Enhanced Production of Thermostable Pullulanase Type 1 Using Bacillus cereus FDTA 13 and Its Mutant

Subhash U. Nair, Rekha S. Singhal and Madhusudan Y. Kamat*

Food Engineering and Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India

Article history:
Received November 30, 2005
Accepted March 14, 2006

Key words:
thermostable pullulanase, Bacillus cereus, orthogonal array, optimization, mutation, polymethylsulphonate fluoride

Summary:
This paper describes optimization of fermentation conditions for the production of thermostable pullulanase from a new strain Bacillus cereus FDTA 13, isolated from tapioca soil. By using one-factor-at-a-time and orthogonal array method, a simple medium with effective and minimal components was optimized for maximal pullulanase production. The optimized medium containing (in g/L): starch 20, yeast extract 5, NaCl 2, MgSO₄ 0.1 and K₂HPO₄ 0.17 gave nearly 3.6-fold increase in pullulanase activity. Addition of Mn²⁺ ions in the optimized medium further enhanced the production by 1.4-fold. Effects of proteolytic inhibitors on stability of pullulanase in the medium were studied. A mutant Bacillus cereus FDTA 13/NTG04-B4, obtained by chemical mutation of the parent strain, further increased the pullulanase production by 2-fold in optimized medium, and gave similar yield as the parent strain in a medium with lower levels of carbon source.

*Corresponding author:            mykamat@udct.org
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