Aspartic protease from Aspergillus niger: Molecular characterization and interaction with pepstatin A

In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneit...

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Veröffentlicht in:International journal of biological macromolecules 2019-10, Vol.139, p.199-212
Hauptverfasser: Purushothaman, Kavya, Bhat, Sagar Krishna, Singh, Sridevi Annapurna, Marathe, Gopal Kedihithlu, Appu Rao, Appu Rao G.
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container_title International journal of biological macromolecules
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creator Purushothaman, Kavya
Bhat, Sagar Krishna
Singh, Sridevi Annapurna
Marathe, Gopal Kedihithlu
Appu Rao, Appu Rao G.
description In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ± 0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ± 1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 μM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in β-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin > defatted soya flour > gluten > gelatin > skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.
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Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ± 0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ± 1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 μM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in β-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin &gt; defatted soya flour &gt; gluten &gt; gelatin &gt; skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. 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Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ± 0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ± 1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 μM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in β-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin &gt; defatted soya flour &gt; gluten &gt; gelatin &gt; skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. 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Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ± 0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ± 1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 μM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in β-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. 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subjects Aspartic Acid Proteases - antagonists & inhibitors
Aspartic Acid Proteases - chemistry
Aspartic Acid Proteases - isolation & purification
Aspartic Acid Proteases - metabolism
Aspartic protease
Aspergillus niger - classification
Aspergillus niger - enzymology
Aspergillus niger - genetics
Catalysis
Chromatography, Liquid
Commercial application
Enzyme Stability
Hydrogen-Ion Concentration
Hydrolysis
Molecular Weight
Pepstatin A interaction
Pepstatins - chemistry
Pepstatins - pharmacology
Phylogeny
Protease Inhibitors - chemistry
Protease Inhibitors - pharmacology
Protein Binding
Structure and stability
Structure-Activity Relationship
Tandem Mass Spectrometry
Temperature
title Aspartic protease from Aspergillus niger: Molecular characterization and interaction with pepstatin A
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