一种酶改制甜菊糖苷的制备用酶及其应用
阅读说明：本技术 一种酶改制甜菊糖苷的制备用酶及其应用 (Enzyme for preparing enzyme-modified stevioside and application thereof ) 是由 杨峰 李山河 张振雷 李艳萍 赵蕾 于 2019-11-12 设计创作，主要内容包括：本发明公开一种酶改制甜菊糖苷的制备用酶及其应用,所述酶来源自果糖苷酶GT29或α-半乳糖苷酶BRT21,果糖苷酶GT29的核苷酸DNA序列如SEQ No.1所示,氨基酸PRT序列如SEQ No 2所示；α-半乳糖苷酶BRT21的核苷酸DNA序列如SEQ No.3所示,氨基酸PRT序列如SEQ No 4所示。利用酶法催化具有后苦味的甜菊苷,生成味道更正的果糖-甜菊苷和半乳糖-甜菊苷,适合工业化生产,为甜菊糖苷的应用和产业链发展具有重要意义。(The invention discloses an enzyme for preparing enzyme-modified stevioside and application thereof, wherein the enzyme is derived from fructosidase GT29 or alpha-galactosidase BRT21, the nucleotide DNA sequence of the fructosidase GT29 is shown as SEQ No.1, and the amino acid PRT sequence is shown as SEQ No. 2; the nucleotide DNA sequence of alpha-galactosidase BRT21 is shown in SEQ No.3, and the amino acid PRT sequence is shown in SEQ No. 4. The enzyme method is used for catalyzing stevioside with the aftertaste to generate fructose-stevioside and galactose-stevioside with more corrected taste, is suitable for industrial production, and has important significance for the application of stevioside and the development of industrial chains.)
技术领域
本发明涉及甜菊糖，具体是一种酶改制甜菊糖苷的制备用酶，以及应用该酶对甜菊糖苷进行改制的方法。
背景技术
甜菊糖苷是从植物甜叶菊提取出来的，是一种甜度很高的天然甜味剂，广泛应用于食品甜味剂。然而，天然甜菊糖的结构苷，无甜味且具有苦涩味，其中甜菊苷（图1）是甜菊糖苷中含量最高的成分之一，后苦味明显，破坏了甜菊糖的口感，也限制了其更为广泛的工业应用。因此，改善甜菊糖的甜味特征具有重要意义。
现有技术，大多是采用化学提纯的方法，除去具有苦味的成分，提高口感，生成味道更正的甜菊糖苷A，但该方法能耗高。或是利用环糊精葡萄糖转移酶在甜菊糖苷分子上随机加一分子或多分子葡萄糖，产物多且复杂，稳定性不高，安全性和口感均不能得到稳定的保障，难以规模化、工业化生产。
发明内容
本发明的目的是针对现有技术的不足，而提供一种酶改制甜菊糖苷的制备用酶，应用该酶对甜菊糖苷进行改制，工艺简单、能耗低、产率高且绿色环保，适合工业化生产。
实现本发明目的的技术方案是：
一种酶改制甜菊糖苷的制备用酶，所述酶来源自果糖苷酶GT29或α-半乳糖苷酶BRT21，果糖苷酶GT29的核苷酸DNA序列如SEQ No.1所示，氨基酸PRT序列如SEQ No 2所示；α-半乳糖苷酶BRT21的核苷酸DNA序列如SEQ No.3所示，氨基酸PRT序列如SEQ No 4所示。
上述酶改制甜菊糖苷的制备用酶的制备方法是：将GT29或BRT21酶基因构建至大肠杆菌或酵母表达系统，将上述工程菌用发酵罐培养，离心收集菌体，所得菌体加入破菌液，均质机破菌，镍柱纯化，冻干分别制得果糖苷酶GT29酶粉和α-半乳糖苷酶BRT21酶粉。
应用制得的果糖苷酶GT29酶粉对甜菊糖苷进行改质的方法是：
称取甜菊苷，加入果糖苷酶GT29酶粉，蔗糖，蒸馏水，搅拌，控制温度25～50℃，用HCl/NaOH调节pH值在7.0~7.5之间，反应8~12h；高效液相色谱检测反应，当转化率达到94%~96%升温至100℃加热30min终止反应；过滤去除蛋白，得滤液，重结晶制得产品。
应用制得的α-半乳糖苷酶BRT21酶粉对甜菊糖苷进行改质的方法是：称取甜菊苷，加入α-半乳糖苷酶BRT21酶粉，半乳糖，蒸馏水，搅拌，控制温度25～50℃，用HCl/NaOH调节pH值在7.0~7.5之间，反应8~12h；高效液相色谱检测反应，当转化率达到93%~95%升温至100℃加热30min终止反应；过滤去除蛋白，得滤液，重结晶制得产品。
本发明的有益效果：
（1）利用酶催化将具有后苦味的甜菊苷分子上加一分子的果糖/半乳糖转化为半乳糖-甜菊苷/果糖-甜菊苷，转化率高达96%，为甜菊糖苷的应用和产业链发展具有重要意义；
（2） 制备所得产品无苦味，口感正，甜度是蔗糖的250~300倍；
（3）本发明方法中采用催化反应温度25～50℃、pH值7.0～7.5，反应温和，操作简单，酶法生产属于绿色合成，适用于工业化应用。
附图说明
图1为甜菊苷的分子结构图。
具体实施方式
下面结合实施例对本发明内容作进一步的说明，但不是对本发明的限定。
实施例：
酶改制甜菊糖苷的制备用酶，所述酶来源自果糖苷酶GT29或α-半乳糖苷酶BRT21，果糖苷酶GT29的核苷酸DNA序列如SEQ No.1所示，氨基酸PRT序列如SEQ No. 2所示；α-半乳糖苷酶BRT21的核苷酸DNA序列如SEQ No.3所示，氨基酸PRT序列如SEQ No. 4所示。
所述的DNA序列均是人工序列，根据氨基酸的序列做了密码子优化，非天然的DNA序列。
所述酶的制备方法是：将GT29或BRT21酶基因构建至大肠杆菌或酵母表达系统，将上述工程菌用发酵罐培养，离心收集菌体，所得菌体加入破菌液，均质机破菌，镍柱纯化，冻干分别制得果糖苷酶GT29酶粉和α-半乳糖苷酶BRT21酶粉。
应用制得的果糖苷酶GT29酶粉对甜菊糖苷进行改质的方法是：称取10g甜菊苷，加入0.1g果糖苷酶GT29酶粉，蔗糖1g，蒸馏水100mL，搅拌，控制温度35℃；用1M HCl/NaOH调节pH值在7.0~7.5之间，反应8~12h；高效液相色谱检测反应，当转化率达到94%~96%升温至100℃加热30min终止反应；过滤去除蛋白，得滤液，重结晶2次制得产品，检测甜度，为蔗糖的300倍。
应用制得的α-半乳糖苷酶BRT21酶粉对甜菊糖苷进行改质的方法是：称取10g甜菊苷，加入0.1g半乳糖苷酶BRT21酶粉，半乳糖1.2g，蒸馏水100mL，搅拌，控制温度35℃；用1MHCl/NaOH调节pH值在7.0~7.5之间，反应8~12h；高效液相色谱检测反应，当转化率达到93%~95%升温至100℃加热30min终止反应；过滤去除蛋白，得滤液，重结晶2次制得产品，检测甜度，为蔗糖的275倍。
通过实施例可以看出，采用本发明酶粉对甜菊糖苷进行催化，是在甜菊苷上加一分子的果糖或半乳糖，经高效液相、质谱和核磁验证为单一产物，产品稳定性高，口感稳定，适合工业化生产。
序列表
<110> 广西师范大学
<120> 一种酶改制甜菊糖苷的制备用酶及其应用
<141> 2019-11-12
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ccccccaccg gcctgctgaa cgaccccaac ggcctgatct tcgacggcga gaactaccac 180
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