Production and biochemical characterization of an alkaline protease from Aspergillus oryzae CH93.
In this study, Aspergillus oryzae CH93 was isolated from soil sample and examined using molecular analysis. Following culture of A. oryzae CH93 under optimal enzyme production, a 47.5kDa extracellular protease was purified using ammonium sulfate precipitation and Q-Sepharose chromatography. The optimal pH 8 and temperature of 50°C obtained for the isolated protease. Sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), H2O2decreased activity, while Triton X-100 and phenylmethanesulfonyl fluoride (PMSF) had no inhibitory effect on the enzyme activity; meanwhile, 2-mercaptoethanol and ethylenediaminetetraacetic acid (EDTA) declined the protease activity. Isoamyl alcohol and acetone (30%) enhanced activity whereas 2-propanol, isopropanol and dimethyl sulfoxide (DMSO) (30%) reduced protease activity. The enzyme exhibited a half-life of 100min at its optimum temperature. Among five substrates of bovine serum albumin (BSA), N-acetyl-l-tyrosine ethyl ester monohydrate (ATEE), casein, azocasein and gelatin results showed that casein is the best substrate with Vmaxof 0.1411±0.004μg/min and Kmof 2.432±0.266μg/ml. In conclusion, the extracted protease from A. oryzae CH93 as a fungal source possessed biochemical features which could be useful in some application usages.
Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
Ahsan
A
Salihi
Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran. Electronic address: asoodeh@um.ac.ir.
Ahmad
A
Asoodeh
Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
Mansour
M
Aliabadian
D019840:2-Propanol / Q000737:chemistry; D001236:Aspergillus oryzae / Q000737:chemistry / Q000145:classification / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002364:Caseins / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D010450:Endopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000737:chemistry; D010802:Phylogeny; D011480:Protease Inhibitors / Q000737:chemistry*; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D002364:Caseins; D005656:Fungal Proteins; D011480:Protease Inhibitors; D008623:Mercaptoethanol; D004492:Edetic Acid; D010450:Endopeptidases; C087822:alkaline protease; D019840:2-Propanol; D004121:Dimethyl Sulfoxide
Alkaline protease; Aspergillus; Characterization; Enzyme kinetic