CN114854722A - 一种季也蒙念珠菌外切β-1,3-葡聚糖酶及其突变体及应用 - Google Patents
一种季也蒙念珠菌外切β-1,3-葡聚糖酶及其突变体及应用 Download PDFInfo
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- CN114854722A CN114854722A CN202210511122.1A CN202210511122A CN114854722A CN 114854722 A CN114854722 A CN 114854722A CN 202210511122 A CN202210511122 A CN 202210511122A CN 114854722 A CN114854722 A CN 114854722A
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Abstract
本发明属于生物工程技术领域，具体涉及一种季也蒙念珠菌外切β‑1,3‑葡聚糖酶及其突变体及应用。一种外切β‑1,3‑葡聚糖酶Exo15，其特征在于所述的外切β‑1,3‑葡聚糖酶Exo15的核苷酸序列如SEQ ID NO.1所示，其氨基酸序列如SEQ ID NO.2所示。本发明提供利用外切β‑1,3‑葡聚糖酶Exo15、其突变体或重组菌株具有高效催化合成赛门苷I的作用，其具有见效快、成本低、操作简单、方便实施、产品无毒无残留等特征，可满足罗汉果赛门苷I的大规模生产需求，具有较强的实用性和推广价值。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种季也蒙念珠菌外切β-1,3-葡聚糖酶及其突变体及应用。
背景技术
天然产物在当今的药物治疗中发挥着重要作用，临床中使用的大量药物要么是天然的，要么是从植物中提取的。中国有许多“极其有效”的水果。因此，这些来源可能有助于开发更安全、更可接受的药物。
罗汉果(Siraitia grosvenorii)是广西道地药材，对生境的要求较严格，主要垂直分布于海拔250～1000m的亚热带有坡地区，要求生长在昼夜温差较大、环境湿润的地区；喜光但不耐强光，怕霜冻；适宜栽培在排水较好、有机质丰富、疏松湿润的红黄壤土中。300多年来，罗汉果一直被用作天然甜味剂，在中国被用作治疗咽炎、咽痛和止咳的传统药物。是中国首批批准的药食同源物种之一。是中国首批批准的药食同源物种之一(“药食同源”的概念在《黄帝内经》中有提及)。作为一种极具开发潜力的天然产物，已被广泛研究。从该植物中分离出三萜、类黄酮和氨基酸等化合物。罗汉果及其活性成分具有广泛的药理作用，如抗氧化、降糖、免疫、抗咳痰、保肝、抗菌等【Gong,Xue,et al.Frontiers inpharmacology(2019):1400.】1。目前，以罗汉果为主要原料已经制成的中药有罗汉果咽喉片、镇咳祛痰合剂、罗汉果止咳露、止咳定喘片、罗汉果止咳剂等(黎霜等，2003)。罗汉果甜苷类物质甜度高、热值低、水溶性与热稳定性好。无毒，食用安全，没有异味，因此广泛应用于食品及保健品工业中。近年来，罗汉果被制成汁粉、果糕、柿饼汤、面包、果凉茶、单晶冰糖等食品和保健品；罗汉果在国外市场也非常受欢迎，日本、韩国、英国、新加坡等国先后批准罗汉果可以作为食品添加剂，特别是用罗汉果制成的高级润喉糖、罗汉果果汁等产品在日本深受人们的喜爱。
西葫芦苷是罗汉果的主要成分，也是罗汉果的有效成分。自1983年Takemoto 等从罗汉果中分离出mogrosides IV、V和VI(Takemoto T,.Yakugaku zasshi:Journal of thePharmaceutical Society of Japan,1983,103(11):1167-1173.)2以来，已从罗汉果中分离出30多种类似化合物(Jia,Zhonghua,.Natural Product Communications 4.6(2009):1934578X0900400606)3。在新鲜水果中，罗汉果糖苷含量为1.19％；而在干果中，罗汉果糖苷含量为可达3.82％(Li H B,et al.Food Sci,2006,27(6):171-173.)4.罗汉果苷V是罗汉果中主要糖苷，干果中占比0.5-1.4％(Hai-Bin L I,et al.Food Science, 2006.)5.罗汉果苷大多具有味甜，统称为罗汉果苷，是罗汉果的主要活性成分。其中,罗汉果苷IV、V和赛门苷I是迄今为止报道的罗汉果甜苷中甜度最高的3种成分，分别为蔗糖甜度的392，425和563倍。赛门苷I是葫芦烷三萜苷中最甜的成分，赛门苷I(0.01％浓度)的为5％蔗糖甜度的563倍(Matsumoto K,et al.Chemical&Pharmaceutical Bulletin,2008,38(7):2030-2032.)6此外，体外试验表明赛门苷I对麦芽糖酶有抑制作用，Suzuki等研究发现，罗汉果苷V，IV，III，赛门苷I对单剂量口服麦芽糖大鼠餐后血糖具有强烈的抑制作用，IC50分别为14,12,10和1.6mM(Suzuki Y A,et al.Journal of agricultural and food chemistry,2005,53(8):2941-2946.)7。
在过去的几十年里，全球对天然甜味剂的需求显著增加，原因是人们对人工甜味剂作为糖替代品的长期消费感到担忧。到目前为止，美国食品和药物管理局只批准了两种高强度天然甜味剂，包括纯化的莱鲍迪苷A(甜菊叶提取物)和罗汉果提取物(从罗汉果的果实提取，通常称为罗汉果:LHK)(FDA,2008,2010)。事实上，赛门苷I目前被认为是最甜的罗汉果苷。此外，据报道，赛门苷I的味道优于罗汉果苷IV和罗汉果苷V(Zhou等，2014)；然而，天然甜果苷提取物中赛门苷I的含量有限(Matsumoto等，1990)。因此，采用分离、纯化或富集的方法获得高浓度的天然赛门苷I将是有益的。人们已经进行了许多尝试，包括化学水解、植物组织培、酶处理和微生物发酵，将罗汉果苷V转化为赛门苷I。不幸的是，罗汉果苷V中葡萄糖分支侧链的复杂性使得生产或分离纯赛门苷I极具挑战性。当前赛门苷I的生产与制备主要是通过植物提取获得，但是植物类含量极低，严重制约着赛门苷I的大规模应用。
本实验室前期从罗汉果内生菌中筛选到一株季也蒙念珠菌Meyerozymaguilliermondii，该菌株的名称为Meyerozyma guilliermondii LHGNSJ-VS01(M.guilliermondii LHGNSJ-VS01) (参见专利CN201910266094.X)。对该菌株发酵获得粗胞外蛋白(蛋白混合物)，发明人发现粗蛋白具有所述的胞外蛋白将罗汉果苷V转化为赛门苷I的作用。但由于粗蛋白提取物并不知道具体哪些蛋白起作用。本发明在该专利基础上做了进一步研究。经分离纯化，纯化所得蛋白进行鉴定，获得全新的单一功能蛋白，经文献检索确定为外切β-1,3-葡聚糖酶。该酶未见催化罗汉果苷V转化为赛门苷I的报道。
发明内容
本发明的目的之一：提供一种全新的来自于季也蒙念珠菌的外切β-1,3-葡聚糖酶Exo15，并发发现催化合成赛门苷I的作用；
本发明的目的之二：为提供提高重组蛋白Exo15可溶性，通过迭代饱和突变提高重组蛋白Exo15催化活性，提供外切β-1,3-葡聚糖酶的突变体Exo15的突变体，该突变体具有更高效制备赛门苷I的催化作用。
技术方案
一种外切β-1,3-葡聚糖酶Exo15在制备将罗汉果苷V转化为赛门苷I的催化剂的应用，其特征在于所述的外切β-1,3-葡聚糖酶Exo15的核苷酸序列如SEQ ID NO.1所示，其氨基酸序列如SEQ ID NO.2所示。
一种含有外切β-1,3-葡聚糖酶Exo15的核苷酸序列的表达载体。
所述重组菌株是含有上述表达载体，所述的菌株为E.coli BL21(DE3)。
一种外切β-1,3-葡聚糖酶Exo15的突变体，所述突变体相对于季也蒙念珠菌外切β-1,3-葡聚糖酶亲本，将将第266位氨基酸残基突变为酪氨酸，或第261位氨基酸残基突变为酪氨酸的同时将第289位突变为精氨酸。其氨基酸序列为SEQ ID NO：4或6所示。
一种重组菌株，其特征在于，表达核苷酸序列为SEQ ID NO.1、SEQ ID NO.3或SEQID NO.5所示的外切β-1,3-葡聚糖酶基因；所述重组菌株是将SEQ ID No.1所示的基因克隆到表达载体上，然后将重组质粒转化到表达菌株，所述菌株为E.coli BL21(DE3)中进行表达。所述的重组工程菌株可以生产重组蛋白Exo15
一种生产赛门苷I中的方法，其特征在于外切β-1,3-葡聚糖酶Exo15、其突变体或重组菌株作为催化剂转化罗汉果苷V生产赛门苷I。
本发明的反应方程式如下:
具体而言：
本发明涉及了一种生物酶法催化罗汉果苷V生产赛门苷I的方法，在添加50g/L的罗汉果苷V的条件下转化18h生成40.5g/L的赛门苷I，转化率达到0.926mo1/mo1。催化剂Exo15制备工艺：重组菌株BL21(DE3)-pGEX-4T-Exo15发酵液，8000rpm，离心20min，收集菌体，菌体用PBS(50mM K2HPO4-KH2PO4，pH＝7.2)溶液洗涤3次，菌体超声破碎(1200W，功率80％，超声4s，暂停10s，超声总时长30min)。8000rpm，离心 20min，收集上清液，上清液即为粗酶液。粗酶液即可用于赛门苷I的制备。通过HPLC监测反应进程；反应样品预处理：反应液加入与反应液同体积的乙醇，沉淀蛋白，并溶解底物(罗汉果苷V)和产(赛门苷I)，10000r/min离心6min，0.45μm滤膜过滤，待用。
罗汉果提取物含量的测定方法：以高效液相色谱法(HPLC)测定赛门苷I和其他罗汉果提取物含量。色谱柱：Ultimate○RXB-C18柱(150mm×4.6mm×3μm，
)；流动相：A 相(0.1％v/v磷酸-水溶液)和B相(甲醇)；梯度变化(B相)：0-20min 10-30％，20-25min 30-90％,25-26min 90-10％，26-30min 10％；流速：1mL/min；波长：205nm处紫外检测；进样量：10μL。
有益效果
1、本发明涉及外切β-1,3-葡聚糖酶Exo15具有高度特异性，见图1D所示，该酶特异性由罗汉果苷V催化形成赛门苷I，而不再继续脱糖。而一般葡聚糖酶会持续脱糖，形成不同糖取代基混合物。这种定向合成，对应生产极具优势。
2、虽然外切β-1,3-葡聚糖酶Exo15的特异性好，但其催化效率欠佳，发明人对外切β- 1,3-葡聚糖酶Exo15进行突变。发明人通过将Exo15蛋白晶体与罗汉果苷V进行分子对接，结果发现，罗汉果苷V与Arg321，Tyr326，Asn314，Tyr264，Glu301，Asn156，Tyr44，Asp155，Glu42，Gln240，Asn242,His263,His26 2,Gln271(如图3所示)；排除可能是催化三联体的Glu或Asp；最终，确定261,265，266，和289四个位点进行迭代饱和突变并测定了突变体的催化活性参数。以质粒pGEX-4T-1-Exo15为模板，在261位、265位、266位和289位通过Quick change的方法引入NNK简并密码子((N:Ade/Cyt/Gua/Thy；K:Gua/Thy) 来替代目的氨基酸。所用简并引物如表3所示，PCR反应扩增条件:94℃预变性5min；随后进行94℃1min,57℃50s,72℃50s，28个循环；最后保存在12℃30min。结果如表4所示，较Exo15(野生型)而言，Exo15-V266Y和Exo15-H261Y/S289R突变体酶活别提高了 3.58和5.72倍。该突变体具有催化效率高，反应条件温和，特异性强等特点，有望为高效合成赛门苷I的理想催化剂。
3、本发明提供利用外切β-1,3-葡聚糖酶Exo15、其突变体或重组菌株具有高效催化合成赛门苷I的作用，其具有见效快、成本低、操作简单、方便实施、产品无毒无残留等特征，可满足罗汉果赛门苷I的大规模生产需求，具有较强的实用性和推广价值。
附图说明
图1、M.guilliermondii LHGNSJ-VS01胞外酶的纯化及纯化Exo15的功能验证；
A、M.guilliermondii LHGNSJ-VS01胞外酶的纯化结果；
B、使用TSK-GEL G3000SWxl(7.5mm×300mm)色谱柱对Exo15蛋白纯度监测。
C、罗汉果苷标准液相结果(罗汉果苷V：17.2min；siamenside I：18.2min；罗汉果苷IIIe： 19.8min；罗汉果苷IIe：22.0min)；
D、用纯化的Exo15蛋白催化罗汉果苷V水解产物的液相结果。(罗汉果苷V：17.2分钟； siamenside I：18.2分钟)；图中缩写：V(罗汉果苷V)，SI(siamenside I)，IIIe(罗汉果苷 IIIe)，IIe(罗汉果苷IIe)；
图2、exo-1,3-β葡聚糖酶在大肠杆菌BL21(DE3)中的异源表达与功能验证；
A.从M.guilliermondii LHGNSJ-VS01 PCR扩增出编码exo-1,3-β葡聚糖酶样的Exo15基因。缩写：M，DNA标记；泳道1-6：Exo15的基因片段；
B.重组质粒pET28a-Exo15图；
C.含有pET28a-Exo15的重组大肠杆菌BL21(DE3)的SDS-PAGE分析。缩写：M，蛋白质标记；泳道1-2：重组大肠杆菌BL21(DE3)-pET28a-Exo15在30℃下表达；泳道3-4：重组大肠杆菌BL21(DE3)-pET28a-Exo15在25℃下表达；泳道5-6：重组大肠杆菌 BL21(DE3)-pET28a-Exo15在16℃下表达；泳道7-8：Exo15的包涵体；
D.重组Exo15(rExo15)的功能验证。通过HPLC监测反应产物；罗汉果苷V标准品和siamanoside I标准品的保留时间分别为15.9分钟和16.69分钟；
图3、Exo15与罗汉果苷V的氢键交互作用图；
图4、通过柱层析纯化所得赛门苷I HPLC图。
具体实施方式
下面结合实施例对本发明做进一步的详细说明，以令本领域技术人员参照说明书能够据以实施。
应当理解，本文所使用的诸如“具有”、“包含”以及“包括”术语并不排出一个或多个其它元件或其组合的存在或添加。
需要说明的是，下述实施方案中所述实验方法，如无特殊说明，均为常规方法，所述试剂和材料，如无特殊说明，均可从商业途径获得。
实施例l罗汉果内生菌胞外蛋白发酵制备与分离纯化
季也蒙念珠菌Meyerozyma guilliermondii LHGNSJ-VS01划线接种于新鲜的YPD(葡萄糖 20g/L、蛋白胨20g/L、酵母浸膏10g/L,，pH＝6.5)斜面培养基上，于25℃培养48h，得到活化的斜面菌种；将得到的活化的菌种，接种于装有50mL种子培养基(葡萄糖20g、蛋白胨 20g、酵母浸膏10g、K2HPO4 0.2g、MgSO4·7H2O 0.2g、Na2CO3 0.5g和水1000mL组成，pH＝6.5)的500mL三角瓶中，于25℃、200rpm摇床培养48h，即得种子液；然后按照2％(v/v)-10％(v/v)的接种量，将种子液接种于装有100mL液体发酵培养基(葡萄糖20g、蛋白胨20g、酵母浸膏10g、K2HPO4 0.2g、MgSO4·7H2O 0.2g、Na2CO3 0.5g和水1000mL组成，pH＝6.5) 的500mL三角瓶中，25℃、200rpm摇床培养48h，得到发酵液；将步骤2得到的发酵液进行离心，收集上清液。上清液于4℃冷藏5h，加入硫酸铵将饱和度调为80％，4000rpm离心15min；收集沉淀，沉淀物为粗LHGNSJ-VS01胞外蛋白。胞外蛋白可通过Toyopearl分子尺寸排阻层析填料HW-75F或HW-55F柱(2×100cm)纯化，流动相为50mM K2HPO4-KH2PO4 (pH＝7.2)。采用HW-55F柱纯化结果如图1A所示，122-140管的洗脱液有酶活。胞外蛋白 Exo15纯度如图1B所示；纯化后的胞外蛋白Exo15具有特异性水解罗汉果苷V制备赛门苷I的功能，如图1C，图1D所示，反应产物很纯净，未见其他类型罗汉果苷生成。
实施例2纯化的胞外蛋白Exo15的LC-MS/MS分析
蛋白质酶解
1)蛋白溶液12,000rpm离心10分钟，取上清(蛋白量大约为15μg)于10kD的超滤管中；
2)加入40μl蛋白还原溶液，37℃反应1小时；
3)加入40μl蛋白烷基化溶液，室温避光反应10分钟，12,000rpm离心20分钟，弃掉收集管底部溶液；
4)加入50μl 50mM NH4HCO3，12,000rpm离心20分钟，弃掉收集管底部溶液，重复3次；
5)更换新收集管，在超滤管中加入40μl浓度为12ng/μl的测序级胰蛋白酶溶液，37℃反应15小时；
6)12,000rpm离心10分钟，收集酶解后肽段。在超滤管中再加入50μl mM NH4HCO3，12,000rpm离心10分钟，收集管底溶液并与前次溶液合并冻干；
质谱操作及数据库检索
将冻干的多肽样品重新溶解于Nano-RPLC Buffer A中。在线Nano-RPLC液相色谱在 Eksigent nanoLC-UltraTM 2D系统(AB SCIEX)进行，溶解后的样品以2μl/min的流速上样到C18预柱上(100μm x 3cm，C18,3μm,
)，然后保持流速冲洗脱盐10min。分析柱是C18反相色谱柱(75μm x 15cm C18-3μm
ChromXP Eksigent)，实验所用梯度为 60min内流动相B由5％升高至35％。
质谱采用TripleTOF5600系统(AB SCIEX)结合纳升喷雾III离子源(AB SCIEX,USA)，喷雾电压为2.5kV，气帘气压为30PSI，雾化气压为5PSI，加热器温度为150℃，质谱扫描方式为信息依赖的采集工作模式下(IDA，Information Dependent Analysis)，一级TOF-MS单张图谱扫描时间为250ms，每次IDA循环下最多采集35个电荷为2+到5+且单秒计数大于150的二级图谱，每次循环时间固定为2.5秒，碰撞室能量设定适用于所有前体离子碰撞诱导解离(CID)，动态排除设置为18秒，约等于色谱半峰宽。数据处理采用Mascot 2.3软件(Matrix Science)进行，数据库为客户提供的数据库，酶为胰蛋白酶，允许最大漏切位点为2；固定修饰为：Carbamidomethyl(C)；可变修饰为：Deamidated(NQ)、 Oxidation(M)；MS容差为±50ppm，MS/MS容差为±0.2Da，Protein score C.I.％大于95％为鉴定成功。结果；经数据库比对，序列与来自卡利比克迈耶氏酵母Meyerozyma caribbica外切-β-1,3-葡聚糖酶同源率达31％，该酶是全新的外切β-1,3-葡聚糖酶，命名为外切-β-1,3-葡聚糖酶Exo15。
实施例3含外切-β-1,3-葡聚糖酶Exo15基因工程菌的构建
基于上述测序所得氨基酸序列，设计如下引物：用正向引物F (ggtaccgaattcatgcttccatacttctttatgat)和反向引物R(ctcgagaagcttttagaatttacattggt tgggat)。用TianGen酵母基因组提取试剂盒(Tiangen,Beijing)提取Meyerozyma guilliermondiiLHGNSJ-VS01基因组，通过分子生物学手段用正向引物F与反向引物R将来自于季也蒙念珠菌Meyerozyma guilliermondii LHGNSJ-VS01的外切-β-1,3-葡聚糖酶基因进行PCR扩增：体系中加入LA Taq酶,94℃预变性3min,94℃变性30s,55℃退火30s,72℃延伸1.2min,30个循环,最后72℃延伸10min。克隆结果如图2A所示。用限制性内切酶EcoRI和HindIII将目的基因和表达载体pET28a 37℃酶切1h；用T4连接酶分别将酶切并胶回收后的目的基因和pET28a 16℃连接20h；将构建好的表达质粒(如图2B所示)导入E.co1i BL21(DE3)，在含有卡纳霉素的 LB平板中培养12h；将平板中长出的菌落进行PCR及酶切验证，将含有目的基因的质粒进行测序验证，选出目的基因完全正确的菌株，获得表达载体高拷贝的外切-β-1,3-葡聚糖酶Exo15 基因工程菌。Exo15基因工程菌表达结果如图2C，所示，在25℃和30℃条件下，表达产物均为包涵体，未检测到催化活性；当表达温度降低到16℃，检测到催化活性，结果如图2C，图 2D所示。该重组蛋白功能与上述分离纯化所得Meyerozyma guilliermondiiLHGNSJ-VS01胞外功能蛋白功能一致。
实施例4不同表达宿主对Exo15酶活力的影响。
为促进Exo15在大肠杆菌中可溶性表达，我们将pET28a-Exo15表达质粒，导入到E.coli BL21(DE3)，E.coli BL21 Star(DE3)，E.coli BL21(DE3)pLysS，E.coli Rosetta2(DE3)等表达宿主中，结果如表1所示，在宿主E.coli BL21 Star(DE3)，E.coli BL21(DE3)pLysS中，可溶性表达情况变化不明显；表达产物酶活未见明显提高；而采用宿主E.coliRosetta2(DE3)，可将Exo15催化活力提高1.5倍。
表1：不同表达宿主度Exo15酶活的影响。
实施例5添加融合标签对Exo15酶酶活的影响。
上述重组工程菌，表达产物主要是包涵体，Exo15催化活力较低，我们尝试添加促溶标签，提高蛋白可溶性表达比例。按实施例4，将具有NusA、GST、TrxA、DsbC、MBP、SUMO 等蛋白标签的表达载体分别与目的蛋白Exo15一起融合表达，分别得到NusA-Exo15、GST-Exo15、TrxA-S tag-Exo15、DsbC-Exo15、MBP-Exo15和SUMO-Exo15等融合蛋白，检测结果如下表2所示。添加促溶标签GST，表达蛋白的酶活提高了4.5倍。
表2.不同促溶标签对重组Exo15酶活的影响
实施例6外切-β-1,3-葡聚糖酶Exo15的突变体构建
通过将Exo15蛋白晶体与罗汉果苷V进行分子对接，结果发现，罗汉果苷V与Arg321， Tyr326，Asn314，Tyr264，Glu301，Asn156，Tyr44，Asp155，Glu42，Gln240，Asn242,His263, His26 2,Gln271(如图3所示)；排除可能是催化三联体的Glu或Asp；最终，确定261,265， 266，和289四个位点进行迭代饱和突变并测定了突变体的催化活性参数。以质粒pGEX-4T- 1-Exo15为模板，在261位、265位、266位和289位通过Quick change的方法引入NNK简并密码子((N:Ade/Cyt/Gua/Thy；K:Gua/Thy)来替代目的氨基酸。所用简并引物如表3所示， PCR反应扩增条件:94℃预变性5min；随后进行94℃1min,57℃50s,72℃50s，28个循环；最后保存在12℃30min。结果如表4所示，较Exo15(野生型)而言，Exo15-V266Y和Exo15-H261Y/S289R突变体酶活别提高了3.58和5.72倍。
表3，迭代饱和突变所用引物
备注：突变位点用大写字母和下划线标注。
表4突变体相对酶活
实施例7利用重组外切-β-1,3-葡聚糖酶Exo15蛋白制备赛门苷I
5g/L粗Exo15重组蛋白，纯度为100％罗汉果苷V50 g/L，0.5M磷酸盐缓冲液
pH＝7.2，30℃，200rpm，反应24h。赛门苷I产量为12g/L。
实施例8利用重组外切-β-1,3-葡聚糖酶Exo15(V266Y)突变体制备赛门苷I
1.5g/L粗Exo15(V266Y)，纯度为98％罗汉果苷V 50g/L，0.5M磷酸盐缓冲液
pH＝7.2，30℃，200rpm，反应24h。赛门苷I产量为31g/L。
实施例9利用重组外切-β-1,3-葡聚糖酶Exo15(H261Y/S289R)突变体制备赛门苷I
催化剂Exo15(H261Y/S289R)突变体的制备：重组菌株BL21(DE3)-pGEX-4T-Exo15发酵液，8000rpm，离心20min，收集菌体，菌体用PBS(50mM K2HPO4-KH2PO4， pH＝7.2)溶液洗涤3次，菌体超声破碎(1200W，功率80％，超声4s，暂停10s，超声总时长30min)。8000rpm，离心20min，收集上清液，上清液即为粗酶液。粗酶液即可用于赛门苷I的制备。通过HPLC监测反应进程；反应样品预处理：反应液加入与反应液同体积的乙醇，沉淀蛋白，并溶解底物(罗汉果苷V)和产(赛门苷I)，10000r/min离心6min，0.45 μm滤膜过滤，待用。
罗汉果提取物含量的测定方法：以高效液相色谱法(HPLC)测定赛门苷I和其他罗汉果提取物含量。色谱柱：Ultimate○RXB-C18柱(150mm×4.6mm×3μm，
)；流动相：A相(0.1％v/v磷酸-水溶液)和B相(甲醇)；梯度变化(B相)：0-20min 10-30％，20- 25min 30-90％,25-26min 90-10％，26-30min 10％；流速：1mL/min；波长：205nm处紫外检测；进样量：10μL。
酶转化反应：5.0g/L粗Exo15(H261Y/S289R)突变体蛋白，纯度为95％罗汉果苷V50 g/L，0.5M磷酸盐缓冲液pH＝7.2，30℃，200rpm，反应24h。底物转化率达81％，赛门苷I产量可达40.5g/L；经过柱层析后，赛门苷I纯度可达98.5％(如图4所示)。
实施例10利用重组外切-β-1,3-葡聚糖酶Exo15(H261Y/S289R)突变体制备赛门苷I
5.0g/L粗Exo15(H261Y/S289R)突变体蛋白，纯度为5％罗汉果苷V 20g/L，0.5M磷酸盐缓冲液pH＝7.2，10℃，200rpm，反应12h。底物转化率达30％。
实施例11利用重组外切-β-1,3-葡聚糖酶Exo15(H261Y/S289R)突变体制备赛门苷I
5.0g/L粗Exo15(H261Y/S289R)突变体蛋白，纯度为50％罗汉果苷V 30g/L，0.5M磷酸盐缓冲液pH＝7.2，70℃，200rpm，反应96h。赛门苷I产量可达5.5g/L。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
序列表
<110> 成都普睿法药物研发有限公司
<120> 一种季也蒙念珠菌外切β-1,3-葡聚糖酶及其突变体及应用
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1224
<212> DNA
<213> 外切β-1,3-葡聚糖酶Exo15(2 Ambystoma laterale x Ambystomajeffersonianum)
<400> 1
atgcttccat acttctttat gatggcagca acatttgcgg ccgcgataac tcgccgaggc 60
ctcaattggg attatgataa cgacaaaata cgaggtgtaa accttggtgg ctggtttgtc 120
ctcgaaccat atatcacccc atcattattt gatgtttttg gttccaatat cccagtggat 180
gagtaccact attgtcagca actaggcaag caagtgtgcc aagaaagact cgaaactcac 240
tggaaaactt ggtacacgga agacgacttc aaatctatca aagatgctgg tctcaatgcc 300
gtaagaatcc ccattggata ttgggcatat gaacttttgg acaatgatcc ttacgtccag 360
ggccaagaca aatacttgga acaggcattg gagtggtgta gaaataacga tcttaaggca 420
tggatcgact tgcacggtgc tcctggatca caaaatgggt ttgataactc gggacttcgt 480
ggtcaagttc aattccaatg gggcaacaat gtccaggtga ctcttgatgc cttgaacaaa 540
atcttcaaaa agtacggtgg atccgattac gaggacgttg tcattggaat tgaggccctc 600
aacgaaccct tgggtcctag tcttgacatg aacaaactca aagactttat taatcaagca 660
tactcaaacc ttcgtgacac aggatctgta caggcattgg ttgtgcagga cgcattccag 720
tcaaatactt actggaatga tcaattgcaa acccccaatg cttggaatgt ggtaattgac 780
caccatcact atcaggtatt ttctccttca caactccaga cttcaaacaa ggacagaata 840
aacaacgcct gtatgtgggg ctggagcctg aaggaagagt cccactggaa tgtggctggc 900
gaatggtcgg ctgcgttaac agattgtgct aggtggctca atggtgttgg ccgtggtgcc 960
agatggtcag gaaactacga taacagtcca tacattggct catgtgaccc atataccgac 1020
gttgccaatt ggcccagtga ttacagaacc gatgtccgca aatatatcga ggctcaattg 1080
gatgcattcg aagttgctgc cggatggttt ttctggaatt ggaaatgtga agatgccatc 1140
gaatgggact ttaagcggtt gacagctgct ggggtatttc caagtcctgt caccgaaaga 1200
acctatccca accaatgtaa attc 1224
<210> 2
<211> 408
<212> PRT
<213> 外切β-1,3-葡聚糖酶Exo15(2 Ambystoma laterale x Ambystomajeffersonianum)
<400> 2
Met Leu Pro Tyr Phe Phe Met Met Ala Ala Thr Phe Ala Ala Ala Ile
1 5 10 15
Thr Arg Arg Gly Leu Asn Trp Asp Tyr Asp Asn Asp Lys Ile Arg Gly
20 25 30
Val Asn Leu Gly Gly Trp Phe Val Leu Glu Pro Tyr Ile Thr Pro Ser
35 40 45
Leu Phe Asp Val Phe Gly Ser Asn Ile Pro Val Asp Glu Tyr His Tyr
50 55 60
Cys Gln Gln Leu Gly Lys Gln Val Cys Gln Glu Arg Leu Glu Thr His
65 70 75 80
Trp Lys Thr Trp Tyr Thr Glu Asp Asp Phe Lys Ser Ile Lys Asp Ala
85 90 95
Gly Leu Asn Ala Val Arg Ile Pro Ile Gly Tyr Trp Ala Tyr Glu Leu
100 105 110
Leu Asp Asn Asp Pro Tyr Val Gln Gly Gln Asp Lys Tyr Leu Glu Gln
115 120 125
Ala Leu Glu Trp Cys Arg Asn Asn Asp Leu Lys Ala Trp Ile Asp Leu
130 135 140
His Gly Ala Pro Gly Ser Gln Asn Gly Phe Asp Asn Ser Gly Leu Arg
145 150 155 160
Gly Gln Val Gln Phe Gln Trp Gly Asn Asn Val Gln Val Thr Leu Asp
165 170 175
Ala Leu Asn Lys Ile Phe Lys Lys Tyr Gly Gly Ser Asp Tyr Glu Asp
180 185 190
Val Val Ile Gly Ile Glu Ala Leu Asn Glu Pro Leu Gly Pro Ser Leu
195 200 205
Asp Met Asn Lys Leu Lys Asp Phe Ile Asn Gln Ala Tyr Ser Asn Leu
210 215 220
Arg Asp Thr Gly Ser Val Gln Ala Leu Val Val Gln Asp Ala Phe Gln
225 230 235 240
Ser Asn Thr Tyr Trp Asn Asp Gln Leu Gln Thr Pro Asn Ala Trp Asn
245 250 255
Val Val Ile Asp His His His Tyr Gln Val Phe Ser Pro Ser Gln Leu
260 265 270
Gln Thr Ser Asn Lys Asp Arg Ile Asn Asn Ala Cys Met Trp Gly Trp
275 280 285
Ser Leu Lys Glu Glu Ser His Trp Asn Val Ala Gly Glu Trp Ser Ala
290 295 300
Ala Leu Thr Asp Cys Ala Arg Trp Leu Asn Gly Val Gly Arg Gly Ala
305 310 315 320
Arg Trp Ser Gly Asn Tyr Asp Asn Ser Pro Tyr Ile Gly Ser Cys Asp
325 330 335
Pro Tyr Thr Asp Val Ala Asn Trp Pro Ser Asp Tyr Arg Thr Asp Val
340 345 350
Arg Lys Tyr Ile Glu Ala Gln Leu Asp Ala Phe Glu Val Ala Ala Gly
355 360 365
Trp Phe Phe Trp Asn Trp Lys Cys Glu Asp Ala Ile Glu Trp Asp Phe
370 375 380
Lys Arg Leu Thr Ala Ala Gly Val Phe Pro Ser Pro Val Thr Glu Arg
385 390 395 400
Thr Tyr Pro Asn Gln Cys Lys Phe
405
<210> 3
<211> 1224
<212> DNA
<213> Exo15-H261Y/S289R(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
atgcttccat acttctttat gatggcagca acatttgcgg ccgcgataac tcgccgaggc 60
ctcaattggg attatgataa cgacaaaata cgaggtgtaa accttggtgg ctggtttgtc 120
ctcgaaccat atatcacccc atcattattt gatgtttttg gttccaatat cccagtggat 180
gagtaccact attgtcagca actaggcaag caagtgtgcc aagaaagact cgaaactcac 240
tggaaaactt ggtacacgga agacgacttc aaatctatca aagatgctgg tctcaatgcc 300
gtaagaatcc ccattggata ttgggcatat gaacttttgg acaatgatcc ttacgtccag 360
ggccaagaca aatacttgga acaggcattg gagtggtgta gaaataacga tcttaaggca 420
tggatcgact tgcacggtgc tcctggatca caaaatgggt ttgataactc gggacttcgt 480
ggtcaagttc aattccaatg gggcaacaat gtccaggtga ctcttgatgc cttgaacaaa 540
atcttcaaaa agtacggtgg atccgattac gaggacgttg tcattggaat tgaggccctc 600
aacgaaccct tgggtcctag tcttgacatg aacaaactca aagactttat taatcaagca 660
tactcaaacc ttcgtgacac aggatctgta caggcattgg ttgtgcagga cgcattccag 720
tcaaatactt actggaatga tcaattgcaa acccccaatg cttggaatgt ggtaattgac 780
taccatcact atcaggtatt ttctccttca caactccaga cttcaaacaa ggacagaata 840
aacaacgcct gtatgtgggg ctggcgcctg aaggaagagt cccactggaa tgtggctggc 900
gaatggtcgg ctgcgttaac agattgtgct aggtggctca atggtgttgg ccgtggtgcc 960
agatggtcag gaaactacga taacagtcca tacattggct catgtgaccc atataccgac 1020
gttgccaatt ggcccagtga ttacagaacc gatgtccgca aatatatcga ggctcaattg 1080
gatgcattcg aagttgctgc cggatggttt ttctggaatt ggaaatgtga agatgccatc 1140
gaatgggact ttaagcggtt gacagctgct ggggtatttc caagtcctgt caccgaaaga 1200
acctatccca accaatgtaa attc 1224
<210> 4
<211> 408
<212> PRT
<213> Exo15-H261Y/S289R(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
Met Leu Pro Tyr Phe Phe Met Met Ala Ala Thr Phe Ala Ala Ala Ile
1 5 10 15
Thr Arg Arg Gly Leu Asn Trp Asp Tyr Asp Asn Asp Lys Ile Arg Gly
20 25 30
Val Asn Leu Gly Gly Trp Phe Val Leu Glu Pro Tyr Ile Thr Pro Ser
35 40 45
Leu Phe Asp Val Phe Gly Ser Asn Ile Pro Val Asp Glu Tyr His Tyr
50 55 60
Cys Gln Gln Leu Gly Lys Gln Val Cys Gln Glu Arg Leu Glu Thr His
65 70 75 80
Trp Lys Thr Trp Tyr Thr Glu Asp Asp Phe Lys Ser Ile Lys Asp Ala
85 90 95
Gly Leu Asn Ala Val Arg Ile Pro Ile Gly Tyr Trp Ala Tyr Glu Leu
100 105 110
Leu Asp Asn Asp Pro Tyr Val Gln Gly Gln Asp Lys Tyr Leu Glu Gln
115 120 125
Ala Leu Glu Trp Cys Arg Asn Asn Asp Leu Lys Ala Trp Ile Asp Leu
130 135 140
His Gly Ala Pro Gly Ser Gln Asn Gly Phe Asp Asn Ser Gly Leu Arg
145 150 155 160
Gly Gln Val Gln Phe Gln Trp Gly Asn Asn Val Gln Val Thr Leu Asp
165 170 175
Ala Leu Asn Lys Ile Phe Lys Lys Tyr Gly Gly Ser Asp Tyr Glu Asp
180 185 190
Val Val Ile Gly Ile Glu Ala Leu Asn Glu Pro Leu Gly Pro Ser Leu
195 200 205
Asp Met Asn Lys Leu Lys Asp Phe Ile Asn Gln Ala Tyr Ser Asn Leu
210 215 220
Arg Asp Thr Gly Ser Val Gln Ala Leu Val Val Gln Asp Ala Phe Gln
225 230 235 240
Ser Asn Thr Tyr Trp Asn Asp Gln Leu Gln Thr Pro Asn Ala Trp Asn
245 250 255
Val Val Ile Asp Tyr His His Tyr Gln Val Phe Ser Pro Ser Gln Leu
260 265 270
Gln Thr Ser Asn Lys Asp Arg Ile Asn Asn Ala Cys Met Trp Gly Trp
275 280 285
Arg Leu Lys Glu Glu Ser His Trp Asn Val Ala Gly Glu Trp Ser Ala
290 295 300
Ala Leu Thr Asp Cys Ala Arg Trp Leu Asn Gly Val Gly Arg Gly Ala
305 310 315 320
Arg Trp Ser Gly Asn Tyr Asp Asn Ser Pro Tyr Ile Gly Ser Cys Asp
325 330 335
Pro Tyr Thr Asp Val Ala Asn Trp Pro Ser Asp Tyr Arg Thr Asp Val
340 345 350
Arg Lys Tyr Ile Glu Ala Gln Leu Asp Ala Phe Glu Val Ala Ala Gly
355 360 365
Trp Phe Phe Trp Asn Trp Lys Cys Glu Asp Ala Ile Glu Trp Asp Phe
370 375 380
Lys Arg Leu Thr Ala Ala Gly Val Phe Pro Ser Pro Val Thr Glu Arg
385 390 395 400
Thr Tyr Pro Asn Gln Cys Lys Phe
405
<210> 5
<211> 1224
<212> DNA
<213> Exo15- V266Y(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
atgcttccat acttctttat gatggcagca acatttgcgg ccgcgataac tcgccgaggc 60
ctcaattggg attatgataa cgacaaaata cgaggtgtaa accttggtgg ctggtttgtc 120
ctcgaaccat atatcacccc atcattattt gatgtttttg gttccaatat cccagtggat 180
gagtaccact attgtcagca actaggcaag caagtgtgcc aagaaagact cgaaactcac 240
tggaaaactt ggtacacgga agacgacttc aaatctatca aagatgctgg tctcaatgcc 300
gtaagaatcc ccattggata ttgggcatat gaacttttgg acaatgatcc ttacgtccag 360
ggccaagaca aatacttgga acaggcattg gagtggtgta gaaataacga tcttaaggca 420
tggatcgact tgcacggtgc tcctggatca caaaatgggt ttgataactc gggacttcgt 480
ggtcaagttc aattccaatg gggcaacaat gtccaggtga ctcttgatgc cttgaacaaa 540
atcttcaaaa agtacggtgg atccgattac gaggacgttg tcattggaat tgaggccctc 600
aacgaaccct tgggtcctag tcttgacatg aacaaactca aagactttat taatcaagca 660
tactcaaacc ttcgtgacac aggatctgta caggcattgg ttgtgcagga cgcattccag 720
tcaaatactt actggaatga tcaattgcaa acccccaatg cttggaatgt ggtaattgac 780
caccatcact atcagtactt ttctccttca caactccaga cttcaaacaa ggacagaata 840
aacaacgcct gtatgtgggg ctggagcctg aaggaagagt cccactggaa tgtggctggc 900
gaatggtcgg ctgcgttaac agattgtgct aggtggctca atggtgttgg ccgtggtgcc 960
agatggtcag gaaactacga taacagtcca tacattggct catgtgaccc atataccgac 1020
gttgccaatt ggcccagtga ttacagaacc gatgtccgca aatatatcga ggctcaattg 1080
gatgcattcg aagttgctgc cggatggttt ttctggaatt ggaaatgtga agatgccatc 1140
gaatgggact ttaagcggtt gacagctgct ggggtatttc caagtcctgt caccgaaaga 1200
acctatccca accaatgtaa attc 1224
<210> 6
<211> 408
<212> PRT
<213> Exo15- V266Y(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
Met Leu Pro Tyr Phe Phe Met Met Ala Ala Thr Phe Ala Ala Ala Ile
1 5 10 15
Thr Arg Arg Gly Leu Asn Trp Asp Tyr Asp Asn Asp Lys Ile Arg Gly
20 25 30
Val Asn Leu Gly Gly Trp Phe Val Leu Glu Pro Tyr Ile Thr Pro Ser
35 40 45
Leu Phe Asp Val Phe Gly Ser Asn Ile Pro Val Asp Glu Tyr His Tyr
50 55 60
Cys Gln Gln Leu Gly Lys Gln Val Cys Gln Glu Arg Leu Glu Thr His
65 70 75 80
Trp Lys Thr Trp Tyr Thr Glu Asp Asp Phe Lys Ser Ile Lys Asp Ala
85 90 95
Gly Leu Asn Ala Val Arg Ile Pro Ile Gly Tyr Trp Ala Tyr Glu Leu
100 105 110
Leu Asp Asn Asp Pro Tyr Val Gln Gly Gln Asp Lys Tyr Leu Glu Gln
115 120 125
Ala Leu Glu Trp Cys Arg Asn Asn Asp Leu Lys Ala Trp Ile Asp Leu
130 135 140
His Gly Ala Pro Gly Ser Gln Asn Gly Phe Asp Asn Ser Gly Leu Arg
145 150 155 160
Gly Gln Val Gln Phe Gln Trp Gly Asn Asn Val Gln Val Thr Leu Asp
165 170 175
Ala Leu Asn Lys Ile Phe Lys Lys Tyr Gly Gly Ser Asp Tyr Glu Asp
180 185 190
Val Val Ile Gly Ile Glu Ala Leu Asn Glu Pro Leu Gly Pro Ser Leu
195 200 205
Asp Met Asn Lys Leu Lys Asp Phe Ile Asn Gln Ala Tyr Ser Asn Leu
210 215 220
Arg Asp Thr Gly Ser Val Gln Ala Leu Val Val Gln Asp Ala Phe Gln
225 230 235 240
Ser Asn Thr Tyr Trp Asn Asp Gln Leu Gln Thr Pro Asn Ala Trp Asn
245 250 255
Val Val Ile Asp His His His Tyr Gln Tyr Phe Ser Pro Ser Gln Leu
260 265 270
Gln Thr Ser Asn Lys Asp Arg Ile Asn Asn Ala Cys Met Trp Gly Trp
275 280 285
Ser Leu Lys Glu Glu Ser His Trp Asn Val Ala Gly Glu Trp Ser Ala
290 295 300
Ala Leu Thr Asp Cys Ala Arg Trp Leu Asn Gly Val Gly Arg Gly Ala
305 310 315 320
Arg Trp Ser Gly Asn Tyr Asp Asn Ser Pro Tyr Ile Gly Ser Cys Asp
325 330 335
Pro Tyr Thr Asp Val Ala Asn Trp Pro Ser Asp Tyr Arg Thr Asp Val
340 345 350
Arg Lys Tyr Ile Glu Ala Gln Leu Asp Ala Phe Glu Val Ala Ala Gly
355 360 365
Trp Phe Phe Trp Asn Trp Lys Cys Glu Asp Ala Ile Glu Trp Asp Phe
370 375 380
Lys Arg Leu Thr Ala Ala Gly Val Phe Pro Ser Pro Val Thr Glu Arg
385 390 395 400
Thr Tyr Pro Asn Gln Cys Lys Phe
405
<210> 7
<211> 46
<212> DNA
<213> H261-F(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
ttggaatgtg gtaattgacn nkcatcacta tcaggtattt tctcct 46
<210> 8
<211> 46
<212> DNA
<213> H261-R(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
aggagaaaat acctgatagt gatgmnngtc aattaccaca ttccaa 46
<210> 9
<211> 44
<212> DNA
<213> Q265-F(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
taattgacca ccatcactat nnkgtatttt ctccttcaca actc 44
<210> 10
<211> 44
<212> DNA
<213> Q265-R(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 10
gagttgtgaa ggagaaaata cmnnatagtg atggtggtca atta 44
<210> 11
<211> 44
<212> DNA
<213> V266-F(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 11
taattgacca ccatcactat cagnnktttt ctccttcaca actc 44
<210> 12
<211> 44
<212> DNA
<213> V266-R(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 12
gagttgtgaa ggagaaaamn nctgatagtg atggtggtca atta 44
<210> 13
<211> 48
<212> DNA
<213> S289-F(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 13
taaacaacgc ctgtatgtgg ggctggnnkc tgaaggaaga gtcccact 48
<210> 14
<211> 47
<212> DNA
<213> S289-R(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 14
acattccagt gggactcttc cttcagmnnc cagccccaca tacaggc 47
<210> 15
<211> 35
<212> DNA
<213> 正向引物F (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 15
ggtaccgaat tcatgcttcc atacttcttt atgat 35
<210> 16
<211> 35
<212> DNA
<213> 反向引物R (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 16
ctcgagaagc ttttagaatt tacattggtt gggat 35
Claims (10)
1.一种外切β-1,3-葡聚糖酶Exo15，其特征在于所述的外切β-1,3-葡聚糖酶Exo15的核苷酸序列如SEQ ID NO.1所示，其氨基酸序列如SEQ ID NO. 2所示。
2.一种外切β-1,3-葡聚糖酶Exo15的突变体，其特征在于所述突变体相对于季也蒙念珠菌外切β-1,3-葡聚糖酶亲本，将将第266位氨基酸残基突变为酪氨酸，或第261位氨基酸残基突变为酪氨酸的同时将第289位突变为精氨酸。
3.根据权利要求2所述的突变体，其特征在于其核苷酸序列为SEQ ID NO：3或5所示；其氨基酸序列为SEQ ID NO：4或6所示。
4.一种表达质粒，其特征在于，所述的的表达质粒含有权利要求1所述的外切β-1,3-葡聚糖酶Exo15或含有权利要求2或3所述的突变体。
5.一种重组菌株，其特征在于，含有权利要求4所述的表达质粒。
6.根据权利要求5所述的重组菌株，其特征在于，所述菌株为E.coli BL21 (DE3)、E.coli BL21 Star (DE3)、E.coli Rosetta2 (DE3) 或E.coli BL21 (DE3) pLysS。
7.一种含有权利要求1所述的外切β-1,3-葡聚糖酶Exo15的核苷酸序列的表达载体。
8.一种重组菌株是含有权利要求1所述的表达载体，所述的菌株为E. coli BL21(DE3)。
9.一种赛门苷I的制备方法，其特征在于，采用权利要求1所述的外切β-1,3-葡聚糖酶Exo15、权利要求2或3所述的突变体或权利要求5所述的重组菌株作为催化剂，将罗汉果苷V转化为赛门苷I。
10. 根据权利要求9所述的方法，其特征在于外切β-1,3-葡聚糖酶Exo15及其突变体含有促溶标签，所述的促溶标签是NusA、GST、TrxA、DsbC、MBP或SUMO；所述酶催化反应温度为10℃~70℃；所述反应时间为12~96 h。
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