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.

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. Graphical abstract .

Sample preparation for the analysis of key metabolites from cannabinoids biosynthesis in phytoplankton using GC-MS

Cannabinoids biosynthesis in phytoplankton has attracted much attention due to the rapid development of genetic tools and the optimization of genetic transformation methods in microalgae. To monitor the biosynthesis process, proper sample preparation and practical instrumental methods are needed to measure the various precursors, intermediates, cannabinoids, and their degradation products. The objective of this study was to develop a sample preparation procedure for the quantification of olivetolic acid (OA), cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), olivetol (OL), cannabidiol (CBD), and tetrahydrocannabinol (THC) using single-quadrupole gas chromatography-mass spectrometry (GC-MS). Isochrysis galbana was used as the model matrix. After methanol extraction, samples were purified using solid phase extraction (SPE), silylated with N-methyl-N-(trimethylsilyl)trifluoroacetamide, and analyzed using GC-MS in electron ionization mode. A strong anion-exchange SPE efficiently recovered OA, CBGA, CBDA, and THCA. A graphitized carbon black SPE was necessary to purify OL, CBD, and THC. Both columns removed amino acids, sugars, polyols, and pigments from the algae extract and prepared samples that are suitable for silylation and GC-MS analysis. The total protocol, including solvent extraction, SPE, silylation, and GC-MS analysis, was validated in accordance with the ICH guidelines. Performance characteristics of our method are superior to existing protocols with similar complexity in the literature.

Uptake Dynamics of Ionic and Elemental Selenium Forms and Their Metabolism in Multiple-Harvested Alfalfa (Medicago sativa L.)

A pot experiment, under greenhouse conditions, was carried out aiming at investigating the agronomic biofortification of alfalfa (Medicago sativa L.) with Se and monitoring the Se uptake and accumulation dynamics within four consecutive harvests within the same growing season. Two ionic Se forms, i.e., sodium selenate (Se (VI)) and sodium selenite (Se (IV)), were applied once at a rate of 1, 10, and 50 mg kg−1 (added on Se basis), while 10 and 50 mg L−1 of a red elemental Se (red Se0) were used; all Se treatments were added as soil application. Application of Se (VI) at the rate of 50 mg kg−1 was toxic to alfalfa plants. The effect of Se forms on Se accumulation in alfalfa tissues, regardless of the applied Se concentration, follows: Se (VI) > Se (IV) > red Se0. The leaf, in general, possessed higher total Se content than the stem in all the treatments. The accumulation of Se in stem and leaf tissues showed a gradual decline between the harvests, especially for plants treated with either Se (VI) or Se (IV); however, the chemically synthesized red Se0 showed different results. The treatment of 10 mg kg−1 Se (VI) resulted in the highest total Se content in stem (202.5 and 98.0 µg g−1) and leaf (643.4 and 284.5 µg g−1) in the 1st and 2nd harvests, respectively. Similar tendency is reported for the Se (IV)-treated plants. Otherwise, the application of red Se0 resulted in a lower Se uptake; however, less fluctuation in total Se content between the four harvests was noticed compared to the ionic Se forms. The Se forms in stem and leaf of alfalfa extracted by water and subsequently by protease XIV enzyme were measured by strong anion exchange (SAX) HPLC-ICP-MS. The major Se forms in our samples were selenomethionine (SeMet) and Se (VI), while neither selenocysteine (SeCys) nor Se (IV) was detected. In water extract, however, Se (VI) was the major Se form, while SeMet was the predominant form in the enzyme extract. Yet, Se (VI) and SeMet contents declined within the harvests, except in stem of plants treated with 50 mg L−1 red Se0. The highest stem or leaf SeMet yield %, in all harvests, corresponded to the treatment of 50 mg L−1 red Se0. For instance, 63.6% (in stem) and 38.0% (in leaf) were calculated for SeMet yield % in the 4th harvest of plants treated with 50 mg L−1 red Se0. Our results provide information about uptake and accumulation dynamics of different ionic Se forms in case of multiple-harvested alfalfa, which, besides being a good model plant, is an important target plant species in green biorefining.

Retention Time Prediction Using Neural Networks Increases Identifications in Crosslinking Mass Spectrometry

Crosslinking mass spectrometry (Crosslinking MS) has developed into a robust technique that is increasingly used to investigate the interactomes of organelles and cells. However, the incomplete and noisy information in the spectra limits the numbers of protein-protein interactions (PPIs) that can be confidently identified. Here, we successfully leveraged chromatographic retention time (RT) information to aid the identification of crosslinked peptides from spectra. Our Siamese machine learning model xiRT achieved highly accurate RT predictions of crosslinked peptides in a multi-dimensional separation of crosslinked E. coli lysate. We combined strong cation exchange (SCX), hydrophilic strong anion exchange (hSAX) and reversed-phase (RP) chromatography and reached R2 0.94 in RP and a margin of error of 1 fraction for hSAX in 94%, and SCX in 85% of the predictions. Importantly, supplementing the search engine score with retention time features led to a 1.4-fold increase in PPIs at a 1% false discovery rate. We also demonstrate the value of this approach for the more routine analysis of a crosslinked multiprotein complexes. An increase of 1.7-fold in heteromeric crosslinked residue-pairs was achieved at 1% residue-pair FDR for Fanconi anaemia monoubiquitin ligase complex, solely using reversed-phase RT. Retention times are a powerful complement to mass spectrometric information to increase the sensitivity of Crosslinking MS analyses.

Label-Free Quantitative Phosphoproteomics of the Fission Yeast Schizosaccharomyces pombe Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies

The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the S. pombe phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe.