Molecular engineering and immobilization of lysine decarboxylase for synthesis of 1, 5-diaminopentane: a review / 生物工程学报

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.

Fusion expression with catalase improves the stability of multicopper oxidase and its efficiency in degrading biogenic amines / 生物工程学报

Some enzymes belonging to the multicopper oxidase (MCO) family can degrade the hazardous biogenic amine (BA) present in food. However, the oxidation of MCO in the process of degrading BAs may reduce its activity and stability, resulting in decreased catalytic efficiency. In this work, an MCO from Lactobacillus fermentum (MCOF) was fused with a Bacillus subtilis catalase (CAT) using different strategies and the fusion enzymes were respectively expressed in Escherichia coli BL21(DE3). The tolerance of eight fused MCOFs to H2O2 increased by 51%-68%, and the stability of CAT&MCOF increased by 17%, compared to the wild type MCOF. Using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate, the substrate affinity (Km), the catalytic efficiency (kcat/Km) and the molar specific activity of CAT&MCOF increased by 1.0-fold, 1.7-fold and 1.2-fold than those of MCOF, respectively. The stability of CAT&MCOF under acidic conditions (pH 2.5-4.5) and moderate temperatures (35-55 °C) also improved. Moreover, the degradation rates of putrescine, cadaverine and histamine catalyzed by CAT&MCOF reached 31.7%, 36.0% and 57.8%, respectively, which increased by 132.5%, 45.7% and 38.9% compared to that of MCOF. The improvement on the stability and catalytic efficiency of MCOF by fusion expression with CAT provides a good example for improving the applicability of enzymes through molecular modifications.

Expression and characterization of a multicopper oxidase from Lactobacillus fermentum / 生物工程学报

Biogenic amines (BAs) are low molecular weight organic compounds that present in fermented foods. Large amount of ingested biogenic amines can cause allergy or significant symptoms. Reduction of BAs by enzymatic reaction in fermented foods is one of the most efficient methods for removal of biohazard compounds and assurance food safety. In this study, the multicopper oxidase (MCO) gene in the genome of Lactobacillus fermentum was successfully cloned in Escherichia coli BL21 and expressed at 484 U/L. The recombinant MCO was purified by the immobilized metal affinity chromatography method. The optimal reaction temperature and pH for this enzyme was detected to be 50 °C and 3.5. The Km and Vmax values of the recombinant MCO was determined to be 1.30 mmol/L and 7.67×10⁻² mmol/(L·min). Moreover, this MCO dramatically degrades histamine and tyramine by 51.6% and 40.9%, and can degrade other BAs including tryptamine, phenylethylamine, putrescine, cadaverine and spermidine, and was found to be tolerant to 18% (W/V) NaCl. The recombinant MCO is also capable of degrading BAs in soy sauce. The degradation rate of total BAs in soy sauce reaches 10.6% though a relatively low level of enzyme (500 U/L) is used. Multicopper oxidase has the potential to degrade biogenic amines in fermented foods, which lays a foundation for the further application of this kind of food enzymes.

A useful procedure for detection of polyamines in biological samples as a potential diagnostic tool in cancer diagnosis

Background: Polyamines present in human body are frequently considered as markers of occurrence of cancer. Therefore, the availability of simple and efficient method for determination of their level in body liquids and tissues is of some interest. Methods: Supported liquid membrane technology coupled with HPLC seems to be an appropriate technique to follow the level of polyamines in human blood and urine. Thus, the membranes of two different geometries: flat sheet and hollow fiber were studied as a mean for separation and enrichment of studied polyamines from urine and tissue samples in order to prepare samples to be analyzed by HPLC. Conclusions: Developed extraction systems offer an interesting alternative to traditional techniques such as: liquid-liquid or solid-phase extraction due to several features, which are: very high enrichment of polyamines without previous work-up, simple procedure of extraction and tiny volume of organic solvents used. This enables efficient determination of their levels in body liquids (AU)

Heterologous expression and characterization of Klebsiella oxytoca lysine decarboxylase / 生物工程学报

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.

Progress in biosythesis of diaminopentane / 生物工程学报

Air pollution and global warming are increasingly deteriorating. Large amounts of polyamides derived from fossil fuel sources are consumed around the world. Cadaverine is an important building monomer block of bio-based polyamides, thus biotechnological processes for these polymers possess enormous ecological and economical potential. Currently, the engineered strains for biological production of cadaverine are Corynebacterium glutamicum and Escherichia coli. We review here the latest research progress of biosynthesis of cadaverine including metabolism of cadaverine in microorganisms, key enzymes and transport proteins in cadaverine synthesis pathway, optimum pathways and cadaverine yields.

Effect of cadaverine on Brassica juncea (L.) under multiple stress.

The cadaverine (Cad), an organic diamine was examined for its response on growth in salinity and metal stressed B. juncea cv RH-30 vis-à-vis compared the response of ammonium nitrate. The Cad (1 mM) application ameliorated the effect caused by salinity and metal stress on seed germination and plant growth. The plant growth recovery (dry biomass accumulation) was dependent on stress and diamine type. The higher growth recovery potential of Cad under both stresses was due to elevation in photosynthetic pigments, nitrate reductase activity and organic nitrogen as well as soluble protein, It is inferred that growth in stressed seedlings was mediated by Cad through lowering endogenous Cd/Pb and Na+/K+ level in leaf and shoot tissues.

Determination and correlation analysis of contents of putrescine, cadaverine, and histamine in necrotic tissue, blood, and urine of patients with diabetic foot / 中华烧伤杂志

<p><b>OBJECTIVE</b>To determine and perform a correlation analysis of the contents of putrescine, cadaverine, and histamine in necrotic tissue, blood, and urine of patients with diabetic foot (DF).</p><p><b>METHODS</b>Ten patients with severe wet necrotizing DF hospitalized from January 2011 to January 2012 were assigned as group DF, and 10 orthopedic patients with scar but without diabetes or skin ulcer hospitalized in the same period were assigned as control group. Samples of necrotic tissue from feet of patients in group DF and normal tissue from extremities of patients in control group, and samples of blood and 24-hour urine of patients in both groups were collected, and the amount of each sample was 10 mL. Contents of putrescine, cadaverine, and histamine were determined with high performance liquid chromatography-mass spectrometry. The data got from the determination of blood and urine were processed with t test, and those from necrotic or normal tissue with Wilcoxon rank sum test. The correlation of contents of polyamines between necrotic tissue and blood, blood and urine were processed with simple linear regression analysis.</p><p><b>RESULTS</b>(1) Contents of putrescine, cadaverine, and histamine in the necrotic tissue of group DF were (186.1 ± 26.8), (78.553 ± 12.441), (33 ± 10) mg/kg, which were significantly higher than those in normal tissue of control group [(2.2 ± 1.2), (1.168 ± 0.014), 0 mg/kg, with Z values respectively -3.780, -3.781, -4.038, P values all below 0.01]. The content of putrescine in necrotic tissue of group DF was significantly higher than those of cadaverine and histamine (with Z values respectively -3.780, -3.630, P values all below 0.01). (2) Contents of putrescine, cadaverine, and histamine in the blood of group DF were (0.075 ± 0.013), (0.022 ± 0.003), (0.052 ± 0.014) mg/L, and they were significantly higher than those in the blood of control group [(0.014 ± 0.009), (0.013 ± 0.003), (0.016 ± 0.008) mg/L, with t values respectively 6.591, 2.207, 3.568, P < 0.05 or P<0.01]. The content of putrescine in the blood of group DF was significantly higher than those of cadaverine and histamine (with t values respectively 13.204, 3.096, P values all below 0.01). (3) Contents of putrescine, cadaverine, and histamine in the urine of group DF were (0.735 ± 0.088), (0.450 ± 0.012), (0.1623 ± 0.0091) mg/L, and only the contents of putrescine and cadaverine were significantly higher than those in the urine of control group [(0.050 ± 0.014), (0.035 ± 0.007) mg/L, with t values respectively 3.270, 4.705, P<0.05 or P<0.01]. The content of putrescine in the urine of group DF was significantly higher than that of cadaverine (t = 6.686, P < 0.01). (4) There were significant and positive correlations in contents of putrescine, cadaverine, and histamine between necrotic tissue and blood in patients of group DF (with r values respectively 0.981, 0.994, 0.821, P values all below 0.01). There were no significant correlations in contents of putrescine, cadaverine, and histamine between blood and urine in patients of group DF (with r values respectively 0.150, 0.239, 0.177, P values all above 0.05).</p><p><b>CONCLUSIONS</b>Putrescine, cadaverine, and histamine exist in the necrotic tissue of patients with DF in high concentrations, among which putrescine predominates. These polyamines can be absorbed into the blood through wound and excreted through the urine.</p>

The Role of HS-1200 Induced Autophagy in Oral Cancer Cells

Bile acids and synthetic bile acid derivatives induce apoptosis in various kinds of cancer cells and thus have anticancer properties. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, few data are available regarding the role of autophagy in oral cancers and there have been no reports of autophagic cell death in OSCCs (oral squamous cell carcinoma cells) induced by HS-1200, a synthetic bile acid derivative. We thus examine whether HS-1200 modulates autophagy in OSCCs. Our findings indicate that HS-1200 has anticancer effects in OSCCs, and we observed in these cells that autophagic vacuoles were visible by monodansylcadaverine (MDC)and acridine orange staining. When we analyzed HS-1200-treated OSCC cells for the presence of biochemical markers, we observed that this treatment directly affects the conversion of LC-3II, degradation of p62/SQSTM1 and full-length beclin-1, cleavage of ATG5-12 and the activation of caspase. An autophagy inhibitor suppressed HS-1200-induced cell death in OSCCs, confirming that autophagy acts as a pro-death signal in these cells. Furthermore, HS-1200 shows anticancer activity against OSCCs via both autophagy and apoptosis. Our current findings suggest that HS-1200 may potentially contribute to oral cancer treatment and thus provide useful information for the future development of a new therapeutic agent.

Influence of exogenous putrescine and cadaverine on pro-inflammatory factors in the peripheral blood of rabbits / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the influence of exogenous putrescine and cadaverine on pro-inflammatory factors in the peripheral blood of rabbits.</p><p><b>METHODS</b>Forty ordinary adult New Zealand rabbits were divided into saline, necrotic tissue homogenate (NTH), putrescine, and cadaverine groups according to the random number table, with 10 rabbits in each group. Saline, NTH, 10 g/L putrescine, and 10 g/L cadaverine were respectively peritoneally injected into rabbits of corresponding group in the amount of 1 mL/kg. The blood sample in the volume of 2 mL was collected from the central artery of rabbit ears before injection and at 2, 6, 12, 24, 30, 36, 48, 60 hours post injection (PIH). Contents of TNF-α, IL-1, and IL-6 in the serum were determined with enzyme-linked immunosorbent assay. Data were processed with repeated measurement data analysis of variance and Spearman correlation analysis, and cubic model curve was applied in curve fitting for the contents of inflammatory factors.</p><p><b>RESULTS</b>(1) The serum contents of TNF-α, IL-1, and IL-6 were increased in NTH, putrescine, and cadaverine groups in different degrees at most post injection time points. There was no significant change in the concentrations of the three pro-inflammatory factors in saline group, and they were significantly lower than those of the other three groups at most post injection time points (with F values from 3.49 to 13.58, P values all below 0.05). The serum contents of TNF-α, IL-1, and IL-6 in putrescine group began to increase at PIH 2, 6, and 6, which was similar to the trend of NTH group, but the changes were delayed compared with those of cadaverine group(all at PIH 2). The peak values of TNF-α, IL-1, and IL-6 in putrescine group were respectively (339 ± 36), (518 ± 44), and (265.9 ± 33.5) pg/mL, which were significantly lower than those of cadaverine group [ (476 ± 86), (539 ± 22), and (309.4 ± 27.1) pg/mL], with F values respectively 5.11, 1.90, and 5.56, P values all below 0.05. (2) The period of time in which contents of TNF-α, IL-1, and IL-6 began to increase (PIH 3-4) and the peaking time of the three pro-inflammatory cytokines (PIH 18-30) in putrescine group appeared later than those of cadaverine group (PIH 2 and 12-30). The duration of peaking time of the three pro-inflammatory cytokines in putrescine group was shorter than that of cadaverine group (PIH 18-30 vs. PIH 12-30). The increasing period and the duration of peaking time of TNF-α, IL-1, and IL-6 in putrescine group were close to those of NTH group (PIH 3-5 and 18-30). The correlation coefficient test analysis showed that the trends of changes in contents of three pro-inflammatory cytokines in putrescine group were significantly correlated with those of NTH group (r(TNF-α) = 0.933, P < 0.01; r(IL-1) = 0.967, P < 0.01; r(IL-6) = 0.950, P < 0.01). The obvious correlation between cadaverine group and NTH group was only found in the contents of IL-1 and IL-6 (r(IL-1) = 0.913, P < 0.01; r(IL-6) = 0.883, P < 0.05).</p><p><b>CONCLUSIONS</b>Both exogenous putrescine and cadaverine can cause inflammatory reaction in rabbits. The trend of the inflammatory reaction induced by putrescine was similar with that by NTH, suggesting that putrescine may play a leading role in the inflammatory reaction induced by necrotic tissue decomposition.</p>

Autophagy of Human Tenon's Capsule Fibroblasts Induced by Mitomycin-C

PURPOSE: The present study investigated whether an autophagic process is involved in the apoptotic death of human tenon's capsule fibroblasts (HTCFs) caused by mitomycin-C. METHODS: An autophagic phenotype was tested using fluorescence microscopy and flow cytometry with specific biological staining dyes including monodansylcadaverine and acridine orange and microtubule-associated protein 1 light chain 3 (LC3). RESULTS: Treatment with mitomycin-C (0.4 mg/ml) increased the acidic vesicular organelles of tenon's capsule fibroblasts in a time dependent manner. Mitomycin-C induced both LC3-II cleavage and beclin-1 expression. 3-MA, a pharmacological inhibitor of autophagy, inhibited the mitomycin-C induced increase of acidic vesicular organelleS. CONCLUSIONS: Autophagy was induced with 0.4 mg/ml mitomycin-C in tenon's capsule fibroblasts. And, autophagic mechanisms may be involved in the early stage of apoptosis of fibroblasts.

Extending shelf life of refrigerated lout fish fillets with some organic acid salts and bifidobacteria

Changes in microbiological, some chemical and sensory characteristics of treated lout fish fillets [Sciaena umbra] were evaluated. Fish fillet samples were treated with organic acid salts with or without culture of bifidobacteria and stored at 4°C for 14 days. The combination of sodium metaphosphate [SMP] 10% or sodium lactate [SL] 2% with bifidobacteria [5% vol/vol] delayed growth of Enterobacteriaceae, Pseudomonas, and psychrotrophic bacteria. Production of biogenic amines was delayed up to 14 days storage at 4°C; pH values were similar to the fresh control for up to 8 days and the sensory shelf life was more than 14 days

Possible role of polyamines in gyrate atrophy.

PURPOSE: Gyrate atrophy (GA) is marked by hyperornithinemia and lowered ornithine amino transferase (OAT). However there are patients of GA without hyperornithinemia and those with hyperornithinemia without GA. Some cases of GA have been reported to have low lysine. The purpose of the study was to determine if polyamines, the metabolites of ornithine, and lysine have any diagnostic role in GA. METHODS: Ornithine in plasma was estimated by two-dimensional paper chromatography, with elution of the coloured spot, and the absorbance measured using a spectrophotometer at 560 nm. OAT assay in lymphocytes was done spectrophotometrically using ornithine as substrate. Blood and urinary polyamines were extracted with n-butanol, benzoylated and analysed with HPLC; putrescine, spermine, spermidine, and cadaverine were assayed individually at 254 nm with the UV detector using ODS, G18 column with 63% methanol as solvent. RESULTS: Of the 7 patients investigated, 6 had features typical of GA. One was diagnosed to have atypical retinitis pigmentosa (case 3). The first five cases had elevated ornithine and diminished OAT, but cases 6 and 7 had near-normal ornithine and case 7 had near-normal OAT. However, all 7 patients had increased levels of total polyamines in urine compared to normals. Five had increased putrescine and three had increased spermine. All the 7 had decreased cadaverine in urine. Thus, though there were inconsistencies with ornithine and OAT, all the 7 patients had elevated polyamines from ornithine and decreased cadaverine. CONCLUSION: In addition to estimating ornithine and OAT in GA, it is suggested that urinary polyamines may be analysed as the latter appears to correlate better with the clinical condition and help in the diagnosis to a greater extent. Moreover, while ornithine is an innocuous amino acid, polyamines are known to damage DNA and proteins.

Urinary Polyamine Profiles and Benign Prostatic Hyperplasia / 대한비뇨기과학회지

Polyamines are non-specific marker of cellular proliferation in many malignant tumors, and it is also increase in certain benign conditions. We measured the urinary polyamines to investigate the possibility as a marker of abnormal prostate growth and the correlation with various clinical parameters. Urinary polyamine concentrations in 27 cases of symptomatic benign prostatic hyperplasia (BPH) were compared with those in 32 cases of age matched normal controls. Urinary concentration of polyamine profiles were quantitatively determined by Gas Chromatography/Nitrogen Phosphorus Detector and they were calculated by the correction of gram creatinine. The concentrations of N-acetyl putrescine, N-acetyl cadaverine, spermidine(spd), N1-acetyl spermidine, N8-acetyl spermidine, and spermine(spm) showed significant increase in BPH compared with normal control(all p<0.05). Level of serum prostate specific antigen(PSA) in BPH patients was negatively correlated with the concentration of urinary spermidine(p=0.049). And the ratio of spm/spd correlated with the level of prostate volume(p=0.046). No significant correlations was found between other clinical parameters such as age, level of hemoglobin or erythrocyte count with polyamine profiles concentration. These data suggested that urinary concentration of polyamines in BPH are elevated compared with those in normal control. Altered regulation of the biosynthesis and metabolism of spermidine and spermine may be involved in BPH.

Crystal structure of cadaverine dihydrochloride monohydrate.

The structure of cadaverine dihydrochloride monohydrate has been determined by X-ray crystallography with the following features: NH3+ (CH2)5NH3+.2Cl-.H2O, formula weight 191.1, monoclinic, P2, a = 11.814(2)A, b = 4.517(2)A, c = 20.370(3)A, beta = 106.56 degrees (1): V = 1041.9(2)A3; lambda = 1.541A; mu = 53.41; T = 296 degrees; Z = 4, Dx = 1.218 g.cm-3, R = 0.101 for 1383 observed reflections. The crystal is highly pseudo-symmetric with 2 molecules of cadaverine, 4 chloride ions and 2 partially disordered water molecules present in the asymmetric unit. Though both the cadaverine molecules in the asymmetric unit have an all trans conformation, the carbon backbones are slightly bent. Between the concave surfaces of two bent cadaverine molecules exists water channels all along the short b axis. The water molecules present in the channels are partially disordered.