New Horizons in Insect Science Towards Sustainable Pest Management

Chitinase Expressed as an Inducible Trait in Pseudomonas aeruginosa Schröter P-15 77

chitin, and peptone ( P < 0.05) for chitinase pro-
duction was considered as significant positive
variables. However, tryptone, K 2 HPO 4 , and
MgSO4.7H 2 O showed no significant influence on
chitinase production.
To obtain above results, Plackett–Burman de-
sign is considered a powerful tool for identifying
factors that have significant influence on chitin-
ase production. The optimal concentration of the
individual factor was further determined by the
subsequent CCD experiment.

Optimization of Screened Medium

Components Using CCD

CCD was used to determine the optimal concen-
tration (level) of the medium components. A total
of 20 experiments with three variables (compo-
nents of the medium) and five coded levels (five
different concentrations) were performed. Model
was run for optimization of significant media

components screened via Plackett–Burman ex-

perimental design without any transformation

for chitinase activity values. Chitinase response

ranged from 1.37 to 3.55 Uml−1 and the ratio of

maximum to minimum was found 2.59.

Based on the results obtained from Plackett–

Burman design, we selected three variables, name-

ly, crude glycerol, colloidal chitin, and peptone

were selected. As they were positive influencing,

increased concentrations were studied for optimi-

zation. The other components of the production

medium were nonsignificant and hence, their con-

centrations were set at middle level in CCD. The

other conditions were: temperature 29 °C, 0.2 %

inoculum size, agitation speed (140 rpm), incuba-

tion period 84 h and pH 7.2. (Fig. 2 )

Table 7 represents the experimental design

matrix for CCD along with the experimental

results of predicted responses for chitinase ac-

tivity in broth. The experimental values for the

regression coefficient were obtained by quadrat-

ic polynomial equation, where only significant

Table 5 Effects of media amendments on chitinase production, growth and in vitro antipathogenic activity against
F. udum Butler
Treatments Chitinase activity (U/ml) Microbial biomass
(Log CFU/ml)

Inhibition zone
(mm)
KB 2.73 8.00 2.69
KB + FP 3.16 1 1.00 11.00
KB + CC 3.51 9.00 20.00
KB + IP 2.96 8.00 9.00
KBCG 2.00 8.00 6.00
KBCG + FP 2.10 8.50 14.00
KBCG + CC 2.72 13.00 13.00
KBCG + IP 2.23 10.00 10.00
SEM 0.08 0.32 0.06
CD 0.25 0.95 0.20
CV % 5.64 5.82 4.27

Table 6 Statistical analysis of process parameters for chitinase activity
Factors Fermentation
parameters

Chitinase activity (Uml−1)

Effect ( Exi) S.E. t( (^) xi) P-value
X 1 Crude glycerol 12.07 0.66 4.58 0.00593
X 2 Colloidal chitin 15.32 14.48 0.00003
X 3 Peptone 11.72 3.52 0.01695
X 4 Tryptone 9.88 −2.09 0.09131
X 5 K 2 HPO 4 11.26 2.12 0.08785
X 6 MgSO4.7H 2 O 11.15 1.78 0.13487