In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.
Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein's genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form.
Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.
Pasteurella multocida is the main cause of haemorrhagic septicaemia (HS) outbreak in livestock, such as cattle and buffaloes. Conventional vaccines such as alum-precipitated or oil-adjuvant broth bacterins were injected subcutaneously to provide protection against HS. However, the immunity developed is only for short term and needed to be administered frequently. In our previous study, a short gene fragment from Pasteurella multocida serotype B was obtained via shotgun cloning technique and later was cloned into bacterial expression system. pQE32-ABA392 was found to possess immunogenic activity towards HS when tested in vivo in rat model. In this study, the targeted gene fragment of ABA392 was sub-cloned into a DNA expression vector pVAX1 and named as pVAX1-ABA392. The new recombinant vaccine was stable and expressed on mammalian cell lines. Serum sample collected from a group of vaccinated rats for ELISA test shows that the antibody in immunized rats was present at high titer and can be tested as a vaccine candidate with challenge in further studies. This successful recombinant vaccine is immunogenic and potentially could be used as vaccine in future against HS.
Biomolecules produced by living organisms can perform vast array of functions and play an important role in the cell. Important biomolecules such as lysozyme, bovine serum albumin (BSA), and bromelain are often studied by researchers due to their beneficial properties. The application of reverse micelles is an effective tool for protein separation from their sources due to the special system structure. Mechanisms of transferring biomolecules and factors that influence the extraction of biomolecules are reviewed in this paper. The enhancement of biomolecule extraction could be achieved depending on the properties of reverse micelles. This paper provides an overall review on lysozyme, BSA, and bromelain extraction by reverse micelle for various applications.
The application of native enzymes may not be economical owing to the stability factor. A smaller protein molecule may be less susceptible to external stresses. Haloalkane dehalogenases (HLDs) that act on toxic haloalkanes may be incorporated as bioreceptors to detect haloalkane contaminants. Therefore, this study aims to develop mini proteins of HLD as an alternative bioreceptor which was able to withstand extreme conditions. Initially, the mini proteins were designed through computer modeling. Based on the results, five designed mini proteins were deemed to be viable stable mini proteins. They were then validated through experimental study. The smallest mini protein (model 5) was chosen for subsequent analysis as it was expressed in soluble form. No dehalogenase activity was detected, thus the specific binding interaction of between 1,3-dibromopropane with mini protein was investigated using isothermal titration calorimetry. Higher binding affinity between 1,3-dibromopropane and mini protein was obtained than the native. Thermal stability study with circular dichroism had proven that the mini protein possessed two times higher Tm value at 83.73 °C than the native at 43.97 °C. In conclusion, a stable mini protein was successfully designed and may be used as bioreceptors in the haloalkane sensor that is suitable for industrial application.
Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.
Over the past few decades, cancer immunotherapy has experienced a significant revolution due to the advancements in immune checkpoint inhibitors (ICIs) and adoptive cell therapies (ACTs), along with their regulatory approvals. In recent times, there has been hope in the effectiveness of cancer vaccines for therapy as they have been able to stimulate de novo T-cell reactions against tumor antigens. These tumor antigens include both tumor-associated antigen (TAA) and tumor-specific antigen (TSA). Nevertheless, the constant quest to fully achieve these abilities persists. Therefore, this review offers a broad perspective on the existing status of cancer immunizations. Cancer vaccine design has been revolutionized due to the advancements made in antigen selection, the development of antigen delivery systems, and a deeper understanding of the strategic intricacies involved in effective antigen presentation. In addition, this review addresses the present condition of clinical tests and deliberates on their approaches, with a particular emphasis on the immunogenicity specific to tumors and the evaluation of effectiveness against tumors. Nevertheless, the ongoing clinical endeavors to create cancer vaccines have failed to produce remarkable clinical results as a result of substantial obstacles, such as the suppression of the tumor immune microenvironment, the identification of suitable candidates, the assessment of immune responses, and the acceleration of vaccine production. Hence, there are possibilities for the industry to overcome challenges and enhance patient results in the coming years. This can be achieved by recognizing the intricate nature of clinical issues and continuously working toward surpassing existing limitations.
The world of pharmaceutical research has been increasingly turning its gaze toward the treasure trove of natural products in search of novel drugs and therapeutic agents. Amidst the vast array of medicinal plants that dot our planet, the Asclepiadaceae family unexplored species have piqued the interest of researchers. Both medicinal plants are indigenous to specific regions and have been integral to traditional medicine systems for centuries. This systematic review aims to provide a comprehensive summary of the current knowledge regarding the phytochemical profile of these plants and their potential implications in the pharmaceutical industry. These plants are rich in phytochemical constituents such as alkaloids, flavonoids, terpenoids, phenolic compounds, glycosides, and saponins. These constituents have been found to exhibit a range of pharmacological activities. They have antimicrobial properties, providing a defense against various microorganisms. They also show anti-inflammatory properties, helping to reduce inflammation in the body. In addition, these plants have antioxidant properties, which help protect cells from damage by harmful free radicals. They have shown anticancer activity, offering potential for cancer treatment. Their neuroprotective properties could be beneficial in treating neurological disorders. The analgesic properties of these plants could be harnessed for pain relief. Furthermore, they have antidiabetic properties, offering potential for diabetes management. The hope is that this review will stimulate further research into these fascinating plants and contribute to discovering new drugs from natural herbs.
Fatty acid desaturase enzymes are capable of inserting double bonds between carbon atoms of saturated fatty acyl-chains to produce unsaturated fatty acids. A gene coding for a putative Δ9-fatty acid desaturase-like protein was isolated from a cold-tolerant Pseudomonas sp. A8, cloned and heterologously expressed in Escherichia coli. The gene named as PA8FAD9 has an open reading frame of 1185 bp and codes for 394 amino acids with a predicted molecular weight of 45 kDa. The enzyme showed high Δ9-fatty acid desaturase-like protein activity and increased overall levels of cellular unsaturated fatty acids in the recombinant E. coli cells upon expression at different temperatures. The results showed that the ratio of palmitoleic to palmitic acid in the recombinant E. coli cells increased by more than twice the amount observed in the control cells at 20 °C using 0.4 mM IPTG. GCMS analysis confirmed the ability of this enzyme to convert exogenous stearic acid to oleic acid incorporated into the recombinant E. coli membrane phospholipids. It may be concluded that the PA8FAD9 gene from Pseudomonas sp. A8 codes for a putative Δ9-fatty acid desaturase protein actively expressed in E. coli under the influence of temperature and an inducer.
The current study focuses on molecular cloning, expression and structural characterization of growth hormone-receptor (GHR) and its extracellular domain as growth hormone binding protein (GHBP) from the liver of Nili-Ravi buffalo (Bubalus bubalis; Bb). RNA was isolated, genes were amplified by reverse transcriptase-polymerase chain reaction and sequence was characterized. The BbGHR sequence showed three amino acid variations in the extracellular domain when compared with Indian BbGHR. For the production of full length BbGHR and BbGHBP in Escherichia coli (E. coli) BL21 (RIPL) Codon Plus, expression plasmids were constructed under the control of T7lac promoter and isopropyl β-D thiogalactopyranoside was used as an inducer. BbGHR and BbGHBP were expressed as inclusion bodies at ~ 40% and > 30% of the total E. coli proteins, respectively. The BbGHBP was solubilized and refolded by dilution method using cysteine-cystine redox potential. The recombinant BbGHBP was purified and biological activity was checked on HeLa cell lines showing increase cell proliferation in the presence of ovine GH (oGH), hence justifying the increase in the half-life of GH in the presence of BbGHBP. For the molecular interactions of oGH-BbGHBP multiple docking programs were employed to explore the subsequent interactions which showed high binding affinity and presence of large number of hydrogen bonds. Molecular Dynamics studies performed to examine the stability of proteins and exhibited stable structures along with favorable molecular interactions. This study has described the sequence characterization of BbGHR in Nili-Ravi buffaloes and hence provided the basis for the assessment of GH-GHR binding in other Bovidae species.
The synergistic effects of antimicrobial nanostructures with antibiotics present a promising solution for overcoming resistance in methicillin-resistant Staphylococcus aureus (MRSA). Previous studies have introduced iron as a novel coating for silver nanoparticles (AgNPs) to enhance both economic efficiency and potency against S. aureus. However, there are currently no available data on the potential of these novel nanostructures to reverse MRSA resistance. To address this gap, a population study was conducted within the MRSA community, collecting a total of 48 S. aureus isolates from skin lesions. Among these, 21 isolates (43.75%) exhibited cefoxitin resistance as determined by agar disk diffusion assay. Subsequently, a PCR test confirmed the presence of the mecA gene in 20 isolates, verifying them as MRSA. These results highlight the cefoxitin disk diffusion susceptibility test as an accurate screening method for predicting mecA-mediated resistance in MRSA. Synergy tests were performed on cefoxitin, serving as a marker antibiotic, and iron-coated AgNPs (Fe@AgNPs) in a combination study using the checkerboard assay. The average minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) of cefoxitin were calculated as 11.55 mg/mL and 3.61 mg/mL, respectively. The findings indicated a synergistic effect (FIC index
Lovastatin is an anti-cholesterol medicine that is commonly prescribed to manage cholesterol levels, and minimise the risk of suffering from heart-related diseases. Aspergillus terreus (ATCC 20542) supplied with carbohydrates or sugar alcohols can produce lovastatin. The present work explored the application of metabolic engineering in A. terreus to re-route the precursor flow towards the lovastatin biosynthetic pathway by simultaneously overexpressing the gene for acetyl-CoA carboxylase (acc) to increase the precursor flux, and eliminate ( +)-geodin biosynthesis (a competing secondary metabolite) by removing the gene for emodin anthrone polyketide synthase (gedC). Alterations to metabolic flux in the double mutant (gedCΔ*accox) strain and the effects of using two different substrate formulations were examined. The gedCΔ*accox strain, when cultivated with a mixture of glycerol and lactose, significantly (p
Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile-Thr-Arg-Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein-solvent interaction.
Seaweeds are photosynthetic marine macroalgae known for their rapid biomass growth and their significant contributions to global food and feed production. Seaweeds play a crucial role in mitigating various environmental issues, including greenhouse gases, ocean acidification, hypoxia, and eutrophication. Tropical seaweeds are typically found in tropical and subtropical coastal zones with warmer water temperatures and abundant sunlight. These tropical seaweeds are rich sources of proteins, vitamins, minerals, fibers, polysaccharides, and bioactive compounds, contributing to their health-promoting properties and their diverse applications across a range of industries. The productivity, cultivability, nutritional quality, and edibility of tropical seaweeds have been well-documented. This review article begins with an introduction to the growth conditions of selected tropical seaweeds. Subsequently, the multifunctional properties of tropical seaweeds including antioxidant and anti-inflammatory, anti-coagulant, anti-carcinogenic and anti-proliferative, anti-viral, therapeutic and preventive properties were comprehensively evaluated. The potential application of tropical seaweeds as functional foods and feeds, as well as their contributions to sustainable cosmetics, bioenergy, and biofertilizer production were also highlighted. This review serves as a valuable resource for researchers involved in seaweed farming as it provides current knowledge and insights into the cultivation and utilization of seaweeds.
A broad range of cell lines with characteristic features are used as bio-factories to produce recombinant proteins for basic research and therapeutic purposes. Genetic engineering strategies have been used to manipulate the genome of mammalian cells, insects, and yeasts for heterologous expression. One reason is that the glycosylation pattern of the expression hosts differs somehow from mammalian cells, which may cause immunogenic reactions upon administration in humans. CRISPR-Cas9 is a simple, efficient, and versatile genome engineering tool that can be programmed to precisely make double-stranded breaks at the desired loci. Compared to the classical genome editing methods, a CRISPR-Cas9 system is an ideal tool, providing the opportunity to integrate or delete genes from the target organisms. Besides broadened applications, limited studies have used CRISPR-Cas9 for editing the endogenous pathways in expression systems for biopharmaceutical applications. In the present review, we discuss the use of CRISPR-Cas9 in expression systems to improve host cell lines, increase product yield, and humanize glycosylation pathways by targeting intrinsic genes.
Solid waste generation is a huge contributor to environmental pollution issues, and food wastes are prominent in this category due to their large generation on a day-to-day basis. Thus, the settlement of daily food waste is one of the major constraints and needs innovative manufacturing sheme to valorize solid waste in sustainable manner. Moreover, these food wastes are rich in organic content, which has promising scope for their value-added products. In the present study, raw mango seed waste has been biotransformed to produce bacterial hydrolytic enzymes as feedstock. On investigating the impact of substrate, the highest bacterial cellulase production was recorded to be 18 IU/gds FP (filter paper) in 24 h of microbial incubation at 5 g of substrate in solid-state fermentation (SSF). Furthermore, at 40 °C and pH 6.0, 23 IU/gds FP enzyme could be produced in 24 h of SSF. Beside this, on comparing the influence of inorganic and organic nitrogen sources, urea has been found to provide better cellulase production, which yielded 28 IU/gds FP in 24 h of incubation, along with 77 IU/gds BG (β-glucosidase) and 89 IU/gds EG (endoglucanase). On the other hand, Tween-40 and Tween-80, two different surfactants, were employed at a 1.0% concentration for 24 h of incubation. It was noticed that Tween-80 showed complete enzyme activity at 24 h, which was found to be relatively superior to that of Tween-40. This study may have potential utility in enzyme production using mango seed as a food waste for various industrial applications.
Cordyceps, an entomopathogenic fungus belonging to the Ascomycota phylum, is a familiar remedial mushroom that is extensively used in the traditional medicinal system, especially in South Asian nations. The significance of this genus' members in a range of therapeutic and biotechnological applications has long been acknowledged. The exceedingly valuable fungus Ophiocordyceps sinensis (Cordyceps sinensis) is found in the alpine meadows of Bhutan, Nepal, Tibet, and India, where it is severely harvested. Driven by market demand and ecological concerns, the study highlights challenges in natural C. sinensis collection and emphasizes the shift towards sustainable artificial cultivation methods. This in-depth review navigates Cordyceps cultivation strategies, focusing on C. sinensis and the viable alternative, C. militaris. The escalating demand for Cordyceps fruiting bodies and bioactive compounds prompts a shift toward sustainable artificial cultivation. While solid-state fermentation on brown rice remains a traditional method, liquid culture, especially submerged and surface/static techniques, emerges as a key industrial approach, offering shorter cultivation periods and enhanced cordycepin production. The review accentuates the adaptability and scalability of liquid culture, providing valuable insights for large-scale Cordyceps production. The future prospects of Cordyceps cultivation require a holistic approach, combining scientific understanding, technological innovation, and sustainable practices to meet the demand for bioactive metabolites while ensuring the conservation of natural Cordyceps populations.
The present study focused on preparing and characterizing magnetite-polyvinyl alcohol (PVA) hybrid nanoparticles using Acanthophora spicifera marine algae extract as a reducing agent. Various analytical techniques, including UV-Visible spectrometry, Fourier-transform infrared (FTIR) analysis, energy-dispersive X-ray (EDX), scanning electron microscopy (SEM), and X-ray diffraction (XRD) analysis, were used to characterize the nanoparticles. The results showed the successful synthesis of nanoparticles with a characteristic color change and absorption peak at 400 nm in UV-Visible spectrometry. FTIR analysis indicated an interaction between the carboxyl group and magnetite-polyvinyl alcohol hybrid ions. SEM analysis revealed spherical nanoparticles with sizes ranging from 20 to 100 nm. EDX analysis confirmed the presence of strong magnetite peaks in Acanthophora spicifera, validating successful preparation. XRD analysis indicated the crystalline nature of the nanoparticles. Furthermore, the antimicrobial potential of As-PVA-MNPs was evaluated, demonstrating a significant zone of inhibition against tested bacterial and fungal samples at a concentration of 100 µg. These findings suggest the promising antimicrobial activity of the synthesized nanoparticles for potential applications in combating pathogenic microorganisms.