KR101722984B1 - A Method for production of cadaverin by immobilization of lysine decarboxylase-overexpressing recombinant E. coli using barium-alginate - Google Patents

Abstract

The present invention relates to Escherichia coli coli), lysine decarboxylase (lysine decarboxylase derived) recombinant E. coli the gene introduction, and high concentration with the manufacture and this in the whole cell enzyme-immobilized insoluble even by using the recombinant protein high concentration cadaverine (cadaverine) production ≪ / RTI > of cadaverine. The present invention introduces a barium-alginate immobilization method that solves the problems of the conventional calcium-alginate immobilization method and utilizes the same to remove cadaverine from high-concentration lysine (L-lysine) It is possible to obtain economical efficiency by overcoming the problems of high efficiency, stability, and reuse in production and making it possible to use it repeatedly.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing high-concentration cadaverine by immobilization of recombinant E. coli overexpressing lysine decarboxylase using a barium-alginate carrier,

The present invention relates to a method for immobilizing whole cells used in the production of cadaverine which is used as a nylon precursor and more particularly to a method for immobilizing lysine decarboxylase gene derived from Escherichia coli And a method of immobilizing a recombinant Escherichia coli strain on a barium-alginate carrier to convert a high concentration of cadaverine.

Development of industrial strains for the production of bio-based plastics produced for the purpose of replacing petroleum-derived plastics have been studied extensively. In this connection, nylon monomers, such as cadverine, putrescine, may be used as an active ingredient in Escherichia coli or Corynebacterium (European Patent Publication No. 2540836; Qian ZG, et al., Biotechnol (2002)), which has been applied to industrial strains such as glutamic acid and glutamicum by fermentation from glucose or xylose by applying a metabolic engineering method Bioeng . 2009 Nov 1; 104 (4): 651-62; Qian ZG, et al., Biotechnol Bioeng . 2011 Jan; 108 (1): 93-103). However, when such a method is used, productivity and concentration are relatively inefficient and thus it has been difficult to utilize it industrially.

Recently, in order to overcome this problem, a case has been reported in which a high concentration of cadaverine is produced by a whole-cell reaction from lysine (L-lysine), which is a mass-produced precursor in several companies (Ma W, et al., Biotechnol Lett . 2015 Apr; 37 (4): 799-806). However, this method has a problem in reusing the enzyme because it tends to lose activity of the enzyme easily although the activity is good and the reaction can be carried out at a high concentration.

On the other hand, in a high concentration reaction, the activity of the whole cell of E. coli overexpressing lysine decarboxylase is inhibited by one or two reactions. In order to overcome this phenomenon, the whole cell of lysine decarboxylase-overexpressing E. coli You need to create a system that can be used many times, and you can use the immobilization method.

In this connection, a calcium-alginate carrier is often used as a generally used method for enzyme immobilization. However, when cadaverine conversion reaction is carried out in L-lysine, when Ca-alginate is immobilized, immobilization with high concentration of lysine or cadaverine Reuse of the immobilized carrier by the dissolution of the carrier was almost impossible.

Therefore, the present inventors have found that, in order to maintain the activity of the enzyme from a high concentration of substrate for the purpose of mass production of cadaverine at a high concentration and to enable stability and repeated reuse, the whole cell enzyme is immobilized on a barium-alginate carrier The present invention has been completed.

It is an object of the present invention to overcome the difficulties of low efficiency of the production method of cadaverine and reuse of enzymes through the existing whole cell reaction and to overcome the difficulty of reusing the enzymes by using whole cells of lysine decarboxylase overexpressing barium-alginate -alginate carrier, and a method for mass-producing cadaverine using the same.

In order to achieve the above object, the present invention provides a recombinant Escherichia coli for producing cadaverine, which is produced by introducing a lysine decarboxylase (cadA) gene into E. coli.

In addition,

1) Escherichia coli) the lysine decarboxylase (lysine decarboxylase, cadA) derived from the step of producing an expression vector comprising the gene; And

2) a step of transforming the expression vector of step 1) into E. coli, which comprises preparing a recombinant E. coli for producing cadaverine.

The present invention also provides a method for immobilizing lysine dicarboxylase overexpressing recombinant Escherichia coli on a barium-alginate carrier according to the present invention.

The present invention also provides immobilized recombinant Escherichia coli, wherein the lysine dicarboxylase overexpressing recombinant Escherichia coli according to the present invention is immobilized on a barium-alginate carrier.

In addition,

1) immobilizing lysine dicarboxylase overexpressing recombinant Escherichia coli according to the present invention on a barium-alginate carrier; And

2) recovering the cadaverine from the culture medium in which the immobilized recombinant E. coli of step 1) has been cultured.

In accordance with the present invention, a high concentration of cadaverine can be produced from L-lysine using recombinant Escherichia coli obtained by introducing lysine decarboxylase (cadA) gene into E. coli.

In addition, when the present invention is applied, when the immobilization method using a conventional calcium-alginate carrier is applied, the carrier dissolves due to high concentrations of lysine and cadaverine, Can be improved, so that the productivity of cadaverine can be increased.

Figure 1 is a schematic representation of an Escherichia coli (+) vector map including a lysine decarboxylase (cadA) derived from E. coli .
Fig. 2 is a graph The lysine decarbpylase expression and size of E. coli- derived lysine decarbpylase were confirmed by SDS-PAGE.
FIG. 3 shows that when the carrier immobilized with BaCl ₂ or CaCl ₂ is subjected to a 1M lysine reaction, dissolution of the carrier occurs in the case of CaCl ₂ , but dissolution does not occur when BaCl ₂ is used as the carrier It is a picture showing.
FIG. 4 is a graph showing the cadaverine conversion rates of immobilized cells and cells immobilized with BaCl ₂ or CaCl ₂ with time. FIG.
FIG. 5 is a graph showing the conversion of cadaverine according to each cycle when 18 immobilized cells and cells immobilized with BaCl ₂ or CaCl ₂ were subjected to repeated experiments.
FIG. 6 is a graph comparing relative enzyme activities when immobilized with various concentrations of BaCl _2. FIG.
Figure 7 is a graph comparing relative enzyme activities when immobilizing carriers of various sizes.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for immobilizing a lysine decarboxylase (cadA) protein overexpressing recombinant Escherichia coli on a barium-alginate carrier.

The present invention also provides immobilized recombinant Escherichia coli having lysine dicarboxylase overexpressing recombinant Escherichia coli immobilized on a barium-alginate carrier.

The recombinant E. coli is a gene encoding lysine decarboxylase ( cadA Gene) into a host E. coli strain.

The cadA gene is expressed by Escherichia It is preferable that the gene is derived from E. coli .

The cadA gene is preferably composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. In the nucleotide sequence of SEQ ID NO: 1, one or two or more genes may be added, deleted or substituted.

The host E. coli preferably is E. coli BL21, but is not limited thereto, and E. coli species are all possible.

The concentration of the barium-alginate carrier used in the immobilization may be 0.1 to 0.6 M. However, when the carrier concentration is 0.1 M, the carrier may be damaged even in the presence of a weak external impact, thereby affecting the reusability of the carrier . Therefore, the concentration of the barium-alginate carrier is preferably 0.2 M to 0.6 M, more preferably 0.2 M.

The size of the barium-alginate carrier used in the immobilization is preferably 1 to 5 mm in diameter, and more preferably 2 mm.

The immobilization method can be performed by culturing the recombinant E. coli to express lysine decarboxylase, mixing the diluted cell solution with the sodium alginate solution, and then adding BaCl ₂ as the immobilization support.

The present invention also provides a method for mass-producing cadaverine using recombinant E. coli overexpressing lysine decarboxylase protein immobilized on a barium-alginate carrier.

The production of the cadaverine can be produced by batch or continuous process of recombinant E. coli.

Specifically, the cadaverine production method

1) culturing the recombinant E. coli according to the present invention to overexpress the lysine decarboxylase protein;

2) immobilizing the recombinant Escherichia coli overexpressing the lysine decarboxylase protein cultured in the step 1) on a barium-alginate carrier; And

3) recovering the cadaverine from the culture solution in which the immobilized recombinant E. coli has been cultured in the step 2).

In this method, the culture of step 1) is preferably carried out under aerobic conditions at a temperature of 20 to 40 DEG C for 2 to 5 days, and it is more preferable to culture for 24 to 72 hours at a temperature of 30 DEG C under an aerobic condition But is not limited thereto.

In the above method, the concentration of BaCl ₂ used in the immobilization of step 2) is preferably 0.1 to 0.6 mM.

In the above method, the size of the immobilization support of step 2) is preferably 1 to 5 mm in diameter, more preferably 2 mm.

The present invention also provides a recombinant Escherichia coli for producing cadaverine, wherein a lysine decarboxylase gene is introduced into E. coli.

In addition,

1) preparing an expression vector containing a lysine decarboxylase gene; And

2) transforming the expression vector of step 1) into Escherichia coli. The present invention also provides a method for producing recombinant Escherichia coli for cadaverine production.

In the above method, the cadA gene of step 1) is preferably composed of the nucleotide sequence of SEQ ID NO: 1, but not limited thereto, and the nucleotide sequence in which one or two or more genes of SEQ ID NO: 1 are added, deleted or substituted Lt; / RTI >

In this method, the expression vector of step 1) is preferably a pET-24ma plasmid, but not limited thereto, and all viral vectors or non-viral vectors expressing in E. coli can be used.

In the above method, the E. coli of step 2) is preferably E. coli BL21, but is not limited thereto, and all E. coli species are possible.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below.

The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art. It is provided to fully inform the category of invention to a knowledgeable person.

Lysine  Dicarboxylase (lysine decarboxylase ) Production of Overexpressed Recombinant Escherichia coli

≪ 1-1 > Preparation of transformant

Echerichia After isolating the genome from E. coli K12 MG1655 (KCCM 41310), a gene encoding lysine decarboxylase (cadA) was isolated and purified. At this time, the gene was composed of the following base sequences.

ATGAACGTTATTGCAATATTGAATCACATGGGGGTTTATTTTAAAGAAGAACCCATCCGT

 GAACTTCATCGCGCGCTTGAACGTCTGAACTTCCAGATTGTTTACCCGAACGACCGTGAC

 GACTTATTAAAACTGATCGAAAACAATGCGCGTCTGTGCGGCGTTATTTTTGACTGGGAT

 SEARCH

 Gt;

 AGCTTCTTTGAATATGCGCTGGGTGCTGCTGAAGATATTGCTAATAAGATCAAGCAGACC

 ACTGACGAATATATCAACACTATTCTGCCTCCGCTGACTAAAGCACTGTTTAAATATGTT

 CGTGAAGGTAAATATACTTTCTGTACTCCTGGTCACATGGGCGGTACTGCATTCCAGAAA

 AGCCCGGTAGGTAGCCTGTTCTATGATTTCTTTGGTCCGAATACCATGAAATCTGATATT

 TCCATTTCAGTATCTGAACTGGGTTCTCTGCTGGATCACAGTGGTCCACACAAAGAAGCA

 GAACAGTATATCGCTCGCGTCTTTAACGCAGACCGCAGCTACATGGTGACCAACGGTACT

 TCCACTGCGAACAAAATTGTTGGTATGTACTCTGCTCCAGCAGGCAGCACCATTCTGATT

 GACCGTAACTGCCACAAATCGCTGACCCACCTGATGATGATGAGCGATGTTACGCCAATC

 TATTTCCGCCCGACCCGTAACGCTTACGGTATTCTTGGTGGTATCCCACAGAGTGAATTC

 CAGCACGCTACCATTGCTAAGCGCGTGAAAGAAACACCAAACGCAACCTGGCCGGTACAT

 Gt

 ACACTGGATGTGAAATCCATCCACTTTGACTCCGCGTGGGTGCCTTACACCAACTTCTCA

 CCGATTTACGAAGGTAAATGCGGTATGAGCGGTGGCCGTGTAGAAGGGAAAGTGATTTAC

 GAAACCCAGTCCACTCACAAACTGCTGGCGGCGTTCTCTCAGGCTTCCATGATCCACGTT

 AAAGGTGACGTAAACGAAGAAACCTTTAACGAAGCCTACATGATGCACACCACCACTTCT

 CCGCACTACGGTATCGTGGCGTCCACTGAAACCGCTGCGGCGATGATGAAAGGCAATGCA

 GGTAAGCGTCTGATCAACGGTTCTATTGAACGTGCGATCAAATTCCGTAAAGAGATCAAA

 CGTCTGAGAACGGAATCTGATGGCTGGTTCTTTGATGTATGGCAGCCGGATCATATCGAT

 ACGACTGAATGCTGGCCGCTGCGTTCTGACAGCACCTGGCACGGCTTCAAAAACATCGAT

 AACGAGCACATGTATCTTGACCCGATCAAAGTCACCCTGCTGACTCCGGGGATGGAAAAAA

 GACGGCACCATGAGCGACTTTGGTATTCCGGCCAGCATCGTGGCGAAATACCTCGACGAA

 CATGGCATCGTTGTTGAGAAAACCGGTCCGTATAACCTGCTGTTCCTGTTCAGCATCGGT

 ATCGATAAGACCAAAGCACTGAGCCTGCTGCGTGCTCTGACTGACTTTAAACGTGCGTTC

 GACCTGAACCTGCGTGTGAAAAACATGCTGCCGTCTCTGTATCGTGAAGATCCTGAATTC

 TATGAAAACATGCGTATTCAGGAACTGGCTCAGAATATCCACAAACTGATTGTTCACCAC

 AATCTGCCGGATCTGATGTATCGCGCATTTGAAGTGCTGCCGACGATGGTAATGACTCCG

 TATGCTGCATTCCAGAAAGAGCTGCACGGTATGACCGAAGAAGTTTACCTCGACGAAATG

 GTAGGTCGTATTAACGCCAATATGATCCTTCCGTACCCGCCGGGAGTTCCTCTGGTAATG

 CCGGGTGAAATGATCACCGAAGAAAGCCGTCCGGTTCTGGAGTTCCTGCAGATGCTGTGT

 GAAATCGGCGCTCACTATCCGGGCTTTGAAACCGATATTCACGGTGCATACCGTCAGGCT

 GATGGCCGCTATACCGTTAAGGTATTGAAAGAAGAAAGCAAAAAATAA (SEQ ID NO: 1)

Then, the purified cadA gene was cloned into pET-24ma plasmid (Hiroshi Sakamoto, Paris) (Fig. 1). The cloned pET-24ma plasmid was then transformed into E. coli BL21 (lambda DE3) (Novagen®).

<1-2> Lysine  Expression of dicarboxylase

The transformed E. coli was streaked on an LB plate. Then, after culturing in an incubator at 37 ° C for 24 hours, a single colony was obtained and pre-cultured in 5 ml of LB medium (aerobic condition, 37 ° C, 200 rpm, 24 hours culture). Then, 500 μl of the preculture was inoculated in 50 ml of LB containing kanamycin (50 μl / ml), IPTG was added to the final concentration of IPTG at 0.1 mM at 30 ° C. and 200 rpm at OD 600 nm 0.6 , 30 ° C, 200 rpm overnight. After 24 hours, 1 ml of cells were collected. After centrifugation at 13000 g, the supernatant was discarded, and then treated with 100 μl of Bugbuster (Novagen®), followed by incubation at 37 ° C for 1 hour.

After centrifuging the cell lysate at 13000 g, 16 μl of the supernatant and 4 μl of 5x SDS-PAGE loading buffer were mixed and boiled for 5 minutes at 100 ° C for sample pretreatment. The pretreated sample was loaded on a 12% SDS-PAGE and developed with 120V 700 mA current for 2 hours. SDS-PAGE gel was then stained with a staining solution (0.1% Coomassie Brilliant Blue R-250, 50% Gt; glacial &lt; / RTI &gt; acetic acid for 1 hour.

The stained SDS-PAGE gel was then decolorized (50% methanol and 10% glacial acetic acid decolorized for 2 hours, centrifuged at 13000 g for 80 kDa cell lysate, and then 16 μl of supernatant and 4 μl of 5x SDS-PAGE loading buffer The pre-treated sample was loaded on 12% SDS-PAGE, developed by flowing 120V 700 mA current for 2 hours, and then subjected to SDS-PAGE gel The staining solution was stained with 0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid for 1 hour.

Then, the stained SDS-PAGE gel was discolored (decolorized in 50% methanol and 10% glacial acetic acid solution for 2 hours to confirm the protein band near 80 kDa, confirming that the strain overexpressed lysine decarboxylase (Fig. 2).

My combination  Barium of E. coli Alginate The carrier  Immobilization using

<2-1> Barium Alginate The carrier  Immobilization using

The transformed E. coli prepared in Example 1 was streaked on an LB plate. Then, after culturing in an incubator at 37 ° C for 24 hours, a single colony was obtained and pre-cultured in 5 ml of LB medium (aerobic condition, 37 ° C, 200 rpm, 24 hours culture). Then, the pre-cultured cells were inoculated with 0.5 ml of LB medium (50 ml) and cultured to express the protein (aerobic condition, 37 ° C, 200 rpm, culture for 24 hours, added with 5 μl of 1 M IPTG after 3 hours of inoculation) . Cells were washed twice with distilled water and diluted to a cell concentration of 15 mg / mL dcw. Then 2.5 ml of the cell solution and 2.5 ml of the 5% sodium alginate solution were mixed to make a 2.5% solution of sodium alginate. Then, it was transferred to a 30 ml syringe (Korean vaccine, korea), and dropped to 0.2 M of CaCl ₂ or BaCl ₂ by using a syringe pump (LSP01, Longer) to form an immobilized carrier. The size of the immobilized carrier was adjusted by adjusting the pump speed to 2 mm.

The resulting calcium alginate-immobilized carrier and barium alginate carrier were used so that the total cell weight was equivalent to 0.3 mg. The final concentration of each component was 0.1 mM Pyridoxal 5'-phosphate, 1 M lysine, 500 mM pH 6.0, and a final volume of 500 μl was made in 1.7 ml microtube (Axygen, USA).

Subsequently, the reaction was carried out in a constant temperature water bath (BioFree, Korea) at 37 ° C. for 1 hour. Subsequently, the substrates immersed in the immobilized carrier twice with distilled water were washed, The reaction proceeded.

 As a result, when the carrier immobilized in the presence of a high concentration of lysine was repeatedly used 9 times or more, it was confirmed that the bead was dissolved in the case of calcium alginate. However, in the case of the barium-alginate carrier, dissolution of the immobilization support did not occur even at a high concentration of 9 or more conversion reaction, and recovery was easy (FIG. 3).

In order to examine the effect of the enzyme activity on the immobilized carrier, the concentration of the calcium alginate-immobilized carrier, barium alginate carrier, and unimmobilized whole cell produced through the above method were compared with each other to determine the concentration of cadaverine over time.

All reactions were carried out so that the total cell weight was equivalent to 0.3 mg. After the final concentration of each component was adjusted to 0.1 mM Pyridoxal 5'-phosphate, 1 M lysine, 500 mM pH 6.0, (Axygen, USA), and the reaction was carried out at 37 ° C in a constant temperature bath (BioFree, Korea). The samples were collected at 0, 30, 60, 120, and 180 minutes to analyze the concentration of the produced catarrhine by HPLC.

As a result, it was confirmed that the activity of the lysine decarboxylase was reduced by 39% in the case of the capsule-alginate-immobilized carrier, as compared with that of the original immobilized cell. On the other hand, in the case of the barium-alginate-immobilized support, the relative conversion of cadaverine to 78% relative to immobilized whole cells was observed, indicating that the decrease in activity of lysine decarboxylase was 17% less than that of calcium-alginate carrier (FIG. 4).

Further, in order to confirm how the enzyme activity decreases depending on the immobilization method, the calcium alginate-immobilized carrier and barium alginate carrier produced through the above method, and the lysine decarboxylase according to the number of re- (lysine decarboxylase).

All reactions were carried out so that the total cell weight was equivalent to 0.3 mg. After the final concentration of each component was adjusted to 0.1 mM Pyridoxal 5'-phosphate, 1 M lysine, 500 mM pH 6.0, (Axygen, USA) to a final volume of 500 μl. Subsequently, the reaction was carried out in a constant temperature water bath (BioFree, Korea) at 37 ° C. for 1 hour. Subsequently, the substrates immersed in the immobilized carrier twice with distilled water were washed, The reaction proceeded.

As a result, it was confirmed that the lysine decarboxylase activity was not observed when the immobilized whole cells and the calcium alginate-immobilized carrier were repeated 10 times or more at a high concentration.

On the other hand, in the case of the barium-alginate-immobilized carrier, it was confirmed that the lysine decarboxylase activity was maintained at about 50% even at the high-concentration conversion reaction repeated 19 times (FIG. 5).

Therefore, it was confirmed that the whole cell immobilization method using barium-alginate was a relatively good immobilization method in the high concentration cadaverine conversion reaction.

&Lt; 2-2 > Alginate (Barium-alginate) carrier  Optimization of manufacturing

One of the factors that may influence the enzyme activity in the preparation of barium-alginate carriers is the BaCl ₂ concentration used for immobilization support formation. After immobilization with various concentrations of BaCl ₂ , relative enzyme activities were compared.

The transformed E. coli prepared in Example 1 was streaked on an LB plate. Then, after culturing in an incubator at 37 ° C for 24 hours, a single colony was obtained and pre-cultured in 5 ml of LB medium (aerobic condition, 37 ° C, 200 rpm, 24 hours culture). Then, the pre-cultured cells were inoculated with 0.5 ml of LB medium (50 ml) and cultured to express the protein (aerobic condition, 37 ° C, 200 rpm, culture for 24 hours, added with 5 μl of 1 M IPTG after 3 hours of inoculation) . Cells were washed twice with distilled water and diluted to a cell concentration of 15 mg / mL dcw. Then 2.5 ml of cell solution and 2.5 ml of 5% sodium alginate solution were added to make a 2.5% solution of sodium alginate. After transferring to a 30-ml syringe (Korean vaccine, korea), a drop of 0.1, 0.2, 0.4, 0.6, and 0.8 M BaCl ₂ was applied to the immobilized carrier using a syringe pump (LSP01, Longer) I dropped it. The size of the immobilized carrier was adjusted by adjusting the pump speed to 2 mm.

E. coli The E. coli solution overexpressing the lysine decarboxylase derived from K12 MG1655 was dissolved in ice to a concentration of 0.3 mg, and the final concentration of each component was 0.1 mM Pyridoxal 5'-phosphate , 1 M lysine, 500 mM pH 6.0, and then a final volume of 500 μl was made in 1.7 ml microtube (Axygen, USA). Then, the reaction was carried out at 37 ° C in a water bath (BioFree, Korea) for 2 hours and analyzed using HPLC.

As a result, it was confirmed that the relative activity of lysine decarboxylase decreased with increasing BaCl ₂ concentration affecting immobilization support formation, and relative activity decreased rapidly to about 26% at 0.8 M . As the concentration of BaCl ₂ decreased, the relative activity of lysine decarboxylase decreased. However, when the BaCl ₂ concentration is less than 0.2 M, the immobilization support is easily damaged even under a slight impact, and recovery of the immobilization support is not easy (FIG. 6).

In addition, another factor that may affect enzyme activity may be the size of the carrier. Thus, immobilized carriers of various sizes were prepared and the relative enzyme activity per weight was compared.

The transformed E. coli prepared in Example 1 was streaked on an LB plate. Then, after culturing in an incubator at 37 ° C for 24 hours, a single colony was obtained and pre-cultured in 5 ml of LB medium (aerobic condition, 37 ° C, 200 rpm, 24 hours culture). Then, the pre-cultured cells were inoculated with 0.5 ml of LB medium (50 ml) and cultured to express the protein (aerobic condition, 37 ° C, 200 rpm, culture for 24 hours, added with 5 μl of 1 M IPTG after 3 hours of inoculation) . Cells were washed twice with distilled water and diluted to a cell concentration of 15 mg / mL dcw. Then 2.5 ml of cell solution and 2.5 ml of 5% sodium alginate solution were added to make a 2.5% solution of sodium alginate. Then, it was transferred to a 30 ml syringe (Korean vaccine, korea), and dropped to 0.2 M of BaCl 2 by using a syringe pump (LSP01, Longer) to form an immobilized carrier. The size of immobilized carrier was 1, 2, 3, 4, 5 mm.

The resulting barium alginate carrier was used in an amount corresponding to 0.3 mg of total cells. The final concentration of each component was adjusted to 0.1 mM Pyridoxal 5'-phosphate, 1 M lysine, 500 mM pH 6.0, A final volume of 500 μl was made in a 1.7 ml microtube (Axygen, USA). Then, the reaction was carried out in a constant temperature water bath (BioFree, korea) at 37 ° C for 1 hour and analyzed by HPLC after completion of the reaction.

As a result, the difference in lysine decarboxylase was found to differ by up to 21% depending on the immobilized carrier size, and the best relative enzyme activity was shown when the immobilized carrier size was 2 mm. However, this result suggests that the size may have a great influence on the enzyme activity, so that it can be utilized in immobilized carriers of various sizes (FIG. 7).

&Lt; 2-3 > Analytical Method for Measurement of Cadaviner Activity

 In order to compare the activity of immobilized whole cells, the derivatization of diethyl ethoxy methyl malonate (DEEMM), which can be used for diamine or amino acid analysis, was performed.

3 μl of diethylethoxymethylmalonic acid was transferred to a 1.7 ml microtube (Axygen, USA), and then 100 μl of methanol, 300 μl of a borate buffer of 50 mM pH 9 and 47 μl of distilled water were mixed, and 10 mM Of diamine or amino acid standard solution was transferred to a 1.7 ml microtube (Axygen, USA) containing the mixture and reacted at 70 ° C for 2 hours.

For the improvement of sample separation and detection, the following HPLC (YL9100 system, Youngin Instrument, Korea) conditions were analyzed. The temperature of the column was set to 35 ° C and the flow rate was set to 1 ml / min, and various organic compounds were added to the column. Among the solvents, acetonitrile having the lowest polarity was used, and 25 mM sodium acetate was used as a buffer solution, and the pH was adjusted to 4.8 using acetic acid.

The peaks of the diamine components of the polyamide-based nylon monomer do not overlap, and conditions for optimum separation are set as shown in Table 1 in consideration of the polarity difference between the fixed phase, the moving phase and the components.

Time (minutes) Acetonitrile (%) 25 mM sodium acetate (%) 0 20 80 2 25 75 32 60 40 35 20 80

Lysine decarboxylase produced by the present invention can be applied to the conversion of cadaverine, a nylon monomer, from L-lysine in the future. Due to the possibility of high concentration of the lysine decarboxylase, And thus has a considerable efficiency. In addition, since the carrier thus produced is easy to recover after the conversion reaction and the enzyme activity can be maintained at a constant level even after several times of reaction, it is expected that the production cost of the nadyl monomer, cadaverine, will be greatly reduced .

<110> Konkuk University Industrial Cooperation Corp          Sejong Industry-Academia Cooperation Foundation Hongik University <120> A method for production of cadaverin by immobilization of lysine          decarboxylase-overexpressing recombinant E. coli using          barium-alginate <130> 10-2015-0166441 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 2148 <212> DNA <213> Artificial Sequence <220> <223> lysine decarboxylase (cadA) of Echerichia coli K12 MG1655 <400> 1 atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60 gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120 gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180 aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240 gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300 agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360 actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420 cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480 gt; tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600 gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660 tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720 gccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780 tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840 cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900 gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960 acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020 ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080 gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140 aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200 ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260 ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320 cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380 acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440 aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500 gcggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560 catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620 atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680 gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740 tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800 aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860 tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920 gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980 ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040 gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100 gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148

Claims (10)

A method for immobilizing recombinant E. coli overexpressing lysine decarboxylase protein on a barium-alginate carrier.
The method according to claim 1, wherein the lysine decarboxylase is derived from E. coli.
2. The method of claim 1, wherein the lysine decarboxylase is encoded by the nucleotide sequence of SEQ ID NO: 1.
The method according to claim 1, wherein the concentration of the barium-alginate carrier is 0.2 to 0.6 M.
The method of claim 1 wherein the size of the barium-alginate carrier is 1 to 5 mm in diameter.
delete A method for mass production of cadaverine using recombinant Escherichia coli overexpressing lysine dicarboxylase protein immobilized on a barium-alginate carrier.
8. The method according to claim 7, wherein the cadaverine is produced through a batch process or a continuous process.
delete delete

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