Caulobactercrescentus蔗糖水解酶受体亚位点分子改造及其在松二糖制备中的应用

doi:10.12211/2096-8280.2021-047

摘要/Abstract

摘要：

松二糖是由葡萄糖与果糖以α-1，3糖苷键连接而成的还原性二糖，具有代替蔗糖成为新型功能性甜味剂的潜力，在食品工业中应用前景广阔。淀粉蔗糖酶能够以蔗糖为底物催化异构（分子内转苷）反应制备松二糖，产率高但易产生副产物麦芽寡糖和海藻酮糖。为解决这一问题，选用前期获得的松二糖产率高并且不产副产物麦芽寡糖的Caulobactercrescentus蔗糖水解酶突变体S271A为研究对象，进一步通过受体亚位点分子改造，获得了反应特异性和松二糖产率提升的突变体S271A/I382Q。在此基础上进行了酶转化条件优化，当以2 mol/L蔗糖溶液为底物，加酶量为40 U/mL，在pH 5.0、30 ℃的条件下，松二糖的产率达到最高为70.3%，松二糖的浓度为480 g/L，并且反应产物中不含副产物海藻酮糖。分子动力学模拟表明，突变体S271A/I382Q可通过氢键相互作用稳定受体果糖参与形成α-1，3糖苷键时的构象，从而更有利于生成松二糖。本研究创新性地将蔗糖水解酶改造为键型特异性强的转苷酶，获得的松二糖产率为目前报道的最高水平，为松二糖的规模化制备与应用奠定了理论和技术基础。

关键词: 松二糖, 蔗糖, 海藻酮糖, 蔗糖水解酶, 突变体, 酶转化

Abstract:

Turanose is a reductive disaccharide which is made from glucose and fructose through the formation of α-1,3 glycosidic bond, and has a potential to replace sucrose as a new functional sweetener with a broad application in food industry. Turanose can also be produced from sucrose with high yield through the isomerization (intramolecular transglycosidation) reaction catalyzed by amylosucrases. However, by-products, maltooligosaccharide and trehalulose, are easily produced in this process. In order to solve this problem, based on previously identified sucrose hydrolase mutant S271A from Caulobacter crescentus for the isomerization reaction with high turanose yield but without the formation of the by-product maltooligosaccharide, we further developed the mutant S271A/I382Q with improved reaction specificity and increased yield of turanose through the molecular modification of the acceptor subsite. Furthermore, conditions of the enzymatic conversion were optimized. When the reaction was performed under its optimal conditions: 2 mol/L sucrose as substrate with 40 U/mL enzyme dosage under pH 5.0 and 30 ℃, the yield of turanose could reach up to 70.3% and its final concentration was 480 g/L. Most significantly, no the by-product trehalulose was detected. Molecular dynamics simulations show that the mutant S271A/I382Q could stabilize the conformation of α-1,3 glycosidic bond formed by the acceptor fructose through hydrogen bond interactions, making it more conducive to the formation of turanose. This study innovates the transformation of sucrose hydrolase into transglucosidase with high reaction specificity, and the production yield of turanose is the highest reported at present, which lays a theoretical and technical foundation for the large-scale production and application of turanose.

Key words: turanose, sucrose, trehalulose, sucrose hydrolase, mutant, enzymatic conversion

中图分类号:

Q814

王蕾, 邢晨晨, 郭志勇, 宿玲恰, 吴敬. Caulobactercrescentus蔗糖水解酶受体亚位点分子改造及其在松二糖制备中的应用[J]. 合成生物学, 2022, 3(3): 602-615.

Lei WANG, Chenchen XING, Zhiyong GUO, Lingqia SU, Jing WU. Molecular modification of acceptor subsite in sucrose hydrolase from Calobacter crescentus and its application in producing turanose[J]. Synthetic Biology Journal, 2022, 3(3): 602-615.

图/表 13

图1 淀粉蔗糖酶催化的反应类型

Fig. 1 Reactions catalyzed by amylosucrase

表1 定点突变引物

Tab. 1 Primers used for the site-directed mutation

引物名称	引物序列（5′—3′）
S271A/I382G-F	TGTCATGATGATTTAGGTTGGAATGCATTAGCA
S271A/I382G-R	TGCTAATGCATTCCAACCTAAATCATCATGACA
S271A/I382S-F	TGTCATGATGATTTAAGTTGGAATGCATTAGCA
S271A/I382S-R	TGCTAATGCATTCCAACTTAAATCATCATGACA
S271A/I382T-F	TGTCATGATGATTTAACTTGGAATGCATTAGCA
S271A/I382T-R	TGCTAATGCATTCCAAGTTAAATCATCATGACA
S271A/I382Q-F	TGTCATGATGATTTACAATGGAATGCATTAGCA
S271A/I382Q-R	TGCTAATGCATTCCATTGTAAATCATCATGACA
S271A/I382K-F	TGTCATGATGATTTAAAGTGGAATGCATTAGCA
S271A/I382K-R	TGCTAATGCATTCCACTTTAAATCATCATGACA
S271A/I382R-F	TGTCATGATGATTTAAGGTGGAATGCATTAGCA
S271A/I382R-R	TGCTAATGCATTCCACCTTAAATCATCATGACA
S271A/V211I-F	CGCGAATTAATCGATATATTTCCGGATACC
S271A/V211I-R	GGTATCCGGAAATATATCGATTAATTCGCG
S271A/P273A-F	CGCTTAGATGCCGCAGCGTTTCTGTGGAAACAG
S271A/P273A-R	CTGTTTCCACAGAAACGCTGCGGCATCTAAGCG
S271A/P273S-F	CGCTTAGATGCCGCATCGTTTCTGTGGAAACAG
S271A/P273S-R	CTGTTTCCACAGAAACGATGCGGCATCTAAGCG
S271A/I209R-F	GATCGCGAATTAAGGGATGTGTTTCCG
S271A/I209R-R	CGGAAACACATCCCTTAATTCGCGATC

图2 结构模拟与序列比对分析

Fig. 2 Structure simulations and sequence alignment analysis

图3 突变体摇瓶发酵情况

Fig. 3 The shake flask fermentation of E.coli BL21(DE3) mutants

图4 突变体制备松二糖的HPLC结果分析

Fig. 4 HPLC analysis of turanose produced from sucrose by the mutants

图5 纯化后突变体S271A/I382Q的SDS-PAGE分析M—低分子量蛋白Marker；1—S271A/I382Q

Fig. 5 SDS-PAGE analysis of the purified S271A/I382QM—Low molecular weight protein marker; 1—Purified S271A/I382Q

表2 S271A/I382Q纯化过程参数

Tab. 2 Parameter for S271A/I382Q purification

步骤	总酶活 /U	总蛋白含量/mg	比活 /(U/mg)	纯化倍数	回收率 /%
粗酶液	2918.5	653.0	4.5	1.0	100.0
镍柱纯化	573.3	49.8	11.5	2.6	20.0

图6 突变体S271A/I382Q的酶学性质

Fig. 6 Characterization of the mutant S271A/I382Q

图7 突变体271A/I382Q中Q382和D380的氢键相互作用

Fig. 7 Hydrogen bond developed between Q382 and D380 in the mutant S271A/I382Q

图8 S271A/I382Q催化制备松二糖的条件优化

Fig. 8 Optimization of reaction conditions for the catalysis of S271A/I382Q to produce turanose

图9 CcSH突变体蛋白骨架原子的均方根偏差（RMSD）随时间演变过程

Fig. 9 Time evolution for the root-mean-square deviation (RMSD) of the backbone atoms of CcSH mutants

图10 CcSH突变体与松二糖/海藻酮糖相关距离随时间的演变过程

Fig. 10 Time evolution of relative distances between CcSH single/double mutants and turanose/trehalulose

图11 CcSH突变体与松二糖/海藻酮糖复合物的相互作用分析

Fig. 11 Interaction analysis between CcSH mutants and turanose/trehalulose complex

参考文献 34

1	DONER L W. The sugars of honey—a review[J]. Journal of the Science of Food and Agriculture, 1977, 28(5): 443-456.
2	COSTA LEITE J M DA, TRUGO L C, COSTA L S M, et al. Determination of oligosaccharides in Brazilian honeys of different botanical origin[J]. Food Chemistry, 2000, 70(1): 93-98.
3	PARK M O, LEE B H, LIM E, et al. Enzymatic process for high-yield turanose production and its potential property as an adipogenesis regulator[J]. Journal of Agricultural and Food Chemistry, 2016, 64(23): 4758-4764.
4	HAN D J, LEE B H, YOO S H. Physicochemical properties of turanose and its potential applications as a sucrose substitute[J]. Food Science and Biotechnology, 2021, 30(3): 433-441.
5	TIAN Y Q, DENG Y, ZHANG W L, et al. Sucrose isomers as alternative sweeteners: properties, production, and applications[J]. Applied Microbiology and Biotechnology, 2019, 103(21/22): 8677-8687.
6	SHIBUYA T, MANDAI T, KUBOTA M, et al. Production of turanose by cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus [J]. Journal of Applied Glycoscience, 2004, 51(3): 223-227.
7	TIAN Y Q, XU W, ZHANG W L, et al. Amylosucrase as a transglucosylation tool: from molecular features to bioengineering applications[J]. Biotechnology Advances, 2018, 36(5): 1540-1552.
8	GUÉRIN F, BARBE S, PIZZUT-SERIN S, et al. Structural investigation of the thermostability and product specificity of amylosucrase from the bacterium Deinococcus geothermalis [J]. Journal of Biological Chemistry, 2012, 287(9): 6642-6654.
9	WANG R, BAE J S, KIM J H, et al. Development of an efficient bioprocess for turanose production by sucrose isomerisation reaction of amylosucrase[J]. Food Chemistry, 2012, 132(2): 773-779.
10	SU L Q, ZHAO Y Q, WU D, et al. Heterogeneous expression, molecular modification of amylosucrase from Neisseria polysaccharea, and its application in the preparation of turanose[J]. Food Chemistry, 2020, 314: 126212.
11	赵雅琪. 松二糖的酶法制备研究[D]. 无锡: 江南大学, 2019.
	ZHAO Y Q. Enzymatic synthesis of turanose[D]. Wuxi: Jiangnan University, 2019.
12	SEO D H, YOO S H, CHOI S J, et al. Versatile biotechnological applications of amylosucrase, a novel glucosyltransferase[J]. Food Science and Biotechnology, 2019, 29(1): 1-16.
13	POTOCKI DE MONTALK G, REMAUD-SIMEON M, WILLEMOT R M, et al. Amylosucrase from Neisseria polysaccharea: novel catalytic properties[J]. FEBS Letters, 2000, 471(2/3): 219-223.
14	ALBENNE C, SKOV L K, MIRZA O, et al. Molecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase[J]. Journal of Biological Chemistry, 2004, 279(1): 726-734.
15	STAM M R, DANCHIN E G J, RANCUREL C, et al. Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of α-amylase-related proteins[J]. Protein Engineering, Design & Selection, 2006, 19(12): 555-562.
16	LOMBARD V, GOLACONDA RAMULU H, DRULA E, et al. The carbohydrate-active enzymes database (CAZy) in 2013[J]. Nucleic Acids Research, 2013, 42(D1): D490-D495.
17	KIM M I, KIM H S, JUNG J, et al. Crystal structures and mutagenesis of sucrose hydrolase from Xanthomonas axonopodis pv. glycines: insight into the exclusively hydrolytic amylosucrase fold[J]. Journal of Molecular Biology, 2008, 380(4): 636-647.
18	邢晨晨, 王蕾, 郭志勇, 等. Caulobacter crescentus蔗糖水解酶突变体S271A的重组表达及其转化蔗糖制备松二糖的研究[J]. 食品与发酵工业, 2022, 48(2): 20-25.
	XING C C, WANG L, GUO Z Y, et al. Recombinant expression of Caulobacter crescentus sucrose hydrolase mutant S271A and its conversion of sucrose to turanose[J]. Food and Fermentation Industries, 2022, 48(2): 20-25.
19	ARNOLD K, BORDOLI L, KOPP J, et al. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling[J]. Bioinformatics, 2006, 22(2): 195-201.
20	WANG Z, SUN H Y, YAO X J, et al. Comprehensive evaluation of ten docking programs on a diverse set of protein-ligand complexes: the prediction accuracy of sampling power and scoring power[J]. Physical Chemistry Chemical Physics, 2016, 18(18): 12964-12975.
21	OLSSON M H M, SØNDERGAARD C R, ROSTKOWSKI M, et al. PROPKA3: consistent treatment of internal and surface residues in empirical pK_a predictions[J]. Journal of Chemical Theory and Computation, 2011, 7(2): 525-537.
22	MAIER J A, MARTINEZ C, KASAVAJHALA K, et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB[J]. Journal of Chemical Theory and Computation, 2015, 11(8): 3696-3713.
23	KIRSCHNER K N, YONGYE A B, TSCHAMPEL S M, et al. GLYCAM06: a generalizable biomolecular force field. carbohydrates[J]. Journal of Computational Chemistry, 2008, 29(4): 622-655.
24	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.
25	RYCKAERT J P, CICCOTTI G, BERENDSEN H J C. Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes[J]. Journal of Computational Physics, 1977, 23(3): 327-341.
26	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.
27	HUMPHREY W, DALKE A, SCHULTEN K. VMD: Visual molecular dynamics[J]. Journal of Molecular Graphics, 1996, 14(1): 33-38.
28	ROE D R, CHEATHAM T E 3rd. PTRAJ and CPPTRAJ: Software for processing and analysis of molecular dynamics trajectory data[J]. Journal of Chemical Theory and Computation, 2013, 9(7): 3084-3095.
29	BENKERT P, BIASINI M, SCHWEDE T. Toward the estimation of the absolute quality of individual protein structure models[J]. Bioinformatics, 2011, 27(3): 343-350.
30	BIASINI M, BIENERT S, WATERHOUSE A, et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information[J]. Nucleic Acids Research, 2014, 42(W1): W252-W258.
31	WATERHOUSE A, BERTONI M, BIENERT S, et al. SWISS-MODEL: homology modelling of protein structures and complexes[J]. Nucleic Acids Research, 2018, 46(W1): W296-W303.
32	SKOV L K, MIRZA O, SPROGØE D, et al. Oligosaccharide and sucrose complexes of amylosucrase: structural implications for the polymerase activity[J]. Journal of Biological Chemistry, 2002, 277(49): 47741-47747.
33	NIELSEN J E, BORCHERT T V, VRIEND G. The determinants of α-amylase pH-activity profiles[J]. Protein Engineering, Design and Selection, 2001, 14(7): 505-512.
34	NIELSEN J E, VRIEND G. Optimizing the hydrogen-bond network in Poisson-Boltzmann equation-based pK_a calculations[J]. Proteins, 2001, 43(4): 403-412.

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