产L-天冬氨酸α-脱羧酶细菌的分离、鉴定及发酵条件优化

Isolation, identification and fermentation optimization of Bacillus tequilensis PanD37 producing L-aspartate α-decarboxylase

摘要:

【目的】从葡萄园土壤中分离L-天冬氨酸α-脱羧酶的产生菌株,对其进行分类鉴定,优化其产生L-天冬氨酸α-脱羧酶的发酵条件,为β-丙氨酸的生物合成提供基础。【方法】采用变色圈法和液体复筛培养基分离筛选具有L-天冬氨酸α-脱羧酶活力的菌株,对菌株进行形态、生理生化特征试验及16SrRNA序列同源性分析鉴定菌株的系统发育学地位,采用单因素及正交设计试验优化培养基及发酵条件。【结果】筛选到一株L-天冬氨酸α-脱羧酶高产菌株PanD37,其亲缘关系和特基拉芽孢杆菌(Bacillustequilensis)较近,且形态与培养特征、生理生化特性与特基拉芽孢杆菌基本相符。研究表明其最佳发酵配方和培养条件为:蔗糖22.5 g/L、富马酸7.5 g/L、蛋白胨20 g/L、L-天冬氨酸6 g/L、Triton X-1002g/L,起始pH为7.0,装液量50 mL/500 mL,摇床转速220 r/min,种子液接种量为5%(V/V), 35℃培养28h。在最优条件下L-天冬氨酸α-脱羧酶活力可达44.57 U/mL,比初筛时提高2.57倍。【结论】分离并获得一株特基拉芽孢杆菌(Bacillus tequilensis) PanD37,经条件优化后具有较高的L-天冬氨酸α-脱羧酶产生能力,有望应用于β-丙氨酸的工业生产。

Abstract:

[Objective] We screened bacteria producing L-aspartate α-decarboxylase from grapery soil and optimized the fermentation conditions.[Methods] L-aspartate α-decarboxylase producing bacteria were screened by color-changing circle and liquid secondary screening culture media. Combination of morphological, physiological and biochemical characteristics and 16S rRNA sequence analysis were used to identify the bacteria. Fermentation conditions were optimized by single factor test and orthogonal experiment.[Results] Strain PanD37 showed high L-aspartate α-decarboxylase producing property and was identified as Bacillus tequilensis. The optimum fermentation conditions of PanD37 were liquid volume of 50 mL in 500 mL flask, 220 r/min at 35℃, inoculation amount of 5% for 28 h with a medium of 22.5 g/L sucrose, 7.5 g/L fumaric acid, 20 g/L peptone, 6 g/L L-aspartic acid, 2 g/L Triton X-100, at initial pH of 7.0. Under the optimal fermentation conditions, the highest L-aspartate α-decarboxylase activity reached 44.57 U/mL, which was 2.57 folds higher than that obtained before optimization.[Conclusion] Strain PanD37 was identified as Bacillus tequilensiswhich was capable of highly producing L-aspartate α-decarboxylase under the optimal fermentation conditions.