庆大霉素产生菌发酵液中抑菌活性小组分ANT-A和ANT-B的初步研究

【作者】 沈洁；

【导师】 陈代杰； 李继安；

【作者基本信息】 上海师范大学 ， 微生物学， 2009， 硕士

【摘要】 氨基糖苷类抗生素是目前临床上常用的抗感染药物,由于该类抗生素具有抗菌谱广,疗效好,性质稳定等优势,在市场上占据相当的份额。在氨基糖苷类抗生素发酵过程中产生的各种次要组分,可能是抗菌作用更好而毒副作用更小的潜在的新抗生素。同时,在研究次要组分的基础上,进一步研究微生物的代谢机制,以及关键酶的基因工程改造等,可探索它们的生物合成机制。本文通过对绛红色小单孢菌SIPI-A-2020的培养、发酵和初步提取鉴定,验证了绛红色小单孢菌SIPI-A-2020为庆大霉素产生菌。再以革兰氏阳性菌枯草芽孢杆菌为筛选模型,对绛红色小单孢菌发酵代谢的次要组分进行活性测定,发现ANT-A和ANT-B两种活性小组分,并对ANT-A和ANT-B两种活性小组分进行了分离纯化的初步研究。ANT-A主要存在于菌丝体中易溶于甲醇。将发酵液放瓶后直接抽滤,滤饼用甲醇抽提,甲醇抽提液进行脱盐脱色,并进一步使用硅胶柱进行分配层析,最后通过结晶沉淀得到纯度为96%以上的样品。该ANT-A组分在DAD-UV检测中有特征吸收,通过质谱检测得到ANT-A的分子量为315,根据1H-NMR数据分析并结合相关代谢途径进行分析,初步推测ANT-A可能为含有不饱和双键的假二糖结构化合物。ANT-B极易溶于水,极性较庆大霉素大,对革兰氏阳性菌以及革兰氏阴性菌都具有抑菌活性,通过D301大孔弱碱性阴离子交换树脂脱色,HD-2大孔弱酸阳离子交换树脂吸附层析,0.125%氨水洗脱,并进一步使用活性炭脱色,CM羧甲基纤维素树脂精制等步骤,得到一定纯度的样品,MS推测其分子量可能为444。通过本课题的研究,我们发现了ANT-A与ANT-B两种活性组分,这两种组分在庆大霉素生物合成途径中均未曾报道,可能为庆大霉素产生菌代谢途径中存在的未知活性组分,并推测在产庆大霉素绛红色小单孢菌的代谢途径中还可能存在其他未发现的代谢支路,这为开发新的药物做出了初步的探索,并为庆大霉素合成代谢途径的深入研究,提供了一些新的依据。更多还原

【Abstract】 Aminoglycoside antibiotics has occupied fairly large market share, it always used on clinical as anti-infective drugs because of its wild antibacterial spectrum, fine curative effect, stable property and any other advantages. The minor component produced durining the fermentation process which has the potential to be some new drugs with high activity and low side effect. Meanwhile, we`ll screening more new activity compounds if we do further research on minor component and metabolic mechanism of the producing strain.In this paper, we validated that Micromonospora purpurea SIPI-A-2020 can produce Gentamicin by strain cultivation, fermentation and extracted Gentamicin from the fermentation broth. Furthermore, we use Bacillus subtilis as screeing model to screening secondary antibiotic activity compounds from the fermentation broth. As a result, we got ANT-A and ANT-B two active components. ANT-A mostly resides in the mycelium and dissolved in the methanol. The extraction process including desalt and decolor from the mycelium methanol soak, and using silica gel column partition chromatographic separation, finally through crystallize and precipitation got 96% purity compounds by TLC and HPLC validated. ANT-A has special character absorption wavelength on DAD-UV detecion.We got its molecular weight is 315 by MS spectra.The chemical structure was analyzed by 1H-NMR.We proposed that ANT-A is an pseud-disaccharide compouds with unsaturated double bond. ANT-B is water soluted with higher polarity than Gentamycin. It has high antibacterial activity against G+ and G- bacterial. We use macropore Lewatit resin D301for decolour and then the decoloured solution was loaded on a column of HD-2 macropore cation exchange resin.As for the adsorbed ANT-B was eluted with 0.125% ammonia water by us. After using activated carbon for secondary decolour and CM carboxymethyl cellulose resin for depurate, we can got comparatively purified compounds, at last. We proposed that ANT-B` s molecular weight is 444 by MS spectra. Through our research we got two active compounds ANT-A and ANT-B which has not been reported before.The two compounds may be developed into any new drugs if we do further research on them.On the other hand, we proposed that there will be anyother metabolic pathway in Micromonospora purpurea.更多还原