MALDI-TOF Analysis Research Articles

Molecular Diversity of Peptide Toxins in the Venom of Spider Heteropoda pingtungensis as Revealed by cDNA Library and Transcriptome Sequencing Analysis.

The venoms of toxic animals are chemical pools composed of various proteins, peptides, and small organic molecules used for predation and defense, in which the peptidic toxins have been intensively pursued mining modulators targeting disease-related ion channels and receptors as valuable drug pioneers. In the present study, we uncovered the molecular diversity of peptide toxins in the venom of the spider Heteropoda pingtungensis (H. pingtungensis) by using a combinatory strategy of venom gland cDNA library and transcriptome sequencing (RNA-seq). An amount of 991 high-quality expressed sequence tags (ESTs) were identified from 1138 generated sequences, which fall into three categories, such as the toxin-like ESTs (531, 53.58%), the cellular component ESTs (255, 25.73%), and the no-match ESTs (205, 20.69%), as determined by gene function annotations. Of them, 190 non-redundant toxin-like peptides were identified and can be artificially grouped into 13 families based on their sequence homology and cysteine frameworks (families A–M). The predicted mature toxins contain 2–10 cysteines, which are predicted to form intramolecular disulfide bonds to stabilize their three-dimensional structures. Bioinformatics analysis showed that toxins from H. pingtungensis venom have high sequences variability and the biological targets for most toxins are unpredictable due to lack of homology to toxins with known functions in the database. Furthermore, RP-HPLC and MALDI-TOF analyses have identified a total of 110 different peptides physically existing in the H. pingtungensis venom, and many RP-HPLC fractions showed potent inhibitory activity on the heterologously expressed NaV1.7 channel. Most importantly, two novel NaV1.7 peptide antagonists, µ-Sparatoxin-Hp1 and µ-Sparatoxin-Hp2, were characterized. In conclusion, the present study has added many new members to the spider toxin superfamily and built the foundation for identifying novel modulators targeting ion channels in the H. pingtungensis venom.

Identification of novel extracellular putative chitinase and hydrolase from Geomyces sp. B10I with the biodegradation activity towards polyesters

Cold-adapted filamentous fungal strain Geomyces sp. B10I has been reported to decompose polyesters such as poly(e-caprolactone) (PCL), poly(butylene succinate) (PBS) and poly(butylene succinate-co-butylene adipate) (PBSA). Here, we identified the enzymes of Geomyces sp. B10I, which appear to be responsible for its biodegradation activity. We compared their amino acid sequences with sequences of well-studied fungal enzymes. Partial purification of an extracellular mixture of the two enzymes, named hydrGB10I and chitGB10I, using ammonium sulfate precipitation and ionic exchange chromatography gave 14.16-fold purity. The amino acid sequence of the proteins obtained from the MALDI-TOF analysis determined the molecular mass of 77.2 kDa and 46.5 kDa, respectively. Conserved domain homology analysis revealed that both proteins belong to the class of hydrolases; hydrGB10I belongs to the glycosyl hydrolase 81 superfamily, while chitGB10I contains the domain of the glycosyl hydrolase 18 superfamily. Phylogenetic analysis suggests a distinct nature of the hydrGB10I and chitGB10I of Geomyces sp. B10I when compared with other fungal polyester-degrading enzymes described to date.

Predictive Potential of MALDI-TOF Analyses for Wine and Brewing Yeast.

The potential of MALDI-TOF profiling for predicting potential applications of yeast strains in the beverage sector was assessed. A panel of 59 commercial yeasts (47 wine and 12 brewing yeasts) was used to validate the concept whereby 2 culture media (YPD agar and YPD broth), as well as two mass ranges m/z 500–4000 and m/z 2000–20,000, were evaluated for the best fit. Three machine learning-based algorithms, PCA, MDS, and UMAP, in addition to a hierarchical clustering method, were employed. Profiles derived from broth cultures yielded more peaks, but these were less well-defined compared with those from agar cultures. Hierarchical clustering more clearly resolved different species and gave a broad overview of potential strain utility, but more nuanced insights were provided by MDS and UMAP analyses. PCA-based displays were less informative. The potential of MALDI-TOF proteomics in predicting the utility of yeast strains of commercial benefit is supported in this study, provided appropriate approaches are used for data generation and analysis.

Remotely controllable supramolecular rotor mounted inside a porphyrinic cage

Remotely controllable supramolecular rotor mounted inside a porphyrinic cage

Simultaneous Production of Antibacterial Protein and Lipopeptides in Bacillus tequilensis, Detected by MALDI-TOF and GC Mass Analyses.

As antibiotic resistance is nowadays one of the important challenges, efforts are crucial for the discovery of novel antibacterial drugs. This study aimed to evaluate antimicrobial/anticancerous activities of halophilic bacilli from the human microbiota. A spore-forming halotolerant bacterium with antibacterial effect against Staphylococcus aureus was isolated from healthy human feces. The antibacterial protein components of the extracted supernatant were identified by SDS-PAGE and zymography. The MALDI-TOF, GC mass, and FTIR analyses were used for peptide and lipopeptide identification, respectively. The stability, toxicity, and anticancerous effects were investigated using MTT and Flow cytometry methods. According to the molecular analysis, the strain was identified as Bacillus tequilensis and showed potential probiotic properties, such as bile and acid resistance, as well as eukaryotic cell uptake. SDS-PAGE and zymography showed that 15 and 10-kDa fragments had antibacterial effects. The MALDI-TOF mass analysis indicated that the 15-kDa fragment was L1 ribosomal protein, which was the first report of the RpL1 in bacilli. GC-mass and FTIR analyses confirmed the lipopeptide nature of the 10-kDa fragment. Both the extracted fractions (precipitation or "P" and chloroform or "C" fractions) were stable at < 100°C for 10min, and their antibacterial effects were preserved for more than 6months. Despite its non-toxicity, the P fraction had anticancer activities against MCF7 cells. The anticancer and antibacterial properties of B. tequilensis, along with its non-toxicity and stability, have made it a potential candidate for studying the beneficial probiotic properties for humans and drug production.

Genome sequence of a multidrug-resistant Campylobacter coli strain isolated from a newborn with severe diarrhea in Lebanon.

A multidrug-resistant (MDR) Campylobacter coli (C. coli) strain was isolated from a 2-month-old newborn who suffered from severe diarrhea in Lebanon. Here, whole-genome sequencing (WGS) analysis was deployed to determine the genetic basis of antimicrobial resistance and virulence in the C. coli isolate and to identify its epidemiological background (sequence type). The identity of the isolate was confirmed using API® Campy, MALDI-TOF, and 16S rRNA gene sequencing analysis. The antimicrobial susceptibility phenotype was determined using the disk diffusion assay. Our analysis showed that resistance to macrolide and quinolone was potentially associated with the presence of multiple point mutations in antibiotic targets on the chromosomal DNA. Furthermore, tetracycline and aminoglycoside resistance were encoded by genes on a pTet plasmid. The blaOXA-61, which is associated with beta-lactam resistance, was also detected in the C. coli genome. A set of 30 genes associated with the virulence in C. coli was detected using WGS analysis. MLST analysis classified the isolate as belonging to a new sequence type (ST-9588), a member of ST-828 complex which is mainly associated with humans and chickens. Taking together, this study provides the first WGS analysis of Campylobacter isolated from Lebanon. The detection of a variety of AMR and virulence determinants strongly emphasizes the need for studying the burden of Campylobacter in Lebanon and the Middle East and North Africa (MENA) region, where information on campylobacteriosis is scant.

Epidemiological significance of microbiote monitoring of arctic settlements along the Northern Sea Route

Monitoring of polar ecosystems is one of the most important areas of research in the use of Arctic territories in economic activities. An important place in such work is occupied by biomedical research aimed at identifying the risks of the occurrence of foci of infectious diseases in the areas of polar settlements.The purpose: to develop an algorithm for microbiological monitoring in the Arctic region.Materials and methods: classical bacteriological, mycological research, molecular-genetic, MALDI-TOF mass spectrometric analysis, phenotypic methods for determining antibiotic resistance, methods of mathematical data processing.Results and their discussion. The paper considers the scientific and methodological principles and the main stages of microbiota monitoring in the areas of polar settlements. The results of the study of samples from several territories along the Northern Sea Route are presented. The main habitats where potentially dangerous microorganisms should be detected are indicated. The role of anthropogenic invasion in the formation of Arctic microbiocenoses is noted. The trends of increasing the number and diversity of pathogenic microorganisms, both in the natural biocenoses of the Arctic and in the areas of polar settlements, are discussed. The necessity of microbiological monitoring as an integral part of epidemiological monitoring in the areas of Arctic settlements along the Northern Sea Route is substantiated.

МОЛЕКУЛЯРНЫЕ МЕХАНИЗМЫ СВЕРХУСТОЙЧИВОСТИ ШТАММА BACILLUS PUMILUS 25 К АНТИБИОТИКАМ И ОКИСЛИТЕЛЬНОМУ СТРЕССУ

Spore-forming bacteria are residents on the International space station (ISS) and can act as potential biodistructors of structural materials with a consequent disturbance of equipment operation. These bacteria demonstrate high resistance to many stress factors. Molecular mechanisms of this resistance are poorly understood. Sanitary/hygienic monitoring resulted in detection and isolation of strain Bacillus pumilus 25. The investigation was aimed to test B. pumilus 25 resistance to oxidative agents, antibiotics, and to analyze expression of genes associated with the cell response to DNA damage and hydrogen sulfide production. The strain was identified by sequencing 16S RNA and MALDI-TOF analysis. Resistance to stresses and antibiotics was tested with the use of standard microbiological methods. Level of mRNA genes was determined with the real-time polymerase chain reaction. Our results evidence for heightened B. pumilus 25 resistance to the oxidative stress and majority of the antibiotics used in the investigation. This resistance seems to be associated with high expression of the ku-ligD operon genes, and elevated production of hydrogen sulfide.

Species identification and antimicrobial susceptibility profile of bacteria associated with cow mastitis in southern Brazil

ABSTRACT: Bovine mastitis is the most common disease in dairy cattle and responsible for economic losses in the milk industry. The present study aimed to identify the main species and to evaluate the antimicrobial susceptibility of bacterial isolates from cow herds with mastitis in dairy farms from southern Brazil. A total of 107 milk samples were collected from different cow herds in one important dairy producing region in southern Brazil, including farms located in ten cities from the Northeast region in the Rio Grande do Sul state. Bacterial strains were isolated and submitted to presumptive identification by classical bacteriological methods. Bacterial species were also identified by MALDI-TOF MS and antimicrobial susceptibility testing was performed with 12 antimicrobials commonly used in dairy farms. Fifty-one bacterial strains were isolated and the presumptive identification demonstrated the occurrence of Staphylococcus spp. (82.3%), Bacillus spp. (3.9%), Klebsiella spp. (3.9%), Streptococcus spp. (3.9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) and Serratia sp. (2%). Forty-one isolates were successfully identified in the MALDI-TOF analysis, including 35 isolates from eleven different bacterial species. Importantly, there were eight different Staphylococcus species, with a high frequency of Staphylococcus chromogenes (48.6%) and Staphylococcus aureus (20%). Overall, bacterial isolates demonstrated resistance to penicillin (46.3%), tetracycline (39%), amoxicillin (36.6%), ampicillin (34.1%) and sulfamethoxazole/trimethoprim (31.7%). Enrofloxacin was the unique antimicrobial that all isolates were susceptible. In addition, there were six multidrug resistant isolates (five S. chromogenes and one S. aureus). This study highlights that bacterial pathogens with resistance to several antimicrobials were identified in cows from dairy farms in a very important milk producing region located in southern Brazil. Microbial identification of the bovine mastitis pathogens and determination of the antimicrobial profile is necessary for the rational use of the medicines.

Isolation and Identification of Lipopeptide-Producing Bacillus velezensis Strains from Wheat Phyllosphere with Antifungal Activity against the Wheat Pathogen Zymoseptoria tritici

Septoria tritici blotch, caused by the fungal pathogen Zymoseptoria tritici, is a highly significant disease on wheat crops worldwide. The objective of the present study was to find out new bacterial strains with bio-antimicrobial activity against Z. tritici. Two phyllospheric bacteria (S1 and S6) were isolated from wheat ears and identified as Bacillus velezensis strains according to 16S rRNA Sanger sequencing. Antagonistic assays performed with either living strains or cell-free culture filtrates showed significant in vitro antifungal activities against Z. tritici. For the culture filtrates, the half-maximal inhibitory dilution and the minimal inhibitory dilution were 1.4% and 3.7% for the strain S1, and 7.4% and 15% for the strain S6, respectively. MALDI—ToF analysis revealed that both strains synthesize cyclic lipopeptides but from different families. Interestingly, only strain S1 produces putative bacillomycin D. Such differential lipopeptide production patterns might explain the difference observed between the antifungal activity of the culture filtrates of the two strains. This study allows the identification of new lipopeptide-producing strains of B. velezensis with a high potential of application for the biocontrol of Z. tritici.

Surface-engineered liposomes for dual-drug delivery targeting strategy against methicillin-resistant Staphylococcus aureus (MRSA)

This study focused on the encapsulation of vancomycin (VAN) into liposomes coated with a red blood cell membrane with a targeting ligand, daptomycin–polyethylene glycol–1,2-distearoyl-sn-glycero-3-phosphoethanolamine, formed by conjugation of DAPT and N-hydroxysuccinimidyl-polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphoethanolamine. This formulation is capable of providing controlled and targeted drug delivery to the bacterial cytoplasm. We performed MALDI-TOF, NMR and FTIR analyses to confirm the conjugation of the targeting ligand via the formation of amide bonds. Approximately 45% of VAN could be loaded into the aqueous cores, whereas 90% DAPT was detected using UV–vis spectrophotometry. In comparison to free drugs, the formulations controlled the release of drugs for > 72 h. Additionally, as demonstrated using CLSM and flow cytometry, the resulting formulation was capable of evading detection by macrophage cells. In comparison to free drugs, red blood cell membrane–DAPT–VAN liposomes, DAPT liposomes, and VAN liposomes reduced the MIC and significantly increased bacterial permeability, resulting in > 80% bacterial death within 4 h. Cytotoxicity tests were performed in vitro and in vivo on mammalian cells, in addition to hemolytic activity tests in human erythrocytes, wherein drugs loaded into the liposomes and RBCDVL exhibited low toxicity. Thus, the findings of this study provide insight about a dual antibiotic targeting strategy that utilizes liposomes and red blood cell membranes to deliver targeted drugs against MRSA.

The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov., and emended description of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish and four discrete genetic groups exist within the species, suggesting that the species designation requires revision. The present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches and included five representative isolates from each genetic group (including type strain ATCC 23463T; genetic group 1). 16S rRNA gene sequence analysis revealed genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT shared less than <98.8 % sequence identity to F. columnare ATCC 23463T. Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463T, genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT were less than 90.84% and 42.7%, respectively. Biochemical and physiological characteristics were similar among the four genetic groups; however, quantitative differences in fatty acid profiles were detected and MALDI-TOF analyses demonstrated numerous distinguishing peaks unique to each genetic group. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. Herein, the names F. covae sp. nov. (AL-02-36T), F. davisii sp. nov. (90-106T), and F. oreochromis sp. nov. (Costa Rica 04-02-TNT) are proposed to represent genetic groups 2, 3, and 4, respectively.

Liquid Chromatographic/Mass Spectrometric Study on the Role of Beech (Fagus sylvatica L.) Wood Polyphenols in Red Heartwood Formation

The present research focused on the analysis of European beech (Fagus sylvatica L.) wood polyphenols in respect to red heartwood formation, which is a significant color and technological defect of the species. For the first time, high-performance liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) were applied for the investigation of the structure and the radial distribution of polyphenols. Altogether 125 compounds were characterized by their MSn spectra, of which 71 were tentatively identified, including procyanidins (dimers to pentamers), flavonoids (taxifolin, naringenin, isorhamnetin, (epi)afzelechin), (+)-catechin, (−)-epicatechin) and their glycoside conjugates, phenolic acids (gallic acid, vanillic acid, syringic acid) and their glycosides as well as gallic acid derivatives, many of which were identified for the first time in beech wood. It was found that the concentration of many compounds increased at the color boundary. In situ polyphenol synthesis and metabolism were clearly evidenced at the color boundary. Red heartwood contained only free aglycones (syringic acid, taxifolin, naringenin, isorhamnetin, naringenin, syringic acid). Contrary to earlier assumptions, the MALDI-TOF analysis did not indicate the presence of oxidized high-molecular-weight polymeric polyphenols in the red heartwood tissues. The role of individual compounds in the formation of the red heartwood chromophores are discussed.

Storage protein composition during germination and its association with physiological seed quality in common bean

Seed reserve mobilization is considered a post-germination process; however, seed storage protein mobilization occurs during germination. Thus, the knowledge of seed protein composition is important to understand various processes during germination, and it be associated with seed vigor. Therefore, this study aimed to characterize landrace genotypes of common bean (Phaseolus vulgaris) through fractionation of seed storage proteins during germination and to verify the association between seed protein composition and seed vigor. Genotypes of the highest (55 and 81) and lowest (23 and 50) physiological quality were selected. Protein content analysis, profiling, and characterization were performed by combining different hydration times. Mass spectrometry involving in-gel digestion and MALDI-ToF analysis was employed for the qualitative identification of proteins. Glutelin extraction detected the enzyme lipoxygenase in genotypes 55 and 81 alone. In plants, this enzyme may be involved in diverse physiological processes, including growth and development, pest resistance, senescence, and response to wounding. In the process of germination, lipoxygenase removes reactive oxygen species during reserve mobilization; therefore, lipoxygenase may be a candidate biochemical marker for high-vigor genotypes.

Isolation, characterization, and antioxidant activity of neutral carbohydrates from Astragalus arbusculinus gum

“Anzaroot” is an exudate gum obtained from Astragalus species in Iran. Plant gum exudates contain hydrophilic biopolymers, and carbohydrates are the most important ingredients in the gums. Carbohydrates possess different biological properties like enhancement of immunity and antioxidant effect. This study aimed to isolate the novel carbohydrates from Astragalus arbusculinus Bornm. & Gauba gum and investigate their monosaccharides composition and antioxidant activity. For this purpose, the crude water-soluble A. arbusculinus extract was fractionated by DEAE-52 column chromatography. The A. arbusculinus carbohydrate (AAC) fraction was analyzed by high-performance liquid chromatography- evaporative light scattering detector (HPLC-ELSD), and eight compounds, including two major, were detected in its chromatogram. Subsequently, preparative-thin layer chromatography (prep-TLC) was used to isolate and purify the two major compounds, named AAC1 and AAC2. The structural features of AAC1 and AAC2 were investigated by a combination of monosaccharide component analysis using fourier-transform infrared spectroscopy (FT-IR), gas chromatography–mass spectrometry (GC–MS), gas chromatography with a flame ionization detector (GC-FID), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The results indicated that AAC1 was composed of six monosaccharide components, including glucose, rhamnose, and arabinose, with a relative molar ratio of 4:1:1 and molecular weight 971.4531 Da approved by GC–MS and MALDI-TOF analyses, respectively. Also, AAC2 was composed of six monosaccharide units, including glucose, pinitol, and arabinose, with a relative molar ratio of 4:1:1 and molecular weight of 987.4084 Da. Radical scavenging activity of the two compounds was also evaluated against DPPH.

LIMITED PROTEOLYSIS OF FIBRINOGEN BY PROTEASE OF Gloydius halys halys SNAKE VENOM

Aim. One of the approaches for studying structure and functions of proteins is their limited proteolysis. Proteolytic fragments of macromolecules can preserve the biological activity and can be used for the study of their structural and functional peculiarities. Thus, the characterization of new proteolytic enzymes and determination of the specificity of their action can be of interest for exploration. In the present work, we focused on the action of protease from the venom of Gloydius halys halys on fibrinogen, the crucial protein of blood coagulation system. Methods. Products of fibrinogen hydrolysis by protease from the venom of G. halys halys were studied by SDS-PAGE electrophoresis and western-blot analysis using monoclonal antibodies ІІ-5 Сand 1-5A targeted to 20‒78 and 549‒610 fragments of fibrinogen Aα-chain. Molecular weights of hydrolytic products were determined using MALDI-TOF analysis on Voyager DE PRO (USA). Sequence of hydrolytic products were predicted by «Peptide Mass Calculator» soft ware. Results. SDS-PAGE showed that protease from the venom of Gloydius halys halys initially cleaved Аα-chain of fibrinogen molecule. Western-blot analysis confirmed that this protease specifically cleaves off fragment of C-terminal parts of Аα-chain with apparent molecular weight of 22 kDa. Cleaved fragment was identified by MALDI-TOF analysis as the 21.1 kDa polypeptide. «Peptide Mass Calculator» predicted that such a fragment corresponded to Аα414-610 residue of fibrinogen molecule. Thus, we showed that studied protease cleaved peptide bond AαK413-L414 with the formation of stable partly hydrolyzed fibrinogen desAα414-610. Conclusions. The use of protease from the venom of Gloydius halys halys would allow obtaining the unique partly hydrolyzed fibrinogen des Aα 414‒610 that is suitable for the study of structure and functions of fibrinogen αС-regions.

One-Pot Heterotelechelic Metathesis Polymers via Regioselective Chain Transfer Agents.

Single chain transfer agents are used to synthesize narrowly distributed heterotelechelic ROMP polymers in one pot, exploiting a new mechanistic and synthetic approach. The chain transfer agents carrying different functional groups are synthesized in a few straightforward steps. Prefunctionalization of commercially available Grubbs' third-generation catalyst is realized in situ using regioselective chain transfer agents within a short reaction period. After monomer consumption, the excess chain transfer agent in the reaction medium automatically end-functionalizes the polymer chain, yielding a heterotelechelic polymer via a ring-opening-ring-closing sequence. 1H NMR, MALDI-ToF, and SEC analyses confirmed end-group functionalization as well as excellent control over molecular weight and dispersity. This strategy highlights a new way of synthesizing one-pot heterotelechelic ROMP polymers straightforwardly and efficiently.

Label-free cell assays to determine compound uptake or drug action using MALDI-TOF mass spectrometry.

Cell-based assays for compound screening and profiling are fundamentally important in life sciences, chemical biology and pharmaceutical research. Most cell assays measure the amount of a single reporter molecule or cellular endpoint, and require the use of fluorescence or other labeled materials. Consequently, there is high demand for label-free technologies that enable multiple biomolecules or endpoints to be measured simultaneously. Here, we describe how to develop, optimize and validate MALDI-TOF mass spectrometry (MS) cell assays that can be used to measure cellular uptake of transporter substrates, to monitor cellular drug target engagement or to discover cellular drug-response markers. In uptake assays, intracellular accumulation of a transporter substrate and its inhibition by test compounds is measured. In drug response assays, changes to multiple cellular metabolites or to abundant posttranslational protein modifications are monitored as reporters of drug activity. We detail a ten-part optimization protocol with every part taking 1-2 d that leads to a final 2 d optimized procedure, which includes cell treatment, transfer, MALDI MS-specific sample preparation, quantification using stable-isotope-labeled standards, MALDI-TOF MS data acquisition, data processing and analysis. Key considerations for validation and automation of MALDI-TOF MS cell assays are outlined. Overall, label-free MS cell-based assays offer speed, sensitivity, accuracy and versatility in drug research.

Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from Plasmodium knowlesi for the Development of Anti-Malarial Drugs.

Plasmodium lactate dehydrogenase (pLH) is one of the enzymes in glycolysis with potential target for chemotherapy. This study aimed to clone, overexpress and characterize soluble recombinant lactate dehydrogenase from Plasmodium knowlesi in a bacterial system. Synthetic P. knowlesi lactate dehydrogenase (Pk-LDH) gene was cloned into pET21a expression vector, transformed into Escherichia coli strain BL21 (DE3) expression system and then incubated for 18 h, 20 °C with the presence of 0.5 mM isopropyl β-d-thiogalactoside in Terrific broth supplemented with Magnesium sulfate, followed by protein purifications using Immobilized Metal Ion Affinity Chromatography and size exclusion chromatography (SEC). Enzymatic assay was conducted to determine the activity of the enzyme. SDS-PAGE analysis revealed that protein of 34 kDa size was present in the soluble fraction. In SEC, a single peak corresponding to the size of Pk-LDH protein was observed, indicating that the protein has been successfully purified. From MALDI-TOF analysis findings, a peptide score of 282 was established, which is significant for lactate dehydrogenase from P. knowlesi revealed via MASCOT analysis. Secondary structure analysis of CD spectra indicated 79.4% α helix and 1.37% β strand structure. Specific activity of recombinant Pk-LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble Pk-LDH that is biologically active was produced, which can be used further in other malaria studies.

Oxidative biodegradation of a solid-solid polyether-urethane phase change material by Penicillium and Aspergillus

Polyurethane-based polymers are widely used in modern materials and as Phase Change Materials (PCMs) to improve the energy efficiency of buildings. The fungal development in buildings is favoured by the degradation of polymeric materials that act as nutritional substrates. In the present study, the fungal biodegradation of a polyether-urethane PCM (PUX1520) was investigated. Two methods of biodegradation by fungal growth on agar and in liquid media were used. Fungal growth on agar plates was monitored by measuring the surface area of each mycelium over a period of 11 days. The degradation products of PUX1520 were analysed by FT-IR and MALDI-TOF. The PUX1520 polymer had no impact on the growth of Aspergillus niger, while the growth of Penicillium sp. was increased in the presence of PUX1520. MALDI-TOF analysis revealed the existence of different soluble forms of the polymer whose concentration decreased after exposure to the fungal strains. From FT-IR analysis on the residual polymer material, an oxidation process is proposed to describe the dissociation of the urethane functions.