关键词: 反式-4-羟基-L-脯氨酸, 脯氨酸羟化酶, 响应面法, 转化率, 工艺条件

Abstract: The conversion rate of L-proline 4-hydroxylase can be increased by the optimizing expression conditions and catalytic reaction conditions. The condition of optimal inducing temperature,inducer concentration,and conversion for L-proline 4-hydroxylase was screened by single factor screening,and the optimal catalytic reaction conditions were further predicted using response surface methodology. Results showed that the optimal protein expression temperature was 28℃ and the concentration of IPTG was 0.2 mmol/L. The optimal conditions for catalytic reaction were as follows：the harvested cells（1 g）were suspended in 10 mL 2-morpholinoethanesulfonic acid buffer（120 mmol/L,pH 6.6）containing 1.5% Nonidet P-40,200 mmol/L L-proline,200 mmol/L α-ketoglutarate,6 mmol/L L-ascorbate and 6.0 mmol/L ferrous sulfate,and incubated at 27℃ with shaking at 152 r/min for 48 h. Under the optimal condition,the conversion rate for trans-4-hydroxy-L-proline reached 100% after 48 h incubation. This study may provide a solid foundation for the synthesis of trans-4-hydroxy-L-proline.

Key words: trans-4-hydroxy-L-proline, L-proline 4-hydroxylase, response surface methodology, conversion rate, reaction condition