Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay

L-Arginine dehydrogenase (L-ArgDH, EC 1.4.1.25) is an amino acid dehydrogenase which catalyzes the reversible oxidative deamination of L-arginine to the oxo analog in the presence of NADP. Although the enzyme activity is detected in the cell extract of Pseudomonas aruginosa , the purification and characterization of the enzyme have not been achieved to date. We here found the gene homolog of L-ArgDH in genome data of Pseudomonas veronii and succeeded in expression of P. veronii JCM11942 gene in E. coli. The gene product exhibited strong NADP-dependent L-ArgDH activity. The crude enzyme was unstable under neutral pH conditions, but was markedly stabilized by the addition of 10% glycerol. The enzyme was purified to homogeneity through a single Ni-chelate affinity ch romatography step and consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed l-arginine oxidation in the presence of NADP with maximal activity at pH 9.5. The apparent K m values for l-arginine and NADP were 2.5 and 0.21 mM, respectively. The nucleotide sequence coding the enzyme gene ( was determined and the amino acid sequence was deduced from the nucleotide sequence. As an application of the enzyme, simple colorimetric microassay for L-arginine using the enzyme was achieved.

Antibiofilm Activities of Biogenic Silver Nanoparticles Against Candida albicans

Biofilms are microbial colonies that are encased in an organic polymeric matrix and are resistant to antimicrobial treatments. Biofilms can adhere to both biotic and abiotic surfaces, allowing them to colonize medical equipment such as urinary and intravenous catheters, mechanical heart valves, endotracheal tubes, and prosthetic joints. Candida albicans biofilm is the major etiological cause of the pathogenesis of candidiasis in which its unobstructed growth occurs in the oral cavity; trachea, and catheters that progress to systemic infections in the worst scenarios. There is an urgent need to discover novel biofilm preventive and curative agents. In the present investigation, an effort is made to observe the role of cyanobacteria-derived AgNPs as a new antibiofilm agent with special reference to candidiasis. AgNPs synthesized through the green route using Anabaena variabilis cell extract were characterized by UV–visible spectroscopy. The nanoparticles were spherical in shape with 11–15 nm size and were monodispersed. The minimum inhibitory concentration (MIC) of AgNPs was obtained at 12.5 μg/mL against C. albicans. AgNPs 25 μg/mL showed 79% fungal cell membrane permeability and 22.2% ROS production. AgNPs (25 μg/mL) also facilitated 62.5% of biofilm inhibition and degradation. Therefore, AgNPs could be considered as a promising antifungal agent to control biofilm produced by C. albicans.

Toxicity of The Microcystin-Producing Cyanobacterium Microcystis Aeruginosa To Litopenaeus Vannamei

Microcystis aeruginosa is a common kind of harmful bloom algae, which was also frequently found as a dominant microalgae specie in shrimp breeding ponds. And it was found that blooms always induced massive death of shrimp, but the toxic effects of M. aeruginosa on Litopenaeus vannamei are still not completely understood. In this paper, the toxicity of M. aeruginosa cells to L. vannamei was examined, and the toxic components in the cells were analyzed through high-pressure liquid chromatography (HLPC). In addition, the immune response of shrimp to the microalgal extract was assessed by measuring the activity of immune-related enzymes, as well as the transcription of the relevant genes. Overall, both M. aeruginosa cells and the algal extract resulted in a 100% mortality rate in shrimp, whereas the cell-free culture medium was ineffective. And HPLC analysis results revealed the presence of microcystin-LR (MC-LR) at a concentration of 190.40 mg/kg of cells. In addition, the activity and gene transcription of two immune related enzymes, SOD and LZM, were both significantly reduced in shrimp hepatopancreas (p<0.05) after injection with cell extract. However, reduced glutathione (GSH) content was slightly increased, but the ratio of GSH to GSSG was down, and the transcription of gst gene function as detoxification, was significantly downregulated (p<0.05). The results demonstrated that M. aeruginosa cell extract was highly toxic to L. vannamei, and exerted a negative effect on shrimp immunity including reduction of antioxidant capacity, antibacterial activity and detoxification activity, due to microcystin-LR.

KLK4T2 Is a Hormonally Regulated Transcript from the KLK4 Locus

The human kallikrein-related peptidase 4 (KLK4) and the transcribed pseudogene KLKP1 are reported to be highly expressed in the prostate. When trying to clone transcripts of KLKP1, we partly failed. Instead, we identified an androgen-regulated transcript, KLK4T2, which appeared to be a splice variant of KLK4 that also contained exons of KLKP1. Expression analysis of KLK4, KLK4T2, and KLKP1 transcripts in prostate cancer cell lines showed high levels of KLKP1 transcripts in the nucleus and in unfractionated cell extract, whereas it was almost completely absent in the cytoplasmatic fraction. This was in contrast to KLK4 and KLK4T2, which displayed high to moderate levels in the cytoplasm. In patient cohorts we found significantly higher expression of both KLK4T2 and KLK4 in benign prostatic hyperplasia compared to both primary prostate cancer and bone metastasis. Analysis of tissue panels demonstrated the highest expression of KLK4T2 in the prostate, but in contrast to the classical KLK4, relatively high levels were also found in placenta. So far, the function of KLK4T2 is still to be explored, but the structure of the translation product indicated that it generates a 17.4 kDa intracellular protein with possible regulatory function.

Biochemical Characterisation of Human Transglutaminase 4

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell–cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.

Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications.

Study of Potential Application of Cyanobacteria Leptolyngbya Valderiana in Skin Cosmeceuticals

Skin is the outermost cover and first line of defense of the human body. Skin cosmeceuticals are therapeutic agents used to maintain flawless skin and prevent skin disorders. Considering the ill effects of synthetic active agents, natural metabolites are preferred for skincare. Cyanobacterium was known to produce various secondary metabolites with therapeutic potential. This study focuses on screening whole-cell extract of Leptolyngbya valderiana for skincare. Isolation and identification of cyanobacteria were made. The whole-cell extract of Leptolyngbya valderiana was studied for its metabolite profile, mainly for proteins, carbohydrate, and lipid content. Screening of whole-cell extract was carried out for its (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS) radical scavenging ability and UV absorption potential. The antimicrobial effect of extracts was determined by agar cup and minimum inhibitory concentration (MIC) method using salicylic acid as a standard. Skin whitening potential was determined using tyrosinase inhibition assay. Leptolyngbya valderiana was isolated from the tree bark and selected for further study. The extract of Leptolyngbya valderiana was found to have 25.22% proteins, 36.65 % carbohydrates and 35.97% lipids. The extract was found to have antioxidant potential with 96.55% ABTS radical scavenging potential. Antimicrobial potency of the extract against Staphylococcus aureus ATCC 6538, Propionibacterium acne MTCC 1351, Candida albicans ATCC 10231 was equivalent to salicylic acid. The test extract was found to inhibit the tyrosinase enzyme by 43.46%. The test extract is rich in proteins, carbohydrates, and lipids. The extract has significant antioxidant potential, antimicrobial efficiency, ability to absorb ultraviolet B and ultraviolet A region, tyrosinase inhibition potential indicative photoprotective, and skin whitening potential. The isolate Leptolyngbya valderiana has a promising potential for skin cosmeceuticals.

Alcohol dehydrogenases AdhE and AdhB with broad substrate ranges are important enzymes for organic acid reduction in Thermoanaerobacter sp. strain X514

Background The industrial production of various alcohols from organic carbon compounds may be performed at high rates and with a low risk of contamination using thermophilic microorganisms as whole-cell catalysts. Thermoanaerobacter species that thrive around 50–75 °C not only perform fermentation of sugars to alcohols, but some also utilize different organic acids as electron acceptors, reducing them to their corresponding alcohols. Results We purified AdhE as the major NADH- and AdhB as the major NADPH-dependent alcohol dehydrogenase (ADH) from the cell extract of the organic acid-reducing Thermoanaerobacter sp. strain X514. Both enzymes were present in high amounts during growth on glucose with and without isobutyrate, had broad substrate spectra including different aldehydes, with high affinities (< 1 mM) for acetaldehyde and for NADH (AdhE) or NADPH (AdhB). Both enzymes were highly thermostable at the physiological temperature of alcohol production. In addition to AdhE and AdhB, we identified two abundant AdhA-type ADHs based on their genes, which were recombinantly produced and biochemically characterized. The other five ADHs encoded in the genome were only expressed at low levels. Conclusions According to their biochemical and kinetic properties, AdhE and AdhB are most important for ethanol formation from sugar and reduction of organic acids to alcohols, while the role of the two AdhA-type enzymes is less clear. AdhE is the only abundant aldehyde dehydrogenase for the acetyl-CoA reduction to aldehydes, however, acid reduction may also proceed directly by aldehyde:ferredoxin oxidoreductase. The role of the latter in bio-alcohol formation from sugar and in organic acid reduction needs to be elucidated in future studies.

Dissecting Highly Mutagenic Processing of Complex Clustered DNA Damage in Yeast Saccharomyces cerevisiae

Clusters of DNA damage, also called multiply damaged sites (MDS), are a signature of ionizing radiation exposure. They are defined as two or more lesions within one or two helix turns, which are created by the passage of a single radiation track. It has been shown that the clustering of DNA damage compromises their repair. Unresolved repair may lead to the formation of double-strand breaks (DSB) or the induction of mutation. We engineered three complex MDS, comprised of oxidatively damaged bases and a one-nucleotide (1 nt) gap (or not), in order to investigate the processing and the outcome of these MDS in yeast Saccharomyces cerevisiae. Such MDS could be caused by high linear energy transfer (LET) radiation. Using a whole-cell extract, deficient (or not) in base excision repair (BER), and a plasmid-based assay, we investigated in vitro excision/incision at the damaged bases and the mutations generated at MDS in wild-type, BER, and translesion synthesis-deficient cells. The processing of the studied MDS did not give rise to DSB (previously published). Our major finding is the extremely high mutation frequency that occurs at the MDS. The proposed processing of MDS is rather complex, and it largely depends on the nature and the distribution of the damaged bases relative to the 1 nt gap. Our results emphasize the deleterious consequences of MDS in eukaryotic cells.

Biochemical characterisation of human transglutaminase 4

Transglutaminases are protein modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is frequently associated with pathological symptoms and particularly with cancer invasiveness. Although rodent TG4 is well characterised, bio-chemical characteristics of human TG4 that could help the understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtiter plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.