Purification and characterization of an extracellular aspartate protease from Phycomyces blakesleeanus.
An acid protease has been found in the culture broth of Phycomyces blakesleeanus growing under standard conditions. It has been induced up to 70-fold with several complex growth media and the enzyme has been purified to homogeneity and characterized. The molecular mass of the native enzyme was estimated by gel filtration to be 40 kDa. The acid protease of Phycomyces migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 35 kDa. The glycoprotein nature for the acid protease was deduced from its binding to a concanavalin A-Sepharose 4B column. The carbohydrate moiety is composed of mannose and rhamnose. Its amino acid composition was determined, and its isoelectric point was estimated to be 4.2, the optimum pH was 2.5 to 3, and the optimum temperature was 70 degrees C, using hemoglobin as a substrate. The enzyme showed thermal stability between 37 and 50 degrees C. The thermodynamic parameters for hemoglobin hydrolysis and thermal inactivation were calculated. With Lys-Pro-Ile-Glu-Phe-Phe(4-N02)-Arg-Leu as the substrate, the Km, kcat, and Vmax values were 8.78 microM, 1.25 s(-1), and 2.12 mumol min(-1) mg(-1), respectively. The protease was insensitive to phenylmethylsulfonyl fluoride, O-phenanthroline, N-ethylmaleimide, iodoacetamide, ethylenediaminetetraacetate, [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid, and trypsin inhibitor. However, pepstatin A established a strong competitive inhibition against it, with a K(i) value of 1.33 nM. The data suggest that this protease has properties of an aspartate-type proteinase.
De Vicente
J I
JI
Departamento de Microbiología y Genética, Facultad de Biología, Universidad de Salamanca, Spain.
De Arriaga
D
D
Del Valle
P
P
Soler
J
J
Eslava
A P
AP
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003470:Culture Media; D004795:Enzyme Stability; D006454:Hemoglobins / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008358:Mannose / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010436:Pepstatins / Q000494:pharmacology; D010800:Phycomyces / Q000201:enzymology* / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D012210:Rhamnose / Q000032:analysis; D013696:Temperature
D000596:Amino Acids; D003470:Culture Media; D006454:Hemoglobins; D010436:Pepstatins; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; D016282:Aspartic Acid Endopeptidases; D008358:Mannose; D012210:Rhamnose; C031375:pepstatin