Journal, genetic engineering & biotechnology最新文献

Background: Pectinase is helpful in food and beverage industries, particularly in the preparation of fruit juice, the extraction of vegetable oil, and the fermentation of coffee. The current work aimed to screen Aspergillus niger LFP-1, a recently identified fungal strain, for its ability to produce pectinase and to ascertain the contribution of various physicochemical factors to pectinase production.

Results: The primary and secondary pectinase activity screenings by Aspergillus niger LFP-1 were performed using pectin screening agar and shake flask system, respectively. The finding revealed that the locally isolated strain is able to secrete favourable pectinase production. Before improvement, the pectinase production was 0.88 ± 0.09 U/mL. However, the improved conditions such as 6 days of the cultivation period, agitation speed of 150 rpm, inoculum size of 1 × 10⁶ spores/mL, 2.5% (w/v) citrus pectin, and 0.4% (w/v) ammonium nitrate could significantly increase pectinase production up to 7.41 ± 0.24 U/mL, representing an 88% increase. In this study, supplementing 2.5% (w/v) citrus pectin to the culture medium as a carbon source increased enzyme production by up to 3.07 ± 0.17 U/mL. Meanwhile, 0.4% (w/v) ammonium nitrate was used as a nitrogen source yielding the highest enzyme activity with a value of 6.86 ± 0.07 U/mL.

Conclusion: Thus, the locally isolated fungal strain, A. niger LFP-1 has outstanding pectinase-producing capability and can be utilized for the commercial production of pectinase. The improved cultural conditions significantly increase pectinase production and shorten the incubation period from 8 days (before improvement) to 6 days (after improvement).

Background: Horseshoe crab (Tachypleus gigas) amebocytes are useful biomedical components for endotoxin detection, and their growing needs for biomedical purposes cause the horseshoe crab population to decline. Factor C synthesis via genetic engineering offers a solution to replace natural horseshoe crab's factor C and prevent its excessive harvest from nature. In response to these concerns, this study aimed to characterize the amebocyte lysates and factor C protein modeling of T. gigas originated from Banyuasin South Sumatra Estuary.

Methods and results: Sampling of T. gigas was carried out in Banyuasin South Sumatra Estuary, Indonesia. The endotoxin test or TAL (Tachypleus amebocyte lysates) assay was performed using gel coagulation method. Protein characterization of protease enzyme was conducted by protease activity, SDS-PAGE, and zymogram analysis. The cDNA of mitochondrial COI gene was amplified for molecular identification followed by cDNA cloning of factor C. Protein modeling was investigated by molecular docking and molecular dynamic (MD) simulation. Endotoxin test results showed that TAL-35 had endotoxin sensitivity in a range of 0.0156-1 EU/ml, while TAL 36 had a sensitivity between 00,625 and 1 EU/ml. T. gigas amebocytes have protease activity in molecular mass sizes less than 60 kDa, with 367 U/ml for TAL 35 and 430 U/ml for TAL 36. The molecular identification revealed 98.68% identity similarity to T. gigas. The docking results suggested three ligands; i.e., diphosphoryl lipid A, core lipid A, and Kdo2 lipid A can be activators of the factor C protein by binding to the region of the receptor to form a ligand-receptor complex.

Conclusions: Endotoxins can be detected using horseshoe crab amebocytes. The presence of proteases is considered responsible for this ability, as evidenced by casein zymogram results. According to docking and MD analysis, we found that lipopolysaccharides (LPS) participate to the binding site of factor C.

Background: MicroRNAs (miRNAs) are small endogenous RNAs with an approximate length of 18-22 nucleotides and involved in the regulation of gene expression in transcriptional or post-transcriptional levels. They were found to be associated with leaf morphogenesis, flowering time, vegetative phase change, and response to environmental cues in plants, where they act as a critical regulatory factor. The nature of high conservancy of plant miRNAs within the plant species made it possible to detect the conserved miRNAs by computational approaches. Expressed Sequence Tags (EST) based comparative genomic approaches provide advantages over wet lab approaches as it is convenient, easy to carry out and less time consuming. EST-based in silico approach can unravel new conserved miRNAs in plants, even when the complete genome sequence is not available.

Results: To identify the novel miRNAs, a total of 46,865 ESTs from Jatropha curcas were searched for homology to all available 6746 mature miRNAs of plant eudicotyledons. Finally, we ended up with 12 novel miRNAs in Jatropha that range from 18 to 19 nucleotides where their respective precursor miRNAs had 54.11-71.76% (A + U) content. The putative miRNAs belong to 12 individual miRNA family and most of them have higher (A + U) content ranging from 47.36 to 77.77% than their respective miRNA homologs. Many of the target genes by the newly identified miRNAs were associated with plant growth and development, stress response, defense and hormone signaling, and oil synthesis pathways.

Conclusion: These findings have the potential to speed up miRNA identification and expand our understanding of miRNA functions in J. curcas.

Background: Gymnema sylvestre (Retz.) R. Br. ex Schult. is a well-known medicinal plant against diabetes in India. There is as such no organized cultivation in India, and the plant is still being collected from the wild for their therapeutic uses. It is, therefore, important to estimate the genetic diversity and population genetic structure of G. sylvestre to ascertain the genetically diverse germplasm. The present study, therefore, was undertaken to analyze the genetic variability in 118 accessions belonging to 11 wild populations of G. sylvestre using directed amplification of minisatellite-region DNA (DAMD) and inter simple sequence repeats (ISSR).

Results: The present genetic analyses of 11 populations with 25 markers (8 DAMD and 17 ISSR) revealed significant genetic diversity (H = 0.26, I = 0.40, PPL = 80.89%) at a species level, while the average genetic diversity at the population level was low. Among the 11 populations studied, PCH and UTK populations showed maximum genetic diversity, followed by KNR and AMB, while TEL population revealed the lowest genetic diversity. AMOVA and G_st values (0.18) revealed that most of the genetic variations are found within populations and very less among populations, and higher gene flow (N_m = 2.29) was found to be responsible for the genetic homogenization of the populations. The clustering pattern resulting from the UPGMA dendrogram was in congruence with STRUCTURE and PCoA, segregating all the 11 populations into two main genetic clusters: cluster I (populations of North and Central India) and cluster II (populations of South India). The clustering patterns obtained from all three statistical methods indicate that the genetic structure in G. sylvestre populations corresponds to the geographical diversity of the populations and represents a strong genetic structure.

Conclusion: The genetically diverse populations identified during the present study could be a potential genetic resource for further prospecting and conserving this important plant resource.

Background: Lichens are complex plants living in symbiotic relationship between fungi and algae. They are used for human and animal nutrition and are used in folk medicine in many countries over a considerable period of time. In the present study, various solvent extracts of Trypethelslium virens and Phaeographis dendritica were tested for their antioxidant and antimicrobial activity.

Results: The phytochemical analysis by GC/MS revealed phenolics (1.273%), terpene (0.963%), hydrocarbons (2.081%), benzofurans (2.081%), quinone (1.273%), alkanes (0.963%), and aliphatic aldehydes (0.963%) as the predominant compounds in Trypethellium virens SPTV02, whereas secondary alcohol (1.184%), alkaloids (1.184%), and fatty acids (4.466) were the major constituents in Phaeographis dendritica. The antioxidant property of methanolic extract of T. virens and P. dendritica revealed the presence of total phenolic and terpenoids. The methanolic extracts of both the lichens exhibited encouraging DPPH antiradical activity, with the IC50 of 62.4 ± 0.76 µg/ml for T. virens and 68.48 ± 0.45 µg/ml for P. dendritica. Similarly, ferric reducing power assay result exhibited higher reducing activity. Further, the lichen extracts (methanolic) indicated promising antimicrobial activities against pathogens showing MIC from 62.5 to 500 µg/ml.

Conclusion: The study results concludes that both the lichens could be used as new natural source of antioxidants and antimicrobial agents which can be exploited for pharmaceutical applications.

Background: The dynamics of mammalian follicular development and atresia is an intricate process involving the cell-cell communication mediated by secreted ovarian factors. These interactions are critical for oocyte development and regulation of follicular atresia which in part are mediated by keratinocyte growth factor (KGF) and kit ligand (KITLG), but their roles in the regulation of apoptosis in buffalo granulosa cells have not yet been defined. During mammalian follicular development, granulosa cell apoptosis triggers the atresia so ~ 1% follicles reach the ovulation stage. In the present study, we used buffalo granulosa cells to examine the effects of KGF and KITLG in apoptosis regulation and investigated potential mechanism on Fas-FasL and Bcl-2 signaling pathways.

Result: Isolated buffalo granulosa cells were cultured with KGF and KITLG proteins using different doses (0, 10, 20, and 50 ng/ml) independently or in combination. Expression analysis for both anti-apoptotic (Bcl-2, Bcl-xL, and cFLIP) and pro-apoptotic (Bax, Fas, and FasL) genes at transcriptional levels were carried out by real-time PCR. Upon treatments, expression levels of anti-apoptotic genes were significantly upregulated in a dose-dependent manner, showing an upregulation at 50 ng/ml (independently), and at 10 ng/ml in combination. Additionally, upregulation of growth-promoting factors, bFGF, and α-Inhibin was also observed.

Conclusions: Our findings suggest the potential roles of KGF and KITLG in determining granulosa cell growth and regulating apoptosis.

Background: Hepatocellular carcinoma (HCC) is among the common cancers, but difficult to diagnose and treat. L-asparaginase has been introduced in the treatment protocol of pediatric acute lymphoblastic leukemia (ALL) since the 1960s with a good outcome and increased survival rates to nearly 90%. Moreover, it has been found to have therapeutic potential in solid tumors. Production of glutaminase-free-L-asparaginase is of interest to avoid glutaminase-related toxicity and hypersensitivity. In the current study, an extracellular L-asparaginase that is free of L-glutaminase was purified from the culture filtrate of an endophytic fungus Trichoderma viride. The cytotoxic effect of the purified enzyme was evaluated in vitro against a panel of human tumor cell lines and in vivo against male Wister albino mice intraperitoneally injected with diethyl nitrosamine (200 mg/kg bw), followed by (after 2 weeks) oral administration of carbon tetrachloride (2 mL/kg bw). This dose was repeated for 2 months, and after that, the blood samples were collected to estimate hepatic and renal injury markers, lipid profiles, and oxidative stress parameters.

Results: L-asparaginase was purified from T. viride culture filtrate with 36 purification folds, 688.1 U/mg specific activity, and 38.9% yield. The highest antiproliferative activity of the purified enzyme was observed against the hepatocellular carcinoma (Hep-G2) cell line, with an IC₅₀ of 21.2 g/mL, which was higher than that observed for MCF-7 (IC₅₀ 34.2 g/mL). Comparing the DENA-intoxicated group to the negative control group, it can be demonstrated that L-asparaginase adjusted the levels of the liver function enzymes and the hepatic injury markers that had previously changed with DENA intoxication. DENA causes kidney dysfunction and altered serum albumin and creatinine levels as well. Administration of L-asparaginase was found to improve the levels of the tested biomarkers including kidney and liver function tests. L-asparaginase treatment of the DENA-intoxicated group resulted in a significant improvement in the liver and kidney tissues to near normal similar to the healthy control group.

Conclusion: The results suggest that this purified T. viride L-asparaginase may be able to delay the development of liver cancer and may be used as a potential candidate for future application in medicine as an anticancer medication.

Thermostable enzymes are enzymes that can withstand elevated temperatures as high as 50 °C without altering their structure or distinctive features. The potential of thermostable enzymes to increase the conversion rate at high temperature has been identified as a key factor in enhancing the efficiency of industrial operations. Performing procedures at higher temperatures with thermostable enzymes minimises the risk of microbial contamination, which is one of the most significant benefits. In addition, it helps reduce substrate viscosity, improve transfer speeds, and increase solubility during reaction operations. Thermostable enzymes offer enormous industrial potential as biocatalysts, especially cellulase and xylanase, which have garnered considerable amount of interest for biodegradation and biofuel applications. As the usage of enzymes becomes more common, a range of performance-enhancing applications are being explored. This article offers a bibliometric evaluation of thermostable enzymes. Scopus databases were searched for scientific articles. The findings indicated that thermostable enzymes are widely employed in biodegradation as well as in biofuel and biomass production. Japan, the United States, China, and India, as along with the institutions affiliated with these nations, stand out as the academically most productive in the field of thermostable enzymes. This study's analysis exposed a vast number of published papers that demonstrate the industrial potential of thermostable enzymes. These results highlight the significance of thermostable enzyme research for a variety of applications.

Background: The prevalence of diarrhoea remains high despite efforts by governments and NGOs to reverse trend. This study investigated the antidiarrhoeal activity and mechanism of Spondias mombin leaf fraction rich in quercetin-3-O-β-D-glucopyranoside (Q3G-RF) because of the acclaimed therapeutic efficacy. Secretory, osmotic, and infectious diarrhoea models using castor oil, magnesium sulphate, and Shigella flexneri respectively were evaluated at the doses of 100, 200, and 400 mg/kg in Wistar rats. Enteropathy was induced with castor oil and magnesium sulphate, while gastrointestinal motility was determined with charcoal meal.

Results: Findings showed no mortality after 14 days of experimental period and no significant changes in behaviour, food, and water consumption. Relative to control, Q3G-RF inhibited the three models of diarrhoea, enteropathy, and gastrointestinal motility; bacterial colonies were reduced by Q3G-RF, while it improved the relative body weight of the animals. Q3G-RF also increased the intestinal concentration/activity of glucose, total protein, and Na⁺-K⁺ ATPase but reduced the concentration of TNF-α, PGE₂, IL-1β, nitric oxide, Na⁺, K⁺, and Cl^- in the diarrhoeal models. The intestinal fluid level of K⁺, Na⁺, and Cl^- was significantly decreased by Q3G-RF in the enteropathy model. Length of the small intestine in the motility model was also increased by Q3G-RF, while peristaltic index and inhibition of peristalsis were reduced.

Conclusion: Overall, quercetin-3-O-β-D-glucopyranoside from Spondias mombin leaves demonstrated efficacy against infectious, secretory, and osmotic form of diarrhoeal and further justified its traditional use in the treatment of diarrhoea due to its antimotility, antisecretory, and antimicrobial properties by mechanism related to enhanced Na⁺-K⁺ ATPase, repressed nitric oxide, and suppressed prostaglandins.