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Table 3 Effects of various inhibitors, reducing agents, and metallic ions on SPPS enzyme stability. Protease activity measured in the absence of any inhibitor or reducing agent was taken as control (100%). The non-treated and dialyzed enzyme was considered as 100% for metallic ion assay. Residual activity was measured at pH 9.5 at 70 °C

From: Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest

Reagent Specificity/origin Concentration Relative protease stability (%) a
Inhibitor/reducing agents Control   100 ± 2.5
PMSF or DFP Serine protease specific inhibitors 5 mM 0 ± 0.0
Benzamidine Competitive inhibitor of serine proteases 10 mM 103 ± 2.6
Aprotinin 5 mM 101 ± 2.5
SBTI Soybean trypsin inhibitor 3 mg/mL 101 ± 2.5
TPCK Chymotrypsin specific inhibitor 1 mM 94 ± 2.2
TLCK Trypsin-like specific inhibitor 1 mM 102 ± 2.5
DTNB Cysteine or thiol-containing protease inhibitors 5 mM 41 ± 0.8
NEM 5 mM 74 ± 1.8
MIA 10 mM 97 ± 2.4
EPNP 2 mM 87 ± 2.1
Iodoacetamide 50 μg/mL 65 ± 1.4
β-ME Thiol reducing agent for cleaving protein disulfide bonds (cystin) 1 mM 66 ± 1.6
ld-DTT 50 μM 61 ± 1.5
EDTA Specific metallo-protease inhibitors 1 mM 91 ± 2.2
EGTA 2 mM 80 ± 2.1
1,10-Phenanthroline monohydrate 10 mM 102 ± 2.5
Leupeptine Acid protease specific inhibitors 10 μg/mL 101 ± 2.5
Pepstatine A 10 mM 104 ± 2.7
Metallic ions Control   100 ± 2.5
Ca2+ CaCl2 2 mM 270 ± 6.0
Mg2+ MgCl2 2 mM 180 ± 3.8
Fe2+ FeCl2 2 mM 145 ± 3.4
Cu2+ CuCl2 2 mM 99 ± 2.5
Mn2+ MnCl2 2 mM 109 ± 2.6
Co2+ CoCl2 2 mM 61 ± 1.2
Ba2+ BaCl2 2 mM 75 ± 1.8
Zn2+ ZnCl2 2 mM 40 ± 0.8
Hg2+ or Cd2+ or Ni2+ HgCl2, CdCl2, or NiCl2 2 mM 0 ± 0.0
  1. a Values represent means of three independent replicates, and ± standard errors are reported
  2. SBTI soybean trypsin inhibitor, TPCK Nα-p-tosyl-l-phenylalanine chloromethyl ketone, TLCK Nα-p-tosyl-l-lysine chloromethyl ketone, DTNB 5,5′-dithio-bis-2-nitro benzoic acid, NEM N-ethylmalemide, MIA monoiodoacetic acid, EPNP 1.2-epoxy-3-(p-nitrophenoloxy) propane, β-ME β-mercaptoethanol, ld-DTT ld-dithiothreitol