糖磷酸酶的挖掘及其酶学性质研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0095

摘要/Abstract

摘要：

【目的】 在以淀粉或糊精为底物合成具有高附加值的单糖的多酶级联反应中，利用糖磷酸酶催化发生不可逆的脱磷反应可以高效拉动整个反应朝产物生成的方向进行。本研究旨在挖掘新的糖磷酸酶并对酶学性质进行鉴定。【方法】 通过基因挖掘的手段筛选得到一种来源于嗜热菌Thermoproteus sp.CIS_19的未知功能的基因TsPase具有糖磷酸酶活性，在E. coli BL21（DE3）中实现异源可溶性表达及纯化，进一步对其酶学性质进行表征。【结果】 最终确定糖磷酸酶TsPase的最适反应温度是70℃，T_m值为（80.3 ± 1.3）℃，最适反应pH为4，金属离子Mg²⁺对TsPase有较强的促进作用。针对底物为塔格糖6磷酸盐的动力学常数K_m为（2.40 ± 0.98）mmol/L，催化常数k_cat为（102.50 ± 8.60）min^-1。将TsPase应用于体外多酶合成体系中，以10 g/L麦芽糊精为底物，一锅法生产D-塔格糖，反应平衡体系中D-塔格糖产量为（4.26 ± 0.03）g/L，转化率可达42.6% ± 0.3%。【结论】 TsPase不仅具有较好的热稳定性和活性，在体外多酶合成体系中也具有优势，这些特征在以后的理论研究及工业生产中具有一定的科学价值。

关键词: 糖磷酸酶, 酶学特性, 热稳定性, 基因挖掘

Abstract:

【Objective】 High value-added monosaccharides can be synthesized through multi-enzyme cascade reactions using starch or dextrin as substrates. In the reaction systems, sugar phosphatase catalyzes the irreversible dephosphorylation reaction of the phosphorylated sugar intermediates to generate the corresponding sugars. This work is aimed to mine the new sugar phosphatase and to characterize this enzyme. 【Method】 In this study, a gene with unknown function, named as TsPase derived from the thermophilic bacterium Thermoproteus sp.CIS_19, was obtained from gene mining and screening. TsPase was found to have sugar phosphatase activity after its heterologous soluble expression with Escherichia coli BL21（DE3）, purification and enzymatic characterization. 【Result】 TsPase has a T_m value of（80.3 ± 1.3）℃, while the optimal reaction temperature of 70℃ and pH of 4. Our study indicated that metal ion Mg²⁺ has a strong promoting effect on TsPase. With the substrate tagatose 6 phosphate kinetic constant K_m is（2.40 ± 0.98）mmol/L, and the catalytic constant k_cat is（102.50 ± 8.60）min^-1. TsPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D-tagatose from maltodextrin. The production of D-tagatose in the reaction equilibrium system was（4.26 ± 0.03）g/L and the conversion rate reach 42.6% ± 0.3%. 【Conclusion】 Overall, TsPase possesses fine thermal stability and activity, as well as high conversation rate in vitro synthetic enzymatic biosystem. These characteristics make it great value in future theoretical research and industrial production.

Key words: sugar phosphatase, enzymatic characterization, thermal stability, gene mining

乔烨, 张楠, 杨建花, 张翠英, 朱蕾蕾. 糖磷酸酶的挖掘及其酶学性质研究[J]. 生物技术通报, 2024, 40(7): 299-306.

QIAO Ye, ZHANG Nan, YANG Jian-hua, ZHANG Cui-ying, ZHU Lei-lei. Identification and Enzymatic Characterization of a Sugar Phosphatase[J]. Biotechnology Bulletin, 2024, 40(7): 299-306.

图/表 5

图1 以AfPase为模板构建系统发育树

Fig. 1 Phylogenetic tree constructed using AfPase as a template

图2 SDS-PAGE分析TsPase的表达（A）和纯化（B）（A）1：pET28a（+）粗酶上清；2：TsPase粗酶上清；3：蛋白标准。（B）1：纯化后的TsPase；2：蛋白标准

Fig. 2 SDS-PAGE analysis of expression（A）and purification（B）of TsPase （A）1: The crude supernatant of pET28a（+）; 2: the crude supernatant of TsPase; 3: protein marker.（B）1: The purified enzyme of TsPase; 2: protein marker

图3 不同底物时TsPase催化活力测定

Fig. 3 Determination of catalytic activity of TsPase with different substrates

图4 TsPase酶学性质检测 A：TsPase催化活力随反应温度变化曲线；B：TsPase催化活力随pH变化的曲线；C：TsPase催化活力随不同金属离子的变化；D：TsPase催化活力随Mg2+浓度的变化

Fig. 4 Enzymatic properties of TsPase A: Catalytic activity curve of TsPase with different temperature. B: pH Curve of TsPase. C: Effect of different metal ions on the activity of TsPase. D: The catalytic activity of TsPase with different concentration of Mg2+

图5 通过体外多酶合成体系从麦芽糖糊精生产D-塔格糖

Fig. 5 D-tagatose bioproduction from maltodextrin by an in vitro synthetic enzymatic biosystem

参考文献 30

[1]	Ahmed A, Khan TA, Dan Ramdath D, et al. Rare sugars and their health effects in humans: a systematic review and narrative synthesis of the evidence from human trials[J]. Nutr Rev, 2022, 80(2): 255-270.
[2]	Li YJ, Shi T, Han PP, et al. Thermodynamics-driven production of value-added D-allulose from inexpensive starch by an in vitro enzymatic synthetic biosystem[J]. ACS Catal, 2021, 11(9): 5088-5099.
[3]	Chahed A, Nesler A, Aziz A, et al. A review of knowledge on the mechanisms of action of the rare sugar D-tagatose against phytopathogenic oomycetes[J]. Plant Pathol, 2021, 70(9): 1979-1986.
[4]	Campbell HR, Alsharif FM, Marsac PJ, et al. The development of a novel pharmaceutical formulation of D-tagatose for spray-drying[J]. J Pharm Innov, 2022, 17(1): 194-206.
[5]	Philippe RN, De Mey M, Anderson J, et al. Biotechnological production of natural zero-calorie sweeteners[J]. Curr Opin Biotechnol, 2014, 26: 155-161.
[6]	Howling D, Callagan JL. RTE cereals and other foods presweetened with D tagatose: US6475540[P]. 2002-11-05.
[7]	Yang JG, Zhang T, Tian CY, et al. Multi-enzyme systems and recombinant cells for synthesis of valuable saccharides: advances and perspectives[J]. Biotechnol Adv, 2019, 37(7): 107406.
[8]	Li ZJ, Gao YH, Nakanishi H, et al. Biosynthesis of rare hexoses using microorganisms and related enzymes[J]. Beilstein J Org Chem, 2013, 9: 2434-2445. doi: 10.3762/bjoc.9.281 pmid: 24367410
[9]	Beerens K, Desmet T, Soetaert W. Enzymes for the biocatalytic production of rare sugars[J]. J Ind Microbiol Biotechnol, 2012, 39(6): 823-834.
[10]	Tian CY, Yang JG, Liu C, et al. Engineering substrate specificity of HAD phosphatases and multienzyme systems development for the thermodynamic-driven manufacturing sugars[J]. Nat Commun, 2022, 13(1): 3582.
[11]	Jia DX, Zhou L, Zheng YG. Properties of a novel thermostable glucose isomerase mined from Thermus oshimai and its application to preparation of high fructose corn syrup[J]. Enzyme Microb Technol, 2017, 99: 1-8.
[12]	Saburi W, Jaito N, Kato K, et al. Biochemical and structural characterization of Marinomonas mediterranea D-mannose isomerase Marme_2490 phylogenetically distant from known enzymes[J]. Biochimie, 2018, 144: 63-73.
[13]	Yang JG, Tian CY, 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]. Biotechnol Bioeng, 2019, 116(4): 745-756.
[14]	Meng DD, Liang AL, Wei XL, et al. Enzymatic characterization of a thermostable phosphatase from Thermomicrobium roseum and its application for biosynthesis of fructose from maltodextrin[J]. Appl Microbiol Biotechnol, 2019, 103(15): 6129-6139.
[15]	Pathira Kankanamge LS, Ruffner LA, Touch MM, et al. Functional annotation of haloacid dehalogenase superfamily structural genomics proteins[J]. Biochem J, 2023, 480(19): 1553-1569.
[16]	Allen KN, Dunaway-Mariano D. Markers of fitness in a successful enzyme superfamily[J]. Curr Opin Struct Biol, 2009, 19(6): 658-665.
[17]	Burroughs AM, Allen KN, Dunaway-Mariano D, et al. Evolutionary genomics of the HAD superfamily: understanding the structural adaptations and catalytic diversity in a superfamily of phosphoesterases and allied enzymes[J]. J Mol Biol, 2006, 361(5): 1003-1034. doi: 10.1016/j.jmb.2006.06.049 pmid: 16889794
[18]	Lahiri SD, Zhang GF, Dai JY, et al. Analysis of the substrate specificity loop of the HAD superfamily cap domain[J]. Biochemistry, 2004, 43(10): 2812-2820. pmid: 15005616
[19]	Kuznetsova E, Nocek B, Brown G, et al. Functional diversity of haloacid dehalogenase superfamily phosphatases from Saccharomyces cerevisiae: biochemical, structural, and evolutionary insights[J]. J Biol Chem, 2015, 290(30): 18678-18698. doi: 10.1074/jbc.M115.657916 pmid: 26071590
[20]	Huang H, Pandya C, Liu CL, et al. Panoramic view of a superfamily of phosphatases through substrate profiling[J]. Proc Natl Acad Sci USA, 2015, 112(16): E1974-E1983.
[21]	Hu CC, Wei XL, Song YH. A thermophilic phosphatase from Methanothermobacter marburgensis and its application to in vitro biosynthesis[J]. Enzyme Microb Technol, 2022, 159: 110067.
[22]	马延和, 孙媛霞, 其他发明人请求不公开姓名. 塔格糖的制备方法.天津市:CN106399427B[P]. 2018-11-13.
	Ma YH, Sun YX and the other inventors asked not to be named. Method for preparing tagatose. Tianjin: CN106399427B[P]. 2018-11-13.
[23]	徐培娟, 刘晶晶. 塔格糖(tagatose)的生产及应用研究现状[J]. 中国食品添加剂, 2007(6): 79-82.
	Xu PJ, Liu JJ. Recent research on manufacturing and application of tagatose[J]. China Food Addit, 2007(6): 79-82.
[24]	Dai YW, Zhang JX, Zhang T, et al. Characteristics of a fructose 6-phosphate 4-epimerase from Caldilinea aerophila DSM 14535 and its application for biosynthesis of tagatose[J]. Enzyme Microb Technol, 2020, 139: 109594.
[25]	温鑫, 刘光文, 宁宇航, 等. D-塔格糖生物合成研究进展[J]. 食品与发酵工业, 2023, 49(22): 326-333. doi: 10.13995/j.cnki.11-1802/ts.035016
	Wen X, Liu GW, Ning YH, et al. Research progress for biosynthesis of D-tagatose[J]. Food Ferment Ind, 2023, 49(22): 326-333.
[26]	Dai YW, Zhang T, Jiang B, et al. Dictyoglomus turgidum DSM 6724 α-glucan phosphorylase: characterization and its application in multi-enzyme cascade reaction for D-tagatose production[J]. Appl Biochem Biotechnol, 2021, 193(11): 3719-3731.
[27]	You C, Percival Zhang YH. Biomanufacturing by in vitro biosystems containing complex enzyme mixtures[J]. Process Biochem, 2017, 52: 106-114.
[28]	Ye XT, Honda K, Sakai T, et al. Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway[J]. Microb Cell Fact, 2012, 11: 120. doi: 10.1186/1475-2859-11-120 pmid: 22950411
[29]	Wang W, Meng DD, Li QZ, et al. Characterization of a hyperthermophilic phosphatase from Archaeoglobus fulgidus and its application in in vitro synthetic enzymatic biosystem[J]. Bioresour Bioprocess, 2019, 6(1): 22.
[30]	Wei X, Li Q, Hu C, et al. An ATP-free in vitro synthetic enzymatic biosystem facilitating one-pot stoichiometric conversion of starch to mannitol[J]. Appl Microbiol Biotechnol, 2021, 105(5): 1913-1924.