简单节杆菌17α-羟基-16β-甲基-孕甾-4,9(11)二烯-3,20-二酮脱氢酶产酶条件研究

Optimization of producing conditions for Δ~1-dehydrogenase of 17α-hydroxy-16β-methyl-pregna-4, 9, (11)-diene-3, 20-dione by Arthrobacter simplex

目的优化简单节杆菌17α-羟基-16β-甲基孕甾-4,9(11)-二烯-3,20-二酮(简称HMPDD)脱氢酶产酶条件。方法采用摇瓶培养方法,考察培养基组成及发酵条件对HMPDD转化率的影响。结果较佳产酶培养基为:葡萄糖0.4%、玉米浆1.2%、KH2PO40.2%、蛋白胨0.3%。较佳产酶条件为:培养基初始pH7.0;接种量20%;摇瓶装量100mL/250mL三角瓶;接种后4h内添加0.05g/L底物作为诱导物对产酶有利;添加10-4mol/LCo2+和Mg2+,可使酶活分别较对照提高12.9%和6.1%;菌体培养12h时酶活达最大值,为24.46μmol/g·min。结论通过培养基组成及产酶条件的优化,酶活力有较大幅度的提高。

OBJECTIVE 17 α -hydroxy-16 β -methyl-pregna-4, 9, (11)-diene-3, 20-dione (HMPDD) was one of the important intermediates used for the production of beta-methasone. In this paper, optimum conditions for the microbial dehydrogenation of HMPDD by Arthrobacter simplex were studies. METHOD Effects of culture medium and bioconversion conditions on dehydrogenase activity were investigated by shake culture. RESULTS Optimized culture medium for dehydrogenase production was as follows: glucose 0.4% , corn steep liquor 1.2%, KH_2PO_4 0.2%, peptone 0.3%. The optimum conditions for enzyme formation were as follows: initial pH of medium 7.0, inoculum volume 20%(V/V), loading amount 100mL for 250mL shake flask. By adding 0.05g/L HMPDD as inducer, the dehydrogenase activity could be enhanced obviously. Added 10 -4 mol/L Co 2+ and Mg 2+ , the enzyme activity was increased by 12.9% and 6.1% respectively, compared to the contrast. Maximum dehydrogenase activity of 24.46μmol/g·min was obtained at 12h for shake culture. CONCLUSION At optimum conditions, the dehydrogenase activity is increased obviously.