低聚果糖生产相关酶的分离纯化及性质研究
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摘要
低聚果糖是一种在高等植物和微生物中分布甚广的天然寡聚糖。通常所指的低聚果糖是通过β-(2-1)糖苷键在蔗糖分子的果糖残基上连接1～3个果糖分子而形成的果糖寡聚体蔗果三糖、蔗果四糖、蔗果五糖及其混合物。由于低聚果糖具有难消化、低热量、抗糖尿病、降血脂、增殖双歧杆菌、抗龋齿及美容等独特的生理功能，现已作为一类重要的保健食品和蔗糖替代品广泛使用。工业上，主要以高浓度蔗糖作底物用微生物酶法生产低聚果糖。
本实验室筛选到了一株低聚果糖高产菌黑曲霉SIPI-602。我们采用凝胶过滤层析的方法对黑曲霉SIPI-602转化蔗糖的产物进行了分离纯化，得到了在HPLC上呈单峰的三聚糖和四聚糖，其保留时间分别与蔗果三糖和蔗果四糖一致。并且用~(13)C-NMR和MS对其结构进行了鉴定，确定它们分别为蔗果三糖和蔗果四糖。
在低聚果糖生产过程中，发生转移反应的同时往往会出现水解反应，影响了低聚果糖的转化率和产品中低聚果糖的含量。但目前对水解反应的本质尚未取得共识。一般认为，催化水解反应和转移反应的是同一个酶，但对于这一酶的归属有不同的观点，有人认为它属于转移酶，也有人称其为糖苷酶；还有个别学者认为水解反应为杂酶污染所致。为了加深对低聚果糖生产相关酶的了解，我们对黑曲霉SIPI-602产生的与低聚果糖生产相关的酶进行了分离纯化和性质研究。分离到了β-果糖基转移酶和β-呋喃果糖苷酶两个酶，其中β-果糖基转移酶可以催化蔗糖分子上果糖基的转移，生成低聚果糖；而β-呋喃果糖苷酶则可以将蔗糖和低聚果糖水解成葡萄糖和果糖。这有助于确定低聚果糖产生酶的归属和解释低聚果糖生产过程中的水解反应。此外，应用β-果糖基转移酶纯酶对提高低聚果糖的转化率以及进一步研究高浓度低聚果糖的生产均有重要意义。
低聚果糖生产相关酶的分离纯化包括用球磨机破碎细胞、硫酸铵分级沉淀、Octyl-Sepharose CL-4B疏水层析、Q-Sepharose Fast Flow阴离子交换层析、Sephacryl S-300凝胶过滤层析等步骤。两个蛋白在SDS-聚丙烯酰胺凝胶电泳上均呈单带，用SDS-PAGE测得β-果糖基转移酶和β-呋喃果糖苷酶的分子量分别为98.86KD和84.92KD。
一
日一果糖基转移酶催化蔗糖转化成蔗果三糖的Km和Vmax分别为0．78mol／L
和斗．30mmol／min／L；将蔗果三糖转化成蔗果四糖的 Km和 Vmax分别为 0．34mol／L
和 0二ommol／min／L。葡萄糖是 p一果糖基转移酶的竞争性抑制剂，其 Ki＝
1．36mol几。6一果糖基转移酶反应的最适pH和最适温度分别为牛5～6．0和 55
’C。6一果糖基转移酶在中性条件、50oC以下稳定性良好。6一果糖基转移酶转化
蔗糖生成低聚果糖的反应进程实验表明，高浓度的底物有利于提高反应速度和转化
率，产物中果糖含量亦较低，说明提高底物浓度可以抑制水解反应的发生。
旦一陕哺果糖苦酶水解蔗糖生成葡萄糖和果糖的 Km和 Vmax 值分别为
0．025mol／L和 l．39。mol／min／L。葡萄糖对6一吱哺果糖昔酶没有影响；而果糖是
6一味g南果糖昔酶的竞争性抑制剂，Ki＝10．05mmol／L。fi一味哺果糖苦酶在酸性
条件稳定，反应活性高。6一映哺果糖昔酶在40℃活力最高，在65℃以下稳定。
通过对菌丝体、6一果糖基转移酶与p一吠哺果糖苦酶的混合物及纯的p一果糖基
转移酶转化蔗糖生成低聚果糖的反应进程的比较，考察了p一陕哺果糖昔酶对p一
果糖基转移酶反应进程的影响。结果表明6一吠哺果糖苦酶对低聚果糖生成速率和
最终产率均有影响。结果还提示在用菌丝体转化蔗糖过程中低聚果糖可能会诱导出
更多的6一味哺果糖苦酶。
Purification and properties of the enzymes related to the production of fructooligosaccharides from Aspergillus niger SIPI-602
ABSTRACT:Fructooligosaccharides(FOS) are a class of natural oligosaccharides existing widely in many kinds of higher plants and microorganisms. They are mainly composed of l-kestose(GF2), nystose(GF3), 1F- 3 -fructofuranosyl nystose (GF4) in which two, three, and four fructosyl units are bound at the $ -2,1 position of sucrose(GF),
respectively.
Because of their specific physiological characteristics, i.e., low caloric, non-digestible, noncariogenic, selectively stimulating bifidobacteria growth, antidiabete, FOS have become a class of important health food and alternative sweetener. FOS can be obtained by enzymatic action of a fungal enzyme system, fructosyl transferase on sucrose.
Previously, we isolated a FOS-producing fungus, Aspergillus niger SIPI-602. The products converted by Aspergillus niger SIPI-602 mycelia were purified and a pure trisaccharide and a pure tetrasaccharide were obtained. The HPLC chromatograms of both the trimer and tetramer showed single peak and their retention times were identical with GF2 and GF3, respectively. In addition, the structures of the trisaccharide and tetrasaccharide were determined by 13C-NMR and MS.
Then, we purified two enzymes related to the FOS production from A.niger SIPI-602, in which P -fructosyltransferase (FTase) converted sucrose into fructooligosaccharides, while ?> -fructofuranosidase hydrolyzes sucrose and fructooligosaccharides into fructose and glucose.
The purification protocol consisted of the following procedures: ball milling, ammonium sulfate precipitation and hydrophobic, anion-exchange and gel filtration chromatographies. Both the two enzymes showed single band on SDS-PAGE and the molecular weights of FTase and FFase were 98.86KD and 84.92KD by SDS-PAGE, respectively.
The properties of FTase and FFase were also investigated. The Km and Vmax values of FTase with sucrose as a substrate were 0.78mol/L and 4.30mmol/min/L by using the Lineweaver-Buek plot, respectively and the Km and Vmax values of FTase using GFi as a substrate were 0.34mol/L and 0.20mmol/min/L. Glucose was a
competitive inhibitor. FTase was most active at pH4.5-6.0 and at 55"C and stable up to 50 at pH5.8 for one hour of incubation and from pH6.0 to 8.0 at 40癈 in one hour of incubation. The time-course of the formation of FOS from sucrose indicated that the reaction rates increased at a high initial sucrose concentration and the fructose concentration increased at a low sucrose concentration.
The Km and Vmax values of FFase for hydrolyzing sucrose into glucose and fructose were 0.025mol/L and 1.39rnmmol/rnin/L respectively. Glucose didn't affect FFase activation and fructose was a competitive inhibitor for FFase. FFase was more active and more stable at acidic conditions. The optimal temperature of FFase was 40癈 and the enzyme was stable below 65癈. The time-course for mycelia, FTase and FFase mixture , and pure FTase were compared respectively and it was confirmed that FFase would slow the time-course of FTase and decrease the FOS concentration in the final product. The results also indicated FOS could induce FFase increase in the reaction course catalyzed by mycelia.
本实验室筛选到了一株低聚果糖高产菌黑曲霉SIPI-602。我们采用凝胶过滤层析的方法对黑曲霉SIPI-602转化蔗糖的产物进行了分离纯化，得到了在HPLC上呈单峰的三聚糖和四聚糖，其保留时间分别与蔗果三糖和蔗果四糖一致。并且用~(13)C-NMR和MS对其结构进行了鉴定，确定它们分别为蔗果三糖和蔗果四糖。
在低聚果糖生产过程中，发生转移反应的同时往往会出现水解反应，影响了低聚果糖的转化率和产品中低聚果糖的含量。但目前对水解反应的本质尚未取得共识。一般认为，催化水解反应和转移反应的是同一个酶，但对于这一酶的归属有不同的观点，有人认为它属于转移酶，也有人称其为糖苷酶；还有个别学者认为水解反应为杂酶污染所致。为了加深对低聚果糖生产相关酶的了解，我们对黑曲霉SIPI-602产生的与低聚果糖生产相关的酶进行了分离纯化和性质研究。分离到了β-果糖基转移酶和β-呋喃果糖苷酶两个酶，其中β-果糖基转移酶可以催化蔗糖分子上果糖基的转移，生成低聚果糖；而β-呋喃果糖苷酶则可以将蔗糖和低聚果糖水解成葡萄糖和果糖。这有助于确定低聚果糖产生酶的归属和解释低聚果糖生产过程中的水解反应。此外，应用β-果糖基转移酶纯酶对提高低聚果糖的转化率以及进一步研究高浓度低聚果糖的生产均有重要意义。
低聚果糖生产相关酶的分离纯化包括用球磨机破碎细胞、硫酸铵分级沉淀、Octyl-Sepharose CL-4B疏水层析、Q-Sepharose Fast Flow阴离子交换层析、Sephacryl S-300凝胶过滤层析等步骤。两个蛋白在SDS-聚丙烯酰胺凝胶电泳上均呈单带，用SDS-PAGE测得β-果糖基转移酶和β-呋喃果糖苷酶的分子量分别为98.86KD和84.92KD。
一
日一果糖基转移酶催化蔗糖转化成蔗果三糖的Km和Vmax分别为0．78mol／L
和斗．30mmol／min／L；将蔗果三糖转化成蔗果四糖的 Km和 Vmax分别为 0．34mol／L
和 0二ommol／min／L。葡萄糖是 p一果糖基转移酶的竞争性抑制剂，其 Ki＝
1．36mol几。6一果糖基转移酶反应的最适pH和最适温度分别为牛5～6．0和 55
’C。6一果糖基转移酶在中性条件、50oC以下稳定性良好。6一果糖基转移酶转化
蔗糖生成低聚果糖的反应进程实验表明，高浓度的底物有利于提高反应速度和转化
率，产物中果糖含量亦较低，说明提高底物浓度可以抑制水解反应的发生。
旦一陕哺果糖苦酶水解蔗糖生成葡萄糖和果糖的 Km和 Vmax 值分别为
0．025mol／L和 l．39。mol／min／L。葡萄糖对6一吱哺果糖昔酶没有影响；而果糖是
6一味g南果糖昔酶的竞争性抑制剂，Ki＝10．05mmol／L。fi一味哺果糖苦酶在酸性
条件稳定，反应活性高。6一映哺果糖昔酶在40℃活力最高，在65℃以下稳定。
通过对菌丝体、6一果糖基转移酶与p一吠哺果糖苦酶的混合物及纯的p一果糖基
转移酶转化蔗糖生成低聚果糖的反应进程的比较，考察了p一陕哺果糖昔酶对p一
果糖基转移酶反应进程的影响。结果表明6一吠哺果糖苦酶对低聚果糖生成速率和
最终产率均有影响。结果还提示在用菌丝体转化蔗糖过程中低聚果糖可能会诱导出
更多的6一味哺果糖苦酶。
Purification and properties of the enzymes related to the production of fructooligosaccharides from Aspergillus niger SIPI-602
ABSTRACT:Fructooligosaccharides(FOS) are a class of natural oligosaccharides existing widely in many kinds of higher plants and microorganisms. They are mainly composed of l-kestose(GF2), nystose(GF3), 1F- 3 -fructofuranosyl nystose (GF4) in which two, three, and four fructosyl units are bound at the $ -2,1 position of sucrose(GF),
respectively.
Because of their specific physiological characteristics, i.e., low caloric, non-digestible, noncariogenic, selectively stimulating bifidobacteria growth, antidiabete, FOS have become a class of important health food and alternative sweetener. FOS can be obtained by enzymatic action of a fungal enzyme system, fructosyl transferase on sucrose.
Previously, we isolated a FOS-producing fungus, Aspergillus niger SIPI-602. The products converted by Aspergillus niger SIPI-602 mycelia were purified and a pure trisaccharide and a pure tetrasaccharide were obtained. The HPLC chromatograms of both the trimer and tetramer showed single peak and their retention times were identical with GF2 and GF3, respectively. In addition, the structures of the trisaccharide and tetrasaccharide were determined by 13C-NMR and MS.
Then, we purified two enzymes related to the FOS production from A.niger SIPI-602, in which P -fructosyltransferase (FTase) converted sucrose into fructooligosaccharides, while ?> -fructofuranosidase hydrolyzes sucrose and fructooligosaccharides into fructose and glucose.
The purification protocol consisted of the following procedures: ball milling, ammonium sulfate precipitation and hydrophobic, anion-exchange and gel filtration chromatographies. Both the two enzymes showed single band on SDS-PAGE and the molecular weights of FTase and FFase were 98.86KD and 84.92KD by SDS-PAGE, respectively.
The properties of FTase and FFase were also investigated. The Km and Vmax values of FTase with sucrose as a substrate were 0.78mol/L and 4.30mmol/min/L by using the Lineweaver-Buek plot, respectively and the Km and Vmax values of FTase using GFi as a substrate were 0.34mol/L and 0.20mmol/min/L. Glucose was a
competitive inhibitor. FTase was most active at pH4.5-6.0 and at 55"C and stable up to 50 at pH5.8 for one hour of incubation and from pH6.0 to 8.0 at 40癈 in one hour of incubation. The time-course of the formation of FOS from sucrose indicated that the reaction rates increased at a high initial sucrose concentration and the fructose concentration increased at a low sucrose concentration.
The Km and Vmax values of FFase for hydrolyzing sucrose into glucose and fructose were 0.025mol/L and 1.39rnmmol/rnin/L respectively. Glucose didn't affect FFase activation and fructose was a competitive inhibitor for FFase. FFase was more active and more stable at acidic conditions. The optimal temperature of FFase was 40癈 and the enzyme was stable below 65癈. The time-course for mycelia, FTase and FFase mixture , and pure FTase were compared respectively and it was confirmed that FFase would slow the time-course of FTase and decrease the FOS concentration in the final product. The results also indicated FOS could induce FFase increase in the reaction course catalyzed by mycelia.
引文
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[62] Duan KJ, Chen JS, Sheu DC. Kinetic studies and mathematical model for enzymatic production of fructooligosaccharides from sucrose[J]. Enzyme Microb Technol, 1994, 16: 334-339．
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[65] 日高秀昌，平山匡男．転移行微生物．植物酵素[J]．化学生物，23(9)：600-605．
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