Applied Biochemistry and Microbiology最新文献

In recent years, the prevalence of intestinal diseases, particularly Vibrio cholerae, has been on the rise, leading to numerous epidemics and pandemics. To effectively manage and treat this disease, the development of a simple and fast diagnostic method is crucial. This research aims to design a colorimetric V. cholerae aptasensor using gold nanoparticles (GNPs) and the Localized Surface Plasmon Resonance (LSPR) method. A specific DNA aptamer was selected and confirmed through bioinformatics tools and docking software for binding to the surface of outer membrane protein U (OMP U) in V. cholerae. The aptamer sequence was synthesized, amplified, and used to coat the surface of GNPs. The effectiveness of the designed aptamer was evaluated for bacterial detection by observing the aggregation or non-aggregation of GNPs in the presence of NaCl. The colorimetric method and LSPR spectrum were used for final evaluation. The results of molecular docking indicated that the selected aptamer exhibited appropriate sensitivity and specificity in detecting the surface protein OMP U of the target bacteria in suspension. Additionally, the aptamer successfully detected bacteria with a detection limit of 10³ CFU/mL within 80 min. It displayed a significant optical shift of 8 nm compared to the absorption peak of GNPs, indicating successful detection of V. cholerae. The designed aptasensor demonstrated high sensitivity and specificity for V. cholerae and the potential for the development of rapid diagnostic kits to detect this bacterium.

The aerobic and rod-shaped FO1 strain of iron-oxidizing bacteria was isolated from electroplating wastewater sludge using colloidal silica plates. Bacterial growth occurred at 25–30°C (optimum temperature, 27°C), pH 2.0–3.5, and in the presence of 1.0–4.0% NaCl. The X-ray diffraction analysis identified the precipitates that were formed during cell growth as jarosite. A biochemical analysis, including the assessments of the whole-cell fatty acids, quinone, and DNA G+C contents, revealed that FO1 was closely related to the genus Acidihalobacter. The 16S rRNA sequence of the FO1 gene was similar to those of the Acidihalobacter prosperus DSM 5130 (97% similarity), Ah. aeolianus DSM 14174 (100%), and Ah. yilgarnensis DSM 105917 (100%) strains. Antibiotic sensitivity tests of strain FO1 revealed that it was tolerant for gentamicin, kanamycin, and tetracycline, whereas it was sensitive to ampicillin and chloramphenicol. Moreover, strain FO1 was able to grow in a microbial electrolysis cell with continuous reduction of Fe³⁺ to Fe²⁺ on the cathode, albeit with a slight inhibition of cell growth. This study provides information regarding the genetics, bioleaching of metals, and electrochemical cultivation of halotolerant iron-oxidizing bacteria in the genus Acidihalobacter.

Conventional gene cloning methods always require tedious processes confined by PCR conditions and restriction sites, which might result in a large number of redundant fragments in the constructs. These shortages can be overcome by using DNA recombination methods. Homologous recombination in Escherichia coli cells was used to develop positive/negative cassette for phage λ red operon recombination. In the bi-functional cassette, the Ala294Gly α-subunit mutant of phenylalanyl-tRNA synthetase (mPheS) was fused with the kanamycin-resistant (KanR) cassette under the control of the promoter of the E. coli gene NEOKAN as a single open reading frame (ORF) by PCR. KanR was utilized as a positive selection marker, while the mutant gene mPheS served as a negative selection marker. Two-step red-mediated recombination was used to replace the third exon of TMEM18 in the BAC clone RP23-25C13 with eGFP. Initially, the third TMEM18 exon in the BAC was replaced with the NEOKAN-mPheS-KanR cassette, and 40% of the clones on the positive selection plate (LB plate with kanamycin) were positive. The second step involved replacing the NEOKAN-mPheS-KanR cassette with eGFP in the modified BAC, and 60% of the clones on the negative plate (p-Cl-phe plate) were positive. Following the two steps, eGFP was used to replace the third exon of TMEM18 in the BAC. The NEOKAN-mPheS-KanR could be a promising choice as a positive/negative selection cassette for red recombination to establish constructs and make gene manipulation on BAC more applicable and effortless.

Fungi estimated to be more than 5.1 million species around the globe represent an invaluable source of innumerable natural products that can be screened for various bioactivities. They have provided society with some of the largest medical breakthroughs in modern era. Repeated isolation of known compounds has been a tough challenge for natural products research. Fungi especially microfungi, have proved themselves as potent producers of novel compounds to adapt themselves to diverse conditions including extreme habitats. This review highlights the importance of microfungal strains in the biosynthesis of polyketides, their biosynthetic pathways, isolation methods, structure elucidation and further their significance focusing on activity against various pathogenic strains, human cancer cell lines and other biological entities.

In the modern era of human progress and industrialization, various agricultural and industrial activities lead to the accumulation of excessive hazardous substances in the environment. These wastes are continuously being concentrated in agricultural soil or water, thereby affecting and threatening the lives of both flora and fauna. To treat these toxic substances, various physico-chemical practices are performed, which are ultimately associated with the production of other toxic chemicals in the environment. Some of the toxic products formed are recalcitrant and inert which further causes hindrances in the proper removal of contaminants from different pollutant sites. However, various biological approaches are being used nowadays for the remediation of these pollutants from the environment. Microbial bioremediation and phytoremediation represent such environment-friendly approaches that have been employed in the degradation of many of these chemicals. Among the microbes, endophytes are now considered a promising means of the remediation of different pollutants from their natural environments. Therefore, the review focuses on the use of endophytes in the treatment of different kinds of pollutants and highlights the potential of these microbial tools in the removal of heavy metals, plastic, and other contaminants from both terrestrial and aquatic habitats.

Garlic suffers from frequent pathogenic diseases, seriously affecting its yield and quality. Biosurfactants have been reported with high antimicrobial activity and wide antimicrobial spectrum. Biosurfactant-producing bacteria existing in garlic farmland soil are expected to be used in control of garlic diseases. In this study, strain FA1 having high yield of biosurfactants was isolated from garlic farmland soil. Combined with morphological analysis and its 16S rDNA sequences analysis, the strain FA1 was identified as Pseudomonas aeruginosa. HPLC-MS analysis showed that biosurfactants produced from P. aeruginosa FA1 were presented rhamnolipids, mainly Rha-C₈-C₁₀, Rha-Rha-C₈-C₁₀, Rha-C₁₀-C₁₀ and Rha-Rha-C₁₀-C₁₀. The proportion of mono-rhamnolipids and di-rhamnolipids was about 1 : 1. P. aeruginosa FA1 can produce 7.96 g/L of rhamnolipids. The FA1 rhamnolipids can decrease the air-water surface tension to 27.5 mN/m with a critical micelle concentration of 50 mg/L. Through the agar diffusion method, P. aeruginosa FA1 exhibited high antimicrobial activity with diameters of inhibition zones larger than 20 mm to both the garlic potential pathogenic bacteria and fungi. Rhamnolipids produced from P. aeruginosa FA1 induced the potential pathogens to generate higher level of intracellular Reactive Oxygen Species (ROS), thus, exerting antimicrobial effects. P. aeruginosa FA1 can highly produce rhamnolipids biosurfactants and exhibit high antimicrobial effect to the garlic potential pathogens. This study verified that indigenous biosurfactant-producing bacteria from garlic farmland soil were feasible and effective to control garlic potential pathogens.

L-Asparaginase (EC 3.5.1.1) primarily catalyzes the hydrolysis of Asn into ammonium and L-Asp and has wide applications in various industrial sectors. This protein has several sources, including microorganisms, plants, and the serum of certain rodents. Currently, L-asparaginase is primarily produced through biotechnology using bacteria and fungi. Genetic engineering methods are employed to create more stable enzymes than those available for the wild type. Given the extensive industrial use of L-asparaginase, optimization, and purification techniques are utilized to increase ex vivo enzyme production. The objective of this study is to comprehensively review the current literature on the production of L-asparaginase from diverse sources and using various methodologies.

Metagenomic fosmid libraries were prepared from soil and water samples collected from diverse places. Clones in the range of (1.1–2.8) × 10³ were isolated from these samples and used for screening and isolation of D-amidase producer using dyes bromthymol green and phenol red. Maximum number of metagenomic libraries were obtained from soil rich in fishery waste while minimum number of libraries were obtained from tap water genome. Five positive clones after two-stage functional screening for D-alanine amidase were isolated on the basis of change in colour of growth medium due to enzyme production. Selected isolate on the basis of maximum enzyme production (2 U/mL) was cloned and overexpressed. Selected isolate from fishery waste metagenomes was found to be constitutive producer of D-amidase enzyme which after optimization led to 32% increase in enzyme activity. Isolated novel enzyme showed catalytic promiscuity with amides of D-Leu, D-Pro, D-Met and D-Val. Optimization of reaction conditions leading to production of 72% D-Ala and 65% D-Leu from their D-amide salts, respectively, in 8 h were confirmed by HPLC. Isolated homologous and putative genes after genomic screening were confirmed to be matching with Rhodococcus and Pseudomonas aeruginosa amidases. Enzyme also had acyl transferase activity with various amide substrates.

Vibrio harveyi is a normal flora present in seawater, but in recent years, with large density aquaculture, V. harveyi has become one of the causative organisms in fish and shrimp. In this study, V. harveyi VieA gene was used as the target to design primers and establish the loop-mediated isothermal amplification (LAMP) combined with visual lateral flow test strip (LFD). The optimal reaction conditions were optimized as 60°C, 45 min, 1.2 mM Mg²⁺, 0.64 mM dNTPs, 0.25 mM betaine, and 16:1 ratio of internal/external primers. The results showed that the established LAMP assay was able to specifically detect V. harveyi with a sensitivity of 3.4 × 10^–5 ng/µL. Clinical applicability analysis revealed that only V. harveyi was detected in the samples for pearl gentian grouper and whiteleg shrimp artificially infected by Vibrio. Therefore, we established a visual, reliable, rapid and sensitive LAMP-LFD method for detecting V. harveyi in aquatic animals.

Lubricant oils are largely responsible for environmental contamination and surfactants can hopefully improve the bioremediation process in soils contaminated with this kind of waste. However, to date only a few studies have elucidated how different surfactants affect the bioremediation process in oil-contaminated soils using the bioaugmentation technique. Thus, this study aimed to compare the effects of surfactants on the bioremediation process of waste lubricant oil in soil in association or not with bioaugmentation. Three biostimulating agents were tested: a chemical detergent, a synthetic surfactant, and a biosurfactant (rhamnolipid). The bioaugmentation process with Pseudomonas aeruginosa showed a faster biodegradation activity with total biodegradation on 181 day after application. The biosurfactant increased the number of microbial and fungal colonies leading to high biodegradation levels and a quicker treatment with or without the bioaugmentation. Bioaugmentation associated with biosurfactant reached 30.43 mg of CO₂ of production on 181 day (90% of total biodegradation of waste lubricant oil) and reduced soil toxicity to 30%. Therefore, results showed that the association of biosurfactant and bioaugmentation with P. aeruginosa represents an ecologically viable strategy for bioremediation of soils contaminated with waste lubricant oil.