Journal, genetic engineering & biotechnology最新文献

Background: The present study focuses on the isolation of Bacillus thuringiensis bacterium from the gut of fresh water fish, Systomus sarana, the innovative optimization of culture parameters to produce maximum protease enzyme, by the isolated bacterium, and the elucidation of peptide profile of the protease. And the experimental data and results were authenticated through the response surface method (RSM) and Box-Behnken design (BBD) model.

Results: During the RSM optimization, the interaction of the highest concentrations (%) of 2.2 maltose, 2.2 beef extract, and 7.0 pH, at 37 °C incubation, yielded a maximum protease enzyme of 245 U/ml by the fish gut-isolated, B. thuringiensis. The spectral analysis of the obtained enzyme revealed the presence of major functional groups at the range of 610-3852 cm^-1 viz., alkynes (-C≡C-H: C-H stretch), misc (P-H phosphine sharp), α, β-unsaturated aldehydes, and through PAGE analysis, its molecular weight was determined as 27 kDa. The enzyme's MALDI-TOF/MS analysis revealed the presence of 15 peptides from which the R.YHTVCDPR.L peptide has been found to be a major one.

Conclusions: The fish gut-isolated bacterium, B. thuringiensis, SS4 exhibited the potential for high protease production under the innovatively optimized culture conditions, and the obtained result provides scope for applications in food and pharmaceutical industries.

Background: Oxidative stress is among the most common risk factors in the pathogenesis of acute myocardial infarction (AMI). Glutathione peroxidase 1 enzyme coded by the GPX1 gene plays an essential role in reducing oxidative stress. Previous studies correlated the GPX1 (Pro200Leu) single nucleotide polymorphism (SNP) with AMI incidence. Elevated homocysteine (Hcy) levels induce oxidative stress and are considered an independent risk factor for AMI. Evidence showed a complex relationship between Hcy and GPx-1 activity. This study examined the association of the common (Pro200Leu) SNP in GPX1 with AMI incidence in an Egyptian population. This study is the first to check this association in an Egyptian population. Moreover, the association between serum Hcy and the incidence of AMI was checked, and the novelty was to statistically correlate GPX1 Pro200Leu genotypes with serum Hcy levels in patients and control subjects. Hundred control subjects and hundred and twenty AMI patients were genotyped using PCR-RFLP analysis. An ELISA was used to measure serum Hcy levels.

Results: The GPX1 (Pro200Leu) genotype distribution and allele frequency were not significantly different between patients and control subjects (P = 0.60 and P = 0.62, respectively). Serum levels of Hcy were significantly elevated in patients compared to control subjects (P ≤ 0.0001). However, no significant difference was observed in serum Hcy levels among different GPX1 genotypes in neither patients nor control subjects.

Conclusions: The minor T allele of GPX1 Pro200Leu is not associated with AMI risk in this Egyptian population. However, high homocysteine serum levels might contribute independently to the risk of AMI. Finally, Hcy levels were not significantly different in homozygous minor TT compared to homozygous wild CC.

Background: This study targets the enhanced production of L-asparaginase, an antitumor enzyme by Acinetobacter baumannii ZAS1. This organism is an endophyte isolated from the medicinal plant Annona muricata. Plackett-Burman design (PBD) and central composite design (CCD) were used for statistical optimization of media components.

Results: The organism exhibited 18.85 ± 0.2 U/mL enzyme activities in unoptimized media. Eight variables: L-asparagine, peptone, glucose, lactose, yeast extract, NaCl, MgSO₄, and Na₂HPO₄ were screened by PBD. Among them, only four factors-L-asparagine, peptone, glucose, and Na₂HPO₄-were found to affect enzyme production significantly (p < 0.05). Furthermore, the best possible concentrations and interactive effects of the components that enhance this enzyme's output were chosen by using CCD on these selected variables. The results revealed that an optimized medium produces a higher concentration of enzymes than the unoptimized medium. After optimizing media components, the maximum L-asparaginase activity was 45.59 ± 0.36 U/mL, around the anticipated value of 45.04 ± 0.42 U/mL. After optimization of process parameters, it showed a 2.41-fold increase in the production of L-asparaginase by the endophyte Acinetobacter baumannii ZAS1.

Conclusion: The findings of this study indicated that an endophyte, Acinetobacter baumannii ZAS1 that produces L-asparaginase could be used to increase enzyme output. However, using the statistical methods Plackett-Burman design and central composite design of response surface methodology is a handy tool for optimizing media components for increased L-asparaginase synthesis.

Background: Wadi El Natrun microorganisms have been considered as a new resource for natural products due to its extreme condition of salinity and alkalinity. Therefore, this study was devoted to generate metagemic library from soils collected from such an extreme environment in order to clone a novel cellulase for physique industrial applications.

Results: Total soil-DNA was successfully extracted, and then digested by different restriction enzymes. Purified fragments ranged ~ 200-6500 bp were ligated and were cloned into plasmid cloning vector (pUC19) by using Escherichia coli DH5α (E. coli) host cells. A constructed metagenomic library composed of 270 clones was screened on carboxymethylcellulose (CMC) agar plate where the active clones had been characterized by the formation of the yellowish halo zone. Thereafter, clone 1 was selected as the most active as being based on cellulase activity quantification (19 μ/ml). Plasmid related to clone 1 encoded cellSNSY gene of approximately 1.5 kb was subjected to molecular characterization; the obtained partial sequence of 861 bps encoded 287 amino acids showing 76% similarity to the endoglucanase gene of Bacillus amyloliquefaciens. The recombinant cellSNSY was expressed under lacz promoter at 1 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG), giving 21 μ/ml cellulase after ~ 27 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and an activity staining of the recombinant cellSNSY which revealed an active band with a molecular mass ~ 59 kDa appeared in the induced sample. The maximum enzyme activity of crude cellSNSY was observed at 45 °C and for a pH of 8.5. Interestingly, the enzyme activity was slightly inhibited by ethylenediamine tetraacetic acid (EDTA) and methanol. It showed high resistance to the tested heavy metals and the surfactant which ordered Zn> (SDS,Fe)>Mn>Cu.

Conclusions: This study established an easy and a skillful way to clone/express a new found cellulase gene(s) under lacZ promoter. The isolated recombinant cellSNSY showed 76% similarity to endoglucanase gene, and the enzyme showed tolerance to the mostly tested agents including heavy metals, surfactant, solvents, and EDTA. Additionally, the studied recombinant showed a high stability up to 55 °C and for alkaline pH 8.5. These features make it an ample and viable for many applications.

Background: A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein.

Results: The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with EcoRI. Digestion by EcoRI revealed a size of 5953 and 1160 bps for both TA cloning petSUMO vector and gene of interest, respectively. Furthermore, the right direction of construct pETSUMO-L1HPV52 was proven by PCR techniques using specific primer pairs then followed by sequencing, which shows 147 base pairs. Characterization of L1 HPV 52 by SDS-PAGE analysis confirms the presence of a protein band at a size of ~55 kDa with 6.12 mg/L of total protein concentration. Observation under by transmission electron microscope demonstrates the formation of VLP-L1 at a size between 30 and 40 nm in assembly buffer under the condition of pH 5.4. Based on bioinformatics studies, we found that there are three B cell epitopes (GFPDTSFYNPET, DYLQMASEPY, KEKFSADLDQFP) and four T cell epitopes (YLQMASEPY, PYGDSLFFF, DSLFFFLRR, MFVRHFFNR). Moreover, an immunogenicity study shows that among all the T cell epitopes, the one that has the highest affinity value is DSLFFFLRR for Indonesian HLAs.

Conclusion: Regarding the achievement on successful formation of L1 HPV52-VLPs, followed by some possibilities found from bioinformatics studies, this study suggests promising results for future development of L1 HPV type 52 vaccine in Indonesia.

Background: The primary amino acid sequence of a protein is a translated version from its gene sequence which carries important messages and information concealed therein. The present study unveils the structure-function and evolutionary aspects of 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) proteins of fungal origin. ACCD, an important plant growth-promoting microbial enzyme, is less frequent in fungi compared to bacteria. Hence, an inclusive understanding of fungal ACC deaminases (fACCD) has brought forth here.

Results: In silico investigation of 40 fACCD proteins recovered from NCBI database reveals that fACCD are prevalent in Colletotrichum (25%), Fusarium (15%), and Trichoderma (10%). The fACCD were found 16.18-82.47 kDa proteins having 149-750 amino acid residues. The enzyme activity would be optimum in a wide range of pH having isoelectric points 4.76-10.06. Higher aliphatic indices (81.49-100.13) and instability indices > 40 indicated the thermostability nature. The secondary structural analysis further validates the stability owing to higher α-helices. Built tertiary protein models designated as ACCNK1-ACCNK40 have been deposited in the PMDB with accessions PM0083418-39 and PM0083476-93. All proteins were found as homo-dimer except ACCNK13, a homo-tetramer.

Conclusions: Hence, these anticipated features would facilitate to explore and identify novel variants of fungal ACCD in vitro aiming to industrial-scale applications.

Castor bean (Ricinus communis L.) is an important cultivated oilseed. Seeds contain ricinoleic acid, a valuable product for a variety of industries. Castor cake is a residue of ricinoleic manufacture and could be used as animal feed due to its high amount of protein. However, castor cake contains ricin and RCA₁₂₀, both highly toxic and allergenic proteins. In 2017, we reported the development of a transgenic event (named TB14S-5D) with an undetectable amount of ricin/RCA_120. In the present work, we evaluate TB14S-5D for tolerance to the herbicide imazapyr, as it contains the selectable marker gene, ahas, which was previously isolated from Arabidopsis thaliana and contains a mutation at position 653 bp. In addition, we demonstrated that the ricin coding genes are stably silenced over three generations.

Background: Association between Helicobacter pylori (H. pylori) and chronic hepatitis C (CHC) still remains controversial. This work is concerned with assessing the potential role of H. pylori in the progression of hepatitis C virus (HCV)-related chronic liver disease.

Results: A total of 449 individuals constituted this study (200 individuals were used to validate the assay while 249 individuals were used to assess the correlation between H. pylori infection and CHC). H. pylori antigen was quantified in serum samples using ELISA. As a consequence, our findings showed that H. pylori positivity was increased significantly (P = 0.021) with liver fibrosis progression as it was found in 44.45% of fibrotic patients and 71.88% of cirrhotic patients. We demonstrated that patients with F4 were accompanied by a significant (P < 0.05) increase in the concentration of H. pylori antigen displaying 16.52-fold and 1.34-fold increase in its level over F0 and F1-F3, respectively. Patients co-infected with H. pylori and HCV are 3.19 times (219%) more likely to experience cirrhosis than those who are mono-infected with HCV. This suggests that the risk for developing F4 was found to increase upon H. pylori co-infection when compared to CHC mono-infected patients.

Conclusion: The elevated levels of H. pylori-antigen in HCV/H. pylori co-infection suggest increased susceptibility of co-infected patients for promoting hepatic fibrosis progression.

Background: The marine environment hosts a wide variety of species that have evolved to live in harsh and challenging conditions. Marine organisms are the focus of interest due to their capacity to produce biotechnologically useful compounds. They are promising biocatalysts for new and sustainable industrial processes because of their resistance to temperature, pH, salt, and contaminants, representing an opportunity for several biotechnological applications. Encouraged by the extensive and richness of the marine environment, marine organisms' role in developing new therapeutic benefits is heading as an arable field. There is currently much interest in biologically active compounds derived from natural resources, especially compounds that can efficiently act on molecular targets, which are involved in various diseases. Studies are focused on bacteria and fungi, isolated from sediments, seawater, fish, algae, and most marine invertebrates such as sponges, mollusks, tunicates, coelenterates, and crustaceans. In addition to marine macro-organisms, such as sponges, algae, or corals, marine bacteria and fungi have been shown to produce novel secondary metabolites (SMs) with specific and intricate chemical structures that may hold the key to the production of novel drugs or leads. The marine environment is known as a rich source of chemical structures with numerous beneficial health effects. Presently, several lines of studies have provided insight into biological activities and neuroprotective effects of marine algae, including antioxidant, anti-neuroinflammatory, cholinesterase inhibitory activity, and neuronal death inhibition.

Conclusion: The application of marine-derived bioactive compounds has gained importance because of their therapeutic uses in several diseases. Marine natural products (MNPs) display various pharmaceutically significant bioactivities, including antibiotic, antiviral, neurodegenerative, anticancer, or anti-inflammatory properties. The present review focuses on the importance of critical marine bioactive compounds and their role in different diseases and highlights their possible contribution to humanity.

Background: Although microbial fuel cells (MFCs) represent a promising technology for capturing renewable energy from wastewater, their scaling-up is significantly limited by a slow-rate cathodic oxygen reduction reaction (ORR) and the development of a resilient anodic microbial community. In this study, mixed transition metal oxides of nickel and copper (Ni and Cu), supported on a graphene (G) (NiO-CuO/G) electrocatalyst, were synthesized and tested as a cost-effective cathode for ORR in MFCs. Electrochemical measurements of electrocatalyst were conducted using a rotating disk electrode (RDE) and linear sweep voltammetry (LSV) in a neutral electrolyte, and compared with a benchmark Pt/C catalyst. Furthermore, the long-term performance of the as-synthesized electrocatalyst was evaluated in a single-chamber MFC by measuring organic matter removal and polarization behavior. The successful enrichment of electroactive biofilm was also monitored using transmission electron microscopy and the Vitek2 compact system technique.

Results: When compared with the benchmark platinum cathode, the NiO-CuO/G electrocatalyst exhibited high selectivity toward ORR. The rotating disk electrode (RDE) experiments reveal that ORR proceeds via a 4-electron ORR mechanism. Furthermore, the NiO-CuO/G electrocatalyst also exhibited a high power density of 21.25 mW m^-2 in an air-cathode MFC, which was slightly lower than that of Pt/C-based MFC (i.e., 50.4 mW m^-2). Biochemical characterization of the most abundant bacteria on anodic biofilms identified four genera (i.e., Escherichia coli, Shewanella putrefaciens, Bacillus cereus, and Bacillus Thuringiensis/mycoides) that belonged to Gammaproteobacteria, and Firmicutesphyla.

Conclusions: This study demonstrates that the NiO-CuO/G cathode had an enhanced electrocatalytic activity toward ORR in a pH-neutral solution. This novel mixed transition metal oxide electrocatalyst could replace expensive Pt-based catalysts for MFC applications.