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*

Carboxy-Lyases ; deficiency ; genetics ; Child, Preschool ; Humans ; Male ; Polymorphism, Single Nucleotide

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

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

PURPOSE: Recently, the whole DNA sequence of Bacillus subtilis (B. subtilis) was identified, revealing the existence of the YvrK gene encoding a 43 kD oxalate decarboxylase (OXDC), which degrades oxalate by a simple pathway. The objective of this study was to develop recombinant Escherichia coli (E. coli) expressing the Yvrk gene from B. subtilis. MATERIALS AND METHODS: After the extraction of total DNA from B. subtilis, the YvrK gene was cloned by polymerase chain reaction. The cloned DNA encoding OXDC was inserted into the pBAD/gIII-A vector, downstream of the L-arabinose promotor. The plasmid vector was transformed into TOP 10 E. coli, and the transformants were selected with ampicillin. The recombinant E. coli, named pBy, was then analyzed by DNA sequencing and Western blot. To evaluate the oxalate-degrading function of pBy, pBy was cultured in LB broth containing oxalate, and then the amount of oxalate in the medium was assessed. The oxalate-degrading activity of homogenates of pBy was evaluated. RESULTS: DNA sequencing showed the successful transformation of the YvrK gene into TOP 10 E. coli. Western blot analyses showed that pBy expressed OXDC. pBy removed oxalate during the overnight culture in oxalate-containing LB broth, and the homogenate of pBy degraded 90% of oxalate under acidic conditions. CONCLUSIONS: A recombinant E. coli expressing the YvrK gene was successfully produced. The bacteria showed potent oxalate-degrading activity. The results of this study will provide a solution to the treatment of calcium oxalate stones and hyperoxaluria, for which there are few medical treatment modalities.
Ampicillin ; Arabinose ; Bacillus subtilis ; Bacteria ; Base Sequence ; Blotting, Western ; Calcium Oxalate ; Carboxy-Lyases ; Clone Cells ; DNA ; Escherichia coli ; Hyperoxaluria ; Oxalates ; Plasmids ; Polymerase Chain Reaction ; Sequence Analysis, DNA

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

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

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

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in cerebral ischemia by maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia. Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane if brain vessels to promote cell death and tissue injury. We previously reported that agmatine, synthesized from L-arginine by arginine decarboxylase (ADC) which is expressed in endothelial cells, has shown a direct increased eNOS expression and decreased MMPs expression in bEnd3 cells. But, there are few reports about the regulation of eNOS by agmatine in ischemic animal model. In the present study, we examined the expression of eNOS and MMPs by agmatine treatment after transient global ischemia in vivo. Global ischemia was induced with four vessel occlusion (4-VO) and agmatine (100 mg/kg) was administered intraperitoneally at the onset of reperfusion. The animals were euthanized at 6 and 24 hours after global ischemia and prepared for other analysis. Global ischemia led severe neuronal damage in the rat hippocampus and cerebral cortex, but agmatine treatment protected neurons from ischemic injury. Moreover, the level and expression of eNOS was increased by agmatine treatment, whereas inducible NOS (iNOS) and MMP-9 protein expressions were decreased in the brain. These results suggest that agmatine protects microvessels in the brain by activation eNOS as well as reduces extracellular matrix degradation during the early phase of ischemic insult.
Agmatine ; Animals ; Arginine ; Basement Membrane ; Brain ; Brain Ischemia ; Capillary Permeability ; Carboxy-Lyases ; Cell Death ; Cerebral Cortex ; Endothelial Cells ; Extracellular Matrix ; Glycosaminoglycans ; Hippocampus ; Ischemia ; Matrix Metalloproteinases ; Microvessels ; Models, Animal ; Neurons ; Nitric Oxide ; Nitric Oxide Synthase Type III ; Rats ; Reperfusion

In ischemic strokes, apoptosis is caused by excitotoxicity, ionic imbalance, oxidative/nitrosative stress, and apoptotic-like pathways. Nitric oxide (NO), a free radical, is elevated after ischemic insult. NO, which is generated primarily by neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS), promotes neuronal damage following ischemia. Evidence obtained in recent years has demonstrated that endoplasmic reticulum (ER)-mediated cell death plays an important role in cerebral ischemia. Agmatine is an endogenous substance synthesized from L-arginine by arginine decarboxylase (ADC) and is present in mammalian brain. We had previously reported that agmatine contributes to neuroprotection against ischemic injury. In continuation of our earlier work, we intended to investigate whether agmatine protects brain from transient global ischemia, and also tried to determine the neuroprotective mechanism of agmatine. Twenty minutes of transient global ischemia was induced by 4 vessel occlusion (4-VO). Agmatine (100 mg/kg, IP) was administered simultaneously with reperfusion. Samplings of brain were done at 6, 24, 48, and 72 h after reperfusion to determine the effect of agmatine on ischemic injured hippocampus. ER-damage was also investigated using electron microscope. Results showed that agmatine treatment prevented delayed neuronal cell death in hippocampal CA1 neurons after global cerebral ischemia. It also blocked NOS expression in the rat brain. Agmatine induced the increased expression of glucose-regulated protein 78 (Grp78). These results suggest that agmatine inhibits the production of NO by decreasing the expression of nNOS and iNOS on global forebrain ischemia and the neuroprotective effect of agmatine were concerned with the ER stress-mediated condition.
Agmatine ; Animals ; Apoptosis ; Arginine ; Brain ; Brain Ischemia ; Carboxy-Lyases ; Cell Death ; Electrons ; Endoplasmic Reticulum ; Glycosaminoglycans ; Hippocampus ; Ischemia ; Neurons ; Neuroprotective Agents ; Nitric Oxide ; Nitric Oxide Synthase ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Prosencephalon ; Rats ; Reperfusion ; Stroke