An efficient DNAzyme for the fluorescence detection of Vibrio cholerae.

Vibrio cholerae (Vc) causes cholera disease. Vc contamination is widely found in water and aquatic products, and therefore is a serious food safety concern, especially for the seafood industry. In this paper, we attempted the rapid detection of V. cholerae. Nine rounds of in vitro selection using an unmodified DNA library were successfully performed to find specific DNAzymes of Vc. Their activity was evaluated based on a fluorescence assay and gel electrophoresis. Finally, a DNAzyme (named DVc1) with good activity and specificity with a detection limit of 7.2 × 103 CFU/mL of Vc was selected. A simple biosensor was constructed by immobilizing DVc1 and its substrate in shallow circular wells of a 96-well plate using pullulan polysaccharide and trehalose. When the crude extracellular mixture of Vc was added to the detection wells, the fluorescent signal was observed within 20 min. The sensor effectively detected Vc in aquatic products indicating its simplicity and efficiency. This sensitive DNAzyme sensor can be a rapid onsite Vc detection tool.

Immobilization of Dextranase Obtained from the Marine Cellulosimicrobium sp. Y1 on Nanoparticles: Nano-TiO2 Improving Hydrolysate Properties and Enhancing Reuse.

Dextranase is widely used in sugar production, drug synthesis, material preparation, and biotechnology, among other fields. The immobilization of dextranase using nanomaterials in order to make it reusable, is a hot research topic. In this study, the immobilization of purified dextranase was performed using different nanomaterials. The best results were obtained when dextranase was immobilized on titanium dioxide (TiO2), and a particle size of 30 nm was achieved. The optimum immobilization conditions were pH 7.0, temperature 25 °C, time 1 h, and immobilization agent TiO2. The immobilized materials were characterized using Fourier-transform infrared spectroscopy, X-ray diffractometry, and field emission gun scanning electron microscopy. The optimum temperature and pH of the immobilized dextranase were 30 °C and 7.5, respectively. The activity of the immobilized dextranase was >50% even after 7 times of reuse, and 58% of the enzyme was active even after 7 days of storage at 25 °C, indicating the reproducibility of the immobilized enzyme. The adsorption of dextranase by TiO2 nanoparticles exhibited secondary reaction kinetics. Compared with free dextranase, the hydrolysates of the immobilized dextranase were significantly different, and consisted mainly of isomaltotriose and isomaltotetraose. The highly polymerized isomaltotetraose levels could reach >78.69% of the product after 30 min of enzymatic digestion.

On-site detection of fish furunculosis by combining DNAzyme and carboxyl-functionalized graphene.

Furunculosis, which is caused by Aeromonas salmonicida, can induce septicemia, leading to the rapid death of fishes belonging to Salmonidae, Cyprinidae, and Fuscheridae, and lamprey. Targeting A. salmonicida, five DNAzyme sequences with the highest enrichment rates were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The enrichment rates were 34.78, 23.60, 8.91, 2.89, and 2.34%, respectively. The DNAzyme with the highest activity, named D-AS-2, showed specificity and sensitivity. D-AS-2 was combined with carboxyl-functionalized graphene to construct a biosensor, which showed good fluorescence response to scabies lesion samples. The diagnostic procedure was completed in <2 min and can be used for the on-site diagnosis of fish diseases. A low-cost, rapid, simple, and highly specific biosensor for the diagnosis of furunculosis was established based on DNAzyme and carboxyl-functionalized graphene.

Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability.

Obtaining high-degree polymerized isomaltose is more difficult while achieving better prebiotic effects. We investigated the mutation specificity and enzymatic properties of SP5-Badex, a dextranase from the GH66 family of Bacillus aquimaris SP5, and determined its mutation sites through molecular docking to obtain five mutants, namely E454K, E454G, Y539F, N369F, and Y153N. Among them, Y539F and Y153N exhibited no enzymatic activity, but their hydrolysates included isomaltotetraose (IMO4). The enzymatic activity of E454G was 1.96 U/ml, which was 3.08 times higher than that before mutation. Moreover, 70% of the enzymatic activity could be retained after holding at 45°C for 180 min, which was 40% higher than that of SP5-Badex. Furthermore, its IMO4 content was 5.62% higher than that of SP5-Badex after hydrolysis at 30°C for 180 min. To investigate the effect of different amino acids on the same mutation site, saturation mutation was induced at site Y153, and the results showed that the enzyme activity of Y153W could be increased by 2 times, and some of the enzyme activity could still be retained at 50°C. Moreover, the enzyme activity increased by 50% compared with that of SP5-Badex after holding at 45°C for 180 min, and the IMO4 content of Y153W was approximately 64.97% after hydrolysis at 30°C for 180 min, which increased by approximately 12.47% compared with that of SP5-Badex. This site is hypothesized to rigidly bind to nonpolar (hydrophobic) amino acids to improve the stability of the protein structure, which in turn improves the thermal stability and simultaneously increases the IMO4 yield.

Fast and sensitive graphene oxide-DNAzyme-based biosensor for Vibrio alginolyticus detection.

DNAzymes have been widely and effectively used for the detection of pathogenic bacteria, which pose a serious public health threat. However, the rapid and cost-effective detection of such bacteria remains a major challenge. In this study, we successfully selected Vibrio alginolyticus-specific DNAzymes. The activity of the candidates was assessed via fluorescence intensity and gel electrophoresis. The DNAzyme DT1 had a detection limit of 31 CFU/ml for V. alginolyticus and exhibited high specificity. Graphene oxide (GO) was used to develop a DNAzyme-based fluorescent sensor for the detection of V. alginolyticus, which significantly improved detection performance and shortened the reaction time as little as 10 s. The proposed method was then validated using crab, shrimp, fish, clam, and oyster samples. This study thus provides a new method for the rapid and sensitive detection of V. alginolyticus.