A sensitive, portable, and smartphone-based whole-cell biosensor device for salicylic acid monitoring.

Considerable effort has been invested in developing salicylic acid (SA) biosensors for various application purposes. Here, by engineering the sensing modules and host cell chassis, we have gradually optimized the NahR-Psal/Pr-based SA biosensor, increasing the sensitivity and maximum output by 17.2-fold and 9.4-fold, respectively, and improving the detection limit by 800-fold, from 80 µM to 0.1 µM. A portable SA sensing device was constructed by embedding a gelatin-based hydrogel containing an optimized biosensor into the perforations of tape adhered to glass slide, which allowed good determination of SA in the range of 0.1 µM-10 µM. Then, we developed a customized smartphone App to measure the fluorescence intensity of each perforation and automatically calculate the corresponding SA concentration so that we could detect SA concentrations in real cosmetic samples. We anticipate that this smartphone-based imaging biosensor, with its compact size, higher sensitivity, cost-effectiveness, and easy data transfer, will be useful for long-term monitoring of SA.

Site-directed display of zearalenone lactonase on spilt-intein functionalized nanocarrier for green and efficient detoxification of zearalenone.

In this study, we prepared a functional organic-inorganic hybrid nanoflower (InHNF) via split intein moiety in a biomineralization process without using organic solvents. InHNF could specifically bind the target enzymes from crude cell lysates within seconds and site-directedly display them on the surface by forming a peptide bond with enzyme's terminal amino acid residue. This unique feature enabled InHNF to increase the specific activity of zearalenone detoxifying enzyme ZHD518 by 40 âˆ¼ 60% at all tested temperatures and prevented enzyme denaturation even under extreme pH conditions (pH 3-11). Furthermore, it exhibited excellent operational stability, with a residual activity of over 70% after eight reaction cycles. Strikingly, InHNF-ZHD518 achieved above 50% ZEN degradation despite the near inactivation of free ZHD518 in beer sample. Overall, InHNF nanocarriers can achieve environmentally friendly, purification-free, and site-directed immobilization of food enzymes and enhance their catalytic properties, making them suitable for a wide range of industrial applications.

The thiolation of uridine 34 in tRNA, which controls protein translation, depends on a [4Fe-4S] cluster in the archaeum Methanococcus maripaludis.

Thiolation of uridine 34 in the anticodon loop of several tRNAs is conserved in the three domains of life and guarantees fidelity of protein translation. U34-tRNA thiolation is catalyzed by a complex of two proteins in the eukaryotic cytosol (named Ctu1/Ctu2 in humans), but by a single NcsA enzyme in archaea. We report here spectroscopic and biochemical experiments showing that NcsA from Methanococcus maripaludis (MmNcsA) is a dimer that binds a [4Fe-4S] cluster, which is required for catalysis. Moreover, the crystal structure of MmNcsA at 2.8 Å resolution shows that the [4Fe-4S] cluster is coordinated by three conserved cysteines only, in each monomer. Extra electron density on the fourth nonprotein-bonded iron most likely locates the binding site for a hydrogenosulfide ligand, in agreement with the [4Fe-4S] cluster being used to bind and activate the sulfur atom of the sulfur donor. Comparison of the crystal structure of MmNcsA with the AlphaFold model of the human Ctu1/Ctu2 complex shows a very close superposition of the catalytic site residues, including the cysteines that coordinate the [4Fe-4S] cluster in MmNcsA. We thus propose that the same mechanism for U34-tRNA thiolation, mediated by a [4Fe-4S]-dependent enzyme, operates in archaea and eukaryotes.

A subclass of archaeal U8-tRNA sulfurases requires a [4Fe-4S] cluster for catalysis.

Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC + C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe-4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe-4S] cluster is coordinated by the three cysteines of the CXXC + C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.

Determination of the Absolute Molar Mass of [Fe-S]-Containing Proteins Using Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS).

Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) is a technique that determines the absolute molar mass (molecular weight) of macromolecules in solution, such as proteins or polymers, by detecting their light scattering intensity. Because SEC-MALS does not rely on the assumption of the globular state of the analyte and the calibration of standards, the molar mass can be obtained for proteins of any shape, as well as for intrinsically disordered proteins and aggregates. Yet, corrections need to be made for samples that absorb light at the wavelength of the MALS laser, such as iron-sulfur [Fe-S] cluster-containing proteins. We analyze several examples of [2Fe-2S] and [4Fe-4S] cluster-containing proteins, for which various corrections were applied to determine the absolute molar mass of both the apo- and holo-forms. Importantly, the determination of the absolute molar mass of the [2Fe-2S]-containing holo-NEET proteins allowed us to ascertain a change in the oligomerization state upon cluster binding and, thus, to highlight one essential function of the cluster.

High-Level Extracellular Expression of a New ß-N-Acetylglucosaminidase in Escherichia coli for Producing GlcNAc.

N-acetyl-ß-D glucosamine (GlcNAc) is wildly used in cosmetics, nutraceuticals and pharmaceuticals. The traditional chemical process for GlcNAc production from chitin causes serious acidic pollution. Therefore, the enzymatic hydrolysis becomes a great promising and alternative strategy to produce GlcNAc. ß-N-acetylglucosaminidase (NAGase) can hydrolyze chitin to produce GlcNAc. Here, a GH3 family NAGase encoding gene BlNagZ from Bacillus licheniformis was expressed extracellularly in Escherichia coli guided by signal peptide PelB. The recombinant BlNagZ presented the best activity at 60°C and pH 5.5 with a high specific activity of 13.05 U/mg. The BlNagZ activity in the fermentation supernatant can reach 13.62 U/mL after optimizing the culture conditions, which is 4.25 times higher than optimization before. Finally, combining BlNagZ with chitinase ChiA we identified before, chitin conversion efficiency to GlcNAc can reach 89.2% within 3.5 h. In all, this study provided not only a high active NAGase, and a secreted expression strategy to reduce the cost of production, which is conducive to the industrial application.

Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc.

Chitinase and chitin-oligosaccaride can be used in multiple field, so it is important to develop a high-yield chitinase producing strain. Here, a recombinant Pichia pastoris with 4 copies of ChiA gene from Bacillus licheniformis and co-expression of molecular chaperon HAC1 was constructed. The amount of recombinant ChiA in the supernatant of high-cell-density fermentation reaches a maximum of 12.7 mg/mL, which is 24-fold higher than that reported in the previous study. The recombinant ChiA can hydrolyze 30% collodidal chitin with 74% conversion ratio, and GlcNAc is the most abundant hydrolysis product, followed by N, N'-diacetylchitobiose. Combined with BsNagZ, the hydrolysate of ChiA can be further transformed into GlcNAc with 88% conversion ratio. Additionally, the hydrolysate of ChiA can obviously accelerate the germination growth of rice and wheat, increasing the seedling height and root length by at least 1.6 folds within 10 days.

Expression and Regulation of pde6h by Thyroid Hormone During Metamorphosis in Paralichthys olivaceus.

PDE6H is a cone cell-specific inhibitory subunit that plays a critical role in the adaptation of the photosensitive system to bright and dark phases of the light environment. Thyroid hormone (TH) is one of the most important factors that control development and metabolism in animals, composed mainly of triiodothyronine (T3), and thyroxine (T4). TH also plays a key role in the metamorphosis of the flounder (Paralichthys olivaceus), wherein exogenous TH can accelerate the behavioral changes of larvae from the pelagic to benthic type accompanying changes in the light environment from bright to dark. In this study, transcriptional analysis showed that pde6h is expressed in adult eye, that its expression peaks at the climax of metamorphosis, and that it can be significantly up-regulated to the highest level by exogenous T4 in the early stages of metamorphosis but is inhibited by thiourea (TU). The rescue experiment showed that metamorphic inhibition of larvae and expression inhibition of pde6h gene in TU groups can be rescued by removing TU. Further, dual-luciferase reporter assay indicated the putative regulatory effect of TH on pde6h expression, mediated directly on the gene promoter by the TRαA gene. Together, we speculated that TH may control physiological adaptation of the photosensitive system to light changes during metamorphosis by acting directly on pde6h. This study can help us further study the physiological function of pde6h during flounder metamorphosis in the future.

Expression of Bacillus licheniformis α-amylase in Pichia pastoris without antibiotics-resistant gene and effects of glycosylation on the enzymic thermostability.

Thermostable α-amylases are widely used in industry. The α-amylase from Bacillus licheniformis (BLA) with six potential glycosylation sites possessed excellent thermal and pH stability and high activity. Here, it was expressed in Pichia pastoris. The Pic-BLA-producing yeast without any antibiotics-resistant gene was cultivated in flasks and the amylase activity in fermentation supernatant reached 900 U/mL. The recombinant α-amylase Pic-BLA produced in P. pastoris was deeply glycosylated with 30% increase in molecular mass (MM). The deglycosylation treatment by Endoglycosidase H (Endo H) reduced the MM of Pic-BLA. Thermostability analysis showed that Pic-BLA and deglycosylated Pic-BLA were similar in heat tolerance. In order to eliminate the extra impact of Endo H, the BLA was also expressed in Escherichia coli to get non-glycosylated Eco-BLA. A comparative study between non-glycosylated Eco-BLA and glycosylated Pic-BLA showed no obvious difference in thermostability. It is speculated that the glycosylation has little effect on the thermostability of α-amylase BLA.

Construction of an octosyl acid backbone catalyzed by a radical S-adenosylmethionine enzyme and a phosphatase in the biosynthesis of high-carbon sugar nucleoside antibiotics.

Unique bicyclic octosyl uronic acid nucleosides include ezomycin, malayamycin, and octosyl acid (OA). They are structurally characterized by OA, an unusual 8-carbon furanosyl nucleoside core proposed to be the precursor to polyoxin and nikkomycin. Despite the well-known bioactivity of these nucleoside antibiotics, the biosynthesis of OA has not been elucidated yet. Here we report the two pivotal enzymatic steps in the polyoxin biosynthetic pathway leading to the identification of OA as a key intermediate. Our data suggest that this intermediate is formed via a free radical reaction catalyzed by the radical S-adenosylmethionine (SAM) enzyme, PolH, and using 3'-enolpyruvyl uridine 5'-monophosphate (3'-EUMP) as a substrate. Subsequent dephosphorylation catalyzed by phosphatase PolJ converts the resulting octosyl acid 5'-phosphate (OAP) to OA. These results provide, for the first time, significant in vitro evidence for the biosynthetic origins of the C8 backbone of OA.

Structural basis of rifampin inactivation by rifampin phosphotransferase.

Rifampin (RIF) is a first-line drug used for the treatment of tuberculosis and other bacterial infections. Various RIF resistance mechanisms have been reported, and recently an RIF-inactivation enzyme, RIF phosphotransferase (RPH), was reported to phosphorylate RIF at its C21 hydroxyl at the cost of ATP. However, the underlying molecular mechanism remained unknown. Here, we solve the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations. LmRPH comprises three domains: an ATP-binding domain (AD), an RIF-binding domain (RD), and a catalytic His-containing domain (HD). Structural analyses reveal that the C-terminal HD can swing between the AD and RD, like a toggle switch, to transfer phosphate. In addition to its catalytic role, the HD can bind to the AD and induce conformational changes that stabilize ATP binding, and the binding of the HD to the RD is required for the formation of the RIF-binding pocket. A line of hydrophobic residues forms the RIF-binding pocket and interacts with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moieties of RIF. The R group of RIF points toward the outside of the pocket, explaining the low substrate selectivity of RPH. Four residues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and Gln337, were found to play essential roles in the phosphorylation of RIF; among these the His825 residue may function as the phosphate acceptor and donor. Our study reveals the molecular mechanism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins.