Cloning, expression and purification of cellobiohydrolase gene from Caldicellulosiruptor bescii for efficient saccharification of plant biomass.

Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme's catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against pNPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60-80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w/v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector.

CRISPR/Cas9: A revolutionary genome editing tool for human cancers treatment.

Cancer is a genetic disease stemming from genetic and epigenetic mutations and is the second most common cause of death across the globe. Clustered regularly interspaced short palindromic repeats (CRISPR) is an emerging gene-editing tool, acting as a defense system in bacteria and archaea. CRISPR/Cas9 technology holds immense potential in cancer diagnosis and treatment and has been utilized to develop cancer disease models such as medulloblastoma and glioblastoma mice models. In diagnostics, CRISPR can be used to quickly and efficiently detect genes involved in various cancer development, proliferation, metastasis, and drug resistance. CRISPR/Cas9 mediated cancer immunotherapy is a well-known treatment option after surgery, chemotherapy, and radiation therapy. It has marked a turning point in cancer treatment. However, despite its advantages and tremendous potential, there are many challenges such as off-target effects, editing efficiency of CRISPR/Cas9, efficient delivery of CRISPR/Cas9 components into the target cells and tissues, and low efficiency of HDR, which are some of the main issues and need further research and development for completely clinical application of this novel gene editing tool. Here, we present a CRISPR/Cas9 mediated cancer treatment method, its role and applications in various cancer treatments, its challenges, and possible solution to counter these challenges.

Multifarious revolutionary aspects of microbial keratinases: an efficient green technology for future generation with prospective applications.

Since the dawn of century, tons of keratin bio-waste is generated by the poultry industry annually, and they end up causing environmental havoc. Keratins are highly flexible fibrous proteins which exist in α- and ß- forms and provide mechanical strength and stability to structural appendages. The finding of broad-spectrum protease, keratinase, from thermophilic bacteria and fungi, has provided an eco-friendly solution to hydrolyze the peptide bonds in highly recalcitrant keratinous substances such as nails, feathers, claws, and horns into valuable amino acids. Microorganisms produce these proteolytic enzymes by techniques of solid-state and submerged fermentation. However, solid-state fermentation is considered as a yielding approach for the production of thermostable keratinases. This review prioritized the molecular and biochemical properties of microbial keratinases, and the role of keratinases in bringing prodigious impact for the sustainable progress of the economy. It also emphasizes on the current development in keratinase production with the focus to improve the biochemical properties related to enzyme's catalytic activity and stability, and production of mutant and cloned microbial strains to improve the yield of keratinases. Recently, multitude molecular approaches have been employed to enhance enzyme's productivity, activity, and thermostability which makes them suitable for pharmaceutical industry and for the production of animal feed, organic fertilizers, biogas, clearing of animal hides, and detergent formulation. Hence, it can be surmised that microbial keratinolytic enzymes are the conceivable candidates for numerous commercial and industrial applications.

Genus Thermotoga: A valuable home of multifunctional glycoside hydrolases (GHs) for industrial sustainability.

Nature is a dexterous and prolific chemist for cataloging a number of hostile niches that are the ideal residence of various thermophiles. Apart from having other species, these subsurface environments are considered a throne of bacterial genus Thermotoga. The genome sequence of Thermotogales encodes complex and incongruent clusters of glycoside hydrolases (GHs), which are superior to their mesophilic counterparts and play a prominent role in various applications due to their extreme intrinsic stability. They have a tremendous capacity to use a wide variety of simple and multifaceted carbohydrates through GHs, formulate fermentative hydrogen and bioethanol at extraordinary yield, and catalyze high-temperature reactions for various biotechnological applications. Nevertheless, no stringent rules exist for the thermo-stabilization of biocatalysts present in the genus Thermotoga. These enzymes endure immense attraction in fundamental aspects of how these polypeptides attain and stabilize their distinctive three-dimensional (3D) structures to accomplish their physiological roles. Moreover, numerous genome sequences from Thermotoga species have revealed a significant fraction of genes most closely related to those of archaeal species, thus firming a staunch belief of lateral gene transfer mechanism. However, the question of its magnitude is still in its infancy. In addition to GHs, this genus is a paragon of encapsulins which carry pharmacological and industrial significance in the field of life sciences. This review highlights an intricate balance between the genomic organizations, factors inducing the thermostability, and pharmacological and industrial applications of GHs isolated from genus Thermotoga.

A Contemporary Appraisal on Impending Industrial and Agricultural Applications of Thermophilic-Recombinant Chitinolytic Enzymes from Microbial Sources.

The ability of chitinases to degrade the second most abundant polymer, chitin, into potentially useful chitooligomers and chitin derivatives has not only rendered them fit for chitinous waste management but has also made them important from industrial point of view. At the same time, they have also been recognized to have an imperative role as promising biocontrol agents for controlling plant diseases. As thermostability is an important property for an industrially important enzyme, various bacterial and fungal sources are being exploited to obtain such stable enzymes. These stable enzymes can also play a role in agriculture by maintaining their stability under adverse environmental conditions for longer time duration when used as biocontrol agent. Biotechnology has also played its role in the development of recombinant chitinases with enhanced activity, thermostability, fungicidal and insecticidal activity via recombinant DNA techniques. Furthermore, a relatively new approach of generating pathogen-resistant transgenic plants has opened new ways for sustainable agriculture by minimizing the yield loss of valuable crops and plants. This review focuses on the potential applications of thermostable and recombinant microbial chitinases in industry and agriculture.

Eminent Industrial and Biotechnological Applications of Laccases from Bacterial Source: a Current Overview.

Laccases are blue multicopper oxidases that oxidize a wide range of phenolic as well as non-phenolic substrates in the presence or absence of mediators. They occur in various species of bacteria, fungi, insects, and plants; bacterial laccases show high substrate specificity. Bacteria produce these enzymes either extracellularly or intracellularly and exhibit stability to a wide range of pH and temperature. Therefore, they are suitable for various industrial processes such as food, textile, and paper and pulp industry. They are also valuable for producing biofuels, pharmaceuticals, biosensors, and degradation of various environmental pollutants and xenobiotics compounds. Since bacterial laccases are more versatile in the sense of nutritional needs and ecological factors, their use can provide a promising solution to various problems related to industry and the field of biotechnology. However, there is a need for a thorough understanding of the chemistry and activity of bacterial laccases to enable their full potential use in bioremediation and biofuel production.

Insights into the evolutionary and prophylactic analysis of SARS-CoV-2: A review.

In late 2019, following the emergence of a ß-originated SARS-CoV-2, phylogenetic and evolutionary approaches have been demonstrated to strengthen the diagnostic and prophylactic stratagem of COVID-19 at an unprecedented level. Despite its clinical prominence, the SARS-CoV-2 gene set remains largely irrefutable by impeding the dissection of COVID-19 biology. However, many pieces of molecular and serological evidence have predicted that SARS-CoV-2 related viruses carry their roots from bats and pangolins of South East Asia. Analysis of viral genome predicts that point mutations at a rate of 10-4 nucleotides per base in the receptor-binding domain allow the emergence of new SARS-CoV-2 genomic variants at regular intervals. Research in the evolution of molecular pathways involved in emergence of pandemic is critical for the development of therapeutics and vaccines as well as the prevention of future zoonosis. By determining the phyletic lineages of the SARS-CoV-2 genomic variants and those of the conserved regions in the accessory and spike proteins of all the SARS-related coronaviruses, a universal vaccine against all human coronaviruses could be formulated which would revolutionize the field of medicine. This review highlighted the current development and future prospects of antiviral drugs, inhibitors, mesenchymal stem cells, passive immunization, targeted immune therapy and CRISPR-Cas-based prophylactic and therapeutic strategies against SARS-CoV-2. However, further investigations on Covid-19 pathogenesis is required for the successful fabrication of successful antivirals.

Biochemical and Immunological implications of Lutein and Zeaxanthin.

Throughout history, nature has been acknowledged for being a primordial source of various bioactive molecules in which human macular carotenoids are gaining significant attention. Among 750 natural carotenoids, lutein, zeaxanthin and their oxidative metabolites are selectively accumulated in the macular region of living beings. Due to their vast applications in food, feed, pharmaceutical and nutraceuticals industries, the global market of lutein and zeaxanthin is continuously expanding but chemical synthesis, extraction and purification of these compounds from their natural repertoire e.g., plants, is somewhat costly and technically challenging. In this regard microbial as well as microalgal carotenoids are considered as an attractive alternative to aforementioned challenges. Through the techniques of genetic engineering and gene-editing tools like CRISPR/Cas9, the overproduction of lutein and zeaxanthin in microorganisms can be achieved but the commercial scale applications of such procedures needs to be done. Moreover, these carotenoids are highly unstable and susceptible to thermal and oxidative degradation. Therefore, esterification of these xanthophylls and microencapsulation with appropriate wall materials can increase their shelf-life and enhance their application in food industry. With their potent antioxidant activities, these carotenoids are emerging as molecules of vital importance in chronic degenerative, malignancies and antiviral diseases. Therefore, more research needs to be done to further expand the applications of lutein and zeaxanthin.

MicroRNA, a Promising Biomarker for Breast and Ovarian Cancer: A Review.

BACKGROUND: MicroRNAs (miRNA) belong to the substantial class of posttranscriptional gene regulators with decisive functions in typical cellular and disease progressions. They are short RNA molecules that are not translated into proteins but bind to the complementary sites of various mRNAs, thus blocking them and leading to translational inhibition. OBJECTIVE: These miRNA molecules act as signatory molecules or biomarkers for various types of malignancies. Different miRNAs are involved in different cancer-linked pathways depending on the nature, stage, and kind of cancer. The objective of this article is to discuss and review the role and significance of various miRNAs in two of the most prominent cancers; breast and ovarian cancer. METHODS: The role of miRNAs in the instigation, propagation, and metastasis of melanoma has been elucidated. RESULTS: This article focuses on the up- and down-regulation of various miRNAs in breast and ovarian cancer, with stress on diversity in their occurrence and specificity in their threshold levels in certain types and stages of cancer, suggesting their potential role as an effective diagnostic and treatment tool for cancer. CONCLUSION: Considering all the aforementioned information, it can be concluded that miRNAs can act as potential biomarkers for the diagnosis of breast and ovarian cancers.