Journal, genetic engineering & biotechnology最新文献

Background: Enhancement of lipid accumulation is the major strategy to improve the commercial feasibility of microalgae as a source for biodiesel production. Pseudochlorella pringsheimii (Formally was named as Chlorella ellipsoidea) green microalgae strain was chosen with respect to their ability as a potential source to produce high lipids content, could be used for the production of biofuel, which can be an alternative renewable energy source instead of fossil fuels.

Results: Initially, the Pseudochlorella pringsheimii microalgae was evaluated on the basis of tested at Lab scales 2 L by applicable different nutrient individual of N, P, Fe conditions in BBM medium concentrations for choosing the best concentrations induce lipid contents and productivity to cultivate in large scale in the 2000 L PBR. The suitable concentrations of nutrients with highest lipid contents were obtained under deficient of nitrogen (1.25 gL^-1, limited N) and phosphorus (0.1 mg L^-1, limited P) coupled with high iron concentration (10 mg L, rich Fe) and CO₂ (6%). Therefore, their collective of nutrients was applied to culture of microalgae cells at large scale in 2000 L photobioreactor (PBR model), which, this techniques was used to quantify high lipid contents (25% w/w) and high lipid productivity (74.07 mgL^-1 day^-1). The inducted lipid conversion to biodiesel via transestrification process was 91.54 ± 1.43%. The fatty acid methyl esters (FAMEs profile by means of GC/MS resulted in C16:0, C18:1, C18:2, C18:3 as a main constituents. With regard to physical-chemical property (such as density, kinematic viscosity, gravity, and certain number), the Pseudochlorella pringsheimii biodiesel have biofuel properties, in accordance with appropriate biodiesel properties, as ASTM and EU standards, that thereby referring to high quality biodiesel.

Conclusions: Pseudochlorella pringsheimii cultured in large scale in photobioreactor under stress condition have a high potential of lipids production with high quality of FAMEs that can be used as a promising biodiesel fuel. It has also a potential to be applied for commercialization based on the techno-economic and environmental impacts.

Background: Biopolymers like polyhydroxyalkanoates (PHA) are the best natural macromolecules to use as alternative to the synthetic polymers. Many prokaryotes accumulate PHA as cytoplasmic intracellular granules and their accumulation is triggered by starving conditions. The PHAs are ecofriendly and used to create biodegradable plastics. The microbial synthesized PHA had acquired global importance in industrial and biomedical sectors.

Results: Ten different bacterial strains were isolated for the screening of PHA producers from the estuarine region of the Bay of Bengal, Suryalanka in Bapatla. A yellowish slimy circular colony known as M4 is actively growing on selective minimal media and was screened for polymeric granules in its cytoplasm using Sudan Black B and confirmed with the fluorescent dye Nile blue A. All of the isolates were biochemically tested and isolate M4 is the most capable of growing at high NaCl concentrations (3.2 percent) and tests positive for catalase, methyl red. The M4 strain revealed clear hydrolysis of gelatin, starch, and casein. The 16S rRNA sequencing revealed that M4 is 99.72% of identity to Brachybacterium paraconglomeratum LMG 19861(T) in BLAST and the obtained strain was assigned with accession no. MTCC 13074 and deposited in NCBI with accession no. MW899045. The chief cellular fatty acids found in M4 were C14:0, C15:0, C16:0, C18:1cis-9, C18:0, iso-C15: 0, iso-C14: 0, anteiso-C17: 0 and C18:1-7. Crotonic acid formation from M4-PHB extract was detected at 235nm in a UV spectrophotometer. Methanolysis was done, and derivatives of polyhydroxybutyric acid (PHB) in the extract were analyzed using GC-MS. Increasing viscosity was seen in the extracts which confirms the presence of polymer in the extracts. Thermogravimetric analysis was studied to determine the thermal profile of the PHB in the extract of M4.

Conclusion: In the study, the selective screening and extraction of ecofriendly PHB from M4 strain was highlighted. Brachybacterium paraconglomeratum is a novel strain showed its uniqueness by producing few monomeric derivatives of PHB. The strain was reporting for the first time as PHA producer. B. paraconglomeratum has promising characteristics according to its metabolic profile. In addition, this study also helps to understand the diversity of bacteria isolated from marine sources.

Background: The biopharmaceutical industry is significantly growing worldwide, and the Chinese hamster ovary (CHO) cells are used as a main expression host for the production of recombinant monoclonal antibodies. Various metabolic engineering approaches have been investigated to generate cell lines with improved metabolic characteristics for increasing longevity and mAb production. A novel cell culture method based on the 2-stage selection makes it possible to develop a stable cell line with high-quality mAb production.

Results: We have constructed several design options of mammalian expression vectors for the high production of recombinant human IgG antibodies. Versions for bipromoter and bicistronic expression plasmids different in promoter orientation and cistron arrangements were generated. The aim of the work presented here was to assess a high-throughput mAb production system that integrates the advantages of high-efficiency cloning and stable cell clones to stage strategy selection reducing the time and effort required to express therapeutic monoclonal mAbs. Development of a stable cell line using bicistronic construct with EMCV IRES-long link gave an advantage in high mAb expression and long-term stability. Two-stage selection strategies allowed the elimination of low-producer clones by using metabolic level intensity to estimate the IgG production in the early steps of selection. The practical application of the new method allows to reduce time and costs during stable cell line development.

Background: Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides.

Results: This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four β sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs.

Conclusion: Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

Background: Superoxide dismutase is an important antioxidative stress enzyme which is found in honeybee venom and has a wide pharmaceutical and medical applications.

Results: We reported the purification and characterization of venom SOD from Egyptian honeybee Apis mellifera lamarckii and termed BVSOD. It was purified to homogeneity from the Egyptian honeybee venom. The purification procedures included crude extraction, DEAE-cellulose anion exchange column chromatography, and Sephacryl S-300 gel filtration column chromatography. The purified BVSOD is found to be homogeneous as investigated by native PAGE. It exhibited homodimeric structure with a molecular weight of native form of 32 kDa and subunits of 16.0 kDa. It displayed the maximum activity at pH 7.4. CuCl₂, ZnCl₂, and MgCl₂ and elevated the activity of BVSOD, while CoCl₂, FeCl₂, and NiCl₂ inhibited BVSOD activity. Potassium cyanide and hydrogen peroxide were most potent inhibitors for BVSOD activity suggesting that it is a Cu/Zn-SOD type.

Conclusions: The purified BVSOD is found to have antimicrobial and antitumor activities which can be used for various medical and clinical applications.

Objective: Canine parvovirus (CPV) is a small virus without an envelope that consists of three viral proteins including VP1, VP2, and VP3. Exclusively, the VP2 can form a typically CPV-sized virus-like particle (CPV-VLP) that can be used as a biological nanocarrier for diagnostic and therapeutic purposes since these VLPs can target cancer cells specially through the transferrin surface receptors (TFRs). Consequently, we aimed to produce these nanocarriers to be used for specific targeting of cancer cells.

Methods: Sf9 insect cells were transfected with constructed recombinant bacmid shuttle vector encoding an enhanced green fluorescent protein (EGFP) and CPV-VP2 by the cationic lipids of Cellfectin II. Subsequently, two recombinant baculoviruses expressing EGFP and VP2 were produced and expression of VP2 was increased under the optimal condition. In consequence, the CPV-VLP nanoparticles composed of recombinant VP2 subunits were extracted. The purity of VLPs was then evaluated by SDS-PAGE, and the structural integrity and quality of the final product were evaluated by TEM and HA methods. Eventually, the size distribution of the produced biological nanoparticles and their uniformity were determined by the DLS method.

Results: The expression of EGFP protein was confirmed by fluorescent microscopy, and the expression of VP2 protein was evaluated by SDS-PAGE and western blotting. Infected Sf9 insect cells also showed cytopathic effects (CPEs), and the maximum expression of VP2 occurred at MOI of 10 (pfu/cell) at the harvest time of 72 h post-infection (hpi). After performing various stages of purification, buffer exchange, and concentration, the quality and structural integrity of the VLP product were confirmed. The results of the DLS technique showed the presence of uniform particles (PdI below 0.5) with an approximate size of 25 nm.

Conclusion: The results indicate BEVS as an appropriate and efficient system for generating CPV-VLPs, and the used method based on two-stage ultracentrifugation was appropriate for purifying these nanoparticles. Produced nanoparticles can be used as the biologic nano-carriers in future studies.

Background: Bone morphogenetic protein receptor 1B (BMPR1B) gene is one of candidate genes for reproductive and growth traits in sheep. The present study was aimed to detect the Booroola (Fec^B) allele in BMPR1B gene and its association with growth traits in MEGA (Merino × Garut) sheep. A total of 82DNA samples collected from individual lamb (mixed-sex) blood were genotyped for allelic polymorphism using a PCR-RFLP method.

Results: The PCR analysis in BMPR1B gene resulted the amplicons with size of140 bp. The RFLP analysis with AvaII restriction enzymeresultedtwo allelic types of wildtype (A/Fec⁺) and mutant or Booroola (G/Fec^B) with frequency of 0.89 and 0.11, respectively. However, the genetic diversity in BMPR1B/AvaII gene of animal studies was categorized tolow category (PIC = 0.18)and under in a genetic equilibrium (χ² = 1.25).

Conclusions: Itshowed us that carrying Fec^B allele in the heterozygous sheep were not associated with growth traits in MEGA sheep.

Background: Applying microbial biostimulants during crop cultivation allows for higher sustainability levels. It reduces the need for fertilizers and environmental contaminants while enhancing plant quality. This study assessed 13 endophytic bacteria, 4 newly isolated, and 9 donated, for plant growth-promoting capabilities. Quantitative assessments of indole acetic acid (IAA), gibberellic acid (GA₃), siderophores, ammonia, exopolysaccharides, volatile HCN, and phosphate solubilization, along with Bray-Curtis cluster analyses were performed.

Results: Upon the results  we selected RhizobiumMAP7, Brevibacillus DesertYSK, Pseudomonas MAP8, BacillusMAP3, Brevibacillus MAP, and Bacillus DeltaYSK to evaluate their effects on Lactuca sativa growth and pigmentation in a 30-day greenhouse pot experiment. Both Brevibacillus DesertYSK and Rhizobium MAP7surpassed other strains in growth promotional effects. They doubled shoot length (12 and 12.3 cm, respectively, when compared with 7 cm for control after 30 days), and fresh weight (0.079 and 0.084 g, respectively, when compared with 0.045 g for control after 30 days), and increased root length by at least 3-fold when compared with control (4.5 and 3.5 cm, respectively, when compared with 1.2 cm for control after 30 days). Chlorophyll content also exhibited at least a 2-fold significant increase in response to bacterization compared with control.

Conclusions: This strain superiority was consistent with the in vitro assays data that showed strains capability as IAA and GA₃producers. Also, strains were highly capable ammonia and siderophore producers and phosphate solubilizers, providing considerable hormone and nutrient levels for L. sativa plantsleading to improved growth parameters and appearance. These data support the notion that nodule-based bacteria are potential plant growth-promoting bacteria (PGPB) that may be used on a wider scale rather than just for legumes.

Background: Flavonoid natural dyes have gained attention because they are nontoxic and eco-friendly. However, they do not work effectively with artificial fibers and require the use of mordants, which are considered as hazardous chemicals. Laccase enzyme catalyzes the oxidation of phenols, forming phenoxyl radicals that undergo a further polymerization process. So, laccase can oxidize flavonoid dyes, and it can be used instead of harmful mordants in flavonoid dye fixation on cotton fabrics. Laccases also are involved in a variety of metabolic processes, and they have anti-proliferative effects toward HepG2 and MCF-7 tumor cells.

Results: Among fifteen fungal isolates, the fungus Ceratorhiza hydrophila isolated from the submerged plant Myriophyllum spicatum was selected as the most potent laccase producer. Optimization of the production medium resulted in a 9.9-fold increase in laccase productivity. The partially purified Ceratorhiza hydrophila laccase could successfully improve the affinity of cotton fabrics toward quercetin (flavonoid) dye with excellent color fastness properties. The partially purified laccase also showed anti-proliferative activity against HepG2 and MCF-7 tumor cells. However, high laccase concentration is required to estimate IC50.

Conclusions: Ceratorhiza hydrophila MK387081 is an excellent laccase producer. The partially purified laccase from Ceratorhiza hydrophila can be used in textile dyeing and printing processes as a safer alternative to the conventional hazardous mordants. Also, it can be used in preparation of cancer treatment drugs. However, further studies are needed to investigate IC50 for both cell types at higher laccase concentrations.