该研究首次在地衣芽孢杆菌中异源表达溶纤瘤胃杆菌H10来源的D-阿洛酮糖3-差向异构酶(D-psicose 3-epimerase,DPE),开拓D-阿洛酮糖生物合成法又一新的表达载体途径。首先,选取不同启动子介导表达该酶,选取表达效果最好的重组菌BL1进行全细胞转化条件和发酵条件的优化。利用优化后的重组菌全细胞转化条件,65 ℃、反应体系细胞OD600值为2、反应10 min、底物D-果糖100 g/L探索出发酵培养条件,碳源为蔗糖75 g/L、初始pH 7.5、37 ℃,最终利用全细胞转化方法,65 ℃、500 g/L D-果糖、反应体系细胞OD600值为30、反应20 min,转化率达30.3%,D-阿洛酮糖产量可达120 g/L,单位酶活力达到33.3 U/mL。采用分4次添加与果糖摩尔质量比为0.4的硼酸至反应体系中的方法,将转化率提高至69.8%。该研究为工业生产D-阿洛酮糖提供一定的参考价值。
D-allulose (D-psicose) is a low-calorie functional sugar, which has many functions such as sucrose substitute, participating in Maillard reaction, improving food gel process, etc.One of the methods of preparing D-allulose, biosynthesis, has many advantages such as simple purification steps, high product concentration, good environmental compatibility, etc.Therefore, the preparation of D-allulose by biosynthesis has become a research hotspot.In this study, Bacillus licheniformis is used to heterologously express D-psicose 3-epimerase (DPEase) from Ruminiclostridium cellulolyticum H10 for the first time, which opens up a new expression vector pathway for D-allulose biosynthesis.Firstly, different promoters were selected to mediate the expression of DPEase, and the recombinant strain BL1 with the best expression effect was selected to optimize the whole-cell transformation and fermentation conditions.The optimized recombinant bacterial whole-cell transformation conditions were that the temperature was 65 ℃, the cell OD600 of the reaction system was 2, the reaction time was 10 minutes, and the substrate D-fructose was 100 g/L, which was utilized to explore the fermentation medium conditions, which was that the carbon source was 75 g/L sucrose, the initial pH was 7.5, and the temperature was 37 ℃.Finally, the whole-cell biosynthesis transformation method was used, which was that the temperature was 65 ℃, D-fructose was 500 g/L, the cell OD600 was 30, and the reaction time was 20 minutes, the conversion rate was achieved at 30.3%, the D-allulose was converted to 120 g/L, and unit enzyme activity reached 33.3 U/mL.The conversion rate was increased to 69.8% by adding boric acid with a molar mass ratio of 0.4 to fructose in four stages of the reaction system.This study provides a certain reference value for the industrial production of D-allulose.
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