Abstract:

Cold-adapted proteases have great superiority in the food, cosmetic, pharmaceutical and detergent industries
due to high catalytic activity at low temperature and thermal instability. In the present study, the Plackett-Burman design
was used to identify glucose, peptone and MgSO4 as the most significant medium components affecting the production of
cold-adapted protease by Pseudomonas sp. W7. Then the steepest ascent experiment was used to approach the optimal
region of the three medium components. Box-Behnken design and response surface analysis were used to determine the
best culture medium containing 4.25 g/L glucose, 7.00 g/L peptone, 4.00 g/L casein, 5.00 g/L K2HPO4, 1.00 g/L KH2PO4,
0.26 g/L CaCl2 and 0.14 g/L MgSO4. After the optimization, the yield of the protease was increased by 21% to 183.9 U/mL.
In addition, the optimal culture conditions for producing cold-adapted protease using the optimized medium were determined
using combination of single factor experiments and orthogonal array design experiments as culture temperature of 20 ℃,
initial medium pH of 7.0, inoculum amount of 3%, medium loading volume of 20% and rotation speed of 100 r/min. Under
these conditions, the enzyme activity was 193.2 U/mL. Based on the growth curve and enzyme production curve, the optimal
culture period was determined to be 48 h.

Key words: cold-adapted protease, Pseudomonas sp. W7, optimization of fermentation conditions, response surface analysis