大蒜蔗糖﹕蔗糖1-果糖基转移酶（1-SST）的酶学特征

doi:10.3864/j.issn.0578-1752.2015.02.13

摘要/Abstract

摘要： 【目的】研究大蒜（Allium sativum L.）中蔗糖﹕蔗糖1-果糖基转移酶（1-SST）的酶学特征，为大蒜保鲜、加工和品质改良提供依据。【方法】采用饱和度10%、20%、30%、40%、50%、60%、70%、80%、90%和100%的硫酸铵分级沉淀1-SST，以确定其所在的硫酸铵部位。分别取1-SST活力最高的硫酸铵部分与底物蔗糖反应，研究其在不同的温度、pH、离子强度、底物质量浓度等条件下合成高果聚糖的能力，即采用高效液相色谱-蒸发光散射（HPLC-ELSD），测定1-SST反应前后的1-蔗果三糖的含量，进而计算出1-SST活力大小并总结出其在不同的温度、pH、离子强度、底物质量浓度等条件下合成高果聚糖反应的规律；采用的色谱柱为Prevail Carbohydrate ES柱（250 mm×4.6 mm，5 μm），色谱柱柱温箱温度为40℃，流动相最大限压20 MP，流动相为乙腈和水，其流速为1.0 mL·min^-1，ELSD撞击器关闭状态，漂移管温度90℃，载气流量2.5 L·min^-1（空气）。其梯度洗脱方式为：0 min，75%乙腈﹕25%水；15 min，65%乙腈﹕35%水；30 min，50%乙腈﹕50%水；35 min，75%乙腈﹕25%水；40 min，75%乙腈﹕25%水。【结果】在上述测定条件下，果糖、葡萄糖、蔗糖、1-蔗果三糖、蔗果四糖、蔗果五糖6种糖完全分离，出峰时间仅为20 min，测定方法可靠、灵敏度高，满足了测定1-SST活性的要求。以50%（w/w）蔗糖为底物时，仅产生1-蔗果三糖，无其它高果聚糖的产生，证实了在所试条件下，大蒜中1-SST反应具有专一性。大蒜1-SST主要存在于30%—40%硫酸铵饱和度部分，其中0—30%，40%—70%有轻微活力，未发现70%—100%硫酸铵组分有1-SST的活力。取30%—40%的硫酸铵饱和度部分作为样品研究1-SST酶学特征发现其最适反应温度为35℃，当温度到达40℃以上时，酶的活力下降的很快，说明1-SST具有热不稳定性，因此在研究大蒜1-SST时，要尽量避免高温，以免影响后续研究；其最适反应pH为5.0，与大麦（Hordeum vulgare）、菊苣（Cichorium intybus L.）、菊芋(Helianthus tuberosus Colombia )、黑麦草（Lolium rigidum）、龙舌兰（Agave americana L.）的1-SST最适pH大致相同，显示不同植物1-SST活力中心部位的结构可能基本一致；在蔗糖浓度为100—600 mg·mL^-1内，酶活力随着底物质量浓度的增加而提高，而当底物浓度超过500 mg·mL^-1时，酶活力增加的幅度减少而趋于平缓，这完全符合米氏方程（Michaelis-menten）的酶动力学性质；对于Na⁺、Cl^-来说，离子强度越低，1-SST活力越强，1-SST在35℃下的米氏常数Km为10⁴ mmol·L^-1。【结论】大蒜1-SST随温度、pH、离子强度、底物质量浓度等变化，不仅影响大蒜贮藏过程中的果聚糖代谢，也影响大蒜加工产品，尤其是大蒜提取物中糖的种类和含量。

关键词: 大蒜, 果聚糖, 蔗糖﹕蔗糖果糖基转移酶, 酶学特征, 1-蔗果三糖

Abstract: 【Objective】 Enzymologic characteristics of sucrose: sucrose 1-fructosyltransferase (1-SST) in garlic (Allium sativum L.) was investigated to provide a basis for the preservation, processing and further quality improvements of fresh garlic.【Method】1-SST was fractional precipitated by ammonium sulfate with different saturations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, successively, in order to determine the fraction of the ammonium sulfate which 1-SST is in. 1-SST catalyses in fructan biosynthesis. After that, the 1-SST was further researched on the catalytic activity under different temperatures, pH, ionic strength and substrate concentrations. Here sucrose was used as substrate. The 1-SST enzymatic activity was revealed through production of 1-kestose which was analyzed by high performance liquid chromatography-evaporation light-scattering detection (HPLC-ELSD). Chromatographic separation of 1-kestose was achieved by the Prevail Carbohydrate ES column (250 mm×4.6 mm, 5 μm) at a flow rate of 1 mL·min^-1 under 20 MP . The mobile phase consisted of a combination of B water and A acetonitrile. The gradient elution was applied as followings: 75% A : 25% B at 0 min, 65% A : 35% B at 15 min, 50% A : 50% B at 30 min, 75% A : 25% B at 35 min, 75% A : 25% B at 40 min. The column temperature was held at 40℃. ELSD impactor was off and drift tube temperature was kept at 90℃. The air was used as carrier gas for ELSD detection with a flow rate of 2.5 L·min^-1.【Result】Fructose, glucose, sucrose, 1-kestose, nystose, 1F-fructofuranosylnystose were separated in 20 min. Thus the HPLC-ELSD method was proved a reliable and highly sensitive method for determining the 1-kestose. As early described, 1-kestose was produced when sucrose incubated with 1-SST and used to represent the enzymatic activity of 1-SST. Only 1-kestose was produced with 50% (w/w) sucrose solution as substrate. This phenomenon indicated that the 1-SST preserved very high substrate specificity under the above experiment conditions. Garlic 1-SST mainly existed in the fraction precipitated with 30%-40% ammonium sulfate solution while exhibited very weak enzymatic activity in the fractions precipitated with 0-30%, 40%-70% ammonium sulfate solution and no 1-SST was detected in the fraction precipitated with 70%-100% ammonium sulfate. Garlic 1-SST from the fraction precipitated with 30%-40% ammonium sulfate solution was further investigated. The optimal temperature was 35℃. The enzymatic activity was decreased dramatically when the temperature reached 40℃ which indicated this enzyme is heat labile. Therefore, high temperature should be avoided for studying the garlic 1-SST. Its optimum pH value was 5, which was similar to the 1-SST from Hordeum vulgare, Cichorium intybus L., Helianthus tuberosus Colombia and Lolium rigidum, Agave americana L. This result shows that the conformation of 1-SST activity site from different plants could be similar. The enzymatic activity of 1-SST was dependent on the sucrose concentration in the range of 100-600 mg·mL^-1 which was increased with the substrate concentration below 500 mg·mL^-1. However, the enzyme was saturated at the high concentration of substrate (over 500 mg·mL^-1). This result fits into the Michaelis-Menten Equation. For Na⁺, Cl^-ion, 1-SST enzymatic activity was reversed with the ion strength. Michaelis constant of 1-SST was 10⁴mmol·L^-1 at 35℃.【Conclusion】 In this study, The effect of different temperatures, pH, ionic strength and substrate concentration on the 1-SST enzymatic activity was investigated. This mechanism will help to understand the change of fructan, species and content of saccharides during processing and storage.

Key words: fructan, sucrose:sucrose 1-Fructosyltransferase (1-SST), enzymologic characteristics, Allium sativum L., 1-kestose

文明，卜利伟，罗紫韵，王佳伟，董芬，黄雪松. 大蒜蔗糖﹕蔗糖1-果糖基转移酶（1-SST）的酶学特征[J]. 中国农业科学, 2015, 48(2): 334-342.

WEN Ming, BU Li-wei, LUO Zi-yun, WANG Jia-wei, DONG Fen, HUANG Xue-song. Characteristics of Sucrose: Sucrose 1-Fructosyltransferase in Garlic[J]. Scientia Agricultura Sinica, 2015, 48(2): 334-342.

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参考文献

[1] 康雅. 大蒜的营养成分及其保健功能. 中国食物与营养, 2010(9): 75-77.

Kang Y. Nutrition and health function of garlic.Food and Nutrition in China, 2010(9): 75-77. (in Chinese)

[2] 刘燕琼, 黄雪松.大蒜中果聚糖的测定. 食品与发酵工业, 2005, 31(8): 84-86.

Liu Y Q,Hunag X S. Determination of fructan in garlic. Food and Fermentation Industries, 2005, 31(8): 84-86. (in Chinese)

[3] Huang G L, Shu S Q, Cai T T, Liu Y Q, Xiao F. Preparation and deproteinization of garlic polysaccharide. International Journal of Food Sciences & Nutrition, 2012, 63(6): 739-741.

[4] 黄雪松. 大蒜多糖的提取分离与分析. 食品科学, 2005, 26(9): 48-51.

Huang X S. Isolation and identification of garlic polysaccharide. Food Science, 2005, 26(9): 48-51. (in Chinese)

[5] Chandrashekar P M, Prashanth K V H, Venkatesh Y P. Isolation, structural elucidation and immunomodulatory activity of fructans from aged garlic extract. Phytochemistry, 2011, 72(2/3): 255-264.

[6] 王文玲, 黄雪松, 曾莉莎. 大蒜多糖的研究综述. 广州食品工业科技, 2004(4): 144-146.

Wang W L, Huang X S, Zeng L S. Review of garlic polysaccharide. Guangzhou Science and Technology of Food Industry, 2004(4): 144-146. (in Chinese)

[7] 赵海燕, 韩啸, 余洁, 白凌子, 何忠伟. 中国大蒜产业国际竞争力研究. 农业展望, 2013(5): 55-59.

Zhao H Y, Han X, Yu J, Bai L Z, He Z W. Study on the international competitiveness of industry China garlic. Agricultural Outlook, 2013(5): 55-59. (in Chinese)

[8] Ende W V D, Coninck B D, Laere A V. Plant Fructan exohydrolases: a role in signaling and defense? Trends in Plant Science, 2004, 9(11): 523-528.

[9] Asega A F, Nascimento J R O.do, Schroeven L, Ende W V D, Carvalho M A M. Cloning, Characterization and Functional Analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby. Plant and Cell Physiology, 2008, 49(8): 1185-1195.

[10] Marx S, Nosberger J, Frehner M. Seasonal variation of fructan-β-fructosidase (FEH) activity and characterization of a β-(2-1)-linkage specific FEH from tubers of Jerusalem artichoke (Helianthus tuberosus). New Phytologist, 1997, 135(2): 267-277.

[11] Kawakami A, Yoshida M, Ende W V D. Molecular cloning and functional analysis of a novel 6&1-FEH from wheat (Triticum aestivum L.) preferentially degrading small graminans like bifurcose. Gene, 2005, 358: 93-101.

[12] Lothier J, Lasseur B, Le Roy K, van Laere A, Prud'homme1 M P, Barre P, van den Ende W, morvan-Bertrand1 A. Cloning,gene mapping,and functional analysis of a fructan 1-FEH from Lolium perenne implicated in fructan synthesis rather than in fructan mobilization. Journal of Experiental Botany, 2007, 58(8): 1969-1983.

[13] Verhaest M, Ende W V D, Roy K L, Ranter C J D, Laere A V, Rabijns A. X-ray diffraction structure of a plant glycosyl hydrolase family 32 protein: fructan 1-exohydrolase IIa of Cichorium intybus. The Plant Journal, 2005, 41(3): 400-411.

[14] van den Ende W, De Coninck B, Clerens S, Vergauwen R, Van Laere A. Unexpected presence of fructan 6-exohydrolases (6-FEHs) in non-fructan plants: characterization, cloning, mass mapping and functional analysis of a novel ‘cell-wall invertase-like’ specific 6-FEH from sugar beet (Beta vulgaris L.). The Plant Journal, 2003, 36(5): 697-710.

[15] Ueno K, Ishiguro Y, Yoshida M, Shiomi N. Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.). Chemistry Central Journal, 2011, 5(1): 16.

[16] Viso F del, Puebla A F, Hopp H E, Heinz R A. Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in the cold tolerant Patagonian species Bromus pictus. Planta, 2009, 231(1): 13-25.

[17] 殷桂香, 佘茂云, 高翔, 王瑾, 叶兴国. 植物果聚糖合成酶基因克隆及特性分析. 中国生物工程杂志, 2009, 29(2): 125-133.

Yin G X, She M Y, Gao X, Wang J, Ye X G. Cloning and analysis of characteristics of fructan synthase genes in plants. China Journal of Biotechnology, 2009, 29(2): 125-133. (in Chinese)

[18] van der Meer I M, Koops A J, Hakkert J C, van Tunen A J. Cloning of the fructan biosynthesis pathway of Jerusalem artichoke. The Plant Journal, 1998, 15(4): 489-500.

[19] Ende W V D, Laere A V. Purification and properties of an invertase with sucrose:sucrose fructosyltransferase (SST) activity from the roots of Cickorium intybus L. New Phytologist, 1993, 123(1): 31-37.

[20] Hellwege E M, Gritscher D, Willmitzer L, Heyer A G. Transgenic potato tubers accumulate high levels of 1-kestose and nystose: functional identification of a sucrose:sucrose 1-fructosyltransferase of artichoke (Cynara scolymus) blossomdiscs. The Plant Journal, 1997, 12(5): 1057-1065.

[21] Fujishima M, Sakai H, Ueno K, Takahashi N, Onodera S, Benkeblia N, Shiomi N. Purification and characterization of a fructosyltransferase from onion bulbs and its key role in the synthesis of fructo- oligosaccharides in vivo. New Phytologist, 2005, 165(2), 513-524.

[22] Koops A J, Jonker H H. Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus Tuberosus Colombia. II. Purificationof sucrose:sucrose 1-fructosyltransferase and reconstitution of fructan synthesis in vitro with purified sucrose: sucrose 1-fructosyltransferase and fructan: fructan1- fructosyltransferase. Plant Physiology, 1996, 110(4): 1167-1175.

[23] Kawakami A, Yoshida M. Molecular characterization of sucrose: sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase associated with fructan accumulation in winter wheat during cold hardening. Bioscience, Biotechnology, and Biochemistry, 2002, 66(11): 2297-2305.

[24] Luscher M, Hochstrasser U, Boller T, Wiemken A. Isolation of sucrose: sucrose 1-fructosyltransferase (1-SST) from barley (Hordeum vulgare). New Phytologist, 2000, 145(2): 225-232.

[25] John A S T, Sims I M, Bonnett G D, Simpson R J. Synthesis of fructans by partially purified fructosyltransferase activities from Lolium rigidum. New Phytologist, 1997, 136(1): 39-51.

[26] Fernández Á Á, Carranza C O, Piñera E R, Cassab G I, Sotelo J N, Munguía A L. Molecular characterization of sucrose: sucrose 1-fructosyltransferase (1-SST) from Agave tequilana Weber var. azul. Plant Science, 2007, 173(4): 478-486.

[27] Vijn I, Smeekens S. Fructan: more than a reserve carbohydrate? Plant Physiology, 1999, 120(2): 351-359.

[28] Ende W V D, Laere A V. Fructan Synthesizing and Degrading Activities in chicory roots (Cichorium intybus L.) during field-growth, storage and forcing. Plant Physiology, 1996, 149(1/2): 43-50.