Study of protease activity from Aspergillus awamori INCQS2B.361U2/1 extracellular fraction and modification of culture medium composition to isolate a novel aspartic protease

• Published in: Brazilian journal of microbiology Vol. 53; no. 3; pp. 1599 - 1611
• Main Authors: da Silva-López, Raquel Elisa, de Araujo, Thayane Aparecida Alves, Monteiro, Hélvio José Jalles, Teixeira, Érika Maria Gomes Ferreira, Tupi, Lucas, da Silva Bon, Elba Pinto
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01-09-2022; Springer Nature B.V
• Subjects: Affinity chromatography; Arginine; Aspartic Acid Proteases - chemistry; Aspartic Acid Proteases - genetics; Aspartic Acid Proteases - metabolism; Aspartic endopeptidase; Aspergillus - metabolism; Aspergillus awamori; Biochemical characteristics; Biochemistry; Biomedical and Life Sciences; Breccia; Food Microbiology; Fungal and Bacterial Physiology - Research Paper; Gelatin; Hydrogen-Ion Concentration; Life Sciences; Medical Microbiology; Microbial Ecology; Microbial Genetics and Genomics; Microbiology; Mycology; Pepstatins - metabolism; Peptidase; Peptidases; Peptide Hydrolases; Peptides; pH effects; Protease; Proteins; Substrates; Thermal stability; Yeasts; Aspartic protease isolation; Extracellular fraction; Culture medium modification supplementation; Enzyme characterization; Aspergillus awamori
• ISSN: 1517-8382; 1678-4405
• DOI: 10.1007/s42770-022-00750-0

Aspergillus awamori
was cultivated in a modified
Breccia
medium, and the extracellular fraction was obtained, which presented 260 ± 15 µg of protein/mg and specific protease activity of 3.87 ± 0.52 mM.min
−1
.mg of protein
−1
using
N
α
-
p
-tosyl-L-arginine methyl ester hydrochloride (
L
-TAME) as substrate. This fraction showed major proteins about 104 and 44 kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that
A. awamori
secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified
Breccia
medium,
A. awamor
i protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75 kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50 °C. Pepstatin inhibited about 80% of ASPA activity, with IC
50
and
Ki
values of 0.154 and 0.072 μM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with
K
M
0.0589 mM and
V
max
1.909 mM.min
−1
.mg protein
−1
, using
L
-TAME as substrate.
A. awamor
i extracellular fraction is a source of proteases with important activity, and the supplementation of modified
Breccia
medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported
A. awamori
aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.