Parametric and kinetic studies of activated sludge dewatering by cationic chitosan-like bioflocculant BF01314 produced from Citrobacter youngae.

Bacterial strains belonging to Citrobacter spp. were reported to produce polysaccharides consisting of N-acetylglucosamine and glucosamine like chitosan, with high flocculation activity. In this work, the flocculation dewatering performance of activated sludge conditioned by a novel cationic chitosan-like bioflocculant (BF) named BF01314, produced from Citrobacter youngae GTC 01314, was evaluated under the influences of flocculant dosage, pH, and temperature. At BF dosage as low as 0.5 kg/t DS, the sludge dewaterability was significantly enhanced in comparison to the raw (untreated) sludge, featuring well-flocculated characteristic (reduction in CST from 22.0 s to 9.4 s) and good sludge filterability with reduced resistance (reduction in SRF by one order from 7.42 × 1011 to 9.59 × 1010 m/kg) and increased compactness of sludge (increase in CSC from 15.2 to 23.2%). Besides, the BF demonstrated comparable high sludge dewatering performance within the pH range between 2 and 8, and temperature range between 25 °C and 80 °C. Comparison between the BF, the pristine chitosan and the commercial cationic copolymer MF 7861 demonstrated equivalent performance with enhanced dewaterability at the dosage between 2.0 and 3.0 kg/t DS. Besides, the BF demonstrated strong flocculation activity (>99%) when added to the sludge suspension using moderate to high flocculation speeds (100-200 rpm) with at least 3-min mixing time. The BF's reaction in sludge flocculation was best fitted with a pseudo first-order kinetic model. Electrostatic charge patching and polymer bridging mechanisms are believed to be the dominant mechanistic phenomena during the BF's sludge conditioning process (coagulation-flocculation).

Repeated Stimulation of Toll-Like Receptor 2 and Dectin-1 Induces Chronic Pancreatitis in Mice Through the Participation of Acquired Immunity.

BACKGROUND: Stimulation of Toll-like receptor 3 (TLR3) induces autoimmune-mediated pancreatitis in susceptible mice, whereas stimulation of TLR4 causes nonautoimmune-mediated pancreatitis. However, the effects of TLR2 stimulation on the pancreas are unknown. AIMS: We investigated the role of TLR2 stimulation on pancreatic damage by repeatedly stimulating mice with TLR2 ligands. METHODS: Wild-type (WT) and interleukin 10-deficient (IL-10-knockout (KO)) mice were administered zymosan and lipoteichoic acid (LTA) intraperitoneally at various doses twice weekly for 4 weeks. Syngeneic T-cell-deficient mice, B-cell-deficient mice, recombination activating gene 2-deficient (RAG2-KO) mice and RAG2-KO mice that had been reconstituted with CD4+ or CD8+ T cells isolated from WT mice were treated with zymosan similarly. Mice were killed, the severity of pancreatitis was graded histologically, and serum cytokine levels were measured. RESULTS: Repeated administration of zymosan induced pancreatitis dose dependently in both WT and IL-10-KO mice. Administration of LTA induced pancreatitis only in IL-10-KO mice. Adoptive transfer of splenocytes obtained from IL-10-KO mice with pancreatitis did not cause pancreatitis in recipient RAG2-KO mice. Pancreatitis was scarcely observed in RAG2-KO mice and was attenuated in T-cell-deficient and B-cell-deficient mice compared with WT mice. A single administration of zymosan significantly increased the serum level of monocyte chemoattractant protein 1 (MCP-1) in WT mice. CONCLUSIONS: Repeated stimulation of TLR2 and dectin-1 induced nonautoimmune-mediated pancreatitis in mice. Participation of acquired immunity seems to play an important role in the pathogenesis of pancreatitis in association with the increase in serum MCP-1 level.

Inhibition of the dephosphorylation of eukaryotic initiation factor 2α ameliorates murine experimental pancreatitis.

BACKGROUND: Endoplasmic reticulum (ER) stress in the pancreas is closely associated with the development of acute pancreatitis. However, the role of the protein kinase RNA-like ER kinase (PERK) in this disease is not fully understood. We investigated whether an inhibitor of the dephosphorylation of eukaryotic initiation factor 2α, salubrinal, could improve murine experimental pancreatitis through the amelioration of ER stress. METHODS: Acute pancreatitis was induced by the intraperitoneal administration of cerulein (50 µg/kg) six times at 1-h intervals followed by lipopolysaccharide (10 mg/kg). Salubrinal was administered intraperitoneally immediately after lipopolysaccharide injection and 3 h later. Mice were sacrificed 24 h after the first injection of cerulein, and serum amylase and proinflammatory cytokines were measured. The severity of pancreatitis was evaluated histologically using a scoring system. The expression levels of ER stress-related proteins were evaluated by Western blotting. RESULTS: The administration of salubrinal significantly attenuated the increase in serum amylase levels and improved histologically assessed pancreatitis. The serum levels of proinflammatory cytokines were significantly suppressed in salubrinal-treated mice, as was the expression of glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein, and cleaved caspase-3. CONCLUSIONS: The amelioration of ER stress through augmentation of the PERK-signaling pathway may be a therapeutic target for the treatment of acute pancreatitis.

[Intussusception Due to Inflammatory Polyp in the Ileum after ESD for Rectal LST].

A 69-year-woman was admitted to the clinic in August 2018 because of general fatigue and low appetite.She had occult blood-positive and was referred to our hospital for further investigations.There was LST in the rectum for which colonoscopy and ESD were performed.She had abdominal pain and slight fever on postoperative day 1.Abdominal CT showed an intussusception in the ileum.We could not achieve endoscopic de-torsion and carried out laparotomy.The intussusception was found to be strangulated due to inflammatory polyp and mesenteric adhesion.The affected portion was resected.Although treatment for low hypoalbuminemia and neurogenic cystitis was required, she was discharged on postoperative day 28.

Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate.

Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD 1 and nylE 1 , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD1) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using α-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE1) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP+ as a cofactor. Phylogenic analysis revealed that NylD1 should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE1 should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD1/NylE1 coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology.

Correction to: Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate.

The original publication of this paper contains mistakes for Tables 1 and 2 legends as well as the sublabels in Figs. 2, 4, 5, 6, and 7.

Preparation of 1L-myo-Inositol 1-Phosphate as a Substrate of Phosphatidylinositol Phosphate Synthase.

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [14C] glucose 6-phosphate ([14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1l-Ino-1P synthase. The activity of 1l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1l-Ino-1P using a solution containing recombinant 1l-Ino-1P synthase derived from the hyperthermophilic archaeon Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1l-Ino-1P using this enzyme. Quantitation of the synthesized 1l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1d-inositol 1-phosphate cannot be a substrate for PIP synthase.

Biosynthesis-inspired deracemizative production of d-luciferin by combining luciferase and thioesterase.

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.

A firefly inspired one-pot chemiluminescence system using n-propylphosphonic anhydride (T3P).

A simple reaction procedure for chemiluminescence of firefly luciferin (D-luc) using n-propylphosphonic anhydride (T3P) is reported. A luminescent photon is produced as a result of one-pot reaction, only requiring mixing with the substrate carboxylic acid and T3P in the presence of a mild organic base.

Inhibition of the dephosphorylation of eukaryotic initiation factor 2α ameliorates murine experimental colitis.

BACKGROUND/AIMS: Endoplasmic reticulum (ER) stress in the intestine is closely associated with the development of inflammatory bowel disease (IBD). However, the role of the protein kinase RNA-like ER kinase in this disease is not fully known. We studied whether an inhibitor of the dephosphorylation of eukaryotic initiation factor 2α, salubrinal, improves murine experimental colitis through the amelioration of ER stress. METHODS: Colitis was induced by the administration of 3% dextran sulfate sodium (DSS) for 5 days. Mice were injected salubrinal intraperitoneally from the commencement of DSS treatment and were sacrificed on day 10. The severity of colitis was evaluated histologically using a scoring system.Myeloperoxidase activity and the expression of proinflammatory cytokine genes in the colon were analyzed. The expression levels of ER stress-related proteins were evaluated by Western blotting. RESULTS: The administration of salubrinal significantly attenuated body weight loss and improved colitis, as assessed histologically. The elevation of myeloperoxidase activity and the expression of proinflammatory cytokine genes were suppressed in salubrinal-treated mice. The expression of glucose-regulated protein 78, activating translation factor 4, and heat-shock protein 70 was elevated in mice treated with salubrinal. CONCLUSION: The amelioration of ER stress may be a therapeutic target for the treatment of IBD.

Enzymatic hydrolysis of nylons: quantification of the reaction rate of nylon hydrolase for thin-layered nylons.

Nylon hydrolase degrades various aliphatic nylons, including nylon-6 and nylon-66. We synthesized a nylon-66 copolymer (M w = 22,900, M n = 7,400), in which a part of an adipoyl unit (32 % molar ratio) of nylon-66 was replaced with a succinyl unit by interfacial polymerization. To quantify the reaction rate of the enzymatic hydrolysis of nylons at the surface of solid polymers, we prepared a thin layer of nylons on the bottom surface of each well in a polystyrene-based micro-assay plate. The thickness of the nylon layer was monitored by imaging analysis of the photographic data. More than 99 % of the copolymer with thicknesses of 260 nm (approximately 600 layers of polymer strands) were converted to water-soluble oligomers by nylon hydrolase (3 mg enzyme ml(-1)) at 30 °C within 60 h. These results were further confirmed by TLC analysis of the reaction products and by assay of liberated amino groups in the soluble fractions. The degradation rate of the thin-layered nylon-6 was similarly analyzed. We demonstrate that this assay enables a quantitative evaluation of the reaction rate of hydrolysis at the interface between the solid and aqueous phases and a quantitative comparison of the degradability for various polyamides.

Proximal superficial femoral artery puncture using an ascending approach for stent re-occlusion in the common femoral artery.

We aimed to describe a technique for approaching the common femoral artery (CFA) in cases where doing so is difficult owing to an occluded lesion caused by a previously implanted stent. A 72-year-old woman had severe stenotic lesions in both iliac arteries that required an approach via the bilateral femoral arteries. The right CFA had a previously implanted stent and a completely occluded lesion that extended from the superficial femoral artery (SFA). A 20G needle was inserted through the proximal SFA, and the needle tip was advanced into the CFA stent and passed through the occluded lesion using a microcatheter and guide wire (GW). This allowed us to insert a guide catheter via the GW into the occluded lesion. No complications, such as bleeding, were observed after the procedure. When the CFA is occluded by a stent, an ascending approach through the proximal SFA is a viable treatment option. Learning objective: An occluded lesion due to a previously implanted stent makes approaching the common femoral artery difficult. Hence, alternative approaches are needed. In this regard, an approach via the proximal superficial femoral artery may prove useful.

Diffuse large B-cell lymphoma of the gallbladder with hepatoduodenal invasion exhibiting a necrotic tendency.

We report a case of diffuse large B-cell lymphoma (DLBCL) of the gallbladder with extensive hepatoduodenal invasion, which was challenging to diagnose histologically due to a strong tendency to be necrotic. An 71 year-old man presented with upper abdominal pain and was referred to our hospital. Computed tomography revealed a distended gallbladder with air within the irregular gallbladder wall and an indistinct border with the hepatoduodenum, suggesting invasion. Esophagogastroduodenoscopy detected an ulceration in the duodenal bulb. However, histologic analysis failed to provide a definitive diagnosis due to the presence of necrotic tissue. Furthermore, direct biopsy from the gallbladder mucosa by endoscopic retrograde cholangiography revealed only necrotic tissue and no diagnosis. Contrast ultrasonography for the hepatic invasion revealed enhancement with blood flow, suggesting non-necrotic tissue. Subsequently, an ultrasound-guided core-needle biopsy was conducted to obtain tissue samples from the described lesion. The pathology showed atypical lymphocytes with irregular nuclei. Immunostaining indicated positive expression of CD10, CD20, Bcl-6, and C-Myc, consistent with a diagnosis of DLBCL. In our case, the lymphoma exhibited a strong tendency to be necrotic, making histologic diagnosis difficult. However, selective biopsy from the site of blood flow made the diagnosis possible and proved to be useful.

Function of a glutamine synthetase-like protein in bacterial aniline oxidation via γ-glutamylanilide.

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl2. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.

Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis.

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αßßα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T(m) = 52 °C, enhanced the protein thermostability by 36 °C (T(m) = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.

Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) from Arthrobacter sp. KI72.

Nylon hydrolase (NylC) encoded by Arthrobacter plasmid pOAD2 (NylCp2) was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylCp2 was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in 0.1 M HEPES buffer pH 7.5 containing 0.2 M NaCl and 25% glycerol. Diffraction data were collected from the native crystal to a resolution of 1.60 Å. The obtained crystal was spindle shaped and belonged to the C-centred orthorhombic space group C2221, with unit-cell parameters a=70.84, b=144.90, c=129.05 Å. A rotation and translation search gave one clear solution containing two molecules per asymmetric unit.

Distribution of chitin/chitosan-like bioflocculant-producing potential in the genus Citrobacter.

Some strains belonging to the genera Citrobacter and Enterobacter have been reported to produce chitin/chitosan-like bioflocculants (BFs) from acetate. In this study, to investigate the distribution of the BF-producing potential in the genus Citrobacter and to screen stably and highly BF-producing strains, we obtained 36 Citrobacter strains from different culture collection centers, which were distributed among seven species in the genus, and tested for the flocculating activities of their culture supernatants using a kaolin suspension method. As a result, 21 strains belonging to C. freundii (17 strains in 23 strains tested), C. braakii (two in two), C. youngae (one in one), and C. werkmanii (one in two) showed flocculating activity, but this ability was limited to cells grown on acetate. Gas chromatography/mass spectrometry (GC/MS) analysis of the hydrolysates from the BFs of five selected strains indicated that they consisted of glucosamine and/or N-acetylglucosamine, such as the chitin/chitosan-like BF (BF04) produced by Citrobacter sp. TKF04 (Fujita et al. J Biosci Bioeng 89: 40-46, 2000). Gel filtration chromatography using a high-performance liquid chromatography system revealed that the molecular weight ranges of these BFs varied, but the average sizes were all above 1.66 × 106Da.

Pseudoaneurysm of mitral-aortic intervalvular fibrosa with rupture: a case report.

BACKGROUND: Mitral-aortic intervalvular fibrosa (MAIVF) is a fibrous region connecting the anterior mitral leaflet (AML) and aortic valve. Pseudoaneurysm of the MAIVF is a rare condition that has been reported as a sequela of infective endocarditis (IE) and surgical trauma. Here, we report a case of a ruptured pseudoaneurysm of the MAIVF, along with some literature reviews. CASE PRESENTATION: A 65-year-old man diagnosed with moderate aortic regurgitation five years previously had a fever of unknown origin. He suddenly developed headache and apraxia and was transported to our hospital. He was diagnosed with intracranial hemorrhage and admitted. One week after admission, echocardiography revealed aorto-mitral discontinuity and protrusion with severe regurgitant flow from left ventricular outflow tract to the left atrium. The AML was suspected to have ruptured. However, intraoperatively, the AML structure was preserved. A ruptured pseudoaneurysm of the MAIVF was also observed. Therefore, we successfully performed pseudoaneurysm repair using a bovine pericardial patch, aortic valve replacement, and mitral annuloplasty. CONCLUSIONS: P-MAIVF is a rare but potentially life-threatening complication of IE, for which timely diagnosis and prompt appropriate therapeutic intervention are required. In the present case, although neither obvious active IE nor history of previous IE could be identified, healed IE was considered based on the clinical course. The patient had intracranial hemorrhage (ICH) with well-controlled heart failure and underwent elective surgical repair more than one month after the onset of ICH, while the clinical course after the surgical procedure was uneventful.

Novel Concepts for Graphene-Based Nanomaterials Synthesis for Phenol Removal from Palm Oil Mill Effluent (POME).

In recent years, the global population has increased significantly, resulting in elevated levels of pollution in waterways. Organic pollutants are a major source of water pollution in various parts of the world, with phenolic compounds being the most common hazardous pollutant. These compounds are released from industrial effluents, such as palm oil milling effluent (POME), and cause several environmental issues. Adsorption is known to be an efficient method for mitigating water contaminants, with the ability to eliminate phenolic contaminants even at low concentrations. Carbon-based materials have been reported to be effective composite adsorbents for phenol removal due to their excellent surface features and impressive sorption capability. However, the development of novel sorbents with higher specific sorption capabilities and faster contaminant removal rates is necessary. Graphene possesses exceptionally attractive chemical, thermal, mechanical, and optical properties, including higher chemical stability, thermal conductivity, current density, optical transmittance, and surface area. The unique features of graphene and its derivatives have gained significant attention in the application of sorbents for water decontamination. Recently, the emergence of graphene-based adsorbents with large surface areas and active surfaces has been proposed as a potential alternative to conventional sorbents. The aim of this article is to discuss novel synthesis approaches for producing graphene-based nanomaterials for the adsorptive uptake of organic pollutants from water, with a special focus on phenols associated with POME. Furthermore, this article explores adsorptive properties, experimental parameters for nanomaterial synthesis, isotherms and kinetic models, mechanisms of nanomaterial formation, and the ability of graphene-based materials as adsorbents of specific contaminants.

X-ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase.

Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10-5  s-1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis.