Journal, genetic engineering & biotechnology最新文献

Background: Cellulose is the most prevalent biomass and renewable energy source in nature. The hydrolysis of cellulosic biomass to glucose units is essential for the economic exploitation of this natural resource. Cellulase enzyme, which is largely generated by bacteria and fungus, is commonly used to degrade cellulose. Cellulases are used in a variety of industries, including bioethanol manufacturing, textiles, detergents, drugs, food, and paper. As part of our quest to find an efficient biocatalyst for the hydrolysis of cellulosic biomass, we describe the amplification, cloning, and sequencing of cellulase (cel9z) from Bacillus licheniformis strain Z9, as well as the characterization of the resulting enzyme.

Results: Cellulase was partially purified from B. licheniformis strain Z9 using (NH₄)₂SO₄ precipitation and Sephadex G-100 gel column chromatography with 356.5 U/mg specific activity, 2.1-purification fold, and 3.07 % yield. The nucleotide sequence of the cellulase gene was deposited to the GenBank, B. licheniformis strain Z9 cellulase (cel9z) gene, under accession number MK814929. This corresponds to 1453 nucleotides gene and encodes for a protein composed of 484 amino acids. Comparison of deduced amino acids sequence to other related cellulases showed that the enzyme cel9z can be classified as a glycoside hydrolase family 9. SDS-PAGE analysis of the purified enzyme revealed that the molecular mass was 54.5 kDa. The optimal enzyme activity was observed at pH 7.4 and 30 °C. The enzyme was found to be strongly inhibited by Mg²⁺ and Na⁺, whereas strongly activated by Fe³⁺, Cu²⁺, and Ca²⁺.

Conclusions: B. licheniformis strain Z9 and its cellulase gene can be further utilized for recombinant production of cellulases for industrial application.

Background: The increased demand for oil and fats to satisfy the ever-increasing human needs has enhanced the research in this field. Single-cell oils or microbial lipids produced by oleaginous microorganisms are being utilized as an alternative to traditional oil sources. Oleaginous yeasts can accumulate lipids above 20% of their biomass when they are grown under controlled conditions.

Results: In the present study, sixty-five yeasts were isolated from different sources. Using Sudan Black B staining technique, five yeast isolates were selected. Under nitrogen-limited cultivation conditions, the Co1 isolate was the best lipid accumulation potential of 39.79%. Isolate (Co1) was characterized morphologically and identified using the ribosomal DNA internal transcribed spacers regions (rDNA-ITS) from their genomic DNA. The sequence alignment revealed a 99.2% similarity with Rhodotorula diobovata. Under the optimized conditions, Rhodotorula diobovata accumulated lipids up to 45.85% on a dry biomass basis. R. diobovata, when grown on different raw materials, accumulated lipid up to 46.68% on sugar beet molasses medium, and the lipid had a high degree of monounsaturated fatty acids which gives biodiesel better quality.

Conclusions: The data suggest that the potent oleaginous yeast, R. diobovata, together with the use of cheap feedstock raw materials such as sugar beet molasses, can be considered as a promising feedstock for biodiesel production.

Background: The B30.2 variants lead to most relevant severity forms of familial Mediterranean fever (FMF) manifestations. The B30.2 domain plays a key role in protein-protein interaction (PPI) of pyrin with other apoptosis proteins and in regulation the cascade of inflammatory reactions. Pyrin-casp1 interaction is mainly responsible for the dysregulation of the inflammatory responses in FMF. Lower binding affinity was observed between the mutant B30.2 pyrin and casp1 without the release of the complete pathogenicity mechanism. The aim of this study was to identify the possible effects of the interface pocked residues in B30.2/SPRY-Casp1/p20 complex using molecular mechanics simulation and in silico analysis.

Results: It was found that Lys671Met, Ser703Ile, and Ala744Ser variants led mainly to shift of the binding affinity (∆G), dissociation constant (K_d), and root mean square deviation (RMSD) in B30.2/SPRY-Casp1/p20 complex leading to dynamic disequilibrium of the p20-B30.2/SPRY complex toward its complex form. The current pathogenicity model and its predicted implementation in the relevant colchicine dosage were delineated.

Conclusion: The molecular mechanics analysis of B30.2/SPRY-p20 complex harboring Lys671Met, Ser703Ile, and Ala744Ser variants showed dynamic disequilibrium of B30.2/SPRY-casp1/p20complex in context of the studied variants that could be a new computational model for FMF pathogenicity. This study also highlighted the specific biochemical markers that could be useful to adjust the colchicine dose in FMF patients.

Background: Cerium-containing materials have wide applications in the biomedical field, because of the mimetic catalytic activities of cerium. The study aims to deeply estimate the biocompatibility of different scaffolds based on Ce-doped nanobioactive glass, collagen, and chitosan using the first passage of rabbit bone marrow mesenchymal stem cells (BM-MSCs) directed to osteogenic lineage by direct and indirect approach. One percentage of glass filler was used (30 wt. %) in the scaffold, while the percentage of CeO₂ in the glass was ranged from 0 to 10 mol. %. Cytotoxicity was evaluated by monitoring of cell morphological changes and reduction in cell proliferation activity of BMMSCs maintained under osteogenic condition using proliferation assays, MTT assay for the direct contact of cells/scaffolds twice in a week, trypan blue and hemocytometer cell counting for indirect contact of cells/scaffolds extracts at day 7. Cell behaviors growth, morphology characteristics were monitored daily under a microscope and cell counting were conducted after 1 week of the incubation of the cells with the extracts of the four composite scaffolds in the osteogenic medium at the end of the week.

Results: Showed that at 24 h after direct contact with composite scaffold, all scaffolds showed proliferation of cells > 50% and increased in cell density on day 7. The scaffold of the highest percentage of CeO₂ in bioactive glass nanoparticles (sample CL/CH/C10) showed the lowest inhibition of cell proliferation (< 25%) at day 7. Moreover, the indirect cell viability test showed that all extracts from the four composite scaffolds did not demonstrate a toxic effect on the cells (inhibition value < 25%).

Conclusion: The addition of CeO₂ to the glass composition improved the biocompatibility of the composite scaffold for the proliferation of rabbit bone marrow mesenchymal stem cells directed to osteogenic lineage.

Background: The role of atrial natriuretic peptide (ANP) in edema formation in idiopathic nephrotic syndrome (INS) was studied before with conflicting results reported; however, the possible contribution of genes regulating ANP expression and receptors was never explored.

Methods: One hundred children (60 with active INS and 40 in remission) were studied for plasma atrial natriuretic peptide (ANP), urinary sodium, ANP gene A2843G and ScaI polymorphisms, and natriuretic peptide receptor clearance C (-55) A polymorphism. For comparative purposes, 20 healthy controls were studied for ANP levels.

Results: ANP was higher in active compared to remission patients (p<0.001). ANP in the healthy control group was significantly lower than the ANP level of active INS (during edema) group (p=0.009) but did not show significant differences when compared to ANP levels of either active INS group after resolution of edema or remission group (p= 0.42 and 0.56, respectively). Urinary sodium levels in edematous patients were significantly lower while ANP levels were significantly higher during edema than after resolution (p< 0.001 for both). Genotypes' frequencies of studied polymorphisms did not differ between active and remission groups. Patients with the A1A1 genotype of ScaI polymorphism had higher ANP levels compared to other genotypes (p =0.01).

Conclusions: During edema, ANP levels are elevated in INS children however this increment is not associated with natriuresis suggesting a blunted renal response to ANP. Polymorphisms of genes regulating ANP levels and receptors don't seem to be implicated in edema formation except for the A1A1 genotype of ScaI polymorphism however, its possible role needs further evaluation.

Background: Durian (Durio zibethinus L.) is a tropical fruit crop which is popular in Southeast Asia but recently gaining popularity in other parts of the world. In this study, we analyzed the resistance gene analogs (RGAs) of durian through mining of the currently available reference genome of its 'Musang King' cultivar (PRJNA400310).

Results: A total of 2586 RGAs were identified in the durian genome consisting of 47 nucleotide binding site proteins (NBS), 158 NBS-leucine rich repeat proteins (NL), 400 coiled-coil NBS-LRR (CNL), 72 toll/interleukin-1 receptor NBS-LRR (TNL), 54 coiled-coil NBS (CN), 10 toll/interleukin-1 receptor NBS (TN), 19 toll/interleukin-1 receptor with unknown domain (TX), 246 receptor-like proteins (RLP), 1,377 receptor-like kinases (RLK), 185 TM-CC, and 18 other NBS-containing proteins with other domains. These RGAs were functionally annotated and characterized via gene ontology (GO) analysis. Among the RGAs with the highest copies in durian genome include the putative disease resistance RPP13-like protein 1, disease resistance protein At4g27190, disease resistance protein RPS6, Probable disease resistance protein At4g27220, and putative disease resistance protein RGA3, while 35 RGAs were found to be novel. Phylogenetic analyses revealed that the genome-wide RGAs were broadly clustered into four major clades based on their domain classification.

Conclusion: To our knowledge, this is the most comprehensive analysis of durian RGAs which provides a valuable resource for genetic, agronomic, and other biological research of this important tropical fruit crop.

Background: The Plant U-box (PUB), ubiquitin ligase gene, has a highly conserved domain in potato. However, little information is available about U-box genes in potato (Solanum tuberosum). In this study, 62 U-box genes were detected in the potato genome using bioinformatics methods. Further, motif analysis, gene structure, gene expression, TFBS, and synteny analysis were performed on the U-box genes.

Results: Based on in silico analysis, most of StU-boxs included a U-box domain; however, some of them lacked harbored domain the ARM, Pkinase_Tyr, and other domains. Based on their phylogenetic relationships, the StU-box family members were categorized into four classes. Analysis of transcription factor binding sites (TFBS) in the promoter region of StU-box genes revealed that StU-box genes had the highest and the lowest number of TFBS in MYB and CSD, respectively. Moreover, based on in silico and gene expression data, variable frequencies of TFBS in StU-box genes could indicate that these genes control different developmental stages and are involved in complex regulatory mechanisms. The number of exons in U-box genes ranged from one to sixteen. For most U-box genes, the exon-intron compositions and conserved motifs composition in most proteins in each group were similar. The intron-exon patterns and the composition of conserved motifs validated the U-box genes phylogenetic classification. Based on the results of genome distribution, StU-box genes were distributed unevenly on the 12 S. tuberosum chromosomes. The results showed that gene duplication may possess a significant role in genome expansion of S. tuberosum. Furthermore, genome evolution of S. tuberosum was surveyed using identification of orthologous and paralogous. We identified 40 orthologous gene pairs between S. tuberosum with Solanum lycopersicum, Oryza sativa, Triticum aestivum, Gossypium hirsutum, Zea maize, Coriaria mytifolia, and Arabidopsis thaliana as well as eight duplicated genes (paralogous) in S. tuberosum. StU-box 51 gene is one of the important gene among other StU-boxes in S. tuberosum under drought stress which was expressed in tuber and leaf under drought stress. Furthermore, StU-box 51 gene has the highest expression levels in four tissue-specific (stem, root, leaf, and tuber) in potato as well as it had the highest number of TFBS in promoter region. Based on our results, StU-box 51 can introduce to researcher to utilize in breeding program and genetic engineering in potato.

Conclusions: The results of this survey will be useful for further investigation of the probable role and molecular mechanisms of U-box genes in response to different stresses.

Background: Plant grows in nature facing various types of abiotic stresses for their normal growth and development. During abiotic stress, plants evolve different types of mechanisms to survive in a hostile environment. Phospholipase D (PLD) plays important role in the regulation of diverse cellular processes including stress responses in plants. Member of PLD genes are well studied in different model plants; however, their functions in the jute are not clear yet.

Result: In the present study, a total of 12 and 11 PLD genes were identified in the genome of C. capsularis and C. olitorius, respectively. The presence of the two conserved HKD motifs in PLD genes except for CoPLDδ-2 in jute suggests their strong lipase activity. Twenty different motifs were found in the identified PLD genes, and PLD-β1, PLD-γ1, and all members of PLD-δ1 of both jute species contained the highest number of motifs. Phylogenetic analysis showed the close evolutionary relationship among the five groups of jute PLD proteins along with the PLD proteins from Arabidopsis. Tissue-specific expression pattern of PLDα1-2, PLD-α2, PLDβ1, PLDγ1, and PLDδ1 of two jute species suggested their involvement in plant growth and development. However, the expression pattern of PLDα1-2, PLDα1-3, PLD-α4, PLDδ1, and PLDδ3 indicated their association during waterlogging stress. In addition, PLD-α2, PLDβ1, and PLDδ2 seemed to be involved in drought stress as well as salinity stress.

Conclusion: This genome-wide identification of jute PLD genes from C. capsularis and C. olitorius will help to further functional characterization of the PLD genes for developing stress-tolerant jute variety.

Background: Amylase is used commercially in food, textiles, sugar syrup, paper, and detergent industries. Bacteria and fungi remain a significant source of industrial enzymes. Pleurotus tuberregium is a macro-fungi that can exist as a fruiting body, sclerotium, mycelium, and spores. Some studies have been conducted on this fungus, with minimal studies on its enzyme activity (s) using the submerged fermentation technique.

Results: The purified amylase has a specific activity of 5.26 U/mg, total activity of 189.20 U, maximally active at 70 °C, pH of 5, and retaining 100% of its activity at 30 ^oC for 4 min. P. tuberregium amylase showed optimal activity with plantain peel, followed by starch and pineapple peel (42, 30, and 29 μg/mL/min respectively). The presence of Ca²⁺, Mg²⁺, and Na⁺ ions in the reaction mixture activated the enzyme activity, but was slightly and moderately inhibited by KCl and Na₂H₂PO₄ respectively. The crude enzyme effectively clarified juice, liquefied soluble cassava starch (with a release of appreciable glucose quantity), and partially de-stained white fabric.

Conclusions: The amylase obtained from the submerged fermentation of Pleurotus tuberregium has potential applications in food and detergent industries.