CN117106685A - 一种利用糖基转移酶高效生物合成莱鲍迪苷m2的方法 - Google Patents
一种利用糖基转移酶高效生物合成莱鲍迪苷m2的方法 Download PDFInfo
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- C12N9/10—Transferases (2.)
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- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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Abstract
本发明公开了一种利用糖基转移酶高效生物合成莱鲍迪苷M2的方法,属于生物催化合成技术领域。本发明通过挖掘得到一种具有催化莱鲍迪苷D合成莱鲍迪苷M2的糖基转移酶UGT94D1,为莱鲍迪苷M2的生产提供了一条高效且绿色的新途径。整个催化反应过程中,不产生副产物,利于下游纯化,进一步降低生产成本。
Description
技术领域
本发明涉及一种利用糖基转移酶高效生物合成莱鲍迪苷M2的方法,属于生物催化合成技术领域。
背景技术
甜菊糖苷是从甜叶菊叶子中提取和纯化的二萜糖苷,具有高甜度、低热量、对人体无副作用等优点。甜菊糖苷已被美国、巴西、韩国、日本和欧洲食品安全局批准为安全食品添加剂。到目前为止,已从甜菊叶中鉴定出64种甜菊醇糖苷,其中,甜菊糖含量最高,占干重的5-10%,其次是莱鲍迪苷A,占干重的2-4%。他们表现出比蔗糖高250-300倍的甜度,但即使是高纯度的甜菊糖苷也保留了苦味的属性,这在很大程度上限制了它们在天然食品添加剂市场中的应用。
近年来研究发现,C-13和C-19所连接的糖基单元的数量及种类对甜菊糖苷的性质有显著影响。例如,莱鲍迪苷A的C-13位置上有一个额外的葡萄糖,使其比甜菊糖更甜更可口。甜叶菊中进一步分离得到的莱鲍迪苷D和莱鲍迪苷M在C-19位置上连接有一个和两个额外的葡萄糖,使其表现出比莱鲍迪苷A更好的甜味品质。因此人们非常关注在这些位置上具有不同的糖基单元的新型甜菊糖苷以挖掘具有更好品质的甜味剂。
UDP-葡萄糖基转移酶(UGTs)是一种天然进化的催化糖基化反应的酶,它们将糖部分从活化的糖供体转移到受体分子上。UGTs在甜菊糖苷的合成中已经有了许多的应用。2014年,Prakash等人首次报道了一种新型甜菊糖苷衍生物-莱鲍迪苷M2,其是糖基转移酶UGTSL2催化莱鲍迪苷A转化为莱鲍迪苷D的次要产物。但是由于产率较低,产物混杂,分离纯化成本高,限制了对莱鲍迪苷M2的进一步研究。因此,鉴定一种新的具有高催化活性和区域选择性的糖基转移酶催化合成莱鲍迪苷M2是非常有必要的。
发明内容
为解决上述问题,本发明挖掘来自芝麻的糖苷转移酶UGT94D1催化莱鲍迪苷D合成莱鲍迪苷M2,在尿苷二磷酸葡萄糖(UDPG)存在的情况下具有催化莱鲍迪苷D合成莱鲍迪苷M2的活性,为莱鲍迪苷M2的进一步研究提供了可能性。
为了解决上述技术问题,本发明的技术方案如下:
本发明的第一个目的是提供一种重组菌,所述重组菌过表达芝麻来源的糖基转移酶UGT94D1。
在一种实施方式中,所述糖基转移酶的氨基酸序列的登录号为XP_011076907.1,核苷酸序列的登录号为XM_011078605.1。
在一种实施方式中,所述重组菌以原核细胞或真核细胞为宿主细胞。
在一种实施方式中,所述原核宿主细胞可以是任何革兰氏阳性或革兰氏阴性细菌。革兰氏阳性细菌包括但不限于:芽孢杆菌属、梭菌属、肠球菌属、土芽孢杆菌属(Geobacillus)、乳杆菌属、乳球菌属、大洋芽孢杆菌属、葡萄球菌属、链球菌属和链霉菌属。革兰氏阴性细菌包括但不限于弯曲杆菌属、大肠杆菌、黄杆菌属、梭杆菌属、螺杆菌属、泥杆菌属、奈瑟氏菌属、假单胞菌属、沙门氏菌属、以及脲原体属;所述真核细胞为真菌细胞。
在一种实施方式中,所述重组菌以大肠杆菌为宿主细胞。
在一种实施方式中,所述重组菌以pET系列为表达载体。
在一种实施方式中,所述重组菌以pET-21b(+)为表达载体。
本发明的第二个目的是提供一种组合物,所述组合物为糖基转移酶UGT94D1、所述重组菌或所述重组菌的细胞裂解液中的一种或多种;所述糖基转移酶的氨基酸序列的登录号为XP_011076907.1。
在一种实施方式中,所述细胞裂解液为所述重组菌经诱导表达后,裂解细胞获得的上清液。
本发明的第三个目的是提供一种催化合成莱鲍迪苷M2的方法,所述方法为以莱鲍迪苷D为底物,利用糖基转移酶UGT94D1、所述重组菌或所述组合物进行催化反应。
在一种实施方式中,所述方法以UDP-葡萄糖为糖基供体。
在一种实施方式中,所述催化体系为0.1~3.0mM Reb D,1~10μM糖基转移酶UGT94D1,1~10mM UDPG,5~20mM MnCl2,20~80mM Tris。
在一种实施方式中,所述催化反应的条件为在pH为5.5-9.0、30~50℃下反应2~10h。
本发明还提供糖基转移酶UGT94D1或上述重组菌或上述组合物或上述方法在制备莱鲍迪苷M2或含有莱鲍迪苷M2的产品中的应用。
在一种实施方式中,所述糖基转移酶UGT94D1的氨基酸序列的登录号为XP_011076907.1,核苷酸序列的登录号为XM_011078605.1。
有益效果:
本发明将编码糖基转移酶UGT94D1的核酸序列用于制备能够催化莱鲍迪苷D生成莱鲍迪苷M2的重组蛋白,制备出的重组蛋白能够以UDPG为糖基供体,使莱鲍迪苷D为底物发生糖基化合成莱鲍迪苷M2,且在催化反应过程中无副产物的产生,利于下游纯化,降低生产成本。莱鲍迪苷M2的产率为90%,产量为2.32g/L。
附图说明
图1显示糖基转移酶UGT94D1催化莱鲍迪苷D产生莱鲍迪苷M2的生物合成途径。
图2为实施例2中糖基转移酶UGT94D1蛋白表达表达及纯化分析。泳道1:Marker;泳道2:无IPTG诱导表达样品;泳道3:粗酶液;泳道4:粗酶液上清;泳道5:粗酶液沉淀;泳道6:纯化穿透液;泳道7:洗涤杂蛋白质样品;泳道8:目的蛋白质洗脱样品。
图3为实施例3中糖基转移酶UGT94D1催化莱鲍迪苷D合成莱鲍迪苷M2的UPLC分析图。
图4为实施例3中莱鲍迪苷D糖基化反应产物莱鲍迪苷M2质谱分析。
图5为实施例4中产物莱鲍迪苷M2的核磁共振波谱分析氢谱。
图6为实施例4中产物莱鲍迪苷M2的核磁共振波谱分析碳谱。
图7为实施例4中产物莱鲍迪苷M2的核磁共振波谱分析COSY谱。
图8为实施例4中产物莱鲍迪苷M2的核磁共振波谱分析TOCSY谱。
图9为实施例4中产物莱鲍迪苷M2的核磁共振波谱分析HSQC谱。
图10为实施例4中产物莱鲍迪苷M2的核磁共振波谱分析HMBC谱。
图11为实施例4中产物莱鲍迪苷M2的核磁共振波谱分析ROESY谱。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市售商品或者可以通过已知方法制备。
下述实施例中涉及到的培养基:
LB固体培养基:10g/L蛋白胨,5g/L酵母粉,10g/L NaCl,20g/L琼脂粉。
2×YT液体培养基:16g/L蛋白胨,10g/L酵母粉,5g/L NaCl。
下述实施例中涉及到的方法:
糖基转移酶酶学性质的测定:糖基转移酶UGT94D1对莱鲍迪苷D的动力学分析是在200μL反应体系中进行的,反应体系含有5mM UDPG、10mM MnCl2、50mM Tris-HClpH 8.0和5μg纯化蛋白样品(糖基转移酶UGT94D1),莱鲍迪苷D的浓度为0-5mM。反应温度为35℃,反应时间为2h;然后在95℃条件下加热5min淬灭反应,并将反应混合物用2倍体积甲醇稀释;20000×g离心5min以去除沉淀物;取上层液体用0.22μm滤膜过滤后用于UPLC分析。
酶活的定义:在1h内合成1μM莱鲍迪苷M2所需的酶量。
莱鲍迪苷M2的产率的测定:配制不同浓度(0mM、0.05mM、0.1mM、0.15mM、0.2mM、0.25mM、0.3mM)的莱鲍迪苷M2标准溶液,利用UPLC对标准溶液进行分析,获得莱鲍迪苷M2浓度标准曲线方程为y=2383564.28571x+1556.96429,R2=0.99664。根据标准曲线换算得到莱鲍迪苷M2的产率。产率=莱鲍迪苷M2实际产量/莱鲍迪苷M2理论产量。
Waters Acquity UPLC系统:采用BEH C18 1.7μM色谱柱(2.1×50mm),液相条件为:有机相--乙腈、水相--超纯水;流速0.3mL/min;柱温40℃;紫外检测波长210nm;检测程序:0-1min 15%有机相、6min40%有机相、7-8min 15%有机相。
实施例1糖基转移酶UGT94D1基因的获取和重组菌株的构建
从Genbank中下载芽孢杆菌糖基转移酶氨基酸序列(登录号:XP_011076907.1)及核苷酸序列(登录号:XM_011078605.1),由亦欣生物科技有限公司进行基因合成并连接至载体pET-21b(+)的多克隆酶切位点,得到重组质粒pET-21b(+)-UGT94D1。
将所得质粒pET-21b(+)-UGT94D1进行测序鉴定并转化至大肠杆菌E.coli BL21(DE3)感受态细胞中,采用含有100μg/mL氨苄青霉素的LB固体平板进行筛选,得到重组菌株E.coli BL2l(DE3)pET-21b(+)-UGT94D1。
实施例2重组菌株的诱导表达及目的蛋白纯化
将实施例1中构建的重组菌株E.coli BL2l(DE3)pET-21b(+)-UGT94D1接种至含有100μg/mL氨苄青霉素的1L2×YT液体培养基中,并在135rpm,37℃条件下培养至OD600为0.6-0.8后,培养温度降至18℃,加入终浓度为0.1mmol/L的异丙基-β-硫代半乳糖苷(IPTG),诱导培养8h。
将诱导表达的菌液离心(7000rpm,7min,4℃),弃上清,收集菌体。将菌体用裂解缓冲液(50mmol/L Tris-HClpH 8.0,300mmol/LNaCl,10mmol/L咪唑,10%甘油)按照1g菌体每10mL裂解缓冲液进行重悬。利用高压匀浆机进行破碎,然后将破碎后的菌液离心(40000×g,30min),取上清即获得粗酶液。
将粗酶液利用Ni+柱进行亲和层析纯化,上样结束后,10倍体积裂解缓冲液冲洗杂蛋白,用洗脱缓冲液(50mmol/L Tris-HClpH 8.0,300mmol/LNaCl,250mmol/L咪唑,10%甘油)进行目的蛋白洗脱。收集洗脱的目的蛋白,通过脱盐柱(HistrpTM 5mL Desalting)进行脱盐,脱盐缓冲液(25mmol/L Tris-HCl,150mmol/LNaCl,10%甘油)。脱盐后浓缩至10mg/mL,进行后续反应。纯化的蛋白通过10%SDS-PAGE凝胶电泳进行检测,结果见图2,成功获得目的条带清晰蛋白大小准确的纯酶,测得UGT94D1对莱鲍迪苷D的Km值为0.89±0.05mM,kcat值为0.33±0.08min-1。并测得纯酶UGT94D1的酶活为2.12U/mg。
实施例3UGT94D1催化莱鲍迪苷D反应合成莱鲍迪苷M2的糖基化反应
将实施例2获得的纯化后的糖基转移酶UGT94D1用于糖基化反应(图1)。
糖基化反应在200μL反应体系中进行,反应体系如下:50mmol/L Tris pH 8.0,5mmol/L UDPG,10mmol/L MnCl2,2mmol/L莱鲍迪苷D,实施例2中所获得纯酶UGT94D1浓度为5μM。在35℃下反应4h。然后在95℃条件下加热5min淬灭反应,并将反应混合物用2倍体积甲醇稀释;20000×g离心5min以去除沉淀物;取上层液体用0.22μm滤膜过滤后用于UPLC和LC-MS分析。WatersAcquity UPLC系统采用BEH C181.7μM色谱柱(2.1×50mm),液相条件为:有机相--乙腈、水相--超纯水;流速0.3mL/min;柱温40℃;紫外检测波长210nm;检测程序:0-1min 15%有机相、6min 40%有机相、7-8min 15%有机相。
通过液相分析可得知,结果如图3所示,与莱鲍迪苷D标准品对比可知,在反应体系中有明显的新产物生成,且无副产物的产生。对反应混合物进行质谱(MS)分析(图4),LC-MS的负离子模式结果显示,在m/z 1289.5421处有一个[M-H]-离子的峰,对应分子式C56H90O33,表明产物是莱鲍迪苷D的单糖基衍生物莱鲍迪苷M2。通过HPLC定量分析,莱鲍迪苷M2的产率为90%,产量为2.32g/L。
实施例4新型莱鲍迪苷D单糖基化衍生物的结构鉴定
利用糖基转移酶UGT94D1进行大规模(100mL)糖基化反应制备新型衍生物,反应体系如下:2mM Reb D,10μM糖基转移酶,5mM UDPG,10mM MnCl2,50mM Tris pH8.0。反应混合物在35℃下反应24h,然后在95℃条件下加热5min淬灭反应,20000×g离心5min以去除沉淀物;取上层液体用0.22μm滤膜过滤后,使用半制备高效液相色谱系统进行纯化,系统采用Shim-pack GIST C18色谱柱(10×250mm,5μm,SHIMADZU,Japan),液相条件为:有机相--乙腈、水相--超纯水;流速5mL/min;时间程序:0-28min 23%有机相;28.5-30.5min 60%有机相;31-35min 23%有机相。柱温:40℃;紫外检测波长210nm。所得样品重水溶解,通过1D(1H和13C)和2D NMR(COSY、TCOSY、HSQC、HMBC和ROESY)波谱对产物的完整结构进行分析。用Bruker Avance III 600MHz spectrometer(Bruker BioSpin,Karlsruhe,Germany)收集数据,1H谱检测频率为600MHz,13C谱为151MHz。
通过1D和2D HMR对新衍生物的H和C的化学位移进行了详细的归属,如表1所示。UGT94D1催化Reb D合成的产物的结构式为13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester]。该产物的结构和文献中Reb M2的结构一致生成的产物与文献所报道的莱鲍迪苷M2一致(图5-11)。
表11H和13C化学位移归属表(D2O)
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。
Claims (10)
1.一种重组菌,其特征在于,表达来源于芝麻的糖基转移酶UGT94D1,所述糖基转移酶UGT94D1的氨基酸序列的NCBI登录号为XP_011076907.1。
2.根据权利要求1所述的重组菌,其特征在于,所述重组菌以大肠杆菌为宿主细胞。
3.根据权利要求1所述的重组菌,其特征在于,所述重组菌以pET系列为表达载体。
4.根据权利要求3所述的重组菌,其特征在于,所述重组菌以pET-21b(+)为表达载体。
5.一种组合物,其特征在于,所述组合物为糖基转移酶UGT94D1、权利要求1~4任一所述重组菌或权利要求1~4任一所述重组菌的细胞裂解液中的一种或多种;所述糖基转移酶UGT94D1的氨基酸序列的登录号为XP_011076907.1。
6.根据权利要求5所述的组合物,其特征在于,所述细胞裂解液为权利要求1~4任一所述重组菌经诱导表达后,裂解细胞获得的上清液。
7.一种催化合成莱鲍迪苷M2的方法,其特征在于,所述方法为以莱鲍迪苷D为底物,利用糖基转移酶UGT94D1、权利要求1~4任一所述重组菌和/或权利要求5或6所述组合物进行催化反应;所述糖基转移酶UGT94D1的氨基酸序列的登录号为XP_011076907.1。
8.根据权利要求7所述的方法,其特征在于,所述方法以UDP-葡萄糖为糖基供体。
9.根据权利要求8所述的方法,其特征在于,所述催化体系为0.1~3.0mM Reb D,1~10μM糖基转移酶UGT94D1,1~10mM UDP-葡萄糖,5~20mM MnCl2,20~80mM Tris。
10.糖基转移酶UGT94D1、权利要求1~4任一所述重组菌,或权利要求5或6所述组合物,或权利要求7~9任一所述方法在制备莱鲍迪苷M2或含有莱鲍迪苷M2的产品中的应用;所述糖基转移酶UGT94D1的氨基酸序列的登录号为XP_011076907.1。
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