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.
[1] HAN Y, KWON E Y, YU M K, et al.A preliminary study for evaluating the dose -dependent effect of D-allulose for fat mass reduction in adult humans:A randomized, double-blind, placebo-controlled trial[J].Nutrients, 2018, 10(2):160.
[2] LEE D, HAN Y, KWON E Y, et al.D-allulose ameliorates metabolic dysfunction in C57BL/KsJ-db/db mice[J].Molecules, 2020, 25(16):3656.
[3] HAYAKAWA M, HIRA T, NAKAMURA M, et al.Secretion of GLP-1 but not GIP is potently stimulated by luminal D-allulose (D-psicose) in rats[J].Biochemical and Biophysical Research Communications, 2018, 496:898-903.
[4] NAGATA Y, MIZUTA N, KANASAKI A, et al.Rare sugars, D-allulose, D-tagatose and D-sorbose, differently modulate lipid metabolism in rats[J].Journal of the Science of Food and Agriculture, 2018, 98(5):2020-2026.
[5] ATES E G, OZVURAL E B, OZTOP M H.Understanding the role of D-allulose and soy protein addition in pectin gels[J].Journal of Applied Polymer Science, 2021, 138(8): e49885.
[6] OGAWA M, INOUE M, HAYAKAWA S, et al.Effects of rare sugar D-allulose on heat-induced gelation of surimi prepared from marine fish[J].Journal of the Science of Food and Agriculture, 2017, 97(14): 5014-5020.
[7] XIA Y, CHENG Q Q, MU W M, et al.Research advances of D-allulose:An overview of physiological functions, enzymatic biotransformation technologies, and production processes[J].Foods, 2021, 10(9):2186.
[8] 李晓波. D-阿洛酮糖3-差向异构酶的表达及其固定化转化D-阿洛酮糖的研究[D].天津:天津科技大学, 2013.
LI X B.Expression and immobilization of D-psicose 3-epimerase and its application in the production of D-psicose[D].Tianjin:Tianjin University of Science and Technology, 2013.
[9] 王琪, 彭超, 周卫强, 等.新型甜味剂阿洛酮糖绿色生物制造工艺研究进展[J].生物化工, 2022, 8(3):170-176.
WANG Q, PENG C, ZHOU W Q, et al.Research progress on green bio-manufacturing technology of new sweetener D-psicose[J].Biological Chemical Engineering, 2022, 8(3):170-176.
[10] 胡梦莹. D-阿洛酮糖3-差向异构酶在枯草芽孢杆菌中的表达[D].无锡:江南大学, 2022.
HU M Y.Expression of D-psicose 3-epimerase in Bacillus subtilis[D].Wuxi:Jiangnan University, 2022.
[11] 李秋凤, 陈静, 赵婧邑, 等.D-阿洛酮糖3-差向异构酶在大肠杆菌内的高效可溶性表达及发酵条件研究[J].食品工业科技, 2022, 43(22):136-143.
LI Q F, CHEN J, ZHAO J Y, et al.Efficient soluble expression and fermentation conditions of D-allulose 3-epimerase in Escherichia coli[J].Science and Technology of Food Industry, 2022, 43(22):136-143.
[12] SHINTANI T, YAMADA T, HAYASHI N, et al.Rare sugar syrup containing D-allulose but not high-fructose corn syrup maintains glucose tolerance and insulin sensitivity partly via hepatic glucokinase translocation in wistar rats[J].Journal of Agricultural and Food Chemistry, 2017, 65(13):2888-2894.
[13] NAGATA Y, KANASAKI A, TAMARU S, et al.D-psicose, an epimer of D-fructose, favorably alters lipid metabolism in Sprague-Dawley rats[J].Journal of Agricultural and Food Chemistry, 2015, 63(12):3168-3176.
[14] HOSSAIN A, YAMAGUCHI F, MATSUO T, et al.Rare sugar D-allulose:Potential role and therapeutic monitoring in maintaining obesity and type 2 diabetes mellitus[J].Pharmacology & Therapeutics, 2015, 155:49-59.
[15] 沈佳颖, 李由然, 石贵阳.重组地衣芽孢杆菌全细胞转化产(R)-柠苹酸的研究[J].食品与发酵工业, 2023, 49(14):9-15.
SHEN J Y, LI Y R, SHI G Y.Whole-cell biosynthesis of (R)-Citramalate by recombinant Bacillus licheniformis[J].Food and Fermentation Industries, 2023, 49(14):9-15.
[16] 袁堂国, 李益民, 杜聪, 等.利用重组大肠杆菌和马克斯克鲁维酵母高效催化合成D-阿洛酮糖[J].食品与发酵工业, 2022, 48(1):15-20.
YUAN T G, LI Y M, DU C, et al.Efficient biocatalytic production of D-allulose combinate with Escherichia coli and Kluyveromyces marxianus[J].Food and Fermentation Industries, 2022, 48(1):15-20.
[17] 赵鑫馨, 李由然, 石贵阳.地衣芽孢杆菌强组成型启动子的鉴定及其表达效果验证[J].基因组学与应用生物学, 2021, 40(Z4):3566-3575.
ZHAO X X, LI Y R, SHI G Y.Identification and application of strong constitutive promoter of Bacillus licheniformis[J].Genomics and Applied Biology, 2021, 40(Z4):3566-3575.
[18] 温宇威. D-阿洛酮糖3-差向异构酶的异源表达及发酵优化[D].无锡:江南大学, 2016.
WEN Y W.Heterologous expression and fermentation optimization of D-psicose 3-epimerase[D].Wuxi:Jiangnan University, 2016.
[19] 朱星星. 马克斯克鲁维酵母D-阿洛酮糖-3-差向异构酶基因工程菌的构建[D].合肥:合肥工业大学, 2018.
ZHU X X.Construction of D-psicose 3-epimerase gene engineered Kluyveromyces marxianus[D].Hefei:Hefei University of Technology, 2018.
[20] PATEL S N, SHARMA M, LATA K, et al.Improved operational stability of D-psicose 3-epimerase by a novel protein engineering strategy, and D-psicose production from fruit and vegetable residues[J].Bioresource Technology, 2016, 216(1):121-127.
[21] CHEN J C, HE P, BAI H M, et al.Poly(β-cyclodextrin)/carbon quantum dots modified glassy carbon electrode:Preparation, characterization and simultaneous electrochemical determination of dopamine, uric acid and tryptophan[J].Sensors & Actuators B:Chemical, 2017, 252:9-16.
[22] 陆一鸣, 李由然, 许银彪, 等.启动子工程提高海藻糖生产用酶在地衣芽孢杆菌中的表达[J].基因组学与应用生物学, 2022, 41(8):1703-1712.
LU Y M, LI Y R, XU Y B, et al.Promoter engineering improves the expression of trehalose production enzyme in Bacillus licheniformis[J].Genomics and Applied Biology, 2022, 41(8):1703-1712.
