Optimized Production of the Allylamine Antifungal “Terbinafine” by Lysinibacillus Isolate MK212927 Using Response Surface Methodology

Figures & data

Table 1 Variables and Levels Used in RSM Central Composite Design for Optimization of Antifungal Production

Variables	Level
	−1	0	+1
Starch (g/L)	1	3	5
Ammonium chloride (g/L)	1	3	5
pH	6	7	8
Temperature (°C)	30	35	40
Agitation rate (rpm)	150	225	300

Table 2 Central Composite Design Runs for the 5 Different Factors Tested Showing the Observed and Predicted Responses

Run No	Starch (g/L)	Ammonium Chloride (g/L)	pH	Temperature (°C)	Agitation (rpm)	Observed Inhibition Zone (mm)	Predicted Inhibition Zone (mm)
1	3	3	6	35	225	21	21.9
2	1	5	6	40	300	17	17.9
3	1	5	8	40	150	12	13.8
4	1	1	8	30	150	0	0.8
5	3	3	7	30	225	26	25.1
6	5	1	6	30	150	22	24
7	5	3	7	35	225	33	33
8	3	3	7	35	225	28	28
9	3	3	8	35	225	15	17.6
10	3	3	7	35	225	28	28
11	5	5	6	30	300	30	30.7
12	3	3	7	40	225	19	23.5
13	3	3	7	35	225	28	28
14	1	5	6	30	150	19	21
15	1	1	8	40	300	14	14.4
16	5	5	6	40	150	23	22.9
17	1	5	8	30	300	11	11
18	3	3	7	35	225	28	28
19	5	1	6	40	300	17	20.5
20	3	3	7	35	225	28	28.1
21	1	1	6	40	150	13	14.9
22	3	5	7	35	225	31	33.1
23	5	5	8	30	150	27	28.1
24	3	3	7	35	150	25	27.1
25	5	1	8	40	150	16	16.3
26	1	3	7	35	225	18	23.1
27	5	5	8	40	300	20	22.5
28	3	1	7	35	225	27	28.5
29	1	1	6	30	300	15	15.1
30	3	3	7	35	300	26	29
31	3	3	7	35	225	28	28.1
32	5	1	8	30	300	24	25.1

Figure 1 Standard calibration curve of terbinafine concentration versus inhibition zones obtained against Candida albicans ATCC 10231.

Figure 2 Time course of antifungal production by Lysinibacillus isolate MK212927 in basal media. The presented data are the means of three readings while the vertical small error bars indicate the standard deviation of the data.

Figure 3 Effect of (A) different Carbon sources and (B) Nitrogen sources on the antifungal activity of Lysinibacillus isolate MK212927 against Candida albicans ATCC 10231.

Table 3 The Analysis of Variance (ANOVA) of the Central Composite Design (CCD) Model for the Effects of Starch (A), Ammonium Chloride (B), pH (C) Temperature (D) and Agitation (E) on the Inhibition Zones Caused by the Antifungal Metabolite

Source	Sum of Squares	Degrees of Freedom (df)	Mean Square	F value	P-value
Model	1587.60	14	113.40	34.20	<0.0001
A-Starch	480.5	1	480.5	144.90	<0.0001
B-NH4Cl	98	1	98	29.55	<0.0001
C-pH	80.22	1	80.22	24.19	0.0001
D-TEMP	29.39	1	29.39	8.86	0.0085
E-Agitation	16.06	1	16.06	4.84	0.0419
AC	30.25	1	30.25	9.12	0.0077
AD	90.25	1	90.25	27.22	<0.0001
BD	12.25	1	12.25	3.69	0.0715
BE	30.25	1	30.25	9.12	0.0077
CD	16	1	16	4.83	0.0422
CE	9	1	9	2.71	0.1178
B^2	21.02	1	21.02	6.34	0.0221
C^2	196.02	1	196.02	59.11	<0.0001
D^2	40.93	1	40.93	12.34	0.0027
Residual	56.37	17	3.32
Lack of fit	56.37	12	4.70		>0.05
Pure error	0	5	0
Corrected total	1643.97	31

Figure 4 Three-dimensional response surfaces representing the effect of the five significant parameters on inhibition zone obtained by the metabolite of Lysinibacillus isolate MK212927. When the effect of two parameters was plotted, the remaining ones were set at central level (A) starch and pH (B) starch and temperature (C) NH₄Cl and agitation.

Figure 5 Contour plots representing the effect of the five significant parameters on inhibition zone obtained by the metabolite of Lysinibacillus isolate MK212927. When the effect of two parameters was plotted, the remaining ones were set at central level (A) starch and pH (B) starch and temperature (C) NH₄Cl and agitation.

Figure 6 Model diagnostic plots. (A) The normal probability plot of residuals, (B) Box Cox plot, (C) Predicted versus actual values plot and (D) Residuals versus Run number plot.

Figure 7 Comparison of antifungal production by Lysinibacillus isolate MK212927 using optimized and standardized conditions.