离子束注入提高Trichosporon lactis T立体拆分布洛芬水平的研究

以从土壤中分离筛选到的可立体选择性水解布洛芬乙酯生成S-布洛芬的菌株Trichosporon lactisT为出发菌株,对其进行能量30KeV,剂量1×1015~5×1015ions/cm2的低能N 注入,筛选立体选择性水解布洛芬乙酯活性高的诱变株。菌株T.lactisT,在4×1015ions/cm2的诱变剂量下突变率最高,正向和负向突变率分别达32.9%和37.1%,因此选定该剂量为T.lactisT的最佳N 离子注入剂量。经离子束诱变,通过初筛和复筛,共筛选到7株水解布洛芬乙酯的高产菌株,其中诱变株K1培养24h时酶活力比出发菌株T高50%,且具有较好的遗传稳定性。将菌株K1和出发菌株T培养24h,分别加入布洛芬乙酯水解24h,二者水解布洛芬乙酯生成S-布洛芬旋光度均为 54.1°,对映体过量值ee%均为98%,K1菌株水解的产量达6.96g/L,而出发株仅为4.24g/L。

Study on ion beam mutagenizing of the Trichosporon lactis T for enantioselective separation of ibuprofen

The initial strain, Trichosporon Lactis T, isolated from soil sample, having the capability of enantioselectively hydrolyzing S-isomer of racemic ibuprofen ethyl-ester into the corresponding S-ibuprofen, was implanted by 30 KeV, 1 x 10(15) ions/cm2 - 5 x 10(15) ions/cm2 low-energy N+ for the purpose of obtaining mutants with high-efficiency hydrolyzing enzyme to produce active S-ibuprofen. Under the dosage of 30 KeV, 4 x 10(15) ions/cm2, the mutation rate is the highest, with 32.9 % positive and 37.1% negative mutant, respectively. Therefore, 30 KeV, 4 x 10(15) ions/cm2 is chosen as the optimal implantation dosage. Under optimal implantation dosage, seven mutants with high-efficiency hydrolyzing enzyme are selected after N+ implantation. The genetic stability test shows that T. lactis K1, one of the seven mutants, has a stable hydrolyzing ability during consecutive five-generation. The enzyme activity of T. lactis K1 is higher with 50% than that of the initial strain after 24 h cultivation, and the highest enzyme activity of T. lactis K1 appears 6h earlier than that of the initial strain. After 24 h cultivation and succeeding 24 h incubation with ibuprofen ethyl ester, the S-ibuprofen production of T. lactis K1 is 6.96 g/L, 64.2% higher than that of T. lactis T, which only produces 4.24 g/L S-ibuprofen at the same time, the specific rotation and enantiomeric excess (ee%) of the S-ibuprofen produced by two stains, however, are the same, + 54.1 degrees and 98%, respectively.