Investigating Toxin Diversity and Abundance in Snake Venom Proteomes

Understanding snake venom proteomes is becoming increasingly important to understand snake venom biology, evolution and especially clinical effects of venoms and approaches to antivenom development. To explore the current state of snake venom proteomics and transcriptomics we investigated venom proteomic methods, associations between methodological and biological variability and the diversity and abundance of protein families. We reviewed available studies on snake venom proteomes from September 2017 to April 2021. This included 81 studies characterising venom proteomes of 79 snake species, providing data on relative toxin abundance for 70 species and toxin diversity (number of different toxins) for 37 species. Methodologies utilised in these studies were summarised and compared. Several comparative studies showed that preliminary decomplexation of crude venom by chromatography leads to increased protein identification, as does the use of transcriptomics. Combining different methodological strategies in venomic approaches appears to maximize proteome coverage. 48% of studies used the RP-HPLC →1D SDS-PAGE →in-gel trypsin digestion → ESI -LC-MS/MS pathway. Protein quantification by MS1-based spectral intensity was used twice as commonly as MS2-based spectral counting (33–15 studies). Total toxin diversity was 25–225 toxins/species, with a median of 48. The relative mean abundance of the four dominant protein families was for elapids; 3FTx–52%, PLA2–27%, SVMP–2.8%, and SVSP–0.1%, and for vipers: 3FTx–0.5%, PLA2–24%, SVMP–27%, and SVSP–12%. Viper venoms were compositionally more complex than elapid venoms in terms of number of protein families making up most of the venom, in contrast, elapid venoms were made up of fewer, but more toxin diverse, protein families. No relationship was observed between relative toxin diversity and abundance. For equivalent comparisons to be made between studies, there is a need to clarify the differences between methodological approaches and for acceptance of a standardised protein classification, nomenclature and reporting procedure. Correctly measuring and comparing toxin diversity and abundance is essential for understanding biological, clinical and evolutionary implications of snake venom composition.

Biosorption and Bioleaching of Heavy Metals from Electronic Waste Varied with Microbial Genera

Industrialization and technological advancements have led to the exploitation of natural resources and the production of hazardous wastes, including electronic waste (E-waste). The traditional physical and chemical techniques used to combat E-waste accumulation have inherent drawbacks, such as the production of harmful gases and toxic by-products. These limitations may be prudently addressed by employing green biological methods, such as biosorption and bioleaching. Therefore, this study was aimed at evaluating the biosorption and bioleaching potential of seven microbial cultures using E-waste (printed circuit board (PCB)) as a substrate under submerged culture conditions. The cut pieces of PCB were incubated with seven microbial cultures in liquid broth conditions in three replicates. Atomic absorption spectroscopy (AAS) analysis of the culture biomass and culture filtrates was performed to evaluate and screen the better-performing microbial cultures for biosorption and bioleaching potentials. The best four cultures were further evaluated through SEM, energy-dispersive X-ray spectroscopy (EDX), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) studies to identify the possible culture that can be utilized for the biological decontamination of E-waste. The study revealed the highest and differential ability of Pleurotus florida and Pseudomonas spp. for biosorption and bioleaching of copper and iron. This can be attributed to bio-catalysis by the laccase enzyme. For both P. florida and Pseudomonas spp. on the 20th day of incubation, laccase exhibited higher specific activity (6.98 U/mg and 5.98 U/mg, respectively) than other microbial cultures. The biomass loaded with Cu2+ and Fe2+ ions after biosorption was used for the desorption process for recovery. The test cultures exhibited variable copper recovery efficiencies varying between 10.5 and 18.0%. Protein characterization through SDS-PAGE of four promising microbial cultures exhibited a higher number of bands in E-waste as compared with microbial cultures without E-waste. The surface topography studies of the E-waste substrate showed etching, as well as deposition of vegetative and spore cells on the surfaces of PCB cards. The EDX studies of the E-waste showed decreases in metal element content (% wt/% atom basis) on microbial treatment from the respective initial concentrations present in non-treated samples, which established the bioleaching phenomenon. Therefore, these microbial cultures can be utilized to develop a biological remediation method to manage E-waste.

Mitochondrial сomplexome of etiolated pea shoots

Studies into mitochondrial сomplexomes in various organisms provide an insight into the native organization of proteins and metabolic pathways in the organelles of the subject under study. “Complexome” is a relatively recent concept describing the proteome of protein complexes, supercomplexes, and oligomeric proteins. Complexome analysis is performed using current electrophoretic and mass spectrometric techniques, in particular, by two-dimensional electrophoresis (2D BN/SDS-PAGE) in combination with mass spectrometry (MS). Unlike 2D IEF/SDS-PAGE, this method enables analysis of not only hydrophilic proteins of the mitochondrial matrix, but also membrane proteins and their associations, thus expanding the possibilities of studying the organelle proteome. In the present work, the complexome of etiolated pea shoots was studied for the first time using 2D BN/SDS-PAGE followed by MALDI-TOF MS. To this end, 145 protein spots excised from the gel were analyzed; 110 polypeptides were identified and assigned to different functional groups. A densitometric analysis revealed that the major protein group comprised the enzymes of the mitochondrial energy system (1), accounting for an average of 43% of the total polypeptide content. The remaining 57% was primarily distributed among the following functional categories: pyruvate dehydrogenase complex and citric acid cycle (2); amino acid metabolism (3); nucleic acid processing (4); protein folding (5); antioxidant protection (6); carrier proteins (7); other proteins (8); proteins having unknown functions (9). The obtained data indicate the complex organization of the pea proteome. In addition to the enzymes of the OXPHOS system, the proteins of other functional categories are found to form supramolecular structures. It is suggested that the presence of proteins from other cellular compartments may indicate the interaction of mitochondria with the enzymes or structures of corresponding organelles. In general, the obtained data on the pea complexome represent a kind of a mitochondrial “passport” that reflects the native state of the proteome of organelles corresponding to their physiological status.

Purification and Enzymatic Properties of a New Thermostable Endoglucanase From Aspergillus Oryzae HML366

Aspergillus oryzae HML366 is a newly screened cellulase-producing strain. The endoglucanase HML ED1 from A. oryzae HML366 was quickly purified by two-step method ammonium sulfate precipitation and strong anion exchange column. SDS-PAGE electrophoresis indicated that the molecular weight of the enzyme was 68 kDa. The optimum temperature of the purified endoglucanase was 60 ℃ and the enzyme activity was stable below 70 ℃. The optimum pH was 6.5, and the enzyme activity was stable at pH between 4.5 to 9.0. The analysis indicated that additional Na+, K+, Ca2+, and Zn2+ reduced the catalytic ability of enzyme to the substrate, but Mn2+ enhanced its catalytic ability to the substrate.The Km and Vmax of the purified endoglucanase was 8.75 mg/mL and 60.24 μg/min·mL, respectively. Our study demonstrated that A. oryzae HML366 can produce a heat-resistant and wide pH tolerance endoglucanase HML ED1, which has potential industrial application value in bioethanol, paper, food, textile, detergent and pharmaceutical industries.

Production and Identification of Polypeptide from Silks by Hydrothermal Method

There are two kinds of proteins in silk, sericin and silk fibroin. Polypeptide compounds from silk sericin and silk fibroin were prepared by hydrothermal method. The process of silk dissolution was investigated under different solid-liquid ratio, reaction temperature and reaction time. By controlling the operating parameters of hydrothermal method, the temperature, material ratio and time were further optimized, and the best experimental results were obtained, the expected decomposition of silks occurred when the ratio of silks to waters was selected as 1 to 10, at 140 degree in 30 min. The molecular weight of polypeptide was detected by SDS-PAGE electrophoresis and analysed by MALDI-TOF-MS. The results showed that the molecular weight of the polypeptide obtained from silks was about 6000-8000Da. After literature research, the polypeptide with such molecular weight could have better performance for some functional additives.

Comparative Evaluation of Adsorption of Major Enzymes in a Cellulase Cocktail Obtained from Trichoderma reesei onto Different Types of Lignin

Cellulase adsorption onto lignin decreases the productivity of enzymatic hydrolysis of lignocellulosic biomass. Here, adsorption of enzymes onto different types of lignin was investigated, and the five major enzymes—cellobiohydrolases (CBHs), endoglucanase (Cel7B), β-glucosidase (Cel3A), xylanase (XYNIV), and mannanase (Man5A)—in a cellulase cocktail obtained from Trichoderma reesei were individually analyzed through SDS-PAGE and zymogram assay. Lignin was isolated from woody (oak and pine lignin) and herbaceous (rice straw and kenaf lignin) plants. The relative adsorption of CBHs compared to the control was in the range of 14.15–18.61%. The carbohydrate binding motif (CBM) of the CBHs contributed to higher adsorption levels in oak and kenaf lignin, compared to those in pine and rice lignin. The adsorption of endoglucanase (Cel7B) by herbaceous plant lignin was two times higher than that of woody lignin, whereas XYNIV showed the opposite pattern. β-glucosidase (Cel3A) displayed the highest and lowest adsorption ratios on rice straw and kenaf lignin, respectively. Mannanase (Man5A) was found to have the lowest adsorption ratio on pine lignin. Our results showed that the hydrophobic properties of CBM and the enzyme structures are key factors in adsorption onto lignin, whereas the properties of specific lignin types indirectly affect adsorption.

Selected Quality Attributes of Freshwater Mussel Powder as a Promising Ingredient for Pet Food

The aim of this study was to describe the quality attributes of a freeze-dried preparation obtained from freshwater mussel Sinanodonta woodiana (SW) soft tissue in respect to its potential as a novel pet food ingredient. After ecotoxicological testing of the raw material with MARA (Microbial Assay for Risk Assessment), the basic physico-chemical properties of the powder, such as approximate composition, bulk density, color parameters, water activity, electrophoretic analysis (SDS-PAGE), solubility, gelling and emulsifying capacity, were analyzed. The powder with a water activity of 0.43 offers a toxically safe preparation that contains over 34% protein/100 g of dry matter (DM). The SDS-PAGE profile showed twelve protein bands with a molecular weight (MW) ranging from >250 to 10 kDa. Taurine content has been estimated at an essential amount above 150 mg/100 g of DM. The powder possessed desirable emulsifying properties with 230 mL per 1 g and demonstrated the ability to form a firmer gel with a strength of 152.9 g at a temperature above 80 °C with at least 10% protein content. The L*, a*, and b* values characterizing powder color were found to be 69.49, 16.33, and 3.86, respectively. The SW mussel powder seems to be a promising ingredient that can be added with other binding or gelling agents in order to improve both the taste and acceptance of the final pet food products.

Analisis Bioinformatika dan Ekspresi Protein Rekombinan Hemagglutinin Domain Globular dari Virus H5N1 Indonesia pada Eschericia coli BL21 (DE3) sebagai Komponen Vaksin Subunit Influenza

Flu burung merupakan salah satu penyakit zoonosis yang patut diwaspadai di Indonesia, khususnya galur High Pathogenic Avian Influenza (HPAI) karena dapat mematikan jika menular kepada manusia. Penggunaan vaksin influenza pada unggas, merupakan langkah preventif terhadap evolusi virus yang berbahaya dan juga penyebarannya. Selama ini, Indonesia masih menggunakan seed vaksin impor yang berasal dari luar Indonesia. Namun, karena Indonesia merupakan negara yang berada di garis khatulistiwa, karakteristik virus bisa berbeda dengan virus dari nothern-hemispere maupun southern-hemispere. Mengingat hal tersebut, Indonesia harus mengembangkan vaksin influenza menggunakan galur virus lokal. Berbeda dengan vaksin whole virus, vaksin rekombinan memiliki keunggulan dari sisi kemurnian, kecepatan produksi, dan kesesuaian galur terhadap virus yang beredar saat diperlukan. Penelitian ini bertujuan untuk menganalisis sekuen hemagglutinin (HA) Indonesia dengan strain lainnya serta mengekspresikkan protein HA1 rekombinan pada Escherichia coli BL21 (DE3). Galur yang digunakan pada studi ini berasal dari virus H5N1 (A/Indonesia/05/05), khususnya bagian domain globular dari HA1. Sekuen HA1 bervariasi antara strain Indonesia dengan nothern-hemispere maupun southern-hemispere dan merupakan protein yang terpapar ke luar virus. Gen HA1 disisipkan pada vektor pET-28a, kemudian plasmid diisolasi menggunakan meoe manniatis, setelah itu diekspresikan dengan induksi 1 mM IPTG selama 4 jam. Protein HA1 telah berhasil diekspresikan secara intraseluler dan telah dikonfirmasi pada berat molekul 40 kDa menggunakan SDS-PAGE. Penelitian ini dapat digunakan untuk mengembangkan vaksin subunit yang lebih spesifik terhadap virus yang beredar di lapangan.