterase nucleic acid molecules from Lactobacillus acidophillus are modified by deletion, substitution or insertion, Lactobacillus ash Cinnamoyl esterase from Lactobacillus Acidophillus (Cinnamoyl esterase) is a concept containing a variant (variants) that can function functionally the same as the nucleic acid molecule. Specifically, the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

On the other hand, the gene encoding the Cinnamoyl esterase protein derived from Lactobacillus Acidophillus of the present invention can be provided included in a recombinant vector for intracellular delivery. The recombinant vector includes pCE21lactob4NX represented by the cleavage map of FIG. 4.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been re-introduced into the cell by an artificial means as modified.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. The recombinant vector is preferably a recombinant yeast expression vector or a recombinant plant expression vector.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinotricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. However, the present invention is not limited thereto.

The present invention also provides a microorganism transformed with a recombinant vector comprising a Cinnamoyl esterase gene derived from Lactobacillus Acidophillus of the present invention. The host to be transformed can be a variety of prokaryotes or eukaryotes, preferably E. coli.

The method for transforming with a recombinant vector may be any method known in the art without particular limitation such as fusion of bacterial protoplasts, electroporation, projectile bombardment, and infection with viral vectors.

By culturing the transformant prepared as described above, cells can be harvested and crushed to obtain cinnamoyl esterase produced in high yield. Therefore, according to another embodiment of the present invention, there is provided a method for producing cinnamoyl esterase, comprising the step of preparing the cinnamoyl esterase expression vector and introducing the transformed vector into a microorganism. do. Preferably, the method may further include culturing the transformed transformant after the transformation step.

The medium used for the culture includes any host cell, including E. coli, and any culture medium, solution, solid, semi-solid or rigid support, which may support or contain the cell contents, preferably LB medium, SOB Medium or TB medium.

Preferably, the culturing step may be incubated by adding 0.5-2.0 mM ITPG at a culture temperature of 27 ° C. to 37 ° C. for efficient expression in cinnamoyl esterase host (preferably E. coli) cells.

In another aspect, the present invention provides a probiotic containing the transforming microorganism.

The transforming microorganism expressing Cinnamoyl esterase derived from Lactobacillus acidophillus of the present invention provides a hydrosinamic acid useful for preventing and treating diabetes, and thus may be usefully used as a probiotic. Can be. The probiotics of the present invention can be used in animals, including livestock, and can be added to food or animal feed. It can also be used in veterinary medicines and medicines.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  One. Cinnamo Cinnamoyl esterase activity  Selection of lactic acid bacteria which we possess

1-1 Diabetic Group and In non-diabetic patients  Detached Lactic acid bacteria  comparison analysis

In order to separate lactic acid bacteria with cinnamoyl esterase activity from human intestinal microorganisms, five pairs of young men and women in their 20s and 30s with similar ages were selected to obtain fecal samples. About 1 g of the recovered fecal sample was transferred to 50 ml of sterile Falcon tube immediately after recovery, and then diluted in 9 ml of peptone water in anaerobic condition. In order to separate the microorganisms from the diluted sample, dilutions were prepared 10 times in succession, and then 100 μl each of the prepared selective medium and normal medium (MRS) were plated. As a selection medium, LAMVAB (Difco) medium, which is a selection medium for lactic acid bacteria, was used. Each plate was plated for 37 hours at 37 ℃ anaerobic culture using BD GasPak Anaerobic Systems (BectomDicKinson and company, USA). In order to provide anaerobic conditions, the inoculated medium in the GasPak Jar was used, and the GasPak pouch was used. After the experiment was activated by heating at 160 ° C. for 2 hours, oxygen was removed using a dried catalyst.

EtFA (Ethyl felurate), an indirect substrate of hydroxycinnamic acid, similar to chlorogenic acid, to isolate strains with high activity cinnamoyl esterase activity from lactic acid bacteria isolated from each fecal sample Feruloyl esterase plate assay was performed. That is, about 200 isolate strains selected from each plate medium were transferred to MRS solid medium containing 0.1% (w / v) ethyl ferulate (analogous substrate), and then each medium was subjected to 72 hours at 37 ° C. as described above. Cultured anaerobicly for a while. After incubation, the strain having a clear zone or halo of 3 mm or more in diameter was estimated as a cinnamoyl esterase active strain.

As a result, as shown in Figure 1, it was confirmed that there is no conventional cinnamoyl esterase activity in the case of Lactobacillus platarum (Lactobacillus plantarum) used as a control, but the transparent ring was not found in the total intestinal lactic acid bacteria isolated Six strains in the diabetic group and about 42 strains in the non-diabetic group showed transparent rings, which were successfully isolated. In particular, F46, which was isolated from normal feces and found to have the best cinnamoyl esterase activity, was found to be 99.5% consistent with Lactobacillus acidophilus NCFM. Therefore, F46 was selected as a strain having cinamoyl esterase potential activity through ethyl ferulate, and used for future experiments.

1-2. ELSD - HPLC Using Cinnamo Estherazade  (cinnamoyl esterase ) Validation

A test was performed to verify the cinnamoyl esterase activity of the isolated F46 lactic acid bacteria selected in Example 1-1. 0.5% (w / v) chlorogenic acid was mixed with MRS liquid medium as a substrate and sterilized at 121 ° C. for 15 minutes, and used as a measuring medium. After inoculating the strain precultured in MRS liquid medium at 1% (v / v) ratio and incubated for 72 hours at 37 ℃, pretreatment of the culture was carried out as follows. First, 5 mL of the culture solution was mixed well by adding 0.1% (w / v) ascorbic acid, and stirred and extracted with 2.5 ml of ethyl acetate, followed by centrifugation (8000 rpm, 20 min, 4 ° C.). It was. After standing at −20 ° C., the supernatant was collected, and the obtained supernatant was filtered through a 0.45 μm membrane filter and used as an analytical sample for HPLC (high performance liquid chromatography, high performance liquid chromatography). Chlorogenic acid and caffeic acid were used as a standard by dissolving in phosphate buffer (PBS). For the HPLC quantitative analysis of each sample, each concentration of standard was compared after performing pretreatment of the sample. HPLC analysis conditions are as shown in Table 1 below. As a result of HPLC analysis, chlorogenic acid showed retention time at 2.3 minutes and caffeic acid at 3.4 minutes, and the F46 strain also showed the same peak at the same retention time (FIG. 2). Through the above experiment, it was confirmed that the cinnamoyl esterase activity of decomposing chlorogenic acid of the F46 strain into caffeic acid.

Instrument Waters 1515 Binary HPLC Pump Detector ELSD 800 (Alltech) Column inersil ^® ODS-3V, 5 μm, 4.0 × 150 mm (GL Sciences Inc. Japan) Mobile phase 0.5% formic acid in water: MeCN = 3: 1 Drift tube Temp. 60 ° C Column Temp. 40 ℃ Flow rate 1 mL / min Injection Vol. 20 μl

Example  2. Cinnamo Estradiol  ( cinnamoyl esterase ) gene Cloning

In order to isolate the gene of cinnamoyl esterase from Lactobacillus ashidophilus F46 isolated in Example 1, a database registered in GENBANK was searched. Therefore, based on the nucleotide sequence of cinnamoyl esterase derived from L. jonnosonii strain N6.2, which is known to date, two motifs (LVGHSQGGVVASMLAG, LLAPAA) are considered to be important for cinnamoyl esterase activity. As a result of searching the gene sequence, alpha / beta fold familly hydrolase, which is assumed to be an esterase in the genome database of the Lactobacillus ashdophilus NCFM strain whose genomic sequence was identified, was identified as L. Cinnamoyl esterase sequence derived from jonnosonii strain N6.2 showed about 68% homology with DNA at the amino acid level. Therefore, based on the putative esterase sequence of the Lactobacillus ashdophyllus NCFM genome sequence, two primers corresponding to the upstream of the start codon and the downstream of the stop codon were designed (Table 2).

primer order CEacidoNde-C 5'-aaaaacatatgtctcgcattacaattgat-3 ' CEacidoXho-N 5'-aaaaactcgagaaataggggcttcaaaaattc-3 '

Amplification of the gene encoding cinnamoyl esterase from Lactobacillus ashidophilus F46 strain was confirmed using the primers CEacidoNde-C and CEacidoXho-N prepared through the above procedure. First, the Lactobacillus ashidophilus F46 strain was incubated in 5 mL of MRS liquid medium at 37 ° C. for 24 hours, and then genomic DNA was isolated using a genomic DNA purification kit, followed by PCR (Polymerase Chain Reaction). It was used as a template for. PCR reaction was carried out with 10 cycles of 1 minute at 95 ° C, 30 seconds at 45 ° C, and 1 minute at 72 ° C with the reaction solution prepared using CEhel-C, CEhel-N primer, taq polymerase, dNTP mixure, and Taq reaction buffer. PCR was carried out under the conditions of 27 cycles of 1 minute at 95 ° C, 30 seconds at 54 ° C, and 1 minute at 72 ° C. Each obtained PCR product was subjected to electrophoresis using a 1.2% agarose gel to confirm amplification of a gene fragment of about 700 bp (FIG. 3). The identified PCR products were purified using a PCR Product Purification Kit and sequenced with an ABI PRISM 3730 DNA analyzer, resulting in an open reading frame consisting of 741 bp of DNA (SEQ ID NO: 1). Cinnamoyl esterase gene was identified and it was confirmed that it encodes 247 amino acids (SEQ ID NO: 2).

Example  3. Preparation of transformants and Cinnamo Estradiol  ( cinnamoyl esterase ) Check activity

Expression cloning was performed to confirm whether the gene of the final PCR product identified in Example 2 encodes cinamoyl esterase. Specifically, the PCR product and the vector (pET21a) purchased from Novagen were cut with the same restriction enzyme and then transformed into E. coli JM109 competent cells after ligation. After transformation, 100 μL of the reaction solution was plated on LB agar plates containing ampicillin (ampicillin, 50 μL / mL) and incubated overnight at 37 ° C. PCR was performed for the selection of cinnamoyl esterase clones into E. coli in the colonies of cultured plates. Primers used the same ones used for template amplification (Table 2). As a control, E. coli JM109 having the same vector but without transformation was used. As a result, it was confirmed that one band appeared at about 700 bp only in the transformed strain and the insertion was successful, and the strain having the best cinnamoyl esterase activity was selected. A transformed vector was obtained from E. coli JM109 transformed using the Plasmid Miniprep kit (Axygen Co.), which was named pCE21lactob4NX (FIG. 4).

After transforming the vector pCE21lactob4NX obtained through the above procedure to the high expression of cinamoyl esterase in Escherichia coli into BL21 (DE3) strain, 16 hours in 5 mL of LB medium containing 50 μg / mL of ampicillin antibiotics. After pre-culturing for 10 minutes, the main culture was incubated with LB medium at 37 ℃ OD600 nm until the absorbance was 0.5. Expression of cinnamoyl esterase, a recombinant protein of interest, was induced by aseptic addition of 1 mM IPTG (isopropyl-1-thio-β-D-galactoside) and incubated for 6 hours. The cultured cells were collected by centrifugation at 12,000 rpm for 5 minutes, washed three times with 50 mM, pH7 phosphate buffer. Cultured cells were disrupted by ultrasound and analyzed by SDS-PAGE, and a new band was found at about 27 kDa compared to the control (FIG. 6). As a result, the molecular weight of this band was also consistent with the expected molecular weight in the amino acid sequence of the gene.

In addition, in order to confirm the conversion of recombinant cinnamoyl esterase to chlorogenic acid to caffeic acid and to confirm the amount of production, a part of the culture medium cultured in the same manner as described above is a coenzyme solution containing cinnamoyl esterase. The obtained cells were suspended in 1 ml of 50 mM PBS buffer (pH 7.0) and subjected to sonication to destroy the cells. The supernatant was used as coenzyme solution by centrifugation at 12,000 rpm for 5 minutes. The activity of cinnamoyl esterase was obtained by adding 10 mM chlorogenic acid to 50 mM PBS (pH 7.0) and reacting the crude enzyme solution (10 μg / mL) for 10 minutes at 37 ° C, followed by filtration with 0.45 μm filter. The solution was analyzed by HPLC. As a control, the enzyme solution from Escherichia coli BL21 (DE3) into which plasmid pET21 (a) was inserted was used. As shown in FIG. 5, the recombinant cinnamoyl esterase was almost hydrolyzed to produce caffeic acid (retention time, 3.44 minutes) after the enzymatic reaction. Could be observed. In addition, it was confirmed that the retention time of 1.4 minutes was the peak of quinic acid as a result of HPLC analysis under the same conditions. In this enzyme reaction, quinic acid was also produced. This result confirms that hydrolysis of caffeic acid and quinic acid by chlorogenic acid and cinnamoyl esterase. On the other hand, in the enzyme reaction solution from Escherichia coli BL21 (DE3) in which the plasmid pET21 (a) was used as a control, the decomposition of chlorogenic acid added as a substrate was not performed at all, so that not only caffeic acid but also quinic acid could be observed. (Fig. 5- (a)).

Example  4. In transformants Cinnamo Estradiol  ( cinnamoyl esterase Establishment of optimal conditions for

In order to establish the optimal condition for the expression of cinnamoyl esterase in Escherichia coli BL21 (DE3) strain transformed with pCE21lactob4NX, the effect on the temperature and the amount of IPTG was confirmed. After pre-incubation at 37 ° C. for 16 hours in a medium containing 50 μg / mL of ampicillin, incubation at 37 ° C. in 5 mL of LB medium was performed at 600 nm until the absorbance was 0.5, and then 1 Culture was based on the addition of mM IPTG.

First, in order to determine the effect of temperature, the culture was incubated for 16 hours after addition of IPTG at 22 ° C, 27 ° C, 32 ° C, and 37 ° C, followed by enzyme activity analysis and SDS-PAGE analysis (FIG. 7). In order to determine the effect of the IPTG was added by concentration (0.1mM, 0.5mM, 1mM, 2mM), the cells were sonicated and subjected to enzymatic activity analysis and SDS-PAGE analysis using chlorogenic acid as a substrate (FIG. 6). As a result of the experiment, the highest expression was observed when cultured for 16 hours after the addition of 1.0 mM IPTG under the culture temperature of 32 ℃.

Example  5. His - Taq Using Cinnamo Esterase  refine

Ampicillin (Ampicillin, 50mg / ml) was inoculated with 0.1% bacteria incubated for 16 hours in 12 4ml LB medium added with antibiotics was incubated for 3 hours in a 37 ℃ shaking incubator so that the OD value is 0.5. After measuring the OD value, the expression of the protein of interest was induced according to the optimal expression conditions (32 ° C., IPTG 1 mM) identified in Example 4. After the incubation was completed, the cells were dispensed into four 15 ml palcon tubes and centrifuged at 8,000 g for 10 minutes at 4 ° C. to recover the cells. After 16 hours storage at -80 ° C, thaw for 40 minutes on ice and washed three times with 5ml of 50 mM PBS. It was collected in one tube and finally suspended in 5 ml. The coenzyme was repeated after sonication (10sec X 20) and centrifuged at 13,000rpm and 40 ° C for 30 minutes to prepare a supernatant. Enzyme purification was carried out by adsorbing the prepared crude enzyme solution using a Ni-sepharose ™ HP column, and then washed with 100 mL of washing buffer (50 mM PBS, 300 mM NaCl, 30 mM Imidazole-pH 7.0) to remove non-adsorbed protein. Adsorbed protein eluted by elution of 2 ml per imidazole concentration 5 times using elution buffer (50mM PBS, 300mM NaCl, 100mM / 150mM / 200mM Imidazole-pH 8.0) according to the concentration of imidazole The purified result was confirmed using a 10% SDS-PAGE gel.

Example  6. Refined Cinnamo Estradiol  For active pH  And influence of temperature

6-1. pH Influence of

To examine the effect of pH on purified cinnamoyl esterase activity, enzyme (100ug / ml) was added to each 50 mM Citrate-citric buffer (pH3-pH6) and Phosphate buffer (pH6-8) containing 10 mM chlorogenic acid. ), Tris-HCl buffer (pH8-pH9) in a buffer buffered for 10 minutes at 37 ℃. To stop the reaction, it was put in ice as soon as the reaction was finished. Activity measurements were performed using HPLC-ELSD analysis under the same conditions.

As a result, as shown in Figure 9, the optimum pH was found to be pH 7.5, showing a relative activity of at least 80% even at pH 7 to pH 8.5. At pH 6.5, the activity was about 70%, but at pH 6.0 and pH 9, the activity was less than 50%, especially at pH 4.0 and pH 3.0, 21.5% and 1.5%, respectively. Therefore, purified L. acidophillus F-46-derived cinnamoyl esterase showed optimal activity in the vicinity of about basic pH between pH 7.5 and pH 8.0.

6-2. Influence of temperature

To examine the effect of temperature on purified cinnamoyl esterase activity, enzymes (100 ug / ml) were used as substrates in 10 mM chlorogenic acid in 50 mM PBS buffer (pH 7.5), respectively. After the reaction for 10 minutes at a temperature of 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, the activity was examined. The culture conditions except for the temperature was kept constant by performing the same. Activity measurements were performed using HPLC-ELSD analysis under the same conditions.

As a result, as shown in Figure 10, the highest activity was shown at 50 ℃ and showed a high relative activity of 97% at 45 ℃. It showed 69.5% relative activity at 37 ℃ and low activity below 23.4% at low temperature of 25 ℃. On the other hand, it showed a relative activity of 70% or more even at the temperature of 40 ℃, 55 ℃ relatively high activity at high temperatures. Therefore, purified L. acidophillus F-46-derived cinnamoyl esterase was found to exhibit optimal activity at 45 to 50 ℃.

Example  7. Refined Cinnamo Esterase  Confirm stability

To test the pH stability of the purified cinnamoyl esterase, the enzyme was clouded at each pH (3-9) and left overnight at 4 ° C. Thereafter, the enzyme of each pH was added to 50mM PBS (pH7.5) containing 10mM chlorogenic acid as a substrate and reacted at 50 ° C for 10 minutes. As a result of the experiment, it was confirmed that the most stable at pH 7, as shown in FIG. Relative activity of over 80% remained at pH5, 6 and pH8, and over 50% at pH4,9. But at pH3 only 17.7% of activity remained (FIG. 11).

In addition, in order to test the thermal stability of the purified cinnamoyl esterase, the enzyme was incubated at 25 ° C., 37 ° C., 45 ° C., 50 ° C., 55 ° C., 60 ° C. for one hour, and 10 mM chlorogenic acid was added as a substrate. After the addition of 50mM PBS (pH7.5) was reacted for 10 minutes at 50 ℃. As a result, as shown in Figure 11, the most stable at 25 ℃ and 37 ℃, 50 ℃ was the most active activity temperature was 41.2% showed a drop in activity. At 60 ° C., only 13.9% of activity remained (FIG. 12).

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Novel Cinnamoyl esterase from Lactobacillus Acidophillus <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 741 <212> DNA <213> Lactobacillus Acidophillus <400> 1 atgtctcgca ttacaattga gagagatggc ctcactttag taggcgatcg tgaagaacct 60 tttggtgaaa tttatgatat ggctattctt atgcatggct ttactgcaaa cagaaatacc 120 cctttattaa ggcaaattgc cgataactta cgcgatgaaa atgtagctag tgttcgtttt 180 gactttaatg gacatggcga aagtgatggt gcgtttgaag atatgactgt ctgcaatgaa 240 attgcggatg cacaaaagat tttagaatac gtacgtactg atccacatgt acgtaatatt 300 tttttggtgg gacattcaca aggtggcgta gttgcttcaa tgctagctgg actttatcca 360 gacattgtta aaaaagtcgt tcttttagca ccggctgctc aattaaagga tgatgcctta 420 aatggtgaca ctcaaggcgc aacttataat cctgaacaca ttccagcagc tattccattt 480 catggtaaaa agcttggcgg cttttattta agaaccgcac aagtattacc aatctatgaa 540 attgctaaac attatactaa tccagtttct ataattgttg gctccaatga tcaagtagtt 600 gcccctaaat actcaaagaa atacgacgaa gtctacgaaa acagtgaact gcacatggtc 660 ccagatgcag atcatagttt cactggtcaa tataaagact ctgctgttga tttaactgca 720 gaatttttga agcccctatt t 741 <210> 2 <211> 247 <212> PRT <213> Lactobacillus Acidophillus <400> 2 Met Ser Arg Ile Thr Ile Glu Arg Asp Gly Leu Thr Leu Val Gly Asp   1 5 10 15 Arg Glu Glu Pro Phe Gly Glu Ile Tyr Asp Met Ala Ile Leu Met His              20 25 30 Gly Phe Thr Ala Asn Arg Asn Thr Pro Leu Leu Arg Gln Ile Ala Asp          35 40 45 Asn Leu Arg Asp Glu Asn Val Ala Ser Val Arg Phe Asp Phe Asn Gly      50 55 60 His Gly Glu Ser Asp Gly Ala Phe Glu Asp Met Thr Val Cys Asn Glu  65 70 75 80 Ile Ala Asp Ala Gln Lys Ile Leu Glu Tyr Val Arg Thr Asp Pro His                  85 90 95 Val Arg Asn Ile Phe Leu Val Gly His Ser Gln Gly Gly Val Val Ala             100 105 110 Ser Met Leu Ala Gly Leu Tyr Pro Asp Ile Val Lys Lys Val Val Leu         115 120 125 Leu Ala Pro Ala Ala Gln Leu Lys Asp Asp Ala Leu Asn Gly Asp Thr     130 135 140 Gln Gly Ala Thr Tyr Asn Pro Glu His Ile Pro Ala Ala Ile Pro Phe 145 150 155 160 His Gly Lys Lys Leu Gly Gly Phe Tyr Leu Arg Thr Ala Gln Val Leu                 165 170 175 Pro Ile Tyr Glu Ile Ala Lys His Tyr Thr Asn Pro Val Ser Ile Ile             180 185 190 Val Gly Ser Asn Asp Gln Val Val Ala Pro Lys Tyr Ser Lys Lys Tyr         195 200 205 Asp Glu Val Tyr Glu Asn Ser Glu Leu His Met Val Pro Asp Ala Asp     210 215 220 His Ser Phe Thr Gly Gln Tyr Lys Asp Ser Ala Val Asp Leu Thr Ala 225 230 235 240 Glu Phe Leu Lys Pro Leu Phe                 245

Claims (9)

Cinnamoyl esterase protein from Lactobacillus acidophillus, consisting of the amino acid sequence of SEQ ID NO: 2.
Gene encoding the Cinnamoyl esterase protein of claim 1.
The gene of claim 2, wherein the gene is SEQ ID NO: 1.
Recombinant vector comprising the gene of claim 2.
The vector of claim 4, wherein the recombinant vector is pCE21lactob4NX represented by a cleavage map of FIG. 4.
Transgenic microorganism transformed with the recombinant vector of claim 4.
The transforming microorganism according to claim 6, wherein the microorganism is Escherichia coli.
Preparing a recombinant cinnamoyl esterase expression vector of claim 4; And
A method for producing cinnamoyl esterase comprising the step of transforming by introducing the prepared expression vector into the microorganism.
Probiotics comprising the transforming microorganism of claim 6 as an active ingredient.

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