Activity- and Enrichment-Based Metaproteomics Insights into Active Urease from the Rumen Microbiota of Cattle

Regulation of microbial urease activity plays a crucial role in improving the utilization efficiency of urea and reducing nitrogen emissions to the environment for ruminant animals. Dealing with the diversity of microbial urease and identifying highly active urease as the target is the key for future regulation. However, the identification of active urease in the rumen is currently limited due to large numbers of uncultured microorganisms. In the present study, we describe an activity- and enrichment-based metaproteomic analysis as an approach for the discovery of highly active urease from the rumen microbiota of cattle. We conducted an optimization method of protein extraction and purification to obtain higher urease activity protein. Cryomilling was the best choice among the six applied protein extraction methods (ultrasonication, bead beating, cryomilling, high-pressure press, freeze-thawing, and protein extraction kit) for obtaining protein with high urease activity. The extracted protein by cryomilling was further enriched through gel filtration chromatography to obtain the fraction with the highest urease activity. Then, by using SDS-PAGE, the gel band including urease was excised and analyzed using LC-MS/MS, searching against a metagenome-derived protein database. Finally, we identified six microbial active ureases from 2225 rumen proteins, and the identified ureases were homologous to those of Fibrobacter and Treponema. Moreover, by comparing the 3D protein structures of the identified ureases and known ureases, we found that the residues in the β-turn of flap regions were nonconserved, which might be crucial in influencing the flexibility of flap regions and urease activity. In conclusion, the active urease from rumen microbes was identified by the approach of activity- and enrichment-based metaproteomics, which provides the target for designing a novel efficient urease inhibitor to regulate rumen microbial urease activity.

Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System v2

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System

Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System v2

This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Quantitation with the Biomek-FX liquid handler system

Automated Protein Quantification with the Biomek-FX Liquid Handler System v2

This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best as part of a full proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System

Optimization of the extraction and precipitation process of a leaf protein concentrate from Moringa oleifera Lam.

This study aimed to determine the best extraction and precipitation conditions of Moringa oleifera Lam. leaf protein. The influence of pH (10, 11, 12) and the concentration of NaCl (0, 0.25, 0.5) for the protein extraction process were studied through a Completely Randomized Design (CRD) with factorial arrange 32. The combination of pH 11 and 12 with 0 M NaCl had the best yield (P<0.05). The treatment of pH 11 without NaCl followed a precipitation stage for its purification, and the effect of different levels of pH (4, 4.5, 5) and temperature (40, 60, 80 °C) were evaluated using a CRD with factorial arrange 22 and 6 central points. The temperature did not affect the yield of the process in a significant way and the amount of precipitate was maximized at pH 4 and 4.5. From 100 g of the dry leaf, 7.26±0.19 g of protein was isolated with a recovery of 26.93±0.22 g 100 g-1 from the total protein. Due to their astringency and bitterness, consuming large amounts of Moringa oleifera Lam leaves is not a solution; therefore, obtaining a leaf proteinconcentrate could be useful for diverse applications in nutritional supplements, and as raw material for functional products development.

Caseinolytic proteases of Lactobacillus and Lactococcus strains isolated from fermented dairy products

Proteases of Lactobacillus and Lactococcus strains catalyze casein degradation in fermented dairy products, which can result in the production of bioactive peptides. Proteolytic properties of a selection of lactic acid bacteria (LAB) strains previously isolated in Croatia, including Lactococcus lactis and Lactobacillus strains, are described. All strains of Lactobacillus and Lactococcus showed an Fmc+ phenotype that can be associated with efficient growth in milk. The significant caseinolytic effect, after incubation of culture supernatant or concentrated cell biomass, was observed for Levilactobacillus brevis D6 and Lactiplantilactobacillus plantarum D13 after growth in the optimal growth medium, while for Lactoccocus lactis ZGBP5-32 and Levilactobacillus brevis SF9B strains after growth in skimmed milk. To assess the LAB growth in skimmed milk, the acidification rate was monitored. Statistically, significant acidification capacity was determined for L. plantarum D13 in the optimal medium and by the proteolytic strain Lactobacillus helveticus M92 in skimmed milk. After extraction of proteinases from the strains with caseinolytic activity, protein samples were analysed by the SDS-PAGE. The protein extract of the Lc. lactis ZGBP5-32 and ZGZA7-10, retained proteolytic activity even at very low concentrations. The ultrafiltration improved protein extraction. The crude extract possibly contained putative protease, as a decrease in contaminating proteins was confirmed by SDS-PAGE in samples of L. brevis D6 and SF9B, L. fermentum D12 and L. plantarum D13.

Water as carrier of information of heat shock and drug effect between two groups of Adhatoda vasica plants

Adhatoda vasica Nees plants were grown in 50 earthen pots, which were divided into 5 batches A, B, C, D, and E. Of these A, B and C, D were arranged into two separate parallel pairs. One leaf of each plant of an adjacent pair was immersed in sterile tap water in a beaker. Adjacent beakers in each pair A B or C D were connected by polythene tubes containing wet cotton threads. One leaf of each plant of A was given heat shock by immersing a leaf in hot water for 5 min. One leaf of each plant of C was treated with Cantharis vesicatoria 200c. Batch E served as the unstressed and untreated control. One hour after heat shock or drug treatment all the leaves were harvested and their proteins were extracted by chilled protein extraction buffer. Proteins were separated by Fast Protein Liquid Chromatography (FPLC). Protein profiles of A, B and C, D showed marked similarity with respect to expression and repression of some proteins. It is concluded that the effect of heat shock and drug treatment is transmitted through water in the capillaries of cotton threads connecting the pairs of plants. It is assumed that heat shock or drug treatment altered locally the water structure in the leaves which was propagated through global network of water structure over the protein network in the whole plants, and from there to the interfacial water in the beakers and cotton threads. A homeopathic potency is thought to be specifically structured water which influences the water structure in the treated organism.