1, 5-diaminopentane, also known as cadaverine, is an important raw material for the production of biopolyamide. It can be polymerized with dicarboxylic acid to produce biopolyamide PA5X whose performances are comparable to that of the petroleum-based polyamide materials. Notably, biopolyamide uses renewable resources such as starch, cellulose and vegetable oil as substrate. The production process does not cause pollution to the environment, which is in line with the green and sustainable development strategy. The biosynthesis of 1, 5-diaminopentane mainly includes two methods: the de novo microbial synthesis and the whole cell catalysis. Lysine decarboxylase as the key enzyme for 1, 5-diaminopentane production, mainly includes an inducible lysine decarboxylase CadA and a constituent lysine decarboxylase LdcC. Lysine decarboxylase is a folded type Ⅰ pyridoxal-5' phosphate (PLP) dependent enzyme, which displays low activity and unstable structure, and is susceptible to deactivation by environmental factors in practical applications. Therefore, improving the catalytic activity and stability of lysine decarboxylase has become a research focus in this field, and molecular engineering and immobilization are the mainly approaches. Here, the mechanism, molecular engineering and immobilization strategies of lysine decarboxylase were reviewed, and the further strategies for improving its activity and stability were also prospected, with the aim to achieve efficient production of 1, 5-diaminopentane.
Escherichia coli/metabolism* ; Carboxy-Lyases/metabolism* ; Catalysis ; Cadaverine/metabolism*

Cadaverine is a biogenic amine that has the potential to become an important platform chemical for the production of industrial polymers, such as polyamides and polyurethanes. We reported here a lysine decarboxylase from Klebsiella oxytoca. The lysine decarboxylase from Klebsiella oxytoca was cloned to Escherichia coli to get the strain LN18. The specific activity of the crude protein from LN18 reached 30 000 U. The molecular weight was about 80 kDa. The optimum temperature and pH of the crude protein were 55 ℃ and 5.5 respectively. The specific activity could keep over 30% at pH 8.0 compared the one at pH 5.5, much difference from Escherichia coli lysine decarboxylase CadA. Mg²⁺ was positive to the specific activity, whereas Fe²⁺, Zn²⁺ and Ca²⁺ were negative.
Bacterial Proteins ; genetics ; metabolism ; Cadaverine ; Carboxy-Lyases ; genetics ; metabolism ; Escherichia coli ; metabolism ; Hydrogen-Ion Concentration ; Klebsiella oxytoca ; enzymology ; genetics ; Temperature

OBJECTIVETo observe the alterations of taurine transport, taurine transporter (TAUT) and cysteine sulfinate decarboxylase (CSD) mRNA in the calcification of myocardial cells in vitro.

METHODS3H-taurine measured the amount of taurine uptake. TAUT and CSD mRNA consents were measured using competitive quantitative RT-PCR in cultured and calcified myocardial cells.

RESULTSIn calcification of myocardial cells, taurine concentration was decreased by 27% (P < 0.05), taurine uptake was markedly reduced, Vmax reduced by 39% (P < 0.01), there were no statistical significance of Km values between the two groups. TAUT mRNA decreased by 45% (P < 0.01), but CSD mRNA increased by 25% (P < 0.05).

CONCLUSIONSThe data suggest that there were impediment of taurine transport in calcification of myocardial cells, as TAUT mRNA level was decreased, but CSD mRNA concentration was improved.

Animals ; Biological Transport ; Calcinosis ; metabolism ; pathology ; Calcium ; metabolism ; Carboxy-Lyases ; metabolism ; Cells, Cultured ; Myocytes, Cardiac ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Rats ; Taurine ; biosynthesis ; genetics ; metabolism

2,3-butanediol (2,3-BD) is a major byproduct of 1,3-propandediol (1,3-PDO) fermentation by Klebsiella pneumoniae. To decrease the formation of 2,3-BD, the budC and budA gene, coding two key enzymes of 2,3-BD synthetic pathway in K. pneumoniae, were knocked out using Red recombination technology. The growth of the two mutants were suppressed in different level. The budC deficient strain fermentation results showed that 1,3-PDO concentration increased to 110% and 2,3-butanediol concentration dropped to 70% of the parent strain. However, the budA deficient strain did not produce 1,3-PDO and 2,3-BD, and the final titer of lactic acid, succinic acid, ethanol and acetic acid increased remarkably compared with the parent strain. Further analysis of budC deficient strain fermentation inferred that K. pneumoniae possessed the 2,3-BD cycle as a replenishment pathway. The consequence provided a new evidence for reforming low-byproduct K. pneumoniae.
Acetolactate Synthase ; genetics ; metabolism ; Bacterial Proteins ; genetics ; Butylene Glycols ; metabolism ; Carboxy-Lyases ; genetics ; Gene Knockout Techniques ; Glycerol ; metabolism ; Klebsiella pneumoniae ; genetics ; metabolism ; Mutation ; Propylene Glycols ; metabolism

The role of surfactant protein A (SP-A) in the recognition and clearance of apoptotic cells is well established, but to date, it is still not clear which surface molecules of apoptotic cells are involved in the process. Here we present evidence that phosphatidylserine (PS) is a relevant binding molecule for human SP-A. The binding is Ca(2+)-dependent and is not inhibited by mannose, suggesting that the sugar-binding site of the carbohydrate recognition domain (CRD) of SP-A is not involved. Flow cytometry studies on apoptotic Jurkat cells revealed apparent inhibition of annexin V binding by increasing concentrations of SP-A in late apoptotic but not early apoptotic cells, and this was consistent for Jurkat cells and neutrophils. Supporting these data, confocal microscopy results show a co-localisation of annexin V and SP-A in late apoptotic but not early apoptotic cells. However, we cannot conclude that this inhibition is exclusively due to the binding of SP-A to PS on the cell surface, as annexin V is not wholly specific for PS and SP-A also interacts with other phospholipids that might become exposed on the apoptotic cell surface.
Annexin A5 ; metabolism ; Apoptosis ; Carboxy-Lyases ; metabolism ; Flow Cytometry ; Humans ; Jurkat Cells ; Microscopy, Confocal ; Neutrophils ; physiology ; Phosphatidylserines ; metabolism ; Pulmonary Surfactant-Associated Protein A ; metabolism

Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures.
Carboxy-Lyases ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; metabolism ; Genetic Vectors ; Huperzia ; enzymology ; genetics ; Lysine ; metabolism ; Plant Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

In this study, we explored the potentiality of human arginine decarboxylase (ADC) to enhance the survival of mesenchymal stem cells (MSCs) against unfavorable milieu of host tissues as the low survival of MSCs is the issue in cell transplantation therapy. To address this, human MSCs overexpressing human ADC were treated with H2O2 and the resultant intracellular events were examined. First, we examined whether human ADC is overexpressed in human MSCs. Then, we investigated cell survival or death related events. We found that the overexpression of human ADC increases formazan production and reduces caspase 3 activation and the numbers of FITC, hoechst, or propidium iodide positive cells in human MSCs exposed to H2O2. To elucidate the factors underlying these phenomena, AKT, CREB, and BDNF were examined. We found that the overexpression of human ADC phosphorylates AKT and CREB and increases BDNF level in human MSCs exposed to H2O2. The changes of these proteins are possibly relevant to the elevation of agmatine. Collectively, our data demonstrate that the overexpression of human ADC stimulates pro-survival factors to protect human MSCs against H2O2 toxicity. In conclusion, the present findings support that ADC can enhance the survival of MSCs against hostile environment of host tissues.
Apoptosis/*drug effects ; Brain-Derived Neurotrophic Factor/metabolism ; Carboxy-Lyases/genetics/*metabolism ; Caspase 3/metabolism ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein/metabolism ; Humans ; Hydrogen Peroxide/*toxicity ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/cytology/drug effects/metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism