This work focused on the immobilization of recombinant Bacillus subtilis expressing D-allulose 3-epimerase (DAE) by using zeolitic imidazolate framework-8 (ZIF-8) as a coating material.The composite B.subtilis-DAE@ZIF-8 was prepared by co-precipitating with zinc acetate, 2-methylimidazole, and recombinant B.subtilis expressing DAE, and the structure of ZIF-8 coated cells was proved by the analysis by scanning electron microscope, Fourier transform infrared spectroscopy, and X-ray diffraction.To improve the catalytic activity of B.subtilis-DAE@ZIF-8, orthogonal experiments were used to study the coprecipitation time, cell concentration, and the ratio of Zn2+ and 2-methylimidazole, and the optimum preparing condition involved a Zn2+∶2-methylimidazole ratio of 1∶4, a bacterial cell concentration of 15 g/L, and a coprecipitation time of 1 h at 4 ℃.By preparing B.subtilis-DAE@ZIF-8 at this condition, the enzyme activity reached the highest value of 344.81 U/g, with a recovery rate of 77.75%, the optimal reaction temperature reached 60 ℃ (5 ℃ higher than free cells) and the optimal pH changed to 7.5.At 60 ℃, the half-life of B.subtilis-DAE@ZIF-8 was 10.75 h, increased by 46.46% compared to free cells.Notably, after 10 cycles of reaction, B.subtilis-DAE@ZIF-8 maintained 45.67% of its initial activity and a D-allulose conversion rate of 20.07%, whereas free cells lost almost all activity.This ZIF-8 immobilization technique significantly improves the biocatalysis stability and reusability of DAE-expressing recombinant cells and provides a reference for efficient D-allulose production.
[1] 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, 216:121-127.
[2] ZHANG W L, CHEN D, CHEN J J, et al.D-allulose, a versatile rare sugar:Recent biotechnological advances and challenges[J].Critical Reviews in Food Science and Nutrition, 2023,63(22):5661-5679.
[3] HOSSAIN M A, KITAGAKI S, NAKANO D, et al.Rare sugar D-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats[J].Biochemical and Biophysical Research Communications, 2011, 405(1):7-12.
[4] WEN X, LIN H B, TESFAY M A, et al.Bioproduction of high-allulose-fructose syrup from D-glucose catalyzed by recombinant E.coli co-expressing glucose isomerase and D-allulose 3-epimerase[J].Food Bioscience, 2023, 56:103222.
[5] GUAN L J, ZHU L, WANG K L, et al.Biochemical characterization, structure-guided mutagenesis, and application of a recombinant D-allulose 3-epimerase from Christensenellaceae bacterium for the biocatalytic production of D-allulose[J].Frontiers in Bioengineering and Biotechnology, 2024, 12:1365814.
[6] WANG Q Y, WANG H Y, ZHANG W G, et al.Economical one-pot synthesis of isoquercetin and D-allulose from quercetin and sucrose using whole-cell biocatalyst[J].Enzyme and Microbial Technology, 2024, 176:110412.
[7] 孙帆, 宿玲恰, 张康, 等.D-阿洛酮糖3-差向异构酶在枯草芽孢杆菌中的高效表达及固定化细胞研究[J].中国生物工程杂志, 2018, 38(7):83-88.
SUN F, SU L Q, ZHANG K, et al.D-psicose 3-epimerase gene overexpression in Bacillus subtilis and immobilization of cells[J].China Biotechnology, 2018, 38(7):83-88.
[8] 孙帆. 解纤维梭菌D-阿洛酮糖3-差向异构酶在枯草芽孢杆菌中的重组表达、应用及固定化研究[D]. 无锡: 江南大学, 2018.
SUN F. Recombinant expression, application and immobilization of Clostridium cellulosum D- psicose 3- epimerase in Bacillus subtilis[D]. Wuxi: Jiangnan University, 2018.
[9] 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. Study on the expression of D-psicose 3-epimerase and its immobilized transformation into D-psicose[D]. Tianjin: Tianjin University of Science & Technology, 2013.
[10] 刘亚, 王雨, 陈莹莹, 等. MOF-5吸附姜黄素及在胃肠中的消化研究[J]. 食品科学技术学报, 2017, 35(5):46-52.
LIU Y, WANG Y, CHEN Y Y, et al. Study on MOF-5 adsorption curcumin and digestion in gastrointestinal tract[J]. Journal of Food Science and Technology, 2017, 35(5):46-52.
[11] LEE Y R, JANG M S, CHO H Y, et al.ZIF-8:A comparison of synthesis methods[J].Chemical Engineering Journal, 2015, 271:276-280.
[12] SADEGH N, DEHCHESHMEH I M, SADEGH F.Review of zeolitic imidazolate framework/graphene oxide:A synergy of synthesis, properties and function for multifaceted applications in nanotechnology[J].FlatChem, 2024, 44:100618.
[13] 张营康,程婷,赵飞扬,等.基于金属有机沸石咪唑骨架的固定化细胞的制备及其在丙谷二肽制备中的应用[J].生物工程学报, 2023, 39(3):1131-1141.
ZHANG Y K, CHENG T, ZHAO F Y, et al.Immobilizing engineered Escherichia coli cells into zeolitic imidazolate framework 8 for efficient biosynthesis of Ala-Gln[J].Chinese Journal of Biotechnology, 2023,39(3):1131-1141.
[14] TIAN F C, ZHOU Y M, MA Z Q, et al.Organismal function enhancement through biomaterial intervention[J].Nanomaterials, 2024, 14(4):377.
[15] 梁鑫, 张成楠, 周威, 等.ZIF-8原位自封装固定化脂肪酶的研究[J].食品研究与开发, 2021, 42(5):1-8.
LIANG X, ZHANG C N, ZHOU W, et al.Study on immobilization of lipase by in-situ encapsulation in ZIF-8[J].Food Research and Development, 2021, 42(5):1-8.
[16] 芦瑜涵, 张成楠, 李秀婷, 等.基于无定形沸石-咪唑框架-8材料固定米根霉源脂肪酶研究[J].食品科学技术学报, 2023, 41(6):39-51.
LU Y H, ZHANG C N, LI X T, et al.Immobilization of lipase derived from Rhizopus oryzae based on amorphous zeolitic imidazolate framework-8 material[J].Journal of Food Science and Technology, 2023, 41(6):39-51.
[17] ATIROĞLU V, ATIROĞLU A, MAHMUT Ö.Immobilization of α-amylase enzyme on a protein @metal-organic framework nanocomposite:A new strategy to develop the reusability and stability of the enzyme[J].Food Chemistry, 2021, 349:129127.
[18] LYU F J, ZHANG Y F, ZARE R N, et al.One-pot synthesis of protein-embedded metal-organic frameworks with enhanced biological activities[J].Nano Letters, 2014, 14(10):5761-5765.
[19] HOWARTH A J, PETERS A W, VERMEULEN N A, et al.Best practices for the synthesis, activation, and characterization of metal-organic frameworks[J].Chemistry of Materials, 2017, 29(1):26-39.
[20] VAIDYA L B, NADAR S S, RATHOD V K.Entrapment of surfactant modified lipase within zeolitic imidazolate framework (ZIF)-8[J].International Journal of Biological Macromolecules, 2020, 146:678-686.
[21] SALIBA D, AMMAR M, RAMMAL M, et al.Crystal growth of ZIF-8, ZIF-67, and their mixed-metal derivatives[J].Journal of the American Chemical Society, 2018, 140(5):1812-1823.
[22] HOU S Y, ZHANG M, HOU Y Q, et al.Mechanistic insight into the removal of aqueous Cd using an immobilized ZIF-8 and microflora cooperative composite[J].Chemosphere, 2022, 293:133582.
[23] PANG S H, HAN C, SHOLL D S, et al.Facet-specific stability of ZIF-8 in the presence of acid gases dissolved in aqueous solutions[J].Chemistry of Materials, 2016, 28(19):6960-6967.
[24] ZHANG W L, CHEN D, CHEN J J, et al.D-allulose, a versatile rare sugar:Recent biotechnological advances and challenges[J].Critical Reviews in Food Science and Nutrition, 2023,63(22):5661-5679.
[25] 黄月园,徐铮,徐虹.生物法生产D-阿洛酮糖的研究进展[J].生物加工过程,2021,19(6):581-588.
HUANG Y Y, XU Z, XU H.Progress in biological production of D-psicose[J].Chinese Journal of Bioprocess Engineering, 2021, 19(6):581-588.
[26] FU G, ZHANG S B, DONG H N, et al.Enhanced production of D-psicose 3-epimerase in Bacillus subtilis by regulation of segmented fermentation[J].Biotechnology and Applied Biochemistry, 2020, 67(5):812-818.
