Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

Isolation, characterization, bacteriocin production and biological potential of Bifidobacteria of ruminants

Bacteriocins exhibited a wide spectrum of antibacterial activity against different pathogens. The aim of current study was to characterize the bacteriocins produced by Bifidobacterium spp. isolated from ruminants. The Bifidobacterium isolates were identified as B. longum, B. pseudolongum, B. bifidum, B. thermophilum, B. boum, B. merycicum and B. ruminantium. Bacteriocins were found to be pH stable, heat resistant, highly diffusible, NaCl tolerant and resistant to UV radiations. SDS, EDTA and urea induced 14%, 21% and 24% bacteriocins activity loss. Modified MRS broth (1% tryptone, 1% yeast extract and 2% glucose) was found to be the best nutrient medium for optimal production of bacteriocins. Minimum inhibitory concentration (MIC) values varied from 300 μl/ml to 500 μl/ml and minimum bactericidal concentration (MBC) values ranged from 500 μl/ml to >500 μl/ml for E. coli and S. aureus respectively. The highest protein concentration (29.0248 mg/ml) was recorded for Bifidobacteria bacteriocin produced by B. longum. Tricine-Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) revealed that molecular weight of isolated bifidobacterial bacteriocins was in the range of 3.6 kDa–30 kDa. Current study indicated that bifidobacterial bacteriocins have considerable potential to be used as biopreservative.

Characterisation and separation of infectious bursal disease virus-like particles using aqueous two-phase systems

Infectious bursal disease (IBD) causes considerable economic losses in the commercial poultry industry worldwide. The principal way to control IBD virus (IBDV), the causative agent of IBD, is still through vaccination programs. Virus-like particles (VLPs) are recognised as a safe and potent recombinant vaccine platform. This research work explores the characterisation and separation of infectious bursal disease virus-like particles (IBD-VLPs) from crude feedstock. Various characteristics were studied with high-performance size-exclusion chromatography (HP-SEC), sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) analyses. Subsequently, the separation of IBD-VLPs using polyethylene glycol (PEG)/sodium citrate-based aqueous two-phase systems (ATPSs) was conducted and optimised. Moreover, a scale-up study of the best ATPS constituted of 15% PEG 6000, 11% sodium citrate and 10% crude feedstock was performed to compare the separation performance of IBD-VLPs with and without centrifugation-assisted. The results indicated that the optimised ATPS with centrifugation-assisted for both 5 g and 50 g systems showed good recovery of IBD-VLPs of >97% in the interphase between the PEG-rich top and salt-rich bottom phases. These optimised systems also showed high removal efficiencies of impurities of >95%. The results demonstrated that aqueous two-phase extraction could be a promising technology for efficient VLPs separation.

Western Blotting of the Endocannabinoid System.

Measuring protein levels of receptors and enzymes involved in endocannabinoid metabolism is an important step for understanding the distribution, function, and regulation of these components of the endocannabinoid system. A common approach for detecting proteins from complex biological systems is western blotting. In this chapter, we describe a general approach to western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, with due attention paid to the validation of the primary antibodies used, it can provide quantitative information on protein expression levels. Additional information can also be inferred from western blotting such as potential pre- and post-translational modifications (e.g., alternative splicing, phosphorylation, or glycosylation) that can be further evaluated by specific analytical techniques.

Study on preparation of chickpea peptide and its effect on blood glucose.

Chickpeas are the third largest bean in the world and are rich in protein. In this study, chickpea peptides were prepared by the enzyme-bacteria synergy method. Taking the peptide yield as the index, we first screened 8 strains suitable for the fermentation of chickpea peptides from 16 strains, carried out sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then screened 4 strains with the best decomposition effect of chickpea protein. The molecular weight, amino acid content, and α-glucosidase inhibitory activity of the chickpea peptides fermented by these four strains were detected. Finally, the strains with the best α-glucosidase inhibitory activity were obtained, and the inhibitory activities of the different molecular weight components of the chickpea peptides fermented by the strains with the best α-glucosidase inhibitory were detected. It was found that Bifidobacterium species had the best fermentation effect, and the highest peptide yield was 52.99 ± 0.88%. Lactobacillus thermophilus had the worst fermentation effect, and the highest peptide yield was 43.22 ± 0.47%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that Bifidobacterium species, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus paracasei have a better effect on the decomposition of chickpea protein in the fermentation process, and the molecular weight of their fermented peptides is basically below 20 KDa. Among the four strains, the α-glycosidase inhibition of chickpea peptide fermented by Lactobacillus acidophilus was the best, which was 58.22 ± 1.10% when the peptide concentration was 5.0 mg/ml. In chickpea peptide fermented by Lactobacillus acidophilus, the influence of molecular weight on the inhibitory activity is not obvious when the molecular weight is <10 kD, and the molecular weight range of the best inhibitory effect is 3–10 kD, and the inhibitory rate of α-glucosidase is 37 ± 1.32% at 2.0 mg/ml. This study provides a theoretical basis for the study of a new preparation method for chickpea peptide and its hypoglycemic effect.

A method to obtain purified free light chain monomers and dimers from urine samples of patients with multiple myeloma

Antibody light chains are synthesized in excess by plasma cells, and this excess can be secreted into biological fluids as dimers or monomers in various proportions. Structural differences between monomers or dimers of free light chains (FLC) can affect their biological functions and possibly their pathogenicity. They also may exhibit differential immune reactivity, perhaps explaining discrepant quantifications when measured by different immunoreagents. Having purified FLC monomers and dimers available can be useful for studying their properties. Here we propose a simple preparatory procedure to purify FLC monomers and dimers from urine samples of patients with plasma cell disorders. Two representative urine samples containing lambda or kappa FLC were loaded into a nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The gel strips containing separate monomers and dimers were excised, electroeluted, and the FLC recovered. The FLC were recovered from SDS-PAGE gel in sufficient amounts to be quantified by UV and two automated nephelometric assays immunochemical. The procedure was found to be simple, reproducible, and with a high yield, thus offering the opportunity to compare different assays. Not all urine samples are suitable for this procedure, but this approach allows for the purification of FLC monomers and dimers from many selected urine samples which maintain their oligomeric organization.

Sulfur uptake and distribution, grain yield, and quality of hybrid and inbred winter wheat (Triticum aestivum L.) varieties under early and late waterlogging

AbstractBackgroundWaterlogging at early and later growth stages could affect sulfur (S) uptake, distribution, yield, and quality of winter wheat. We hypothesized that the hybrid winter wheat variety (Hyvento), with a high degree of heterosis and genetic diversity, is likely to uptake and utilize S more efficiently and withstands waterlogging better than the inbred wheat variety (Kredo).AimsThe objective of the present study was to investigate the effect of 2‐week waterlogging at the end of tillering stage (Feekes Stage 6) and/or ear emergence stage (Feekes Stage 10.1) on grain yield, yield components, S‐uptake and its partitioning, and composition of grain storage proteins (glutenins and gliadins) of hybrid (Hyvento) and inbred (Kredo) winter wheat varieties.MethodsPlants were grown in large containers (120 L) in a semi‐controlled greenhouse and harvested three times immediately after each waterlogging treatment and at agronomic maturity. Grain yield, S concentration in different plant organs, total protein content, and patterns of gluten proteins (gliadin and glutenin) and their subunits (by sodium dodecyl sulfate–polyacrylamide gel electrophoresis) in grains were evaluated.ResultsGrain yield of the inbred variety was more negatively affected under early and late waterlogging than the hybrid variety and was associated with reduced grain size. The decline in grain yield was significantly higher under late waterlogging (30%–31%) than early waterlogging (17%–18%). Compared to the hybrid variety Hyvento, the waterlogging‐induced decline in S concentration in grains, husk, and straw was markedly lower in the inbred variety Kredo. While early waterlogging did not affect total grain protein content in either of the varieties, late waterlogging significantly reduced the total grain protein content (18%) in the inbred variety Kredo. Similarly, the grain gliadin content of the hybrid variety was not affected by both waterlogged conditions, whereas 32% and 23% reductions were recorded for the inbred variety under early and late waterlogging, respectively. Nevertheless, waterlogging significantly increased the proportion of S‐poor high‐molecular‐weight glutenin subunits and ω‐gliadin in the inbred variety.ConclusionIt could be inferred from the present study that hybrid winter wheat variety is relatively resistant to waterlogging than the inbred wheat variety, and thereby produces higher grain yield. Efficient uptake and metabolism of S could be one of the mechanisms conferring waterlogging resistance in winter wheat.

Effects of Radiation on Cross-Linking Reaction, Microstructure, and Microbiological Properties of Whey Protein-Based Tissue Adhesive Development.

Whey proteins are mainly a group of small globular proteins. Their structures can be modified by physical, chemical, and other means to improve their functionality. The objectives of this study are to investigate the effect of radiation on protein–protein interaction, microstructure, and microbiological properties of whey protein–water solutions for a novel biomaterial tissue adhesive. Whey protein isolate solutions (10%, 27%, 30%, 33%, and 36% protein) were treated by different intensities (10–35 kGy) of gamma radiation. The protein solutions were analyzed for viscosity, turbidity, soluble nitrogen, total plate count, and yeast and mold counts. The interactions between whey proteins were also analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and scanning electron microscopy. The viscosity of protein solution (27%, w/w) was increased by the treatment of gamma radiation and by the storage at 23 °C. The 35 kGy intensity irradiated soluble nitrogen (10%, w/w) was reduced to about half of the sample treated by 0 kGy gamma radiation. The effects of gamma radiation and storage time can significantly increase the viscosity of whey protein solutions (p < 0.05). Radiation treatment had significant impact on soluble nitrogen of whey protein solutions (p < 0.05). SDS-PAGE results show that the extent of oligomerization of whey protein isolate solutions are increased by the enhancement in gamma radiation intensity. Photographs of SEM also indicate that protein–protein interactions are induced by gamma radiation in the model system. Consistent with above results, the bonding strength increases by the addition of extent of gamma radiation and the concentration of glutaraldehyde. Our results revealed that the combination of gamma-irradiated whey protein isolate solutions and glutaraldehyde can be used as a novel biomaterial tissue adhesive.

Tropomyosin micelles are the major components contributing to the white colour of boiled shellfish soups

Basket clam soup, a popular Asian dish, is prepared by boiling clams in hot water. The soup is generally cloudy, and it is considered that increased cloudiness enhances taste. However, the composition of the whitening ingredients and their association with taste enhancement remains unclear. In this study, we aimed to identify the components contributing to the white colour of the boiled soup. The white component upon precipitation with trichloroacetic acid reacted positively with ninhydrin, indicating the presence of proteins. The separation of proteins using sodium dodecyl sulphate–polyacrylamide gel electrophoresis revealed an intense band of size 33 kDa. Peptide mass fingerprinting of the identified protein using matrix-assisted laser desorption/ionisation-time-of-flight tandem mass spectrometry revealed the protein as tropomyosin. To validate the involvement of tropomyosin in the turbidity of the soup, tropomyosin was expressed and extracted from Escherichia coli. As expected, the purified protein suspended in water resulted in turbid appearance. To determine whether lipids have any association with the observed cloudiness of the soup, the amounts of fatty acids were measured. The proportion of estimated fatty acids was very low compared to that of proteins. Overall, we identified the major component contributing to soup cloudiness as tropomyosin forming micelles.

A Novel Thermal-driven Self-assembly Method to Prepare Albumin Nanoparticles: Formation Kinetics, Degradation Behavior and Formation Mechanism.

Nanoparticles based on bovine serum albumin (BSA), which shares 76% homology with human serum albumin (HSA), have emerged as a promising candidate for the efficient delivery of anticancer drugs. Thermal-driven self-assembly is a novel organic solvent-free approach to produce albumin nanoparticles. In our previous study, some features of this nanoparticle such as drug loading efficiency, drug encapsulation efficiency and drug release kinetics have been evaluated. However, the formation mechanism that determines the above nanoparticle properties remains unclear. Here, we investigated the formation kinetics and mechanism using spectroscopic methods including fluorescence spectroscopy, circular dichroism (CD) and differential scanning calorimetry (DSC). We also applied chemical analysis methods that measured the content changes of albumin active groups and vanillin. To verify the covalent networks in the nanoparticles, trypsin and glutathione (GSH) were used separately to cleave the peptide bonds and disulfide bridges, and dynamic light scattering (DLS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to analyze the degraded samples. BSA nanoparticles started to form at 10min and were completely formed at 120min. With the digestion of trypsin, more than 50% of the nanoparticles were degraded within 60min. CD spectra showed that α-helical structure of BSA decreased from 42.3% to 39.8% and 37.7% after heating for 10 and 60min, respectively. In the DSC thermogram, the melting peak of BSA nanoparticles was 229.14℃, which is about 12℃ higher than the physical mixture of BSA and vanillin, indicating that chemical reactions occurred during the nanoparticle formation and formed a new more stable substance. Moreover, the results of active group assay, GSH degradation and SDS-PAGE experiments also proved that disulfide bonds and peptide bonds were formed between BSA molecules, whereas Schiff bases were formed between BSA and vanillin molecules. Formation kinetics and degradation behavior are important properties to characterize albumin nanoparticles and should be paid attention to. Not only that, this study also provides an effective way to study the formation mechanism of protein-based nanodrug delivery systems.

Using T4 genetics and Laemmli’s development of high-resolution SDS gel electrophoresis to reveal structural protein interactions controlling protein folding and phage self-assembly

One of the most transformative experimental techniques in the rise of modern molecular biology and biochemistry was the development of high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis, which allowed separation of proteins—including structural proteins—in complex mixtures according to their molecular weights. Its development was intimately tied to investigations of the control of virus assembly within phage-infected cells. The method was developed by Ulrich K. Laemmli working in the virus structural group led by Aaron Klug at the famed Medical Research Council Laboratory for Molecular Biology at Cambridge, UK. While Laemmli was tackling T4 head assembly, I sat at the next bench working on T4 tail assembly. To date, Laemmli’s original paper has been cited almost 300,000 times. His gel procedure and our cooperation allowed us to sort out the sequential protein–protein interactions controlling the viral self-assembly pathways. It is still not fully appreciated that this control involved protein conformational change induced by interaction with an edge of the growing structure. Subsequent efforts of my students and I to understand how temperature-sensitive mutations interfered with assembly were important in revealing the intracellular off-pathway aggregation processes competing with productive protein folding. These misfolding processes slowed the initial productivity of the biotechnology industry. The article below describes the scientific origin, context, and sociology that supported these advances in protein biochemistry, protein expression, and virus assembly. The cooperation and collaboration that was integral to both the Laboratory for Molecular Biology culture and phage genetics fields were key to these endeavors.

Ompk36 disruption and expression of down-regulated blaKPC-2 in Klebsiella pneumoniae clinical isolates carbapenem resistant

This work investigated the resistance mechanisms that produce different susceptibility profiles in Klebsiella pneumoniae clinical isolates. The genes that encode the main porins in K. pneumoniae, ompK35 and ompK36, and several beta-lactamase genes were analyzed. The expression of these genes was evaluated by quantitative real time PCR (polymerase chain reaction) with reverse transcriptase (RT-qPCR). SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) was performed to analyze the outer membrane proteins. The analysis of the ompK36 genetic environment found the presence of the IS903 insertion sequence interrupting this gene in the ertapenem resistant isolate (KPN133). The blaKPC-2 gene showed down-regulated expression in both isolates.

Protease activities of extracts from kiwi of various colors and their use as a tenderizer in cured pork loins

The aim of this study was to evaluate protease activities of kiwi extracts with various colors (red, green, gold) and determine physicochemical and textural properties of pork loins added with such kiwi extracts during storage at 8°C for 24 h. Green and gold kiwi extracts exhibited higher protease activities than red kiwi extract using casein as a substrate. Pork loin was injected with 5% or 10% of red, green, or gold kiwi extract. Shear force were was decreased when kiwi extracts were added. When 10% of green or gold kiwi extract was added to pork loin, the tenderness increased with increased incubation time, which was confirmed by myosin heavy chain (MHC) band in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). These results indicate that green and gold kiwi extract could be used as a tenderizer for though meat through proteolytic activities of enzymes contained in kiwi. Novelty impact statement Green and gold kiwi extracts exhibited higher protease activities than red kiwi extract. When 10% of green and gold kiwi extracts were added to pork loin, the tenderness increased with incubation time. This study demonstrates that green and gold kiwi extracts could be used as a meat tenderizer without affecting the physicochemical properties of meat products.

Evaluation of the effect of sage seed gum (Salvia macrosiphon) conjugation on physicochemical and antimicrobial properties of egg white protein

SummarySage seed gum (SSG), a galactomannan hydrocolloid, possesses excellent thickening, stabilising and antimicrobial properties. This study aimed to investigate the effect of SSG conjugation on the functional, antimicrobial and antioxidant properties of EWP. EWP‐SSG conjugates were prepared by dry heat treatment of EWP‐SSG mixture at 60 °C and 79% relative humidity for 3‐, 5‐, 7‐ and 10‐day incubation. Interaction of reducing‐end SSG with amino groups of EWP was confirmed by melanoidin compounds content, free amino groups (FAG), Fourier transform infrared (FT‐IR), sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) and scanning electron microscopy (SEM). The results indicated solubility, foaming capacity, foaming stability, emulsification activity index and emulsification stability index remarkably increased after 5 days of incubation. Conjugation led to enhanced antimicrobial activity in 10‐day conjugated samples, so higher antibacterial activity against Bacillus cereus and Staphylococcus aureus was obtained compared with antibiotic disc. Comparison of emulsions (O/W) stabilised by 5‐day conjugate, EWP‐SSG mixture and unmodified EWP revealed better physical stability behaviour in 5‐day conjugate against ionic, pH and thermal stresses, suggesting its potential application in different food processing conditions. The findings showed dry heat‐treated SSG‐EWP conjugate could enhance the functional, antioxidant and antimicrobial properties of EWP.

Dithiothreitol-induced reassembly of soybean lipophilic protein as a carrier for resveratrol: Preparation, structural characterization, and functional properties

We employed dithiothreitol (DTT) to reassemble soy lipophilic protein (LP) and increased its solubility for encapsulating resveratrol (Res); we subsequently added hydroxypropyl methylcellulose (HPMC) to further stabilize Res. Physicochemical characterization, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and spectral analysis revealed that DTT triggered the breakage and reassembly of the disulfide bond. Consequently, the solubility of LP increased from 38.64 % to 71.49 %, and the number of free sulfhydryl groups increased to 7.84 mol·g−1. Furthermore, the encapsulation efficiency and structure of reassembled LP nanoparticles loaded with Res were found to be closely related to the DTT concentration used for induction. When HPMC was added, the LP-Res complex demonstrated spontaneous self-assembly, and the pH and temperature stability of the Res in the nanoparticles improved. An in vitro digestion simulation revealed that the reassembled LP was an efficient carrier for Res delivery. Particularly, HPMC improved the bioavailability and sustained release of Res.

Improving the antithrombogenicity of nylon monofilament for surgical suture by coating a protease from Euphorbia resinifera latex

This study developed a method to improve the antithrombogenicity of a blue nylon monofilament suture (USP 3/0) by coating a plant serine protease purified from Euphorbia resinifera latex (EuRP-61) onto the surface of nylon using a combination of dipping and ultrasonication methods. EuRP-61 was a selected protease because the enzyme had been previously reported to have antithrombotic and anticoagulant activities, high stability and biological safety. Analyses of the fibrinogenolytic and fibrinolytic activities using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and microwell plate assays revealed that the proper concentration of protease was 1 mg/ml for each coating of 20 strips of 10-mm nylon. The enzyme-coated nylon was disinfected by ethylene oxide (EO) sterilization. After EO sterilization, the enzyme-coated nylon presented fibrinogenolytic and fibrinolytic activities. Comparing the enzyme-coated and bare nylon, the enzyme-coated nylon prevented the human fibrin clot from adhering to its surface. In contrast, the fibrin clot stuck and was unable to be removed from the surface of the bare nylon. The antithrombogenicity of the protease-coated nylon evaluated in this study suggests its potential application as a surgical suture, especially for vessel anastomosis.

Safe production of Aspergillus terreus xylanase from Ricinus communis: gene identification, molecular docking, characterization, production of xylooligosaccharides, and its biological activities

BackgroundThe production of industrial enzymes such as xylanase using sufficient cost-effective substrates from potent microorganisms is considered economically feasible. Studies have reported castor cake (Ricinus communis) as the most potent and inexpensive alternative carbon source for production of xylanase C by using Aspergillus terreus (A. terreus). ResultsA. terreus strain RGS Eg-NRC, a local isolate from agro-wastes, was first identified by sequencing the internal transcribed spacer region of a nuclear DNA encoding gene cluster deposited in GenBank (accession number MW282328). Before optimization of xylanase production, A. terreus produced 20.23 U/g of xylanase after 7 days using castor cake as a substrate in a solid-state fermentation (SSF) system that was employed to achieve ricin detoxification and stimulate xylanase production. Physicochemical parameters for the production of xylanase were optimized by using a one-variable-at-a-time approach and two statistical methods (two-level Plackett–Burman design and central composite design, CCD). The maximum xylanase yield after optimization was increased by 12.1-fold (245 U/g). A 60–70% saturation of ammonium sulfate resulted in partially purified xylanase with a specific activity of 3.9 IU/mg protein. At 60 °C and pH 6, the partially purified xylanase had the highest activity, and the activation energy (Ea) was 23.919 kJmol. Subsequently, antioxidant capacity and cytotoxicity tests in normal Ehrlich ascites carcinoma human cells demonstrated xylooligosaccharides produced by the xylanase degradation of xylan as a potent antioxidant and moderate antitumor agent. Further investigations with sodium dodecyl sulfate polyacrylamide gel electrophoresis then determined the molecular weight of partially purified xylanase C to be 36 kDa. Based on the conserved regions, observations revealed that xylanase C belonged to the glycosyl hydrolase family 10. Next, the xylanase-encoding gene (xynC), which has an open reading frame of 981 bp and encodes a protein with 326 amino acids, was isolated, sequenced, and submitted to the NCBI GenBank database (accession number LC595779.1). Molecular docking analysis finally revealed that Glu156, Glu262, and Lys75 residues were involved in the substrate-binding and protein-ligand interaction site of modeled xylanase, with a binding affinity of −8.7 kcal. mol−1. ConclusionThe high production of safe and efficient xylanase could be achieved using economical materials such as Ricinus communis.

High-level ertapenem resistance in Klebsiella pneumoniae is due to RamA downregulation of ompK35 through micF

An ertapenem-resistant Klebsiella pneumoniae clinical isolate (KP20) without carbapenemase and negative for the efflux pump inhibition test was resistant to ertapenem at a high level [minimum inhibitory concentration (MIC)=64 mg/L] but susceptible to meropenem and imipenem. Second-generation sequencing was performed and a termination mutation was found in ramR. Complementation of ramR in KP20 reduced the ertapenem MIC by 128 times (from 64 mg/L to 0.5 mg/L). Overexpression of ramA and loss of OmpK35 were discovered in strain KP20 by quantitative reverse transcription PCR (RT-qPCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Furthermore, ramA deletion in strain KP20 resulted in a 128-fold decrease in the MIC of ertapenem (from 64 mg/L to 0.5 mg/L), and expression of OmpK35 was observed in KP20ΔramA by SDS-PAGE. Complementation of ramA in KP20ΔramA led to a 45.45-fold downregulation of ompK35. Complementation of ompK35 in KP20 could restore susceptibility to ertapenem (MIC reduced from 64 mg/L to 0.25 mg/L). Furthermore, results of the electrophoretic mobility shift assay showed that RamA could bind to the promoter of micF. These results showed that the termination mutation in ramR resulted in overexpression of ramA causing loss of OmpK35 expression through upregulation of micF, revealing the mechanism of ertapenem resistance only in K. pneumoniae.

Development and characterization of wheat–Aegilops kotschyi 1Uk(1A) substitution line with positive dough quality parameters

Exploring novel high molecular weight glutenin subunits (HMW-GSs) from wild related species is a strategy to improve wheat processing quality. The objective of the present investigation was to identify the chromosomes of the wheat-alien introgression line N124, derived from the hybridization between Triticum aestivum with Aegilops kotschyi, and characterize the effects on quality-related traits. Fluorescence in situ hybridization karyotypes showed that N124 is a disomic 1Uk(1A) substitution line. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high-performance liquid chromatography verified N124 expressed two HMW-GSs of the Ae. kotschyi parent. PacBio RNA sequencing and phylogenetic analysis confirmed that the two HMW-GSs were Ukx and Uky. Compared to the wheat parent, the substitution line had no obvious agronomic defects except fewer grains per spike but improved several major quality parameters. It can be served as a donor or bridge material for wheat quality improvement.

Enhancing SDS-PAGE Detection of Dilute Extracellular Polystyrene Degrading Enzymes Expressed by Bacillus megaterium Strain via Centrifugal Freeze Concentration Method

This study aims to enhance the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) detection and visualization of extracellular polystyrene degrading enzyme expressed by Bacillus megaterium as a result of utilizing polystyrene as a sole carbon source. Dilute polystyrene degrading enzyme expressed in the culture supernatant could not be directly visualized from SDS-PAGE stained with colloidal Coomassie blue stain as it falls below its sensitivity or detection limit level. To overcome this problem, the crude enzyme extract was concentrated via the centrifugal freeze concentration method (cryoconcentration). The extracellular enzyme profiles were determined by SDS-PAGE. The SDS-PAGE analysis of the concentrated enzyme extract revealed 6 visible protein bands ranging in size from 10 to 60 kDa. Three distinct protein bands at approximately 20, 45 and 60 kDa were observed to be highly expressed in the culture supernatant. Polystyrene degrading enzyme extracted from the culture supernatant was closely related to several polymer degrading enzymes which suggest that the extracted enzyme from Bacillus megaterium also belongs to the hydrolase’s enzyme family. GC-MS analysis of the extracted samples significantly contains benzene derivatives due to the breaking down of the long-chain aromatic hydrocarbon polymer. Hydroxylation of the aromatic ring formed phenolic substrate took place which suggests being facilitated by intracellular enzyme hydroxylases. Hence, the biodegradation of polystyrene by Bacillus megaterium was believed to incorporate both intracellular and extracellular enzymes.&#x0D; HIGHLIGHTS&#x0D; &#x0D; Bacillus megaterium strain was first reported to contribute to polystyrene biodegradation&#x0D; Polystyrene degrading enzyme expressed by Bacillus megaterium was detected through SDS-PAGE&#x0D; Cryoconcentration enhanced SDS-PAGE protein detection&#x0D; The protein bands were most distinct visualized at approximately 20, 45 and 60 kDa by SDS-PAGE&#x0D; GC-MS analysis detected benzene derivatives which confirmed enzymatic degradation activity&#x0D; &#x0D; GRAPHICAL ABSTRACT

Regulation of Amylose Content by Single Mutations at an Active Site in the Wx-B1 Gene in a Tetraploid Wheat Mutant.

The granule-bound starch synthase I (GBSSI) encoded by the waxy gene is responsible for amylose synthesis in the endosperm of wheat grains. In the present study, a novel Wx-B1 null mutant line, M3-415, was identified from an ethyl methanesulfonate-mutagenized population of Chinese tetraploid wheat landrace Jianyangailanmai (LM47). The gene sequence indicated that the mutated Wx-B1 encoded a complete protein; this protein was incompatible with the protein profile obtained using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which showed the lack of Wx-B1 protein in the mutant line. The prediction of the protein structure showed an amino acid substitution (G470D) at the edge of the ADPG binding pocket, which might affect the binding of Wx-B1 to starch granules. Site-directed mutagenesis was further performed to artificially change the amino acid at the sequence position 469 from alanine (A) to threonine (T) (A469T) downstream of the mutated site in M3-415. Our results indicated that a single amino acid mutation in Wx-B1 reduces its activity by impairing its starch-binding capacity. The present study is the first to report the novel mechanism underlying Wx-1 deletion in wheat; moreover, it provided new insights into the inactivation of the waxy gene and revealed that fine regulation of wheat amylose content is possible by modifying the GBSSI activity.