Abstract: The fermentation medium for the clutivation of cold-active amylase producing strain Pseudomonas sp. D5-2, which was isolated in previous studies, was optimized to improve its enzyme production capacity. The effect of medium factor was determined by single factor test. Then, base on surface methodology, a Box-Benhnken design was used to identify the optimal level and interaction of significant factors to obtain the best combination. The results revealed that lactose, peptone and Cu2+were important factors affecting fermentation. The optimal conditions for producing of the maximal amylase activity by strain pseudomones sp. were as follows: 19.715 g/L lactose, 14.191 g/L peptone and 2.417 g/L CuSO4. Under the conditions, the highest amylase activity was predicted to be 98.876 U/mL, and the actual value was 99.2 U /mL, which was highly consistent with the theoretical model. The Box-Benhnken response surface method is feasible to optimize the fermentation medium of D5-2, which can provide guidance for the explanation of catalytic mechanism and industrial application of cold-active enzyme.

Key words: cold- active amylase, fermentation medium, response surface methodology, Box-Benhnken design