一株蟾毒灵转化菌株的筛选鉴定与转化条件优化

Isolation and characterization of a fungus for bufalin transformation

摘要:

【背景】微生物转化是对天然产物进行结构修饰的重要手段，具有催化反应快、选择性强、反应条件易控制和污染小等特点。许多微生物能够对蟾毒配基类化合物进行生物转化，在不同位置进行结构修饰，并且产生毒性减弱的活性衍生物。【目的】筛选蟾毒灵生物转化菌株，以期发现活性更好、毒性降低的化合物。【方法】利用蟾毒灵为底物筛选其生物转化菌株，通过HPLC (High performance liquid chromatography)/LC-MS (Liquid chromatograph-mass spectrometer)鉴定转化产物；对可生物转化蟾毒灵的菌株进行菌落形态观察和分子生物学鉴定，并优化发酵条件提高转化率，同时测试该菌株对其他甾体化合物的转化作用。【结果】筛选获得一株蟾毒灵转化菌，经形态学与ITS (Internal transcribed spacer)分析鉴定为Naganishia属菌，转化产物为3-去氢蟾毒灵。该菌株生物转化的培养基最适底物浓度为8 mg/L，最适初始pH值为6.5，最佳接种量为3%，最佳转化时间为96 h，最终转化率为48.3%。该菌株可将雌酮E1转化为雌二醇E2，也可将雌二醇E2逆向转化为E1。【结论】首次发现Naganishia属菌株对甾体类化合物具有转化活性，其产生的弱细胞毒性的转化产物3-去氢蟾毒灵有望成为高效、安全的强心药物。微生物转化法选择性高、反应条件温和、操作简单，为大量制备活性化合物3-去氢蟾毒灵提供了一条简便易行的道路，也为更多甾体化合物结构修饰提供了可能。

Abstract:

[Background] Microbial transformation is an important means for structural modification of natural products, featured with fast reaction, high selectivity, easy-controlled conditions and little pollution. Many microorganisms have been isolated for transformation of bufadienolides and several derivatives of reduced toxicity can be obtained by the modification at different positions. [Objective] The aim of this work is to find compounds with better bioactivity or reduced toxicity by screening the converting strains towards bufalin. [Methods] Substrate transformation experiments were carried out with bufalin as substrate to screen strains, and the products were identified by HPLC (High performance liquid chromatography) and LC-MS (Liquid chromatograph-mass spectrometer). The selected strain was determined in terms of colony morphology observation and molecular biological identification. The fermentation conditions were optimized to increase the conversion rate. The transformation of other steroids were also determined. [Results] One bufalin-transforming strain was obtained and identified as Naganishia sp. by the morphological characterisitics and evolutionary analysis on the basis of ITS sequence. The transformation product of this strain to bufalin was 3-ketobufalin. The optimal conversion conditions were determined with the initial pH value of 6.5, substrate concentration of 8 mg/L, the inoculation amount of 3% and the transformation time of 96 hours. About 48.3% of bufalin was converted into 3-ketobufalin. In addition, this strain could also transform the reversible reaction of estrone and 17β-estradiol. [Conclusion] It is first reported that Naganishia strain can transform bufalin into 3-ketobufalin, with less toxicity and great potential for safe drug to heart failure. The capability of transforming steroids of this strain also makes it possible to bioconvert and modify other steroidic compounds. The microbial conversion can provide a convenient path for large-scale production of the valuable compound because of high selectivity, mild reaction conditions and simple operation process.