Advances in Therapeutic Cancer Vaccines, Their Obstacles, and Prospects Toward Tumor Immunotherapy.

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.

Recent insight into the advances and prospects of microbial lipases and their potential applications in industry.

Enzymes play a crucial role in various industrial sectors. These biocatalysts not only ensure sustainability and safety but also enhance process efficiency through their unique specificity. Lipases possess versatility as biocatalysts and find utilization in diverse bioconversion reactions. Presently, microbial lipases are gaining significant focus owing to the rapid progress in enzyme technology and their widespread implementation in multiple industrial procedures. This updated review presents new knowledge about various origins of microbial lipases, such as fungi, bacteria, and yeast. It highlights both the traditional and modern purification methods, including precipitation and chromatographic separation, the immunopurification technique, the reversed micellar system, the aqueous two-phase system (ATPS), and aqueous two-phase flotation (ATPF), moreover, delves into the diverse applications of microbial lipases across several industries, such as food, vitamin esters, textile, detergent, biodiesel, and bioremediation. Furthermore, the present research unveils the obstacles encountered in employing lipase, the patterns observed in lipase engineering, and the application of CRISPR/Cas genome editing technology for altering the genes responsible for lipase production. Additionally, the immobilization of microorganisms' lipases onto various carriers also contributes to enhancing the effectiveness and efficiencies of lipases in terms of their catalytic activities. This is achieved by boosting their resilience to heat and ionic conditions (such as inorganic solvents, high-level pH, and temperature). The process also facilitates the ease of recycling them and enables a more concentrated deposition of the enzyme onto the supporting material. Consequently, these characteristics have demonstrated their suitability for application as biocatalysts in diverse industries.

Essential factors, advanced strategies, challenges, and approaches involved for efficient expression of recombinant proteins in Escherichia coli.

Producing recombinant proteins is a major accomplishment of biotechnology in the past century. Heterologous hosts, either eukaryotic or prokaryotic, are used for the production of these proteins. The utilization of microbial host systems continues to dominate as the most efficient and affordable method for biotherapeutics and food industry productions. Hence, it is crucial to analyze the limitations and advantages of microbial hosts to enhance the efficient production of recombinant proteins on a large scale. E. coli is widely used as a host for the production of recombinant proteins. Researchers have identified certain obstacles with this host, and given the growing demand for recombinant protein production, there is an immediate requirement to enhance this host. The following review discusses the elements contributing to the manifestation of recombinant protein. Subsequently, it sheds light on innovative approaches aimed at improving the expression of recombinant protein. Lastly, it delves into the obstacles and optimization methods associated with translation, mentioning both cis-optimization and trans-optimization, producing soluble recombinant protein, and engineering the metal ion transportation. In this context, a comprehensive description of the distinct features will be provided, and this knowledge could potentially enhance the expression of recombinant proteins in E. coli.

Current achievements, strategies, obstacles, and overcoming the challenges of the protein engineering in Pichia pastoris expression system.

Yeasts serve as exceptional hosts in the manufacturing of functional protein engineering and possess industrial or medical utilities. Considerable focus has been directed towards yeast owing to its inherent benefits and recent advancements in this particular cellular host. The Pichia pastoris expression system is widely recognized as a prominent and widely accepted instrument in molecular biology for the purpose of generating recombinant proteins. The advantages of utilizing the P. pastoris system for protein production encompass the proper folding process occurring within the endoplasmic reticulum (ER), as well as the subsequent secretion mediated by Kex2 as a signal peptidase, ultimately leading to the release of recombinant proteins into the extracellular environment of the cell. In addition, within the P. pastoris expression system, the ease of purifying recombinant protein arises from its restricted synthesis of endogenous secretory proteins. Despite its achievements, scientists often encounter persistent challenges when attempting to utilize yeast for the production of recombinant proteins. This review is dedicated to discussing the current achievements in the usage of P. pastoris as an expression host. Furthermore, it sheds light on the strategies employed in the expression system and the optimization and development of the fermentative process of this yeast. Finally, the impediments (such as identifying high expression strains, improving secretion efficiency, and decreasing hyperglycosylation) and successful resolution of certain difficulties are put forth and deliberated upon in order to assist and promote the expression of complex proteins in this prevalent recombinant host.

Biodegradable Carbonate Apatite Nanoparticle as a Delivery System to Promote Afatinib Delivery for Non-Small Cell Lung Cancer Treatment.

Nanomedicine-based drug-delivery systems have significant interest in cancer treatment, such as improving the stabilities and biocompatibilities, precise targeting, and reducing toxicities for non-cancerous cells. Herein, this study presents the synthesis and characterisation of carbonate apatite nanoparticles (nCA) and encapsulated afatinib (AFA) as promising drug delivery candidates for lung cancer treatment. nCA/AFA was synthesised and physicochemically characterised, then the encapsulation capacity, drug loading, and cumulative drug release profile were evaluated. Powder X-ray diffraction (PXRD) confirmed that the synthesised nCA is apatite. Fourier-transform infrared spectroscopy (FTIR) results confirmed the drug loading into the nanoparticles. High-resolution transmission electron microscopy (HR-TEM) determined the morphology of nCA and nCA/AFA and the diameters of 47.36 ± 3.16 and 42.97 ± 2.78 nm, respectively, without an unaltered nCA phase. Encapsulation efficiency (%) and drug loading (%) were 55.08% ± 1.68% and 8.19% ± 0.52%. Brunauer-Emmett-Teller (BET) and dynamic light-scattering (DLS) results revealed that the synthesised nCA is mesoporous, with a surface area of 55.53 m2/g, and is negatively charged. Atomic force microscopy (AFM) showed increasing roughness of nCA/AFA compared to nCA. The drug release from the nano-formulation nCA/AFA demonstrated slow and sustained release compared to the pure drug. Accordingly, nCA/AFA represents a promising drug delivery system for NSCLC treatment.

Discovery of new inhibitor for the protein arginine deiminase type 4 (PAD4) by rational design of α-enolase-derived peptides.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.24 % of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC50 measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35 % with IC50 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors.

The Therapeutic Effect and In Vivo Assessment of Palmitoyl-GDPH on the Wound Healing Process.

The standard treatment of open wounds via the direct usage of therapeutic agents is not without limitations with respect to healing. Small peptides can create a favorable milieu for accelerating the healing of wounds. This study presents the potential of a novel fatty acid conjugated tetrapeptide (palmitic acid-glycine-aspartic acid-proline-histidine; Palmitoyl-GDPH) in alleviating wound healing. Tetracycline was employed as a standard control drug following its significance in wound healing including biologically active and antimicrobial effects. The peptide in liquid form was applied on to a 4 cm2 full thickness wound surgically induced at the dorsum of Sprague Dawley (SD) rats. The in vivo wound treatment with Palmitoyl-GDPH for eighteen days, histologically demonstrated an almost perfect healing exhibited by increased re-epithelialization, enhanced collagen deposition, and diminished scar formation compared to the controls. In addition, the well-developed epidermal-dermal junction and ultimate stimulation of hair follicle-growth in the Palmitoyl-GDPH treated group indicated the wound to have healed as functionally viable tissues. In general, the much lower hemogram values in the Palmitoyl-GDPH group indicated that the ongoing healing is en route to an earlier recovery. Additionally, the liver, kidney, and pancreas function biomarkers being within normal limits indicated the relatively non-toxic nature of Palmitoyl-GDPH at the used dosage. These results indisputably supported the great potential of this newly synthesized Palmitoyl-GDPH to be used as an effective therapeutic agent for wound healing (this actually means creating a new wound).

Antifreeze Proteins and Their Practical Utilization in Industry, Medicine, and Agriculture.

Antifreeze proteins (AFPs) are specific proteins, glycopeptides, and peptides made by different organisms to allow cells to survive in sub-zero conditions. AFPs function by reducing the water's freezing point and avoiding ice crystals' growth in the frozen stage. Their capability in modifying ice growth leads to the stabilization of ice crystals within a given temperature range and the inhibition of ice recrystallization that decreases the drip loss during thawing. This review presents the potential applications of AFPs from different sources and types. AFPs can be found in diverse sources such as fish, yeast, plants, bacteria, and insects. Various sources reveal different α-helices and ß-sheets structures. Recently, analysis of AFPs has been conducted through bioinformatics tools to analyze their functions within proper time. AFPs can be used widely in various aspects of application and have significant industrial functions, encompassing the enhancement of foods' freezing and liquefying properties, protection of frost plants, enhancement of ice cream's texture, cryosurgery, and cryopreservation of cells and tissues. In conclusion, these applications and physical properties of AFPs can be further explored to meet other industrial players. Designing the peptide-based AFP can also be done to subsequently improve its function.

Metallointercalator [Ru(dppz)2(PIP)]2+ Renders BRCA Wild-Type Triple-Negative Breast Cancer Cells Hypersensitive to PARP Inhibition.

There is a need to improve and extend the use of clinically approved poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), including for BRCA wild-type triple-negative breast cancer (TNBC). The demonstration that ruthenium(II) polypyridyl complex (RPC) metallointercalators can rapidly stall DNA replication fork progression provides the rationale for their combination alongside DNA damage response (DDR) inhibitors to achieve synergism in cancer cells. The aim of the present study was to evaluate use of the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyrido[3,2-a:2',3'-c]phenazine, PIP = (2-(phenyl)imidazo[4,5-f][1,10]phenanthroline, Ru-PIP) alongside the PARPi olaparib and NU1025. Cell proliferation and clonogenic survival assays indicated a synergistic relationship between Ru-PIP and olaparib in MDA-MB-231 TNBC and MCF7 human breast cancer cells. Strikingly, low dose Ru-PIP renders both cell lines hypersensitive to olaparib, with a >300-fold increase in olaparib potency in TNBC, the largest nongenetic PARPi enhancement effect described to date. A negligible impact on the viability of normal human fibroblasts was observed for any combination tested. Increased levels of DNA double-strand break (DSB) damage and olaparib abrogation of Ru-PIP-activated pChk1 signaling are consistent with PARPi-facilitated collapse of Ru-PIP-associated stalled replication forks. This results in enhanced G2/M cell-cycle arrest, apoptosis, and decreased cell motility for the combination treatment compared to single-agent conditions. This work establishes that an RPC metallointercalator can be combined with PARPi for potent synergy in BRCA-proficient breast cancer cells, including TNBC.

Microwave synthesis, crystal structure, antioxidant, and antimicrobial study of new 6-heptyl-5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazoline compound.

BACKGROUND: Although the development of antibiotic and antioxidant manufacturing, the problem of bacterial resistance and food and/or cosmetics oxidation still needs more efforts to design new derivatives which can help to minimize these troubles. Benzimidazo[1,2-c]quinazolines are nitrogen-rich heterocyclic compounds that possess many pharmaceutical properties such as antimicrobial, anticonvulsant, immunoenhancer, and anticancer. RESULTS: A comparative study between two methods, (microwave-assisted and conventional heating approaches), was performed to synthesise a new quinazoline derivative from 2-(2-aminophenyl)-1H-benzimidazole and octanal to produce 6-heptyl-5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazoline (OCT). The compound was characterised using FTIR, 1H and 13C NMR, DIMS, as well as X-ray crystallography. The most significant peak in the 13C NMR spectrum is C-7 at 65.5 ppm which confirms the cyclisation process. Crystal structure analysis revealed that the molecule grows in the monoclinic crystal system P21/n space group and stabilised by an intermolecular hydrogen bond between the N1-H1A…N3 atoms. The crystal packing analysis showed that the molecule adopts zig-zag one dimensional chains. Fluorescence study of OCT revealed that it produces blue light when expose to UV-light and its' quantum yield equal to 26%. Antioxidant activity, which included DPPH· and ABTS·+ assays was also performed and statistical analysis was achieved via a paired T-test using Minitab 16 software with P < 0.05. Also, the antimicrobial assay against two Gram-positive, two Gram-negative, and one fungus was screened for these derivatives. CONCLUSIONS: Using microwave to synthesise OCT have drastically reduced reaction time, and increased yield. OCT show good antioxidant activity in one of the tests and moderate antimicrobial activity.

Optimization of Quercetin loaded Palm Oil Ester Based Nanoemulsion Formulation for Pulmonary Delivery.

In this research, the palm oil ester (POE)- based nanoemulsion formulation containing quercetin for pulmonary delivery was developed. The nanoemulsion formulation was prepared by high energy emulsification method and then further optimized using D-optimal mixture design. The concentration effects of the mixture of POE:ricinoleic acid (RC), ratio 1:1 (1.50-4.50 wt.%), lecithin (1.50-2.50 wt.%), Tween 80 (0.50-1.00 wt.%), glycerol (1.50-3.00 wt.%), and water (88.0-94.9 wt.%) towards the droplet size were investigated. The results showed that the optimum formulation with 1.50 wt.% POE:RC, 1.50 wt.% lecithin, 1.50 wt.% Tween 80, 1.50 wt.% glycerol and 93.90 % water was obtained. The droplet size, polydispersity index (PDI) and zeta potential of the optimized formulation were 110.3 nm, 0.290 and －37.7 mV, respectively. The formulation also exhibited good stability against storage at 4℃ for 90 days. In vitro aerosols delivery evaluation showed that the aerosols output, aerosols rate and median mass aerodynamic diameter of the optimized nanoemulsion were 99.31%, 0.19 g/min and 4.25 µm, respectively. The characterization of physical properties and efficiency for aerosols delivery results suggest that POE- based nanoemulsion containing quercetin has the potential to be used for pulmonary delivery specifically for lung cancer treatment.

Biochemical Characterization of the Cytochrome P450 CYP107CB2 from Bacillus lehensis G1.

The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.

Danger lurking in the "unknowns": structure-to-function studies of hypothetical protein Bleg1_2437 from Bacillus lehensis G1 alkaliphile revealed an evolutionary divergent B3 metallo-beta-lactamase.

The effectiveness of ß-lactam antibiotics as chemotherapeutic agents to treat bacterial infections is gradually threatened with the emergence of antibiotic resistance mechanism among pathogenic bacteria through the production metallo-ß-lactamase (MBL). In this study, we discovered a novel hypothetical protein (HP) termed Bleg1_2437 from the genome of alkaliphilic Bacillus lehensis G1 which exhibited MBL-like properties of B3 subclass; but evolutionary divergent from other circulating B3 MBLs. Domain and sequence analysis of HP Bleg1_2437 revealed that it contains highly conserved Zn2+-binding residues such as H54, H56, D58, H59, H131 and H191, important for catalysis, similar with the subclass B3 of MBL. Built 3-D Bleg1_2437 structure exhibited an αßßα sandwich layer similar to the well-conserved global topology of MBL superfamily. Other features include a ceiling and floor in the model which are important for accommodation and orientation of ß-lactam antibiotics docked to the protein model showed interactions at varying degrees with residues in the binding pocket of Bleg1_2437. Hydrolysis activity towards several ß-lactam antibiotics was proven through an in vitro assay using purified recombinant Bleg1_2437 protein. These findings highlight the presence of a clinically important and evolutionary divergent antibiotics-degrading enzyme within the pools of uncharacterized HPs.

Synthesis and in vitro Bioactivity Evaluation of New Galactose and Fructose Ester Derivatives of 5-Aminosalicylic Acid.

Inflammatory bowel disease (IBD) is the main risk factor for developing colorectal cancer which is common in patients of all ages. 5-Aminosalicylic acid (5-ASA), structurally related to the salicylates, is highly active in the treatment of IBD with minor side effects. In this study, the synthesis of galactose and fructose esters of 5-ASA was planned to evaluate the role of glycoconjugation on the bioactivity of the parent drug. The antibacterial activity of the new compounds were evaluated against two Gram-negative and two Gram-positive species of bacteria, with a notable effect observed against Staphylococcus aureus and Escherichia coli in comparisons with the 5-ASA. Cytotoxicity testing over HT-29 and 3T3 cell lines indicated that the toxicity of the new products against normal cells was significantly reduced compared with the original drug, whereas their activity against cancerous cells was slightly decreased. The anti-inflammatory activity test in RAW264.7 macrophage cells indicated that the inhibition of nitric oxide by both of the monosaccharide conjugated derivatives was slightly improved in comparison with the non-conjugated drug.

Hepatitis B virus peptide inhibitors: solution structures and interactions with the viral capsid.

Hepatitis B virus (HBV) infection remains a health problem globally despite the availability of effective vaccines. In the assembly of the infectious virion, both the preS and S regions of the HBV large surface antigen (L-HBsAg) interact synergistically with the viral core antigen (HBcAg). Peptides preS and S based on the L-HBsAg were demonstrated as potential inhibitors to block the viral assembly. Therefore, the objectives of this study were to determine the solution structures of these peptides and study their interactions with HBcAg. The solution structures of these peptides were solved using (1)H, (13)C, and (15)N NMR spectroscopy. Peptide preS has several structured regions of ß-turns at Ser7-Pro8-Pro9, Arg11-Thr12-Thr13 and Ser22-Thr23-Thr24 sequences whereas peptide S has only one structured region observed at Ser3-Asn4-His5. Both peptides contain bend-like structures surrounding the turn structures. Docking studies revealed that both peptides interacted with the immunodominant region of HBcAg located at the tip of the viral capsid spikes. Saturation Transfer Difference (STD) NMR experiments identified several aromatic residues in peptides preS and S that interact with HBcAg. This study provides insights into the contact regions of L-HBsAg and HBcAg at atomic resolution which can be used to design antiviral agents that inhibit HBV morphogenesis.

Molecular characterization, modeling and docking of CYP107CB2 from Bacillus lehensis G1, an alkaliphile.

Cytochrome P450s are a superfamily of heme monooxygenases which catalyze a wide range of biochemical reactions. The reactions involve the introduction of an oxygen atom into an inactivated carbon of a compound which is essential to produce an intermediate of a hydroxylated product. The diversity of chemical reactions catalyzed by cytochrome P450s has led to their increased demand in numerous industrial and biotechnology applications. A recent study showed that a gene sequence encoding a CYP was found in the genome of Bacillus lehensis G1, and this gene shared structural similarity with the bacterial vitamin D hydroxylase (Vdh) from Pseudonocardia autotrophica. The objectives of present study was to mine, for a novel CYP from a new isolate B. lehensis G1 alkaliphile and determine the biological properties and functionalities of CYP in this bacterium. Our study employed the usage of computational methods to search for the novel CYP from CYP structural databases to identify the conserved pattern, functional domain and sequence properties of the uncharacterized CYP from B. lehensis G1. A computational homology model of the protein's structure was generated and a docking analysis was performed to provide useful structural knowledge on the enzyme's possible substrate and their interaction. Sequence analysis indicated that the newly identified CYP, termed CYP107CB2, contained the fingerprint heme binding sequence motif FxxGxxxCxG at position 336-345 as well as other highly conserved motifs characteristic of cytochrome P450 proteins. Using docking studies, we identified Ser-79, Leu-81, Val-231, Val-279, Val-383, Ala-232, Thr-236 and Thr-283 as important active site residues capable of stabilizing interactions with several potential substrates, including vitamin D3, 25-hydroxyvitamin D3 and 1α-hydroxyvitamin D3, in which all substrates docked proximally to the enzyme's heme center. Biochemical analysis indicated that CYP107CB2 is a biologically active protein to produce 1α,25-dihydroxyvitamin D3 from 1α-hydroxyvitamin D3. Based on these results, we conclude that the novel CYP107CB2 identified from B. lehensis G1 is a putative vitamin D hydroxylase which is possibly capable of catalyzing the bioconversion of parental vitamin D3 to calcitriol, or related metabolic products.

Monte Carlo simulation of mixed nonionic Brij surfactants in water.

Nonionic surfactants such as the Brij® series are important in the preparation of transdermal drug nanodelivery products using nanoemulsions because of their low toxicity and low irritancy. Here, Monte Carlo (MC) simulation was used to examine the physical behavior of the model deterministic system by using sampling procedures. Metropolis MC simulations were run on three mixtures of two different nonionic surfactants, Brij92 and Brij96, with different compositions in aqueous solution. The system was simulated in the canonical ensemble with constant temperature, volume and number of molecules. Hence, the acceptance ratio for single atom moves of the mixed surfactants increased as the concentration of surfactants increased from 0.494 to 0.591. The lowest total energy for the mixed surfactant systems was -99,039 kcal mol(-1) due to the interaction between all molecules in the system simulated. The physicochemical properties of models such as the radius of gyration and radial distribution function, were also determined. These observations indicate that the behavior and physicochemical of mixed surfactant and PKOEs nanoemulsion systems were described adequately during the simulation.

Tetrabutylammonium bromide (TBABr)-based deep eutectic solvents (DESs) and their physical properties.

Density, viscosity and ionic conductivity data sets of deep eutectic solvents (DESs) formed by tetrabutylammonium bromide (TBABr) paired with ethlyene glycol, 1,3-propanediol, 1,5-pentanediol and glycerol hydrogen bond donors (HBDs) are reported. The properties of DES were measured at temperatures between 303 K and 333 K for HBD percentages of 66.7% to 90%. The effects of HBDs under different temperature and percentages are systematically analyzed. As expected, the measured density and viscosity of the studied DESs decreased with an increase in temperature, while ionic conductivity increases with temperature. In general, DESs made of TBABr and glycerol showed the highest density and viscosity and the lowest ionic conductivity when compared to other DESs. The presence of an extra hydroxyl group on glycerol in a DES affected the properties of the DES.

A Sco protein among the hypothetical proteins of Bacillus lehensis G1: Its 3D macromolecular structure and association with Cytochrome C Oxidase.

BACKGROUND: At least a quarter of any complete genome encodes for hypothetical proteins (HPs) which are largely non-similar to other known, well-characterized proteins. Predicting and solving their structures and functions is imperative to aid understanding of any given organism as a complete biological system. The present study highlights the primary effort to classify and cluster 1202 HPs of Bacillus lehensis G1 alkaliphile to serve as a platform to mine and select specific HP(s) to be studied further in greater detail. RESULTS: All HPs of B. lehensis G1 were grouped according to their predicted functions based on the presence of functional domains in their sequences. From the metal-binding group of HPs of the cluster, an HP termed Bleg1_2507 was discovered to contain a thioredoxin (Trx) domain and highly-conserved metal-binding ligands represented by Cys69, Cys73 and His159, similar to all prokaryotic and eukaryotic Sco proteins. The built 3D structure of Bleg1_2507 showed that it shared the ßαßαßß core structure of Trx-like proteins as well as three flanking ß-sheets, a 310 -helix at the N-terminus and a hairpin structure unique to Sco proteins. Docking simulations provided an interesting view of Bleg1_2507 in association with its putative cytochrome c oxidase subunit II (COXII) redox partner, Bleg1_2337, where the latter can be seen to hold its partner in an embrace, facilitated by hydrophobic and ionic interactions between the proteins. Although Bleg1_2507 shares relatively low sequence identity (47%) to BsSco, interestingly, the predicted metal-binding residues of Bleg1_2507 i.e. Cys-69, Cys-73 and His-159 were located at flexible active loops similar to other Sco proteins across biological taxa. This highlights structural conservation of Sco despite their various functions in prokaryotes and eukaryotes. CONCLUSIONS: We propose that HP Bleg1_2507 is a Sco protein which is able to interact with COXII, its redox partner and therefore, may possess metallochaperone and redox functions similar to other documented bacterial Sco proteins. It is hoped that this scientific effort will help to spur the search for other physiologically relevant proteins among the so-called "orphan" proteins of any given organism.

Enzymatic production of a solvent-free menthyl butyrate via response surface methodology catalyzed by a novel thermostable lipase from Geobacillus zalihae.

Most substrate for esterification has the inherent problem of low miscibility which requires addition of solvents into the reaction media. In this contribution, we would like to present an alternative and feasible option for an efficient solvent-free synthesis of menthyl butyrate using a novel thermostable crude T1 lipase. We investigated the effects of incubation time, temperature, enzyme loading and substrate molar ratio and determined the optimum conditions. The high conversion of menthyl butyrate catalyzed by crude T1 lipase in a solvent-free system is greatly affected by temperature and time of the reaction media. The highest yield of menthyl butyrate was 99.3% under optimized conditions of 60 °C, incubation time of 13.15 h, 2.53 mg, 0.43% (w/w) enzyme to substrate ratio and at molar ratio of butyric anhydride/menthol 2.7:1. Hence, the investigation revealed that the thermostable crude T1 lipase successfully catalyzed the high-yield production of menthyl butyrate in a solvent-free system. The finding suggests that the crude T1 lipase was a promising alternative to overcome shortcomings associated with solvent-assisted enzymatic reactions.