Purification and biochemical characterization of aspartic peptidase produced by novel isolate mucor circinelloides (von tieghem) using ssf process

Souhila Bensmail, Fethia Naimi-Fazouane

Purification and biochemical characterization of aspartic peptidase produced by novel isolate mucor circinelloides (von tieghem) using ssf process

Číslo: 3/2019/2020
Periodikum: Journal of Microbiology, Biotechnology and Food Sciences
DOI: 10.15414/jmbfs.2019/20.9.3.590-598

Klíčová slova: Milk clotting activity, peptidases, Mucor circinelloides, purification, cheese making

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Anotace: Microbial peptidases are among the most important hydrolytic enzymes which have a potential application in a wide number of

industrial processes. In this study, the milk-clotting enzyme of a newly isolated strain Mucor circinelloides (von Tieghem) MG603064
was produced by solid-state fermentation using wheat bran as the substrate. The crude extract exhibited a maximum milk-clotting
activity of 1500 ± 50.94 SU/mL after 72 h of incubation at 25 °C. Purification of the enzyme using fractionation at 20‒70% (NH4)2SO4
followed by size exclusion chromatography on Sephadex G-100 allowed us to obtain a 20-fold purified peptidase with a recovery of
18.41%. The highest activity of the purified enzyme (30 kDa) was obtained in 25 mM CaCl2, at pH 5.0 and temperature of 60 °C. The
enzyme was stable between pH 3.0‒4.5 for 24 h at 4 °C in 0.1 M citrate buffer and retained more than 80% of its maximum activity at
45 °C for 1 h with complete inactivation at 75 °C. The enzyme inhibition of 94.5 and 98.6% by 0.02 and 0.1 mM Pepstatin A,
respectively, confirmed that the enzyme is an aspartyl peptidase. A partial inhibition of 78.17% was noted for EDTA at 14 mM. The
enzyme activity was improved significantly by Mg2+, Fe2+, Mn2+ and Zn2+ by 40.5, 59.5, 75.6 and 85%, respectively, and strongly
inhibited by Al3+ (93.1%) and Hg2+(94.4%), at a concentration of 10 mM. Activity stimulation of 120% was maximum in the presence of
Ba2+ using the same concentration. The crude and pre-purified extracts were applied in a trial of semi-hard cheese making (type Edam)
as possible rennet substitutes.