Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents

Figures & data

Table 1. Summary of the alkaline protease from A. pallidus C10 purification procedure.

Purification methods	Purification steps	Total activity(U)	Total protein(mg)	Specific activity(U/mg)	Yield (%)	Purificationfold
DE52 anion exchange chromatography	Culture supernatant	2148	42960	0.056	100	1
	(NH4)2SO4 precipitation and dialysis	283.6	1610	0.176	13.02	3.14
	Anion exchange chromatography	76.3	280	0.272	26.9	4.85
Probond affinity chromatography	Culture supernatant	2473.8	40380	0.061	100	1
	(NH4)2SO4 precipitation and dialysis	332.5	4088	0.081	13.44	1.33
	Affinity chromatography	65.04	61.05	1.057	19.56	17.32

Figure 1. Neighbor-joining phylogenetic tree on the basis of 16S rRNA gene sequence data of the thermophilic bacteria. Alicyclobacillus acidiphilus DSM 14558 was used as out-group. Bootstrap values based on 1000 replications are listed as percentages at branching points. Only bootstrap values >50% are shown at nodes. The scale bar represented 1% divergence.

Figure 2. SDS-PAGE of the purified protease from A. pallidus C10. (A) Lane 1: Standard protein molecular mass markers, lane 2: (NH₄)₂SO₄ precipitated proteins, lane 3–4: Purified protease from DE52 anion exchange chromatography, (B) Lane 1–2: Purified enzyme from Probond affinity chromatography, lane 3: Zymography of the purified enzyme.

Figure 3. Effect of pH on activity (A) and stability (B) of the purified protease from A. pallidus C10.

Figure 4. Effect of temperature on activity (A) and stability (B) of the purified protease from A. pallidus C10.

Table 2. The effect of inhibitors on A. pallidus C10 alkaline protease activity.

Inhibitor	Concentration(mM)	Residual activity (%)	p Values
Control	0	100	–
PMSF	1	69.54 ± 0.29	<0.0001^a
	5	0	–
EDTA	1	79.92 ± 0.08	<0.0001^a
	5	100.4 ± 0.42	0.1277^ns
DTNB	1	92.43 ± 0.26	<0.0001^a
	5	77.21 ± 0.05	<0.0001^a
β-mercaptoethanol	1	119 ± 0.57	<0.0001^a
	5	605.7 ± 0.33	<0.0001^a

p > 0.05 (not significant, ns).

^a p < 0.0001.

Table 3. The effect of metal ions on A. pallidus C10 alkaline protease activity.

Metal ions	Concentration(mM)	Residual activity (%)	p Values
Control	0	100	–
Fe^2+	1	109 ± 2.50	0.0032^b
	5	95.41 ± 5.68	0.2692^ns
	10	101.2 ± 1.50	0.1647^ns
Mg^2+	1	120.8 ± 0.38	<0.0001^d
	5	113.1 ± 1.83	0.0003^c
	10	113.6 ± 0.52	<0.0001^d
Ca^2+	1	115.3 ± 2.17	0.0003^c
	5	120.6 ± 0.24	<0.0001^d
	10	108.8 ± 0.69	<0.0001^d
Zn^2+	1	83.11 ± 7.28	0.0172^a
	5	114.8 ± 1.45	<0.0001^d
	10	111.6 ± 2.67	0.0016^b
Co^2+	1	96.61 ± 0.97	0.0093^b
	5	87.45 ± 1.67	0.0003^c
	10	83.08 ± 7.77	0.0212^a
Ni^2+	1	107.1 ± 0.77	0.0002^c
	5	94.30 ± 1.79	0.0079^b
	10	91.54 ± 1.64	0.0013^b
Mn^2+	1	83.08 ± 7.77	0.0212^a
	5	84.92 ± 0.69	<0.0001^d
	10	80.58 ± 4.39	0.0017^b
Ag^+	1	108.2 ± 0.57	<0.0001^d
	5	124.3 ± 2.70	0.0001^c
	10	90.78 ± 8.61	0.1501^ns
K^+	1	149.3 ± 5.23	<0.0001^d
	5	129.9 ± 4.61	0.0004^c
	10	91.60 ± 2.29	0.0041^b

p > 0.05 (not significant, ns).

^a p < 0.05.

^b p < 0.01.

^c p < 0.001.

^d p < 0.0001.

Table 4. The effect of organic solvents on A. pallidus C10 alkaline protease activity.

Organic solvents	Concentration (%)	Residual activity (%) (1h)	p Values	Residual activity (%) (24h)	p Values
Control	0	100	–	100	–
Methanol	15	68.42 ± 1.83	<0.0001^d	83.38 ± 1.77	0.0001^c
	25	114 ± 2.79	0.0009^c	50.27 ± 1.39	<0.0001^d
	50	80.24 ± 1.74	<0.0001^d	0	–
Ethanol	15	93.59 ± 1.12	0.0011^b	90.28 ± 1.98	0.0014^b
	25	107.8 ± 1.52	0.0009^c	75.47 ± 2.95	0.0002^c
	50	103.7 ± 2.07	0.0303^a	0	–
Acetone	15	86.73 ± 1.29	<0.0001^d	102.9 ± 1.92	0.0472^a
	25	88.50 ± 2.02	0.0008^c	72.67 ± 1.45	<0.0001^d
	50	72.74 ± 1.89	<0.0001^d	0	–
DMSO	15	84.73 ± 0.93	<0.0001^d	85.86 ± 3.65	0.0030^b
	25	78.10 ± 1.75	<0.0001^d	102.2 ± 1.53	0.0522^ns
	50	95.33 ± 2.19	0.0299^a	0	–
Butanol	15	93.74 ± 2.09	0.0091^b	58.88 ± 2.20	<0.0001^d
	25	73.66 ± 1.85	<0.0001^d	106.5 ± 3.21	0.0217^a
	50	52.01 ± 2.44	<0.0001^d	0	–
Chloroform	15	95.94 ± 2.58	0.0724^ns	74.56 ± 0.13	<0.0001^d
	25	85.02 ± 1.73	0.0002^c	96.41 ± 0.87	0.0056^b
	50	59.59 ± 1.44	<0.0001^d	0	–
Isopropanol	15	90.21 ± 1.91	0.0012^b	90.13 ± 1.73	0.0008^c
	25	84.46 ± 1.00	<0.0001^d	62.91 ± 2.42	<0.0001^d
	50	50.50 ± 1.038	<0.0001^d	0	–

p > 0.05 (not significant, ns).

^a p < 0.05.

^b p < 0.01.

^c p < 0.001.

^d p < 0.0001.

Table 5. The effect of various surfactants and the oxidant agent on A. pallidus C10 alkaline protease activity.

Surfactants/oxidizingagent	Concentration(%) (v/v)	Residual activity (%)	p Values
Control	0	100	–
SDS	1 (w/v)	79.33 ± 2.65	0.0002^a
SDS	5 (w/v)	116.7 ± 1.76	<0.0001^b
Triton X-100	1	41.33 ± 1.85	<0.0001^b
Triton X-100	5	63.0 ± 1.52	<0.0001^b
Tween-20	1	20.0 ± 1.15	<0.0001^b
Tween-20	5	37.67 ± 1.45	<0.0001^b
Tween-80	1	31.0 ± 2.08	<0.0001^b
Tween-80	5	29.0 ± 2.08	<0.0001^b
H2O2	1	9.66 ± 0.88	<0.0001^b
H2O2	5	11.67 ± 0.88	<0.0001^b

^a p < 0.001.

^b p < 0.0001.

Figure 5. Stability of the alkaline protease from A. pallidus C10 in the presence of various commercial detergents.