WO2017128529A1 - 一种高效合成双果糖酐iii的方法 - Google Patents

一种高效合成双果糖酐iii的方法 Download PDF

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WO2017128529A1
WO2017128529A1 PCT/CN2016/079428 CN2016079428W WO2017128529A1 WO 2017128529 A1 WO2017128529 A1 WO 2017128529A1 CN 2016079428 W CN2016079428 W CN 2016079428W WO 2017128529 A1 WO2017128529 A1 WO 2017128529A1
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inulin
fructose
iii
controlled
sucrose
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French (fr)
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沐万孟
江波
郁书怀
朱莺莺
张涛
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江南大学
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Priority to US15/777,932 priority Critical patent/US10351888B2/en
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01009—Inulosucrase (2.4.1.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02017—Inulin fructotransferase (DFA-I-forming) (4.2.2.17)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02018—Inulin fructotransferase (DFA-III-forming) (4.2.2.18)

Definitions

  • the invention relates to a method for efficiently synthesizing bis-fructose III, in particular to achieve high-efficiency production of Difructose anhydride (DFA III) by two enzyme two-step methods, and belongs to the technical field of functional food bioprocessing.
  • DFA III Difructose anhydride
  • Bis-fructose III is a novel functional sweetener whose structure is mainly formed by the removal of two water molecules from two fructose molecules. Since the hydroxyl group on the anomeric carbon is a glycosidic bond, the bis-fructose anhydride III is a novel non-reducing disaccharide. Due to the presence of such a special disaccharide bond form, the nature of the bis-fructose III is very stable. It is quite stable to heat and acid, and is resistant to high temperature. It does not cause browning or decomposition of bis-fructose III during high-temperature processing of general foods.
  • bis-fructose III The selectivity of bis-fructose III is only 1/2 of that of sucrose, but the calorie is very low compared with sucrose, only 1/15 of sucrose (0.263kcal/g), which has good function for diabetic and obese patients. Therefore, bis-fructose III It can be used as an ideal sucrose replacement sugar in low-energy foods and diabetic foods to prevent and treat diabetes. At the same time, bis-fructose III is more hygroscopic than sucrose and can not absorb moisture at 74% relative humidity, which facilitates the storage of sugar. In vivo, bis-fructose III is not absorbed by the intestines and thus does not produce energy, so it can be used as a sweetener for weight-loss adjuvant therapy.
  • the bis-fructose III can be metabolized by the probiotics in the intestine to produce a large number of prebiotics, thereby promoting the growth of probiotics and health in the human body.
  • Double fructose III can promote the absorption of mineral elements such as calcium, magnesium, zinc and copper as well as flavonoids, thereby enhancing bone growth.
  • Caries are mainly caused by the metabolism of sucrose and other sugars in the cavity to produce acid, which is caused by corrosion of teeth.
  • bis-fructose cannot be metabolized by Streptococcus mutans and therefore has an anti-caries effect.
  • the bis-fructose III has various physiological functions such as lowering cholesterol and hypertension. In view of these excellent properties of bis-fructose III, it can be applied as a food additive to baked goods, beverages, confectionery, and the like. Therefore, bis-fructose III has broad prospects in the food industry.
  • bis-fructose anhydrides mainly in the compositae including chicory and Jerusalem artichoke, but the content is small.
  • the bis-fructose III is also present in a small amount in the processing of honey, coffee, and the like. Because of the low content in nature, the cost of extracting and separating the bis-fructose III is high.
  • chemical methods can also produce bis-fructose III, there are many disadvantages in chemical synthesis, such as environmental pollution.
  • Bis-fructose can also be obtained by biocatalysis.
  • the present inventors propose to use a cheaper sucrose as a substrate, first converting sucrose into inulin by inulin sucrase, and then converting the synthesized bio-inulin directly into bis-fructose III by using stevioside transferase.
  • the separation and purification of inulin is not required, which saves energy consumption and reduces production costs.
  • the yeast is used to remove other sugars present in the reaction solution, and a relatively pure bis-fructose III is obtained to facilitate subsequent separation and purification.
  • the technical problem to be solved by the present invention is to provide an efficient synthesis method of an inexpensive bis-fructose anhydride III, which has a high purity.
  • the whole synthesis process is simple in process, high in efficiency and low in cost.
  • the process for converting sucrose to inulin is: completely dissolving sucrose in water, the concentration is controlled to 300-500 g/l, the pH is adjusted to 5.0-6.0, the temperature is controlled at 20-30 ° C, and the inulin sucrase is added. It is 1-15 U/g and is reacted at a constant temperature for 25 to 60 minutes.
  • the preferred process is as follows: the process of converting sucrose to synthesize inulin is preferred: the amount of inulin sucrase is controlled at 5-15 U/g, the pH is controlled at 5.0-6.0, the temperature is controlled at 20-30 ° C, and the temperature is controlled. The reaction was carried out for 30 to 60 minutes.
  • the process for synthesizing inulin to synthesize bis-fructose III is: heating the sucrose conversion solution to 50-60 ° C, and then adding chrysanthemum candy transferase to the solution, and adding the concentration of chrysanthemum candy transferase is 1-20 U/g, pH The value is still controlled at 5.0-6.0 and the temperature is reacted for 2-20 hours.
  • the preferred process is as follows: the process of converting inulin to synthesize bis-fructose III is preferred: the concentration of chrysanthemum candy transferase is 5-10 U/g; the pH is controlled at 5.0-6.0; and the temperature is controlled at 55-60. °C, constant temperature reaction for 6-12 hours.
  • a residual sugar elimination step may be added to the conversion solution to remove sucrose, glucose, fructose and a part of oligofructose, and the residual sugar may be removed by adding yeast or the like.
  • the specific scheme is: cooling the reaction solution, and then adding yeast cells to absorb and remove sucrose, glucose, fructose and partial oligofructose in the reaction solution, and controlling pH 5.0-6.0, temperature The reaction was controlled at 28 ° C, shaking at 200 rpm for 24-36 hours.
  • the inulin sucrase amino acid sequence is shown in SEQ ID NO.
  • the inulin glycosyltransferase amino acid sequence is shown in SEQ ID NO.
  • the supernatant after the reaction was filtered through a micropore filter (0.22), and the filtrate was analyzed by HPLC equipped with a refractive index display.
  • the HPLC conditions were: Sugarpak 1, 6.5 mm id x 300 mm calcium type cation exchange column, pure water as mobile phase, column temperature of 85 ° C, flow rate of 0.4 ml / min, and injection volume of 10 ul.
  • the concentration of the difructose III standard was 0.5%.
  • the concentration of the synthesized bis-fructose III in the reaction solution was calculated by measuring the ratio of the peak area of the bis-fructose III in the solution after the (3) reaction to the peak area of the 0.5% bis-fructose III standard.
  • the invention utilizes inulin sucrase to first convert the cheap substrate sucrose into inulin, and then converts the synthetic bio-inulin into disucrose anhydride III with chrysanthemum candy transferase, thereby avoiding the separation and purification process of inulin. Since the market price of phyto inulin is higher than that of sucrose, the direct conversion of sucrose to inulin of the present invention reduces the cost.
  • the conversion rate of inulin to bis-fructose III catalyzed by chrysanthemum candy transferase in the present invention can be as high as 54%.
  • the yeast is used to get out of the other sugars in the reaction system to obtain relatively pure bis-fructose III. The whole process is relatively simple, the production cost is low, and the energy consumption is low, which is beneficial to realize industrial production.
  • Figure 1 Inulin sucrase synthesizes inulin (sweet solution) using sucrose as a substrate.
  • sucrose substrate was completely dissolved in deionized water, the concentration was controlled to be 400 g/L, the pH was adjusted to 5.5, the temperature was controlled at 20 ° C, the inulin sucrase was added, the amount was 10 U/g sucrose, and the reaction was carried out for 30 min at a constant temperature;
  • the temperature of the reaction system is raised to 55 ° C, and then the chrysanthemum candy transferase is added thereto, the concentration of the added chrysanthemum sugar transferase is 5 U / g, the pH value is still controlled at 5.5, and the constant temperature reaction is 12 h;
  • the solution was centrifuged, and the supernatant was filtered through a microporous membrane (0.22), and the filtrate was analyzed by HPLC equipped with a refractive index display.
  • HPLC conditions were: Sugarpak 1, 6.5 mm id x 300 mm calcium type cation exchange column, pure water as mobile phase, column temperature of 85 ° C, and tassel of 0.4 ml / min.
  • the injection volume was 10 ul and the concentration of the di fructose III standard was 0.5%.
  • the concentration of the synthesized bis-fructose III in the reaction solution was calculated by using the ratio of the peak area of the bis-fructose III in the solution to the peak area of the 0.5% bis-fructose III standard.
  • the conversion of the obtained bis-fructose III reached 54%.
  • sucrose substrate was completely dissolved with deionized water, the concentration was controlled to 300 g/L, the pH was adjusted to 5.0, the temperature was controlled at 22 ° C, the inulin sucrase was added, the amount was 15 U/g sucrose, and the reaction was kept at a constant temperature for 60 min;
  • the temperature of the reaction system was raised to 60 ° C, and then the chrysanthemum candy transferase was added thereto, and the concentration of the added chrysanthemum sugar transferase was 10 U/g, the pH was still controlled at 5.0, and the constant temperature reaction was 6 h; The conversion of fructose III reached 48%.
  • sucrose substrate was completely dissolved in deionized water, the concentration was controlled to 500 g / L, the pH was adjusted to 6.0, the temperature was controlled at 30 ° C, the inulin sucrase was added, the amount was 5 U / g sucrose, and the reaction was kept at a constant temperature for 60 min;
  • the temperature of the reaction system was raised to 50 ° C, and then the chrysanthemum candy transferase was added thereto, and the concentration of the chrysanthemum candy transferase was 20 U/g, the pH was still controlled at 6.0, and the constant temperature reaction was carried out for 20 h; The conversion rate of fructose III is up to To 43%.
  • the bis-fructose III was produced according to the reaction and detection conditions in Example 1, and the reaction system was naturally cooled to 28 ° C, and then the yeast cells were added thereto, pH 5.0-6.0, placed at 28 ° C, and shaken at 200 rpm for 24-36 hours.
  • This embodiment mainly uses yeast fermentation to remove sucrose, glucose, fructose and partial oligofructose in the reaction solution, thereby obtaining relatively pure bis-fructose III for subsequent detection and separation and purification in industrial practice.
  • the conversion rate of the final bis-fructose III was similar to that of Example 1, but the solution no longer contained sucrose, glucose, fructose and partial oligofructose in the solution, thereby improving the purity of the bis-fructose.

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Abstract

本发明公开了一种合成双果糖酐III的方法，先使用菊糖蔗糖酶将蔗糖转化为菊糖，再使用菊糖果糖转移酶转化菊糖合成双果糖酐III。本发明的菊糖合成双果糖酐III的转化率为40％-54％。为了获得较纯的双果糖酐III，用酵母除去反应液中的小分子单糖。

Description

一种高效合成双果糖酐III的方法 技术领域
本发明涉及一种高效合成双果糖酐III的方法，特别是两种酶两步法实现双果糖酐III(Difructose anhydride,DFA III)的高效生产，属于功能性食品生物加工技术领域。
背景技术
近年来由于生活水平的提高,糖尿病、肥胖以及心血管疾病逐年增加并且出现了低龄化。因此，开发出能量较低、具有多功能的营养特性的甜味剂成为了消费者关注的热点。
双果糖酐III就是一种新型的功能性甜味剂，其结构主要是由两个果糖分子脱去两个水分子形成。由于异头碳上的羟基都成糖苷键，因此双果糖酐III是一种新型的非还原性双糖。由于存在着这样特殊的双糖苷键形式，因此，双果糖酐III的性质十分的稳定。对热和酸相当稳定，耐高温，在一般食品的高温加工过程中不会出现双果糖酐III的褐变或者是分解现象。双果糖酐III甜度只有蔗糖的1/2,但是热量相对蔗糖很低，只有蔗糖的1/15(0.263kcal/g)，对于糖尿病和肥胖病患者具有良好的功能，因此，双果糖酐III可以作为一种理想的蔗糖替代糖而应用到低能量食品及糖尿病人食品中，防治糖尿病。同时，双果糖酐III比蔗糖难吸湿，能够在74％的相对湿度下不吸湿，这便于糖的存储。在体内，双果糖酐III不会被肠道吸收，从而不会产生能量，因此可以作为一种甜味剂用于减肥辅助治疗。尽管不被人体的肠道直接消化吸收，但是双果糖酐III可以被肠道内的益生菌利用代谢，产生大量的益生元，从而促进人体内的益生菌生长以及身体健康。双果糖酐III能促进人体对钙、镁、锌和铜等矿物质元素以及黄酮类物质的吸收，从而有增进骨骼生长等作用。龋齿主要是由于空腔中的突变链球菌代谢蔗糖等糖产酸，从而腐蚀牙齿所致。但是双果糖酐不能被突变链球菌代谢，因此具有抗龋齿作用。另外，双果糖酐III有降低胆固醇,、高血压等多种生理功能。鉴于双果糖酐III的这些优良性质，可以将其作为食品添加剂应用到焙烤食品、饮料、糖果等中。因此，双果糖酐III在食品行业中具有广阔的前景。
自然界中就存在双果糖酐，主要在包括菊苣、菊芋等菊科植物中，但是含量较少。另外，双果糖酐III也会少量的存在于蜂蜜、咖啡等的加工过程中。由于在自然界中的含量较少，因此提取分离双果糖酐III的成本会很高。虽然化学方法也能够生产双果糖酐III，但是化学合成法有诸多的不利因素，如环境的污染等。也可以用生物催化法获得双果糖酐，1973年Uchiyama T首次从产脲节杆菌中发现了菊糖果糖转移酶(inulin fructotransferase,EC4.2.2.18)，此后发现了十余种微生物可以产生菊糖果糖转移酶。生物转化法具有原料来源广泛且价格低 廉，转化率高,适于工业化生产的优点。但是目前的方法都是直接转换植物菊糖来获得双果糖酐III，首先还需要植物菊糖，菊糖相对蔗糖价格较高，因此不具有经济性。
本发明人提出以更为廉价的蔗糖为底物，首先通过菊糖蔗糖酶将蔗糖转化为菊糖，再将合成的生物菊糖直接用菊糖果糖转移酶转化为双果糖酐III，此步骤中不需要菊糖的分离纯化，节省了能源消耗，降低生产成本。最后用酵母出去除去反应液中存在的其它糖，获得较为纯的双果糖酐III便于后续的分离纯化。
发明内容
本发明要解决的技术问题是提供一种廉价的双果糖酐III的高效合成方法，所制备的双果糖酐III具有较高的纯度。整个合成过程工艺简单，效率高，成本低。
本发明的技术方案：
一种双果糖酐III的高效合成步骤为：
1)先使用菊糖蔗糖酶将蔗糖转化为菊糖，不分离聚糖；
2)再使用菊糖果糖转移酶转化菊糖合成功能性双糖双果糖酐III。
转化蔗糖合成菊糖的工艺为：将蔗糖完全溶解于水中，其浓度控制为300～500g/l，调节pH值为5.0-6.0，温度控制在20-30℃，加入菊糖蔗糖酶，加入量为1-15U/g，恒温反应25～60min。
为了获得较好的效果，优选工艺为：转化蔗糖合成菊糖的工艺优选：菊糖蔗糖酶加入量控制在5-15U/g，pH控制在5.0-6.0，温度控制在20-30℃，恒温反应30～60min。
转化菊糖合成双果糖酐III的工艺为：将蔗糖转化液升温至50-60℃，再将向其中加入菊糖果糖转移酶，添加的菊糖果糖转移酶浓度为1-20U/g，pH值仍然控制在5.0-6.0，恒温反应2-20小时。
为了获得较好的效果，优选工艺为：转化菊糖合成双果糖酐III的工艺优选：菊糖果糖转移酶浓度为5-10U/g；pH值控制在5.0-6.0；温度控制在55-60℃，恒温反应6-12小时。
为了进一步降低成本，还可以对转化液添加残糖消除步骤，清除其中的蔗糖，葡萄糖，果糖以及部分低聚果糖，通过添加酵母菌等其他方式清除残糖。
当使用酵母菌清除残糖时，具体方案为：冷却反应液，再向其中加入酵母菌体吸收除掉反应液中的蔗糖，葡萄糖，果糖以及部分低聚果糖，控制pH5.0-6.0，温度控制在28℃，200rpm摇床反应24-36小时。
所述菊糖蔗糖酶氨基酸序列如SEQ ID NO.1所示。
所述菊糖果糖转移酶氨基酸序列如SEQ ID NO.2所示。
SEQ ID NO.1:
Figure PCTCN2016079428-appb-000001
SEQ ID NO.2:
Figure PCTCN2016079428-appb-000002
检测双果糖酐III的方法：
反应后的上清液用微孔滤膜(0.22)过滤，滤液用配备有示差折光显示器的HPLC分析。HPLC条件为：Sugarpak1，6.5mm id×300mm钙型阳离子交换柱，纯水作流动相，柱温为85℃，流速为0.4ml/min，进样量为10ul。双果糖酐III标样的浓度为0.5％。利用检测(3)反应后溶液中双果糖酐III的峰面积与0.5％的双果糖酐III标样的峰面积的比值，计算得到反应液中的合成的双果糖酐III的浓度。
本发明利用菊糖蔗糖酶先将廉价底物蔗糖转化为菊糖，再用菊糖果糖转移酶将合成的生物菊糖转化为双果糖酐III，从避免了菊糖的分离纯化过程。由于植物菊糖的市场价格较蔗糖高，因此，本发明直接转化蔗糖为菊糖降低了成本。本发明中用菊糖果糖转移酶催化菊糖为双果糖酐III的转化率能够高达54％。最后利用酵母出去反应体系中的其它糖，获得较为纯的双果糖酐III。整个工艺较为简单，生产成本低，能耗低，有利于实现工业化生产。
附图说明
图1菊糖蔗糖酶以蔗糖为底物合成菊糖(稀释后溶液)。
图2菊糖果糖转移酶以转化合成的菊糖为双果糖酐III(稀释后溶液)。
图3酵母处理合成的双果糖酐III溶液。
具体实施方式
以下是用菊糖蔗糖酶和菊糖果糖转移酶实现转化蔗糖为双果糖酐III的实例，说明本发明的方法，但本发明并不限于所列出的几个实例。
实施例1
用去离子水将蔗糖底物完全溶解，其浓度控制为400g/L,调节pH值为5.5，温度控制在20℃，加入菊糖蔗糖酶，加入量为10U/g蔗糖，恒温反应30min；
将反应体系的温度提升至55℃，再将向其中加入菊糖果糖转移酶，添加的菊糖果糖转移酶的浓度为5U/g，pH值仍然控制在5.5，恒温反应12h；
取反应后溶液离心，上清液用微孔滤膜(0.22)过滤，滤液用配备有示差折光显示器的HPLC分析。HPLC条件为：Sugarpak1，6.5mm id×300mm钙型阳离子交换柱，纯水作流动相，柱温为85℃，流苏为0.4ml/min。进样量为10ul,双果糖酐III标样的浓度为0.5％。利用反应后溶液中双果糖酐III的峰面积与0.5％的双果糖酐III标样的峰面积的比值，计算得到反应液中的合成的双果糖酐III的浓度。所获得的双果糖酐III的转化率达到54％。
实施例2
用去离子水将蔗糖底物完全溶解，其浓度控制为300g/L,调节pH值为5.0，温度控制在22℃，加入菊糖蔗糖酶，加入量为15U/g蔗糖，恒温反应60min；
将反应体系的温度提升至60℃，再将向其中加入菊糖果糖转移酶，添加的菊糖果糖转移酶的浓度为10U/g，pH值仍然控制在5.0，恒温反应6h；所获得的双果糖酐III的转化率达到48％。
实施例3
用去离子水将蔗糖底物完全溶解，其浓度控制为500g/L,调节pH值为6.0，温度控制在30℃，加入菊糖蔗糖酶，加入量为5U/g蔗糖，恒温反应60min；
将反应体系的温度提升至50℃，再将向其中加入菊糖果糖转移酶，添加的菊糖果糖转移酶的浓度为20U/g，pH值仍然控制在6.0，恒温反应20h；所获得的双果糖酐III的转化率达 到43％。
实施例4
按照实施例1中反应和检测条件生产双果糖酐III，将反应体系自然冷却至28℃，再向其中加入酵母菌体，pH 5.0-6.0，置于28℃，200rpm摇床反应24-36小时。此实施例子主要是用酵母发酵除去反应液中的蔗糖，葡萄糖，果糖、部分低聚果糖，从而得到较为纯的双果糖酐III，以便于工业实施中的后续检测和分离纯化。最终双果糖酐III的转化率与实施例1相近，但是反应后溶液中不再含有蔗糖，葡萄糖，果糖、部分低聚果糖，实现双果糖酐纯度的提高。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2016079428-appb-000003
Figure PCTCN2016079428-appb-000004
Figure PCTCN2016079428-appb-000005
Figure PCTCN2016079428-appb-000006
Figure PCTCN2016079428-appb-000007
Figure PCTCN2016079428-appb-000008
Figure PCTCN2016079428-appb-000009
Figure PCTCN2016079428-appb-000010
Figure PCTCN2016079428-appb-000011
Figure PCTCN2016079428-appb-000012

Claims (10)

  1. 一种高效合成双果糖酐III的方法，其特征在于，先使用菊糖蔗糖酶将蔗糖转化为菊糖，不分离聚糖，再使用菊糖果糖转移酶转化菊糖合成功能性双糖双果糖酐III。
  2. 权利要求1所述的方法，其特征在于转化蔗糖合成菊糖的工艺为：将蔗糖完全溶解于水中，其浓度控制为300-500g/l，调节pH值为5.0-6.0，温度控制在20-30℃，加入菊糖蔗糖酶，加入量为1-15U/g，恒温反应25-60min。
  3. 权利要求1或2所述的方法，其特征在于转化蔗糖合成菊糖的工艺优选：菊糖蔗糖酶加入量控制在5-15U/g，pH控制在5.0-6.0，温度控制在20-30℃，恒温反应30-60min。
  4. 权利要求1或2所述的方法，其特征在于所述转化菊糖合成双果糖酐III的工艺为：将蔗糖转化液升温至50-60℃，再将向其中加入菊糖果糖转移酶，菊糖果糖转移酶浓度为1-20U/g，pH值控制在5.0-6.0，恒温反应2-20小时。
  5. 权利要求4所述的方法，其特征在于所述转化菊糖合成双果糖酐III的工艺优选：菊糖果糖转移酶浓度为5-10U/g；pH值控制在5.0-6.0；温度控制在55-60℃，恒温反应6-12小时。
  6. 权利要求1所述的方法，其特征在于还包括残糖消除步骤，清除其中的蔗糖、葡萄糖、果糖以及部分低聚果糖。
  7. 权利要求6所述的方法，其特征在于通过添加酵母菌清除残糖。
  8. 权利要求1或4所述的方法，其特征在于所述菊糖蔗糖酶氨基酸序列如SEQ ID NO.1所示。
  9. 权利要求1或4所述的方法，其特征在于所述菊糖果糖转移酶氨基酸序列如SEQ ID NO.2所示。
  10. 权利要求6所述的方法，其特征在于残糖消除步骤为：冷却反应液，再向其中加入酵母菌体吸收除掉反应液中的蔗糖，葡萄糖，果糖以及部分低聚果糖，控制pH5.0-6.0，温度控制在28℃，200rpm摇床反应24-36小时。
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