Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Abstract

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40 degrees C. The enzyme retained activity after incubation at pHs ranging from 8 to 11 for 12 h at 37 degrees C and 6 to 8 for 24 h at 37 degrees C. It retained 80% of its activity after incubation at 30 to 70 degrees C for 30 min and lost 50% of its activity after incubation for 15 min at 80 degrees C. Noticeable activation of the enzyme is observed when Fe(2+) ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu(2+), Fe(3+), Hg(2+), and Zn(2+) ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.

ANCUT2, an Extracellular Cutinase from Aspergillus nidulans Induced by Olive Oil

“…Olive oil was substituted by the indicated compound at indicated concentration bands showed CEH activity (37,29, and 22 kDa). These results correspond to those previously reported [14], where it was also stated that the 37-kDa protein corresponds to the alkaline protease PrtA. In the present study, the use of mass spectrometry allowed the identification of the other two bands, which correspond to new cutinases: ANCUT2 and its post-translational processing product, as described in detail below.…”

“…CEHM was prepared as a minimal nitrate medium with the following modifications: 1.5% starch was added as a carbon source instead of glucose, 0.5% yeast extract was used as an organic nitrogen source, and 0.5% olive oil was added as an inducer [14].…”

“…Little is known about how CEH production is regulated, their physiological importance, or the overall features of these enzymes .We have previously reported that A. nidulans produces three proteins that have CEH activity when grown in a medium with olive oil (CEHM) that was designed to induce lipase production. One of these enzymes had a molecular weight of 37 kDa, and was purified and identified as the alkaline protease encoded by the prtA gene while the other two esterases remained unidentified [14].…”

Effect of activators and inhibitors on extracellular thermostable alkaline protease isolated from Bacillus subtilis obtained from eastern province of Saudi Arabia

“…Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License through thermal stability (Kotlova et al, 2007 , Fe +2, on the contrary, found to inhibit pretense activity (Pena-Montes et al, 2008). and molybdenum and the source of the enzyme detect the important ions heeded.…”

Semi Purification and Identifications Molecule Protein Weigh of Alkaline Protease Enzyme from Bacillus cereus LS2B

“…is one of the best commercial enzymes products that exist in the market. Each alkaline protease enzyme produced must have a high catalytic ability (Pena-Montes et al, 2008) [4]. So that the analysis of the degree of purity and characterization of enzymes becomes necessary to support the ability of the catalysis enzyme, so it can be maximum applied.…”