D-allulose is a rare ketose found in nature, which has attracted significant attention for its similar sweetness to sucrose but lower caloric content.However, the efficient biosynthesis of D-allulose still poses a challenge, primarily attributed to the low thermostability of the ketose 3-epimerase utilized in production.In this study, the optimal mutant M03 with higher thermostability was developed by rational design, through the introduction of a disulfide bond and attribute grafting strategy.The Tm value of the best mutant M03 increased from 52.7 ℃ to 69.4 ℃.At 55 ℃, the half-life was extended to 254.3 min without metal ions, which was 54.1 times that of the wild-type.The detailed mechanism of performance improvement was analyzed by molecular dynamics simulation and structure analysis.This study provided a potential mutant strain for the industrial production of D-allulose.
GUO Dingyu
,
WEI Wanqing
,
SONG Wei
,
WU Jing
,
WEN Jian
,
HU Guipeng
,
LIU Liming
. Rational design and mechanism analysis of D-tagatose 3-epimerase[J]. Food and Fermentation Industries, 2025
, 51(4)
: 58
-64
.
DOI: 10.13995/j.cnki.11-1802/ts.038982
[1] FUKUNAGA K, YOSHIMURA T, IMACHI H, et al.A pilot study on the efficacy of a diabetic diet containing the rare sugar D-allulose in patients with type 2 diabetes mellitus:A prospective, randomized, single-blind, crossover study[J].Nutrients, 2023, 15(12):2802.
[2] PONGKAN W, JINAWONG K, PRATCHAYASAKUL W, et al.D-allulose provides cardioprotective effect by attenuating cardiac mitochondrial dysfunction in obesity-induced insulin-resistant rats[J].European Journal of Nutrition, 2021, 60(4):2047-2061.
[3] MATSUO T, TANAKA T, HASHIGUCHI M,et al.Metabolic effects of D-psicose in rats:Studies on faecal and urinary excretion and caecal fermentation[J].Asia Pacific Journal of Clinical Nutrition, 2003, 12(2):225-231.
[4] ZHANG X J, LI A, WANG Y, et al.D-Psicose intake exacerbates dextran sulfate sodium-induced colitis in mice through alteration in the gut microbiota and dysfunction of mucosal barrier[J].Food Science and Human Wellness, 2024, 13(1):173-182.
[5] ANDREANA P R, MCLELLAN J S, CHEN Y C, et al.Synthesis of 2,6-dideoxysugars via ring-closing olefinic metathesis[J].Organic Letters, 2002, 4(22):3875-3878.
[6] NORTHRUP A B, MACMILLAN D W C.Two-step synthesis of carbohydrates by selective aldol reactions[J].Science, 2004, 305(5691):1752-1755.
[7] DONER L W.Isomerization of D-fructose by base:Liquid-chromatographic evaluation and the isolation of D-psicose[J].Carbohydrate Research, 1979, 70 (2):209-216.
[8] IZUMORI K.Izumoring:A strategy for bioproduction of all hexoses[J].Journal of Biotechnology, 2006, 124(4):717-722.
[9] WANG H Y, CHEN J, ZHAO J Y, et al.Improved thermostability of D-allulose 3-epimerase from Clostridium bolteae ATCC BAA-613 by proline residue substitution[J].Protein Expression and Purification, 2022, 199:106145.
[10] ZHU Z L, LI L, ZHANG W, et al.Improving the enzyme property of D-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense through rational design[J].Enzyme and Microbial Technology, 2021, 149:109850.
[11] CHEN J J, CHEN D, CHEN Q M, et al.Computer-aided targeted mutagenesis of Thermoclostridium caenicola D-allulose 3-epimerase for improved thermostability[J].Journal of Agricultural and Food Chemistry, 2022, 70(6):1943-1951.
[12] BOSSHART A, PANKE S, BECHTOLD M.Systematic optimization of interface interactions increases the thermostability of a multimeric enzyme[J].Angewandte Chemie International Edition, 2013, 52(37):9673-9676.
[13] ZHANG W L, ZHANG Y M, HUANG J W, et al.Thermostability improvement of the D-allulose 3-epimerase from Dorea sp.CAG317 by site-directed mutagenesis at the interface regions[J].Journal of Agricultural and Food Chemistry, 2018, 66(22):5593-5601.
[14] ZHU Z L, GAO D K, LI C, et al.Redesign of a novel D-allulose 3-epimerase from Staphylococcus aureus for thermostability and efficient biocatalytic production of D-allulose[J].Microbial Cell Factories, 2019, 18(1):59.
[15] MAO S H, CHENG X T, ZHU Z L, et al.Engineering a thermostable version of D-allulose 3-epimerase from Rhodopirellula baltica via site-directed mutagenesis based on B-factors analysis[J].Enzyme and Microbial Technology, 2020, 132:109441.
[16] CASE D A, BETZ R M, CERUTTI D S, et al.Amber 16[M].San Francisco:University of California, 2016.
[17] JAKALIAN A, BUSH B L, JACK D B, et al. Fast, efficient generation of high-quality atomic charges. AM1-BCC model: I. Method[J]. Journal of Computational Chemistry, 2000, 21(2): 132-146.
[18] JAKALIAN A, JACK D B, BAYLY C I.Fast, efficient generation of high-quality atomic charges.AM1-BCC model:Ⅱ.Parameterization and validation[J].Journal of Computational Chemistry, 2002, 23(16):1623-1641.
[19] WANG J M, WOLF R M, CALDWELL J W, et al.Development and testing of a general amber force field[J].Journal of Computational Chemistry 2004, 25(9):1157-1174.
[20] JORGENSEN W L, CHANDRASEKHAR J, MADURA J D, et al.Comparison of simple potential functions for simulating liquid water[J].The Journal of Chemical Physics, 1983, 79(2):926-935.
[21] DARDEN T, YORK D, PEDERSEN L.Particle mesh Ewald:An N.log(N) method for Ewald sums in large systems[J].The Journal of Chemical Physics, 1993, 98(12):10089-10092.
[22] TSENG W C, HSU C T, CHANG H C, et al.Fusion of the peptide derived from the acidic tail of alpha-synuclein improves the thermostability and soluble expression of recombinant Agrobacterium sp.D-allulose 3-epimerase[J].Biochemical Engineering Journal, 2021, 165:107828.
