Lysine decarboxylase having improved stability with a pH change, microorganism comprising a polynucleotide encoding the same, and method for producing cadaverine using the same

Abstract

The present invention relates to: a novel lysine decarboxylase; a microorganism transformed with a gene coding for the activity concerned; and a method for producing cadaverine by using the same.

Claims

1. A microorganism which is transformed with a polynucleotide encoding a protein having lysine decarboxylase activity, comprising the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence having 90% or more homology therewith to express the protein, wherein the protein having lysine decarboxylase activity is from a Pseudomonas sp. and wherein the microorganism is an Escherichia sp. microorganism.

2. The microorganism of claim 1, wherein the polynucleotide has the nucleotide sequence of SEQ ID NO: 2.

3. The microorganism of claim 1, wherein the protein having lysine decarboxylase activity maintains 90% or more of the activity at pH 9 compared to that at pH 6.

4. A method of preparing cadaverine, comprising the steps of: converting lysine into cadaverine by using the microorganism of claim 1; and recovering the cadaverine.

5. The method of preparing cadaverine of claim 4, wherein the step of converting comprises: culturing the microorganism in a medium; and recovering cadaverine from the microorganism or the medium.

6. The method of preparing cadaverine of claim 4, the protein having the lysine decarboxylase activity comprises the amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 1.

7. The method of preparing cadaverine of claim 4, wherein the polynucleotide has a nucleotide sequence of SEQ ID NO: 2.

8. The microorganism of claim 1, wherein the protein having the lysine decarboxylase activity comprises the amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 1.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a reaction mechanism of lysine decarboxylase which produces cadaverine from lysine;

(2) FIG. 2 is an image of SDS-PAGE gel showing expression results of PtLDC, and PtLDC with an N-terminal his-tag;

(3) FIG. 3 shows PtLDC reactivity of converting lysine into cadaverine;

(4) FIG. 4 shows a relative enzymatic activity of PtLDC according to varying temperature;

(5) FIG. 5 shows a relative enzymatic activity of PtLDC according to varying pH;

(6) FIG. 6 is an image of SDS-PAGE gel showing PaLDC and PrLDC expression;

(7) FIG. 7 shows PaLDC reactivity of converting lysine into cadaverine;

(8) FIG. 8 shows a relative enzymatic activity of PaLDC according to varying temperature;

(9) FIG. 9 shows a relative enzymatic activity of PaLDC according to varying pH;

(10) FIG. 10 shows PrLDC reactivity of converting lysine into cadaverine;

(11) FIG. 11 shows a relative enzymatic activity of PrLDC according to varying temperature;

(12) FIG. 12 shows a relative enzymatic activity of PrLDC according to varying pH;

(13) FIG. 13 is an image of SDS-PAGE gel showing expression results of EcLDC, PpLDC, PtLDC, and PxLDC;

(14) FIG. 14 shows PpLDC reactivity of converting lysine into cadaverine;

(15) FIG. 15 shows a relative enzymatic activity of PpLDC according to varying temperature;

(16) FIG. 16 shows a relative enzymatic activity of PpLDC according to varying pH;

(17) FIG. 17 shows PxLDC reactivity of converting lysine into cadaverine;

(18) FIG. 18 shows a relative enzymatic activity of PxLDC according to varying pH;

(19) FIG. 19 shows respective relative enzymatic activities of EcLDC and PtLDC according to varying temperature; and

(20) FIG. 20 shows respective relative enzymatic activities of EcLDC and PtLDC according to varying pH.

MODE OF THE INVENTION

(21) Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the scope of the present invention is not intended to be limited by these Examples.

Example 1

Selection of Novel Lysine Decarboxylase for Producing Cadaverine

(22) 1-1. Selection of Lysine Decarboxylase Derived from Pseudomonas Thermotolerans

(23) To select a novel lysine decarboxylase to be used in the production of cadaverine, a BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK.sub.LOC=blasthome) provided by the National Center for Biotechnology Information (NCBI), USA was used to search for lysine decarboxylase derived from a thermophilic bacterium which has high similarity to a peptide sequence of an active site of lysine decarboxylase derived from E. coli. In detail, a BLAST search was carried out, based on a total of 31 peptide sequences (GRVEGKVIYETQSTHKLLAAFSQASMIHVKG: SEQ ID NO: 12) each including 15 amino acids at the N-terminus and the C-terminus centered around the 367.sup.th lysine which is the main amino acid of lysine decarboxylase derived from E. coli. As a result, it was confirmed that Escherichia, Shigella, Enterobacteria, Edwardsiella, Klebsiella, Serratia, Yersinia, Yokenella, Raoultella, Ceratitis, Salmonella, Sutterella, Shimwellia, Vibrio, and Pseudomonas sp. microorganisms have high homology. The search was aimed at finding lysine decarboxylase having high thermal stability while having activity similar to that of lysine decarboxylase of E. coli. In general, proteins found in thermophilic bacteria are known to have high thermal stability, and therefore, of the microorganisms found from the search, Pseudomonas thermotolerans known as a thermophilic (4660 C.) microorganism was selected.

(24) 1-2. Selection of Lysine Decarboxylase Derived from Various Pseudomonas sp. Microorganisms

(25) To select lysine decarboxylases derived from Pseudomonas sp. microorganisms other than Pseudomonas thermotolerans, four microorganisms (Pseudomonas alcaligenes, Pseudomonas resinovorans, Pseudomonas putida, and Pseudomonas synxantha) showing low homology between Pseudomonas sp. were selected. Nucleotide and genome programs provided by the National Center for Biotechnology Information (NCBI), USA (http://www.ncbi.nlm.nih.gov/) were used to identify nucleotide and amino acid sequences of lysine decarboxylases derived from the four Pseudomonas sp. microorganisms selected as above.

(26) The following Table 1 shows amino acid sequence homology of lysine decarboxylase derived from Pseudomonas sp.

(27) TABLE-US-00001 PtLDC PaLDC PrLDC PpLDC PxLDC PtLDC 87% 86% 81% 84% PaLDC 87% 89% 80% 85% PrLDC 86% 89% 77% 83% PpLDC 81% 80% 77% 84% PxLDC 84% 85% 83% 84% PtLDC: lysine decarboxylase derived from Pseudomonas thermotolerans (P. thermotolerans) PaLDC: lysine decarboxylase derived from Pseudomonas alcaligenes (P. alcaligenes) PrLDC: lysine decarboxylase derived from Pseudomonas resinovorans (P. resinovorans) PpLDC: lysine decarboxylase derived from Pseudomonas putida (P. putida) PxLDC: lysine decarboxylase derived from Pseudomonas synxantha (P. synxantha)

Example 2

Preparation of E. coli Introduced with Lysine Decarboxylase Gene Derived from Pseudomonas Thermotolerans and Analysis of Activity of Lysine Decarboxylase Expressed Therefrom

(28) 2-1. Transformation of E. coli with Lysine Decarboxylase Gene Derived from Pseudomonas thermotolerans

(29) To introduce the lysine decarboxylase gene derived from Pseudomonas thermotolerans into E. coli and express the gene therefrom, cloning of a recombinant gene was performed. Genetic information on Pseudomonas thermotolerans was obtained from genomic data of the NCBI (http://www.ncbi.nlm.nih.gov/genome/).

(30) The genomic DNA of Pseudomonas thermotolerans was obtained, and then used as a template to amplify a Pseudomonas thermotolerans-derived lysine decarboxylase gene (ptldc) by polymerase chain reaction (PCR). To perform PCR, primers of 5_LDC_Ndel (AATATACATATGTACAAAGACCTCCAATTCCCC) (SEQ ID NO: 13) and 3_LDC_Xhol (AATATACTCGAGTCAGATCTTGATGCAGTCCACCG) (SEQ ID NO: 14) and PfuUltra DNA polymerase (Stratagene, USA) were used to perform PCR for 30 cycles under conditions of 94 C.: 30 sec, 55 C.: 30 sec, and 72 C.: 2 min. As a result, amplified ptldc (SEQ ID NO: 2) was obtained. Further, to express Pseudomonas thermotolerans-derived lysine decarboxylase with an N-terminal His-tag, primers of 5_LDC_BamHI (AATATAGGATCCGTACAAAGACCTCCAATTCCCC) (SEQ ID NO: 15) and 3_LDC_Sacl (AATATAGAGCTCTCAGATCTTGATGCAGTCCACCG) (SEQ ID NO: 16) were used to perform PCR in the same manner as the above PCR method. Next, each ptldc gene obtained from PCR was inserted into an E. coli expression vector, pET-Deut1. Thereafter, each plasmid cloned with the ptldc gene was inserted into E. coli Rosetta by a heat shock transformation method. Each of the transformed E. coli was cultured in a 50 ml liquid LB medium (containing 50 mg/ml ampicillin) at 37 C. When an OD600 value reached 0.8, 0.4 mM isopropyl -D-1-thiogalactopyranoside (IPTG) was added thereto and incubated at 18 C. for 48 hours to induce expression. Each Pseudomonas thermotolerans-derived lysine decarboxylase (PtLDC) thus completely expressed was identified by SDS-PAGE (FIG. 2). The results of SDS-PAGE showed that PtLDC and PtLDC with His-tag expressed at a low temperature were overexpressed as soluble proteins (Lanes 2 and 4 of FIG. 2).

(31) The E. coli Rosetta transformed with the plasmid including the ptldc (SEQ ID NO: 2) was designated as Escherichia coli CC04-0055, and deposited at the Korean Culture Center of Microorganisms (KCCM) on Jul. 24, 2014 under the Accession number KCCM11559P.

(32) 2-2. Analysis of Activity of Pseudomonas thermotolerans-Derived Lysine Decarboxylase Expressed in E. coli

(33) (1) Analysis of Reactivity of Lysine Decarboxylase

(34) To investigate reactivities of PtLDC and PtLDC with the His-tag, 50 ml of soluble protein, 100 mM pyridoxal-phosphate (pyridoxal-phosphate, PLP), and 250 mM lysine were reacted in a volume of 200 ml at 46 C. for 2 hours. A reaction buffer solution was 50 mM sodium phosphate at pH 6.2. A microorganism into which an empty vector was introduced was used as a control, and amounts of lysine and cadaverine were analyzed (FIG. 3). High-performance liquid chromatography (Waters, Milford, Mass.) was performed to analyze accurate amounts of lysine and cadaverine in a 2414 Refractive Indes Detector (Waters, Milford, Mass.). Lysine-HCl reagent and 1,5-diaminopentane (cadaverine) reagent were purchased from Sigma (St. Louis, Mo.), and a mobile phase consisting of 1 mM citric acid, 10 mM tartaric acid, 24 mM ethylenediamine, and 5% acetonitrile was used to separate and quantify the two materials in an IonoSpher C3-100 mm, 5 mm column. The control showed no production of cadaverine. PtLDC with the N-terminal His-tag showed 72% lysine conversion, and PtLDC showed 100% lysine conversion, indicating production of cadaverine.

(35) (2) Analysis of Activity of Lysine Decarboxylase According to Temperature and pH

(36) To analyze enzymatic characteristics of PtLDC under various temperature conditions (30 C., 42 C., 50 C., 60 C., 70 C., and 80 C.), relative activities were compared. When PtLDC was diluted and reacted with 250 mM lysine substrate at 60 C. for 30 minutes, 42 mM cadaverine was found to be produced. In this regard, concentrations of cadaverine were analyzed by using 50 mM sodium phosphate buffer (pH 6.2) as a buffer solution, and an equal amount of the enzyme under the same reaction conditions, except that temperature conditions were 30 C., 42 C., 50 C., 70 C., and 80 C., and compared with the amount of cadaverine produced at a reaction temperature of 60 C. (FIG. 4). As shown in FIG. 4, PtLDC showed the highest activity at 60 C. Further, PtLDC maintained 80% or more of the activity at a temperature of 55 C.65 C.

(37) Additionally, activity of lysine decarboxylase was evaluated under various pH conditions (6.2, 7.0, 8.0, and 9.0). The temperature condition was fixed at 60 C., and an equal amount of the enzyme was used under the same reaction conditions, except that 50 mM sodium phosphate buffer (pH 6.2), 50 mM tris buffer (pH 7.0), 100 mM potassium phosphate buffer (pH 8.0), and 50 mM tris buffer (pH 9.0) were used. Reactivities of lysine decarboxylase at different pHs were compared (FIG. 5). PtLDC showed the highest activity at pH 8.0, and maintained 90% or more of the activity at pH 6 to pH 9. An amount of cadaverine produced at each pH condition was compared with that of cadaverine produced at pH 8 (FIG. 5). The experimental result showed that PtLDC has high stability against pH change or high pH.

Example 3

Preparation of E. coli Introduced with Lysine Decarboxylase Gene Derived from Pseudomonas Alcaligenes and Analysis of Activity of Lysine Decarboxylase Expressed Therefrom

(38) 3-1. Transformation of E. coli with Lysine Decarboxylase Gene Derived from Pseudomonas alcaligenes

(39) To clone a lysine decarboxylase gene (paldc) derived from Pseudomonas alcaligenes, primers of 5_PaLDC_Ndel (AATATACATATGTACAAAGACCTGAA GTTCCCCATCC) (SEQ ID NO: 17) and 3_PaLDC_Xhol (AATATACTCGAGTCACTCCCTTATGCAATCAACGGTATAGC) (SEQ ID NO: 18) and purified genomic DNA of Pseudomonas alcaligenes as a template were used to perform PCR. Pfu DNA polymerase was used as a polymerase, and PCR was performed for 30 cycles under conditions of 94 C.: 30 sec, 55 C.: 30 sec, and 72 C.: 2 min. As a result, an amplified paldc gene (SEQ ID NO: 4) was obtained.

(40) The obtained paldc gene was expressed at a low temperature in E. coli in the same manner as in Example 2-1, and identified by SDS-PAGE (FIG. 6). As shown in FIG. 6, Pseudomonas alcaligenes-derived lysine decarboxylase (PaLDC) was mostly expressed as insoluble protein, and no soluble protein was observed on the SDS-PAGE gel (FIG. 6, see Lanes 1 and 2).

(41) 3-2. Analysis of Activity of Pseudomonas alcaligenes-Derived Lysine Decarboxylase Expressed in E. coli

(42) (1) Analysis of Reactivity of Lysine Decarboxylase

(43) To investigate reactivity of PaLDC, a cell lysate of PaLDC obtained in Example 3-1 was centrifuged at 13,000 rpm for 15 minutes to obtain a supernatant (soluble protein), which was used in conversion of lysine. 50 l of the soluble protein, 100 mM PLP, and 250 mM lysine were reacted in 50 mM sodium phosphate buffer (pH 6.2) in a reaction volume of 200 l at 46 C. for 2 hours. As a result, 70% lysine was found to be converted into cadaverine by PaLDC (FIG. 7).

(44) (2) Analysis of Activity of Lysine Decarboxylase According to Temperature and pH

(45) To find an optimum temperature condition for activity of Pseudomonas alcaligenes-derived lysine decarboxylase, enzymatic activities were evaluated under temperature conditions of 30 C., 40 C., 46 C., and 60 C. in the same manner as in Example 2-2. As a result, PaLDC was found to have the highest activity at 50 C. (FIG. 8).

(46) The activity of Pseudomonas alcaligenes-derived lysine decarboxylase under different pH conditions was evaluated in the same manner as in Example 2-2. As a result, PaLDC had the highest stability at pH 8 and pH 9, and maintained 95% or more of the activity at pH 6 (FIG. 9).

Example 4

Preparation of E. coli Introduced with Lysine Decarboxylase Gene Derived from Pseudomonas Resinovorans and Analysis of Activity of Lysine Decarboxylase Expressed Therefrom

(47) 4-1. Transformation of E. coli with Lysine Decarboxylase Gene Derived from Pseudomonas resinovorans

(48) To clone a lysine decarboxylase gene (prldc) derived from Pseudomonas resinovorans, primers of 5_PrLDC_Ndel (AATATACATATGTACAAAGAGCTC AAGTTCCCCGTCCTC) (SEQ ID NO: 19) and 3_PrLDC_Xhol (AATATACTCGAG TTATTCCCTGATGCAGTCCACTGTA TAGC) (SEQ ID NO: 20) and purified genomic DNA of Pseudomonas resinovorans as a template were used to perform PCR. PCR was performed by using the same polymerase under the same PCR conditions as in Example 3-1. As a result, amplified prldc (SEQ ID NO: 6) was obtained.

(49) The obtained prldc gene was expressed at a low temperature in E. coli in the same manner as in Example 2-1, and identified by SDS-PAGE (FIG. 6; Lanes 3 and 4). As a result, PrLDC was found to be hardly expressed at a low temperature.

(50) 4-2. Analysis of Activity of Pseudomonas resinovorans-Derived Lysine Decarboxylase Expressed in E. coli

(51) (1) Analysis of Reactivity of Lysine Decarboxylase

(52) To investigate reactivity of lysine decarboxylase (PrLDC) derived from Pseudomonas resinovorans, a cell lysate of PrLDC obtained in Example 4-1 was centrifuged at 13,000 rpm for 15 minutes to obtain a supernatant, which was used in conversion of lysine. 50 l of the soluble protein, 100 mM PLP, and 250 mM lysine were reacted in 50 mM sodium phosphate buffer (pH 6.2) in a reaction volume of 200 l at 46 C. for 2 hours. As a result, 66% cadaverine was produced (FIG. 10).

(53) (2) Analysis of Activity of Lysine Decarboxylase According to Temperature and pH

(54) To find an optimum temperature condition for activity of PrLDC, enzymatic activities were evaluated under temperature conditions of 30 C., 40 C., 46 C., and 60 C. in the same manner as in Example 2-2. As a result, PrLDC was found to have the highest activity at 60 C. (FIG. 11).

(55) The activity of PrLDC under different pH conditions was evaluated in the same manner as in Example 2-2. As a result, PrLDC had the highest stability at pH 6, and maintained 90% or more of the activity at pH 9 (FIG. 12).

Example 5

Preparation of E. coli Introduced with Lysine Decarboxylase Gene Derived from Pseudomonas putida and Analysis of Activity of Lysine Decarboxylase Expressed Therefrom

(56) 5-1. Transformation of E. coli with Lysine Decarboxylase Gene Derived from Pseudomonas putida

(57) To clone a lysine decarboxylase gene (ppldc) derived from Pseudomonas putida, primers of 5_PpLDC_Ndel (AATATACATATGTACAAAGACCTCCAA TTCCCC) (SEQ ID NO: 21) and 3_PpLDC_Xhol (AATATACTCGAGTCACTCCCTTATGCAATCAACGGTATAGC) (SEQ ID NO: 22) and purified genomic DNA of Pseudomonas putida as a template were used to perform PCR. Pfu DNA polymerase was used as a polymerase, and PCR was performed for 30 cycles under conditions of 94 C.: 30 sec, 55 C.: 30 sec, and 72 C.: 2 min. As a result, an amplified ppldc gene (SEQ ID NO: 8) was obtained.

(58) The obtained ppldc gene was expressed at a low temperature in E. coli in the same manner as in Example 2-1, and identified by SDS-PAGE (FIG. 13). As shown in Lanes 3 and 4 of FIG. 13, Pseudomonas putida-derived lysine decarboxylase (PpLDC) was found to be hardly expressed at a low temperature.

(59) A cell lysate was centrifuged at 13,000 rpm for 15 minutes, and a supernatant was used in a conversion reaction of lysine.

(60) 5-2. Analysis of Activity of Pseudomonas putida-Derived Lysine Decarboxylase Expressed in E. coli

(61) (1) Analysis of Reactivity of Lysine Decarboxylase

(62) To investigate reactivity of PpLDC, the cell lysate of PpLDC obtained in Example 5-1 was centrifuged at 13,000 rpm for 15 minutes to obtain a supernatant, which was used in conversion of lysine. 50 l of the soluble protein, 100 mM PLP, and 250 mM lysine were reacted in 50 mM sodium phosphate buffer (pH 6.2) in a reaction volume of 200 l at 46 C. for 2 hours. As a result, 16% cadaverine was produced (FIG. 14).

(63) (2) Analysis of Activity of Lysine Decarboxylase According to Temperature and pH

(64) To find an optimum temperature condition for activity of PpLDC, enzymatic activities were evaluated under temperature conditions of 50 C., 60 C., and 70 C. in the same manner as in Example 2-2. As a result, PpLDC was found to have the highest activity at 50 C. (FIG. 15).

(65) The activity of PpLDC under different pH conditions was evaluated in the same manner as in Example 2-2. As a result, PpLDC showed the highest activity at pH 6, and its reactivity decreased with increasing pH (FIG. 16).

Example 6

Preparation of E. coli Introduced with Lysine Decarboxylase Gene Derived from Pseudomonas synxantha and Analysis of Activity of Lysine Decarboxylase Expressed Therefrom

(66) 6-1. Transformation of E. coli with Lysine Decarboxylase Gene Derived from Pseudomonas synxantha

(67) To clone a lysine decarboxylase gene (pxldc) derived from Pseudomonas synxantha, primers of 5_PxLDC_Ndel (AATATACATATGTACAAAGACCTCCAA TTCCCC) (SEQ ID NO: 23) and 3_PxLDC_Xhol (AATATACTCGAGTCACTCCCTTATGCAATCAACGGTATAGC) (SEQ ID NO: 24) and purified genomic DNA of Pseudomonas synxantha as a template were used to perform PCR. Pfu DNA polymerase was used for gene amplification, and PCR was performed for 30 cycles under conditions of 94 C.: 30 sec, 45 C.: 30 sec, and 72 C.: 2 min to obtain amplified pxldc (SEQ ID NO: 10).

(68) The obtained pxldc gene was expressed at a low temperature in E. coli in the same manner as in Example 2-1, and identified by SDS-PAGE (FIG. 13). As shown in Lanes 7 and 8 of FIG. 13, Pseudomonas synxantha-derived lysine decarboxylase (PxLDC) was found to be overexpressed as soluble protein at a low temperature.

(69) 6-2. Analysis of Activity of Pseudomonas synxantha-derived Lysine Decarboxylase Expressed in E. coli

(70) (1) Analysis of Reactivity of PxLDC

(71) To investigate reactivity of PxLDC, the cell lysate of PxLDC obtained in Example 6-1 was centrifuged at 13,000 rpm for 15 minutes to obtain a supernatant, which was used in conversion of lysine. 50 l of the soluble protein, 100 mM PLP, and 250 mM lysine were reacted in 50 mM sodium phosphate buffer (pH 6.2) in a reaction volume of 200 l at 46 C. for 2 hours. As a result, 25% cadaverine was produced (FIG. 17).

(72) (2) Analysis of Activity of Lysine Decarboxylase According to pH

(73) To find an optimum pH condition for PxLDC, enzymatic activities were evaluated under different pH conditions in the same manner as in Example 2-2 (FIG. 18). As a result, PxLDC showed the highest activity at pH 6, and its reactivity decreased with increasing pH.

Example 7

Comparison of Activity between E. coli-derived Lysine Decarboxylase and Pseudomonas thermotolerans-Derived Lysine Decarboxylase

(74) 7-1. Cloning and Expression of E. coli-derived Lysine Decarboxylase

(75) An E. coli lysine decarboxylase gene, cadA, was cloned to express EcLDC (SEQ ID NO: 11). Homology between PtLDC and EcLDC amino acid sequences is 36%. A cadA gene-cloned plasmid was inserted into E. coli K-12 BL21, and incubated at 37 C. for 4 hours to induce expression. EcLDC thus completely expressed was identified by SDS-PAGE (FIG. 13; Lanes 1 and 2). As a result, EcLDC was found to be overexpressed as soluble protein.

(76) 7-2. Comparison of Relative Enzymatic Activity Between EcLDC and PtLDC

(77) (1) Comparison of Activity According to Temperature

(78) Relative enzymatic activity (relative activity) was compared between EcLDC and PtLDC under various temperature conditions (37 C., 42 C., 50 C., 60 C., 70 C., and 80 C.) in the same manner as in Example 2-2 (FIG. 19).

(79) As a result, both EcLDC and PtLDC were found to show the highest activity at 60 C. EcLDC had 54% relative activity at 50 C. (when the activity of EcLDC at 60 C. was taken as 100%), and EcLDC had 12% relative activity at 80 C. PtLDC had 76% relative activity at 50 C. (when the activity of PtLDC at 60 C. was taken as 100%), and PtLDC had 19% relative activity at 80 C. The activity of PtLDC was found to be well maintained at a high temperature. In conclusion, both of the two enzymes showed a great difference in their activities depending on temperature, and the relative activity of PtLDC was well maintained, compared to EcLDC.

(80) (2) Comparison of Activity According to pH

(81) Additionally, activity was evaluated under various pH conditions (6.2, 7.4, 8.0, and 9.0) in the same manner as in Example 2-2 (FIG. 20). As a result, EcLDC showed the highest activity at pH 6, and enzymatic activity of EcLDC greatly decreased with increasing pH. At pH 9, EcLDC maintained 50% of the activity. In contrast, PtLDC showed no great change in the activity according to pH, and PtLDC maintained 90% or more of the activity at pH 6.2-9. Accordingly, it was evaluated that PtLDC showed higher stability against temperature and pH than EcLDC.

(82) (3) Comparison of Activity Between PtLDC and EcLDC

(83) When PtLDC and EcLDC proteins were quantified to evaluate specific activity (unit/mg), PtLDC showed a value of 10060 (unit/mg), and EcLDC showed a value of 36335 (unit/mg). When their reactivities were compared, EcLDC showed about 3.6 times higher activity than PtLDC. Further, when an optimal temperature was compared between the enzymes, both two enzymes showed optimal reactions at 60 C., and their activities greatly decreased with varying temperature. However, when optimal pH conditions were compared, EcLDC showed higher specific inactivity with increasing pH, and PtLDC showed no great change in the enzymatic activity according to pH change.

(84) EcLDC has higher activity than PtLDC. However, activity of EcLDC may be greatly influenced by pH change, as pH increases by reaction of lysine decarboxylase. PtLDC has higher pH stability than EcLDC, which is advantageous in the lysine conversion reaction. Commercial production of cadaverine by bioconversion of lysine requires pH adjustment by acid treatment, but PtLDC may mitigate the need for pH titration. It is expected that production costs required for the bioconversion of cadaverine may be reduced.

(85) Depositary institution: Korean Culture Center of Microorganisms (KCCM)

(86) Accession number: KCCM11559P

(87) Date of deposit: Jul. 24, 2014