摘要
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OD_(600)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OD_(600)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 OD_(600) 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 OD_(600) 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.
作者
魏雨
李由然
石贵阳
WEI Yu;LI Youran;SHI Guiyang(National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2024年第16期1-9,共9页
Food and Fermentation Industries
基金
国家重点研发计划项目(2020YFA0907704)
国家自然科学基金项目(32172174)。
