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

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

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 lactis T为出发菌株，对其进行能量30KeV，剂量1×10^15-5×10^15 ions／cm^2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．1actis T，在4×10^15 ions／cm^2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％，因此选定该荆量为T．lactis T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。

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 30KeV, 1× 10^15 ions/cm^2 - 5 × 10^15 ions/cm^2 low-energy N^＋ for the purpose of obtaining mutants with high-efficiency hydrolyzing enzyme to produce active S-ibuprofen. Under the dosage of 30KeV, 4 × 10^15 ions/cm^2, the mutation rate is the highest, with 32.9 % positive and 37. 1 % negative mutant, respectively. Therefore, 30KeV, 4 × 10^15 ions/cm^2 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 24h cultivation, and the highest enzyme activity of T. lactis K1 appears 6h earlier than that of the initial strain. After 24h cultivation and succeeding 24h incubation with ibuprofen ethyl ester, the S-ibuprofen production of T. lactis K1 is 6.96g/L, 64.2% higher than that of T. lactis T, which only produces 4.24g/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°and 98%, respectively.