胆固醇转化菌株的筛选及发酵条件优化

Screening and optimization of cholesterol conversion strain

摘要:

摘要:【目的】从土壤中筛选及鉴定具有转化胆固醇能力的菌株SE-1，对转化产物进行结构鉴定，并通过一定的工艺条件优化提高转化产率。【方法】利用胆固醇为唯一碳源筛选能转化胆固醇的菌株SE-1，对菌株进行形态、生理生化特征试验及16S rRNA基因序列同源性分析确定该菌株的系统发育学地位。发酵转化产物经氯仿萃取，对转化产物进行硅胶板薄层层析法分析，用硅胶柱层析法、Sephadex LH20分离产物，通过1H-NMR、13C-NMR分析确定转化产物的化学结构。对菌株转化胆固醇的发酵培养基的碳源、氮源、底物添加方式及发酵条件进行优化。【结果】菌株SE-1为革兰氏阴性菌，生理生化特征与洋葱伯克霍尔德氏菌(Burkholderia cepacia)相似，16S rRNA序列与洋葱伯克霍尔德氏菌(GenBank No．U96927)相似性为99%。硅胶薄层层析显示转化产物为两种产物。发酵转化时，在胆固醇-吐温乳化液的添加量为1 g/L，碳源糖蜜5%，氮源(NH4) 2 SO4 0.3%，接种量4%，发酵液pH7.5，36℃发酵的条件下，7β-羟基胆固醇的产率最高，达到34.4%。【结论】分离得到的菌株SE-1鉴定为Burkholderia cepacia。菌株SE-1转化胆固醇的主产物为7β-羟基胆固醇，次产物为7-酮基胆固醇，胆固醇7β-羟基化转化率在最适的转化条件下比优化前提高了20.8%。

Abstract:

Abstract:［Objective］Bacterial strain SE-1 capable of transforming cholesterol was isolated from soil and characterized．The transformation products were identified．Fermentation conditions were optimized for conversion.［Methods］Cholesterol was used as sole carbon source to isolate strain SE-1．Morphology， physiological and biochemical characteristics of strain SE-1 were studied．16S rRNA gene was sequenced and subjected to phylogenetic analysis．Fermentation supernatants were extracted with chloroform，the transformation products were analyzed by silica gel thin layer chromatography and Sephadex LH20．Their structures were identified by 1H-NMR and 13C-NMR．Fermentation medium including carbon and nitrogen，methods of adding substrates and fermentation conditions for Strain SE-1 were optimized．［Results］Strain SE-1 was a Gram-negative bacterium，exhibiting the highest homologs to Burkholderia cepacia based on the physiological analysis．The sequence analysis of 16S rＲNA gene of SE-1 strain and comparison with related Burkholderia show that SE-1 strain was very close to B．cepacia (Genbank No．U96927)．The similarity was 99%．The result of silica gel thin layer chromatography shows that strain SE-1 transformed cholesterol to two products，7β-hydroxycholesterol and the minor product was 7-oxocholesterol．The optimum culture conditions were: molasses 5%，(NH4) 2 SO4 0.3%，4% of inoculation，pH 7.5 and 36℃．Under the optimum culture condition，the conversion rate reached 34. 4% when concentration of cholesterol-Tween 80 was 1 g/L．Cholesterol 7β-hydroxylation conversion rate under optimal conditions was improved by 20.8%．［Conclusion］Strain SE-1 isolated from soil is capable of converting cholesterol at lab-scale．