Production of a new non-specific nuclease from Yersinia enterocolitica subsp. palearctica: optimization of induction conditions using response surface methodology

Figures & data

Table 1. Coded and non-coded levels of independent variables used in the RSM design.

	Levels
Independent variables	−2	−1	0	1	2
A:OD600 (nm)	0.6	0.7	0.8	0.9	1.0
B: induction temperature (°C)	28	31	34	37	40
C: IPTG concentration (mmol/L)	0.5	1	1.5	2	2.5
D: induction time (h)	16	18	20	22	24

Table 2. Response values for relative enzyme activity obtained from RSM.

	Coded values				Actual values
Run	A	B	C	D	A	B	C	D	Response values (relative enzyme activity)
1	−1	1	1	1	0.7	37	2	22	48
2	2	0	0	0	1.0	34	1.5	20	75
3	0	0	−2	0	0.8	34	0.5	20	75
4	0	0	0	0	0.8	34	1.5	20	100
5	0	0	0	−2	0.8	34	1.5	16	87
6	1	1	1	−1	0.9	37	1.5	18	45
7	0	0	0	0	0.8	34	1.5	20	100
8	−1	1	1	−1	0.7	37	2	18	60
9	1	−1	−1	1	0.9	31	1	22	85
10	0	0	2	0	0.8	34	2.5	20	68
11	0	2	0	0	0.8	40	1.5	20	38
12	−1	1	−1	−1	0.7	37	1	18	72
13	−1	−1	1	1	0.7	31	2	22	85
14	0	0	0	2	0.8	34	1.5	24	84
15	0	−2	0	0	0.8	28	1.5	20	93
16	0	0	0	0	0.8	34	1.5	20	100
17	1	1	1	1	0.9	37	2	22	50
18	0	0	0	0	0.8	34	1.5	20	100
19	1	−1	1	−1	0.9	31	2	18	73
20	1	−1	−1	−1	0.9	31	1.0	18	77
21	1	−1	1	1	0.9	31	2	22	86
22	1	1	−1	1	0.9	37	1.0	22	42
23	−1	−1	1	−1	0.7	31	2	18	78
24	−1	1	−1	1	0.7	37	1.0	22	50
25	1	1	−1	−1	0.9	37	1.0	18	60
26	0	0	0	0	0.8	34	1.5	20	90
27	−1	−1	−1	−1	0.7	31	1.0	18	82
28	−2	0	0	0	0.6	34	1.5	20	85
29	−1	−1	−1	1	0.7	31	1.0	22	82
30	0	0	0	0	0.8	34	1.5	20	100

Table 3. Results of the analysis of variance to response surface quadratic model.

Source	Sum of squares	Degree of freedom	Mean square	F-value	p-value
Model	9745.08	14	696.08	20.02	<0.0001
A: OD600	145.04	1	145.04	4.17	0.0591
B: temperature	4565.04	1	4565.04	131.28	<0.0001
C: IPTG	63.38	1	63.38	1.82	0.1970
D: induction time	26.04	1	26.04	0.75	0.4004
AB	45.56	1	45.56	1.31	0.2703
AC	1.56	1	1.56	0.045	0.8350
AD	76.56	1	76.56	2.20	0.1586
BC	18.06	1	18.06	0.52	0.4822
BD	351.56	1	351.56	10.11	0.0062
CD	126.56	1	126.56	3.64	0.0757
A^2	984.00	1	984.00	28.30	<0.0001
B^2	2535.50	1	2535.50	72.92	<0.0001
C^2	1806.07	1	1806.07	51.94	<0.0001
D^2	584.07	1	584.07	16.80	0.0009
Residual	521.58	15	34.77
Lack of fit	438.25	10	42.82	2.63	0.1488
Pure error	83.33	5	16.67
Total	10,266.67	29

R² = 0.9492, adjusted R² = 0.9018 and coefficient of variation (CV) = 7.79%

Figure 1. Predicted and experimental values for relative enzyme activities (R² = 0.949).

Figure 2. Response surface plots showing the effect of interaction factors on enzyme activity: (A) OD600 and induction temperature (IPTG concentration 1.5 mmol, induction time 20 h); (B) OD600 and IPTG concentration (induction temperature 34 °C, induction time 20 h); (C) IPTG concentration and induction temperature (OD600 0.8, induction time 20 h); (D) induction temperature and induction time (IPTG concentration 1.5 mmol, induction temperature 34 °C); and (E) induction time and IPTG concentration (OD600 0.8, induction temperature 34 °C).