Statistical optimization of cold-active chitinase production by mutagenized cells of multi-enzyme producing Bacillus cereus GA6

Abstract

In the present study, four cold-adapted bacterial isolates were screened for multiple-enzyme production at low temperature (15 °C). The most potent isolate, Bacillus cereus GA6 (HQ832575), was subjected to mutation by UV radiation to obtain a mutant strain with elevated enzyme production. The mutant strain, designated as CUVGA6, with higher chitinase activity at low temperature was selected for enzyme production optimization using factorial design and response-surface methodology (RSM). Two statistically significant parameters (colloidal chitin and KH₂PO₄) for response were selected (p value = 0.008 and 0.004, respectively) along with pH and temperature and utilized to optimize the process. Central composite design of RSM was used to optimize the levels of key ingredients for the best yield of chitinase. Maximum chitinase production was predicted to be 428.57 U/ml for a 4.4-fold increase in medium containing 2 % colloidal chitin, 6.0 g/L K₂HPO₄ and pH 9.0 at 25 °C when incubated for 7 days in submerged fermentation. ANOVA of CCD suggested that the quadratic interaction effect of K₂HPO₄ with chitin, temperature and pH has high impact on the production of chitinase (p value = 0.007, 0.002, 0.035, respectively), although its linear effect was not significant as observed. The closeness of optimized values (R ² = 82.28 %) to experimental values (R ² = 80.13 %) proved the validity of statistical model. Thus, multi-enzyme producing cold-adapted mutant B. cereus GA6 (CUVGA6) could be exploited for the production of chitinase which is of industrial significance.