KR20180053631A - The novel Lysine Decarboxylase and Process for producing cadeverine using the same - Google Patents

Abstract

The present invention relates to a novel lysine decarboxylase, a microorganism transformed with a gene coding the activity and a method for producing a cadaverine using the same. The method for producing a cadaverine comprises the following steps of: converting a lysine to a cadaverine; and recovering the converted cadaverine.

Description

The novel Lysine Decarboxylase and Process for producing cadeverine using the same}

The present invention relates to a novel lysine decarboxylase, a microorganism transformed with a gene encoding the activity, and a method for producing cadaverine using the same.

The general production method of nylon using diamine is by a chemical process using 1,4-diaminobutane and hexamethylenediamine as raw materials. Since these raw materials are made from petroleum-based organic compounds, as environmental regulations are strengthened, the market demand for alternative materials through bio-based pathways is increasing.

Meanwhile, cadaverine is a diamine organic compound composed of five carbons having a molecular formula _{of NH 2} (CH ₂ ) ₅ NH _{2, and may be a raw material for nylon 5,6.} If bio-based cadaverine is manufactured, it is expected that various nylon production will be possible while satisfying the bio-based market demand.

With regard to the bio-based production of cadaverine, studies on bioconversion of lysine were widely known before the 1940s (Gale EF, Epps HM 1944. Studies on bacterial amino-acid decarboxylases. Biochem J. 38, 232-242). Lysine decarboxylase, a key step in biotransformation, is an enzyme that produces cadaverine from lysine (FIG. 1). The activity of lysine decarboxylase has been reported in various microorganisms, and four types of lysine decarboxylase with known specific activity (mmol/min/mg) of the enzyme ( Escherichia coli ), Bacterium cadaveris , Glycine max , Selenomonas ruminantium ). Among them, E. coli-derived lysine decarboxylase is evaluated as a lysine decarboxylase showing the highest activity, and the enzyme used in actual production is also limited to CadA derived from E. coli (Japanese Patent No. 2005-147171, European Patent 2004-010711, and Japanese Patent 2002-257374). However, when lysine decarboxylase is reacted with lysine to produce cadaverine, carbon dioxide is generated by decarboxylation of lysine, and cadaverine, a divalent cation, is generated from lysine, a monovalent cation, so that the pH is increased during the reaction. Rises. Accordingly, as the pH of the lysine decarboxylase changes during the enzymatic reaction, the efficiency decreases. In addition, the enzyme may be denatured by an acid or a base generated in the reaction solution, resulting in loss of activity.

Accordingly, the present inventors discovered a novel lysine decarboxylase having stability against high temperature and pH, and confirmed that the lysine decarboxylase can be expressed in a strain of the genus Escherichia, thereby completing the present invention.

An object of the present invention is to provide a novel lysine decarboxylase and a polynucleotide encoding a protein having the activity.

Another object of the present invention is to provide a microorganism transformed to express the lysine decarboxylase.

Another object of the present invention is to provide a method for producing cadaverine using the enzyme and a microorganism containing the same.

In a specific aspect of the present invention, there is provided a protein having a novel lysine decarboxylase activity, comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 75% or more sequence homology thereto.

In the present invention, the term "protein having lysine decarboxylase activity" refers to a decarboxylation reaction of lysine by using pyridoxal-5'-phosphate as a coenzyme. , It refers to a protein that has the activity of decarboxylating lysine to produce cadaverine and carbon dioxide.

The protein having lysine decarboxylase activity comprising an amino acid sequence having 75% or more sequence homology thereto or SEQ ID NO: 1 may be a protein having lysine decarboxylase activity newly discovered from microorganisms derived from the genus Pseudomonas, and As long as it is a protein having the activity, all microorganisms discovered from microorganisms derived from Pseudomonas genus may be included. For example, the strains of the genus Pseudomonas are Pseuodomonas thermotolerans, Pseuodomonas alcaligenes, Pseuodomonas resinovorans, Pseuodomonas resinovorans, Pseuodomonas putida and Pseuodomonas synsa, Pseuodomonas synsa, and Pseuodomonas synsa, Pseuodomonas alcaligenes. ) Can be.

Specifically, the protein having novel lysine decarboxylase activity derived from Pseudomonas thermotolerance microorganisms has an amino acid sequence of SEQ ID NO: 1 or a sequence homology thereof of 75% or more, 80% or more, 85% or more, 90% or more, or 95 It may have an amino acid sequence greater than or equal to %. The protein having lysine decarboxylase activity derived from the Pseudomonas alkaline genes strain may have an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having a sequence homology thereof of 75% or more, 80% or more, 90% or more, or 95% or more. have. The protein having lysine decarboxylase activity derived from the Pseudomonas reginoborans strain has an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having a sequence homology thereof of 75% or more, 80% or more, 90% or more, or 95% or more. I can. The lysine decarboxylase derived from the Pseudomonas putida strain may have an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a sequence homology thereof of 75% or more, 80% or more, 90% or more, or 95% or more. The protein having lysine decarboxylase activity derived from the Pseudomonas sinsansa strain may have an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a sequence homology thereof of 75% or more, 80% or more, 90% or more, or 95% or more. have. However, it is not limited to the above amino acid sequence, and may include all possible amino acid sequences as long as it maintains lysine decarboxylase activity.

In addition, in another specific aspect of the present invention, a polynucleotide encoding a protein having the newly discovered lysine decarboxylase activity, specifically, a polynucleotide having 75% or more sequence homology with the nucleotide sequence of SEQ ID NO: 2 Provides.

The polynucleotide sequence encoding the protein having the lysine decarboxylase activity can be obtained from the published genomic sequence of a strain derived from the genus Pseudomonas. Specifically, it may be derived from the genomic sequence of one or more strains selected from the group consisting of Pseudomonas thermotolerance Pseudomonas Alkaline Genes, Pseudomonas reginovo lance, Pseuodomonas putida, and Pseudomonas synsansa. The lysine decarboxylase gene sequence derived from the Pseudomonas thermotolerance strain may have the nucleotide sequence of SEQ ID NO: 2, and also have 75% or more, 80% or more, 90% or more, homology with the nucleotide sequence of SEQ ID NO: 2, Or it may have a base sequence of 95% or more. The lysine decarboxylase gene sequence derived from the Pseudomonas alkaline genes strain may have the nucleotide sequence of SEQ ID NO: 4, and also have 75% or more, 80% or more, 90% or more, or It may have an amino acid sequence that is 95% or more. The lysine decarboxylase gene sequence derived from the Pseudomonas reginoborans strain can be obtained from the published genomic sequence of Pseudomonas reginoborans, and specifically may have a nucleotide sequence of SEQ ID NO: 6. In addition, it may have a nucleotide sequence having 75% or more, 80% or more, 90% or more, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 6. The lysine decarboxylase gene sequence derived from the Pseudomonas putida strain may have the nucleotide sequence of SEQ ID NO: 8, and also have 75% or more, 80% or more, 90% or more, or It may have a base sequence of 95% or more. The L-lysine decarboxylase gene sequence derived from the Pseudomonas sinsansa strain may have the nucleotide sequence of SEQ ID NO: 10, and also has 75% or more, 80% or more, 90% or more homology with the nucleotide sequence of SEQ ID NO: 10 , Or may have a base sequence of 95% or more. However, the polynucleotide encoding the protein having lysine decarboxylase activity is not limited thereto, and any polynucleotide capable of encoding the protein having the newly discovered lysine decarboxylase activity of the present invention may be included without limitation. have.

In the present invention, the term "homology" means the degree to which a given amino acid sequence or nucleotide sequence matches, and may be expressed as a percentage. In the present specification, a homologous sequence thereof having the same or similar activity as a given amino acid sequence or base sequence is indicated as "% homology". For example, using standard software that calculates parameters such as score, identity, and similarity, specifically BLAST 2.0, or by using hybridization experiments under defined stringent conditions. It can be confirmed by comparing and defined appropriate hybridization conditions can be determined by a method well known to those skilled in the art (eg, see Sambrook et al., 1989, infra).

More specifically, the lysine decarboxylase may be at least one selected from the group consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. In addition, the polynucleotide encoding the protein having L-lysine decarboxylase activity is at least one selected from the group consisting of the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 I can.

In an embodiment of the present invention, it was confirmed that the lysine decarboxylase derived from strains of the genus Pseudomonas has stability against changes in pH since the activity does not change significantly even at high pH.

In another specific aspect of the present invention, there is provided a microorganism transformed to express the protein having the novel lysine decarboxylase activity. The transformed microorganism includes both prokaryotic microorganisms and eukaryotic microorganisms as long as it is transformed so that the protein having the decarboxylase activity is expressed. For example, Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Coryneform microorganism strains may be included. The microorganism may specifically be a microorganism belonging to the genus Escherichia or the genus Corynebacterium, and more specifically, may be E. coli or Corynebacterium glutamicum, but is not limited thereto.

In addition, the parent strain of the transformed microorganism may be a microorganism having improved lysine-producing ability compared to the wild type. However, it is not limited thereto. The term "microorganisms having improved lysine-producing ability compared to the wild type" of the present invention means a microorganism in a natural state or a microorganism having an increased lysine-producing ability compared to the parent strain, and the microorganism having improved lysine-producing ability is It is not particularly limited as long as it is an improved microorganism.

In order to impart improved lysine-producing ability compared to wild-type as described above, a method of obtaining an auxotrophic mutant, a strain resistant to analogs, or a metabolic control mutant having the ability to produce lysine, and lysine biosynthetic enzyme activity Conventional methods for growing microorganisms, such as a method for producing the recombinant strain prepared, can be used. Upon growth of lysine producing microorganisms, properties such as auxotrophic, analog resistance and metabolic control mutations may be imparted alone or in combination. The increased lysine biosynthetic enzyme activity may be alone or in combination. In addition, it is also possible to increase lysine biosynthetic enzyme activity together while conferring properties such as auxotrophic, analog resistance and metabolic control mutations. Specifically, the gene encoding the lysine biosynthetic enzyme is dihydrodipicolinate synthase gene (dapA), aspartokinase gene (lysC), dihydrodipicolinate reductase gene (dapB), diaminopimelate Decarboxylase gene (lysA), diaminopimelate dehydrogenase gene (ddh), phosphoenolpyruvate carboxylase gene (ppc, aspartate aminotransferase gene (aspC), and aspartate semialdehyde dehydrogenase gene ( asd), etc. The method of conferring or increasing the ability to produce lysine by increasing the lysine biosynthetic enzyme activity is by inducing a mutation in the gene encoding the enzyme or amplifying the gene. It can be carried out by increasing the intracellular activity of the enzyme These can be carried out by genetic recombination, but are not limited thereto.

Any microorganism having an improved lysine-producing ability compared to the wild type may include both prokaryotic microorganisms and eukaryotic microorganisms. Specifically, it may be a microorganism of the genus Escherichia or a coryneform microorganism. The Escherichia microorganisms are Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii Or it may be Escherichia vulneris (Escherichia vulneris), but is not limited thereto. More specifically, the strain of the genus Escherichia may be E. coli. The coryneform microorganisms include microorganisms of the genus Corynebacterium or Brevibacterium. Further, the coryneform microorganism Specifically Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium thermo amino to Ness (Corynebacterium thermoaminogenes), Brevibacterium Plastic boom (Brevibacterium flavum), Brevibacterium Menthum ( Brevibacterium lactofermentum ), but is not limited thereto.

In order for the microorganism to be transformed to express the protein having the lysine decarboxylase activity, the lysine decarboxylase gene of the present invention is included as a lysine decarboxylase protein or a gene expression unit in the microorganism intended for transformation. I can. The gene expression unit of the lysine decarboxylase may be operably linked to a vector to be transformed and included in the microorganism, or may be inserted into the chromosome of the microorganism. Specifically, the lysine decarboxylase gene may be operably linked to be overexpressed by a promoter linked upstream of an initiation codon.

In the present invention, the term "expression unit" refers to a fragment in which a promoter and a polynucleotide encoding a protein are operably linked, and 3'-UTL, 5'-UTL, poly A tail, and the like may be additionally included. In the present invention, the term "expression unit" may be used interchangeably with "expression cassette".

In the present invention, the term "operably linked" means that the gene sequence and a nucleic acid sequence having promoter activity are functionally linked to initiate and mediate transcription of a gene encoding a lysine decarboxylase. This means that the nucleic acid sequence having promoter activity is operably linked to the lysine decarboxylase gene, and thus the transcriptional activity of the lysine decarboxylase gene can be regulated.

In the present invention, the term "transformation" refers to the introduction of a lysine decarboxylase gene derived from the Pseudomonas sp. strain into a host cell, specifically a strain of the genus Escherichia or a coryneform microorganism to be expressed in the host cell. It refers to the overall action. At this time, the lysine decarboxylase gene is a polynucleotide capable of encoding lysine decarboxylase, and includes DNA and RNA. The gene may be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the gene may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct that itself contains all elements necessary for expression of the gene. The expression cassette usually includes a promoter operably linked to the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the gene may be introduced into a host cell by itself or in the form of a polynucleotide structure, and may be operably linked to a sequence required for expression of the host cell. The recombinant vector is a means for expressing a protein by introducing DNA into a host cell, and a known expression vector such as a plasmid vector, a cozmid vector, or a bacteriophage vector can be used. The vector can be easily prepared by a person skilled in the art according to any known method using DNA recombination technology, but is not limited thereto.

The method of transformation includes any method of introducing a polynucleotide into a cell, and can be performed by selecting a suitable standard technique known in the art. For example, electroporation, calcium phosphate co-precipitation, retroviral infection, microinjection, DEAE-dextran, cationic liposome ( cationic liposome) method, etc., but is not limited thereto.

In one embodiment, the microorganism having the improved lysine-producing ability is transformed to express the protein having the lysine decarboxylase activity of the present invention, thereby having excellent cadaverine-producing ability.

Another specific aspect of the present invention provides the use of a microorganism transformed to express a protein having novel lysine decarboxylase or novel lysine decarboxylase activity for producing cadaverine.

The novel lysine decarboxylase and the microorganism transformed to express a protein having the novel lysine decarboxylase activity are as described above, respectively. In one embodiment, it was confirmed that the lysine decarboxylase of the present invention has higher stability than the E. coli-derived lysine decarboxylase that was used for the production of cadaverine conventionally with respect to changes in temperature or pH. In particular, the novel lysine decarboxylase of the present invention has relatively higher pH stability than that of E. coli-derived lysine decarboxylase, and is advantageous in the conversion reaction from lysine to cadaverine. Accordingly, the novel lysine decarboxylase of the present invention and the microorganism transformed to express a protein having the novel lysine decarboxylase activity can be utilized for the production of cadaverine.

Another aspect of the present invention provides a method for producing cadaverine.

A specific aspect of the method for producing cadaverine of the present invention includes the steps of converting from lysine to cadaverine using a protein having a novel lysine decarboxylase activity or a microorganism transformed to express the protein having the activity; And recovering the converted cadaverine.

The protein having the novel lysine decarboxylase activity and the transformed microorganism are as described above, respectively. Specifically, the transformed microorganism may be a microorganism of the genus Escherichia.

In the step of converting lysine to cadaverine, cadaverine can be produced by decarboxylating lysine using an enzyme extracted from a microorganism expressing a protein having novel lysine decarboxylase activity and purified. Alternatively, by adding lysine to a culture medium in which the transformed microorganism is cultured, lysine can be converted to cadaverine by decarboxylating lysine using the microorganism itself.

In addition, another specific aspect of the cadaverine production method of the present invention is a microorganism having a cadaverine-producing ability transformed so that a microorganism having an improved lysine-producing ability compared to the wild-type expresses a protein having a novel lysine decarboxylase activity. Culturing in a medium; And it provides a method for producing cadaverine comprising the step of recovering cadaverine from the microorganism or medium.

The protein having the novel L-lysine decarboxylase activity and the microorganism having an improved lysine-producing ability compared to the wild-type are as described above.

The culture may be performed according to a suitable medium and culture conditions known in the art. Those of ordinary skill in the art can easily adjust and use the medium and culture conditions according to the selected microorganism. The culture method may include, but is not limited to, a batch type, continuous type, fed-batch type, or a combination culture thereof.

The medium may contain various carbon sources, nitrogen sources, and trace element components.

In a specific example, the carbon source is glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, palmitic acid, stearic acid, fatty acids such as linoleic acid, glycerol, and An alcohol such as ethanol, an organic acid such as acetic acid, or a combination thereof may be included, and specifically, it may be performed using glucose as a carbon source, but is not limited thereto. The nitrogen source is an organic nitrogen source and urea such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL), and soybean meal, an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, or It may include a combination of these, but is not limited thereto. The medium as a source of phosphorus may include, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a corresponding sodium-containing salt, a metal salt such as magnesium sulfate or iron sulfate, but is not limited thereto. In addition, amino acids, vitamins, and suitable precursors may be included in the medium. The medium or individual components may be added to the medium in a batch or continuous manner, and the examples are not limited thereto.

In addition, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the microbial medium in an appropriate manner during the cultivation. In addition, the generation of air bubbles can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester during culture. In order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas (eg, air) may be injected into the culture medium. The temperature of the culture medium may be usually 20°C to 45°C, for example 25°C to 40°C. The incubation period may be continued until a desired amount of lysine decarboxylase is obtained, and may be, for example, 10 to 160 hours.

The method for recovering cadaverine may be, for example, a batch, continuous, or fed-batch culture method, using a suitable method known in the art to collect or recover cadaverine produced from the medium. In addition, methods such as centrifugation, filtration, ion exchange chromatography, and crystallization may be used for such a recovery method. For example, the medium may be centrifuged at low speed to remove biomass, and the obtained supernatant may be separated through ion exchange chromatography.

In addition, the method for producing cadaverine may further include the step of recovering lysine decarboxylase from the microorganism or medium.

The method for recovering lysine decarboxylase from the microorganism or medium is a lysine decarboxylase produced from a microorganism or medium using a suitable method known in the art according to a culture method, for example, a batch, continuous, or fed-batch culture method. Reboxylase can be collected or recovered. In addition, methods such as centrifugation, filtration, ion exchange chromatography, and crystallization may be used for such a recovery method. For example, the culture medium may be centrifuged at a low speed to remove biomass, and the obtained supernatant may be separated through ion exchange chromatography. In addition, lysine decarboxylase can be recovered from cell lysate obtained by destroying microorganisms in the medium. The cell lysate can be obtained using an appropriate method known in the art. For example, a physical grinder or a suitable commercially available cell lysis buffer can be used. Lysine decarboxylase can be recovered from such cell pulverization solution through a method known in the art such as centrifugation.

The protein having a novel lysine decarboxylase activity derived from a strain of the genus Psuedomonas provided by the present invention can have a stable activity even with a change in pH increase, and thus can be efficiently used in the conversion reaction from lysine to cadaverine. , It can be widely used in the production of cadaverine.

1 shows the reaction mechanism by which lysine decarboxylase produces cadaverine from lysine.
2 is an SDS-PAGE gel photograph showing the expression results of PtLDC and PtLDC having his-tag at the N-terminus.
Figure 3 shows the reactivity of converting PtLDC lysine to cadaverine.
4 shows the relative enzymatic activity of PtLDC according to various temperatures.
5 shows the relative enzymatic activity of PtLDC according to various pHs.
6 is an SDS-PAGE gel photograph showing the expression of PaLDC and PrLDC.
7 shows the reactivity of converting PaLDC lysine to cadaverine.
8 shows the relative enzymatic activity of PaLDC according to various temperatures.
9 shows the relative enzymatic activity of PaLDC according to various pHs.
Figure 10 shows the reactivity of converting lysine of PrLDC to cadaverine.
11 shows the relative enzymatic activity of PrLDC according to various temperatures.
12 shows the relative enzymatic activity of PrLDC according to various pHs.
13 is an SDS-PAGE gel photograph showing the expression results of EcLDC, PpLDC, PtLDC and PxLDC.
14 shows the reactivity of PpLDC to convert lysine to cadaverine.
15 shows the relative enzymatic activity of PpLDC according to various temperatures.
16 shows the relative enzymatic activity of PpLDC according to various pHs.
Figure 17 shows the reactivity of converting lysine of PxLDC to cadaverine.
18 shows the relative enzymatic activity of PxLDC according to various pHs.
19 shows the relative activities of EcLDC and PtLDC according to various temperatures.
20 shows the relative activities of EcLDC and PtLDC according to various pHs.

Hereinafter, the present invention will be described in more detail by examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example One. Cadaverine New lysine for production Decarboxylase Selection

1-1. Pseudomonas Thermotolerance Derived lysine Decarboxylase Selection

To screen for novel lysine decarboxylase for use in cadaverine production, the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi?) provided by the National Center for Biological Information (NCBI) PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) was used to search for a lysine decarboxylase derived from a thermophilic strain having a high similarity to the active site peptide sequence of E. coli-derived lysine decarboxylase. Specifically, based on a total of 31 peptide sequences (GRVEGKVIYETQSTHKLLAAFSQASMIHVKG: SEQ ID NO: 12), containing 15 amino acids at each of the N-terminal and C-terminus around the 367th lysine, which is the main amino acid of E. coli-derived lysine decarboxylase, BLAST search was conducted. Results Escherichia (Escherichia), Shh Gela (Shigella), Enterobacter bacteria (Enterobacteria), Ed woji Ella (Edwardsiella), keulrep when Ella (Klebsiella), Serratia marcescens (Serratia), Yersinia (Yersinia), yoke Nella (Yokenella), Raul telra (Raoultella), ceramide entity's (Ceratitis), Salmonella (Salmonella), sute Pasteurella (Sutterella), swipwel Liao (Shimwellia), Vibrio (Vibrio), Pseudomonas (Pseudomonas) in microorganisms, such as a high homology Was confirmed to have. Among them, the purpose of the study was to search for a lysine decarboxylase capable of having an activity comparable to that of E. coli lysine decarboxylase and at the same time having high thermal stability. In general, since proteins present in thermophilic strains are known to have high thermal stability, Pseudomonas thermotolerance, known as thermophilic (46-60° C.) microorganisms, was selected from among the searched strains.

1-2. Lysine from various Pseudomonas genus strains Decarboxylase Selection

In order to select lysine polycarboxylase derived from strains of the genus Pseudomonas in addition to Pseudomonas thermotolerance strains, four strains with low homology between Pseudomonas species (Pseudomonas Alkaline Genes, Pseudomonas Reginovorans, Pseudomonas Putida, Pseudomonas Synsssa) were selected. I did. Genes and amino acids of lysine decarboxylase derived from the four selected Pseudomonas species using nucleotide and genome programs provided by the US National Center for Biological Information (http://www.ncbi.nlm.nih.gov/) The sequence was confirmed.

Table 1 below shows amino acid sequence homology to lysine decarboxylase derived from Pseudomonas species, respectively.

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 P. thermotolerans

PaLDC: Lysine decarboxylase derived from P. alcaligenes

PrLDC: Lysine decarboxylase derived from P. resinovorans

PpLDC: Lysine decarboxylase derived from P. putida

PxLDC: Lysine decarboxylase derived from P. synxantha

Example 2. Preparation of Escherichia coli into which the Pseudomonas thermotolerance- derived lysine decarboxylase gene was introduced and analysis of lysine decarboxylase activity expressed therefrom

2-1. Transformation of Escherichia coli by lysine decarboxylase gene derived from Pseudomonas thermotolerance

The lysine decarboxylase gene derived from Pseudomonas thermotolerance was introduced into E. coli and the recombinant gene was cloned for expression in E. coli. Genetic information about Pseudomonas thermotolerance was obtained from NCBI's Genome (http://www.ncbi.nlm.nih.gov/genome/) data information.

After securing the genomic DNA of Pseudomonas thermotolerance, the lysine decarboxylase gene (ptldc) derived from Pseudomonas thermotolerance was amplified through polymerization chain reaction (PCR) using this as a template. To perform PCR, 5_LDC_NdeI (AATATACATATGTACAAAGACCTCCAATTCCCC) (SEQ ID NO: 13) and 3_LDC_XhoI (AATATACTCGAGTCAGATCTTGATGCAGTCCACCG) (SEQ ID NO: 14) primers were used, and PfuUltraTM DNA polymerase (Stratagene Corporation, US for 30 seconds, 94° C., US) : 30 seconds, 72° C.: As a result of repeating the conditions for 2 minutes 30 times, amplified ptldc (SEQ ID NO: 2) was obtained. In addition, for the expression of Pseudomonas thermotolerance-derived lysine decarboxylase having a His-tag at the N-terminus, using 5_LDC_BamHI (AATATAGGATCCGTACAAAGACCTCCAATTCCCC) (SEQ ID NO: 15) and 3_LDC_SacI (AATATAGAGCTCTCAGATCTTGATGCAGTCTCAGATCTTGATGCAGTCTCAGATTGATGCAGTC) as primers. PCR was performed in the same manner as the PCR method. Then, each ptldc gene obtained from PCR was inserted into pET-Deut1, an E. coli expression vector. Thereafter, the plasmid in which the ptldc gene was cloned was inserted into E. coli Rosetta by a heat shock transformation method. The transformed strain was cultured at 37° C. using 50 ml liquid LB medium (including 50 mg/ml ampicillin), and when the OD600 value reached 0.8, isopropyl β-D-1-thiogal at a concentration of 0.4 mM Lactopyranoside (IPTG) was added and incubated at 18° C. for 48 hours to induce expression. Each Pseudomonas thermotolerance-derived lysine decarboxylase (PtLDC) whose expression was completed was confirmed through SDS-PAGE gel results (FIG. 2). Through the SDS-PAGE gel results, it was confirmed that PtLDC expressed at low temperature and PtLDC containing His-tag were overexpressed as soluble proteins (lanes 2 and 4 of FIG. 2).

The ptldc (SEQ ID NO: 2) an E. coli Rosetta strain transformed with a plasmid containing the 'Escherichia coli It was named'CC04-0055' and was deposited with the Korea Microbial Conservation Center (KCCM) on July 24, 2014, and was given the accession number KCCM11559P.

2-2. Analysis of the activity of lysine decarboxylase derived from Pseudomonas thermotolerance expressed in E. coli

(1) Confirmation of reactivity of lysine decarboxylase

In order to verify the reactivity of PtLDC and PtLDC containing His-tag, 50 ml of soluble protein, 100 mM pyridoxal-phosphate (PLP), and 250 mM lysine were added, and a volume of 200 ml was 46 ℃. Reacted for 2 hours. As the reaction buffer solution, 50 mM sodium phosphate, pH 6.2 was used. The amount of lysine and cadaverine was analyzed using the strain into which the empty vector was introduced as a control (FIG. 3). The exact amount of lysine and cadaverine was analyzed by 2414 Refractive Indes Detector (Waters, Milford, MA) using high performance liquid chromatography (Waters, Milford, MA). Lysine® HCl reagent and 1,5-diamino pentane (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 ethylene diamine, 5% acetonitrile. ) By using IonoSpher C3-100mm, 5mm column (column) to separate and quantify the two materials. In the case of the control group, it was confirmed that cadaverine was not produced at all. In the case of PtLDC having a His-tag inserted at the N-terminus, it was confirmed that 72% of lysine was converted, and in PtLDC, 100% of lysine was converted to produce cadaverine.

(2) Analysis of lysine decarboxylase activity according to temperature and pH

In order to determine the enzymatic properties of PtLDC under various temperature conditions (30, 42, 50, 60, 70, 80 ℃), the relative activity was compared. When PtLDC was diluted and reacted at 60° C. for 30 minutes using a 250 mM lysine substrate, it was confirmed that 42 mM cadaverine was produced. At this time, the buffer solution used was 50mM sodium phosphate buffer (pH6.2), and the concentration of cadaverine was analyzed by treating only the temperature conditions at 30, 42, 50, 70, 80 °C under the same reaction conditions as the enzyme of the same amount. And it was compared relatively with the amount of cadaverine produced in the temperature reaction at 60 ℃ (Fig. 4). As can be seen in Figure 4, PtLDC showed the highest activity at 60 ℃. In addition, it was evaluated to maintain 80% or more activity under the temperature condition of 55 to 65°C.

Additionally, evaluation of the activity of lysine decarboxylase for various pHs (6.2, 7.0, 8.0, 9.0) was performed. The temperature conditions were fixed at 60° C. and 50 mM sodium phosphate buffer (pH6.2), 50 mM Tris buffer (pH 7.0), 100 mM potassium phosphate buffer (pH 8.0), 50 mM Tris buffer (pH 9.0) under the same reaction conditions as the enzyme. ) Was used to compare the reactivity at different pHs (FIG. 5). PtLDC showed the highest activity at pH 8.0, but it was confirmed that more than 90% activity was maintained even under the conditions of pH 6 to pH 9. The amount of cadaverine produced under each pH condition was compared with the amount of cadaverine produced under the pH 8.0 condition (FIG. 5). Through the experimental results, PtLDC is evaluated to have high stability against a change in pH or a high pH.

Example 3. Preparation of Escherichia coli into which Pseudomonas Alkaline Genes- derived lysine decarboxylase gene was introduced and analysis of lysine decarboxylase activity expressed therefrom

3-1. Transformation of Escherichia coli by the lysine decarboxylase gene derived from Pseudomonas Alkaline Genes

To clone the lysine decarboxylase gene (paldc) derived from Pseudomonas alkaline genes, 5_PaLDC_NdeI (AATATACATATGTACAAAGACCTGAA GTTCCCCATCC) (SEQ ID NO: 17) and 3_PaLDC_XhoI (AATATACTCGAGTCACTCCCTTATGCAATCAACGGTTATGCAATCAACGGT) PCR was performed using DNA as a template. Pfu DNA polymerase was used as the polymerase, and PCR was carried out by repeating the conditions of 94° C.: 30 seconds, 55° C.: 30 seconds, 72° C.: 2 minutes 30 times, resulting in the amplified paldc gene (SEQ ID NO: 4). Secured.

The obtained paldc gene was expressed at low temperature in E. coli by the same method as in Example 2-1, and the results were confirmed by SDS-PAGE gel (FIG. 6). As can be seen in Figure 6, Pseudomonas Alkaline Genes-derived lysine decarboxylase (PaLDC) was mostly expressed as an insoluble protein, and the soluble protein was not identified on the SDS-PAGE gel (see Figure 6, lanes 1 and 2). .

3-2. Analysis of the Activity of Lysine Decarboxylase Derived from Pseudomonas Alkaline Genes Expressed in E. coli

(1) Confirmation of reactivity of lysine decarboxylase

In order to verify the reactivity of PaLDC, the cell lysate of PaLDC obtained in Example 3-1 was centrifuged at 13,000 rpm for 15 minutes to perform a lysine conversion reaction using a supernatant (soluble protein) obtained. 50 μl of soluble protein, 100 mM PLP, and 250 mM lysine were filled with 50 mM sodium phosphate (pH 6.2) buffer, and reacted at 46° C. for 2 hours in a reaction volume of 200 μl. As a result, it was confirmed that 70% lysine was converted to cadaverine by PaLDC (FIG. 7).

(2) Analysis of lysine decarboxylase activity according to temperature and pH

In order to find the optimum temperature conditions for the activity of Pseudomonas Alkaline Genes-derived lysine decarboxylase, the enzyme activity was evaluated at 30, 40, 46, and 60 ℃ temperature conditions in the same manner as in Example 2-2. As a result, it was confirmed that PaLDC has the best activity at 50°C (FIG. 8).

In addition, the active conditions for the pH of the lysine decarboxylase derived from Pseudomonas Alkaline Genes were evaluated in the same manner as in Example 2-2. As a result, it was confirmed that PaLDC exhibited high stability at pH 8 and pH 9, and maintained 95% or more activity even at pH 6 (FIG. 9).

Example 4. Preparation of E. coli into which Pseudomonas reginoborans- derived lysine decarboxylase gene was introduced and analysis of lysine decarboxylase activity expressed therefrom

4-1. Transformation of Escherichia coli by the lysine decarboxylase gene derived from Pseudomonas reginoborans

5_PrLDC_NdeI (AATATACATATGTACAAAGAGCTC AAGTTCCCCGTCCTC)) (SEQ ID NO: 19) and 3_PrLDC_XhoI (AATATACTCGAG TTATTCCCTGAG) in order to clone the lysine decarboxylase gene (prldc) derived from Pseudomonas reginoborance PCR was performed using Pseudomonas reginoborans genomic DNA as a template. By performing PCR under the same polymerase and PCR conditions as in Example 3-1, amplified prldc (SEQ ID NO: 6) could be obtained.

The obtained prldc gene was expressed at low temperature in E. coli by the same method as in Example 2-1, and the results were confirmed by SDS-PAGE gel (FIG. 6; lanes 3 and 4). As a result, it was confirmed that PrLDC was hardly expressed even under low temperature expression conditions.

4-2. Analysis of the activity of lysine decarboxylase derived from Pseudomonas reginoborans expressed in E. coli

(1) Confirmation of reactivity of lysine decarboxylase

In order to verify the reactivity of Pseudomonas reginoborans-derived lysine decarboxylase (PrLDC), the cell lysate of PrLDC obtained in 4-1 was centrifuged at 13,000 rpm for 15 minutes, and lysine conversion reaction was performed using the supernatant. . 50 μl of soluble protein, 100 mM PLP, and 250 mM lysine were filled with 50 mM sodium phosphate (pH 6.2) buffer, and reacted at 46° C. for 2 hours in a reaction volume of 200 μl. As a result of the lysine conversion reaction, 66% cadaverine was produced by PrLDC (FIG. 10).

(2) Analysis of lysine decarboxylase activity according to temperature and pH

In order to find the optimum temperature condition for the activity of PrLDC, the enzyme activity was evaluated at 30, 40, 46, 60 ℃ temperature conditions in the same manner as in Example 2-2. As a result, it was confirmed that PrLDC has the best activity at 60°C (FIG. 11).

In addition, the activity according to the pH of PrLDC was evaluated in the same manner as in Example 2-2. As a result, it was confirmed that PrLDC exhibited the highest activity at pH 6, but retained 90% or more activity even at pH 9. (FIG. 12).

Example 5. Preparation of Escherichia coli into which Pseudomonas putida- derived lysine decarboxylase gene was introduced and analysis of lysine decarboxylase activity expressed therefrom

5-1. Transformation of Escherichia coli by Pseudomonas putida- derived lysine decarboxylase gene

In order to clone the lysine decarboxylase gene (ppldc) derived from Pseudomonas putida, 5_PpLDC_NdeI (AATATACATATGTACAAAGACCTCCAA TTCCCC)) (SEQ ID NO: 21) and 3_PpLDC_XhoI (AATATACTCGAGTCACTCCCTTATGCAATCAACGGT) were purified using primers of Pseudomonas putida (AATATACTCGAGTCACTCCCTTATGCAATCAACGGT). PCR was performed using DNA as a template. Pfu DNA polymerase was used as the polymerase, and PCR was performed by repeating the conditions of 94° C.: 30 seconds, 55° C.: 30 seconds, 72° C.: 2 minutes 30 times, resulting in the amplified ppldc gene (SEQ ID NO: 8). Secured.

The obtained ppldc gene was expressed at low temperature in E. coli by the same method as in Example 2-1, and the result was confirmed by SDS-PAGE gel (FIG. 13). As can be seen in lanes 3 and 4 of FIG. 13, it was confirmed that Pseudomonas putida-derived lysine decarboxylase (PpLDC) was hardly expressed even under low temperature expression conditions.

The cell lysate was centrifuged at 13,000 rpm for 15 minutes, and a lysine conversion reaction was performed using the supernatant.

5-2. Lysine decarboxylase derived from Pseudomonas putida expressed in E. coli Activity assay

(1) Confirmation of reactivity of lysine decarboxylase

In order to verify the reactivity of PpLDC, the cell lysate of PpLDC obtained in 5-1 was centrifuged at 13,000 rpm for 15 minutes, and lysine conversion reaction was performed using the supernatant. 50 μl of soluble protein, 100 mM PLP, and 250 mM lysine were filled with 50 mM sodium phosphate (pH 6.2) buffer, and reacted at 46° C. for 2 hours in a reaction volume of 200 μl. As a result, 16% cadaverine was produced (Fig. 14).

(2) Analysis of lysine decarboxylase activity according to temperature and pH

In order to find the optimum temperature condition for the activity of PpLDC, the enzyme activity was evaluated under the temperature conditions of 50, 60, 70 ℃ in the same manner as in Example 2-2. As a result, it was confirmed that PpLDC has the best activity at 50°C (FIG. 15).

In addition, the activity conditions for the pH of PpLDC were evaluated in the same manner as in Example 2-2. As a result, it showed the highest activity at pH 6, and the reactivity was evaluated to be low when the pH was increased (FIG. 16).

Example 6. Preparation of Escherichia coli into which the Pseudomonas sinsansa- derived lysine decarboxylase gene was introduced and analysis of lysine decarboxylase activity expressed therefrom

6-1. Transformation of Escherichia coli by the lysine decarboxylase gene derived from Pseudomonas sinsansa

In order to clone the lysine decarboxylase gene (pxldc) derived from Pseudomonas sinsantha, 5_PxLDC_NdeI (AATATACATATGTACAAAGACCTCCAA TTCCCC) (SEQ ID NO: 23) and 3_PxLDC_XhoI (AATATACTCGAGTCACTCCCTTATGCAATCAACGGTATA) were used and the genome of the purified pseudomonas (Sinsadomonas 24) was used. PCR was performed using DNA as a template. Pfu DNA polymerase was used for gene amplification, and the amplified pxldc was obtained by repeating the conditions of 94° C.: 30 seconds, 45° C.: 30 seconds, and 72° C.: 2 minutes 30 times (SEQ ID NO: 10).

The obtained pxldc gene was expressed at low temperature in E. coli by the same method as in Example 2-1, and the result was confirmed by SDS-PAGE gel (FIG. 13). As can be seen in lanes 7 and 8 of FIG. 13, it was confirmed that Pseudomonas sinsansa-derived lysine decarboxylase (PxLDC) was overexpressed as a soluble protein under low temperature expression conditions.

6-2. Lysine decarboxylase derived from Pseudomonas sinsanssa expressed in E. coli Activity assay

(1) Confirmation of reactivity of PxLDC

In order to verify the reactivity of PxLDC, the cell lysate of PxLDC obtained in 6-1 was centrifuged at 13,000 rpm for 15 minutes, and a lysine conversion reaction was performed using the supernatant. 50 μl of soluble protein, 100 mM PLP, and 250 mM lysine were filled with 50 mM sodium phosphate (pH 6.2) buffer, and reacted at 46° C. for 2 hours in a reaction volume of 200 μl. As a result, 25% cadaverine was produced (Fig. 17).

(2) Analysis of lysine decarboxylase activity according to pH

In order to find the optimum pH condition for PxLDC, various pH enzyme activities were evaluated in the same manner as in Example 2-2 (FIG. 18). As a result, PxLDC showed the highest activity at pH 6, and the higher the pH, the lower the reactivity was evaluated.

Example 7. Lysine decarboxylase derived from E. coli and Pseudomonas thermotolerance Comparative analysis of the activity of derived lysine decarboxylase

7-1. Lysine from E. coli Decarboxylase Cloning And expression

EcLDC (SEQ ID NO: 11) was expressed by cloning cadA, an E. coli lysine decarboxylase gene. The homology of the amino acid sequence of PtLDC and EcLDC is 36%. The plasmid in which the cadA gene was cloned was inserted into E. coli K-12 BL21 and incubated at 37°C to induce expression for 4 hours. As a result of confirming the EcLDC having completed expression through an SDS-PAGE gel (FIG. 13; lanes 1 and 2), it was confirmed that EcLDC was overexpressed as a soluble protein.

7-2. EcLDC And PtLDC Comparative analysis of the relative enzyme activity of

(1) Comparison of activity according to temperature

In the same manner as in Example 2-2, the relative activity of EcLDC and PtLDC was compared under various temperature conditions (37, 42, 50, 60, 70, 80°C) (Fig. 19).

As a result, it was confirmed that both EcLDC and PtLDC showed the highest activity at 60°C. At 50 °C, EcLDC was evaluated to have a relative activity of 54% (the activity of EcLDC is fixed at 100% at 60 °C), and at 80 °C, it was evaluated to have a relative activity of 12%. PtLDC was evaluated to have a relative activity of 76% at 50°C (fixing the activity of PtLDC to 100% at 60°C) and 19% at 80°C. It was found that PtLDC better retains the activity under high temperature conditions. In conclusion, both enzymes showed a large difference in activity according to temperature, and the relative activity was evaluated to maintain PtLDC better.

(2) Comparison of activity according to pH

In addition, evaluation for various pHs (6.2, 7.4, 8.0, 9.0) was performed in the same manner as in Example 2-2 (FIG. 20). As a result, EcLDC was evaluated to exhibit the highest activity at pH 6, and as the pH increased, the EcLDC enzyme activity significantly decreased. At pH 9, EcLDC maintained 50% activity. On the other hand, PtLDC showed no significant change in activity due to pH, and it was confirmed that more than 90% activity was maintained at a pH between pH 6.2 and 9. Accordingly, it was evaluated that the stability of PtLDC was higher than that of EcLDC with respect to temperature and pH.

(3) Comparison of the activities of PtLDC and EcLDC

When the protein amounts of PtLDC and EcLDC were quantified to evaluate specific activity (unit/mg), PtLDC had a value of 10060 (unit/mg) and EcLDC had a value of 36335 (unit/mg). When comparing the reactivity, EcLDC showed about 3.6 times higher activity than PtLDC. In addition, when comparing the optimum temperature, it was confirmed that both enzymes reacted optimally at 60° C., and the activity significantly decreased as the temperature was changed. However, comparing the optimal pH conditions, in the case of EcLDC, the specific activity increases as the pH increases, but in the case of PtLDC, it was observed that the enzyme activity did not change significantly with respect to the pH change.

Although EcLDC has a higher activity than PtLDC, if the pH increases due to the reaction of lysine decarboxylase, the activity of EcLDC can be greatly affected by the pH change. PtLDC is evaluated to have a higher relative pH stability than EcLDC, and has an advantage in lysine conversion reaction. In addition, when lysine is bioconverted to commercially produce cadaverine, pH adjustment is necessary through acid treatment, but PtLDC can alleviate the pH titration part, and this part can be expected to lower the production cost in the bioconversion of cadaverine. It is evaluated as.

Korea Microorganism Conservation Center (overseas) KCCM11559P 20140724

<110> CJ CheilJedang Corporation <120> The novel Lysine Decarboxylase and Process for producing cadeverine using the same <130> PN122256 <160> 24 <170> KopatentIn 2.0 <210> 1 <211> 751 <212> PRT <213> Pseudomonas thermotolerans <220> <221> PEPTIDE <222> (1)..(751) <223> Lysine decarboxylase from Pseudomonas thermotolerans (PtLDC) <400> 1 Met Tyr Lys Asp Leu Gln Phe Pro Ile Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Asn Arg Val Arg Glu Ile Ala Arg Glu 20 25 30 Leu Glu Gln Asp Gly Phe Ala Ile Leu Ser Thr Ala Ser Ala Ser Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Arg Ser Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Ala Met 100 105 110 Ala Asp Leu Asn Gln Leu Arg Gly Leu Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Gly Tyr Leu 130 135 140 Glu Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Thr Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Tyr Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Asp Asp Leu 245 250 255 Val Leu Val Asp Arg Asn Cys His Lys Ser Ile Leu His Ala Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Cys Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Lys Glu Ala Ile 290 295 300 Gln Ala Lys Ile Ala Ala Ser Pro Leu Ala Arg Gly Arg Glu Pro Arg 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Met Val Lys Gln Ala Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Ala Gly Arg Tyr Gly Met Gly Thr Arg Leu Glu Ala Asp Ser Pro Leu 370 375 380 Val Phe Ala Thr His Ser Thr His Lys Leu Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Arg Asp Gly Gly Ser Arg Lys Leu Asp Arg 405 410 415 Tyr Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Ser Ile Leu Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Leu Arg Gln Asn Leu Pro Ala Asp Asp 465 470 475 480 Trp Trp Phe Asp Ile Trp Gln Pro Pro Arg Ala Ala Gly Val Glu Glu 485 490 495 Val Ala Thr Arg Asp Trp Leu Leu Glu Pro Asn Ala Glu Trp His Gly 500 505 510 Phe Gly Glu Val Asn Asp Asp Tyr Val Leu Leu Asp Pro Val Lys Val 515 520 525 Thr Leu Val Thr Pro Gly Leu Ser Ala Gly Gly Arg Leu Asp Glu His 530 535 540 Gly Ile Pro Ala Ala Val Val Ser Lys Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Leu Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Leu Tyr Asp Ala Asn Val Ala Leu Ala Glu Ala Leu Pro 595 600 605 Ser Ile Ala Arg Ala Gly Gly Thr Arg Tyr Ala Gly Met Gly Leu Arg 610 615 620 Asp Leu Cys Asp Glu Leu His Ala Cys Tyr Arg Glu Asn Ala Thr Ala 625 630 635 640 Lys Ala Leu Lys Arg Met Tyr Thr Thr Leu Pro Glu Val Val Met Lys 645 650 655 Pro Ala Asp Ala Tyr Asp Arg Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Asp Arg Leu Glu Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Gln 690 695 700 Ala Thr Arg Ser Ile Ile Asp Tyr Leu Gly Phe Ala Arg Asp Phe Asp 705 710 715 720 Arg Arg Phe Pro Gly Phe Asp Ala Asp Val His Gly Leu Gln Ser Glu 725 730 735 Glu Arg Gly Gly Glu Arg Cys Tyr Thr Val Asp Cys Ile Lys Ile 740 745 750 <210> 2 <211> 2256 <212> DNA <213> Pseudomonas thermotolerans <220> <221> gene <222> (1)..(2256) <223> Lysine decarboxylase gene from Pseudomonas thermotolerans (ptldc) <400> 2 atgtacaaag acctccaatt ccccattctc atcgtccatc gcgacatcaa ggccgatacc 60 gtggccggca accgcgtccg tgagatcgcc cgcgaactgg aacaggacgg cttcgccatc 120 ctctccacgg caagtgccag cgaagggcgc atcgtcgctt ccacccacca tggtctggcc 180 tgcatcctgg tcgccgccga gggagcggga gagaatcgaa gcctgctgca ggacgtggtg 240 gagctgatcc gcgtggccag ggtccgtgcg ccgcagctgc cgatcttcgc tctcggcgag 300 caggtgacca tcgagaacgc tccggccgag gccatggccg acctcaacca actgcgcggg 360 ctgctctacc tgttcgagga caccgtgccc ttcctcgccc gtcaggtggc gcgggccgcg 420 cgtggctacc tggaggggct gctgccgccg ttcttccgcg ccctggtgga gcacaccgcg 480 cagtccaact attcctggca caccccgggg cacggcggcg gtgtggctta ccgcaagagc 540 ccggtggggc aggcctttca ccagttcttc ggcgagaaca ccctgcgctc cgacttgtcg 600 gtttccgtac cggagctggg ttcgctgctg gatcacaccg ggccgctggc cgaggcggaa 660 acccgcgcgg cgcgcaactt cggcgccgac catacctatt tcgtgatcaa cggcacctcg 720 acggccaaca agatcgtctg gcactccatg gtcggccgcg acgatctggt gttggtggac 780 cgcaactgcc acaagtcgat cctccacgcc atcatcatga ccggtgccat ccccctgtac 840 ctgtgcccgg agcgcaacga gctgggcatc atcgggccga ttccgctctc cgagttcagc 900 aaggaggcga tccaggcgaa gatcgccgcc agcccgctgg ccagagggcg cgagccgcgg 960 gtcaagctgg cggtggtgac caattccacc tacgacggcc tctgttacaa cgccgagatg 1020 gtcaagcagg ccctcggcga cagcgtcgag gtgctgcact tcgacgaggc ctggtacgcc 1080 tacgcggcct tccacgagtt ctacgcgggg cgttacggca tgggcactcg cctggaggcg 1140 gactcgcctc tggtctttgc cacccattcc acccacaagc tgctggctgc cttcagccag 1200 gcctcgatga ttcacgtgcg cgacggcggt agccgcaagc tggaccggta ccgcttcaac 1260 gaggccttca tgatgcacat ctccacctcg ccgcagtaca gcatcctcgc ctcgctggac 1320 gtggcctcgg cgatgatgga gggaccggcc gggcgctcgc tgatccagga aaccttcgat 1380 gaagcgctga gcttccgccg tgctctggcc aacctgcgcc agaacctgcc ggcggacgac 1440 tggtggttcg acatctggca gccgccgcgc gctgctggtg tcgaggaggt ggcgacccgc 1500 gactggctgc tggagccgaa tgccgagtgg cacggcttcg gcgaggtgaa cgacgactac 1560 gtgctgctcg atccggtcaa ggtcaccctg gtcaccccgg ggctgagcgc cggcgggcgc 1620 ctggacgagc acggcattcc cgccgcggtg gtcagcaagt tcctctggga acgcggcctg 1680 gtggtggaga agaccggcct gtactccttc ctggtgttgt tctccatggg gatcaccaag 1740 ggcaagtgga gcaccctgct caccgagctg ctggagttca agcgcctcta cgacgccaac 1800 gtggcgcttg ccgaggcgtt gccgagcatc gcccgcgccg gtggcacccg ctatgccggc 1860 atgggcctgc gcgacctgtg cgacgagctg cacgcctgct accgggagaa cgccaccgcc 1920 aaggccctca agcgcatgta caccacgctc cccgaggtgg tgatgaagcc tgccgatgcc 1980 tacgaccggc tggtccgcgg agaggtggag gcggtgccca tcgaccgtct ggaaggacga 2040 atcgccgcgg tgatgctggt gccctatccg ccgggcattc cgctgatcat gccgggcgag 2100 cgcttcaccc aggcgacccg ttcgatcatc gactacctgg gtttcgcccg cgatttcgat 2160 cgccgcttcc ccggcttcga cgcggacgtg cacggcctgc agagcgagga gcgcggcggc 2220 gagcgatgct acacggtgga ctgcatcaag atctga 2256 <210> 3 <211> 751 <212> PRT <213> Pseudomonas alcaligenes <220> <221> PEPTIDE <222> (1)..(751) <223> Lysine decarboxylase from Pseudomonas alcaligenes (PaLDC) <400> 3 Met Tyr Lys Asp Leu Lys Phe Pro Ile Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Asp Arg Val Arg Gly Ile Ala Arg Glu 20 25 30 Leu Glu Gln Asp Gly Phe Val Ile Leu Ser Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Val Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Ala Met 100 105 110 Ala Asp Leu Asn Gln Leu Arg Gly Leu Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Asn Tyr Leu 130 135 140 Glu Gly Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Ala Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Ala Arg Asp Asp Leu 245 250 255 Val Leu Val Asp Arg Asn Cys His Lys Ser Ile Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Ser Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Arg Glu Ser Ile 290 295 300 Gln Ala Lys Ile Asp Ala Ser Pro Leu Ala Arg Gly Arg Ala Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Ala Leu Gly Asp Thr Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Asp Gly Arg Tyr Gly Met Gly Thr Ala Arg Ser Glu Glu Gly Pro Leu 370 375 380 Val Phe Thr Thr His Ser Thr His Lys Leu Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Ala Arg Gln Leu Asp Arg 405 410 415 Asp Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Val Trp Gln Thr Leu Asp Ala Lys Asp 465 470 475 480 Trp Trp Phe Asp Ile Trp Glu Pro Pro Gln Val Glu Gly Ala Glu Ala 485 490 495 Val Ala Thr Gly Asp Trp Val Leu Glu Pro Gly Ala Asp Trp His Gly 500 505 510 Phe Gly Glu Val Ala Asp Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Val Thr Pro Gly Leu Ser Ala Asp Gly Lys Leu Gly Glu Gln 530 535 540 Gly Ile Pro Ala Ala Val Val Gly Lys Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Leu Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Ser Tyr Asp Ala Asn Ala Pro Leu Thr Ser Ala Leu Pro 595 600 605 Ser Val Ala Arg Ala Asp Ala Ala Arg Tyr Gln Gly Leu Gly Leu Arg 610 615 620 Asp Leu Cys Asp Gln Leu His Ala Cys Tyr Arg Asp Asn Ala Thr Ala 625 630 635 640 Lys Ala Met Arg Arg Met Tyr Thr Ala Leu Pro Glu Leu Ala Ile Lys 645 650 655 Pro Ser Glu Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Pro Ile Glu Gln Leu Gln Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Ala 690 695 700 Gln Thr Arg Ser Ile Ile Asp Tyr Leu Ala Phe Ala Arg Thr Phe Asp 705 710 715 720 Ser Ala Phe Pro Gly Phe Asp Ser Asp Val His Gly Leu Gln His Asp 725 730 735 Asp Ser Pro Met Gly Arg Cys Tyr Thr Val Asp Cys Ile Arg Glu 740 745 750 <210> 4 <211> 2256 <212> DNA <213> Pseudomonas alcaligenes <220> <221> gene <222> (1)..(2256) <223> Lysine decarboxylase gene from Pseudomonas alcaligenes (paldc) <400> 4 atgtacaaag acctgaagtt ccccatcctc atcgtccacc gcgacatcaa ggccgatacg 60 gtcgccggtg atcgcgtgcg cggcatcgcc cgcgaactgg agcaggacgg tttcgtcatc 120 ctctccaccg ccagttccgc cgaagggcgc atcgtcgcct ccacccacca cggcttggcc 180 tgcatcctcg tcgccgccga aggggcgggc gagaaccagc gcctgctgca ggacgtggtc 240 gagctgatcc gcgtggcccg ggtgcgtgcg ccgcaactgc cgatcttcgc cctgggcgaa 300 caggtgacca tcgagaacgc cccggccgaa gccatggccg acctcaacca gctgcgcggc 360 ctgctctacc tcttcgaaga caccgtgccc ttcctcgccc gccaggtcgc ccgcgccgcg 420 cgcaactacc tcgaaggcct gctgccgccg ttcttccgtg ccctggtgga gcacaccgcg 480 caatccaact actcctggca cacgcccggt cacggcggtg gcgtcgccta ccgcaagagc 540 ccggtggggc aggccttcca ccagttcttc ggcgaaaaca ccctgcgctc ggacctctcc 600 gtctcggtgc ccgagctggg ctcgctgctg gatcacaccg gccccctggc cgaagccgag 660 gcccgcgccg cgcgcaactt cggtgctgac cacaccttct tcgtgatcaa cggcacctcc 720 accgcgaaca agatcgtctg gcactccatg gtcgcccgcg acgacctggt gctggtggac 780 cgcaactgcc acaagtcgat cctccactcg atcatcatga ccggcgccat cccgctctac 840 ctgagccccg agcgcaacga actgggcatc atcgggccca tccccctgag cgagttcagt 900 cgcgaatcga tccaggccaa gatcgacgcc agcccactgg cccggggccg cgcgcccaag 960 gtcaagctgg cggtggtgac caactccacc tacgacggcc tctgctacaa cgccgagctg 1020 atcaagcagg cgctgggcga caccgttgag gtgctgcact tcgacgaagc ctggtacgcc 1080 tacgccgcct tccacgagtt ctacgacggc cgctacggca tgggtacggc gcgcagcgaa 1140 gaaggcccgc tggtgttcac cacccactcc acccacaagc tgctggcggc cttcagtcag 1200 gcctcgatga tccatgtgca ggacggcggc gcccgccagc tggaccggga tcgcttcaac 1260 gaggcgttca tgatgcacat ctccacttcg ccccagtacg gcatcatcgc ctcgctggac 1320 gtcgcctcgg cgatgatgga aggccccgcc gggcgctcgc tgatccagga aaccttcgac 1380 gaggccctga gcttccgccg tgccctggcc aacgtctggc agaccctgga tgccaaggat 1440 tggtggttcg atatctggga gccgccccag gtggaaggcg ccgaggcggt ggccaccggc 1500 gactgggtgc tggagcccgg cgccgactgg cacggcttcg gcgaggtggc ggacgactac 1560 gtgctgctcg acccgatcaa ggtcaccctg gtcacccccg ggctcagtgc cgacggcaag 1620 ctcggcgagc agggcatccc ggcggcggtg gtgggcaagt tcctctggga gcgtggcctg 1680 gtggtggaga agaccggtct ctactccttc ctcgtgctgt tctccatggg catcaccaag 1740 ggcaaatgga gcaccctgct caccgagctg ctggagttca aacgctccta cgacgccaac 1800 gccccgctga ccagtgcact gccctcggtg gcccgggccg atgccgcccg ctaccagggg 1860 ctgggcctgc gcgacctctg cgaccagctg cacgcctgct accgcgacaa cgccacggcc 1920 aaggccatgc ggcgcatgta caccgcgctt ccggagctgg ccatcaagcc gtcggaggct 1980 tacgacaagc tggtgcgtgg cgaggtcgag gcggtgccca tcgagcagct gcaagggcgc 2040 attgccgcgg tgatgctggt gccgtacccg ccgggcatcc cgctgatcat gccgggggag 2100 cgtttcactg cgcagacccg ctcgatcatt gactacctgg ccttcgcccg gaccttcgac 2160 agcgccttcc ccggcttcga ttccgatgtc cacggcctgc agcacgacga cagcccaatg 2220 gggcgctgct ataccgttga ttgcataagg gagtga 2256 <210> 5 <211> 751 <212> PRT <213> Pseudomonas resinovorans <220> <221> PEPTIDE <222> (1)..(751) <223> Lysine decarboxylase from Pseudomonas resinovorans (PrLDC) <400> 5 Met Tyr Lys Glu Leu Lys Phe Pro Val Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Glu Arg Val Arg Ser Ile Ala Arg Glu 20 25 30 Leu Glu Gln Asp Gly Phe Thr Ile Leu Pro Thr Ala Ser Ser Ala Glu 35 40 45 Gly Arg Ile Val Ala Ser Thr His His Gly Leu Ala Cys Ile Leu Val 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Gln Arg Leu Leu Gln Asp Met Val 65 70 75 80 Glu Leu Ile Arg Val Ala Arg Val Arg Ala Pro Gln Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Ile Glu Asn Ala Pro Ala Glu Ala Met 100 105 110 Ala Asp Leu Asn Gln Leu Arg Gly Leu Leu Tyr Leu Tyr Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala Arg Gly Tyr Leu 130 135 140 Glu Ala Leu Leu Pro Pro Phe Phe Arg Ala Leu Val Glu His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Ala Arg Ala Ala 210 215 220 Gln Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Asp Asp Leu 245 250 255 Val Leu Val Asp Arg Asn Cys His Lys Ser Ile Val His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Thr Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ala Glu Phe Ser Arg Glu Ser Ile 290 295 300 Gln Ala Lys Ile Asp Ala Ser Pro Leu Ala Lys Gly Arg Ala Ala Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Ala Leu Gly Asp Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Tyr 355 360 365 Ala Gly Arg Tyr Gly Met Cys Thr His Arg Glu Ala His Ser Pro Leu 370 375 380 Val Phe Thr Thr His Ser Thr His Lys Leu Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Ala Arg Gln Leu Asp Arg 405 410 415 His Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Gly Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Ile Gln Glu Thr Phe Asp Glu Ala Leu Arg 450 455 460 Phe Arg Arg Ala Leu Ala Asn Leu Arg Gln Asn Leu Ala Ala Asp Asp 465 470 475 480 Trp Trp Phe Asp Ile Trp Gln Ser His Leu Ala Glu Gly Ala Asp Thr 485 490 495 Val Ala Thr Glu Asp Trp Leu Leu Arg Pro Asp Ala Asp Trp His Gly 500 505 510 Phe Gly Asp Val Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Val Thr Pro Gly Leu Thr Ala Asp Gly Lys Leu Gly Glu Arg 530 535 540 Gly Ile Pro Ala Ala Val Val Ser Lys Phe Leu Trp Glu Arg Gly Val 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Leu Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Gly Tyr Asp Thr Asn Leu Pro Leu Ala Glu Ala Leu Pro 595 600 605 Ser Ile Ala Arg Asp His Gly Ala Arg Tyr Ala Gly Met Gly Leu Arg 610 615 620 Asp Leu Cys Asp Ala Leu His Gly Cys Tyr Arg Asn Ser Ala Thr Pro 625 630 635 640 Lys Ala Leu Arg Arg Met Tyr Thr Gln Leu Pro Glu Leu Ala Met Lys 645 650 655 Pro Ala Asp Ala Tyr Asp Lys Leu Val Arg Gly Glu Val Glu Pro Val 660 665 670 Ser Leu Asp Leu Leu Gln Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Ala 690 695 700 Glu Thr Arg Ala Ile Ile Asp Tyr Leu Glu Phe Ala Arg Thr Phe Asp 705 710 715 720 Leu Ser Phe Pro Gly Phe Asp Ile Asp Val His Gly Leu Asn Cys Gln 725 730 735 Glu Ser Pro Thr Gly Arg Cys Tyr Thr Val Asp Cys Ile Arg Glu 740 745 750 <210> 6 <211> 2256 <212> DNA <213> Pseudomonas resinovorans <220> <221> gene <222> (1)..(2256) <223> Lysine decarboxylase gene from Pseudomonas resinovorans (prldc) <400> 6 atgtacaaag agctcaagtt ccccgtcctc atcgtccatc gtgacatcaa ggccgatacc 60 gtcgccggcg agcgggtccg cagcatcgcc cgcgagctgg agcaggacgg cttcaccatc 120 ctccccaccg ccagctccgc cgaaggccgt atcgtcgcct ccacccacca tggcctcgcc 180 tgcatcctgg tggccgccga aggcgccggg gaaaaccagc ggctgctgca ggacatggtg 240 gagctgatcc gcgtggcgcg ggtgcgcgcg ccgcagttgc cgatcttcgc cctgggggaa 300 caggtcacca tcgagaacgc gccggccgag gccatggccg acctcaacca gttgcgcggc 360 ctgctctatc tctacgaaga caccgtgcct ttcctcgccc gccaggtggc ccgagccgcc 420 cgcggctacc tggaagccct gttgccgcca ttcttccgcg ccctggtcga gcacaccgcg 480 cagtccaact actcctggca caccccgggc cacggcggtg gcgtggccta ccgcaagagt 540 ccggtggggc aggccttcca ccagttcttc ggggaaaaca ccctgcgctc ggacctctcg 600 gtatcggtgc cggaactggg ctcgctgctg gaccacaccg ggcccctggc cgaagccgag 660 gcgcgtgcgg cgcagaactt cggcgccgac cacaccttct tcgtgatcaa tggcacttcc 720 accgccaaca agatcgtctg gcactccatg gtcggccgcg atgacctggt gctggtggac 780 cgcaactgcc acaagtccat cgtccactcg atcatcatga ccggcgccat ccccctgtac 840 ctgacgccgg agcgcaacga actgggcatc atcggaccca tccccctcgc cgaattcagc 900 cgtgagtcga tccaggcgaa gatcgacgcc agccccctgg ccaaggggcg agccgccaag 960 gtcaagctgg cggtggtgac caactccacc tacgacggcc tctgctacaa cgccgagctg 1020 atcaagcagg cactgggcga ctcggtggag gtgctgcact tcgacgaggc ctggtacgcc 1080 tacgctgcct tccacgagtt ctatgccggg cgctacggca tgtgcaccca ccgcgaggcg 1140 cactcgccgc tggtcttcac cacccattcc acccacaagc tgctggccgc cttcagccag 1200 gcctcgatga tccatgtgca ggacggcggc gcgcgccagc tcgaccggca ccgcttcaac 1260 gaagccttca tgatgcacat ctccacctcg ccgcagtacg gcatcatcgc ttccctggac 1320 gtggcctcgg ccatgatgga ggggcccgcc gggcgctcgt tgatccagga gactttcgac 1380 gaggcgctgc gttttcgccg cgccctggcc aacctgcggc agaacctggc ggcggacgac 1440 tggtggttcg atatctggca gtcgcacctg gcggaaggcg ccgacacggt cgccaccgag 1500 gattggctgt tgcgtcccga cgccgactgg cacggattcg gcgatgtggc cgaggactac 1560 gtgctgctcg atccgatcaa ggtcaccctg gtgacgccgg gcctgaccgc cgatggcaag 1620 ctgggggagc ggggcattcc cgcggcggtg gtcagcaagt tcctctggga gcgtggggtg 1680 gtggtggaga agaccggcct ctattccttc ctggtgctgt tctccatggg tatcaccaag 1740 ggcaagtgga gcaccctgct caccgagttg ctggagttca agcgcggcta tgacaccaac 1800 ctgcccctgg ccgaggcgct gccctccatc gcccgggacc acggcgcgcg gtacgccggc 1860 atgggcctgc gcgatctctg cgacgccctg catggctgct accgcaacag cgccacgccc 1920 aaggccctgc ggcgcatgta cacacagctg ccggaactgg cgatgaagcc cgccgacgct 1980 tacgacaagc tggtgcgcgg cgaggtggaa ccggtgtccc tggacctgct gcaagggcgg 2040 atcgcggcgg tgatgctggt gccctatcca ccgggcatac cgctgatcat gccgggggag 2100 cgcttcaccg ccgagactcg cgcgatcatc gattacctgg aattcgcccg caccttcgac 2160 ctgagcttcc ccggcttcga tatcgatgtg catggcctca actgtcagga aagtcctacc 2220 gggcgctgct atacagtgga ctgcatcagg gaataa 2256 <210> 7 <211> 749 <212> PRT <213> Pseudomonas putida <220> <221> PEPTIDE <222> (1)..(749) <223> Lysine decarboxylase from Pseudomonas putida (PpLDC) <400> 7 Met Tyr Lys Asp Leu Lys Phe Pro Ile Leu Ile Val His Arg Ala Ile 1 5 10 15 Lys Ala Asp Ser Val Ala Gly Glu Arg Val Arg Gly Ile Ala Glu Glu 20 25 30 Leu Arg Gln Asp Gly Phe Ala Ile Leu Ala Ala Ala Asp His Ala Glu 35 40 45 Ala Arg Leu Val Ala Ala Thr His His Gly Leu Ala Cys Met Leu Ile 50 55 60 Ala Ala Glu Gly Val Gly Glu Asn Thr His Leu Leu Gln Asn Met Ala 65 70 75 80 Glu Leu Ile Arg Leu Ala Arg Leu Arg Ala Pro Asp Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Leu Glu Asn Ala Pro Ala Glu Ala Met 100 105 110 Ser Glu Leu Asn Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ala Ala His Thr Tyr Leu 130 135 140 Asp Gly Leu Leu Pro Pro Phe Phe Lys Ala Leu Val Gln His Thr Ala 145 150 155 160 Gln Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr His Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Ala Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ala Met Val Gly Arg Asp Asp Leu 245 250 255 Val Leu Val Asp Arg Asn Cys His Lys Ser Val Val His Ala Ile Ile 260 265 270 Met Thr Gly Ala Val Pro Leu Tyr Leu Cys Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Pro Glu Ala Ile 290 295 300 Glu Ala Lys Ile Gln Ala Asn Pro Leu Ala Arg Asp Arg Gly Arg Arg 305 310 315 320 Ile Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 His Ala Gly Met Ile Lys Gln Cys Leu Gly Ala Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Phe Ala Tyr Ala Ala Phe His Asp Phe Phe 355 360 365 Thr Gly Arg Tyr Ala Met Gly Thr Ala Cys Thr Ala Gly Ser Pro Leu 370 375 380 Val Phe Ser Thr His Ser Thr His Lys Leu Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Ala Arg Arg Gln Leu Asp Arg 405 410 415 Asp Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Ser Ile Leu Ala Ser Leu Asp Val Ala Ser Ser Met Met Glu Gly 435 440 445 Pro Ala Gly His Ser Leu Leu Gln Glu Met Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Leu Arg Glu His Ile Ala Ala Asp Asp 465 470 475 480 Trp Trp Phe Ser Ile Trp Gln Pro Pro Gly Thr Glu Gly Ile Gln Arg 485 490 495 Leu Ala Ala Gln Asp Trp Leu Leu Gln Pro Gly Ala Glu Trp His Gly 500 505 510 Phe Gly Glu Val Val Asp Asp Tyr Val Leu Leu Asp Pro Leu Lys Val 515 520 525 Thr Leu Val Met Pro Gly Leu Ser Ala Gly Gly Val Leu Gly Glu His 530 535 540 Gly Ile Pro Ala Ala Val Val Ser Lys Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Leu Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg His Tyr Asp Gly Asn Thr Ala Leu Ser Ser Cys Leu Pro 595 600 605 Ser Val Val Ala Ala Asp Ala Ser Arg Tyr Gln Arg Met Gly Leu Arg 610 615 620 Asp Leu Cys Asp Gln Leu His Asp Cys Tyr Arg Ala Asn Ala Thr Ala 625 630 635 640 Lys Gln Leu Lys Arg Leu Phe Thr Arg Leu Pro Glu Val Ala Val Ser 645 650 655 Pro Ala Arg Ala Tyr Asp Gln Met Val Arg Gly Asp Val Glu Ala Val 660 665 670 Pro Ile Glu Ala Leu Leu Gly Arg Val Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690 695 700 Ala Thr Arg Ser Ile Leu Asp Tyr Leu Ala Phe Ala Arg Ala Phe Asn 705 710 715 720 Gln Gly Phe Pro Gly Phe Val Ala Asp Val His Gly Leu Gln Asn Glu 725 730 735 Ser Gly Arg Tyr Thr Val Asp Cys Ile Thr Glu Cys Glu 740 745 <210> 8 <211> 2250 <212> DNA <213> Pseudomonas putida <220> <221> gene <222> (1)..(2250) <223> Lysine decarboxylase gene from Pseudomonas putida (ppldc) <400> 8 atgtacaagg acctcaagtt cccgatcctc atcgtccacc gggctatcaa ggctgacagt 60 gtcgccgggg agcgcgtgcg gggcatcgcc gaggaactgc gccaggacgg tttcgccatt 120 ctggccgccg ccgaccacgc cgaagcgcgc ctggttgccg ccactcacca cggcctggcc 180 tgcatgctga ttgccgccga aggagttggc gaaaacaccc acctgctgca gaacatggcc 240 gagctgatcc gcctggcgcg catgcgcgcg cccgacttgc cgatcttcgc cttgggcgag 300 caggtgaccc tggaaaacgc ccctgccgaa gccatgagcg agctcaacca actgcgtggc 360 atcctttacc tgttcgaaga caccgtgccg tttctcgccc gccaggtggc gcgtgccgca 420 cacacctacc ttgacggtct gctgccaccg ttcttcaagg ccctggtgca gcataccgcg 480 cagtccaact attcctggca taccccgggc catggcggtg gcgtggccta tcataaaagc 540 ccggtaggcc aggccttcca ccagttcttc ggggaaaaca ccctgcgctc ggacctgtct 600 gtttcagtgc cggagctggg ctcgctgctc gaccacacag gccccttggc cgaagccgag 660 gccagggcgg cgcgcaactt cggtgccgac cacaccttct tcgtcatcaa tggcacctcc 720 acagccaaca agattgtctg gcacgccatg gtcggtcgcg acgacctggt gttggtggac 780 cgcaactgcc ataagtcagt ggtgcacgcg atcatcatga ccggcgccat tccgctgtac 840 ctgtgcccag agcgcaacga gctgggcatc atcggcccga tcccgctcag cgagttcagc 900 cccgaggcaa tcgaggcgaa gatccaggcc aacccccttg cccatggccg tgggcaacgt 960 atcaagctgg cggtagtgac caactccacc tatgacgggc tgtgctacca cgccgggatg 1020 atcaagcagg ccctgggtgc cagcgtggaa gtactgcact tcgacgaggc ctggttcgct 1080 tatgcggcgt ttcacggctt cttcaccggg cgctatgcca tgggcactgc ctgcgcagcc 1140 gacagcccgt tggtgttcag cacccattcc acccacaagc tgctggcggc gttcagccag 1200 gcctcgatga tccatgtgca ggacggggcc aggcggcagc tggaccggga ccgcttcaac 1260 gaagcgttca tgatgcatat ctcgacttcg ccgcagtaca gcatccttgc ctcgctggac 1320 gtggcctcga ccatgatgga agggcaggcc gggcattcgc tgttgcaaga aatgttcgat 1380 gaggcgctga gttttcgtcg tgccctggcc aacctgcgcg agcacattgc tgcggatgac 1440 tggtggttca gtatttggca gccgcccagc actgaaggca tccagccctt ggccgcgcag 1500 gactggctgc tgcagccggg ggcgcagtgg catggctttg gtgaggtggc ggacggctac 1560 gtgttgctcg accctctgaa ggtgaccctg gtaatgccgg ggctgagtgc gggcggtgtg 1620 ctgggtgagc gtggcatccc ggcggcggtg gtcagcaagt ttctctggga gcgcgggctg 1680 gtggtggaaa aaaccggctt gtacagcttc ctggtgctgt tttccatggg catcaccaag 1740 ggcaagtgga gcaccttgct caccgaactg ctggagttca agcgccacta tgacggcaat 1800 acaccgctga gcagttgcct gccgagtgtg ggggttgccg atgcctcacg ctaccggggc 1860 atgggcctgc gcgacctgtg tgaacagttg catgactgct accgtgccaa tgccacggcc 1920 aagcagctga agcgggtgtt cacgcgtttg ccggaggtgg ccgtgagccc cgctcgggct 1980 tatgaccaga tggtacgtgg cgaggtggaa gcggtgccga tcgaagcttt gctgggccgt 2040 gtggccgcgg tgatgctggt gccgtacccg cccggtattc cgttgatcat gccgggagag 2100 cggttcaccg aggcgacccg ctcgatactt gactacttgg ccttcgcccg agccttcaac 2160 caaggctttc cggggtttgt cgcggatgtt cacggcctgc agaacgaaaa tggccgctac 2220 accgtggatt gcatcatgga atgcgagtga 2250 <210> 9 <211> 751 <212> PRT <213> Pseudomonas synxantha <220> <221> PEPTIDE <222> (1)..(751) <223> Lysine decarboxylase from Pseudomonas synxantha (PxLDC) <400> 9 Met Tyr Lys Asp Leu Lys Phe Pro Ile Leu Ile Val His Arg Asp Ile 1 5 10 15 Lys Ala Asp Thr Val Ala Gly Asp Arg Val Arg Gly Ile Ala Arg Glu 20 25 30 Leu Glu Gln Glu Gly Phe Ser Ile Phe Ser Ala Val Asp Tyr Ala Glu 35 40 45 Gly Arg Leu Val Ala Ser Thr His His Gly Leu Ala Cys Met Leu Ile 50 55 60 Ala Ala Glu Gly Ala Gly Glu Asn Thr His Leu Leu Gln Asn Met Val 65 70 75 80 Glu Leu Ile Arg Leu Ala Arg Val Arg Ala Pro Asn Leu Pro Ile Phe 85 90 95 Ala Leu Gly Glu Gln Val Thr Leu Glu Asn Ala Pro Ala Asp Ala Met 100 105 110 Ser Glu Leu Asn Gln Leu Arg Gly Ile Leu Tyr Leu Phe Glu Asp Thr 115 120 125 Val Pro Phe Leu Ala Arg Gln Val Ala Arg Ser Ala Arg Thr Tyr Leu 130 135 140 Asp Gly Leu Leu Pro Pro Phe Phe Lys Ala Leu Val Gln His Thr Ala 145 150 155 160 Asp Ser Asn Tyr Ser Trp His Thr Pro Gly His Gly Gly Gly Val Ala 165 170 175 Tyr Arg Lys Ser Pro Val Gly Gln Ala Phe His Gln Phe Phe Gly Glu 180 185 190 Asn Thr Leu Arg Ser Asp Leu Ser Val Ser Val Pro Glu Leu Gly Ser 195 200 205 Leu Leu Asp His Thr Gly Pro Leu Ala Glu Ala Glu Ala Arg Ala Ala 210 215 220 Arg Asn Phe Gly Ala Asp His Thr Phe Phe Val Ile Asn Gly Thr Ser 225 230 235 240 Thr Ala Asn Lys Ile Val Trp His Ser Met Val Gly Arg Asp Asp Leu 245 250 255 Val Leu Val Asp Arg Asn Cys His Lys Ser Val Leu His Ser Ile Ile 260 265 270 Met Thr Gly Ala Ile Pro Leu Tyr Leu Cys Pro Glu Arg Asn Glu Leu 275 280 285 Gly Ile Ile Gly Pro Ile Pro Leu Ser Glu Phe Ser Pro Glu Ser Ile 290 295 300 Arg Ala Lys Ile Asp Ala Ser Pro Leu Ala Tyr Gly Arg Pro Pro Lys 305 310 315 320 Val Lys Leu Ala Val Val Thr Asn Ser Thr Tyr Asp Gly Leu Cys Tyr 325 330 335 Asn Ala Glu Leu Ile Lys Gln Gln Leu Gly Asn Ser Val Glu Val Leu 340 345 350 His Phe Asp Glu Ala Trp Tyr Ala Tyr Ala Ala Phe His Glu Phe Phe 355 360 365 Ala Gly Arg Tyr Gly Met Gly Thr Ser Arg Thr Pro Asp Ser Pro Leu 370 375 380 Val Phe Thr Thr His Ser Thr His Lys Leu Leu Ala Ala Phe Ser Gln 385 390 395 400 Ala Ser Met Ile His Val Gln Asp Gly Gly Ala Arg Gln Leu Asp Arg 405 410 415 Asp Arg Phe Asn Glu Ala Phe Met Met His Ile Ser Thr Ser Pro Gln 420 425 430 Tyr Ser Ile Ile Ala Ser Leu Asp Val Ala Ser Ala Met Met Glu Gly 435 440 445 Pro Ala Gly Arg Ser Leu Leu Gln Glu Met Phe Asp Glu Ala Leu Ser 450 455 460 Phe Arg Arg Ala Leu Ala Asn Leu Arg Gln His Ile Ala Ala Glu Asp 465 470 475 480 Trp Trp Phe Ser Ile Trp Gln Pro Gln Ser Val Ala Gly Ile Asp Arg 485 490 495 Val Ala Thr Ala Asp Trp Leu Leu His Pro Gln Asp Asp Trp His Gly 500 505 510 Phe Gly Asp Val Ala Glu Asp Tyr Val Leu Leu Asp Pro Ile Lys Val 515 520 525 Thr Leu Val Met Pro Gly Leu Asn Ala Gly Gly Ala Leu Ser Asp Cys 530 535 540 Gly Ile Pro Ala Ala Val Val Ser Lys Phe Leu Trp Glu Arg Gly Leu 545 550 555 560 Val Val Glu Lys Thr Gly Leu Tyr Ser Phe Leu Val Leu Phe Ser Met 565 570 575 Gly Ile Thr Lys Gly Lys Trp Ser Thr Leu Leu Thr Glu Leu Leu Glu 580 585 590 Phe Lys Arg Ser Tyr Asp Ala Asn Val Ser Leu Ala Ser Cys Leu Pro 595 600 605 Ser Val Tyr Ala Gln Gly Pro Val Arg Tyr Gln Gly Leu Gly Leu Arg 610 615 620 Asp Leu Cys Asp Gln Leu His Ser Cys Tyr Arg Ser Asn Ala Thr Ala 625 630 635 640 Lys His Leu Lys Arg Met Tyr Thr Val Leu Pro Gln Ile Ala Met Lys 645 650 655 Pro Ala Asp Ala Tyr Asp Gln Leu Val Arg Gly Glu Val Glu Ala Val 660 665 670 Ser Ile Asp Ala Leu Pro Gly Arg Ile Ala Ala Val Met Leu Val Pro 675 680 685 Tyr Pro Pro Gly Ile Pro Leu Ile Met Pro Gly Glu Arg Phe Thr Glu 690 695 700 Ser Thr Arg Ser Ile Ile Asp Tyr Leu Ala Phe Ala Arg Thr Phe Asp 705 710 715 720 Ser Ser Phe Pro Gly Phe Val Ala Asp Val His Gly Leu Gln His Glu 725 730 735 Asp Asp Gly Ser Gly Arg Arg Tyr Thr Val Asp Cys Ile Lys Gly 740 745 750 <210> 10 <211> 2256 <212> DNA <213> Pseudomonas synxantha <220> <221> gene <222> (1)..(2256) <223> Lysine decarboxylase gene from Pseudomonas synxantha (pxldc) <400> 10 atgtacaaag acctcaagtt ccctattctt atcgtgcacc gtgacatcaa ggccgacacc 60 gttgccggtg accgggttcg aggcatcgcc agggagttgg aacaagaggg cttcagtata 120 ttttctgcgg tggattacgc cgaagggcgg ttggtggcct ccacccatca tggtttggcg 180 tgcatgttga tcgcagcaga aggcgccggg gaaaataccc acctgctgca aaacatggtc 240 gagctgatcc gcctggcgcg ggtaagggca cccaacctgc cgatctttgc cctgggtgag 300 caagtcaccc ttgaaaacgc gccggccgat gcgatgagcg agcttaacca gctacgcggc 360 attctttatc tgttcgaaga caccgtgccg ttcctggcgc gccaggtcgc ccgctctgcc 420 cgcacttacc tggacggcct gttaccgccg ttcttcaagg ccttggtgca gcacaccgcc 480 gattccaatt attcctggca cacccctggc catggcggtg gcgtggcgta tcgtaaaagc 540 ccggtggggc aggcgtttca ccagttcttc ggggagaaca ccctgcgctc ggacttgtct 600 gtttctgtcc ctgaactggg ctcgctgctc gatcataccg ggcccctggc cgaagccgag 660 gcccgcgccg cgcgcaactt tggcgccgac cataccttct tcgtcatcaa tggcacctcc 720 accgccaaca agatcgtctg gcattccatg gtcggtcgcg acgacctggt gttggtggac 780 cgcaactgcc acaagtcagt gctgcactcg atcatcatga ccggcgcgat cccgctgtat 840 ctgtgcccgg agcgcaacga actggggatc atcggcccga tccccttgag tgaattcagc 900 cccgaatcaa tccgcgccaa gatcgacgcc agcccgttgg catatggccg gccacccaag 960 gtgaagctgg cggtggtgac caattccacc tacgacggcc tgtgctacaa cgccgaactg 1020 atcaagcagc aattgggtaa tagcgtagag gtgctgcact tcgacgaagc ctggtatgcc 1080 tatgcggcat ttcacgagtt tttcgccggg cgctatggca tgggcacctc gcgcacaccg 1140 gacagcccgc tggtatttac cacccactcc acccacaaac tgctggccgc attcagccag 1200 gcatcgatga ttcatgtgca ggatggcggc gcacggcagc tggaccgtga ccgtttcaac 1260 gaagccttca tgatgcacat ctcgacttca ccgcaataca gcatcatcgc ttcgctggat 1320 gtcgcttcgg cgatgatgga aggccccgcc gggcgctcgc tgttgcagga aatgttcgac 1380 gaggccctga gtttccgccg cgcgctggcc aacctgcgcc agcatatcgc tgccgaggat 1440 tggtggtttt cgatctggca gccacaatcg gtggcgggta tcgaccgcgt tgccacggcg 1500 gactggctat tgcatcccca ggatgattgg cacggctttg gcgatgtggc tgaagattat 1560 gtcttgctgg acccgatcaa agtcaccctg gtgatgcctg gcctcaatgc aggtggcgcc 1620 ttgagcgatt gtgggattcc cgccgcggtg gtcagcaagt ttctctggga gcgcggcctc 1680 gtggtggaaa aaaccgggct ttattcgttc ctcgtgttgt tttccatggg gatcaccaaa 1740 ggcaagtgga gcaccttgct caccgagttg ctggagttca agcgcagtta cgatgccaac 1800 gtcagcctgg ccagttgttt gccctcggtg tacgcccagg ggccggtacg ttatcagggc 1860 ttgggcctgc gcgatctttg cgaccagttg cacagctgtt accgtagcaa cgccaccgcc 1920 aagcatctca agcgcatgta cacagtattg ccgcagatcg cgatgaaacc cgccgatgcc 1980 tacgaccaac tggtcagagg cgaagttgaa gcggtatcca tcgatgcctt gccaggacgc 2040 atcgcagccg taatgctggt gccttatcca ccgggcattc cattgataat gcccggcgag 2100 cgctttactg aatcaacgcg ttcaatcatc gactacctgg catttgcccg cacgttcgat 2160 agcagtttcc ccggttttgt cgccgatgtt catgggctgc aacacgaaga tgatggcagt 2220 ggccgtcgtt acaccgtcga ttgcatcaag ggttaa 2256 <210> 11 <211> 715 <212> PRT <213> Escherichia coli <220> <221> PEPTIDE <222> (1)..(715) <223> Lysine decarboxylase from Escherichia coli (EcLDC) <400> 11 Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu 1 5 10 15 Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln 20 25 30 Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn 35 40 45 Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu 50 55 60 Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu 85 90 95 Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp 100 105 110 Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile 115 120 125 Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys 145 150 155 160 Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met 165 170 175 Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe 195 200 205 Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn 210 215 220 Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp 245 250 255 Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu 260 265 270 Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg 275 280 285 Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys 305 310 315 320 Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly 340 345 350 Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu 355 360 365 Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val 370 375 380 Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser 385 390 395 400 Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met 405 410 415 Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala 420 425 430 Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys 450 455 460 Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp 465 470 475 480 Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro 485 490 495 Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser 500 505 510 Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe 545 550 555 560 Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu 565 570 575 Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn 580 585 590 Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg 595 600 605 Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe 610 615 620 Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met 625 630 635 640 Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val 660 665 670 Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr 690 695 700 Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys 705 710 715 <210> 12 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> 15 amino acid of Lysine decarboxylase from Escherichia coli <400> 12 Gly Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys 1 5 10 15 Leu Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly 20 25 30 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 5 LDC NdeI <400> 13 aatatacata tgtacaaaga cctccaattc ccc 33 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 3 LDC XhoI <400> 14 aatatactcg agtcagatct tgatgcagtc caccg 35 <210> 15 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 5 LDC BamHI <400> 15 aatataggat ccgtacaaag acctccaatt cccc 34 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 3 LDC SacI <400> 16 aatatagagc tctcagatct tgatgcagtc caccg 35 <210> 17 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> 5 PaLDC NdeI <400> 17 aatatacata tgtacaaaga cctgaagttc cccatcc 37 <210> 18 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> 3 PaLDC XhoI <400> 18 aatatactcg agtcactccc ttatgcaatc aacggtatag c 41 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> 5 PrLDC NdeI <400> 19 aatatacata tgtacaaaga gctcaagttc cccgtcctc 39 <210> 20 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> 3 PrLDC XhoI <400> 20 aatatactcg agttattccc tgatgcagtc cactgtatag c 41 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 5_LDC_NdeI_ppldc <400> 21 aatatacata tgtacaaaga cctccaattc ccc 33 <210> 22 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> 3_PaLDC_XhoI_ppldc <400> 22 aatatactcg agtcactccc ttatgcaatc aacggtatag c 41 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 5_LDC_NdeI_pxldc <400> 23 aatatacata tgtacaaaga cctccaattc ccc 33 <210> 24 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> 3_PaLDC_XhoI_pxldc <400> 24 aatatactcg agtcactccc ttatgcaatc aacggtatag c 41

Claims (4)

A protein having lysine decarboxylase activity, which is produced from a microorganism transformed to express a protein having lysine decarboxylase activity, which is composed of the amino acid sequence of SEQ ID NO: 1, 3, 5, 7 or 9, Converting from lysine to cadaverine, and
Recovering the converted cadaverine,
Wherein the microorganism is Escherichia coli or Corynebacterium glutamicum, and the step of converting the lysine to cadaverine proceeds at a pH of from 6 to 9. The method according to claim 1, wherein the lysine decarboxylase activity has an activity of 70% or more based on the highest activity of lysine decarboxylase activity at a pH of 6 to 9. Culturing the microorganism transformed in the medium to express a protein having the lysine decarboxylase activity and consisting of the amino acid sequence of SEQ ID NO: 1, 3, 5, 7 or 9, and
Recovering the cadaverine in the microorganism or medium,
Wherein the microorganism is Escherichia coli or Corynebacterium glutamicum, and the reaction of lysine decarboxylase proceeds at a pH of 6 to 9 during the step of culturing the transformed microorganism in a medium. 4. The method according to claim 3, wherein the lysine decarboxylase activity has an activity of 70% or more based on the highest activity of the lysine decarboxylase activity in the range of pH 6 to 9.

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