Pseudomonas sp. W7产低温蛋白酶培养基及培养条件的优化

采用Plackett-Burman法对影响假单胞菌（Pseudomonas sp.）W7产低温蛋白酶的因素进行显著性分析，筛选出对产酶影响显著的3 个因素：葡萄糖、蛋白胨和MgSO4；然后用最陡爬坡试验逼近最大产酶区域；应用Box-Behnken原理设计和响应面分析确定产酶培养基的最佳组合为：葡萄糖4.25 g/L、蛋白胨7.00 g/L、干酪素4.00 g/L、K2HPO4 5.00 g/L、KH2PO4 1.00 g/L、CaCl2 0.26 g/L、MgSO4 0.14 g/L，低温蛋白酶的酶活力达到了183.9 U/mL，比优化前提高了21%。另外，对Pseudomonas sp. W7的产酶条件进行了优化，在单因素的基础上，利用正交试验设计，最终确定产酶的最优培养条件为：培养温度为20 ℃、初始pH值为7.0、接种量为3%、装液量为20%、摇床转速为100 r/min，在最佳条件时测得的酶活力为193.2 U/mL。通过生长曲线和产酶曲线的测定，确定产酶的培养时间为48 h。

Optimization of Medium Components and Culture Conditions for Cold-Adapted Protease Produced by Pseudomonas sp.W7

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.