The use of nitrocellulose filters to study DNA binding proteins in crude cell lysates: Effect of competing DNA

A nitrocellulose-filter-binding assay system for DNA-protein interactions, suitable for use with crude cell lysates, is described. Such an assay system will detect DNA-binding activities, provided that close attention is paid to the overall concentration of proteins and DNA in the reaction system. The extent of the reduction of generalized DNA-binding by the addition of unlabeled competing DNA is shown to be a function of the source of the competing DNA, since the addition of equal quantities of DNA isolated from different organisms produces drastically different effects. A careful choice of labeled and unlabeled DNA permits preferential binding of sequences from labeled DNA and allows the use of the filter-binding assay as an analytical tool during protein purification.     

Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles

Proteases are the most important group of industrial enzymes and they can be used in several fields including biorefineries for the valorization of industrial byproducts. In this study, we purified and characterized novel extremophilic proteases produced by a Pseudomonas aeruginosa strain isolated from Mauritia flexuosa palm swamps soil samples in Peruvian Amazon. In addition, we tested their ability to hydrolyze distillers dried grains with solubles (DDGS) protein. Three alkaline and thermophilic serine proteases named EI, EII, and EIII with molecular weight of 35, 40, and 55 kDa, respectively, were purified. EI and EIII were strongly inhibited by EDTA and Pefabloc being classified as serine-metalloproteases, while EII was completely inhibited only by Pefabloc being classified as a serine protease. In addition, EI and EII exhibited highest enzymatic activity at pH 8, while EIII at pH 11 maintaining almost 100% of it at pH 12. All the enzymes demonstrated optimum activity at 60°C. Enzymatic activity of EI was strongly stimulated in presence of Mn²⁺ (6.9-fold), EII was stimulated by Mn²⁺ (3.7-fold), while EIII was slightly stimulated by Zn²⁺, Ca²⁺, and Mg²⁺. DDGS protein hydrolysis using purified Pseudomonas aeruginosa M211 proteases demonstrated that, based on glycine released, EIII presented the highest proteolytic activity toward DDGS. This enzyme enabled the release 63% of the total glycine content in wheat DDGS protein, 2.2-fold higher that when using the commercial Pronase®. Overall, our results indicate that this novel extremopreoteases have a great potential to be applied in DDGS hydrolysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2728, 2019     

Microplate well coverage mixing using superhydrophobic contact

Two important challenges in microplate instrumentation are to achieve full well sample coverage and complete mixing. An effective approach of using superhydrophobic rods to accomplish these challenges is reported here. Experiments conducted showed that analytes above 50μl could be made to completely cover the bottom of 96-well standard and transparency microplates. Complete mixing was accomplished by moving the rod parallel to the well bottom while contacting the liquid. The approach is simple and controlled, and it minimizes the problems of spillage and cross-contamination. It works with analytes with varied volumes and of different viscosities present in each well of the microplate.