Optimization of chitinase production using statistics based experimental designs

N.N. Nawani and B.P. Kapadnis

Process Biochemistry 2004

a) Statistical based experimental designs were used to optimize the medium

components and environmental factors for chitinase production.
b) Three strains Streptomyces sp. NK1057, NK528 and NK951 were used
c) The variables that could affect chitinase production were identified by 2-level
fractional factorial and optimized using CCD.
2-level factorial
8 variables which were expected to have an effect on chitinase production were identified
by preliminary experiments.
Variables having most significant effect on chitinase production were then identified
using a 2-level fractional factorial design.
According to the 2-level eight variable concept, a complete matrix would have been
based on 28 = 256 runs which was not feasible.
Fractional factorial designs have been useful in such cases where a 1/8 fraction was
considered, i.e. 256/8 = 32 runs and two runs were centre point runs for statistical reasons.
Thus a fractional factorial matrix of 34 ruins was used.
Table 1 listed the variables considered for the design and Table 2 shows the design
matrix covering eight variables to evaluate their effect on chitinase production.
Table 1
Variables in real values for screening by the 2-level fractional factorial design
Variable

A: Chitin
B: Yeast Extract
C: K2HPO4
D: NH4SO4
E: Trace element solution
F: Inoculum density
G: pH
H: Temperature

Unit

Low level
(-1)

High level
(+1)

g/L
g/L
g/L
g/L
mL/L
--o
C

10.0
0.1
0.3
0.1
0.5
0.1
6.0
30.0

20.0
0.4
0.9
0.4
1.5
0.25
8.0
40.0

Table 2: 2 level fractional factorial design of variables

Std

A
:Chitin

B:
Yeast
Extract

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
15.00
15.00

0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.25
0.25

C:
K2HPO4

0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.60
0.60

D:
NH4SO4

0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.25
0.25

E: Trace
element
solution

0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.00
1.00

F:
Inoculum
density

0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.17
0.17

G:
pH

6.00
8.00
8.00
6.00
6.00
8.00
8.00
6.00
8.00
6.00
6.00
8.00
8.00
6.00
6.00
8.00
6.00
8.00
8.00
6.00
6.00
8.00
8.00
6.00
8.00
6.00
6.00
8.00
8.00
6.00
6.00
8.00
7.00
7.00

H:
Temperature

40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
30.00
30.00
40.00
40.00
40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
40.00
40.00
30.00
30.00
40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
35.00
35.00

Respon
se
U/mL

Table 3: Result fot NK 1057

Std

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

A
:Chitin

B:
Yeast
Extract

C:
K2HPO4

D:
NH4SO4

E: Trace
element
solution

10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
15.00
15.00

0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.25
0.25

0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.60
0.60

0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.25
0.25

0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.00
1.00

F:
Inoculum
density

0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.17
0.17

G:
pH

6.00
8.00
8.00
6.00
6.00
8.00
8.00
6.00
8.00
6.00
6.00
8.00
8.00
6.00
6.00
8.00
6.00
8.00
8.00
6.00
6.00
8.00
8.00
6.00
8.00
6.00
6.00
8.00
8.00
6.00
6.00
8.00
7.00
7.00

H:
Temperature

40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
30.00
30.00
40.00
40.00
40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
40.00
40.00
30.00
30.00
40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
35.00
35.00

Response
U/mL
NK1057

271
294
135
147
168
292
136
166
196
217
137
131
189
213
130
131
145
233
138
136
148
212
135
148
128
211
131
136
155
201
139
87
257
245

Table 4: Results for NK 528

Std

A:
Chitin

B:
Yeast
Extract

C:
K2HPO4

D:
NH4SO4

E: Trace
element
solution

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
15.00
15.00

0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.25
0.25

0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.60
0.60

0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.25
0.25

0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.00
1.00

F:
Inoculum
density

0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.17
0.17

G:
pH

6.00
8.00
8.00
6.00
6.00
8.00
8.00
6.00
8.00
6.00
6.00
8.00
8.00
6.00
6.00
8.00
6.00
8.00
8.00
6.00
6.00
8.00
8.00
6.00
8.00
6.00
6.00
8.00
8.00
6.00
6.00
8.00
7.00
7.00

H:
Temperature

40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
30.00
30.00
40.00
40.00
40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
40.00
40.00
30.00
30.00
40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
35.00
35.00

Response
U/mL
NK528

126
117
122
118
165
161
118
115
127
134
93
97
113
128
119
132
165
158
125
123
132
133
127
133
121
123
125
131
119
114
93
89
141
130

Table 5: Results for NK951

Std

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

A
:Chitin

B:
Yeast
Extract

C:
K2HPO4

D:
NH4SO4

E: Trace
element
solution

10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
15.00
15.00

0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.10
0.10
0.40
0.40
0.25
0.25

0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.30
0.30
0.30
0.30
0.90
0.90
0.90
0.90
0.60
0.60

0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.25
0.25

0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.00
1.00

F:
Inoculum
density

0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.10
0.25
0.25
0.10
0.25
0.10
0.10
0.25
0.17
0.17

G:
pH

6.00
8.00
8.00
6.00
6.00
8.00
8.00
6.00
8.00
6.00
6.00
8.00
8.00
6.00
6.00
8.00
6.00
8.00
8.00
6.00
6.00
8.00
8.00
6.00
8.00
6.00
6.00
8.00
8.00
6.00
6.00
8.00
7.00
7.00

H:
Temperature

40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
30.00
30.00
40.00
40.00
40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
40.00
40.00
30.00
30.00
40.00
40.00
30.00
30.00
30.00
30.00
40.00
40.00
35.00
35.00

Response
U/mL
NK951

78
101
57
46
68
62
72
59
80
89
72
84
98
83
59
60
84
78
80
66
73
98
58
48
100
73
50
61
74
85
66
83
112
116

ANOVA ANALYSIS FOR NK1057

Source
Model
A
B
D
E
AB
BE
Curvature
Residual
Lack of Fit
Pure Error
Cor Total

Sum of
Squares
67411.87
7021.12
38503.13
4324.50
6903.13
6962.00
3698.00
12388.26
12765.63
12693.63
72.00
92565.76

DF
6
1
1
1
1
1
1
1
26
25
1
33

Mean
Square
11235.31
7021.12
38503.13
4324.50
6903.13
6962.00
3698.00
12388.26
490.99
507.75
72.00

F
Value
22.88
14.30
78.42
8.81
14.06
14.18
7.53
25.23

Prob > F
< 0.0001
0.0008
< 0.0001
0.0064
0.0009
0.0009
0.0108
< 0.0001

7.05

0.2903

significant

significant
not significant

The Model F-value of 22.88 implies the model is significant. There is only a 0.01% chance that a "Model F-Value" this large could occur due to noise.
Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case A, B, D, E, AB, BE are significant model terms. Values greater than
0.1000 indicate the model terms are not significant.
The "Curvature F-value" of 25.23 implies there is significant curvature (as measured by difference between the average of the center points and the average of
the factorial points) in the design space. There is only a 0.01% chance that a "Curvature F-value" this large could occur due to noise.
The "Lack of Fit F-value" of 7.05 implies the Lack of Fit is not significant relative to the pure error. There is a 29.03% chance that a "Lack of Fit F-value" this
large could occur due to noise. Non-significant lack of fit is good -- we want the model to fit.
Std. Dev.
Mean
C.V.
PRESS

22.16
174.65
12.69
21085.24

R-Squared
Adj R-Squared
Pred R-Squared
Adeq Precision

0.8408
0.8040
0.7722
14.107

The "Pred R-Squared" of 0.7722 is in reasonable agreement with the "Adj R-Squared" of 0.8040.
"Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 14.107 indicates an adequate signal. This model can be
used to navigate the design space.

ANOVA ANALYSIS FOR NK 528

Source
Model
B
D
H
AD
AG
CD
CE
DE
Curvature
Residual
Lack of Fit
Pure Error
Cor Total

Sum of
Squares
10314.75
2380.50
2450.00
2888.00
112.50
180.50
171.13
1922.00
210.13
212.50
451.25
390.75
60.50
10978.50

DF
8
1
1
1
1
1
1
1
1
1
24
23
1
33

Mean
Square
1289.34
2380.50
2450.00
2888.00
112.50
180.50
171.13
1922.00
210.13
212.50
18.80
16.99
60.50

F
Value
68.57
126.61
130.30
153.60
5.98
9.60
9.10
102.22
11.18
11.30

Prob > F
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.0221
0.0049
0.0060
< 0.0001
0.0027
0.0026

0.28

0.9282

significant

significant
not significant

The Model F-value of 68.57 implies the model is significant. There is only a 0.01% chance that a "Model F-Value" this large could occur due to noise.
Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case B, D, H, AD, AG, CD, CE, DE are significant model terms. Values
greater than 0.1000 indicate the model terms are not significant. If there are many insignificant model terms (not counting those required to support hierarchy),
model reduction may improve your model.
The "Curvature F-value" of 11.30 implies there is significant curvature (as measured by difference between the average of the center points and the average of
the factorial points) in the design space. There is only a 0.26% chance that a "Curvature F-value" this large
could occur due to noise.
The "Lack of Fit F-value" of 0.28 implies the Lack of Fit is not significant relative to the pure error. There is a 92.82% chance that a "Lack of Fit F-value" this
large could occur due to noise. Non-significant lack of fit is good -- we want the model to fit.
Std. Dev.
Mean
C.V.
PRESS

4.34
125.50
3.46
998.39

R-Squared
Adj R-Squared
Pred R-Squared
Adeq Precision

0.9581
0.9441
0.9091
33.222

The "Pred R-Squared" of 0.9091 is in reasonable agreement with the "Adj R-Squared" of 0.9441.
"Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 33.222 indicates an adequate signal. This model can be
used to navigate the design space.

ANOVA ANALYSIS OF NK951

Source
Model
B
C
D
G
H
BH
CD
CE
DE
EF
GH
Curvature
Residual
Lack of Fit
Pure Error
Cor Total

Sum of
Squares
6866.59
2869.03
87.78
247.53
675.28
1610.28
108.78
81.28
47.53
175.78
306.28
657.03
3120.97
207.87
199.87
8.00
10195.44

DF
11
1
1
1
1
1
1
1
1
1
1
1
1
21
20
1
33

Mean
Square
624.24
2869.03
87.78
247.53
675.28
1610.28
108.78
81.28
47.53
175.78
306.28
657.03
3120.97
9.90
9.99
8.00

F
Value
63.06
289.84
8.87
25.01
68.22
162.67
10.99
8.21
4.80
17.76
30.94
66.37
315.29

Prob > F
< 0.0001
< 0.0001
0.0072
< 0.0001
< 0.0001
< 0.0001
0.0033
0.0093
0.0398
0.0004
< 0.0001
< 0.0001
< 0.0001

1.25

0.6184

significant

significant
not significant

The Model F-value of 63.06 implies the model is significant. There is only a 0.01% chance that a "Model F-Value" this large could occur due to noise.
Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case B, C, D, G, H, BH, CD, CE, DE, EF, GH are significant model terms.
Values greater than 0.1000 indicate the model terms are not significant. If there are many insignificant model terms (not counting those required to support
hierarchy), model reduction may improve your model.
The "Curvature F-value" of 315.29 implies there is significant curvature (as measured by difference between the average of the center points and the average of
the factorial points) in the design space. There is only a 0.01% chance that a "Curvature F-value" this large could occur due to noise.
The "Lack of Fit F-value" of 1.25 implies the Lack of Fit is not significant relative to the pure error. There is a 61.84% chance that a "Lack of Fit F-value" this
large could occur due to noise. Non-significant lack of fit is good -- we want the model to fit.

Std. Dev.
Mean
C.V.
PRESS

3.15
75.68
4.16
543.68

R-Squared
Adj R-Squared
Pred R-Squared
Adeq Precision

0.9706
0.9552
0.9467
33.283

The "Pred R-Squared" of 0.9467 is in reasonable agreement with the "Adj R-Squared" of 0.9552.
"Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 33.283 indicates an adequate signal. This model can be
used to navigate the design space.

RESPONSE SURFACE METHODOLOGY

The most popular response surface method (RSM) design is the central composite design (CCD).
A CCD has three groups of design points:
(a)
plus and minus two-level factorial or fractional factorial design points
(b)
plus and minus axial points (sometimes called "star" points)
(c)
center points
For NK 1057 and NK528 the variables significantly affecting chitinase production are shown in
Table 6, together with the real values of variables selected for CCD.
Strains
NK 1057

NK 528

Unit

-2

-1

+1

+2

A: Chitin

Variables

g/L

5.0

10.0

15.0

20.0

25.0

B: Yeast Extract

g/L

0.0

0.1

0.2

0.3

0.4

D: NH4SO4

g/L

0.1

0.2

0.3

0.4

0.5

E: Trace element

mL/L

0.25

0.5

0.75

1.0

1.25

B: Yeast Extract

g/L

0.1

0.15

0.2

0.25

0.3

D: NH4SO4

g/L

0.1

0.15

0.2

0.25

0.3

26.0

28.0

30.0

32.0

34.0

H: Temperature

Design for NK 1057 with response values

Run

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
10.00
20.00
5.00
25.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00
15.00

Others (at mid-point i.e. 0 value):

K2HPO4 (g/L) = 0.6
Inoculum density = 0.175
pH = 7.0
Temperature oC = 35

0.10
0.10
0.30
0.30
0.10
0.10
0.30
0.30
0.10
0.10
0.30
0.30
0.10
0.10
0.30
0.30
0.20
0.20
0.00
0.40
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20

0.20
0.20
0.20
0.20
0.40
0.40
0.40
0.40
0.20
0.20
0.20
0.20
0.40
0.40
0.40
0.40
0.30
0.30
0.30
0.30
0.10
0.50
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30

0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.75
0.75
0.75
0.75
0.75
0.75
0.25
1.25
0.75
0.75
0.75
0.75
0.75
0.75

NK 1057
Response
257
301
234
223
238
266
199
209
240
281
188
204
222
251
164
182
219
266
204
107
264
241
269
233
241
252
250
247
257
246

Design for NK 528 with response values

Run

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

0.15
0.25
0.15
0.25
0.15
0.25
0.15
0.25
0.10
0.30
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20

Others (at mid-point i.e. 0 value):

Chitin (g/L) = 15
Trace element (mL/L) = 1.0
K2HPO4 (g/L) = 0.6
Inoculum density = 0.175
pH = 7.0

0.15
0.15
0.25
0.25
0.15
0.15
0.25
0.25
0.20
0.20
0.10
0.30
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20

NK 528 Response

28.00
28.00
28.00
28.00
32.00
32.00
32.00
32.00
30.00
30.00
30.00
30.00
26.00
34.00
30.00
30.00
30.00
30.00
30.00
30.00

97
129
126
138
119
142
130
158
81
100
145
158
113
172
146
136
143
140
128
141

Final model equations in terms of Actual Factors

NK 1057:
Response (Chitinase Activity) =
+215.96875
+5.15417
+943.54167
-205.62500
-72.75000
-0.041250
-2278.12500
+146.87500
+17.50000
-13.62500
-0.62500
+1.65000
+43.75000
-147.50000
+22.50000

* Chitin
* Yeast Extract
* NH4SO4
* Trace
* Chitin2
* Yeast Extract2
* NH4SO42
* Trace2
* Chitin * Yeast Extract
* Chitin * NH4SO4
* Chitin * Trace
* Yeast Extract * NH4SO4
* Yeast Extract * Trace
* NH4SO4 * Trace

NK 528
Response (Chitinase Activity) =
-113.60795
+2008.29545
+190.79545
-5.23011
-4886.36364
+1213.63636
+0.19602
-750.00000
+8.75000
-13.75000

* Yeast Extract
* NH4SO4
* Temp
* Yeast Extract2
* NH4SO42
* Temp2
* Yeast Extract * NH4SO4
* Yeast Extract * Temp
* NH4SO4 * Temp

Surface Plot Activity (Chitinase) of NK 1057 as a function of Chitin and Yeast Extract

DESIGN-EXPERT Pl ot
Response 1
X = A: Chi ti n
Y = B: Yeast Extract
Actual Factors
C: NH4SO4 = 0.30
D: T race = 0.75

280

255

Response1

230

205

180

0.40

30.00
0.30

23.75
0.20

17.50

B: Y east Extract 0.10

11.25
0.00

5.00

A: Chitin

Surface Plot Activity (Chitinase) of NK 528 as a function of Yeast Extract and ammonium sulphate

DESIGN-EXPERT Pl ot
Response 1
X = A: Yeast Extract
Y = B: NH4SO4
Actual Factor
C: T em p = 30.00

150

137.5

R
e
sp
o
n
se1

125

112.5

100

0.30

0.30
0.24

0.24
0.17

B: NH4SO4

0.17
0.11

0.11
0.05

0.05

A: Y east Extract

Optimisation of chitinase by NK1057

10.00

20.00

0.30

Y east Extract = 0.13

C hitin = 20.00

0.20

0.10

0.40

0.50

1.00

Trace = 0.50

N H4SO4 = 0.20

Desirability = 0.953

107

301

R esponse 1 = 291.858

Optimisation of chitinase by NK528

0.15

0.25

0.15

NH4SO4 = 0.25

Y east Extract = 0.21

28.00

32.00

Temp = 32.00

Desirability = 0.858

0.25

81

172

Response 1 = 159.106

Chitinase activity predicted based on quadratic models in CCD

NK 1057
Chitin (g/L) = 20
Yeast Extract (g/L) = 0.13
NH4SO4 (g/L) = 0.20
Trace element (mL/L) = 0.50
Others (at mid-point i.e. 0 value):
K2HPO4 (g/L) = 0.6
Inoculum density = 0.175
pH = 7.0
Temperature oC = 35

Response
Predicted
291.8 U/mL

Response
Obtained
325.6 U/mL

NK 528
Yeast Extract (g/L) = 0.21
NH4SO4 (g/L) = 0.25
Temperature oC = 32
Others (at mid-point i.e. 0 value):
Chitin (g/L) = 15
Trace element (mL/L) = 1.0
K2HPO4 (g/L) = 0.6
Inoculum density = 0.175
pH = 7.0

Response
Predicted
159.1U/mL

Response
Obtained
175.3 U/mL