Purification and some properties of an extracellular acid protease from Aspergillus clavatus

Abstract

Acid proteases represent an important group of enzymes, widely used in food, beverage and pharmaceutical industries. For most of these applications the enzymatic preparation must be at least partially purified and free of substances that could change the characteristics of the product or the process. Fungal proteases have replaced other sources because they are easily obtained mainly from Mucor, Rhizopus, Penicillium and Aspergillus species. A strain of Aspergillus clavatus was selected by producing high level of acid protease activity. An extracellular aspartatic protease from this strain was purified 37.2 times with 37% recovery using (NH 4 ) 2 SO 4 fractionation and ionexchange chromatography. The enzyme was found to be monomeric having a molecular mass of 30.4 kDa. The purified enzyme is an acid protease with optimum pH of 5.5 and temperature for optimum activity of 50°C. Its high pH stability was verified in the range of 3.5-6.5. The acid protease was strongly inhibited by Hg ?2 and partially inhibited by Cu ?2 , Zn ?2 and Mn ?2 . The enzyme was sensitive to denaturing agent SDS and activated by thiolcontaining reducing agent dithiotreitol (DTT). The protease activity was not influenced by iodoacetic acid, E-64 and PMSF, while it was lightly actived by EDTA and totally inhibited by pepstatin, with a K i of 7.8 lM, indicating that is an aspartic protease. A. clavatus acid protease presents interesting characteristics for biotechnological process, such as cheese and flavor manufacture and dietary supplements, in which activity and stability in acid pH are required.

The psychrotrophic yeast Sporobolomyces roseus LOCK 1119 as a source of a highly active aspartic protease for the in vitro production of antioxidant peptides

“…Also in terms of pH‐stability, other authors provide examples of proteins with profiles similar to that of protease G8 from S. roseus LOCK 1119, such as proteases from the filamentous fungi Aspergillus oryzae and Penicillium camemberti . None of the mono‐ and divalent cations tested in the current experiments activated protease G8 from S. roseus LOCK 1119, which is consistent with data for other proteins in this family . On the other hand, the activity of protease G8 was significantly decreased by reducing agents (DTT and 2‐mercaptoethanol) and the anionic surfactant SDS.…”

“…Protease G8 synthesized by S. roseus LOCK 1119 is an aspartic protease and belongs to the A1 family of pepsin‐type enzymes, as confirmed by inhibitor tests, in which the protein underwent 93% inactivation under the influence of pepstatin A, a hexapeptide specifically and irreversibly binding to aspartic acid in the active site. It should be noted that most microbial proteases of this type are synthesized by filamentous fungi , while there is little information on such proteins produced by yeasts .…”

“…In turn, the total inactivation of the studied protein in the presence of an anionic detergent (SDS) indicates an important function of hydrophobic interactions in this respect. SDS has also been reported to inactivate acid proteases from C. humicola , Aspergillus clavatus , and Synergistes sp. .…”

“…Currently, aspartic proteases are used in the pharmaceutical industry as digestive aids in gastrointestinal disorders and as anti‐inflammation agents . In addition, these enzymes are applied in the food industry, mostly in cheese and soy sauce production, meat tenderization, and as bread additives.…”

Identification and magnetic immobilization of a pyrophilous aspartic protease from Antarctic psychrophilic fungus

“…Similar to most aspartic proteases, P10 displays high activity in the pH range of 1.0-4.0, and shows good stability when incubated at acidic conditions. It is worth mentioning that P10 originates from psychrophilic fungi; however, its optimum temperature approaches 60°C, retaining approximately 80% activity at 50-70°C, which is much higher than that of mesophile aspartic proteases from Aspergillus niger (Yin, Hsu, & Jiang, 2013 ) and Aspergillus clavatus (Sampaio e Silva, Knob, Tremacoldi, Brochetto-Braga, & Carmona, 2011 ), and even as high as that of aspartic proteases from thermophilic fungi, such as Thermomucor indicae-seudaticae (Silva, Geraldes, Murari, Gomes, & Da-Silva, 2014 ). This indicates toward the abundant enzyme reserves in microorganisms and their robust resistance to harsh environments.…”