Plastic additives as a new threat to the global environment: Research status, remediation strategies and perspectives.

Discharge or leaching of plastic additives, which are an essential part of the plastic production process, can lead to environmental pollution with serious impacts on human and ecosystem health. Recently, the emission of plastic additives is increasing dramatically, but its pollution condition has not received enough attention. Meanwhile, the effective treatment strategy of plastic additive pollution is lack of systematic introduction. Therefore, it is crucial to analyze the harm and pollution status of plastic additives and explore effective pollution control strategies. This paper reviews the latest research progress on additives in plastics, describes the effects of their migration into packaged products and leaching into the environment, presents the hazards of four major classes of plastic additives (i.e., plasticizers, flame retardants, stabilizers, and antimicrobials), summarizes the existing abiotic/biotic strategies for accelerated the remediation of additives, and finally provides perspectives on future research on the removal of plastic additives. To the best of our knowledge, this is the first review that systematically analyzes strategies for the treatment of plastic additives. The study of these strategies could (i) provide feasible, cost-effective abiotic method for the removal of plastic additives, (ii) further enrich the current knowledge on plastic additive bioremediation, and (iii) present application and future development of plants, invertebrates and machine learning in plastic additive remediation.

The Screening and Identification of a Dextranase-Secreting Marine Actinmycete Saccharomonospora sp. K1 and Study of Its Enzymatic Characteristics.

In this study, an actinomycete was isolated from sea mud. The strain K1 was identified as Saccharomonospora sp. by 16S rDNA. The optimal enzyme production temperature, initial pH, time, and concentration of the inducer of this actinomycete strain K1 were 37 °C, pH 8.5, 72 h, and 2% dextran T20 of medium, respectively. Dextranase from strain K1 exhibited maximum activity at 8.5 pH and 50 °C. The molecular weight of the enzyme was <10 kDa. The metal ions Sr2+ and K+ enhanced its activity, whereas Fe3+ and Co2+ had an opposite effect. In addition, high-performance liquid chromatography showed that dextran was mainly hydrolyzed to isomaltoheptose and isomaltopentaose. Also, it could effectively remove biofilms of Streptococcus mutans. Furthermore, it could be used to prepare porous sweet potato starch. This is the first time a dextranase-producing actinomycete strain was screened from marine samples.

Aptamer-Based Sensor for Rapid and Sensitive Detection of Ofloxacin in Meat Products.

Ofloxacin (OFL) is widely used in animal husbandry and aquaculture due to its low price and broad spectrum of bacterial inhibition, etc. However, it is difficult to degrade and is retained in animal-derived food products, which are hazardous to human health. In this study, a simple and efficient method was developed for the detection of OFL residues in meat products. OFL coupled with amino magnetic beads by an amination reaction was used as a stationary phase. Aptamer AWO-06, which showed high affinity and specificity for OFL, was screened using the exponential enrichment (SELEX) technique. A fluorescent biosensor was developed by using AWO-06 as a probe and graphene oxide (GO) as a quencher. The OFL detection results could be obtained within 6 min. The linear range was observed in the range of 10-300 nM of the OFL concentration, and the limit of the detection of the sensor was 0.61 nM. Furthermore, the biosensor was stored at room temperature for more than 2 months, and its performance did not change. The developed biosensor in this study is easy to operate and rapid in response, and it is suitable for on-site detection. This study provided a novel method for the detection of OFL residues in meat products.

Dextranase Production Using Marine Microbacterium sp. XD05 and Its Application.

Dextranase, also known as glucanase, is a hydrolase enzyme that cleaves α-1,6 glycosidic bonds. In this study, a dextranase-producing strain was isolated from water samples of the Qingdao Sea and identified as Microbacterium sp. This strain was further evaluated for growth conditions, enzyme-producing conditions, enzymatic properties, and hydrolysates. Yeast extract and sodium chloride were found to be the most suitable carbon and nitrogen sources for strain growth, while sucrose and ammonium sodium were found to be suitable carbon and nitrogen sources for fermentation. The optimal pH was 7.5, with a culture temperature of 40 °C and a culture time of 48 h. Dextranase produced by strain XD05 showed good thermal stability at 40 °C by retaining more than 70% relative enzyme activity. The pH stability of the enzyme was better under a weak alkaline condition (pH 6.0-8.0). The addition of NH4+ increased dextranase activity, while Co2+ and Mn2+ had slight inhibitory effects on dextranase activity. In addition, high-performance liquid chromatography showed that dextran is mainly hydrolyzed to maltoheptanose, maltohexanose, maltopentose, and maltootriose. Moreover, it can form corn porous starch. Dextranase can be used in various fields, such as food, medicine, chemical industry, cosmetics, and agriculture.

Multiple Bioactivities of Peptides from Hydrolyzed Misgurnus anguillicaudatus.

Misgurnus anguillicaudatus (loach) is a widely distributed benthic fish in Asia. In this study, the alkaline protease was used to hydrolyze loach, and the hydrolysate products of different molecular weights were obtained by membrane separation. In vitro antioxidant assays showed that the <3 kDa fraction (SLH-1) exhibited the strongest antioxidant activity (DPPH, hydroxyl radical and superoxide radical scavenging ability, and reducing power), while SLH-1 was purified by gel filtration chromatography, and peptide sequences were identified by LC-MS/MS. A total of six peptides with antioxidant activity were identified, namely SERDPSNIKWGDAGAQ (D-1), TVDGPSGKLWR (D-2), NDHFVKL (D-3), AFRVPTP (D-4), DAGAGIAL (D-5), and VSVVDLTVR (D-6). In vitro angiotensin-converting enzyme (ACE) inhibition assay and pancreatic cholesterol esterase (CE) inhibition assay, peptide D-4 (IC50 95.07 µg/mL, 0.12 mM) and D-2 inhibited ACE, and peptide D-2 (IC50 3.19 mg/mL, 2.62 mM), D-3, and D-6 acted as pancreatic CE inhibitors. The inhibitory mechanisms of these peptides were investigated by molecular docking. The results showed that the peptides acted by binding to the key amino acids of the catalytic domain of enzymes. These results could provide the basis for the nutritional value and promote the type of healthy products from hydrolyzed loach.

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.

Optimal Secretory Expression of Acetaldehyde Dehydrogenase from Issatchenkia terricola in Bacillus subtilis through a Combined Strategy.

Acetaldehyde dehydrogenases are potential enzyme preparations that can be used to detoxify acetaldehyde and other exogenous aldehydes from pharmaceuticals, food, and biofuel production. In this study, we enhanced the expression of acetaldehyde dehydrogenase sourced from Issatchenkia terricola (istALDH) in Bacillus subtilis using a combinatorial strategy for the optimization of signal peptides, promoters, and growth conditions. First, a library of various signal peptides was constructed to identify the optimal signal peptides for efficient istALDH secretion. The signal peptide yqzG achieved the highest extracellular istALDH activity (204.85 ± 3.31 U/mL). Second, the aprE promoter was replaced by a constitutive promoter (i.e., P43) and an inducible promoter (i.e., Pglv), resulting in 12.40% and 19.97% enhanced istALDH, respectively. Furthermore, the tandem promoter P43-Pglv provided a better performance, resulting in 30.96% enhanced istALDH activity. Third, the production of istALDH was optimized by testing one factor at a time. Physical parameters were optimized including the inducer (e.g., maltose) concentrations, incubation temperatures, and inoculation amounts, and the results were 2.0%, 35 ∘C, and 2.0%, respectively. The optimized medium results were 2.0% glucose, 1.5% peptone, 2.5% yeast extract, 1% NaCl, and 0.5% (NH4)2SO4. The extracellular istALDH activity was 331.19 ± 4.19 U/mL, yielding the highest production reported in the literature to date.

Screening and Characteristics of Marine Bacillus velezensis Z-1 Protease and Its Application of Enzymatic Hydrolysis of Mussels to Prepare Antioxidant Active Substances.

Bacillus velezensis is a type of microorganism that is beneficial to humans and animals. In this work, a protease-producing B. velezensis strain Z-1 was screened from sludge in the sea area near Qingdao (deposit number CGMCC No. 25059). The response surface methodology was used to analyze protease production, and the optimal temperature was 37.09 °C and pH 7.73 with the addition of 0.42% NaCl, resulting in maximum protease production of 17.64 U/mL. The optimum reaction temperature and pH of the protease of strain Z-1 were 60 °C and 9.0, respectively. The protease had good temperature and pH stability, and good stability in solvents such as methanol, ethanol and Tween 80. Ammonium, NH4+,and Mn2+ significantly promoted enzyme activity, while Zn2+ significantly inhibited the enzyme activity. The protease produced by strain Z-1 was used for the enzymolysis of mussel meat. The mussel hydrolysate exhibited good antioxidant function, with a DPPH free radical removal rate of 75.3%, a hydroxyl free radical removal rate of 75.9%, and a superoxide anion removal rate of 84.4%. This study provides a reference for the application of B. velez protease and the diverse processing applications of mussel meat.

Alkalic dextranase produced by marine bacterium Cellulosimicrobium sp. PX02 and its application.

The enzyme dextranase is widely used in the sugar and food industries, as well as in the medical field. Most land-derived dextranases are produced by fungi and have the disadvantages of long production cycles, low tolerance to environmental conditions, and low safety. The use of marine bacteria to produce dextranases may overcome these problems. In this study, a dextranase-producing bacterium was isolated from the Rizhao seacoast of Shandong, China. The bacterium, denoted as PX02, was identified as Cellulosimicrobium sp. and its growing conditions and the production and properties of its dextranase were investigated. The dextranase had a molecular weight of approximately 40 kDa, maximum activity at 40°C and pH 7.5, with a stability range of up to 45°C and pH 7.0-9.0. High-performance liquid chromatography showed that the dextranase hydrolyzed dextranT20 to isomaltotriose, maltopentaose, and isomaltooligosaccharides. Hydrolysis by dextranase produced excellent antioxidant effects, suggesting its potential use in the health food industry. Investigation of the action of the dextranase on Streptococcus mutans biofilm and scanning electron microscopy showed that it to be effective both for removing and inhibiting the formation of biofilms, suggesting its potential application in the dental industry.

The Two Classic Pb2+ -Selective DNAzymes Are Related: Rational Evolution for Understanding Metal Selectivity.

In 1994, the first DNAzyme named GR5 was reported, which specifically requires Pb2+ for its RNA cleavage activity. Three years later, the 8-17 DNAzyme was isolated. The 8-17 DNAzyme and the related 17E DNAzyme are also most active with Pb2+ , although other divalent metals can work as well. GR5 and 17E have the same substrate sequence, and their catalytic loops in the enzyme strands also have a few similar and conserved nucleotides. Considering these, we hypothesized that 17E might be a special form of GR5. To test this hypothesis, we performed systematic rational evolution experiments to gradually mutate GR5 toward 17E. By using the activity ratio in the presence of Pb2+ and Mg2+ for defining these two DNAzymes, the critical nucleotide was identified to be T12 in 17E for metal specificity. In addition, G9 in GR5 is a position not found in most 17E or 8-17 DNAzymes, and G9 needs to be added to rescue GR5 activity if T12 becomes a cytosine. This study highlights the links between these two classic and widely used DNAzymes, and offers new insight into the sequence-activity relationship related to metal selectivity.

Expression, purification and characterization of a cold-adapted dextranase from marine bacteria and its ability to remove dental plaque.

Dental plaque is a high-incidence health concern, and it is caused by Streptococcus mutans. Dextranase can specifically hydrolyze ɑ-1,6-glycosidic linkages in dextran. It is commonly used in the sugar industry, in the production of plasma substitutes, and the treatment and prevention of dental plaque. In this research work, we successfully cloned and expressed a cold-adapted dextranase from marine bacteria Catenovulum sp. DP03 in Escherichia coli. The recombinant dextranase named Cadex2870 contained a 2511 bp intact open reading frame and encoded 836 amino acids. The expression condition of recombinant strain was 0.1 mM isopropylthio-galactoside (IPTG), and the reduced temperature was 16 °C. The purified enzyme activity was 16.2 U/mg. The optimal temperature and pH of Cadex2870 were 45 °C and pH 8, and it also had catalytic activity at 0 °C. The hydrolysates of Cadex2870 hydrolysis Dextran T70 are maltose, maltotetraose, maltopentose, maltoheptaose and higher molecular weight maltooligosaccharides. Interestingly, 0.5% sodium benzoate, 2% xylitol, 0.5% sodium fluoride, 5% propanediol, 5% glycerin and 2% sorbitol can enhance stability Cadex2870, which are additives in mouthwashes. Additionally, Cadex2870 reduced the formation of dental plaque and effectively degraded formed plaque. Therefore, Cadex2870 shows great promise in commercial applications.

Sensitivity of a classic DNAzyme for Pb2+ modulated by cations, anions and buffers.

GR5 is the first reported DNAzyme, which has RNA cleavage activity in the presence of Pb2+. Due to its excellent selectivity, GR5 has been a popular DNAzyme for developing biosensors for Pb2+. The activity of DNAzymes is often affected by pH and salt, which may in turn affect the sensitivity of related sensors. Although pH can be readily controlled by using a buffer, the effect of salt is more complex. To have a systematic understanding, we herein measured the cleavage activity of GR5 in various concentrations of Na+ and Mg2+. Both metals inhibited the DNAzyme with Pb2+, and the inhibition constants were 1.8 mM Mg2+ and 33.4 mM Na+. For anions, F- inhibited GR5 more strongly than Br-, while Cl- was the least inhibiting anion, which was consistent with the solubility of their lead salts. The reaction can work similarly in many Good's buffers, while phosphate buffer should be avoided. Finally, GR5 is an optimal sequence based on the truncation and elongation studies. This study reveals important solution conditions that should be considered when designing and testing related biosensors for metal detection.

Selection of DNAzymes for Sensing Aquatic Bacteria: Vibrio Anguillarum.

Vibrio anguillarum is a bacterial pathogen that causes serious damage to aquatic fish, and its rapid detection and prevention are critical. DNAzymes are DNA-based catalysts with excellent stability. In this study, in vitro selection of DNAzymes was performed using the crude extracellular matrix (CEM) of V. Anguillarum as the target. Different from previous selections targeting bacterial CEM, this work used an unmodified DNA library, allowing easier adoption of the technology. After seven rounds of selection, a DNAzyme named VAE-2 with high activity and specificity was obtained. It showed the highest activity toward V. Anguillarum among eight types of tested bacterial strains. Polyvalent metal ions are needed for its activity. Protease treatment of the CEM and filtration studies indicated that the target is a protein with a molecular weight between 50 k and 100 k Da. A fluorescent biosensor was designed for V. anguillarum with a detection limit down to 4000 cfu/mL, and detection was demonstrated for real fish tissue and feeding water samples. Being the first work of DNAzyme-based sensing of aquatic bacteria, this study indicates that unmodified DNA can be used for targeting bacterial CEM, and it provides a new framework for developing other RNA-cleaving DNAzymes for rapid detection of pathogenic bacteria and water pollution.

Regenerative NanoOctopus Based on Multivalent-Aptamer-Functionalized Magnetic Microparticles for Effective Cell Capture in Whole Blood.

Isolation of specific rare cell subtypes from whole blood is critical in cellular analysis and important in basic and clinical research. Traditional immunomagnetic cell capture suffers from suboptimal sensitivity, specificity, and time- and cost-effectiveness. Mimicking the features of octopuses, a device termed a "NanoOctopus" was developed for cancer cell isolation in whole blood. The device consists of long multimerized aptamer DNA strands, or tentacle DNA, immobilized on magnetic microparticle surfaces. Their ultrahigh sensitivity and specificity are attributed to multivalent binding of the tentacle DNA to cell receptors without steric hindrance. The simple, quick, and noninvasive capture and release of the target cells allows for extensive downstream cellular and molecular analysis, and the time- and cost-effectiveness of fabrication and regeneration of the devices makes them attractive for industrial manufacture.

Recognition of Helicobacter pylori by protein-targeting aptamers.

BACKGROUND: Helicobacter pylori (H pylori) is a disease-causing pathogen capable of surviving under acidic conditions of the human stomach. Almost half of the world's population is infected with H pylori, with gastric cancer being the most unsatisfactory prognosis. Although H pylori has been discovered 30 years ago, the effective treatment and elimination of H pylori continue to be problematic. MATERIALS AND METHODS: In our study, we screened nucleic acid aptamers using H pylori surface recombinant antigens as targets. Trypsin was used for separating aptamers that were bound to proteins. Following nine rounds of screening, we performed sequence similarity analyses to assess whether the aptamers can recognize the target protein. Two sequences with desirable recognition ability were selected for affinity detection. Aptamer Hp4 with the strongest binding ability to the H pylori surface recombinant antigen was chosen. After optimization of the binding conditions, we conducted specificity tests for Hp4 using Escherichia coli, Staphylococcus aureus, Vibrioanguillarum, and H pylori. RESULTS: The data indicated that the aptamer Hp4 had an equilibrium dissociation constant (Kd ) of 26.48 ± 5.72 nmol/L to the target protein. This aptamer was capable of exclusively detecting H pylori cells, without displaying any specificity for other bacteria. CONCLUSIONS: We obtained a high-affinity aptamer for H pylori, which is expected to serve as a new molecular probe for detection of H pylori.

The Marine Catenovulum agarivorans MNH15 and Dextranase: Removing Dental Plaque.

Dextranase, a hydrolase that specifically hydrolyzes α-1,6-glucosidic bonds, has been used in the pharmaceutical, food, and biotechnology industries. In this study, the strain of Catenovulum agarivorans MNH15 was screened from marine samples. When the temperature, initial pH, NaCl concentration, and inducer concentration were 30 °C, 8.0, 5 g/L, and 8 g/L, respectively, it yielded more dextranase. The molecular weight of the dextranase was approximately 110 kDa. The maximum enzyme activity was achieved at 40 °C and a pH of 8.0. The enzyme was stable at 30 °C and a pH of 5-9. The metal ion Sr2+ enhanced its activity, whereas NH4+, Co2+, Cu2+, and Li+ had the opposite effect. The dextranase effectively inhibited the formation of biofilm by Streptococcus mutans. Moreover, sodium fluoride, xylitol, and sodium benzoate, all used in dental care products, had no significant effect on dextranase activity. In addition, high-performance liquid chromatography (HPLC) showed that dextran was mainly hydrolyzed to glucose, maltose, and maltoheptaose. The results indicated that dextranase has high application potential in dental products such as toothpaste and mouthwash.

Trypsin enhances aptamer screening: A novel method for targeting proteins.

A novel screening method for protein aptamer selection was developed in this study. Aptamers with high affinity and specificity to the surface recombinant antigen of Helicobacter pylori (HP-Ag) and to tumor markers carcinoembryonic antigen (CEA), cancer antigen 125 (CA125) and cancer antigen 19-9(CA19-9) were screened using trypsin enhanced screening method. Briefly, the target proteins above were immobilized onto 96-well polystyrene plates and incubated with a single-stranded DNA (ssDNA) library for aptamer selection. Then, trypsin was introduced to digest the proteins and obtain ssDNA that bound to the target proteins with high specificity. The concentration of ssDNA that shed from protein-ssDNA complexes was detected. After sequencing, the enrichment of target-specific aptamers was monitored and the affinity of each aptamer was analyzed. Urea, which has been reported in other article, was used to compare with trypsin. The results revealed that trypsin was more effective than urea for protein aptamer selection. The protocol used in this study provided a novel method for generating aptamers.

Purification and Characterization of a Biofilm-Degradable Dextranase from a Marine Bacterium.

This study evaluated the ability of a dextranase from a marine bacterium Catenovulum sp. (Cadex) to impede formation of Streptococcus mutans biofilms, a primary pathogen of dental caries, one of the most common human infectious diseases. Cadex was purified 29.6-fold and had a specific activity of 2309 U/mg protein and molecular weight of 75 kDa. Cadex showed maximum activity at pH 8.0 and 40 °C and was stable at temperatures under 30 °C and at pH ranging from 5.0 to 11.0. A metal ion and chemical dependency study showed that Mn2+ and Sr2+ exerted positive effects on Cadex, whereas Cu2+, Fe3+, Zn2+, Cd2+, Ni2+, and Co2+ functioned as inhibitors. Several teeth rinsing product reagents, including carboxybenzene, ethanol, sodium fluoride, and xylitol were found to have no effects on Cadex activity. A substrate specificity study showed that Cadex specifically cleaved the α-1,6 glycosidic bond. Thin layer chromatogram and high-performance liquid chromatography indicated that the main hydrolysis products were isomaltoogligosaccharides. Crystal violet staining and scanning electron microscopy showed that Cadex impeded the formation of S. mutans biofilm to some extent. In conclusion, Cadex from a marine bacterium was shown to be an alkaline and cold-adapted endo-type dextranase suitable for development of a novel marine agent for the treatment of dental caries.

Identification of a highly specific DNA aptamer for Vibrio vulnificus using systematic evolution of ligands by exponential enrichment coupled with asymmetric PCR.

Vibrio vulnificus is an important bacterial pathogen that causes serious infections in fish and is also highly pathogenic to humans. Many effective detection methods targeting this pathogen have previously been designed, but many of these methods are time-consuming, complicated and expensive. Thus, these approaches cannot be widely used by small aqacultural concerns. Although DNA aptamers have been used to detect pathogenic bacteria, these have not been applied to marine bacteria, including V. vulnificus. Therefore, we developed a highly specific DNA aptamer for V. vulnificus detection using systematic evolution of ligands by exponential enrichment (SELEX), coupled with asymmetric PCR. After 13 rounds of cross-selection, we identified a novel DNA aptamer (Vapt2). We evaluated the affinity, specificity and limit of detection (LOD) of this aptamer for V. vulnificus. We found that Vapt2 had a high affinity for V. vulnificus (Kd  = 26.8 ± 5.3 nM) and detected this pathogen at a wide range of concentrations (8-2.0 × 108  cfu/ml). Vapt2 bound to V. vulnificus with high selectivity in the presence of other pathogenic bacteria. Our study increases our knowledge of the possible applications of aptamers with respect to marine bacteria. Moreover, our work might provide a framework for the rapid detection of pathogenic bacteria and water pollution.

Novel Angiotensin-Converting Enzyme-Inhibitory Peptides Obtained from Trichiurus lepturus: Preparation, Identification and Potential Antihypertensive Mechanism.

Peptides possessing antihypertensive attributes via inhibiting the angiotensin-converting enzyme (ACE) were derived through the enzymatic degradation of Trichiurus lepturus (ribbonfish) using alkaline protease. The resulting mixture underwent filtration using centrifugation, ultrafiltration tubes, and Sephadex G-25 gels. Peptides exhibiting ACE-inhibitory properties and DPPH free-radical-scavenging abilities were isolated and subsequently purified via LC/MS-MS, leading to the identification of over 100 peptide components. In silico screening yielded five ACE inhibitory peptides: FAGDDAPR, QGPIGPR, IFPRNPP, AGFAGDDAPR, and GPTGPAGPR. Among these, IFPRNPP and AGFAGDDAPR were found to be allergenic, while FAGDDAPRR, QGPIGPR, and GPTGPAGP showed good ACE-inhibitory effects. IC50 values for the latter peptides were obtained from HUVEC cells: FAGDDAPRR (IC50 = 262.98 µM), QGPIGPR (IC50 = 81.09 µM), and GPTGPAGP (IC50 = 168.11 µM). Peptide constituents derived from ribbonfish proteins effectively modulated ACE activity, thus underscoring their therapeutic potential. Molecular docking and modeling corroborated these findings, emphasizing the utility of functional foods as a promising avenue for the treatment and prevention of hypertension, with potential ancillary health benefits and applications as substitutes for synthetic drugs.