• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2110-2115
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• 2015

A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.289-296
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• 2017

Lysine decarboxylase (CadA) converts ${\small{L}}-lysine$ into cadaverine (1,5-pentanediamine), which is an important platform chemical with many industrial applications. Although there have been many efforts to produce cadaverine through the soluble CadA enzyme or Escherichia coli whole cells overexpressing the CadA enzyme, there have been few reports concerning the immobilization of the CadA enzyme. Here, we have prepared a cross-linked enzyme aggregate (CLEA) of E. coli CadA and performed bioconversion using $CadA^{CLEA}$. $CadA^{free}$ and $CadA^{CLEA}$ were characterized for their enzymatic properties. The optimum temperatures of $CadA^{free}$ and $CadA^{CLEA}$ were $60^{\circ}C$ and $55^{\circ}C$, respectively. The thermostability of $CadA^{CLEA}$ was significantly higher than that of $CadA^{free}$. The optimum pH of both enzymes was 6.0. $CadA^{free}$ could not be recovered after use, whereas $CadA^{CLEA}$ was rapidly recovered and the residual activity was 53% after the $10^{th}$ recycle. These results demonstrate that $CadA^{CLEA}$ can be used as a potential catalyst for efficient production of cadaverine.

• BMB Reports
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• v.33 no.1
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• pp.69-74
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• 2000

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA ($GenBank^{TM}$ U63633) was cloned, then the AdoMetDC protein was expressed and purified. The purified AdoMetDC was inactivated by salicylaldehyde in a pseudo first- order kinetics. The secondorder rate constant for inactivation was 126 $M^{-1}min^{-1}$ with the slope of n=0.73, suggesting that inactivation is the result of the reaction of one lysine residue in the active site of AdoMetDC. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations in conserved $lysine^{81}$ residues. These were chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. Changing $Lys^{81}$ to alanine showed an altered optimal pH. The substrate also provided protection against inactivation by salicylaldehyde. Considering these results, we suggest that the $lysine^{81}$ residue may be involved in catalytic activity.

• Journal of Plant Biology
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• v.32 no.2
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• pp.89-99
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• 1989

The effect of IAA on lysine decarboxylase(LDC)activity, diamine oxidase (DAO) activity and the polyamine level in seedlings of soybean were investigated. The seedlings of soybean were treated with IAA (10-8M, 10-6M, 10-4M) and LDC activity, DAO activity and polyamine levels of cotyledon, elongating hypocotyl, elongated hypocotyl and the root were measured after 4, 6, 8, 10 days. Increased concentration of IAA stimulated the LDC activity and increased the level of polyamine in the seedlings, which reducing the DAO activity and polyamine level was increased in the seedlings. The light also stimulated the IAA effect on polyamine level and DAO activity, and polyamine level was higher in the seedlings grown in the light than those in the dark. Cadaverine was the most abundant polyamine in the seedlings of soybean. The levels of diamines, cadaverine, putrescine were higher in the elongated hypocotyl and root than in the elongating hypocotyl, but the levels of spermidine and spermine were relatively higher in the cotyledon than in the other parts. Accumulation of diamines by high concentration of IAA is postulated to be a phenomenon of inhibition in seedlings growth.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1108-1113
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• 2015

Cadaverine (1,5-diaminopentane) is an important industrial chemical with a wide range of applications. Although there have been many efforts to produce cadaverine through fermentation, there are not many reports of the direct cadaverine production from lysine using biotransformation. Whole-cell reactions were examined using a recombinant Escherichia coli strain overexpressing the E. coli MG1655 cadA gene, and various parameters were investigated for the whole-cell bioconversion of lysine to cadaverine. A high concentration of lysine resulted in the synthesis of pyridoxal-5'-phosphate (PLP) and it was found to be a critical control factor for the biotransformation of lysine to cadaverine. When 0.025 mM PLP and 1.75 M lysine in 500 mM sodium acetate buffer (pH6) were used, consumption of 91% lysine and conversion of about 80% lysine to cadaverine were successfully achieved.

• BMB Reports
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• v.34 no.5
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• pp.408-414
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• 2001

Ornithine decarboxylase (EC 4.1.1.17) is an essential enzyme for polyamine synthesis and growth in mammalian cells and plants. We compared the biochemical and immunological properties of rat and Nicotiana glutinosa ODC by cloning and expressing the recombinant proteins. The primary amino acid sequence between rat and N. glutinosa ODC had a 40% homology The molecular weight of the overexpressed rat ODC was 53 kDa, and that of N. glutinosa was 46.5 kDa. Adding 1 mM of putrescine to the enzyme reaction mixture inhibited both rat and N. glutinosa ODC activity to 30%. Agmatine had an inhibitory effect only on N. glutinosa ODC. Cysteine and lysine modifying reagents reduced both ODC activities, verifying the key roles of cysteine and lysine residues in the catalytic mechanism of ODC. ELISA was performed to characterize the immunological difference between the rat and plant ODC. Both the rat and N. glutinosa ODC were recognized by the polyclonal antibody that was raised against purified N. glutinosa ODC, but the rat ODC was 50-fold less sensitive to the antibody binding. These results indicate that even though both ODCs have the same evolutionary origin, there seems to be a structural distinction between the species.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.176-179
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• 2007

An electron paramagnetic resonance (EPR) signal characteristic of the 5,5'-dimethyl-l-pyrroline-N-oxide (DMPO)-OH spin adduct, which is formed from the reaction of DMPO with superoxide radicals generated by xanthine oxidasemediated reaction, was significantly reduced by the cadaverine or Escherichia coli Mn-containing superoxide dismutase (MnSOD). Likewise, cytochrome c reduction by superoxide was inhibited by cadaverine, and the inhibition level increased in proportion to the level of cadaverine. The cadA mutant of Vibrio vulnificus, which does not produce cadaverine because of the lack of lysine decarboxylase, exhibits less tolerance to superoxide stress in comparison with wild type. The results indicate that cadaverine scavenges superoxide radicals, and protects cells from oxidative stress.

• Korean Journal of Microbiology
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• v.37 no.4
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• pp.259-264
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• 2001

It has been well known that the expression of S. typhimurium cadBA operon requires at least two extracellular signals: low pH and high concentration of lysine. To better understand the nature of pH-dependent and lysine dependent signal transduction, mutants were isolated in JF2238(cadA-lacZ) by Tn10 insertion, spontaneous mutagenesis, and EMS treatment. Two mutants were isolated from JF2238, expressed as a cadA-lacZ operon fusion in various growth conditions, and analyzed to have mutations in cadC, a gene encoding a function necessary for transcriptional activation of cadBA. One isolate (cadC6) conferred pH-independent and lysine-independent cadBA expression and the other(cadC4) showed pH-independent and lysine-dependent cadBA expression. cadR::Tn10 and cadR4 mutants were expressed in the absence of exogenously added lysine. They were also resistant to thiosine and complemented by lysP clone from E. coli. Thus, in the absence of exogenous lysine, cadR is a negative regulator of cadBA expression. Cadaverine, the product of lysine decarboxylation, was shown to inhibit expression of cadA-lacZ fusion in cad $C^+$ cell.

• Journal of Sensor Science and Technology
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• v.6 no.6
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• pp.437-444
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• 1997

A potentiometric L-lysine-selective sensor is described for the direct determination of lysine. The sensor system is based on a carbon dioxide gas sensing electrode and an L-lysine decarboxylase immobilized to CNBr-activated sepharose 4B. A highly selective L-lysine sensor has been prepared with immobilizing enzyme slurry put into reaction buffer solution. The optimum conditions for the measurement were evaluated by various experiments. This sensor exhibits a linear response to L-lysine concentrations from $10^{-4}M$ to $10^{-1}M$. Response time of this lysine sensor is shorter than 30secs and the immobilized enzyme slurry is stable over one year.

• Animal cells and systems
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• v.10 no.4
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• pp.197-201
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• 2006

Diaminopimelate decarboxylase (DAPDC, EC 4.1.1.20) catalyzes the conversion of diaminopimelate into lysine (Lys), which is the last step in Lys biosynthetic pathway. The genes for DAPDC have been reported in many bacteria, and more recently in Arabidopsis. Here we report characterization of a gene for DAPDC from rice (OsDAPDC). Sequence analysis of a cDNA clone revealed a full-length open reading frame for OsDAPDC that encoded 490 amino acids, approximately 53.2 kDa protein. The OsDAPDC protein contains a consensus binding site for pyridoxal-5'-phosphate as a cofactor and has a sequence at the amino terminus that resembles a transit peptide for localization to plastids, similar to that of Arabidopsis. Single gene encoding DAPDC was found in chromosome II in rice. The predicted amino acid sequence of OsDAPDC is highly homologous to that of the enzymes for DAPDC encoded by lysA of many bacteria. Expression of OsDAPDC in lysA mutants of Escherichia coli shows that the gene is able to functionally complement the mutants. These results suggest that OsDAPDC encodes a protein for diaminopimelate decarboxylase in rice.