CN109750072A - 一种酶法制备莱鲍迪苷e的方法 - Google Patents
一种酶法制备莱鲍迪苷e的方法 Download PDFInfo
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- CN109750072A CN109750072A CN201910095404.6A CN201910095404A CN109750072A CN 109750072 A CN109750072 A CN 109750072A CN 201910095404 A CN201910095404 A CN 201910095404A CN 109750072 A CN109750072 A CN 109750072A
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Abstract
本发明公开了一种酶法制备莱鲍迪苷E的方法，该方法利用番茄来源UDP‑糖基转移酶和马铃薯来源蔗糖合成酶，以甜菊甙为原料一步糖基化反应生产莱鲍迪苷E，同时采用定点突变技术，改造UDP‑糖基转移酶，进一步提高莱鲍迪苷E的产率。本发明的方法无需添加UDP‑葡萄糖和UDP，利用粗提液中UDP及额外添加的蔗糖在蔗糖合成酶分解作用下获得UDP‑葡萄糖作为糖基化反应的原材料，建立双酶循环反应体系，有效催化甜菊甙生产莱鲍迪苷E。其中，莱鲍迪苷E产率可达65%以上；后期通过定点突变技术，经筛选获得N358F有益突变体，其催化莱鲍迪苷E产率提高，最高可达85%以上。与已有的莱鲍迪苷E制备方法相比，工艺步骤简单，成本低廉，且产率高，具有重要的应用价值。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种酶法制备莱鲍迪苷E的方法。
背景技术
甜菊糖作为目前已知的最甜的甜味剂，其甜度是蔗糖的250～450倍，而热量只有蔗糖的1/300，略带有轻微涩味。除此之外，甜菊糖还具备对高血压、肥胖症、糖尿病等辅助药理作用[1,2]，因此，越来越受到人们的关注。甜菊糖是甜菊糖甙(Steviol glycosides)的总称，目前已从甜叶菊中分离鉴定出超过35种甜菊糖甙[3]，包括甜菊甙(Stevioside)和莱鲍迪苷A、B、C、D、E、M(Rebsudioside A/B/C/D/E/M)等。甜菊糖甙结构围绕一个双萜甜菊醇为骨架，在C13位羟基和C19位羧基分别连接了不等的葡萄糖基团，其中，骨架上C16-C17之间的碳碳双键是提供甜菊糖甙甜味及功能的药理性基团[3,4]。
不同甜菊糖甙的甜度有所差异[5]，莱鲍迪苷E(Rebaudioside E)的口感无后苦味，甜度与蔗糖相似，其结构上较甜菊甙在骨架上的C19位侧链上多了一个葡萄糖基团，在甜叶菊的干叶片中含量甚微(远小于1％)，直接通过常规物理手段从甜菊糖甙中分离莱鲍迪苷E，难度大且收率极低。
专利申请CN 201110379733.7(一种用β-环糊精葡萄糖基转移酶提高甜菊糖苷味质的方法)和专利申请CN 201010131126.4(一种用微波辅助环糊精葡萄糖基转移酶催化合成改性甜菊糖苷的方法)中均采用的是环糊精葡萄糖基转移酶催化合成甜菊糖苷，但是该酶不能只将单一的葡萄糖残基添加到甜菊糖苷底物分子上，其催化产物是较为复杂的混合物，后期分离难度较大。
专利申请CN 201410185097.8(甜菊苷生成莱鲍迪苷E的生物转化方法)和专利申请CN 201210288703.X(甜菊苷转化为莱鲍迪苷E的方法)中采用的是利用黑曲霉菌CICC40430发酵，每24h添加甲醇诱导表达糖基转移酶(70～75kDa)并纯化，其转化率≥70％，但是黑曲霉菌生长周期长，且需要在培养过程中添加乙醇作为诱导剂，操作复杂，产量不高。本方法通过生物酶催化手段，以甜菊甙纯品为原料一步糖基化反应制备莱鲍迪苷E，工艺步骤简单，成本低廉，且产率高，具有重要的应用价值。
参考文献
[1]KOVYLYAEVA G I,BAKALEINIK G A,STROBYKINA I Y,et al.Glycosides fromStevia rebaudiana[J].Chemistry of Natural Compounds,2007,43(1):81-85.
[2]KINGHORN A D,KINGHORN AD.Stevia:the genus Stevia[J].Crc Press,2002.
[3]OLSSON K,CARLSEN S,SEMMLER A,et al.Microbial production of next-generation stevia sweeteners[J].Microb Cell Fact,2016,15(1):207-220.
[4]UPRETI M,DUBOIS G,PRAKASH I.Synthetic study on the relationshipbetween structure and sweet taste properties of steviol glycosides[J].Molecules,2012,17(4):4186-4196.
[5]BRANDLE J E,TELMER P G.Steviol glycoside biosynthesis[J].Phytochemistry,2007,68:1855-1863.
发明内容
本发明的目的是针对目前直接从甜叶菊干叶片中，仅通过叶片碾碎、物理吸附、提纯等手段难以大量获取制备莱鲍迪苷E且经济效率低下的问题，提供一种酶法制备莱鲍迪苷E的方法，利用糖基转移酶UGTSL2糖基化作用，一步催化甜菊甙合成莱鲍迪苷E，为莱鲍迪苷E的制备及精制提供有力基础。
为了实现上述目的，本发明采用的技术方案为：
一种酶法制备莱鲍迪苷E的方法，利用分子克隆技术，获得异源表达UDP-糖基转移酶和蔗糖合成酶的基因工程菌，经自诱导发酵产酶后以细胞粗提液直接进行催化反应，利用粗提液中本身含有的微量UDP及额外添加的蔗糖，建立双酶循环反应体系，有效催化甜菊甙生产莱鲍迪苷E；其中，所述UDP-糖基转移酶为番茄来源糖基转移酶UGTSL2或者其突变体。
所述自诱导发酵过程中诱导剂在发酵培养液中浓度为0.01～0.5％，诱导剂可以为乳糖。
所述UDP-糖基转移酶为番茄来源的UDP-糖基转移酶基因UGTSL2，为NCBIReference Sequence:NC_015448.3，基因序列见SEQ.No.1所示，氨基酸序列(Uniprotnumber:K4D508)见SEQ.No.3所示；所述蔗糖合成酶为马铃薯来源蔗糖合成酶StSUS1，为NCBI Reference Sequence:M18745，基因序列见SEQ.No.2所示，氨基酸序列(Uniprotnumber:P10691)见SEQ.No.4所示。
所述基因工程菌为含有UDP-糖基转移酶和蔗糖合成酶的共表达的载体的重组大肠杆菌；UGTSL2基因片段插入在质粒载体的Nde I和Xho I酶切位点之间，StSUS1基因片段插入在质粒载体的Nco I和EcoR I酶切位点之间，最终提供了可外源共表达糖基转移酶UGTSL2和蔗糖合成酶StSUS1的重组质粒，pRSFDuet-UGTSL2-StSUS1，将重组质粒热击转化至大肠杆菌BL21(DE3)感受态细胞中，转化产物涂布于含有50mg/L的卡那霉素抗性的平板上，37℃过夜培养，获得共表达的重组大肠杆菌基因工程菌。
所述大肠杆菌基因工程菌诱导表达条件为：将重组大肠杆菌接种到LB培养基中，于16～37℃、200rpm振荡培养8～10h，再将培养菌液按2％接种量接入TB培养基中，其中诱导剂乳糖在TB培养液中浓度为0.01～0.5％，于16～37℃诱导培养16～36h，离心收集菌体。
所述双酶循环反应催化甜菊甙生产莱鲍迪苷E的体系为：底物甜菊甙的起始反应浓度为2～60g/L，蔗糖与甜菊甙的质量比为1～10，由共表达上述两个酶的基因工程菌制备的粗酶液在反应体系中的总蛋白浓度为1～25mg/mL，加水定容至1～100mL，pH值为6～8，于20～50℃反应1～48h。
所述对UDP-糖基转移酶采用定点突变技术，获取并筛选有益突变体，是通过将目标氨基酸序列与多组其他UDP-糖基转移酶氨基酸序列(包括：UniProtKB/Swiss-Prot:Q6VAB4、Q7XJ50、A6XNC6和Q9LK73)进行比对分析，在活性中心部位区间所选定的一个最有可能的关键位点；最终UDP-糖基转移酶定点突变的位点确定为N358；所述突变的氨基酸位点发生突变包括：N358A，N358C，N358D，N358E，N358F，N358G，N358H，N358I，N358K，N358L，N358M，N358P，N358Q，N358R，N358S，N358T，N358V，N358W，N358Y。其中，N358F突变体氨基酸序列见SEQ.No.5。
本发明发现了番茄来源糖基转移酶UGTSL2(51kDa)可一步催化甜菊甙糖基化合成莱鲍迪苷E，其可以在甜菊甙特定的羟基部位进行一分子葡萄糖的添加，产物更为单一，利于后期分离。至此，未有相关报道。
本发明将番茄来源糖基转移酶UGTSL2与马铃薯来源蔗糖合成酶StSUS1共表达于同一质粒载体上，于大肠杆菌中表达，提高双酶制备效率，且双酶在催化甜菊甙生产莱鲍迪苷E过程中，可利用细胞粗提液中本身含有的微量UDP和UDP-葡萄糖，在添加蔗糖的情况下，实现双酶循环反应，有效省去昂贵的糖基供体UDP-葡萄糖这一原料。本发明采用的是以大肠杆菌作为外源基因表达宿主，生长周期较短，且无需在培养过程中添加额外的诱导剂，降低工作量，并且将糖基转移酶和蔗糖合成酶同时构建到一个质粒载体上进行共表达，有效提高双酶制备效率，同时，细胞粗提液可直接用于催化反应，简化材料准备步骤。
本发明在实现酶法催化甜菊甙合成莱鲍迪苷E的基础上，对糖基转移酶UGTSL2进行定点突变，并发现一株更为高效的突变体(N358F)，其催化合成莱鲍迪苷E的产率由原始菌株得66％提高至87％，效果明显。
有益效果
本发明对于目前直接从甜叶菊干叶片中，仅通过叶片碾碎、物理吸附、提纯等手段难以大量获取制备莱鲍迪苷E且经济效率低下的问题，采用生物酶法，通过糖基转移酶UGTSL2一步糖基化反应催化甜菊甙合成莱鲍迪苷E，其中，甜菊甙的转化率可达94％，莱鲍迪苷E的产率≥65％；同时，经定点突变筛选有益突变体后，突变体N358F催化甜菊甙生产莱鲍迪苷E，甜菊甙转化率为94％，莱鲍迪苷E的产率大大提升至87％。
附图说明
图1莱鲍迪苷E结构式；
图2UGTSL2和StSUS1双酶偶联催化甜菊甙合成莱鲍迪苷E；
图3催化甜菊甙合成莱鲍迪苷E的HPLC检测图(a)为1g/L甜菊甙标准品；(b)为1g/L莱鲍迪苷E标准品；(c)为催化反应起始0h的样品；(d)为原始糖基转移酶UGTSL2催化反应24h的样品；(e)为突变体N358F催化反应24h的样品。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制要求书中所详细描述的本发明。
实施例1：重组大肠杆菌基因工程菌的构建
1)、共表达UGTSL2和StSUS1的重组质粒的获得
UGTSL2和StSUS1基因全序列由南京金斯瑞公司合成，并构建到质粒pRSFDuet-1(Novagen公司)上，其中，UGTSL2基因片段插入在质粒载体的Nde I和Xho I酶切位点之间，StSUS1基因片段插入在质粒载体的Nco I和EcoR I酶切位点之间，最终提供了可外源共表达糖基转移酶UGTSL2和蔗糖合成酶StSUS1的重组质粒，pRSFDuet-UGTSL2-StSUS1。
2)、重组大肠杆菌基因工程菌的获得
添加40uL ddH2O至重组质粒pRSFDuet-UGTSL2-StSUS1干粉中，充分溶解，取5ul热击转化至大肠杆菌BL21(DE3)感受态细胞中，转化产物涂布于含有50mg/L的卡那霉素抗性的平板上，37℃过夜培养，获得重组大肠杆菌基因工程菌。
实施例2：基因工程菌双酶的共表达
将含有质粒pRSFDuet-UGTSL2-StSUS1的重组基因工程菌接种到含有50mg/L卡那霉素抗性的LB培养基(0.5g/L酵母粉，1g/L氯化钠，1g/L胰蛋白胨)，于37℃、200rpm振荡过夜培养，再将培养菌按2％接种量接入含有100mL TB培养基(2.5g/L酵母粉，1g/L氯化钠，1.5g/L胰蛋白胨，0.2g/L葡萄糖，0.05g/L乳糖)的500mL摇瓶中，于200rpm，37℃培养2h，随后转移至25℃继续培养24h，离心收集菌体。超声破碎菌体，离心取上清液即为细胞粗提液，置于4℃保存待用。
实施例3酶法制备莱鲍迪苷E的方法
1)、催化反应体系
添加20g/L甜菊甙、60g/L蔗糖、3mM Mg2+及适量共表达UGTSL2和StSUS1的细胞粗提液(～6mg/mL总蛋白)于50mM磷酸钾缓冲(pH 7.2)中，总体积定量20mL。30℃，200rpm反应24h，定时取样500uL，置于95℃水浴加热15min，12000rpm室温离心1min，分离上清液至新1.5mLEP管中，4℃保存，待HPCL检测。就结果来看(表1)，糖基转移酶UGTSL2偶联蔗糖合成酶催化甜菊甙合成莱鲍迪苷E是可行的，且于反应24h时，甜菊甙的转化率已达到94.12％，莱鲍迪苷E的产率为66.32％。
2)、PHLC检测方法
色谱柱：Agilent TC-C18(4.6*250mm，5um，120A)；紫外检测波长：210nm；柱温：55℃；流动相A为含0.1％甲酸的乙腈，流动相B为含0.1％甲酸的娃哈哈水，液相检测条件：0.000min(25％A,75％B),15.000min(47％A,53％B),20.000min(100％A,0％B),20.010-25.000min(100％A,0％B),25.010min(25％A,75％B),25.010-30.000min(25％A,75％B)；样品进样10uL，流速1mL/min。
表1：双酶催化甜菊甙合成莱鲍迪苷E
实施例4：糖基转移酶定点突变
根据糖基转移酶UGTSL2基因序列及定点突变需求设计相应的引物，见表2。
表2：定点突变引物序列
所有引物均由南京金斯瑞公司合成。
定点突变全程采用南京诺唯赞生物技术公司提供的Mut ExpressR II FastMutagenesis Kit试剂盒进行操作。操作如下：以质粒pRSFDuet-UGTSL2-StSUS1为模板，用各自的引物进行PCR扩增全质粒，结束后在体系中添加1uL的Dpn I消化原始质粒模板，使用DNA连接酶(Exnase II)将线性质粒环化。取环化产物20uL热击转化至大肠杆菌BL21(DE3)感受态细胞中。转化物涂布于含有50mg/L卡那霉素抗性的平板上，37℃过夜培养，筛选阳性克隆，获得对应的、正确的突变体。
实施例5糖基转移酶及其突变体的酶活测定
1)、催化反应体系
添加1.2mM甜菊甙、2mM UDPG、3mM Mg2+及适量共表达UGTSL2和StSUS1或共表达UGTSL2突变体和StSUS1的细胞粗提液(～1mg总蛋白)于50mM磷酸钾缓冲(pH 7.2)中，总体积定量3mL。反应中，添加等粗酶液体积的磷酸缓冲作为空白对照。30℃，200rpm反应30min，取样500uL，置于95℃水浴加热15min，12000rpm室温离心1min，分离上清液至新1.5mL EP管中，4℃保存，待HPCL检测。结果发现(表3)，糖基转移酶催化甜菊甙合成莱鲍迪苷E的比酶活为9.36mU/mg，而大多突变体的比酶活并没有提高，只有突变体N358F的比酶活是明显高于原始UGTSL2的，其比酶活提高了约8％。
2)、PHLC检测方法
色谱柱：Agilent TC-C18(4.6*250mm，5um，120A)；紫外检测波长：210nm；柱温：55℃；流动相A为含0.1％甲酸的乙腈，流动相B为含0.1％甲酸的娃哈哈水，液相检测条件：0.000min(25％A,75％B),15.000min(47％A,53％B),20.000min(100％A,0％B),20.010-25.000min(100％A,0％B),25.010min(25％A,75％B),25.010-30.000min(25％A,75％B)；样品进样10uL，流速1mL/min。
3)、酶活力单位定义为：在上述反应条件下，每分钟每生成1umol的产物莱鲍迪苷E所需要的酶量为1个活力单位(U)。
表3：糖基转移酶UGTSL2及其突变体的比酶活
| 酶 | 比酶活(mU/mg) |
| UGTSL2 | 9.36±0.23 |
| N358C | 3.50±0.06 |
| N358F | 10.10±0.27 |
| N358L | 5.11±0.14 |
实施例6糖基转移酶突变体偶联蔗糖合成酶催化甜菊甙合成莱鲍迪苷E
1)、催化反应体系
添加20g/L甜菊甙、60g/L蔗糖、3mM Mg2+及适量共表达UGTSL2突变体和StSUS1的细胞粗提液(～6mg/mL总蛋白)于50mM磷酸钾缓冲(pH 7.2)中，总体积定量20mL。30℃，200rpm反应24h，定时取样500uL，置于95℃水浴加热15min，12000rpm室温离心1min，分离上清液至新1.5mL EP管中，4℃保存，待HPCL检测。就结果来看(表4)，UGTSL2的突变体N358F偶联蔗糖合成酶催化甜菊甙合成莱鲍迪苷E效果更为显著，于反应24h时，甜菊甙的转化率为94.52％，莱鲍迪苷E的产率则提高至87.19％。
2)、HPLC检测方法
色谱柱：Agilent TC-C18(4.6*250mm，5um，120A)；紫外检测波长：210nm；柱温：55℃；流动相A为含0.1％甲酸的乙腈，流动相B为含0.1％甲酸的娃哈哈水，液相检测条件：0.000min(25％A,75％B),15.000min(47％A,53％B),20.000min(100％A,0％B),20.010-25.000min(100％A,0％B),25.010min(25％A,75％B),25.010-30.000min(25％A,75％B)；样品进样10uL，流速1mL/min。
表4：双酶催化甜菊甙合成莱鲍迪苷E
序列表
<110> 南京工业大学
<120> 一种酶法制备莱鲍迪苷E的方法
<141> 2019-01-30
<160> 43
<170> SIPOSequenceListing 1.0
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tatctctcat ttcactctat acatagagaa atcccaaatc aacttatttc aatatggaca 60
catttcacca tttctaaaca tagccaagca actcgcggat agaggattct tgatttacct 120
ctgttccacg cgaatcaatc ttgaatccat catcaagaaa atccctgaaa agtatgctga 180
ttcgattcat cttatcgaac ttcaattgcc agaattaccc gaacttcctc ctcattacca 240
tacgacgaat ggcctcccac cccatctcaa tcccaccctt cataaggccc tgaaaatgtc 300
caaaccaaac ttttcgagaa tcttgcaaaa tctgaaacct gatttattga tttacgatgt 360
attgcagccg tgggctgaac atgttgcgaa tgaacaaaac attccagcag gtaagctgct 420
aacttcgtgt gcagctgtgt tttcgtattt tttcagcttt cgaaagaatc caggggttga 480
attccctttc cctgctattc atctcccgga agttgagaaa gtaaagatca gagaaatact 540
cgcgaaagaa cctgaagaag ggggtcgtct agacgaggga aataagcaaa tgatgttgat 600
gtgtacgtct agaactatcg aggctaaata catagattac tgcactgaat tgtgcaattg 660
gaaagttgtt ccagttggtc caccattcca agatttgatc actaatgatg cggacaacaa 720
ggagcttatc gattggcttg gaacaaaaca tgaaaattca actgttttcg tctcctttgg 780
gagtgagtat ttcttgtcaa aagaagatat ggaagaagta gctttcgcgc tggagttaag 840
taatgttaat ttcatatggg ttgcaagatt tccgaaaggt gaagagcgaa atctcgaaga 900
tgcattacca aaaggttttc ttgaaagaat tggagaaagg ggaagagttt tggacaaatt 960
tgcaccacaa ccaagaattc taaatcatcc gagcaccgga ggatttataa gtcattgtgg 1020
ttggaattca gcaatggaaa gtatagattt cggggttcct ataatcgcaa tgcctattca 1080
taatgatcaa cctataaatg ctaagttgat ggtagaattg ggagtcgcgg tggagattgt 1140
tagagatgat gatgggaaaa ttcacagagg agaaatcgcg gaaactctta aaagtgtcgt 1200
aacaggggaa acaggggaaa ttttgagggc taaagtgaga gaaatcagca agaatttgaa 1260
atctataaga gatgaagaga tggatgctgt tgctgaagag ctaattcaac tttgtaggaa 1320
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atggctgaac gtgttttgac tcgtgttcat agtcttcgtg agcgtgttga tgcaacttta 60
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aaactgaacg aacatgcatt cgaagaactc ctgaaatcca ctcaggaagc gattgttctg 240
cccccttggg ttgcacttgc tattcgtttg aggcctggtg tctgggaata catccgtgtg 300
aacgtcaacg cactagttgt tgaggagctg tctgtccctg agtatttgca attcaaggaa 360
gaacttgtcg acggagcctc gaatggaaat ttcgttctcg agttggattt cgagcctttt 420
actgcatcct ttcctaaacc aaccctcacc aaatctattg gaaatggagt tgaattcctc 480
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tttcttcgcg ctcaccatta taagggcaag acaatgatgc tgaatgatag gatacagaat 600
tcgaatactc ttcaaaatgt cctaaggaag gcagaggaat acctcattat gcttcccccg 660
gaaactccat atttcgaatt cgaacacaag ttccaagaaa tcggattgga gaaaggatgg 720
ggggacacgg cggagcgtgt gctagagatg gtatgcatgc ttcttgatct ccttgaggct 780
cctgactcat gtactcttga gaagttcttg gggagaattc ctatggtttt caatgtggtt 840
atcctttccc ctcatggata ttttgctcaa gaaaatgtct tgggttatcc tgacaccggt 900
ggccaggttg tctacatttt agatcaagtt cccgccttgg agcgtgaaat gcttaagcgc 960
ataaaggagc aaggacttga tatcatcccc cgtattctta ttgttactcg tctgctcccc 1020
gatgcagttg gaaccacttg tggtcagagg attgagaagg tgtatggagc agaacactca 1080
catattctta gggtcccttt taggactgag aagggcattg ttcgcaaatg gatctctcgc 1140
tttgaagtgt ggccatacat ggagacattc attgaggatg ttgcaaaaga aatttctgca 1200
gaactgcagg ccaagccaga tttgataatc ggaaactaca gtgagggcaa tcttgctgct 1260
tctttgctag ctcacaagtt aggcgtaacg cagtgcacca ttgcccacgc gttggagaaa 1320
acgaagtatc ctgattccga catttactgg aaaaagtttg atgaaaaata ccatttctcg 1380
tcccagttta ccgctgatct cattgcaatg aatcacactg atttcatcat caccagcacc 1440
ttccaggaga tagcaggaag caaggacact gtgggacaat atgagagcca tatggcattc 1500
acaatgcctg gattgtacag agttgttcac ggcattaatg tgttcgaccc caaattcaac 1560
attgtctcac ctggagctga tattaacctc tacttctcgt actccgaaac ggagaagaga 1620
cttacagcat ttcaccctga aattgatgag ctgctgtata gtgatgttga gaatgacgaa 1680
catctgtgcg tgctcaagga caggactaaa ccaattttat tcacaatggc gaggttggat 1740
cgtgtgaaga atttaactgg acttgttgag tggtacgcca aaaatccacg gctaagggga 1800
ttggttaacc tggttgtagt tggcggagat cgaaggaagg aatccaaaga tttggaagag 1860
caggcagaga tgaagaagat gtatgagcta attgagactc ataatttgaa tggccaattc 1920
agatggattt cttcccagat gaaccgagtg aggaatggtg agctctaccg atacattgct 1980
gacactaagg gagctttcgt tcagcctgca ttctacgagg cttttggtct gactgttgtc 2040
gaagcaatga cttgtggttt gcctacattt gcaactaatc acggtggtcc agctgagatc 2100
atcgttcatg gaaagtccgg cttccacatt gatccatatc acggtgagca agctgctgat 2160
ctgctagctg atttctttga gaaatgcaag aaagatcctt cacattggga aaccatttcg 2220
atgggtggcc tgaagcgcat cgaagagaag tacacttggc aaatctactc cgaaagccta 2280
ttgacactgg ctgctgttta tgggttctgg aaacatgttt ctaagcttga tcgtctagaa 2340
atccgtcgct atcttgaaat gttttatgct ctcaagtacc gtaagatggc tgaagctgtt 2400
ccattggctg ctgagtga 2418
<210> 3
<211> 442
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly
1 5 10 15
His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly
20 25 30
Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile
35 40 45
Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu
50 55 60
Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn
65 70 75 80
Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met
85 90 95
Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu
100 105 110
Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu
115 120 125
Gln Asn Ile Pro Ala Gly Lys Leu Leu Thr Ser Cys Ala Ala Val Phe
130 135 140
Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe
145 150 155 160
Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile
165 170 175
Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys
180 185 190
Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile
195 200 205
Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro
210 215 220
Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile
225 230 235 240
Asp Trp Leu Gly Thr Lys His Glu Asn Ser Thr Val Phe Val Ser Phe
245 250 255
Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Val Ala Phe
260 265 270
Ala Leu Glu Leu Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe Pro
275 280 285
Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu
290 295 300
Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln
305 310 315 320
Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys
325 330 335
Gly Trp Asn Ser Ala Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile
340 345 350
Ala Met Pro Ile His Asn Asp Gln Pro Ile Asn Ala Lys Leu Met Val
355 360 365
Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile
370 375 380
His Arg Gly Glu Ile Ala Glu Thr Leu Lys Ser Val Val Thr Gly Glu
385 390 395 400
Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu
405 410 415
Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile
420 425 430
Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys
435 440
<210> 4
<211> 805
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Ala Glu Arg Val Leu Thr Arg Val His Ser Leu Arg Glu Arg Val
1 5 10 15
Asp Ala Thr Leu Ala Ala His Arg Asn Glu Ile Leu Leu Phe Leu Ser
20 25 30
Arg Ile Glu Ser His Gly Lys Gly Ile Leu Lys Pro His Glu Leu Leu
35 40 45
Ala Glu Phe Asp Ala Ile Arg Gln Asp Asp Lys Asn Lys Leu Asn Glu
50 55 60
His Ala Phe Glu Glu Leu Leu Lys Ser Thr Gln Glu Ala Ile Val Leu
65 70 75 80
Pro Pro Trp Val Ala Leu Ala Ile Arg Leu Arg Pro Gly Val Trp Glu
85 90 95
Tyr Ile Arg Val Asn Val Asn Ala Leu Val Val Glu Glu Leu Ser Val
100 105 110
Pro Glu Tyr Leu Gln Phe Lys Glu Glu Leu Val Asp Gly Ala Ser Asn
115 120 125
Gly Asn Phe Val Leu Glu Leu Asp Phe Glu Pro Phe Thr Ala Ser Phe
130 135 140
Pro Lys Pro Thr Leu Thr Lys Ser Ile Gly Asn Gly Val Glu Phe Leu
145 150 155 160
Asn Arg His Leu Ser Ala Lys Met Phe His Asp Lys Glu Ser Met Thr
165 170 175
Pro Leu Leu Glu Phe Leu Arg Ala His His Tyr Lys Gly Lys Thr Met
180 185 190
Met Leu Asn Asp Arg Ile Gln Asn Ser Asn Thr Leu Gln Asn Val Leu
195 200 205
Arg Lys Ala Glu Glu Tyr Leu Ile Met Leu Pro Pro Glu Thr Pro Tyr
210 215 220
Phe Glu Phe Glu His Lys Phe Gln Glu Ile Gly Leu Glu Lys Gly Trp
225 230 235 240
Gly Asp Thr Ala Glu Arg Val Leu Glu Met Val Cys Met Leu Leu Asp
245 250 255
Leu Leu Glu Ala Pro Asp Ser Cys Thr Leu Glu Lys Phe Leu Gly Arg
260 265 270
Ile Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly Tyr Phe
275 280 285
Ala Gln Glu Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln Val Val
290 295 300
Tyr Ile Leu Asp Gln Val Pro Ala Leu Glu Arg Glu Met Leu Lys Arg
305 310 315 320
Ile Lys Glu Gln Gly Leu Asp Ile Ile Pro Arg Ile Leu Ile Val Thr
325 330 335
Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Gln Arg Ile Glu
340 345 350
Lys Val Tyr Gly Ala Glu His Ser His Ile Leu Arg Val Pro Phe Arg
355 360 365
Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu Val Trp
370 375 380
Pro Tyr Met Glu Thr Phe Ile Glu Asp Val Ala Lys Glu Ile Ser Ala
385 390 395 400
Glu Leu Gln Ala Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser Glu Gly
405 410 415
Asn Leu Ala Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr Gln Cys
420 425 430
Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser Asp Ile
435 440 445
Tyr Trp Lys Lys Phe Asp Glu Lys Tyr His Phe Ser Ser Gln Phe Thr
450 455 460
Ala Asp Leu Ile Ala Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr
465 470 475 480
Phe Gln Glu Ile Ala Gly Ser Lys Asp Thr Val Gly Gln Tyr Glu Ser
485 490 495
His Met Ala Phe Thr Met Pro Gly Leu Tyr Arg Val Val His Gly Ile
500 505 510
Asn Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Ile
515 520 525
Asn Leu Tyr Phe Ser Tyr Ser Glu Thr Glu Lys Arg Leu Thr Ala Phe
530 535 540
His Pro Glu Ile Asp Glu Leu Leu Tyr Ser Asp Val Glu Asn Asp Glu
545 550 555 560
His Leu Cys Val Leu Lys Asp Arg Thr Lys Pro Ile Leu Phe Thr Met
565 570 575
Ala Arg Leu Asp Arg Val Lys Asn Leu Thr Gly Leu Val Glu Trp Tyr
580 585 590
Ala Lys Asn Pro Arg Leu Arg Gly Leu Val Asn Leu Val Val Val Gly
595 600 605
Gly Asp Arg Arg Lys Glu Ser Lys Asp Leu Glu Glu Gln Ala Glu Met
610 615 620
Lys Lys Met Tyr Glu Leu Ile Glu Thr His Asn Leu Asn Gly Gln Phe
625 630 635 640
Arg Trp Ile Ser Ser Gln Met Asn Arg Val Arg Asn Gly Glu Leu Tyr
645 650 655
Arg Tyr Ile Ala Asp Thr Lys Gly Ala Phe Val Gln Pro Ala Phe Tyr
660 665 670
Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly Leu Pro
675 680 685
Thr Phe Ala Thr Asn His Gly Gly Pro Ala Glu Ile Ile Val His Gly
690 695 700
Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Glu Gln Ala Ala Asp
705 710 715 720
Leu Leu Ala Asp Phe Phe Glu Lys Cys Lys Lys Asp Pro Ser His Trp
725 730 735
Glu Thr Ile Ser Met Gly Gly Leu Lys Arg Ile Glu Glu Lys Tyr Thr
740 745 750
Trp Gln Ile Tyr Ser Glu Ser Leu Leu Thr Leu Ala Ala Val Tyr Gly
755 760 765
Phe Trp Lys His Val Ser Lys Leu Asp Arg Leu Glu Ile Arg Arg Tyr
770 775 780
Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Lys Met Ala Glu Ala Val
785 790 795 800
Pro Leu Ala Ala Glu
805
<210> 5
<211> 442
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly
1 5 10 15
His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly
20 25 30
Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile
35 40 45
Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu
50 55 60
Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn
65 70 75 80
Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met
85 90 95
Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu
100 105 110
Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu
115 120 125
Gln Asn Ile Pro Ala Gly Lys Leu Leu Thr Ser Cys Ala Ala Val Phe
130 135 140
Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe
145 150 155 160
Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile
165 170 175
Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys
180 185 190
Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile
195 200 205
Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro
210 215 220
Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile
225 230 235 240
Asp Trp Leu Gly Thr Lys His Glu Asn Ser Thr Val Phe Val Ser Phe
245 250 255
Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Val Ala Phe
260 265 270
Ala Leu Glu Leu Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe Pro
275 280 285
Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu
290 295 300
Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln
305 310 315 320
Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys
325 330 335
Gly Trp Asn Ser Ala Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile
340 345 350
Ala Met Pro Ile His Phe Asp Gln Pro Ile Asn Ala Lys Leu Met Val
355 360 365
Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile
370 375 380
His Arg Gly Glu Ile Ala Glu Thr Leu Lys Ser Val Val Thr Gly Glu
385 390 395 400
Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu
405 410 415
Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile
420 425 430
Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys
435 440
<210> 6
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccgatccacg ccgaccaacc gattaacgcg aaactgatgg t 41
<210> 7
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cggttggtcg gcgtggatcg gcatcgcaat gatcggaacg c 41
<210> 8
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccgatccact gcgaccaacc gattaacgcg aaactgatgg t 41
<210> 9
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cggttggtcg cagtggatcg gcatcgcaat gatcggaacg c 41
<210> 10
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccgatccacg acgaccaacc gattaacgcg aaactgatgg t 41
<210> 11
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cggttggtcg tcgtggatcg gcatcgcaat gatcggaacg c 41
<210> 12
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccgatccacg aagaccaacc gattaacgcg aaactgatgg t 41
<210> 13
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cggttggtct tcgtggatcg gcatcgcaat gatcggaacg c 41
<210> 14
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ccgatccact tcgaccaacc gattaacgcg aaactgatgg t 41
<210> 15
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cggttggtcg aagtggatcg gcatcgcaat gatcggaacg c 41
<210> 16
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ccgatccacg gcgaccaacc gattaacgcg aaactgatgg t 41
<210> 17
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cggttggtcg ccgtggatcg gcatcgcaat gatcggaacg c 41
<210> 18
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cggttggtcg ccgtggatcg gcatcgcaat gatcggaacg c 41
<210> 19
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ccgatccacc acgaccaacc gattaacgcg aaactgatgg t 41
<210> 20
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ccgatccaca tcgaccaacc gattaacgcg aaactgatgg t 41
<210> 21
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
cggttggtcg atgtggatcg gcatcgcaat gatcggaacg c 41
<210> 22
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ccgatccaca aagaccaacc gattaacgcg aaactgatgg t 41
<210> 23
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
cggttggtct ttgtggatcg gcatcgcaat gatcggaacg c 41
<210> 24
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ccgatccacc tggaccaacc gattaacgcg aaactgatgg t 41
<210> 25
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
cggttggtcc aggtggatcg gcatcgcaat gatcggaacg c 41
<210> 26
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ccgatccaca tggaccaacc gattaacgcg aaactgatgg t 41
<210> 27
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
cggttggtcc atgtggatcg gcatcgcaat gatcggaacg c 41
<210> 28
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ccgatccacc cggaccaacc gattaacgcg aaactgatgg t 41
<210> 29
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
cggttggtcc gggtggatcg gcatcgcaat gatcggaacg c 41
<210> 30
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ccgatccacc aggaccaacc gattaacgcg aaactgatgg t 41
<210> 31
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
cggttggtcc tggtggatcg gcatcgcaat gatcggaacg c 41
<210> 32
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
ccgatccacc gcgaccaacc gattaacgcg aaactgatgg t 41
<210> 33
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
cggttggtcg cggtggatcg gcatcgcaat gatcggaacg c 41
<210> 34
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ccgatccaca gcgaccaacc gattaacgcg aaactgatgg t 41
<210> 35
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
cggttggtcg ctgtggatcg gcatcgcaat gatcggaacg c 41
<210> 36
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ccgatccaca ccgaccaacc gattaacgcg aaactgatgg t 41
<210> 37
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
cggttggtcg gtgtggatcg gcatcgcaat gatcggaacg c 41
<210> 38
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ccgatccacg tcgaccaacc gattaacgcg aaactgatgg t 41
<210> 39
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
cggttggtcg acgtggatcg gcatcgcaat gatcggaacg c 41
<210> 40
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
ccgatccact gggaccaacc gattaacgcg aaactgatgg t 41
<210> 41
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
cggttggtcc cagtggatcg gcatcgcaat gatcggaacg c 41
<210> 42
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ccgatccact acgaccaacc gattaacgcg aaactgatgg t 41
<210> 43
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
cggttggtcg tagtggatcg gcatcgcaat gatcggaacg c 41
Claims (10)
1.一种酶法制备莱鲍迪苷E的方法，其特征在于，利用分子克隆技术，获得异源表达UDP-糖基转移酶和蔗糖合成酶的基因工程菌，经自诱导发酵产酶后以细胞粗提液直接进行催化反应，利用粗提液中本身含有的微量UDP及额外添加的蔗糖，建立双酶循环反应体系，有效催化甜菊甙生产莱鲍迪苷E；其中，所述UDP-糖基转移酶为番茄来源糖基转移酶UGTSL2或者其突变体。
2.根据权利要求1所述的一种酶法制备莱鲍迪苷E的方法，其特征在于，所述番茄来源糖基转移酶UGTSL2的基因序列见SEQ.No.1所示，氨基酸序列见SEQ.No.3所示；所述蔗糖合成酶为马铃薯来源蔗糖合成酶StSUS1，基因序列见SEQ.No.2所示，氨基酸序列见SEQ.No.4所示。
3.根据权利要求1所述的一种酶法制备莱鲍迪苷E的方法，其特征在于，所述基因工程菌为含有UDP-糖基转移酶和蔗糖合成酶的共表达的载体的重组大肠杆菌；所述自诱导发酵过程中诱导剂在发酵培养液中浓度为0.01~0.5%。
4.根据权利要求1所述的一种酶法制备莱鲍迪苷E的方法，其特征在于，所述细胞粗提液为发酵得到的菌体经超声或低温高压破碎所获得的含有UDP-糖基转移酶和蔗糖合成酶的粗酶液。
5.根据权利要求1所述的一种酶法制备莱鲍迪苷E的方法，其特征在于，所述双酶循环反应体系为：蔗糖经蔗糖合成酶分解，与细胞粗提液中的UDP反应生成果糖和UDP-葡萄糖，UDP-葡萄糖再与甜菊甙经UDP-糖基转移酶催化生成莱鲍迪苷E。
6.根据权利要求1所述的一种酶法制备莱鲍迪苷E的方法，其特征在于，所述底物甜菊甙的起始反应浓度为2~60 g/L，蔗糖与甜菊甙的质量比为1~10，粗酶液在反应体系中的总蛋白浓度为1~25 mg/mL。
7.根据权利要求1所述的一种酶法制备莱鲍迪苷E的方法，其特征在于，所述催化反应采用水相反应体系，水溶液pH值为6~8。
8.根据权利要求1所述的一种酶法制备莱鲍迪苷E的方法，其特征在于，所述催化反应的反应温度为20~50℃，反应时间为1~48 h。
9.根据权利要求1所述的一种酶法制备莱鲍迪苷E的方法，其特征在于，所述UDP-糖基转移酶突变体突变的氨基酸位点包括：N358A，N358C，N358D，N358E，N358F，N358G，N358H，N358I，N358K，N358L，N358M，N358P，N358Q，N358R，N358S，N358T，N358V，N358W，N358Y。
10.一种催化合成莱鲍迪苷E的UDP-糖基转移酶突变体，其特征在于，所述突变体是番茄来源糖基转移酶UGTSL2在位点N358突变而成。
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| CN111019918A (zh) * | 2019-11-20 | 2020-04-17 | 广东广业清怡食品科技有限公司 | 一种糖基转移酶突变体及其应用 |
| CN112375750A (zh) * | 2020-12-02 | 2021-02-19 | 南京工业大学 | 一种糖基转移酶突变体及其催化合成莱鲍迪苷a的方法 |
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| CN115975972A (zh) * | 2022-12-20 | 2023-04-18 | 杭州力文所生物科技有限公司 | 一种糖基转移酶突变体及其编码基因 |
| CN115975972B (zh) * | 2022-12-20 | 2023-07-25 | 杭州力文所生物科技有限公司 | 一种糖基转移酶突变体及其编码基因 |
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