Proteases from Canavalia ensiformis: Active and Thermostable Enzymes with Potential of Application in Biotechnology

Table 3

Effects of different types of protease inhibitors on C. ensiformis extracts peptidase activity.

Target protease	Residual activity (%)
	Serine			Serine/cysteine	Cysteine	Aspartic	Metalloprotease
Extract	Inhibitor
	TPCK (100 μM)	BZA (10 μM)	PMSF (1 mM)	Iodo-acetamide (100 μM)	E-64 (10 μM)	Pepstatin (10 μM)	EDTA (10 mM)	PHE (10 mM)
CE-A	81.14 ± 13.7	21.22 ± 0.4	100.00 ± 0.0	100.00 ± 0.0	88.89 ± 5.0	99.70 ± 0.8	43.57 ± 5.7	58.33 ± 2.1
CE-D	71.7 ± 4.6	100.00 ± 0.0	100.00 ± 0.0	100.00 ± 0.0	94.90 ± 4.0	100.00 ± 0.0	100.00 ± 0.0	44.30 ± 8.6
CE-P	44.0 ± 2.8	34.45 ± 2.4	100.00 ± 0.5	100.00 ± 0.0	91.66 ± 0.7	87.50 ± 9.8	49.15 ± 6.4	66.29 ± 3.3
CE-T	100.0 ± 0.0	40.09 ± 3.5	93.08 ± 0.4	100.00 ± 0.0	82.88 ± 3.8	78.70 ± 0.3	79.22 ± 1.9	28.57 ± 0.0
CE-CA	79.8 ± 1.2	14.60 ± 0.9	97.34 ± 2.9	98.82 ± 1.2	97.11 ± 8.0	90.86 ± 5.8	49.64 ± 2.5	42.14 ± 9.2
CE-RA	95.3 ± 13.6	100.00 ± 2.8	100.00 ± 2.5	59.00 ± 7.3	100.00 ± 2.1	91.00 ± 0.0	38.00 ± 0.8	0.00 ± 0.0
CE-SA	14.5 ± 21.8	100.00 ± 0.0	100.00 ± 0.0	100.00 ± 0.0	100.00 ± 0.0	8.90 ± 4.5	100.00 ± 0.0	100.00 ± 0.0

The extracts were preincubated with each reagent for 30 min at room temperature. The remaining activity was assayed by incubation with 0.125 mM -TAME. Values are the remaining activity of serine protease as percentage of the activity on L-TAME without compounds, measured as described in “Materials and Methods,” and represent the average of 3 separate experiments carried out in duplicate. BZA: benzamidine; E-64: -trans-epoxysuccinyl-leucylamido-(4-guanidino) butane; EDTA: ethylenediaminetetraacetic acid; PHE: 1,10-phenanthroline; PMSF: phenylmethanesulfonyl fluoride; TPCK: N-tosyl-L-phenylalanine chloromethyl ketone.