提出了一种高效、低成本的两阶段生产D-阿洛酮糖的生产工艺。第一阶段,构建异源表达Flavonifractor plautii D-阿洛酮糖3-差向异构酶的重组大肠杆菌用于转化果糖,实现D-阿洛酮糖的全细胞生物合成。重组大肠杆菌在pH 7.5、65 ℃条件下反应最优,且在不添加金属离子、60 ℃反应条件下半衰期达到2.7 h,具有高热稳定性。在添加干重2.4 g/L重组大肠杆菌的D-果糖纯水溶液中,产物D-阿洛酮糖的终质量浓度为231 g/L,转化率达到33%。在添加硼酸根离子的条件下,D-阿洛酮糖的终质量浓度为378 g/L,转化率可以达到63%。在第二阶段,利用马克斯克鲁维酵母消耗混合体系中的D-果糖生产乙醇,降低分离成本。在不添加任何营养物质的情况下,在2种不同体系内,D-果糖均可以完全被消耗并产出约0.4 g/g的乙醇。该文研究结果为D-阿洛酮糖的低成本工业化生产奠定了良好的基础。
An efficient and low cost process with two-stage for D-allulose production was proposed. In the first stage, Escherichia coli with the gene D-allulose 3-epimerase (DAEase) from Flavonifractor plautii were constructed. The conditions were optimized to pH 7.5, 65 ℃, and the cell was extremely thermo-stable with the half-life 2.7 h at 60 ℃ without metal ions. The final concentration of D-allulose was 231 g/L with the conversion rate at 33%. With boric acid added, the conversion rate was as high as 63% with 378 g/L D-allulose. In the second stage, the Kluyveromyces marxianus was used to consume the residual D-fructose. Without adding any nutrients, the D-fructose can be completely consumed in 18 hours and 0.4 g/g of ethanol can be produced. The results lay a good foundation for low cost industrial production of D-allulose.
[1] MATSUO T,IZUMORI K.Effects of dietary D-psicose on diurnal variation in plasma glucose and insulin concentrations of rats[J].Bioscience Biotechnology and Biochemistry,2006,70(9):2 081-2 085.
[2] DO G Y,KWON E Y,KIM Y J,et al.Supplementation of non-dairy creamer-enriched high-fat diet with D-allulose ameliorated blood glucose and body fat accumulation in C57BL/6 J mice[J].Applied Sciences,2019,9(13):2750.
[3] 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):2 888-2 894.
[4] HAN Y,HAN H J,KIM A H,et al.D-Allulose supplementation normalized the body weight and fat-pad mass in diet-induced obese mice via the regulation of lipid metabolism under isocaloric fed condition[J].Molecular Nutrition and Food Research,2016,60(7):1 695-1 706.
[5] AZAD M B,ABOU-SETTA A M,CHAUHAN B F,et al.Nonnutritive sweeteners and cardiometabolic health:A systematic review and meta-analysis of randomized controlled trials and prospective cohort studies[J].CMAJ,2017,189(28):E929-E939.
[6] ZHANG W L,YU S H,ZHANG T,et al.Recent advances in D-allulose:Physiological functionalities,applications,and biological production[J].Trends in Food Science and Technology,2016,54:127-137.
[7] LI X B,ZHU Y M,ZENG Y,et al.Overexpression of D-psicose 3-epimerase from Clostridium cellulolyticum H10 in Bacillus subtilis and its prospect for D-psicose production[J].Advance Journal of Food Science and Technology,2013,5(3):264-269.
[8] YANG J G,TIAN C Y,ZHANG T,et al.Development of food-grade expression system for D-allulose 3-epimerase preparation with tandem isoenzyme genes in Corynebacterium glutamicum and its application in conversion of cane molasses to D-allulose[J].Biotechnology and Bioengineering,2019,116(4):745-756.
[9] TSENG W C,CHEN C N,HSU C T,et al.Characterization of a recombinant D-allulose 3-epimerase from Agrobacterium sp. ATCC 31749 and identification of an important interfacial residue[J].International Journal of Biological Macromolecules,2018,112:767-774.
[10] LI C,ZHANG C J,LIN J Q,et al.Enzymatic fructose removal from D-psicose bioproduction model solution and the system modeling and simulation.Journal of Chemical Technology and Biotechnology,2017,93(5):1 249-1 260.
[11] 郭明,胡昌华.生物转化—从全细胞催化到代谢工程[J].中国生物工程杂志,2010,30(4):110－115.
GUO M,HU C H.Bioconversion-from whole cell biocatalysis to metabolic engineering[J].China Biotechnology,2010,30(4):110-115.
[12] PARK C S,KIM T,HONG S H,et al.D-allulose production from D-fructose by permeabilized recombinant cells of Corynebacterium glutamicum cells expressing D-allulose 3-epimerase Flavonifractor plautii[J].PLoS One,2016,11(7):e0160044.
[13] MEN Y,ZHU Y M,ZENG Y,et al.Co-expression of D-glucose isomerase and D-psicose 3-epimerase:Development of an efficient one-step production of D-psicose[J].Enzyme and Microbial Technology,2014,64-65:1-5.
[14] LI C,LIN J Q,GUO Q Q,et al.D-psicose 3-epimerase secretory overexpression,immobilization,and D-psicose biotransformation,separation and crystallization[J].Journal of Chemical Technology and Biotechnology,2017,93(2):350-357.
[15] 周锦怡, 武国庆,郭元亨,等.模拟移动床分离D-果糖与D-阿洛酮糖的色谱模型参数及传质系数测定[J].现代食品,2020,10(19):119-125.
ZHOU J Y,WU G Q,GUO Y H,et al.Chromatographic model parameters and mass transfer determination of D-fructose and D-psicose during the simulated moving bed separation process[J].Modern Food,2020,10(19):119-125.
[16] RAN G Q,TAN D,ZHAO J P,et al.Functionalized polyhydroxyalkanoate nano-beads as a stable biocatalyst for cost-effective production of the rare sugar D-allulose[J].Bioresource Technology,2019,289:121673.
[17] YANG P Z,ZHU X X,ZHENG Z,et al.Cell regeneration and cyclic catalysis of engineered Kluyveromyces marxianus of a D-psicose-3-epimerase gene from Agrobacterium tumefaciens for D-allulose production[J].World Journal of Microbiology and Biotechnology,2018,34(5):1-7.
[18] LIM B C,KIM H J,OH D K.A stable immobilized D-psicose 3-epimerase for the production of D-psicose in the presence of borate[J].Process Biochemistry,2009,44(8):822-828.
[19] KIM N H,KIM H J,KANG D I,et al.Conversion shift of D-fructose to D-psicose for enzyme-catalyzed epimerization by addition of borate[J].Applied and Environmental Microbiology,2008,74(10):3 008-3 013.
[20] SONG Y,NGUYEN Q A,WI S G,et al.Strategy for dual production of bioethanol and D-psicose as value-added products from cruciferous vegetable residue[J].Bioresource Technology,2017,223:34-39.
[21] 邢庆超, 沐万孟,江波,等.D-阿洛酮糖的分离纯化[J].食品工业科技,2011,32(9):236-238,242.
XING Q C,MU W M,JIANG B,et al.Separation and purification of D-psicose[J].Science and Technology of Food Industry,2011,32(9):236-238;242.
[22] 牛莉慧. 硫酸法制硼酸工艺原理及关键控制[J].盐科学与化工,2019,48(5):9-11.
NIU L H.Principle and key control of boric acid production by sulfuric acid method[J].Journal of Salt Science and Chemical Industry,2019,48(5):9-11.
