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

Abstract 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.

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

Scientific Reports ◽

10.1038/s41598-021-93279-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Doaa A. Goda ◽

Ahmad R. Bassiouny ◽

Nihad M. Abdel Monem ◽

Nadia A. Soliman ◽

Yasser R. Abdel-Fattah

Keyword(s):

Amino Acids ◽

Partial Purification ◽

Tetraacetic Acid ◽

Optimal Conditions ◽

Cultivation Temperature ◽

Purification And Characterization ◽

Keratinolytic Activity ◽

Molecular Masses ◽

Keratin Azure ◽

Protein Lysate

AbstractIncubation 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.

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N-Acetyl-ß-ᴅ-hexosaminidases of the Brine Shrimp Artemia: Partial Purification and Characterization

Zeitschrift für Naturforschung C ◽

10.1515/znc-1991-9-1010 ◽

1991 ◽

Vol 46 (9-10) ◽

pp. 781-788 ◽

Cited By ~ 5

Author(s):

Klaus-Dieter Spindler ◽

Brigitte Funke-Höpfner

Keyword(s):

Brine Shrimp ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Partial Purification ◽

Purification And Characterization ◽

Strong Inhibitor ◽

Molecular Masses ◽

Dose Dependent ◽

Enzyme I

Abstract N-Acetyl-β-ᴅ-hexosaminidases (EC 3.2.1.52) from Artemia nauplii were isolated and characterized. Three different enzymes I, II1 and II2 were separated according to their behaviour on anion exchange chromatography and gel filtration columns. Their apparent molecular masses were 83,000 ± 7000, 110,000 ± 10,000 and 56,000 ± 5000 Da with corresponding S-values of 8.6, 11.9 and 7.9. All three enzymes also differ in their apparent pH-optima (5.1, 4.5 and 6.1) and they all bind to concanavalin A. The three enzymes have about the same affinities (app. Km between 0.16 and 0.72 mmol/1) for the three substrates (p-nitrophenyl-N-acetyl-β-ᴅ-glucosamine or p-nitrophenyl-N-acetyl-β-ᴅ-galactosamine and N ,N′-diacetyl-chitobiose) and are therefore N-acetyl-β-ᴅ-hexosaminidases. In contrast, the three enzymes behave quite differently, both in terms of their inhibitor constants and the type of inhibition. The substrates inhibit both enzymes II1 and II2 but not enzyme I. On the other hand, N-acetyl-β-ᴅ-galactosamine inhibits enzyme I in a non-competitive way but not enzymes II1 and II2. All three enzymes are inhibited by the end product N-acetyl-β-ᴅ-glucosamine, enzyme I in a competitive manner, both enzymes II1 and II2 in a non-competitive way. 2-Acetamido-2-deoxy-ᴅ-galactonolactone is a strong inhibitor for enzyme I (Ki = 13 μtmol/l) with much lower affinities towards enzymes II1 and II2 (Ki = 0.63 and 1.03 mmol/l). All three enzymes are inhibited in a dose-dependent way and completely reversible by α-methyl-mannoside.

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Partial purification and characterization of ribonucleases from roots, stem and leaves of cowpea

Revista Brasileira de Fisiologia Vegetal ◽

10.1590/s0103-31312001000300010 ◽

2001 ◽

Vol 13 (3) ◽

pp. 357-364 ◽

Cited By ~ 4

Author(s):

OCTÁVIO LUIZ FRANCO ◽

LORRANCE ABREU GONDIM ◽

KÁTIA REGINA BEZERRA ◽

MARIA ELANE DE CARVALHO GUERRA ◽

CARMEM ROGÉLIA FARIAS MACHADO LIMA ◽

...

Keyword(s):

Exchange Chromatography ◽

Partial Purification ◽

Ionic Exchange ◽

Purification And Characterization ◽

Chemical Denaturation ◽

Molecular Masses ◽

Deoxyribonuclease Activity ◽

Different Tissues ◽

Optimal Ph

Partial purification and characterization of ribonucleases (RNase; EC 3.1.27.1) present in roots, stem and leaves of 5 day-old Pitiúba cowpea [Vigna unguiculata (L.) Walp.] seedlings are described. Crude extracts from the different tissues were precipitated with ammonium sulfate followed by ionic exchange chromatography (CM-Cellulose) resulting in purification factors of 48-fold for roots, 21 for stem and 42 for leaves. No deoxyribonuclease activity was practically observed. The molecular masses of the RNases did not significantly differ, averaging 16.3 kDa. Leaf RNase was stable up to 50ºC while the others were inactivated at this temperature. The maximal inactivation for both stem and roots RNases was reached at 70ºC while for leaf it occurred at 80ºC. The addition of KCl to the assay medium caused a shift of optimal pH from 6.0 toward the range of 5.2 - 5.6 for the enzymes extracted from the different tissues. RNase activities were strongly inhibited by Hg2+, Zn2+ and Cu2+, partially inhibited by Co2+ and Fe2+ and were not affected by EDTA, Ca2+ or Mg2+. In contrast to the leaf RNase, roots and stem enzymes were inactivated by urea and 2-mercaptoethanol (2-ME). Although there is a great similarity among the enzymes studied, leaf RNase appears to be more stable to heat and to chemical denaturation than root and stem RNases. The results also suggest that the enzymes extracted from different tissues of Pitiúba cowpea seedlings are ribonucleases and not nucleases.

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