Feather protein lysate optimization and feather meal formation using YNDH protease with keratinolytic activity afterward enzyme partial purification and characterization

Incubation parameters used for the creation of a protein lysate from enzymatically degraded waste feathers using crude keratinase produced by the Laceyella sacchari strain YNDH were optimized using the Response Surface Methodology (RSM); amino acids quantification was also estimated. The optimization elevated the total protein to 2089.5 µg/ml through the application of the following optimal conditions: a time of 20.2 h, a feather concentration (conc.) of 3 g%, a keratinase activity of 24.5 U/100 ml, a pH of 10, and a cultivation temperature of 50 °C. The produced Feather Protein Lysate (FPL) was found to be enriched with essential and rare amino acids. Additionally, this YNDH enzyme group was partially purified, and some of its characteristics were studied. Crude enzymes were first concentrated with an Amicon Ultra 10-k centrifugal filter, and then concentrated proteins were applied to a "Q FF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranging from 6 to 10 kDa. The maximum enzyme activity was observed at 70 °C and for a pH of 10.4. Most characteristics of this protease/keratinase group were found to be nearly the same when the activity was measured with both casein and keratin-azure as substrates, suggesting that these three protein bands work together in order to degrade the keratin macromolecule. Interestingly, the keratinolytic activity of this group was not inhibited by ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), or iron-caused activation, indicating the presence of a mixed serine–metallo enzyme type.

Feather Biodegradation For Feather Protein Lysate/ Feather Meal Mutual Formations: Optimization Using Crude YNDH Protease/Keratinase; Enzyme Partial Purification and Characterization

Incubation parameters used for the production of a protein lysate from enzymatically degraded waste feather using crude keratinase produced by laceyella sacchari YNDH was optimized using RSM, amino acids quantification was estimated as well. The optimization resulted in total soluble protein 2089.5µg/ml. The optimal conditions were, time 20.2h, feather concentration 3g% and keratinase activity 24.5U/100ml at pH, 10; and cultivation temperature 50oC. The FPL was found enriched with essential and rare amino acids.In parallel, this YNDH enzyme group had been partially purified and some characteristics of it were studied. Crude enzymes were first concentrated with Amicon Ultra 10k centrifugal filter, and then concentrated proteins were applied to "QFF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranged 6 to 10 kDa. The maximum enzyme activity was observed at 70°C and pH 10.4 when measured by both casein and keratin azure as substrates. Interestingly, keratinolytic activity of this group was not affect by EDTA, PMSF and DTT. Generally the overall characters of this group protease/keratinase nearly the same when its activity was measured with both substrates suggesting that all these 3 protein bands working together as a group of keratinases.

Immunoprecipitation of protein lysate from culture cells using Src antibody

It is a procedure for immunoprecipitation using protein lysate from a neuronal cell line, NSC34, and a muscle cell line, C2C12 using DynabeadsTM Protein G Immunoprecipitation Kit (ThermoFisher).