CN115094074A - 双功能udp-糖基转移酶及其应用 - Google Patents
双功能udp-糖基转移酶及其应用 Download PDFInfo
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- CN115094074A CN115094074A CN202210751919.9A CN202210751919A CN115094074A CN 115094074 A CN115094074 A CN 115094074A CN 202210751919 A CN202210751919 A CN 202210751919A CN 115094074 A CN115094074 A CN 115094074A
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- Prior art keywords
- rebaudioside
- udp
- glycosyltransferase
- reb
- expression vector
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- Granted
Links
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Abstract
本发明公开了一种双功能UDP‑糖基转移酶及其应用,属于基因工程领域。其双功能是甜菊醇糖苷糖基转移酶和黄酮糖基转移酶。所述糖基转移酶能够在甜菊糖苷的C13/C19位形成β‑1,3‑糖苷键,并且具有C19位偏好性,或在黄酮类化合物羟基上发生糖基化。本发明也提供使用这种重组多肽制备多种稀有甜菊醇糖苷组合物的方法。本发明也公开了被称为莱鲍迪苷E2、甜菊苷B、莱鲍迪苷E6的甜菊醇糖苷。
Description
技术领域
本发明涉及一种双功能UDP-糖基转移酶及其应用,属于基因工程领域。
背景技术
甜菊糖苷(Steviol glycosides,SGs)是一类从甜叶菊(Stevia rebaudiana)中提取的二萜类天然产物,具有高甜度、低热量等特点,被称为继蔗糖、甜菜糖外的“世界第三糖源”,并且还具有广泛的生物活性,包括抗炎、抑菌以及降血糖等。由于甜菊糖苷及其衍生物价格相对低廉且易于大量提取制备,已被广泛应用于食品饮料、医药和有机化学等领域。
目前已分离鉴定了超过60种二萜甜菊糖苷,总糖苷含量占叶片干重的15%-20%。甜菊苷(Stevioside,STV)和莱鲍迪苷A(Rebaudioside A,Reb A)是甜菊糖苷的主要成分,约占叶片干重的5-10%和2-4%,甜度是蔗糖的150-250倍,具有一定的苦涩后味。莱鲍迪苷D(Rebaudioside D,Reb D)和莱鲍迪苷M(Rebaudioside M,Reb M)的甜度约是蔗糖的350倍,甜味阈值低,且几乎不具有苦涩后味,但其含量仅占叶片干重0.4%-0.5%,高昂的纯化成本降低了Reb D/M商业应用的价值。
在Reb M的生物合成通路中,需要利用具有β-1,3-糖基化功能的UDP-糖基转移酶(Uridine-diphosphate dependent glycosyltransferases,UGTs)在Reb D的C19位的第一个葡萄糖C3位上连接一个新的葡萄糖,此前认为UGT76G1在该过程中发挥功能,但是体外实验表明,UGT76G1在该过程中催化效率较低。在甜叶菊中过表达UGT76G1也不能提高Reb M的累积量。因此,开发高效制备的Reb D/M的方法,对开拓甜菊糖苷市场尤为重要。
另外,具有C19位β-1,3-糖苷键的甜菊糖苷,如Reb AM,含量远低于具有C13位β-1,3-糖苷键的甜菊糖苷,如Reb A,表明具有C19位偏好性的β-1,3-糖基转移酶的研究缺失。
挖掘具有C19位偏好性的β-1,3糖苷键糖基转移酶,解决C19位糖基化效率低的瓶颈,不仅有利于Reb M的制备,也有助于合成新型或稀有甜菊糖苷,丰富甜菊糖苷甜味剂库,实现廉价甜菊糖苷向高附加值糖苷的高效转化,提升甜菊糖苷竞争力。
在植物中,UGTs能立体特异性或空间特异性的转移一个UDP活化的糖到一类受体分子上。其中,GT1家族的糖基转移酶的受体分子是植物次级代谢物,包括抗生素、酚类、黄酮类、生物碱和萜类。研究表明UGTs具有广泛的底物耐受性,但也具有一定底物特异性,即糖基化结构相似的多种物质。
目前文章或专利报道的双功能糖基转移酶主要分为两类,一类双功能体现在供体底物识别多样性,即能够利用不同的供体糖;另一类双功能体现在糖基化类型多样,即C-、O-或S-的糖基化。甜叶菊中尚未发现能够糖基化不同类别受体底物的UGTs。
黄酮类化合物是植物中广泛分布的一大类次级代谢物,结构多样且部分具有抗癌、抗炎等重要的药理活性。黄酮糖苷类天然产物是植物中黄酮类化合物的主要存在形式,糖基化提高这些化合物的生物活性、稳定性、水溶性等,使植物能迅速应对生物或非生物胁迫。多种黄酮糖苷有重要的药用价值。寻找甜叶菊体内的能够实现黄酮糖基化的UGTs,能够得到更多的黄酮糖基化工具酶,有利于制备新型黄酮糖苷。能实现黄酮和萜类物质糖基化的双功能UGTs的挖掘也为UGTs底物识别机制解析提供了材料基础。
发明内容
本发明的目的在于解决UGT76G1不具备甜菊糖苷C19位偏好性,Reb M及其他稀有糖苷合成效率低的技术问题,开发一种高效糖基化甜菊醇糖苷C19位的UGT。
为实现上述目的,本发明提供了一种UDP糖基转移酶,命名为UGT76G4,该酶不仅具备甜菊醇糖苷C19位糖基化偏好性,高效制备Reb M及其他稀有糖苷,也能糖基化黄酮类物质。为糖基转移酶结构功能关系研究提供了材料。
本发明提供了一种UDP-糖基转移酶基因,其核酸序列具有如下特征中的一种或二种以上:
1)具有SEQ ID No.1所示的脱氧核糖核酸序列;
2)编码SEQ ID NO.2氨基酸序列的脱氧核糖核酸序列;
3)对SEQ ID NO.1所示的脱氧核糖核酸序列进行一个或两个以上核苷酸取代和/或缺失和/或添加而得到的编码具有UDP-糖基转移酶活性的脱氧核糖核酸序列;
4)与SEQ ID NO.1限定的脱氧核糖核酸序列的同源性达到80%及以上,且能编码UDP-糖基转移酶的脱氧核糖核酸序列。
本发明还提供了一种UDP-糖基转移酶基因编码的UDP-糖基转移酶,其氨基酸序列具有如下特征中的一种或二种以上:
1)具有SEQ ID NO.2所示的氨基酸序列;
2)具有如SEQ ID NO.2从氨基端开始第1-458位氨基酸残基序列;
3)对SEQ ID NO.2所示的氨基酸序列进行一个或两个以上氨基酸取代和/或缺失和/或添加而形成的具有UDP-糖基转移酶活性的氨基酸序列;
4)与SEQ ID NO.2具有至少80%同一性的氨基酸序列。
本发明还提供了一种含有所述的UDP-糖基转移酶基因的重组表达质粒。
本发明还提供了一种含有所述的UDP-糖基转移酶基因的重组基因工程菌。
本发明还提供了一种所述的UDP-糖基转移酶的制备方法,将所述的UDP-糖基转移酶基因克隆入重组表达载体,导入宿主细胞,获得重组表达的UDP-糖基转移酶。
进一步地,上述技术方案中,所述的重组表达载体包括大肠杆菌表达载体、酵母表达载体、枯草杆菌表达载体、乳酸菌表达载体、链霉菌表达载体、噬菌体载体、丝状真菌表达载体、植物表达载体、昆虫表达载体、或哺乳动物细胞表达载体中的一种或二种以上。
进一步地,上述技术方案中,所述宿主细胞包括大肠杆菌宿主细胞(如Escherichia coli BL21、Escherichia coli JM109、Escherichia coli DH5α等)、酵母菌宿主细胞(如Saccharomyces cerevisiae、Pichiapastoris、Kluyveromyceslactis等)、枯草杆菌宿主细胞(如Bacillus subtilis R25、Bacillus subtilis9920等)、乳酸菌宿主细胞(如Lactic acid bacteria COCC101等)、放线菌宿主细胞(如Streptomyces spp.等)、丝状真菌宿主细胞(如Trichodermaviride、Trichodermareesei、Aspergillusniger、Aspergillusnidulans等)、昆虫细胞(如Bombyxmori、Antharaea eucalypti等),哺乳动物细胞(如中国仓鼠卵巢细胞CHO、幼小仓鼠肾脏细胞BHK、中国仓鼠肺细胞CHL等)中的一种。
本发明还提供了一种所述UDP-糖基转移酶在糖基化甜菊醇糖苷或黄酮类化合物的应用。
进一步地,本发明也涉及制备本文所述稀有甜菊醇糖苷的方法,所述方法包括用包含与SEQ ID NO.2具有至少80%同一性的氨基酸序列的重组多肽孵育底物。
所述底物为甜菊醇糖苷,所述甜菊醇糖苷包括:甜菊单糖苷、甜菊单糖苷A、甜菊双糖苷、悬钩子苷、甜菊双糖A、甜菊苷、莱鲍迪苷G、甜菊苷A、甜菊苷B、莱鲍迪苷A、莱鲍迪苷E、莱鲍迪苷E2、莱鲍迪苷E4、莱鲍迪苷E3、莱鲍迪苷D、莱鲍迪苷AM等。
进一步地,上述技术方案中,糖基化所得甜菊醇糖苷的产物包括甜菊双糖苷D、甜菊双糖苷B、莱鲍迪苷B、莱鲍迪苷G、甜菊苷B、甜菊苷C、莱鲍迪苷A、莱鲍迪苷E2、莱鲍迪苷E3、莱鲍迪苷E4、莱鲍迪苷E6、莱鲍迪苷I、莱鲍迪苷D、莱鲍迪苷AM、莱鲍迪苷D7、莱鲍迪苷M。所述甜菊醇糖苷产物,除莱鲍迪苷A外,在甜叶菊中均为低丰度糖苷。
所述底物为黄酮类化合物,所述黄酮类化合物包括:槲皮素、山奈酚、7-羟基黄酮。
进一步地,上述技术方案中,糖基化所得黄酮类化合物的产物包括槲皮素-3-O-葡萄糖苷、槲皮素-5-O-葡萄糖苷、槲皮素-7-O-葡萄糖苷、山奈酚-3-O-葡萄糖苷。
在一个示例性实施方式中,在本文所述的方法中使用的重组多肽的氨基酸序列与SEQ ID NO.2具有至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%或甚至100%的同一性。
本发明也涉及新型甜菊糖苷即莱鲍迪苷E2、甜菊苷B、莱鲍迪苷E6。莱鲍迪苷E2在本文所述条件下一级质谱m/z为965.4137,二级质谱主要子离子m/z为641.3148;甜菊苷B在本文所述条件下一级质谱m/z为803.3627,二级质谱主要子离子m/z为479.2587;莱鲍迪苷E6在本文所述条件下一级质谱m/z为965.4137,二级质谱主要子离子m/z为641.3091。
所述莱鲍迪苷E2具有如下结构式:
所述甜菊苷B具有如下结构:
所述莱鲍迪苷E6具有如下结构式:
本发明还提供了莱鲍迪苷E2、甜菊苷B、莱鲍迪苷E6在作为甜味剂中的应用。
本发明还提供了所述的核苷酸或编码所述蛋白质的核苷酸在甜叶菊遗传育种中的应用,所述核苷酸在甜叶菊中过表达。
附图说明
图1为SrUGT76G4在甜菊醇糖苷生物合成中的作用。a中底物仅有单一的糖基化位点,b中底物具有两个糖基化位点,13位和19位交替糖基化制备出双糖基化产物。
图2为SrUGT76G4的体外功能验证液相检测。a为以甜菊单糖苷(steviolmonoside)为底物,1表示灭活SrUGT76G4糖基化steviolmonoside,2/3/4表示SrUGT76G4糖基化steviolmonoside;b为以甜菊双糖苷(steviolbioside)为底物,1表示灭活SrUGT76G4糖基化steviolbioside,2/3/4表示SrUGT76G4糖基化steviolbioside;c为以悬钩子苷(rubusoside,Rub)为底物,1表示灭活SrUGT76G4糖基化Rub,2/3/4表示SrUGT76G4糖基化Rub;d为以STV为底物,1表示灭活SrUGT76G4糖基化STV,2/3/4表示SrUGT76G4糖基化STV;e为以Reb A为底物,1表示灭活SrUGT76G4糖基化Reb A,2/3/4表示SrUGT76G4糖基化Reb A;f为以Reb E为底物,1表示灭活SrUGT76G4糖基化Reb E,2/3/4表示SrUGT76G4糖基化Reb E;g为以Reb D为底物,1表示灭活SrUGT76G4糖基化Reb D,2/3/4表示SrUGT76G4糖基化Reb D。以上反应均以UDP-葡萄糖为供体底物。
图3为甜菊糖苷Reb E2的合成路线及质谱结果。a为以甜菊苷STV为底物制备甜菊糖苷Reb E2的反应路线;b为STV的一级质谱结果;c为STV的二级质谱结果;d为Reb E2的一级质谱结果;e为Reb E2的二级质谱结果。
图4为悬钩子苷、甜菊苷B和Reb E6的MS/MS结果。a为悬钩子苷的一级质谱结果;b为悬钩子苷的二级质谱结果;c为甜菊苷B的一级质谱结果;d为甜菊苷B的二级质谱结果;e为Reb E6的一级质谱结果;f为Reb E6的二级质谱结果。
图5为SrUGT76G4(a)和SrUGT76G1(b)糖基化Reb E产物随时间变化情况。
图6为SrUGT76G4(a)和SrUGT76G1(b)糖基化Reb D的酶动力学拟合结果。
图7为SrUGT76G4(a)和SrUGT76G1(b)糖基化5mM和10mM Reb D的产物随时间变化情况。
图8为SrUGT76G4糖基化黄酮类物质的HPLC分析。a为以槲皮素为底物,1表示槲皮素标准品,2表示SrUGT76G4糖基化槲皮素;b为以7-羟基黄酮为底物,1表示7-羟基黄酮标准品,2表示SrUGT76G4糖基化7-羟基黄酮;c为以山奈酚为底物,1表示山奈酚标准品,2表示SrUGT76G4糖基化山奈酚;d为以木犀草素为底物,1表示木犀草素标准品,2表示SrUGT76G4糖基化木犀草素。以上反应均以UDP-葡萄糖为供体底物。
图9为纯化的重组SrUGT76G4多肽的SDS-PAGE分析。
具体实施方式
以下结合实例详细说明本发明。实例是为更好的理解本发明,但不限定于本发明。以下实施方法中的实验方法均为常规方法,所涉及的实验试剂均为常规生化试剂。
实施例1候选UGT基因选择
根据NCBI中公布的SrUGT76G1(AY345974.1)的核酸序列设计引物(表1,正向引物带有组氨酸标签),从不同品种甜叶菊cDNA中扩增目的基因,电泳检测条带长度正确后,使用Gibson Assembly方法将目的基因连接在NdeI、XhoI双酶切后的pET21a(+)载体上。取5μL连接产物转化E.coli TOP10感受态细胞,涂布在含100μg/mL氨苄青霉素的固体Luria-Bertani培养基上,37℃培养12-16h。挑取单克隆,使用通用引物进行菌落PCR验证,将扩增产物长度正确的单克隆接入含有100μg/mL氨苄青霉素的液体Luria-Bertani培养基中培养,提取质粒;使用内切酶NdeI和XhoI对提取的质粒进行双酶切,结果正确的重组质粒送华大基因测序。测序结果表明,在pET21a的NdeI和XhoI酶切位点之间插入了候选基因,且插入方向正确,证明重组质粒构建成功。构建所得重组质粒的核苷酸序列如SEQ ID NO.1所示。
表1 SrUGT76G基因扩增使用引物
SEQ ID NO.1与UGT76G1相比,部分氨基酸位点发生变化。包括,第29位异亮氨酸变为甲硫氨酸,第191位赖氨酸变为谷氨酰胺,第192位丝氨酸变为苯丙氨酸,第193位丙氨酸变为甘氨酸,第194位酪氨酸变为苯丙氨酸,第198位谷氨酰胺变为赖氨酸,第199位异亮氨酸变为谷氨酰胺,第200位亮氨酸变为甘氨酸,第204位亮氨酸变为苯丙氨酸,第205位甘氨酸变为谷氨酸,第206位赖氨酸变为天冬酰胺,第207位甲硫氨酸变为异亮氨酸,第208位异亮氨酸变为苏氨酸,第266位谷氨酰胺变为脯氨酸,第273位脯氨酸变为丝氨酸,第274位丝氨酸变为精氨酸,第284位苏氨酸变为丙氨酸,第285位丝氨酸变为苏氨酸。
实施例2候选UGT酶活性筛选
将实施例1构建好的重组质粒转化入E.coli BL21(DE3),其随后在37℃含有50μg/mL氨苄霉素的LB培养基中生长直至达到OD600为0.4-0.6。通过添加1mM异丙基β-D-1-硫代半乳糖苷(IPTG)诱导蛋白表达并且培养物在16℃进一步生长22小时。通过离心(3,000x g;10min;4℃)收集细胞。收集细胞团立即使用或者在-80℃贮存。
细胞团在裂解缓冲液(20mM Tris-HCl缓冲液、pH 8.0,300mM NaCl,10%丙三醇)中重悬。通过超声法在4℃下裂解细胞,并且通过离心(13,000rpm;30min)使细胞碎片澄清。将上清上样到已平衡(平衡缓冲液:20mM的Tris-HCl缓冲液,pH8.0,300mM NaCl,10%丙三醇)的Ni-NTA(Qiagen)亲和柱上。蛋白样品上样后,用平衡缓冲液、含20mM、40mM咪唑缓冲液洗脱非特异性结合的杂蛋白,用含有250mM咪唑的缓冲液洗脱带有组氨酸标签的UGT重组多肽。得到纯化的候选UGT重组多肽。
用聚丙烯酰胺凝胶电泳检测候选UGT的表达及纯化情况,纯化后的糖基转移酶在电泳胶上呈单一条带,且位置与预测的分子量(52.1K Da)相吻合(图7)。
通过使用多种甜菊醇糖苷(steviolmonoside、steviolbioside、Rub、STV、Reb E、Reb D)作为供体底物测定纯化的候选UGT重组多肽的1,3-O-葡萄糖糖基化活性(图2)。通常,重组多肽(10μg)在100μl体外反应体系中进行活性检测。该反应体系含有20mM的Tris-HCl缓冲液,pH8.25,100mM NaCl,1mM甜菊醇糖苷,1mM UDP-葡萄糖。该反应在30℃进行并通过添加100μL无水乙醇终止反应,震荡混匀后,反应体系过0.22μm有机滤膜用于高效液相色谱(HPLC)分析。
高效液相色谱使用仪器型号,Waters e2695。进样量10μL,色谱柱:依力特superilODS2(5μm,250×4.6mm),柱温:40℃。色谱条件:UV 210nm,流动相:(A)水(含0.1%甲酸),(B)乙腈(1%甲酸),流速:1mL/min,洗脱程序:0-4min,20%B,;4-25min,线性增长至30%B;25-40min,30%B。
由SEQ ID NO.1编码的重组多肽(SEQ ID NO.2)能糖基化所有测试糖苷底物,根据其糖基化steviolmonoside、steviolbioside、STV、Reb E,得到steviolbioside D、Rebaudioside B、Reb A、Reb D,推断该重组多肽具有C13位1,3-O-葡萄糖糖基化活性(图2)。并且,糖基化Rub、STV、Reb A、Reb E产物中还具有stevioside B、Reb E6、Reb E2、RebI、Reb AM,推断该重组多肽也具有C19位1,3-O-葡萄糖糖基化活性。另外,Rub的糖基化产物中,stevioside B的比例高于Reb G;Reb E糖基化产物中,Reb AM比例高于Reb D,推断该酶具有C19偏好性。该基因命名为SrUGT76G4。
实施例3使用SrUGT76G4生物合成多种稀有甜菊醇糖苷
纯化的糖基转移酶蛋白在体外按照以下反应体系进行功能验证(100μL):20mMTris(pH 8.25),100mM氯化钠,20μg纯化的蛋白,2mM UDP-Glucose,2mM的受体底物,受体底物包括:steviolmonoside、steviolmonoside A、steviolbioside、steviolbioside A、rubusoside、stevioside、stevioside A、rebaudioside E反应12小时后,加入等体积无水丁醇终止反应,震荡混匀,体系过0.22μm有机滤膜,利用高效液相色谱定性定量检测反应产物组成。检测条件同实施例2。
液相色谱检测结果显示SrUGT76G4能够糖基化所有实验用底物,所得产物质谱特征与理论产物一致,该重组多肽具有催化多种甜菊醇糖苷C13位和C19位形成β-1,3糖苷键的功能,可用于制备多种稀有甜菊醇糖苷。
图1为SrUGT76G4在糖基化以上甜菊醇糖苷过程中产物合成路线。所制备产物包括steviolbioside D、steviolbioside B、rebaudioside B、stevioside C、rebaudioside G、stevioside B、rebaudioside A、rebaudioside E2、rebaudioside E3、rebaudioside E4、rebaudioside E6、rebaudioside D7、rebaudioside I、rebaudioside D、rebaudiosideAM、rebaudioside M。葡萄糖之间水平方向的链接代表β-1,2-糖苷键,竖直方向的链接代表β-1,3-糖苷键。其中,stevioside B、rebaudioside E2、rebaudioside E6为新型甜菊糖苷。
实施例4 MS/MS分析Reb E2的结构
用SrUGT76G4糖基化STV,并通过HPLC纯化用于Reb E2表征的材料。
用Agilent Q-TOF 6540液相色谱高分辨飞行时间质谱产生质谱数据,其分辨率设为30k;在阴离子电喷雾模式中扫描的数据从m/z 150至1500。ESI源参数设置如下:毛细管电压为-2.8K V,采样锥电压为40V,二级锥孔提取电压为3.5V,离子源温度为90℃,溶媒挥散温度为250℃,碰撞气(N2)流速120lh-1。碰撞区参数设置如下:碰撞气流速为0.45lh-1,碰撞能为20-50eV。
显示为Reb E2化合物结构显示于图3a中。基于其高分辨质谱,化合物STV的m/z803.3627对应于[STV-H]-的离子(图3b),作为母离子进行二级质谱分析,其二级质谱显示子离子在m/z 641.3104对应于[STV-Glc-H]-(图3c)。化合物Reb E2的分子式已被推导为C44H70O23,其高分辨质谱显示了在m/z 965.4137对应于[M-H]-的离子(图3d),该离子作为母离子进行二级质谱分析,其二级质谱显示子离子在m/z 641.3148对应于[M-2Glc-H]-(图3e)。在甜菊糖苷分子结构中,C19位酯键最不稳定。实验所使用的二级质谱能量条件下,该位置化学键是主要的断裂位点,因此,STV的子离子[STV-Glc-H]-641.3104丰度较高。同理,Reb E2的子离子[M-2Glc-H]-641.3148丰度较高,表明Reb E2与STV具有相同的子离子结构,所以Reb E2的C19位连接两个葡萄糖。并且,UGT76G4具有1,3-O-葡萄糖糖基化活性。综上表明,UGT76G4是具有C19位偏好性1,3-O葡萄糖基转移酶。
实施例5 SrUGT76G4利用Reb E高效制备Reb M
纯化的SrUGT76G4蛋白和SrUGT76G1蛋白(由SrUGT76G1基因编码的蛋白)在体外按照以下反应体系检测其对Reb E的糖基化能力:20mM Tris,100mM氯化钠,5μg纯化的蛋白,2mM UDP-Glucose,0.5mM Reb E。反应10min、20min、30min、60min后,加入等体积无水丁醇终止反应,震荡混匀,12000rpm离心10分钟,取上清过0.22μm有机滤膜,利用液相色谱定性定量检测反应产物组成。液相检测条件与实施实例2中相同。
实验结果表明,SrUGT76G4在10min内Reb E的糖基化产物不仅包括Reb D、Reb AM,还包括Reb M,且Reb AM为主要产物,表明SrUGT76G4具有C19位偏好性。在30min时就已经实现Reb E向Reb M的完全转化,其主要以优先C19,同时C13位的糖基化方式在短时间内迅速生产Reb M。SrUGT76G1以优先C13位糖基化的路线从Reb E得到Reb D,继而缓慢糖基化RebD得到Reb M。
实施例6 SrUGT76G4与SrUGT76G1利用Reb D制备Reb M的催化效率比较
纯化的SrUGT76G4蛋白和SrUGT76G1蛋白在体外按照以下反应体系检测其对Reb D的酶动力学数据:20mM Tris,100mM氯化钠,5μg纯化的蛋白,2mM UDP-Glucose,不同浓度的Reb D(0.1mM、0.2mM、0.3mM、0.4mM、0.5mM、0.8mM、1mM、2mM)。反应10min后,加入等体积无水丁醇终止反应,震荡混匀,12000rpm离心10分钟,过0.22μm有机滤膜,利用液相色谱检测定性定量检测反应产物组成。液相检测条件与实施实例2中相同。
收集液相检测数据,计算不同底物浓度下反应速率,利用GraphPad Prism 7.00软件进行Michaelis-Menten拟合,得到酶动力学数据(图5)。
实验结果表明,SrUGT76G4对Reb D的Km值为561.4±16.98μM,Kcat值为132.628±1.731min-1,Kcat/Km值为0.236μM-1min-1;SrUGT76G1对Reb D的Km值为185±25.13μM,Kcat值为3.897±0.146min-1,Kcat/Km值为0.021μM-1min-1。2mM Reb D条件下,1mg SrUGT76G4酶的比活力为12.8U,1mg SrUGT76G1酶的比活力为0.7U。SrUGT76G4糖基化Reb D得到Reb M的能力较SrUGT76G1提高了约18倍,表明SrUGT76G4具有更强的糖基化Reb D制备Reb M的能力。
实施例7 SrUGT76G4能高效糖基化高浓度Reb D制备Reb M
纯化的SrUGT76G4蛋白和SrUGT76G1蛋白在体外按照以下反应体系检测其对高浓度Reb D糖基化结果:20mM Tris,100mM氯化钠,不同浓度(0.025g/L、0.05g/L、0.15g/L)纯化的蛋白,10mM UDP-Glucose,不同浓度的Reb D(5mM、10mM)。反应1h、2h、3h、12h后,加入等体积无水丁醇终止反应,震荡混匀,12000rpm离心10分钟,过0.22μm有机滤膜,利用液相色谱检测定性定量检测反应产物组成。液相检测条件与实施实例2中相同。
实验结果表明,在5mM Reb D浓度下,0.05g/L或0.15g/L SrUGT76G4反应2小时,即可实现90%以上的Reb D转化(图6a);在10mM Reb D浓度下,0.15g/L SrUGT76G4反应2小时可实现70%以上Reb D转化(图6b)。SrUGT76G1在实验所用3种蛋白浓度和系列反应时间内,没有实现大于50%的Reb D转化(图6a-b),效率远低于SrUGT76G4。表明SrUGT76G4的C19位选择性有利于制备甜味强度更高的Reb M。
实施例8 SrUGT76G4能糖基化黄酮类化合物
纯化的SrUGT76G4在体外按照以下反应体系进行功能验证(200μL):10mM Tris(pH7.5),10μg纯化的蛋白,1mM UDP-Glucose,1mM的受体底物,受体底物包括:7-羟基黄酮、槲皮素、山奈酚、木犀草素。加入等体积无水乙醇终止反应,震荡混匀,12000rpm离心10分钟,取上清过0.22μm有机滤膜,利用液相色谱检测反应产物组成。仪器型号,Waters e2695。进样量10μL,色谱柱:依力特superil ODS2(5μm,250×4.6mm),柱温:40℃。色谱条件:UV256nm,流动相:(A):水(含0.1%甲酸),(B):乙腈(1%甲酸),流速:1mL/min,洗脱程序:0-1.5min,20%B,;1.5-16min,线性增长至50%B。
液相结果显示,SrUGT76G4能糖基化7-羟基黄酮、槲皮素、山奈酚、木犀草素。槲皮素和山奈酚均含有多个羟基,所以有多种产物产生。槲皮素产生至少8种产物,而槲皮素仅含有5个羟基,表明产物中含有多糖基化产物。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 中国科学院大连化学物理研究所;诸城市浩天药业有限公司
<120> 双功能UDP-糖基转移酶及其应用
<130> 2022
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1377
<212> DNA
<213> 糖基转移酶基因(DNA)
<400> 1
atggaaaata aaacggagac caccgttcgc cggcgccgga gaataatatt attcccggta 60
ccatttcaag gtcacataaa cccaatgctt cagctagcca atgtgttgta ctccaaagga 120
ttcagtatca ccatctttca caccaacttc aacaaaccca aaacatctaa ttaccctcac 180
ttcactttca gattcatcct cgacaacgac ccacaagacg aacgcatttc caatctaccg 240
actcatggtc cgctcgctgg tatgcggatt ccgattatca acgaacacgg agctgacgaa 300
ttacgacgcg aactggaact gttgatgtta gcttctgaag aagatgaaga ggtatcgtgt 360
ttaatcacgg atgctctttg gtacttcgcg caatctgttg ctgacagtct taacctccga 420
cggcttgttt tgatgacaag cagcttgttt aattttcatg cacatgtttc acttcctcag 480
tttgatgagc ttggttacct cgatcctgat gacaaaaccc gtttggaaga acaagcgagt 540
gggtttccta tgctaaaagt gaaagacatc cagtttggtt tttcgaattg gaaacaaggc 600
aaagagatat tcgagaacat tacgaaacaa acaaaagcat cttcaggagt catctggaac 660
tcatttaagg aactcgaaga gtctgagctc gaaactgtta tccgtgagat cccggctcca 720
agtttcttga taccactccc caagcatttg acagcctctt ccagcagctt actagaccac 780
gatcgaaccg tttttccatg gttagaccaa caaccgtcac gttcggtgct gtatgttagt 840
tttggtagtg ctactgaagt ggatgagaaa gatttcttgg aaatagctcg tgggttggtt 900
gatagcaagc agtcgttttt atgggtggtt cgacctgggt ttgtcaaggg ttcgacgtgg 960
gtcgaaccgt tgccagatgg gttcttgggt gaaagaggac gtattgtgaa atgggttccg 1020
cagcaagaag tgctagctca tggagcaata ggcgcattct ggactcatag cggatggaac 1080
tctacgttgg aaagcgtttg tgaaggtgtt cctatgattt tctcggattt tgggctcgat 1140
caaccgttga atgctagata catgagtgat gttttgaagg taggggtgta tttggaaaat 1200
gggtgggaaa gaggagagat agcaaatgca ataagaagag ttatggtgga tgaagaagga 1260
gaatacatta gacagaatgc aagagttttg aaacaaaagg cagatgtttc tttgatgaag 1320
ggtggttcat cttacgaatc attagagtct ctagtttctt acatttcatc gttgtaa 1377
<210> 2
<211> 458
<212> PRT
<213> 糖基转移酶(PRT)
<400> 2
Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile
1 5 10 15
Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Met Leu Gln Leu
20 25 30
Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr
35 40 45
Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg
50 55 60
Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro
65 70 75 80
Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His
85 90 95
Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser
100 105 110
Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr
115 120 125
Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu
130 135 140
Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln
145 150 155 160
Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu
165 170 175
Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Gln Phe
180 185 190
Gly Phe Ser Asn Trp Lys Gln Gly Lys Glu Ile Phe Glu Asn Ile Thr
195 200 205
Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu
210 215 220
Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro
225 230 235 240
Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser
245 250 255
Leu Leu Asp His Asp Arg Thr Val Phe Pro Trp Leu Asp Gln Gln Pro
260 265 270
Ser Arg Ser Val Leu Tyr Val Ser Phe Gly Ser Ala Thr Glu Val Asp
275 280 285
Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln
290 295 300
Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp
305 310 315 320
Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val
325 330 335
Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala
340 345 350
Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu
355 360 365
Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn
370 375 380
Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn
385 390 395 400
Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val
405 410 415
Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln
420 425 430
Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu
435 440 445
Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu
450 455
Claims (10)
1.一种UDP-糖基转移酶基因,其特征在于:其核苷酸序列具有如下特征中的一种或二种以上:
1)具有SEQ ID NO.1所示的脱氧核糖核酸序列;
2)编码SEQ ID NO.2氨基酸序列的脱氧核糖核酸序列;
3)对SEQ ID NO.1所示的脱氧核糖核酸序列进行一个或两个以上核苷酸取代和/或缺失和/或添加而得到的编码具有UDP-糖基转移酶活性的脱氧核糖核酸序列;
4)与SEQ ID NO.1限定的脱氧核糖核酸序列的同源性达到80%及以上,且能编码UDP-糖基转移酶的脱氧核糖核酸序列。
2.一种权利要求1所述的UDP-糖基转移酶基因编码的UDP-糖基转移酶,其特征在于:其氨基酸序列具有如下特征中的一种或二种以上:
1)具有SEQ ID NO.2所示的氨基酸序列;
2)具有如SEQ ID NO.2从氨基端开始第1-458位氨基酸残基序列;
3)对SEQ ID NO.2所示的氨基酸序列进行一个或两个以上氨基酸取代和/或缺失和/或添加而形成的具有UDP-糖基转移酶活性的氨基酸序列;
4)与SEQ ID NO.2具有至少80%同一性的氨基酸序列。
3.一种含有权利要求1所述的UDP-糖基转移酶基因的重组表达质粒。
4.一种含有权利要求1所述的UDP-糖基转移酶基因的重组基因工程菌。
5.一种权利要求2所述的UDP-糖基转移酶的制备方法,其特征在于:将权利要求1所述的UDP-糖基转移酶基因克隆入重组表达载体,导入宿主细胞,获得重组表达的UDP-糖基转移酶。
6.根据权利要求5所述的制备方法,其特征在于:所述的重组表达载体包括大肠杆菌表达载体、酵母表达载体、枯草杆菌表达载体、乳酸菌表达载体、链霉菌表达载体、噬菌体载体、丝状真菌表达载体、植物表达载体、昆虫表达载体、或哺乳动物细胞表达载体中的一种或二种以上;
所述宿主细胞包括大肠杆菌宿主细胞、酵母菌宿主细胞、枯草杆菌宿主细胞、乳酸菌宿主细胞、放线菌宿主细胞、丝状真菌宿主细胞、昆虫细胞,哺乳动物细胞中的一种。
7.一种权利要求2所述的UDP-糖基转移酶在糖基化甜菊醇糖苷或黄酮类化合物的应用。
9.莱鲍迪苷E2、甜菊苷B、莱鲍迪苷E6在作为甜味剂中的应用。
10.权利要求1所述的核苷酸或编码权利要求2所述蛋白质的核苷酸在甜叶菊遗传育种中的应用,所述核苷酸在甜叶菊中过表达。
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