Purification and biochemical characterization of a novel intestinal protease from Scorpaena notata

Figures & data

Table 1. Purification of the intestinal protease of Scorpaena notata.

Step	Total protein (mg)	Total activity (10^3 U)	Specific activity (10^3 U mg^−1)	Purification fold	Recovery (%)
Crude extract	13.500	1863.813	138.060	1	100
Ammonium sulfate precipitation	6.600	1008.000	152.727	1.10	54.08
Gel filtration	0.780	166.483	445.141	3.22	8.93
Ion exchange	0.440	124.787	675.949	4.89	6.69

Figure 1. (A) Elution profile of S. not-protease after gel filtration chromatography on Toyopearl HW-50 column (0.5 mg of the protein extract was loaded to the column). (B1) Elution profile of S. not-protease on Mono Q-Sepharose anion-exchange chromatography column (0.2 mg of the protein extract was loaded to the column). Elution was carried out with a linear gradient of 0–1 mol L⁻¹ NaCl. (B2) Zymogram detection of proteolytic activity of different fractions (F68, F69, F70, F71, F72, F73, F74) of the purified S. not-protease.

Figure 2. (A) Elution profile of S. not-protease after gel filtration chromatography on Toyopearl HW-40 column using an FPLC system (15 µg of the purified protease was loaded to the column). Elution volume of S. not-protease is showed by an arrow in the calibration curve. Protein content is shown in continuous line and activity in discontinuous line. (B) SDS–PAGE analysis of the purified S. not-protease. Lane 1, standard molecular weight markers; Lane 2, purified S. not-protease (2 µg).

Figure 3. Effect of temperature on enzyme activity (A) and stability (B). Remaining protease activity was measured under standard assay conditions. Effect of pH on purified S. not-protease activity (C) and stability (D). The stability was estimated as residual enzyme activity after pre-incubation in different buffers for 12 h at 4°C. The results represent mean values of three independent experiments each performed in triplicates and error bars indicate standard deviation.

Table 2. Effect of various enzyme inhibitors on the activity of S. not-protease.^†

	Residual activity (%)
Inhibitors	2 mmol L^−1	5 mmol L^−1
None (control)	100	100
PMSF	18.93	9.09
EDTA	97.72	85.60
β-Mercaptoethanol	99.24	96.96

^†Purified enzyme was preincubated with various enzyme inhibitors for 30 min at 4°C and the remaining activity was determined with azocasein substrate at pH 10.0 and 40°C. Enzyme activity measured in the absence of any inhibitor was taken as 100%.

Table 3. Effect of surfactants on enzyme activity of S. not-protease.^†

Surfactant	Residual activity (%)
	1%	5%
None (control)	100	100
Tween 20	100	99.22
Triton X-100	91.47	89.14
SDS	65.89	59.68

^†Purified enzyme was preincubated with various surfactants for 30 min at 4°C and the remaining activity was determined with azocasein substrate at pH 10.0 and 40°C. Enzyme activity measured in the absence of surfactant was taken as 100%.

Table 4. Effect of metal ions on enzyme activity.^†

Metal ions	Residual activity (%)
	5 mmol L^−1	10 mmol L^−1
None (control)	100	100
Mn^2+	99.28	99.33
Zn^2+	60.34	42.35
Ca^2+	106.89	115.23
Mg^2+	96.55	95.23
Cu^2+	39.65	28.36
Na^+	96.55	95.55
K^+	91.37	93.25

^†The protease activity was determined by preincubating the enzyme in the presence of various metal ions for 30 min at 4°C.

Figure 4. Effect of NaCl concentrations on activity and stability of purified S. not-protease.

Table 5. Biochemical and kinetic properties of S. not-protease and different fish proteases.

Fish	pH	Temperature (°C)	Thermal stability at 40°C (% of residual activity)	Km (mmol L^−1)	Kcat/Km	Reference
Small red scorpionfish	10	40	95.16	0.36	4.47	–
Zebra blenny	9.5	60	100	0.6	2.3	[13]
Sardinelle	9	55	70	1.67	2.31	[26]
Flatfish	7.5	70	100	0.017	1.42	[39]
Barbel	10	55	95	0.6	2.3	[14]
Striped seabream	10	50	100	0.29	4.68	[31]
Lane snapper	9	45	100 (15 min)	0.66	–	[24]
Bogue	9	55	80	0.13	12	[12]

Table 6. Substrate specificity of S. not-protease toward natural substrates.

Substrate (3%)	Degree of hydrolysis (%)
Wheat gluten	6.99
Casein	5.87
Egg albumin	3.14
BSA	5.12
Gelatin	6.58

Figure 5. (A) Enzymatic hydrolysis of wheat gluten at two different concentrations (1% and 3%, w/v) with S. not-protease at an enzyme–substrate ratio of 5:1 (U mg⁻¹). (B) SDS–PAGE analysis of gluten hydrolysate at a concentration of 3% at different reaction times. Lane 1, standard molecular weight markers; Lane 2, gluten supernatant at t = 0; Lanes 3–8, gluten hydrolysate supernatant at t = 1, 2, 4, 6, 7, and 24 h, respectively.

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