Purification and characterization of α-amylase in Moroccan locust, Dociostaurus maroccanus Thunberg (Orthoptera: Acrididae) and its inhibition by inhibitors from Phaseolus vulgaris L.

Figures & data

Figure 1. (a) Elution profile on sephacryl 100-HR column of midgut crude extract of D. Maroccanus; (b) Chromatogram obtained from ionic exchange DEAE cellulose chromatography of D. Maroccanus midgut extract. (♦) protein absorbance at 280 nm; and (dashed line) NaCl gradient; (c) Analysis of purified α-amylase by SDS-PAGE. Lane a: Crude extract of midgut extract of D. maroccanus, Lane b: purified α-amylase Lane c: Molecular weight of markers: lysozyme (14.4 kDa); lactoglobulin (18.4 kDa); restriction endonuclease bsp 981 (25 kDa); lactate dehydrogenase (35.5 kDa); ovalbumin (45 kDa); bovine serum albumin (66 kDa); α-galactosidase (116 kDa).

Table 1. Purification of α-amylase from digestive system of D. maroccanus.

Purification step	Total protein (mg)	Total activity (U) (Mean ± SE)	Specific activity (U/mg) (Mean ± SE)	Recovery (%)
Crude extract	4.25	3.71 ± 0.36	0.87 ± 0.06	100
Ammonium sulfate	3.66	1.97 ± 0.21	0.96 ± 0.08	53.09
Sephacryl 100-HR	0.319	0.71 ± 0.06	2.22 ± 0.07	19.13
DEAE-cellulose	0.083	0.40 ± 0.02	4.8 ± 0.09	10.78

Figure 2. (a) The effect of pHs on the mean relative activities of α-amylase purified from the digestive system of D. maroccanus; (b) The effect of temperatures on the mean relative activities of α-amylase purified from the digestive system of D. maroccanus.

Table 2. Effect of some organic and inorganic compounds on α-amylase activity (iso-enzyme 3) of D. maroccanus..

Salt/compound added	Concentrations (mM)	Mean activity (±SE) at different salt and chemical
EDTA	Control	100 a
	0.1	50.32 ± 0.1 b
	1	45.81 ± 0.23 b
	2	26.56 ± 0.35 bc
	5	19.78 ± 0.29 c
Na^+	Control	100 b
	0.1	114.02 ± 1.6 a
	1	118.06 ± 1.9 a
	2	116.93 ± 3.9 a
	5	116.36 ± 0.46 a
Mn^2+	Control	100 a
	0.1	42.53 ± 0.32 b
	1	38.09 ± 0.33 b
	2	35.81 ± 0.3 b
	5	33.86 ± 0.3 b
K^+	Control	100 b
	0.1	113.41 ± 2 b
	1	124 ± 1.18 ab
	2	132.56 ± 2.7 a
	5	146.32 ± 0.37 a
Fe^2+	Control	100 c
	0.1	105.61 ± 1.2 bc
	1	118.49 ± 0.28 ab
	2	123.09 ± 0.52 a
	5	125.77 ± 0.54 a
Hg^+	Control	100 a
	0.1	43.85 ± 0.2 b
	1	36.91 ± 0.32 b
	2	23.64 ± 0.63 cd
	5	11.22 ± 0.40 c
Co^2+	Control	100 c
	0.1	103.71 ± 0.4 bc
	1	131.49 ± 0.47 a
	2	149.62 ± 0.51 a
	5	151.11 ± 1.34 a
Ba^+	Control	100 c
	0.1	110.09 ± 0.85 bc
	1	117.50 ± 0.26 ab
	2	123.67 ± 0.24 a
	5	125.06 ± 0.32 a
Mg^2+	Control	100 bc
	0.1	113.41 ± 2 b
	1	158.35 ± 2.8 ab
	2	188.03 ± 2.1 a
	5	173.41 ± 3.7 a
Ca^2+	Control	100 c
	0.1	160.51 ± 3b
	1	189.81 ± 3.6 ab
	2	199.21 ± 4.1 a
	5	199.45 ± 3.8 a
Zn^2+	Control	100 a
	0.1	84.06 ± 0.6 ab
	1	75.43 ± 0.46 ab
	2	64.05 ± 0.46 ab
	5	46.04 ± 0.51 bc

All the metal ions were added as chloride salts.

Different letters indicate that the relative activity of enzymes is significantly different from each other by Tukey's test (p < 0.05).

Figure 3. (a) Elution profile for inhibitor extracted from seeds of P. vulgaris using sephacryl 100-HR column chromatography; (b) Elution profile for inhibitor extracted from seeds of P. vulgaris using ionic exchange DEAE cellulose as bed, equilibrated with 20 mM Tris-HCl buffer. (♦) protein absorbance at 280 nm; and (dashed line) NaCl gradient; (c) SDS–PAGE of proteins at different process of α-amylase inhibitor purification. Lane a: purified P. vulgaris amylase inhibitor (Fraction 17), lane b: crude plant extract, Lane c: purified P. vulgaris amylase inhibitor (Fraction 24), lane d: Molecular weight markers: lysozyme (14.4 kDa); lactoglobulin (18.4 kDa); restriction endonuclease bsp 981 (25 kDa); lactate dehydrogenase (35.5 kDa); ovalbumin (45 kDa); bovine serum albumin (66 KDa); α-galactosidase (116 kDa); (d) molecular weights-relative mobility of proteins for determination of purified α-amylase inhibitor.

Figure 4. Zymogram of α-amylases extracted from the digestive system of D. maroccanus. Enzyme extract was pre-incubated with P. vulgaris inhibitor (AI1) for 30 min and then gel was run at 4 °C. Lane numbers are as follows: (1) Crude enzyme extract treated with AI1, (2) Crude enzyme extract with no inhibitor, (3) Crude enzyme extract treated with AI2, (4) Purified α- amylase incubated with AI1, (5) Control (Purified α- amylase with no inhibitor), (6) Purified α- amylase incubated with AI2.