Microbial Production of 7-Aminocepahlosporanic Acid and New Generation Cephalosporins (Cephalothin) by Different Processing Strategies

Figures & data

Table 1. CCD (central composite design) experimental plan for enzyme characterization

S.No.	X1 (pH)	X2 (Temperature, °C)	X3 (Agitation, rpm)
1	0 (6.5)	0 (34.5)	0 (110)
2	0 (6.5)	0 (34.5)	0 (110)
3	1 (8.0)	−1 (21.0)	1 (160)
4	0 (6.5)	0 (34.5)	0 (110)
5	0 (6.5)	0 (34.5)	1.68 (194)
6	−1 (5.0)	1 (48.0)	−1 (60)
7	0 (6.5)	0 (34.5)	−1.68 (25)
8	0 (6.5)	0 (34.5)	0 (110)
9	−1 (5.0)	1 (48.0)	1 (160)
10	0 (6.5)	0 (34.5)	0 (110)
11	1.68 (8.0)	−1 (21.0)	−1 (60)
12	1 (9.0)	0 (34.5)	0 (110)
13	0 (6.5)	0 (34.5)	0 (110)
14	1 (8.0)	1 (48.0)	1 (160)
15	1 (8.0)	1 (48.0)	−1 (60)
16	−1.68 (3.9)	0 (34.5)	0 (110)
17	−1 (5.0)	−1 (21.0)	−1 (60)
18	0 (6.5)	−1.68 (11.0)	0 (110)
19	−1 (5.0)	−1 (21.0)	1 (160)
20	0 (6.5)	1.68 (57.0)	0 (110)

The number in parenthesis is the actual value of variables.

Figure 1 Growth profile and enzyme activity.

Table 2. CCD plan and measured response

Run No.	X1 (pH)	X2 (Temperature, °C)	C3 (Agitation, rpm)	Yexp (U/mL)	Ypred (U/mL)
1	6.5	34.5	110	8.2	8.1
2	6.5	34.5	110	8.2	8.1
3	8.0	21.0	160	4.6	4.6
4	6.5	34.5	110	8.2	8.1
5	6.5	34.5	194	6.8	6.7
6	5.0	48.0	60	7.2	6.9
7	6.5	34.5	25	7.9	7.2
8	6.5	34.5	110	8.2	8.1
9	5.0	48.0	160	5.4	5.1
10	6.5	34.5	110	8.2	8.1
11	8.0	21.0	60	6.4	6.3
12	9.0	35.4	110	5.8	6.1
13	6.5	34.5	110	8.2	8.1
14	8.0	48.0	160	5.4	5.6
15	8.0	48.0	160	5.2	5.5
16	3.9	34.5	110	4.2	4.7
17	5.0	21.0	60	4.5	4.3
18	6.5	11.0	110	6.0	5.8
19	5.0	21.0	160	4.3	4.5
20	6.5	57.0	110	4.1	4.2

Table 3. Result of regression analysis of the 2³ factorial design

Term	Coefficient	T-statistic	P-value
Intercept	8.20902	26.339	0.000
X1	0.21168	1.024	0.330
X2	0.01498	0.072	0.944
X3	−0.39906	−1.930	0.082
	−1.19033	−5.913	0.000
	−1.17266	−5.826	0.000
	−0.35948	−1.786	0.104
X1X2	−0.52500	−1.943	0.081
X2X3	0.05000	0.185	0.857
X1X3	0.05000	0.185	0.857

R² = 87.8%; R²(adj) = 76.8%.

Table 4. Analysis of variance for characterization of cephalosporin-C acylase by Pseudomonas diminuta using central composite design criterion

Source	DF	Seq SS	Adj SS	Adj MS	FP
Regression	9	41.9505	41.9505	4.6612	7.98
0.002
Linear	3	2.7899	2.7899	0.9300	1.59
0.252
Square	3	86.9157	36.9157	12.3052	21.07
0.00
Interaction	3	2.2450	2.2450	0.7483	1.28
0.33
Residual	10	5.8395	5.8395	0.5839
Lack of fit	5	5.8395	5.8395	1.1679	*
*
Pure error	5	0.000	0.000	0.000
Total	19	47.790

Figure 2 Contour plot of enzyme activity vs temperature, pH.

Figure 3 Contour plot of enzyme activity vs temperature, agitation.

Figure 4 Contour plot of enzyme activity vs agitation, pH.

Figure 5 Lineweaver-Burk plot for determination of K_m and V_max for cephalosporin-c acylase enzyme.

Figure 6 Chromatogram of semisynthetic cephalosporin (Cephalothin).