CN114657227B - 一种高纯度莱鲍迪苷d的制备方法 - Google Patents
一种高纯度莱鲍迪苷d的制备方法 Download PDFInfo
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- CN114657227B CN114657227B CN202210180976.6A CN202210180976A CN114657227B CN 114657227 B CN114657227 B CN 114657227B CN 202210180976 A CN202210180976 A CN 202210180976A CN 114657227 B CN114657227 B CN 114657227B
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- rebaudioside
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- glycosyltransferase
- ethanol
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Abstract
本发明公开了一种高纯度莱鲍迪苷D的制备方法,使用糖基转移酶催化生产莱鲍迪苷D,所述糖基转移酶如下所述:(1)糖基转移酶的氨基酸序列如SEQ ID NO.1所示;(2)以SEQ ID NO.1所示的氨基酸序列中,经过取代、缺失或者添加一个或一个以上氨基酸,获得比SEQ ID NO.1更高催化活性的糖基转移酶。该方法利用生物酶催化技术制备高纯度莱鲍迪苷D的方法,可以突破天然提取法的限制,实现莱鲍迪苷D的规模化生产,得到纯度高于95%的莱鲍迪苷D。
Description
技术领域
本发明属于莱鲍迪苷D技术领域,具体涉及一种高纯度莱鲍迪苷D的制备方法。
背景技术
随着人们对健康的日渐关注,全球多国纷纷针对减糖推出各项政策,各类代糖在近年来得到了快速发展。甜菊糖是一种高甜度、低热量的天然甜味剂。国内外大量毒理学实验结果表明,甜菊糖苷摄入人体后以原形经粪便和尿液排出,无致畸形,致突变,致癌性,且不被体内微生物代谢,而且对糖尿病、肥胖症、心脏病和高血压有明显的药理作用和辅助疗效,已经FDA批准使用,是目前国内外公认的可替代蔗糖的理想健康新糖源,在国际上被誉为“世界第三糖源”和目前已知的最甜的天然糖料,现已广泛应用于保健饮料、低热值食品和医药工业中。甜菊糖在我国生产应用已经有近30年的历史,我国甜菊糖产业发展迅速,目前已经成为世界最大的甜菊糖生产国和出口国。然而,市售甜菊糖是多种糖苷的混合物,其组分复杂,甜味质相对较差,后味略带甘苦味和甘草异味,在食品工业中的应用和出口都受到了较大的限制。
莱鲍迪苷D是甜叶菊中一种含量较低的糖苷,与甜菊糖组份中其它糖苷相比,其甜度高,约为蔗糖的400倍,且甜味纯正,口感也更接近蔗糖,甘苦味和甘草异味低,稳定性好,是一种理想的天然高倍甜味剂产品,因此成为多家国际甜味剂大公司的重点研究和推广产品。然而,在甜叶菊中,莱鲍迪苷D的含量仅占0.5%,利用天然提取法生产莱鲍迪苷D成本昂贵,限制了莱鲍迪苷D在食品中的广泛应用。利用生物酶法生产莱鲍迪苷D,是目前国内外甜菊糖厂家重点突破方向。
发明内容
本发明的目的在于提供一种高纯度莱鲍迪苷D的制备方法,该方法利用糖基转移酶催化生产高纯度莱鲍迪苷D,可以突破天然提取法的限制,实现莱鲍迪苷D的规模化生产,得到纯度高于95%的莱鲍迪苷D。
本发明的上述目的可以通过以下技术方案来实现:一种高纯度莱鲍迪苷D的制备方法,使用糖基转移酶催化生产莱鲍迪苷D,所述糖基转移酶如下所述:
(1)糖基转移酶的氨基酸序列如SEQ ID NO.1所示;
(2)以SEQ ID NO.1所示的氨基酸序列中,经过取代、缺失或者添加一个或一个以上氨基酸,获得比SEQ ID NO.1更高催化活性的糖基转移酶。
优选的,所述SEQ ID NO.1所示的氨基酸序列至少有如下一个或一个以上位点的突变:第88、92、95、121、150、181、191、364位。
优选的,所述SEQ ID NO.1所示的氨基酸序列中至少有如下一个或一个以上突变:T88S、T88Q、T88L、H92N、H92S、H92V、R95H、R95A、R95G、F121V、F121P、F121M、F150T、Y181C、Y181L、Y181I、F191S、F191L、F191E、F364S。
进一步的,本发明提供的高纯度莱鲍迪苷D的制备方法,包括以下步骤:
(1)将表达有上述糖基转移酶的基因工程菌进行发酵,得发酵液,将发酵液离心,收集菌体,将菌体破碎,离心,取上清,得到含有糖基转移酶的粗酶液;
(2)选取莱鲍迪苷A和蔗糖混合溶液作为反应底物,加入步骤(1)中的粗酶液,在20~40℃温度条件下反应10~48h,然后高温灭酶,终止反应,得到含莱鲍迪苷D反应液;
(3)选取步骤(2)中的含莱鲍迪苷D反应液,离心取沉淀,加入纯水搅拌水洗,然后离心,取沉淀,得到莱鲍迪苷D粗品;
(4)选取步骤(3)得到的莱鲍迪苷D粗品,加入乙醇水溶液,搅拌溶解,离心取上清,得到莱鲍迪苷D乙醇混合溶液;
(5)将步骤(4)中的莱鲍迪苷D乙醇混合溶液蒸发回收乙醇,得到莱鲍迪苷D水溶液,将莱鲍迪苷D水溶液离心,取下层沉淀,干燥,得到高纯度莱鲍迪苷D。
优选的,步骤(1)中将表达有糖基转移酶的基因工程菌进行发酵的过程为:将表达有糖基转移酶的基因工程菌接种至LB培养基中,150~250r/min摇床培养6~10h,以2~5%(V/V)的接种量接种至TB发酵培养基中进行发酵培养,通气量为0.5~1.5VVM,搅拌转速为350~600rpm,在37℃发酵2~6h后,降温至15~25℃继续诱导8~24h,得到发酵液。
优选的,步骤(1)中将发酵液离心时采用碟片式离心机离心,离心转速为2500~4000rpm/min,离心温度5~10℃,离心时间5~10min;步骤(1)中将菌体破碎时采用高压均质机破碎,高压均质机工作时的压力为700~1000Bar,温度5~10℃,循环破碎2~4次。
优选的,步骤(2)中所述莱鲍迪苷A和蔗糖混合溶液中,所述莱鲍迪苷A的浓度为20~100g/L,其中采用的莱鲍迪苷A原料的质量百分含量为90%以上,所述蔗糖的浓度为20~200g/L;步骤(2)中所述反应底物与所述粗酶液的体积比为1:0.5~5;步骤(2)中高温灭酶是在90℃高温下灭酶10~20分钟。
优选的,步骤(3)中沉淀与纯水的用量关系为1g:1~5mL;步骤(4)中所述莱鲍迪苷D粗品与所述乙醇水溶液的用量关系为1g:1~10mL,其中所述乙醇水溶液中乙醇的体积百分含量为40~80%。
优选的,步骤(5)中蒸发回收乙醇时温度为30~45℃;步骤(5)中干燥是在50~60℃温度条件下鼓风干燥6~12h;步骤(5)中高纯度莱鲍迪苷D的纯度≥95%(w/w)。
优选的,步骤(1)中离心时离心机转速为4000~6000rpm/min;步骤(3)~(5)中离心时离心机的转速为4000~6000rpm/min。
本发明具有以下优点:
(1)本发明通过筛选获得SEQ ID NO.1所示的糖基转移酶的氨基酸及其突变体,通过构建基因工程菌并发酵,获得含有如SEQ ID NO.1所示序列的糖基转移酶粗酶液,采用该粗酶液对莱鲍迪苷A进行催化转化,可以获得较高产率和纯度的莱鲍迪苷D;
(2)本发明方法利用生物酶催化技术制备高纯度莱鲍迪苷D的方法,可以突破天然提取法的限制,实现莱鲍迪苷D的规模化生产,得到纯度高于95%的莱鲍迪苷D。
具体实施方式
下面结合具体实施例对本发明做进一步说明,以便本领域技术人员可以更好的了解本发明,但不因此限制本发明。
以下实施例中,莱鲍迪苷D含量检测方法依照GB 8270-2014进行测定。
实施例1酶液制备
取表达有SEQ ID NO.1所示序列糖基转移酶的基因工程菌(在ncbi数据库通过基因挖掘筛选得到糖基转移酶目的基因片段,目的基因片段编码的氨基酸序列如SEQ IDNO.1所示,合成目的基因片段后将其连接入pET28a,两端酶切位点分别为NcoI和BamHI,得到重组表达载体,采用常规方法,将含SEQ ID NO.1所示序列的重组表达载体转移至常规工程菌比如大肠杆菌中,采用工程菌发酵产生糖基转移酶,用糖基转移酶进行催化生产莱鲍迪苷D,基因工程菌的获取过程也可参考但不限于CN111518782A中的描述获取)接种于LB培养基中,200r/min摇床培养8h,以3%接种量(体积百分含量)接入TB培养基中发酵培养,通气量1VVM,搅拌转速350rpm,37℃发酵4h后,降温至25℃,继续发酵18h,得到发酵液,发酵液经碟式离心机离心,4000rpm/min,离心温度5℃,离心时间10min,收集菌体,菌体经高压均质机破碎,压力800bar,温度5℃,循环破碎2次,6000rpm/min离心取上清,得到含有糖基转移酶的粗酶液。
LB培养基:胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,pH 7.0。
TB培养基:胰蛋白胨15g/L,酵母提取物20g/L,氯化钠10g/L,葡萄糖2g/L,乳糖2g/L,pH 7.0。
实施例2
与实施例1不同的是,基因工程菌为表达有SEQ ID NO.1所示序列的T88Q突变体。
实施例3
与实施例1不同的是,基因工程菌为表达有SEQ ID NO.1所示序列的H92S突变体。
实施例4
与实施例1不同的是,基因工程菌为表达有SEQ ID NO.1所示序列的R95H突变体。
实施例5
与实施例1不同的是,基因工程菌为表达有SEQ ID NO.1所示序列的F121V突变体。
实施例6
与实施例1不同的是,基因工程菌为表达有SEQ ID NO.1所示序列的F191S突变体。
实施例7酶活力测定
配制含40g/L莱鲍迪苷A(其中莱鲍迪苷A原料的质量百分含量为90%以上),80g/L蔗糖水溶液的反应底物,以反应底物:粗酶液(实施例1-实施例6中的粗酶液)=1:1(v/v)的比例混合,30℃条件下反应12h,90℃高温10min终止反应,取样进行HPLC检测,不同突变体莱鲍迪苷D合成量如表1所示。
表1不同突变体莱鲍迪苷D产量
实施例8酶催化反应
配制含40g/L莱鲍迪苷A,80g/L蔗糖水溶液的反应底物,以反应底物:粗酶液(实施例5中F121V突变体)=1:1(v/v)的比例混合,30℃条件下反应24h,90℃高温10min终止反应,得到莱鲍迪苷D反应液,转化率为89.5%(以莱鲍迪苷A计)。
实施例9酶催化产物分离
取实施例8中的酶催化反应液离心取沉淀,加入1~5倍(沉淀与纯水的用量关系为1g:1~5mL)纯水搅拌水洗,离心,4000rpm/min离心,取沉淀,得到莱鲍迪苷D粗品,不同纯水倍数水洗,莱鲍迪苷D粗品含量如表2所示。纯水水洗倍数越高,莱鲍迪苷D粗品含量越高,有利于后续纯化。但水洗倍数越高,终产品收率将受到影响,因此不宜采用高于5倍纯水水洗。
表2莱鲍迪苷D粗品含量
实施例10莱鲍迪苷D纯化
取实施例9中的莱鲍迪苷D粗品加入5倍体积40~80%(v/v)浓度乙醇(莱鲍迪苷D粗品与所述乙醇水溶液的用量关系为1g:1~10mL),搅拌溶解,4000rpm/min离心取上清,得到莱鲍迪苷D乙醇混合溶液。莱鲍迪苷D乙醇混合溶液40℃条件下蒸发回收乙醇,得到莱鲍迪苷D水溶液。莱鲍迪苷D水溶液经4000rpm/min离心,60℃鼓风干燥12h,得到莱鲍迪苷D产品。不同浓度乙醇提纯,莱鲍迪苷D产品纯度及收率如表3所示。乙醇浓度越高,莱鲍迪苷D产品纯度越高,但产品收率降低。综合成本因素考虑,乙醇浓度为40~60%最适宜。
表3莱鲍迪苷D产品纯度及收率
乙醇浓度(%,v/v) | 40 | 50 | 60 | 70 | 80 |
莱鲍迪苷D产品纯度(%,w/w) | 95.2 | 96.5 | 96.6 | 96.8 | 97.1 |
收率(%) | 91.8 | 91.1 | 90.5 | 88.7 | 88.5 |
以上实施例仅用于阐述本发明,而本发明的保护范围并非仅仅局限于以上实施例。所属技术领域的普通技术人员依据以上本发明公开的内容均可实现本发明的目的,任何基于本发明构思基础上做出的改进和变形,均落入本发明的保护范围之内,具体保护范围以权利要求书记载的为准。
序列表
<110> 翁源广业清怡食品科技有限公司
广东广业清怡食品科技股份有限公司
<120> 一种高纯度莱鲍迪苷D的制备方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 443
<212> PRT
<213> 糖基转移酶(glycosyltransferases)
<400> 1
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Claims (7)
1.一种高纯度莱鲍迪苷D的制备方法,其特征在于:使用糖基转移酶催化生产莱鲍迪苷D,所述糖基转移酶如下所述:
(A)糖基转移酶的氨基酸序列如SEQ ID NO.1所示;
(B)在SEQ ID NO.1所示的氨基酸序列的基础上,仅发生以下突变中任意一种:T88Q、H92S、R95H、F121V、F191S;
所述制备方法包括以下步骤:
(1)将表达有所述糖基转移酶的基因工程菌进行发酵,得发酵液,将发酵液离心,收集菌体,将菌体破碎,离心,取上清,得到含有糖基转移酶的粗酶液;
(2)选取莱鲍迪苷A和蔗糖混合溶液作为反应底物,加入步骤(1)中的粗酶液,在20~40℃温度条件下反应10~48h,然后高温灭酶,终止反应,得到含莱鲍迪苷D反应液;
(3)选取步骤(2)中的含莱鲍迪苷D反应液,离心取沉淀,加入纯水搅拌水洗,然后离心,取沉淀,得到莱鲍迪苷D粗品;
(4)选取步骤(3)得到的莱鲍迪苷D粗品,加入乙醇水溶液,搅拌溶解,离心取上清,得到莱鲍迪苷D乙醇混合溶液;
(5)将步骤(4)中的莱鲍迪苷D乙醇混合溶液蒸发回收乙醇,得到莱鲍迪苷D水溶液,将莱鲍迪苷D水溶液离心,取下层沉淀,干燥,得到高纯度莱鲍迪苷D。
2.根据权利要求1所述的高纯度莱鲍迪苷D的制备方法,其特征是:步骤(1)中将表达有糖基转移酶的基因工程菌进行发酵的过程为:将表达有糖基转移酶的基因工程菌接种至LB培养基中,150~250r/min摇床培养6~10h,以2~5%(V/V)的接种量接种至TB发酵培养基中进行发酵培养,通气量为0.5~1.5VVM,搅拌转速为350~600rpm,在37℃发酵2~6h后,降温至15~25℃继续诱导8~24h,得到发酵液。
3.根据权利要求1所述的高纯度莱鲍迪苷D的制备方法,其特征是:步骤(1)中将发酵液离心时采用碟片式离心机离心,离心转速为2500~4000rpm,离心温度5~10℃,离心时间5~10min;步骤(1)中将菌体破碎时采用高压均质机破碎,高压均质机工作时的压力为700~1000Bar,温度5~10℃,循环破碎2~4次。
4.根据权利要求1所述的高纯度莱鲍迪苷D的制备方法,其特征是:步骤(2)中所述莱鲍迪苷A和蔗糖混合溶液中,所述莱鲍迪苷A的浓度为20~100g/L,其中采用的莱鲍迪苷A原料的质量百分含量为90%以上,所述蔗糖的浓度为20~200g/L;步骤(2)中所述反应底物与所述粗酶液的体积比为1:0.5~5;步骤(2)中高温灭酶是在90℃高温下灭酶10~20分钟。
5.根据权利要求1所述的高纯度莱鲍迪苷D的制备方法,其特征是:步骤(3)中沉淀与纯水的用量关系为1g:1~5mL;步骤(4)中所述莱鲍迪苷D粗品与所述乙醇水溶液的用量关系为1g:1~10mL,其中所述乙醇水溶液中乙醇的体积百分含量为40~80%。
6.根据权利要求1所述的高纯度莱鲍迪苷D的制备方法,其特征是:步骤(5)中蒸发回收乙醇时温度为30~45℃;步骤(5)中干燥是在50~60℃温度条件下鼓风干燥6~12h;步骤(5)中高纯度莱鲍迪苷D的纯度≥95%(w/w)。
7.根据权利要求1所述的高纯度莱鲍迪苷D的制备方法,其特征是:步骤(1)中离心时离心机转速为4000~6000rpm;步骤(3)~(5)中离心时离心机的转速为4000~6000rpm。
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