Optimised production of protein elicitor AMEP412 by Bacillus subtilis BU412 through response surface methodology

Figures & data

Table 1. PBD for the screening of significant factors affecting AMEP412 production.

Run	Factors							Protein yield (mg/mL)
	X1	X2	X3	X4	X5	X6	X7
1	4	4	6.5	18	32	190	1	0.413
2	16	16	6.5	26	32	190	0.1	1.064
3	4	16	7.5	26	25	190	1	0.665
4	16	4	6.5	26	25	160	1	0.371
5	16	16	6.5	18	25	190	0.1	0.525
6	4	16	6.5	26	32	160	1	0.98
7	4	4	7.5	26	25	190	0.1	0.322
8	16	16	7.5	18	25	160	1	0.588
9	4	16	7.5	18	32	160	0.1	0.434
10	16	4	7.5	18	32	190	1	0.399
11	16	4	7.5	26	32	160	0.1	0.504
12	4	4	6.5	18	25	160	0.1	0.203

With X₁ = carbon source (g/L); X₂ = nitrogen source (g/L); X₃ = initial pH; X₄ = culture time (h); X₅ =culture temperature (°C); X₆ = rotation speed (rpm); X₇ = inoculum size (%, vol/vol).

Table 2. Levels of the factors tested in the experimental CCD.

Factor	Units	Symbol code	Levels
			−1.68	−1	0	1	1.68
Yeast Extract	(g/L)	A	6.59	10	15	20	23.41
Culture temperature	(°C)	B	21.27	24	28	32	34.73
Culture time	(h)	C	19.91	24	30	36	40.09

Table 3. CCD matrixes for the experimental design and observed response for AMEP412.

Run order	A	B	C	Protein yield (mg/mL)
1	0	1.68	0	1.035
2	0	0	0	1.902
3	0	0	0	2.205
4	−1	1	1	0.456
5	1	−1	−1	0.495
6	1	1	1	2.052
7	0	0	−1.68	0.544
8	1	−1	1	0.975
9	0	−1.68	0	0.720
10	1.68	0	0	2.130
11	−1	1	−1	0.636
12	1	1	−1	1.572
13	0	0	0	2.160
14	0	0	0	1.914
15	−1.68	0	0	0.633
16	0	0	1.68	1.056
17	0	0	0	2.082
18	−1	−1	−1	0.690
19	0	0	0	2.043
20	−1	−1	1	0.828

Figure 1. SDS-PAGE detection of AMEP412 before and after purification.1, AMEP412 before purification; 2, AMEP412 after purification; M, low molecular weight protein marker.

Table 4. Effect of various carbon and nitrogen sources on AMEP412 production.

Nutrition source	AMEP412 yield (mg/mL)
Carbon source (0.4%)
Sucrose	0.066 ± 0.006
Glucose	0.111 ± 0.006
Mannitol	0.170 ± 0.006
Maltose	0.124 ± 0.003
Lactose	0.077 ± 0.006
Nitrogen source (0.4%)
(NH4)2SO4	0
Soybean tryptone	0.038 ± 0.005
Yeast extract	0.148 ± 0.007
Urea	0
Peptone	0.054 ± 0.008
NH4Cl	0

Table 5. Analysis of variance (ANOVA) for the PBD results.

Factors	Code	Low level (−1)	High level (1)	F	P
Carbon source (g/L)	X1	0.5	1.5	1.21	0.3339
Nitrogen source (g/L)	X2	0.5	1.5	26.74	0.0066*
pH	X3	6.5	7.5	2.65	0.1786
Culture time (h)	X4	20	28	8.03	0.0472*
Culture temperature (°C)	X5	24	32	11.56	0.0273*
Rotation speed (rpm)	X6	120	180	0.61	0.4794
Inoculum size (%)	X7	0.1	1	0.85	0.4093

R² = 92.81%, Adj R² = 80.23%.

* p < 0.05 corresponds to significance.

Table 6. Analysis of variance (ANOVA) for the CCD experimental results.

Source	Sum of squares	df	Mean squares	F	P
Model	8.57	9.00	0.95	46.53	<0.0001*
A	1.83	1.00	1.83	89.53	<0.0001*
B	0.37	1.00	0.37	18.24	0.0016*
C	0.23	1.00	0.23	11.33	0.0072*
AB	0.83	1.00	0.83	40.67	<0.0001*
AC	0.13	1.00	0.13	6.13	0.0327*
BC	0.01	1.00	0.01	0.62	0.4501
A^2	0.80	1.00	0.80	39.05	<0.0001*
B^2	2.47	1.00	2.47	120.52	<0.0001*
C^2	2.80	1.00	2.80	137.02	<0.0001*
Residual	0.20	10.00	0.02
Lack of fit	0.13	5.00	0.03	1.64	0.3009
Pure error	0.08	5.00	0.02
Cor total	8.77	19.00

R² = 97.67%, Adj R² = 95.57%, Pred R² = 87.50%, coefficient of variation = 10.95.

* p < 0.05 corresponds to significance.

Figure 2. Response surface plot for AMEP412 yield as a function of yeast extract and culture temperature.

Figure 3. Response surface plot for AMEP412 yield as a function of yeast extract and culture time.

Figure 4. Response surface plot for AMEP412 yield as a function of temperature and culture time.

Data availability statement

All data that support the findings of this study are available from the corresponding authors upon reasonable request.