红河传统发酵豆豉中高纤溶活性菌株的分离筛选及其纤溶酶基因分析

Screening of High-yield Fibrinolytic Enzyme-producing Strains from Traditional Fermented Douchi (Honghe, Yunnan) and Analysis of the Gene Encoding the Fibrinolytic Enzyme

摘要:

运用脱脂乳固体培养基及纤维蛋白固体培养基的两步分离筛选法，从云南红河传统发酵豆豉中分离筛选具有高产豆豉纤溶酶活性的菌株，同时对它们的豆豉纤溶酶基因进行克隆分析，以期为新型功能性豆豉的研发提供备选菌株及理论依据。研究结果表明，两步分离筛选法能有效地从云南红河传统发酵豆豉中筛选到高产豆豉纤溶酶的菌株Bacillus subtilis LC-2-1，对该高产菌株的豆豉纤溶酶成熟肽基因分析及预测结果表明，菌株B subtilis LC-2-1确实能分泌一种由825个碱基编码275个氨基酸残基且分子量约为27.4 kDa的豆豉纤溶酶，与纳豆激酶及其他豆豉纤溶酶相比差异显著，同源性仅为85.1%。同时其豆豉纤溶酶活性分析结果则表明，菌株B subtilis LC-2-1所产纤溶酶活性较高，可达79.84 U/mL。因此，本研究结果将为新型且具有溶血栓功能发酵豆豉的研发提供备选菌株及理论依据。

Abstract:

High-yield fibrinolytic enzyme-producing bacterial strains were isolated and screened from traditionally fermented douchi from Honghe, by a two-step screening method, in which skim milk agar and fibrin agar were used. The bacterial gene encoding the fibrinolytic enzyme was cloned and analyzed to provide bacterial reference strains and a theoretical basis to develop a new version of functional douchi. Using these methods, a high-yield fibrinolytic enzyme-producing strain Bacillus subtilis LC-2-1 was successfully isolated, which secreted a kind of douchi fibrinolytic enzyme. The molecular mass of the enzyme was 27.4 kDa that contained 275 amino acids encoded by 825-bp. Compared to other douchi fibrinolytic enzymes and nattokinase, the enzyme produced by B. subtilis LC-2-1 showed significant differences, and the amino acid sequence homology was 85.1%. Additionally, the enzyme had high fibrinolytic activity, which was up to 79.84 U/mL. Thus, these results could provide a bacterial reference strain and theoretical basis to develop a new version of fermented douchi with thrombolytic activity.