Rapid Degradation of Hazardous Amides by Immobilized Engineered Pseudomonas putida KT2440 Based on a Novel Gene Expression Vector.

The amides 4-trifluoromethylnicotinamide, acrylamide, and benzamide are widely used in agriculture and industry, posing hazards to the environment and animals. Immobilized bacteria are preferred in wastewater treatment, but degradation of these amides by immobilized engineered bacteria has not been explored. Here, engineered Pseudomonas putida KT2440 pLSJ15-amiA was constructed by introducing a new amidase gene expression vector into environmentally safe P. putida KT2440. P. putida KT2440 pLSJ15-amiA had high amidase activity, even at 80 °C. P. putida KT2440 pLSJ15-amiA immobilized with calcium alginate exhibited a greater environmental tolerance than free cells. The amides were rapidly degraded by the immobilized cells, but the activity was inhibited by high concentrations of substrates. The substrate inhibition model revealed that the optimum initial concentrations of 4-trifluoromethylnicotinamide, acrylamide, and benzamide for degradation by immobilized cells were 197.65, 350.76, and 249.40 µmol/L, respectively. This study develops a novel and excellent immobilized biocatalyst for remediation of wastewater containing hazardous amides.

Novel biodegradation pathway of insecticide flonicamid mediated by an amidase and its unusual substrate spectrum.

The insecticide flonicamid (FLO) and its main degradation intermediate 4-trifluoromethylnicotinamide (TFNA-AM) are hazardous to the environment and animals. Microbial transformation of FLO has been well studied, but no study has yet reported on TFNA-AM degradation by a microorganism. Here, Pseudomonas stutzeri CGMCC 22915 effectively degraded TFNA-AM to 5-trifluoromethylnicotinic acid (TFNA). P. stutzeri CGMCC 22915 degraded 60.0% of TFNA-AM (1154.44 µmol/L) within 6 h with a half-life of just 4.5 h. Moreover, P. stutzeri CGMCC 22915 significantly promoted TFNA-AM decomposition in surface water. The reaction was catalyzed by an amidase, PsAmiA. PsAmiA is encoded in a novel nitrile-converting enzyme gene cluster. The enzyme shared only 20-44% identities with previously characterized signature amidases. PsAmiA was successfully expressed in Escherichia coli and its enzymatic properties were investigated using TFNA-AM as the substrate. PsAmiA was more active toward amides without hydrophilic groups, and did not hydrolyze another amide metabolite of FLO, N-(4-trifluoromethylnicotinoyl)glycinamide (TFNG-AM), which is structurally very similar to TFNA-AM. Molecular docking of PsAmiA and TFNA-AM indicated that hydrophobic residues Leu148, Ala150, Ala195, Ile225, Trp341, Leu460, and Ile463 may affect its substrate spectrum. This study provides new insights of the environmental fate of FLO at the molecular level and the structure-function relationships of amidases.

Characterization of nitrilases from Variovorax boronicumulans that functions in insecticide flonicamid degradation and ß-cyano-L-alanine detoxification.

AIMS: To characterize the functions of nitrilases of Variovorax boronicumulans CGMCC 4969 and evaluate flonicamid (FLO) degradation and ß-cyano-L-alanine (Ala(CN)) detoxification by this bacterium. METHODS AND RESULTS: Variovorax boronicumulans CGMCC 4969 nitrilases (NitA and NitB) were purified, and substrate specificity assay indicated that both of them degraded insecticide FLO to N-(4-trifluoromethylnicotinoyl)glycinamide (TFNG-AM) and 4-(trifluoromethyl)nicotinol glycine (TFNG). Ala(CN), a plant detoxification intermediate, was hydrolysed by NitB. Escherichia coli overexpressing NitA and NitB degraded 41.2 and 93.8% of FLO (0.87 mmol·L-1 ) within 1 h, with half-lives of 1.30 and 0.25 h, respectively. NitB exhibited the highest nitrilase activity towards FLO. FLO was used as a substrate to compare their enzymatic properties. NitB was more tolerant to acidic conditions and organic solvents than NitA. Conversely, NitA was more tolerant to metal ions than NitB. CGMCC 4969 facilitated FLO degradation in soil and surface water and utilized Ala(CN) as a sole nitrogen source for growth. CONCLUSIONS: CGMCC 4969 efficiently degraded FLO mediated by NitA and NitB; NitB was involved in Ala(CN) detoxification. SIGNIFICANCE AND IMPACT OF THE STUDY: This study promotes our understanding of versatile functions of nitrilases from CGMCC 4969 that is promising for environmental remediation.

Nitroreduction of imidacloprid by the actinomycete Gordonia alkanivorans and the stability and acute toxicity of the nitroso metabolite.

The insecticide imidacloprid (IMI), which is used worldwide, pollutes environments and has significant ecotoxicological effects. Microbial metabolism and photolysis are the major pathways of IMI degradation in natural environments. Several studies have reported that the metabolites of IMI nitroreduction are more toxic to some insects and mammals than IMI itself. Thus, environmental degradation of IMI may enhance the ecotoxicity of IMI and have adverse effects on non-target organisms. Here, we report that an actinomycete-Gordonia alkanivorans CGMCC 21704-transforms IMI to a nitroreduction metabolite, nitroso IMI. Resting cells of G. alkanivorans at OD600 nm = 10 transformed 95.7% of 200 mg L-1 IMI to nitroso IMI in 4 d. Nitroso IMI was stable at pH 4-9. However, it rapidly degraded under sunlight via multiple oxidation, dehalogenation, and oxidative cleavage reactions to form 10 derivatives; the half-life of nitroso IMI in photolysis was 0.41 h, compared with 6.19 h for IMI. Acute toxicity studies showed that the half maximal effective concentration (EC50) values of IMI, nitroso IMI, and its photolytic metabolites toward the planktonic crustacean Daphnia magna for immobilization (exposed to the test compounds for 48 h) were 17.70, 9.38, 8.44 mg L-1, respectively. The half-life of nitroso IMI in various soils was also examined. The present study reveals that microbial nitroreduction accelerates IMI degradation and the nitroso IMI is easily decomposed by sunlight and in soil. However, nitroso IMI and its photolytic products have higher toxicity toward D. magna than the parent compound IMI, and therefore increase the ecotoxicity of IMI.

Ultrasound examination of the normal fetal duodenum.

Objective: To use the fetal pylorus as a reference point to conveniently display the normal fetal duodenum by ultrasound. Methods: This study was designed in cross-section. A total of 450 healthy singleton pregnant women at 19-39 weeks of gestation who underwent prenatal screening at our hospital from January 2019 to February 2020 were selected. They were divided into three groups according to gestational weeks: the 19-23 gestational weeks group, 29-32 gestational weeks group, and 34-39 gestational weeks group. The duodenal bulb was identified. Its movement and course were continuously and dynamically observed. The descending part of the duodenum was identified, and the duodenal course was traced. Results: The fluid-filled in the fetal duodenum was discontinuous. The overall detection rates of the duodenum in the 19-23 gestational weeks group, 29-32 gestational weeks group, and the 34-39 gestational weeks group were 82.2%, 26.2%, and 13.8%, respectively. The detection rates of the bulbar, descending, horizontal, and ascending parts of the duodenum were 94.4%, 58.2%, 58.0%, and 52.0%, respectively. The anatomical structures of the duodenum as a whole and the pancreas were most easily recognized in the 19-23 gestational weeks group; while in the 34-39 gestational weeks group, the bulbar part had a maximum detection rate of 98.8%, and it had the longest filling time and the shortest examination time. Conclusion: The pylorus is an ideal starting point for tracing the fetal duodenum. The overall detection rate of the fetal duodenum decreases with gestational age. The duodenal bulb is the most easily detected site.

Biotransformation of flonicamid and sulfoxaflor by multifunctional bacterium Ensifer meliloti CGMCC 7333.

Flonicamid is a novel, selective, systemic pyridinecarboxamide insecticide that effectively controls hemipterous pests. Sulfoxaflor, a sulfoximine insecticide, effectively controls many sap-feeding insect pests. Ensifer meliloti CGMCC 7333 transforms flonicamid into N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM). Resting cells of E. meliloti CGMCC 7333 (optical density at 600 nm [OD600] = 5) transformed 67.20% of the flonicamid in a 200-mg/L solution within 96 h. E. meliloti CGMCC 7333 transforms sulfoxaflor into N-(methyl(oxido){1-[6-(trifluoromethyl) pyridin-3-yl] ethyl}-k4-sulfanylidene) urea (X11719474). E. meliloti CGMCC 7333 resting cells (OD600 = 5) transformed 89.36% of the sulfoxaflor in a 200 mg/L solution within 96 h. On inoculating 2 mL of E. meliloti CGMCC 7333 (OD600 = 10) into soil containing 80 mg/kg flonicamid, 91.1% of the flonicamid was transformed within 9 d (half-life 2.6 d). On inoculating 2 mL of E. meliloti CGMCC 7333 (OD600 = 10) into soil containing 80 mg/kg sulfoxaflor, 83.9% of the sulfoxaflor was transformed within 9 d (half-life 3.4 d). Recombinant Escherichia coli harboring the E. meliloti CGMCC 7333 nitrile hydratase (NHase)-encoding gene and NHase both showed the ability to transform flonicamid or sulfoxaflor into their corresponding amides, TFNG-AM and X11719474, respectively. These findings may help develop a bioremediation agent for the elimination of flonicamid and sulfoxaflor contamination.

Bioinformatics of a Novel Nitrile Hydratase Gene Cluster of the N2-Fixing Bacterium Microvirga flocculans CGMCC 1.16731 and Characterization of the Enzyme.

Microvirga flocculans CGMCC 1.16731 can degrade many cyano group-containing neonicotinoid insecticides. Here, its genome was sequenced, and a novel nitrile hydratase gene cluster was discovered in a plasmid. The NHase gene cluster (pnhF) has gene structure ß-subunit 1, α-subunit, and ß-subunit 2, which is different from previously reported NHase gene structures. Phylogenetic analysis of α-subunits indicated that NHases containing the three subunit (ß1αß2) structure are independent from NHases containing two subunits (αß). pnhF was successfully expressed in Escherichia coli, and the purified PnhF could convert the nitrile-containing insecticide flonicamid to N-(4-trifluoromethylnicotinoyl)glycinamide. The enzymatic properties of PnhF were investigated using flonicamid as a substrate. Homology models revealed that amino acid residue ß1-Glu56 may strongly affect the catalytic activity of PnhF. This study expands our understanding of the structures and functions of NHases and the enzymatic mechanism of the environmental fate of flonicamid.

Maturation Mechanism of Nitrile Hydratase From Streptomyces canus CGMCC 13662 and Its Structural Character.

Nitrile hydratases have received significant interest both in the large-scale industrial production of acrylamide and nicotinamide, and the remediation of environmental contamination with nitrile-containing pollutants. Almost all known nitrile hydratases include an α-subunit (AnhA) and ß-subunit (AnhB), and a specific activator protein is crucial for their maturation and catalytic activity. Many studies exist on nitrile hydratase characteristics and applications, but few have reported their metal insertion and post-translational maturation mechanism. In this study, we investigated the cobalt insertion and maturation mechanism of nitrile hydratase from Streptomyces canus CGMCC 13662 (ScNHase) bearing three subunits (AnhD, AnhE, and AnhA). ScNHase subunits were purified, and the cobalt content and nitrile hydratase activity of the ScNHase subunits were detected. We discovered that cobalt could insert into the cobalt-free AnhA of ScNHase in the absence of activator protein under reduction agent DL-dithiothreitol (DTT) environment. AnhD not only performed the function of AnhB of NHase, but also acted as a metal ion chaperone and self-subunit swapping chaperone, while AnhE did not act as similar performance. A cobalt direct-insertion under reduction condition coordinated self-subunit swapping mechanism is responsible for ScNHase post-translational maturation. Molecular docking of ScNHase and substrates suggested that the substrate specificity of ScNHase was correlated with its structure. ScNHase had a weak hydrophobic interaction with IAN through protein-ligand interaction analysis and, therefore, had no affinity with indole-3-acetonitrile (IAN). The post-translational maturation mechanism and structure characteristics of ScNHase could help guide research on the environmental remediation of nitrile-containing waste contamination and three-subunit nitrile hydratase.