CN110177881B - 一种酶法制备瑞鲍迪甙j的方法 - Google Patents
一种酶法制备瑞鲍迪甙j的方法 Download PDFInfo
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Abstract
提供了一种酶法制备瑞鲍迪甙J的方法,该方法以瑞鲍迪甙A为底物,在糖基供体存在下,在含有UDP‑糖基转移酶的重组细胞和/或其制备的UDP‑糖基转移酶的催化下,反应生成瑞鲍迪甙J。
Description
技术领域
本发明涉及一种瑞鲍迪甙J的制备方法,特别涉及一种瑞鲍迪甙J的生物制备方法。
背景技术
甜味剂是一类广泛应用于饮料及糖果等食品生产的食品添加剂,其既可以在食品的生产过程中添加,也可以在家庭烘焙时经过适当稀释作为蔗糖的替代品使用。甜味剂包括天然甜味剂和人工甜味剂,前者包括蔗糖、高果糖玉米糖浆、蜜糖等,后者包括阿斯巴甜、糖精等。甜菊糖是一类从植物甜菊中提取出来的天然甜味剂,目前已被广泛用在食品及饮料中。甜菊的提取物中具有包含瑞鲍迪甙在内的多种甜菊糖,天然提取的甜菊糖不同的批次成分差异较大,需要后续的提纯。
瑞鲍迪甙J在甜菊叶中甜菊糖的比例不超过0.5%,用传统提取的方法得到高纯度的瑞鲍迪甙J极其困难,限制了对瑞鲍迪甙J的深入研究,也阻碍了其商业化的应用。
发明内容
本发明所要解决的技术问题是克服现有技术的不足,提供一种酶法制备瑞鲍迪甙J的方法,该方法可以较低的成本、较短的周期生产出高纯度的瑞鲍迪甙J产品。
为解决以上技术问题,本发明采取如下技术方案:
一种酶法制备瑞鲍迪甙J的方法,以瑞鲍迪甙A为底物,在糖基供体存在下,在含有UDP-糖基转移酶的重组细胞和/或其制备的UDP-糖基转移酶的催化下,反应生成瑞鲍迪甙J。UDP-糖基转移酶即尿苷二磷酸糖基转移酶,简称UGT,是已知的。
优选地,所述糖基供体为鼠李糖基供体。
更优选地,所述鼠李糖基供体为UDP-鼠李糖。
优选地,所述UDP-糖基转移酶为来自水稻(Oryza sativa)的UGT-B。
优选地,所述来自水稻的UGT-B的氨基酸序列与序列表中所示的序列2具有至少60%的一致性。
更优选地,所述来自水稻的UGT-B的氨基酸序列与序列表中所示的序列2具有至少70%的一致性。
进一步地,所述来自水稻的UGT-B的氨基酸序列与序列表中所示的序列2具有至少80%的一致性。
更进一步地,所述来自水稻的UGT-B的氨基酸序列与序列表中所示的序列2具有至少90%的一致性。
根据一个具体实例,所述来自水稻的UGT-B的氨基酸序列与序列表中所示的序列2完全一致。
根据本发明,可以使所述反应在温度4-50℃以及pH5.0-9.0的水相体系中进行。优选地,反应在35-45℃温度以及pH7.5-8.5的水相体系中进行。更优选地,反应在温度40℃下进行,反应在pH 8.0下进行。
更优选地,反应在磷酸缓冲溶液中进行。
更优选地,反应体系含有UDP-糖基转移酶的重组细胞和细胞通透剂,是反应在细胞通透剂的存在下进行。进一步地,所述细胞通透剂为甲苯,且甲苯在反应体系中的体积比浓度为1-3%。更进一步地,甲苯的体积比浓度为2%。
更优选地,将反应所用全部原料加入到反应釜中,混合均匀后置于设定温度下,搅拌反应。反应完毕后,通过提纯处理即可获得达到使用要求的瑞鲍迪甙J产品。一个具体的提纯方方法是经包括树脂分离在内的后处理,按照该提纯方法,可获得纯度高达95%的瑞鲍迪甙J产品。
优选地,所述重组细胞为微生物细胞。
更优选地,所述微生物为大肠埃希氏杆菌、酿酒酵母或毕赤酵母。
由于以上技术方案的实施,本发明与已有技术相比具有如下优势:
本发明提供的酶法制备瑞鲍迪甙J的方法具有重要的应用价值。由于酶法生产的底物瑞鲍迪甙A可大量获得,瑞鲍迪甙J的产量不受原材料的限制,极大的降低了生产成本。此外,植物中的甜菊糖含量低,且有较多不同结构的甜菊糖,很难提取较纯的产品,与已有从甜菊叶中提取瑞鲍迪甙J的技术相比,采用本发明的酶法合成方法能够提供纯度更高的产品,必将推动对新型甜菊糖苷瑞鲍迪甙J的研究和应用。
具体实施方式
瑞鲍迪甙A、瑞鲍迪甙J的结构式分别参见式I和II。
本发明主要提供的瑞鲍迪甙J的合成路线如下:
本发明所用的UGT-B可以酶冻干粉形式存在或存在于重组细胞中。
UGT-B的获得方法如下:
利用分子克隆技术、基因工程技术获得UGT-B的重组大肠埃希氏杆菌(或其它微生物菌)表达菌株,然后将重组大肠埃希氏杆菌发酵,制备得到含有UGT-B的重组细胞,或由上述重组细胞制备得到UGT-B的冻干粉。
本发明所述的分子克隆技术和基因工程技术均是已知的。分子克隆技术可参见《分子克隆实验指南》第三版(J.沙姆布鲁克著,2005)。
采用基因工程技术构建本发明重组菌株的表达步骤如下:
(1)(根据序列表中所示的序列1及序列2)基因合成所需的基因片段,连入pUC57载体,两端分别加上NdeI和BamHI酶切位点;
(2)通过双酶切、连接,将各基因片段插入表达载体pET30a相应的酶切位点中,使各基因置于T7启动子的控制之下;
(3)将重组质粒转化进入大肠埃希氏杆菌BL21(DE3)中,利用IPTG诱导目的蛋白表达,得到UGT-B的重组大肠埃希氏杆菌表达菌株。
利用含有UGT-B的重组大肠埃希氏杆菌表达菌株制备含有UGT-B的重组细胞、UGT-B的冻干粉的步骤如下:
以1%比例将含有UGT-B的重组大肠埃希氏杆菌表达菌株接种到4ml液体LB培养基中,37℃振荡培养(200rpm)过夜,取过夜培养物以1%接种量转接于50ml液体LB培养基,37℃振荡培养(200rpm)至OD600值达到0.6-0.8,加入终浓度0.4mM IPTG于20℃振荡培养过夜。诱导结束后离心收集细胞(8,000rpm,10min),用5ml 2mmol/L磷酸缓冲液(pH7.0)重悬细胞,获得所述重组细胞,进一步于冰浴中超声波破碎细胞,将破碎液离心(8,000rpm,10min),收集上清液冻干24h,获得所述冻干粉。
下面结合具体的实施例对本发明作更为详细的描述。
实施例1:制备含UGT-B的重组大肠杆菌细胞
根据序列1及序列2,基因合成UGT-B基因片段,两端分别加上NdeI和BamHI酶切位点,连入pUC57载体(苏州金唯智生物技术有限公司生产)。将UGT基因片段用限制性内切酶NdeI和BamHI酶切,回收纯化片段,加入T4连接酶将片段连入pET30a对应酶切位点,转化BL21(DE3)菌株。
以1%比例将UGT菌种接种到4ml液体LB培养基,37℃振荡培养(200rpm)过夜,取过夜培养物以1%接种量转接于50ml液体LB培养基,37℃振荡培养(200rpm)至OD 600值达到0.6-0.8,加入终浓度0.4mM IPTG于20℃振荡培养过夜。诱导结束后离心收集细胞(8,000rpm,10min),用5ml 2mmol/L磷酸缓冲液(pH7.0)重悬细胞,获得含UGT-B的重组细胞用于催化。
实施例2:制备UGT-B冻干粉
将实施例3中制得的UGT-B的重组细胞于冰浴中超声波破碎细胞,将破碎液离心(8,000rpm,10min),收集上清液冻干24h,获得UGT-B的冻干粉。
实施例3:以瑞鲍迪甙A为底物在UDP-糖基转移酶的催化下合成瑞鲍迪甙J
在本实施例中,按照实施例2方法制备的UGT-B冻干粉被用于催化合成瑞鲍迪甙J。
在反应体系中依次加入1L 0.05mol/L磷酸缓冲液(pH8.0),2g UDP鼠李糖,1g瑞鲍迪甙A,UGT-B冻干粉10g混合均匀后置于40℃水浴,300rpm搅拌反应24h。反应结束后,取500μl反应液加入等体积无水甲醇混匀,8,000rpm离心10min取上清液过滤膜后用高效液相色谱检测(色谱条件:色谱柱:Agilent eclipse SB-C18 4.6×150mm;检测波长:210nm;流动相:乙腈∶去离子水=24%∶76%;流速:1.0mL/min;柱温:30℃)。瑞鲍迪甙A的转化率为90%以上。经硅胶树脂分离、结晶等后处理纯化后得到瑞鲍迪甙J 0.52g,纯度大于95%。
实施例4:以瑞鲍迪甙A为底物在UDP-糖基转移酶的重组细胞的催化下合成瑞鲍迪
甙J
在本实施例中,按照实施例1方法制备的含UGT-B的重组细胞用于催化合成瑞鲍迪甙J。
在反应体系中依次加入1L 0.05mol/L磷酸缓冲液(pH8.0),2g UDP-鼠李糖,1g瑞鲍迪甙A,20ml甲苯,UGT-B全细胞40g混合均匀后置于40℃水浴,300rpm搅拌反应24h。反应结束后,取500μl反应液离心,上清加入等体积无水甲醇混匀,8,000rpm离心10min取上清液过滤膜后用高效液相色谱检测(色谱条件:色谱柱:Agilent eclipse SB-C18 4.6×150mm;检测波长:210nm;流动相:乙腈∶去离子水=24%∶76%;流速:1.0mL/min;柱温:30℃)。瑞鲍迪甙A的转化率为90%以上。经硅胶树脂分离、结晶等后处理纯化后得到瑞鲍迪甙J 0.49g,纯度大于95%。
上述实施例只为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围,凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
序列表
<110> 苏州汉酶生物技术有限公司;百事可乐公司
<120> 一种酶法制备瑞鲍迪甙J的方法
<160> 2
<210> 1
<211> 1381
<212> DNA
<213> 水稻
<400> 1
1 ATGGACAGCG GTTACTCTTC TAGCTATGCT GCGGCAGCCG GTATGCACGT AGTTATTTGT
61 CCGTGGCTCG CTTTCGGTCA CCTCCTGCCG TGCCTGGACC TGGCGCAGCG CCTGGCATCT
121 CGTGGTCACC GTGTCAGTTT CGTTAGCACG CCGCGTAACA TCTCACGTCT GCCGCCGGTC
181 CGTCCGGCTC TGGCCCCGCT GGTTGCGTTC GTTGCGCTAC CTCTGCCGCG CGTTGAAGGC
241 TTACCGGATG GCGCAGAGTC TACCAACGAC GTGCCGCACG ATCGCCCGGA TATGGTTGAA
301 CTCCACCGCC GTGCATTTGA CGGTCTGGCA GCTCCGTTCT CCGAATTTCT GGGTACCGCG
361 TGTGCCGACT GGGTCATCGT AGACGTATTC CACCACTGGG CAGCTGCAGC GGCTTTAGAA
421 CACAAAGTAC CGTGCGCAAT GATGCTGCTG GGCTCTGCTC ACATGATCGC GTCTATTGCC
481 GACCGTCGTC TGGAACGTGC AGAGACCGAA TCTCCAGCGG CAGCCGGTCA GGGCCGTCCT
541 GCAGCTGCTC CGACCTTCGA AGTTGCTCGT ATGAAGCTCA TCCGCACTAA AGGTTCTTCC
601 GGTATGTCAC TGGCAGAGCG TTTCTCGCTG ACGCTCTCCC GTAGCAGCCT GGTTGTGGGG
661 CGCTCCTGCG TGGAATTCGA ACCGGAAACT GTGCCGCTAC TGTCTACCCT GCGTGGCAAG
721 CCGATCACTT TTCTGGGTCT CATGCCGCCA CTGCACGAAG GTCGCCGCGA AGACGGTGAA
781 GATGCTACGG TTCGTTGGTT GGACGCCCAG CCGGCTAAAA GCGTCGTGTA CGTAGCTCTG
841 GGCAGTGAAG TTCCATTGGG TGTCGAGAAA GTGCATGAAC TGGCTTTGGG TCTGGAGCTG
901 GCTGGCACCC GTTTCCTCTG GGCACTGCGT AAGCCGACTG GTGTGTCTGA TGCTGACCTT
961 CTGCCGGCTG GTTTCGAAGA ACGTACCCGT GGTCGCGGCG TAGTGGCAAC CCGCTGGGTA
1021 CCGCAGATGT CCATCCTGGC ACACGCTGCT GTTGGCGCGT TTCTTACCCA CTGCGGGTGG
1081 AACTCTACAA TCGAAGGCCT GATGTTCGGC CATCCTCTGA TTATGCTGCC AATCTTCGGT
1141 GATCAGGGTC CGAACGCTCG TCTGATCGAA GCCAAAAACG CCGGCTTACA AGTCGCACGC
1201 AACGACGGCG ATGGTTCTTT CGATCGTGAA GGTGTTGCGG CAGCTATCCG TGCAGTGGCT
1261 GTAGAAGAAG AGTCGAGCAA AGTGTTCCAG GCAAAAGCCA AAAAGCTGCA GGAAATCGTT
1321 GCGGACATGG CGTGCCACGA ACGTTACATC GATGGCTTTA TCCAGCAGCT GCGCTCCTAC
1381 AAAGATTAA
<210> 2
<211> 421
<212> PRT
<213> 水稻
<400> 2
1 METAspSerGlyTyrSerSerSerTyrAlaAlaAlaAlaGlyMETHisValValIleCys
21 ProTrpLeuAlaPheGlyHisLeuLeuProCysLeuAspLeuAlaGlnArgLeuAlaSer
41 ArgGlyHisArgValSerPheValSerThrProArgAsnIleSerArgLeuProProVal
61 ArgProAlaLeuAlaProLeuValAlaPheValAlaLeuProLeuProArgValGluGly
81 LeuProAspGlyAlaGluSerThrAsnAspValProHisAspArgProAspMETValGlu
101 LeuHisArgArgAlaPheAspGlyLeuAlaAlaProPheSerGluPheLeuGlyThrAla
121 CysAlaAspTrpValIleValAspValPheHisHisTrpAlaAlaAlaAlaAlaLeuGlu
141 HisLysValProCysAlaMETMETLeuLeuGlySerAlaHisMETIleAlaSerIleAla
161 AspArgArgLeuGluArgAlaGluThrGluSerProAlaAlaAlaGlyGlnGlyArgPro
181 AlaAlaAlaProThrPheGluValAlaArgMETLysLeuIleArgThrLysGlySerSer
201 GlyMETSerLeuAlaGluArgPheSerLeuThrLeuSerArgSerSerLeuValValGly
221 ArgSerCysValGluPheGluProGluThrValProLeuLeuSerThrLeuArgGlyLys
241 ProIleThrPheLeuGlyLeuMETProProLeuHisGluGlyArgArgGluAspGlyGlu
261 AspAlaThrValArgTrpLeuAspAlaGlnProAlaLysSerValValTyrValAlaLeu
281 GlySerGluValProLeuGlyValGluLysValHisGluLeuAlaLeuGlyLeuGluLeu
301 AlaGlyThrArgPheLeuTrpAlaLeuArgLysProThrGlyValSerAspAlaAspLeu
321 LeuProAlaGlyPheGluGluArgThrArgGlyArgGlyValValAlaThrArgTrpVal
341 ProGlnMETSerIleLeuAlaHisAlaAlaValGlyAlaPheLeuThrHisCysGlyTrp
361 AsnSerThrIleGluGlyLeuMETPheGlyHisProLeuIleMETLeuProIlePheGly
381 AspGlnGlyProAsnAlaArgLeuIleGluAlaLysAsnAlaGlyLeuGlnValAlaArg
401 AsnAspGlyAspGlySerPheAspArgGluGlyValAlaAlaAlaIleArgAlaValAla
421 ValGluGluGluSerSerLysValPheGlnAlaLysAlaLysLysLeuGlnGluIleVal
441 AlaAspMETAlaCysHisGluArgTyrIleAspGlyPheIleGlnGlnLeuArgSerTyr
461 LysAsp***
Claims (11)
1.一种酶法制备瑞鲍迪甙J的方法,所述方法包括使瑞鲍迪甙A与鼠李糖基供体在包含含有UDP-糖基转移酶的重组细胞和/或其制备的UDP-糖基转移酶的反应体系中反应,其中所述UDP-糖基转移酶包含SEQ ID NO:2的氨基酸序列。
2.根据权利要求1所述的方法,其中所述鼠李糖基供体为UDP-鼠李糖。
3.根据权利要求1所述的方法,其中所述UDP-糖基转移酶为来自水稻的UGT-B。
4.根据权利要求1所述的方法,其中所述反应体系包含35-45℃温度以及pH7.5-8.5的水相体系。
5.根据权利要求4所述的方法,其中所述水相体系为磷酸缓冲溶液。
6.根据权利要求4所述的方法,其中所述反应体系还包含细胞通透剂。
7.根据权利要求6所述的方法,其中所述细胞通透剂为甲苯,并且其中所述甲苯的体积比浓度为1-3%。
8.根据权利要求1所述的方法,其中所述重组细胞为微生物细胞。
9.根据权利要求8所述的方法,其中所述微生物为大肠埃希氏杆菌、酿酒酵母或毕赤酵母。
10.根据权利要求1所述的方法,其还包括通过树脂分离纯化所述瑞鲍迪甙J。
11.根据权利要求10所述的方法,其中通过树脂分离纯化的所述瑞鲍迪甙J的纯度大于95%。
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AU2016427128A1 (en) | 2019-05-30 |
BR112019007882A2 (pt) | 2019-07-02 |
CN110177881A (zh) | 2019-08-27 |
US20190338332A1 (en) | 2019-11-07 |
US11359222B2 (en) | 2022-06-14 |
AU2016427128C1 (en) | 2023-01-05 |
JP2019536444A (ja) | 2019-12-19 |
JP6842800B2 (ja) | 2021-03-17 |
AU2016427128B2 (en) | 2022-08-18 |
WO2018072211A1 (zh) | 2018-04-26 |
MX2019004630A (es) | 2019-07-15 |
US20230022453A1 (en) | 2023-01-26 |
EP3543346A4 (en) | 2020-07-22 |
CA3041147A1 (en) | 2018-04-26 |
RU2736352C1 (ru) | 2020-11-16 |
CN116574775A (zh) | 2023-08-11 |
EP3543346A1 (en) | 2019-09-25 |
US11952604B2 (en) | 2024-04-09 |
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