α-Amylase Production in Fed Batch Cultivation of Bacillus caldolyticus: An Interpretation of Fermentation Course Using 2-D Gel Electrophoresis

J. Bader, B. Neumann, K. Schwab, C. Scheler and R. Bajpai

Abstract
The conditions for increased production of thermostable α-amylase from Bacillus caldolyticus DSM 405 were investigated. Preliminary experiments in batch shake flasks led to an optimized initial cultivation medium. Shake flask experiments in extended-batch and in fed-batch mode of operation indicated that the α-amylase production was enhanced by continuous feeding of starch. The activity of the α-amylase with optimized initial medium in batch-operated shake flasks was 5.7 U mL–1 compared to 15.4U mL–1 in the extended-batch culture and 21 U mL–1 in fed-batch culture. The improvements were achieved by avoiding any excess of starch in medium that led to accumulation of glucose followed by acetate formation. Adding casitone as the second component of the feeding solution in an aerated and agitated fed-batch bioreactor (3-liter working volume) led to an increased α-amylase activity of up to 163.7 U mL.–1 All phases of cultivation were analyzed using 2D-gel electrophoresis in combination with nano LC-ESI-MS/MS for identification of altered proteins. Pyruvate kinase, 6-phosphofructokinase, GltC, anti-sigma F factor, glycogen synthase and several important variable proteins were detected. With help of these results, potential improvements of a two-component feeding strategy are discussed.

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Keywords
Bacillus caldolyticus, α-amylase, thermophilic microorganism, fermentation, 2D-gel-elektrophoresis