Figure 5 - Molecular cloning, expression and characterization of a serine proteinase from Japanese edible mushroom, Grifola frondosa: solving the structure - function anomaly of a reported aminopeptidase

Figure 5. Subtilisin treatment and revival of proteolytic activity of GF-Spr1.

(a) Enzyme activity profiles at different stages of subtilisin treatment. The proteolytic activity of the purified enzyme was assayed before and after subtilisin treatment using Succinyl-Ala-Ala-Pro-Phe-pNA as the substrate, as described in Experimental Procedures. Lane-1, enzyme activity before subtilisin treatment; Lane-2, activity of subtilisin alone; Lane-3, enzyme after subtilisin treatment; Lane-4, activity after inactivation of the subtilisin treated enzyme with PMSF. The proteolytic activity of the subtilisin treated sample was defined as 100%.

(b) SDS-PAGE of subtilisin-treated GF-Spr1. Lane-M, molecular weight marker; Lane-1, Ni-NTA-purified enzyme; Lane-2, 1 hr subtilisin-treated enzyme; Lane-3, 3 hrs subtilisin-treated enzyme; Lane- 4, 16 hrs subtilisin-treated enzyme. The 27 kDa faint band in lanes 2-4 was derived from the subtilisin itself.