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

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

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	Competitive inhibitor of serine proteases	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	Acid protease specific inhibitors	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

^a Values represent means of three independent replicates, and ± standard errors are reported
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

ISSN: 1472-6750