Final Purification Step Research Articles

Validation of a radiosynthesis method and a novel quality control system for [68 Ga]Ga-MAA: is TLC enough to assess radiopharmaceutical quality?

BackgroundTechnetium-99 m-labelled macroaggregated human serum albumin ([99mTc]Tc-MAA) is commonly used for lung perfusion scintigraphy. The European Pharmacopoeia (Eu.Ph.) specifies thin-layer chromatography (TLC) as the only method to assess its radiochemical purity (RCP). Similarly, TLC is the sole method reported in the literature to evaluate the RCP of Gallium-68-labelled MAA [68 Ga]Ga-MAA, recently introduced for lung perfusion PET/CT imaging. Since [68 Ga]Ga-MAA is prepared from commercial kits originally designed for the preparation of [99mTc]Tc-MAA, it is essential to optimize and validate the preparation methods for [68 Ga]Ga-MAA.ResultsWe tested a novel, simplified method for the preparation of [68 Ga]Ga-MAA that does not require organic solvents, prewash or final purification steps to remove radioactive impurities. We assessed the final product using radio-TLC, radio-UV-HPLC, and radio SDS-PAGE. Overall, our quality control (QC) method successfully detected [68 Ga]Ga-MAA along with all potential impurities, including free Ga-68, [68 Ga]Ga-HSA, unlabeled HSA, which may occur during labelling process and HEPES residual, a non-toxic but non-human-approved contaminant, used as buffer solution. We then applied our QC system to [68 Ga]Ga-MAA prepared under different conditions (25°–40°–75°–95 °C), thus defining the optimal temperature for labelling. Scanning Electron Microscopy (SEM) analysis of the products obtained through our novel method confirmed that most [68 Ga]Ga-MAA particles preserved the morphological structure and size distribution of unlabeled MAA, with a particle diameter range of 25–50 μm, assuring diagnostic efficacy.ConclusionsWe optimized a novel method to prepare [68 Ga]Ga-MAA through a QC system capable of monitoring all impurities of the final products.

Purification and characterization of α-amylase from Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae)

Acanthoscelides obtectus is one of the most notorious pests of stored kidney beans (Phaseolus vulgaris) worldwide. Kidney beans are an important source of food for these insects. α-Amylase is the main carbohydrate-digesting enzyme in animals including insects. In the current study, the biochemical analysis revealed higher α-amylase activity (U/ml) in 3rd and 4th larval instars but decreased gradually in subsequent developmental stages. However, the specific activity (U/mg) interestingly was highest in 1st instar and decreased in further developmental stages. During qualitative analysis of α-amylase using starch-agar and native PAGE, the maximum zone of starch lysis and a prominent band on the gel was observed in 3rd and 4th larval stages. The molecular mass of the native enzyme was also estimated and found to be 30.34 kDa. The crude α-amylase was further purified by ammonium sulfate precipitation, gel filtration on a Sephadex G-75, and ion exchange chromatography on the DEAE cellulose column. The purified amylase was found to be a monomer with a molecular mass of 15 kDa. The specific activity of the purified enzyme increased from 1.74 U/mg in the crude sample to 166.35 U/mg in the final purification step resulting in 95-fold purification with a yield of 11.14%. Further characterization of purified α-amylase revealed a pH optimum of 7.0 and a temperature optimum of 35 °C. Lineweaver-Burk plot analysis revealed Km and Vmax to be 0.09% and 3.3 U/mL, respectively. Oxalic acid, tannic acid, and HgCl2 significantly inhibited the enzyme, while the Na+, Ca++, and Mg++ ions acted as activators. In conclusion, the study revealed, the highest α-amylase activity in 3rd and 4th larval stages of Acanthoscelides obtectus followed by native and SDS PAGE resulting in molecular mass of 30.34 and 15 kDa respectively.

Access to Diverse Glutamic Acid Dendrons and a Janus Peptide Dendrimer Using an Atypical Solid Phase Synthesis.

The negligible cytotoxicity of anion surface-linked dendrons makes glutamic acid-based dendrons a potential candidate for materials and biological applications. Despite the inherent drawbacks of the conventional solution phase synthesis of glutamic acid-based dendrons, there have been no advancements in these protocols. Herein, we demonstrate the first-ever convergent solid phase synthesis of dendrons, up to fourth generation, having glutamic acid branching points produced by preactivation of dicarboxylic acid groups with N-hydroxysuccinimide and simultaneous coupling with amine groups of two growing peptide chains, with excellent yields (30-70%). In addition to the general advantages, such as the easy workup, a final single purification step, and an overall short synthesis duration, the convergent solid phase synthesis allowed us to chemically synthesize glutamic acid branching-based dendrons that cannot be accessed by standard divergent solid phase synthesis. This method has also been validated for its application in synthesizing hard-to-achieve Janus peptide dendrimers in a single stretch on a solid support. Our work corroborates the efficacy of controlled -COOH activation to accomplish an atypical solid phase synthesis of diverse glutamic acid dendrons in a convergent fashion. This is the first example of a Janus peptide dendrimer being synthesized on a solid support, utilizing both convergent and divergent approaches simultaneously.

Purification and Characterization of Recombinant Levansucrase From Bacillus lichniformans MJ8

The ability to produce levansucrase was estimated in fifty Bacillus spp. isolates obtained from various sources. One isolate was found to be the higher producer of this enzyme through primary and secondary screening, which was performed by estimating levansucrase activity. It was found to be 6.05 U/ml. The results show, after identification by the Vitek 2 compact system with 92% probability and 16S ribosomal RNA gene sequencing, that this isolate is Bacillus licheniformis, designated in this study as MJ8, and registered in the Gene Bank under the accession number OM672244.1. Then, the gene of (SacB) was studied in detail. It contained 1449 nucleotides (accession number ON811641.1), expressed for 482 amino acids and 29 amino-acid as signal peptides. A recombinant plasmid, pET28a(+), with the levansucrase (SacB) gene, was over expressed in high efficiency E. coli BL21 (DE3). Then the enzyme was purified in three steps; firstly, it was precipitated with ammonium sulfate 40-80%. Secondly, DEAE cellulose ion exchange chromatography was applied then the gel filtration chromatography. The activity of levansucrase at the final purification steps reached 48.65 U/ml with a purification fold of 18.0 and a recovery of 48.9%. The levansucrase characterization was also studied. It was found that the molecular weight by SDS-PAGE and gel filtration methods was 43 kDa and 47 kDa, respectively, and the optimum temperature activity and stability were 45 ◦C, 30-35 ◦C, respectively while the pH activity and stability were 6.0, and 7.0, The levan was produced from cloned (SacB) levansucrase, separated, purified, and identified by NMR, AFM, and SEM analyses showed that its structure mostly consists of fructose residues.

Solid-Phase Synthesis of s-Tetrazines.

An efficient synthesis of s-tetrazines by solid-phase methods is described. This synthesis route was compatible with different solid-phase resins and linkers and did not require metal catalysts or high temperatures. Monosubstituted tetrazines were routinely synthesized using thiol-promoted chemistry, using dichloromethane as a carbon source, while disubstituted unsymmetrical aryl or alkyl tetrazines were synthesized using readily available nitriles. This efficient approach enabled the synthesis of s-tetrazines in high yields (70-94%), eliminating the classical solution-phase problems of mixtures of symmetrical and unsymmetrical tetrazines, with only a single final purification step required, and paves the way to the rapid synthesis of s-tetrazines with various applications in bioorthogonal chemistry and beyond.

Purification of recombinant vesicular stomatitis virus-based HIV vaccine candidate.

In this work, laboratory- and large-scale methods were tested for purification of a human immunodeficiency virus (HIV) vaccine candidate, based on recombinant vesicular stomatitis virus (rVSV). First step of the purification, the clarification of the rVSVs produced in serum-free cell culture medium, was tested by centrifugation and filtration using different filtration media and pore sizes (0.45 to 30µm). To reduce the supernatant volume and process time, the clarified sample was concentrated by ultrafiltration either using tangential flow filtration or centrifugal-based filtration units, depending on the process scale. The final purification step at laboratory-scale, was carried out by density gradient ultracentrifugation, the recovery of which was compared with chromatographic purification at large-scale. The virus preparations were analyzed using dynamic light scattering to verify the virus size and transmission electron microscopy for purity and virus morphology. Density gradient ultracentrifugation allowed the recovery of≥80% infectious particles and reduced the contaminant DNA and host cell proteins relatively to standard ultracentrifugation pelleting using a sucrose cushion. At large-scale, weak and strong anion-exchangers were tested and compared. The best columns allowed infectious virus recoveries as high as 77% and eliminated 92% of host cell proteins.

Enhancement of microsphere specificity to purify human serum albumin from blood plasma

Efforts to rapidly and easily supply purified human serum albumin (HSA) in remote areas during a pandemic are challenging. Here, we developed an HSA-imprinted microsphere (HSAIM) as a matrix to purify HSA from blood plasma on a small scale. HSAIMs were generated by encapsulating silica-3-(2-imidazoline-1-yl) propyltriethoxysilane-Cu2+-HSA (SiO2-IMEO-Cu2+-HSA) into agarose gel, and the stereospecific template for HSA was obtained by eluting the agarose gel. The physicochemical properties and performance of HSAIM were evaluated, and HSAIMs were applied to purify HSA from human blood plasma. Spherical HSAIMs had an average diameter of 51.2 ± 6.1 μm. HSAIMs had a maximum adsorption of 8.77 × 10–2 μmol HSA g–1 with an imprinting factor of 2.83, and the selectivity factors of BSA, thrombin and IgG were 0.96, 0.59 and 0.26, respectively. HSAIMs had a dynamic binding capacity (DBC10%) of 5.94 × 10–2 μmol HSA g–1 and could be reused up to 10 cycles with an ultimate recovery of 55.92%. HSA adsorption kinetics of HSAIM fitted to a pseudo‐second‐order mechanism, and HSA binding characteristics fit with a Sips isotherm model. For practical purposes, an initial blood plasma sample containing 24.9 ± 2.5 mg protein was pretreated with ethanol yielding 14.5 ± 4.6 mg protein, and further purification with HSAIM yielded 3.6 ± 1.1 mg protein. Starting with a blood plasma sample containing 149 type proteins, a single protein identified as HSA was obtained after final purification step with the HSAIM column, indicating that HSAIMs stereospecifically bound HSA. Hence, HSAIM was promising for blood plasma purification on a small scale.

Optimization of the production process for the anticancer lead compound illudin M: downstream processing

BackgroundSecondary metabolites have played a key role as starting points for drug development programs due to their often unique features compared with synthetically derived molecules. However, limitations related to the discovery and supply of these molecules by biotechnological means led to the retraction of big pharmaceutical companies from this field. The reasons included problems associated with strain culturing, screening, re-discovery, purification and characterization of novel molecules from natural sources. Nevertheless, recent reports have described technical developments that tackle such issues. While many of these reports focus on the identification and characterization of such molecules to enable subsequent chemical synthesis, a biotechnological supply strategy is rarely reported. This may be because production processes usually fall under proprietary research and/or few processes may meet the requirements of a pharmaceutical development campaign. We aimed to bridge this gap for illudin M—a fungal sesquiterpene used for the development of anticancer agents—with the intention to show that biotechnology can be a vital alternative to synthetic processes dealing with small molecules.ResultsWe used µL-scale models to develop an adsorption and extraction strategy for illudin M recovery from culture supernatant of Omphalotus nidiformis and these findings were successfully transferred into lab-scale. By adsorbing and eluting the product using a fixed resin-bed we reduced the working volume by ~ 90% and removed the aqueous phase from the process. After a washing step, a highly concentrated illudin M fraction was obtained by isocratic elution with 80% methanol. The fraction was dried and extracted using a water/heptane mixture, enriching illudin M in the heptane phase. From heptane illudin M could be instantly crystalized by concentrating the solution, achieving a final purity > 95%.ConclusionWe have developed a robust, scalable and low-cost downstream process to obtain highly pure illudin M. By using solid phase extraction we reduced the production of solvent waste. Heptane from the final purification step could be recycled. The reduced amounts of solvents required, and the short purification time render this method a very economic and ecologic alternative to published processes.

Chemical Isolation of Isomerically Purified Sc3n@C68

Metallofullerenes with fused pentalenes are rare in arc soot. By far, the majority of endohedral fullerene cages obey the isolated pentagon rule (IPR). In a recent landmark paper (Santiago et al., JACS, 2021, 143, 6037-6042) by the Echegoyen group, they experimentally demonstrated the use of Sc3N@C68 as a new molecular catalyst for Hydrogen Evolution Reactions. Indeed, this fused pentalene metallofullerene (Sc3N@C68) successfully outperformed six other “conventional” (i.e., IPR) endohedrals. Thus, application development for fused pentalene metallofullerenes may open new doors.In this presentation, we discuss our chemical-based purification approach specifically designed for fused pentalene systems such as Sc3N@C68. We find that after two types of selective chemical reaction, we obtain a highly enriched sample of primarily two species, Sc3N@D3(6140)-C68 and Sc3N@Ih(7)-C80. Due to the large retention time difference between C68 and C80 endohedral cages, only a single injection is necessary for a final purification step.

New Fully Automated Preparation of High Apparent Molar Activity 68Ga-FAPI-46 on a Trasis AiO Platform.

A large number of applications for fibroblast activation protein inhibitors (FAPI)-based PET agents have been evaluated in conditions ranging from cancer to non-malignant diseases such as myocardial infarction. In particular, 68Ga-FAPI-46 was reported to have a high specificity and affinity for FAP-expressing cells, a fast and high accumulation in tumor lesions/injuries together with a fast body clearance when investigated in vivo. Due to the increasing interest in the use of the agent both preclinically and clinically, we developed an automated synthesis for the production of 68Ga-FAPI-46 on a Trasis AiO platform. The new synthetic procedure, which included the processing of the generator eluate using a strong cation exchange resin and a final purification step through an HLB followed by a QMA cartridge, yielded 68Ga-FAPI-46 with high radiochemical purity (>98%) and apparent molar activity (271.1 ± 105.6 MBq/nmol). Additionally, the in vitro and in vivo properties of the product were assessed on glioblastoma cells and mouse model. Although developed for the preparation of 68Ga-FAPI-46 for preclinical use, our method can be adapted for clinical production as a reliable alternative to the manual (i.e., cold kit) or modular systems preparations already described in the literature.

Leakage of Endotoxins through the Endotoxin Retentive Filter: An in vitro Study

Introduction: Ultrapurification of dialysis fluid has enabled highly efficient dialysis treatments. Online hemodiafiltration is one such treatment that uses a purified dialysis fluid as a supplemental fluid. In this method, an endotoxin retentive filter (ETRF) is used in the final step of dialysis fluid purification, with the aim of preventing leakage of endotoxins. Sodium hypochlorite and peracetic acid are used as disinfecting agents for the dialysis fluid pipes containing the ETRF; however, the effects of these agents on ETRF membrane pores have not been fully clarified. Methods: Water permeability (flux) and endotoxin permeability were assessed in 3 types of ETRFs made with different membrane materials: polyester polymer alloy (PEPA), polyether sulfone (PES), and polysulfone (PS). High-concentration sodium hypochlorite and 2 types of peracetic acid were used as disinfecting agents, and the changes in flux and the endotoxin sieving coefficient (SC) were measured. Results: After repeated use of high concentrations of sodium hypochlorite and peracetic acid, the PEPA and PES ETRFs did not permit passage of endotoxins, regardless of their flux. However, in the PS ETRF, the flux and endotoxin SC increased with the number of cleaning cycles. No differences were observed according to the concentration of peracetic acid disinfecting agents. Conclusion: PEPA and PES ETRFs completely prevent endotoxin leakage and can be disinfected at concentrations higher than the conventionally recommended concentration without affecting pore expansion. Even new PS ETRFs have low levels of endotoxin leakage, which increase after disinfection cycles using sodium hypochlorite and peracetic acid.

Isolation of Synaptic Vesicles from Mammalian Brain.

Synaptic vesicles (SVs) store neurotransmitters and undergo a fine-tuned regulatory and dynamic cycle of exo- and endocytosis, which is essential for neurotransmission at chemical synapses. The development of protocols for isolating SVs from biological extracts was a fundamental accomplishment since it allowed for characterizing the molecular properties of SVs using biochemical methods. In this chapter, we describe a modified procedure for isolating SVs from a few g of rodent brain and that can be completed within ~12h. The protocol involves the preparation of isolated nerve terminals from which SVs are released by osmotic shock and then enriched via various centrifugation steps, followed by size exclusion chromatography as final purification step. The final vesicle fraction is 22-fold enriched in SVs over the starting material, and the final yield of SVs obtained using this protocol is approximately 20μg of protein per gram of mouse brain. The degree of contamination by other organelles and particles monitored by morphology and immunolabeling compares well with that of the classical protocols.

Protein Delivery by PTDs/CPPs.

The ability to deliver or transduce proteins into cells allows for the manipulation of cell biology in culture, preclinical models, and potentially human disease. Fusion proteins containing the TAT peptide transduction domain (PTD), also known as cell-penetrating peptide (CPP), allow for delivery of a wide variety of proteins, including enzymes, transcription factors, tumor suppressor proteins, and many more. TAT-fusion proteins are generated cloning in-frame into the pTAT-HA plasmid, then transformed into E. coli for expression, and purified by the 6-His affinity tag over Ni-NTA column, followed by a final IEX FPLC purification step.

Purification, Characterization, and Inhibition of Tyrosinase from Jerusalem Artichoke (Helianthus Tuberosus L.) Tuber.

Because it tends to cause deterioration in the quality of food and appearance, food browning is unacceptable. Tyrosinase, which catalyzes the transformation of mono phenolic compounds into o-quinones, has been associated with this phenomenon. Natural anti-browning agents were used to help avoid the enzymatic browning that occurs in many foods. Tyrosinase of Jerusalem Artichoke tubers was purified through (NH4)2SO4 sedimentation, dialysis, chromatography, and finally gel electrophoresis. The purified enzyme was characterized and inhibited by rosemary extracts. Purification of tyrosinase from Jerusalem Artichoke tuber were accomplished. The specific activity at the final step of purification increased to 14115.76 U/mg protein with purification fold 32.89 using CM-Cellulose chromatography. The molecular mass was evaluated by electrophoresis and found to be 62 KDa. Maximum tyrosinase activity was found at 30 °C, pH 7.2, and higher affinity towards L-tyrosine. Inhibition percentage of heated extracts for leaves and flowers on tyrosinase activity was better than nonheated with 29.65% and 23.75%, respectively. The kinetic analysis exposed uncompetitive inhibition by leaves and flowers heated extracts. In this study, we concluded the usage of natural anti-browning inhibitors like rosemary extract be able to be castoff to substitute the chemical agents which might be dangerous to social healthiness. Natural anti-browning agents can be used to prevent the browning of many foods.

From chemostats to high‐density perfusion: the progression of continuous mammalian cell cultivation

AbstractContinuous cultivation of mammalian cells originated in chemostats in the 1950s and, through a series of technological advancements, has evolved into modern‐day high‐density perfusion processes with volumetric productivities >5 g L–1 day–1over extended cultivation periods. With appropriate process development, these productivities can be achieved with no adverse impact on cell line stability and product quality. Developments in downstream processing and automation have enabled the integration of drug substance manufacturing processes suitable for next‐generation plants that require lower capital deployment, with the potential for rapid construction and validation for clinical and commercial production. Perfusion also has been successfully used to generate high‐density cell banks (> 50 × 106cells mL–1) and for seed‐train optimization. They have resulted in significant productivity increases (10‐fold increase in volumetric productivity) from fed‐batch bioreactors by providing a high‐density seeding culture that elevates the inoculation density in a production bioreactor (> 5 × 106cells mL–1). Related advancements in cell culture medium formulations and cell line performance typically are independent of bioreactor format and have broad applicability for biotherapeutic manufacturing. The last decade has seen renewed interest in high‐density perfusion cultures and technological advances spanning the entire space of drug substance manufacturing – from cell line development to the final purification step. Continually evolving, these new technologies now are being introduced into clinical and commercial manufacturing. We anticipate that the general principles and knowledge gleaned from process intensification will extend well beyond classical perfusion cultivation and into all aspects of mammalian cell culture. © 2021 Society of Chemical Industry (SCI).

Expression of Antimicrobial Peptide (AMP), Cecropin B, in a Fused Form to SUMO Tag With or Without Three-Glycine Linker in Escherichia coli and Evaluation of Bacteriolytic Activity of the Purified AMP

Current antibiotics have limited action mode, which makes it difficult for the antibiotics dealing with the emergence of bacteria resisting the existing antibiotics. As a need for new bacteriolytic agents alternative to the antibiotics, AMPs have long been considered substitutes for the antibiotics. Cecropin B was expressed in a fusion form to six-histidine and SUMO tags in Escherichia coli. Six-histidine tag attached to SUMO was for purification of SUMO-cecropin B fusion proteins and removal of the SUMO tag from cecropin B. Chimeric gene was constructed into pKSEC1 vector that was designed to be functional in both Escherichia coli and chloroplast. To maximize translation of the fusion protein, sequences were codon-optimized. Four different constructs were tested for the level of expression and solubility, and the construct with a linker, 6xHisSUMO3xGly-cecropin B, showed the highest expression. In addition, cleavage of the SUMO tag by SUMOase in the three fusion constructs which have no linker sequence (3xGly, three glycines) was not as efficient as the construct with the linker between SUMO and cecropin B. The cleaved cecropin B showed bacteriolytic activity against Bacillus subtilis at a concentration of 0.0625 μg/μL, while cecropin B fused to SUMO had no activity at a higher concentration, 0.125 μg/μL. As an expression system for AMPs in prokaryotic hosts, the use of tag proteins and appropriate codon-optimization strategy can be employed and further genetic modification of the fusion construct should help the complete removal of the tag proteins from the AMP in the final step of purification.

Integrated Lab-Scale Process Combining Purification and PEGylation of l-Asparaginase from Zymomonas mobilis

During the last quarter of a century, PEGylation has been used to enhance the clinical effectiveness of various therapeutic proteins. However, the conjugation process significantly increases the costs of the process. This parameter is critical, especially for biosimilar products that do not have patent protection. The integration of protein purification to its conjugation could potentially reduce total costs. Following this hypothesis, in this work, the purification of l-asparaginase from Zymomonas mobilis, produced recombinantly in Escherichia coli, linked to its conjugation with a PEG of 12 kDa, was studied. The effect of operating parameters on cell rupture by high-pressure homogenization was analyzed, and the optimal values for the conditions in this study were determined. The chromatographic capture and final purification steps were also studied. It was possible to elute the protein from ion-exchange chromatography, used as final purification, in the optimal buffer for the PEGylation step. This result allowed us to eliminate the buffer change step that is usually carried out at the beginning of the PEGylation process. The conjugate obtained by this continuous process showed higher thermal stability and slower protein degradation compared to the native protein. This continuous process not only has great potential to reduce costs but could also be useful for studying the effect of different parameters on the overall performance of the process. This knowledge is especially important to establish a quality by design approach at the industrial scale.

Extraction, Purification and Characterization of β-Galactosidase from Tomato (Lycopersicom esculentum)

β-galactosidase enzyme EC (3.2.1.23), lactase, can be described as an enzyme of glycoside hydrolase which performs the catalyzing of β-galactosidas hydrolysis to monosaccharides by breaking glycosidic bond. The objective of this study was to extraction and purification β-galactosidase from tomato (Lycopersicom esculentum.), ten Different types of extraction were investigated to selection of the best extraction of the enzyme, The Na. phosphate buffer (0.1M and pH6) had given a highest Specific activity of crude enzyme has been 212.27 U/mg. protein. The purification procedures were performed with the use of the precipitation of ammonium sulfate, Ion-exchange and gel filtration chromatographic techniques. ammonium sulfate (70) % saturation has been the best method for precipitation and partially purification of enzyme with a purification fold 1.83 and enzymatic yield 88.31%. This followed by the use of ion exchange chromatography by DEAE sephadex A50 column, the purification times of the enzymatic extract were 2.36, with an enzymatic yield 25.48%. After the final purification step of gel filtration chromatography using SephadexG-100 column, the enzyme has been purified 3.92 fold with 16.33% of enzymatic yield. The optimum enzymatic activity was found at pH (6). The plant extracts tomato (Lycopersicom esculentum) were used to characterize the enzyme in the term of pH, temperature,. The enzyme activity measured by its ability to hydrolyze the substrate 2-nitrophenyl β-D-galactopyranoside (ONPG). The enzyme activity was reached maximum at 45°C and at pH 5.5. The enzyme’s molecular weight has been estimated to 74 KDa by the gel filtration chromatography method, and 73 k D on SDS-PAGE.

Design, Dimerization, and Recombinant Production of MCh-AMP1-Derived Peptide in Escherichia coli and Evaluation of Its Antifungal Activity and Cytotoxicity.

Fungal species resistant to current antifungal agents are considered as a serious threat to human health, the dilemma that has dragged attentions toward other sources of antifungals such as antimicrobial peptides (AMPs). In order to improve biological activity of a recently described antifungal peptide MCh-AMP1 from Matricaria chamomilla flowers, MCh-AMP1dimer (DiMCh-AMP1), containing 61 amino acid residues connected by flexible linker (GPDGSGPDESGPDES), was designed and expressed in Escherichia coli, and its structure was analyzed using bioinformatics tools. DiMCh-AMP1 synthetic gene was cloned into pET-28a expression vector, which was then used to transform E. coli BL21 (DE3) strain. His-tag purification was achieved using metal-chelate affinity chromatography. Because there is no methionine residue in the DiMCh-AMP1 sequence, cyanogen bromide was successfully used to separate the target product from the tag. Reverse-phase high-performance liquid chromatography was used as the final step of purification. Results showed that recombinant peptide was produced in considerable amounts (0.9 mg/L) with improved antifungal activity toward both yeasts and molds compared to its monomeric counterpart. The minimum inhibition concentration and minimum fungicidal concentration values of DiMCh-AMP1 against Candida and Aspergillus species were reported in the range of 1.67-6.66 μM and 3.33-26.64 μM, respectively. Our results showed that while antifungal activity of dimerized peptide was improved considerably, its cytotoxicity was decreased, implying that DiMCh-AMP1 could be a potential candidate to design an effective antifungal agent against pathogenic yeasts and molds.

Fuel-grade sunflower oil butyl esters: synthesis, purification, oxidation stability

Current paper deals with production, purification and oxidative stability enhancement of fuel-grade sunflower oil butyl esters as more ecological alternative of methyl esters as biodiesel. The oil feedstock, used in this study, included refined sunflower oil (acid value – 0.05 mg KOH/g; 25.3 % of oleic and 61.2 % of linoleic acids) and wasted frying high-oleic sunflower oil (acid value – 1.20 mg KOH/g; 6.1 % of linoleic and 81.7 % of oleic acids). Butanolysis was carried out using potassium butoxide, obtained from KOH and alcohols via original patent-pending method, under mild reaction conditions (alcohol-to-oil molar ratio – 4.5-5.0, 15°C, 1.4-1.6 %еq. KOH of butoxide, 20-30 min). High molar yield of butyl esters (93-96 %) was achieved, while glycerol and vast majority of alkaline catalyst formed the separate reaction products phase mainly in the course of reaction. Ester enriched phases were purified in order to obtain fuel-grade butanol-based biodiesel. Samples after removing of butanol under vacuum followed by water washing and drying were characterized by not enough high butyl esters content (about 94-95 %), as well as higher than allowed content of unconverted glycerides. Vacuum distillation as final purification step allowed fitting butyl esters samples composition within the requirements for biodiesel fuel. Distilled samples contained about 99 % of butyl esters, 0.4-0.5 % of monoglycerides and almost no n-butanol, glycerol, di- and triglycerides. Oxidative treatment (110°C, 6 h, air bubbling) revealed the high oxidation stability of the sample, originated from wasted high-oleic oil, due to the predominance of oleic acid in its fatty acid composition. The sample, obtained from refined sunflower oil (mainly linoleic acid in fatty acid composition), demonstrated very low stability. Addition of at least 2000 mg/kg of antioxidant 2,6-di-tert-butyl-4-methylphenol was shown to be able to improve this characteristic to the level of biodiesel requirements.