利用Pseudonocardia sp.微生物转化制备骨化醇类化合物

Preparation of calcified alcohols analogues by bioconversion using Pseudonocardia sp.

【背景】近年来骨化醇类化合物(活性维生素D及其类似物)在肿瘤治疗、免疫调节中的作用逐渐被发现,药用价值显著,但目前化学合成法制备困难,因此高效制备骨化醇类化合物成为研究热点。【目的】利用假诺卡氏菌(Pseudonocardiasp.)转化骨化醇类底物分子并提高转化率。【方法】从化学合成方法常用的原料及中间体中筛选能被有效转化的底物分子,利用单因素及正交试验对转化条件进行优化。【结果】筛选到阿法骨化醇(Alfacalcidol,1α-(OH)VD3)、艾地骨化醇中间体(Eldecalcitolintermediate, AD-M07)、帕立骨化醇中间体(Paricalcitol intermediate,PC-M07) 3种底物分子,分别被转化为骨化三醇(Calcitriol,1α,25(OH)2VD3)、艾地骨化醇中间体(Eldecalcitol intermediate,AD-M08)、帕立骨化醇(Paricalcitol,PC)。确定最优转化条件:蛋白胨20.0 g/L,葡萄糖15.0 g/L,部分甲基化的β-环糊精(PMCD) 0.5%(质量体积比),转化温度25-30°C,接种量5%-10%(体积比),转化时间72、72、96 h,底物浓度1.2、0.6、0.6mg/mL,初始pH6.0-8.0。在此条件下3种底物的最高转化率分别达到85%、96%、75%。【结论】通过转化条件的优化3种产物的转化率大幅提高,为更加快速高效地利用微生物转化法制备骨化醇类化合物提供参考。

[Background] Recently, the medicinal value of calcified alcohols analogues（active vitamin D and its analogues） in the treatment of tumors therapy and immune regulation has gradually been discovered and examined. However, as the current method for chemical synthesis is complex and difficult, more efficient preparation method of calcified alcohols analogues becomes a hot research focus. [Objective] In order to bioconvert the substrates of calcified alcohols analogues using Pseudonocardia sp. and further improve the bioconversion rate. [Methods] Raw materials and intermediates commonly used in chemical synthesis were bioconverted with Pseudonocardia sp. to find appropriate substrates. The bioconversion conditions were optimized by single factor method and orthogonal test. [Results] Alfacalcidol（1α-（OH）VD3）, eldecalcitol intermediate（AD-M07） and paricalcitol intermediate（PC-M07） were bioconveted into calcitriol（1α,25（OH）2 VD3）, eldecalcitol intermediate（AD-M08） and paricalcitol（PC）. The optimal bioconversion conditions of the above three substrates（1α-（OH）VD3, AD-M07, PC-M07） were as follows： peptone 20.0 g/L, glucose 15.0 g/L, PMCD 0.5%, temperature 25 °C to 30 °C, inoculation quantity 5% to 10%, bioconversion time 72, 72, 96 h, substrates concentration 1.2, 0.6, 0.6 mg/mL, initial pH 6.0 to 8.0. Under the above conditions, the maximum bioconversion rate of the three products is 85%, 96%, 75% respectively. [Conclusion] By optimizing the bioconversion conditions, the bioconversion rate of the three products was markedly increased, which provides a good reference for more efficient preparation of calcified alcohols analogues by bioconversion.