CN117106835A - 用于多酶级联催化合成gdp-l-岩藻糖的生物酶组合物及其制备方法和应用 - Google Patents
用于多酶级联催化合成gdp-l-岩藻糖的生物酶组合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物技术和酶工程领域,具体公开了一种多酶级联催化合成GDP‑岩藻糖的方法。本发明合成了分别表达SlSUS酶、SeC2E酶、SeGMD酶和SeGER酶的4种质粒,并分别将表达载体导入至大肠杆菌表达宿主中,经诱导表达、两步纯化、脱盐,并进行了酶分子量大小的鉴定及酶活性鉴定。本方法用E.coli表达系统和鸟苷二磷酸和蔗糖为原料,实现了级联催化反应中各种酶的高效表达,进而达到低成本大量生产GDP‑岩藻糖;此发明不仅为批量生产人乳寡糖提供廉价原料,而且也为天然寡糖药物的生产提供廉价中间体。
Description
技术领域
本发明涉及生物技术和酶工程领域,特别涉一种多酶级联催化合成GDP-L-岩藻糖的方法,具体为重组大肠杆菌表达载体的构建及转化方法、蛋白表达及纯化方法、酶活性测定和GDP-L-岩藻糖制备方法。
背景技术
母乳作为婴儿的最佳食物来源,除了为婴儿提供最基本的营养外,还富含大量对婴幼儿的健康发育有着积极影响的活性物质。其中,人乳寡糖(HMOs)是母乳中仅次于乳糖和脂肪的第三大固体成分,母乳中的寡糖含量是其它动物乳汁的上百倍,如是牛乳的100-300倍。现如今已发掘200多种HMOs,主要分为岩藻糖基化寡糖和唾液酸化寡糖,其中岩藻糖基化寡糖占77%,唾液酸化寡糖占28%。而岩藻糖基化寡糖中岩藻糖基乳糖(FL)的含量最高,占据HMOs总量的36%,在母乳中的浓度为0.5-3g/L。作为母乳中含量最丰富的功能性低聚糖。FL具有益生元、抗感染、免疫调节以及促进婴幼儿大脑发育等重要生理功能,在婴幼儿的免疫力改善、肠道疾病预防、智力发育等领域效果明显。
FL的合成需要GDP-岩藻糖作为前体,GDP-岩藻糖的前体是L-岩藻糖。L-岩藻糖可以通过化学合成,如从D-半乳糖、L-阿拉伯糖、D-甘露糖或D-半乳糖醛酸开始合成L-岩藻糖,比较而言D-半乳糖是制造L-岩藻糖的最合适原料。但化学合成常常要求选择性技术的诸多保护/脱保护步骤使这些方法变得冗长而繁重;另外,化学合成需要进行费力的色谱分离以使中间物与副产物分离,得率低、成本高,限制了其在L-岩藻糖制备方面的应用。目前岩藻糖的来源主要是天然提取,通过萃取而从生物质如从藻类中分离出包含岩藻糖的寡糖,寡糖经水解后产生含岩藻糖以及相关糖类和/或其衍生物的复杂混合物,再从混合物中通过离子交换层析、透析、分步结晶等方法纯化回收岩藻糖,但这样获取的岩藻糖价格高昂。目前人工制备FL时使用的岩藻糖主要用细胞转化的方法,但成本较高,因此仍然迫切需要提供可以增加扩大规模的机会并促进产生低成本方法的替代性L-岩藻糖合成路径。采用酶促合成可以解决成本高昂的问题,最为重要的是如何快速获得大量廉价的GDP-岩藻糖,以实现人工规模化制备岩藻糖基化寡糖。
发明内容
为了解决上述问题,本发明提供了一种生物酶催化合成GDP-岩藻糖的方法,以获得低成本、高纯度GDP-岩藻糖的方法,为岩藻糖基乳糖的合成提供重要的原料。
生物酶组合物及其相关生物材料在制备GDP-岩藻糖中的应用,所述生物酶组合物包括蔗糖合酶(SlSUS)、CDP-泰威糖2-差向异构酶(SeC2E)、GDP-甘露糖脱水酶(SeGMD)和异构还原酶(SeGER);
所述蔗糖合酶(SlSUS)来源于番茄(Solanum locopersicum),氨基酸序列的GenBank序列号为NP_001234655;
所述CDP-泰威糖2-差向异构酶(SeC2E)来源于肠道沙门氏菌(Salmonellaenterica),氨基酸序列的GenBank序列号为DW4091412.1;
所述GDP-甘露糖脱水酶(SeGMD)来源于肠道沙门氏菌(Salmonella enterica),氨基酸序列的GenBank序列号为ACN45760.1;
所述异构还原酶(SeGER)来源于肠道沙门氏菌(Salmonella enterica),氨基酸序列的GenBank序列号为WP_001041701.1。
其中,所述相关生物材料为表达所述生物酶组合物中各酶的核酸分子,或含有所述核酸分子的表达盒、重组载体、重组菌或重组细胞。
其中,所述重组菌为向所述宿主菌中导入能够表达述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的核酸分子,经诱导培养后获得表达所述述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的重组菌;所述重组细胞为向所述宿主细胞中导入能够表达述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的核酸分子,经诱导培养后获得表达所述述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的重组细胞。
上述的生物酶组合物或相关生物材料也应在本发明的保护范围之内。
本发明提供一种多级多酶级联催化合成GDP-岩藻糖的方法,包括如下步骤:
1)以鸟苷二磷酸和蔗糖为底物,经权利要求1中所述的蔗糖合酶催化反应生成GDP-葡萄糖;
2)以GDP-葡萄糖为底物,经权利要求1中所述的CDP-泰威糖2-差向异构酶催化反应生成GDP-甘露糖;
3)以GDP-甘露糖为底物,经权利要求1中所述的DP-甘露糖脱水酶和异构还原酶催化反应生成GDP-岩藻糖。
其中,所述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶均是以粗酶液、粗酶粉、纯酶或全细胞的形式发生催化作用的。
其中,所述催化反应为经蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶共表达的全细胞催化反应。
其中,所述粗酶液、粗酶粉和纯酶均按照包括如下步骤的方法制备得到:在宿主细胞中表达所述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和/或异构还原酶,得到重组细胞;裂解所述重组细胞获得所述粗酶液、粗酶粉或纯酶。
其中,所述全细胞均按照包括如下步骤的方法制备得到:在宿主细胞中表达所述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶,得到的重组细胞即为所述全细胞。
其中,所述重组细胞是按照包括如下步骤的方法制备获得的:向所述宿主细胞中导入能够表达述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的核酸分子,经诱导培养后获得表达所述述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的重组细胞;更进一步,所述“能够表达述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的核酸分子”是通过重组载体的形式导入到所述宿主细胞中的;
所述重组载体为携带有述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶或异构还原酶的编码基因的细菌质粒、噬菌体、酵母质粒或逆转录病毒包装质粒;和/或所述宿主细胞为原核细胞或低等真核细胞;
具体的,所述原核细胞为细菌;所述低等真核细胞为酵母细胞;
更加具体的,所述细菌为大肠杆菌。
本发明的有益效果在于:本方法所采用的大肠表达系统具有表达效率高、表达量大、成本低、易操作等特点,所表达的4种参与级联反应的酶纯度高、产率高等优点。本发明提供了一种以鸟苷二磷酸和蔗糖为原料,利用4种酶级联催化体外制备低成本GDP-岩藻糖的新方法。因利用E.coli表达系统和廉价的鸟苷二磷酸和蔗糖为原料,实现了级联催化反应中各种酶的高效表达,进而达到低成本大量生产GDP-岩藻糖;本发明不仅为批量生产人乳寡糖提供廉价原料,而且也为天然寡糖药物的生产提供廉价中间体。
附图说明
图1为一釜多酶法相关酶的SDS-PAGE分析结果。
图2为SlSUS酶促反应产物的TLC检测。
图3为SeC2E酶促反应产物的UPLC检测。
图4为SeGMD和SeGER酶促反应产物的UPLC检测。
图5为基于UPLC的HpFucT的酶促反应产物分析。
图6为GDP-岩藻糖产物的HpFucT酶偶联分析结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:4种酶的基因克隆、异源表达、纯化和SDS-PAGE鉴定
1、重组质粒转化到E.coli BL21
本发明从不同来源的菌中挖掘出合成GDP-岩藻糖的相关酶(SUS,C2E,GMD,GER)的基因;分解蔗糖的蔗糖合酶(SlSUS)来源于Solanum lycopersicum;可以将核苷酸活化后的葡萄糖差向异构成甘露糖的CDP-泰威糖2-差向异构酶(SeC2E)来源于Salmonellaenterica;可以将GDP-甘露糖转化生成GDP-岩藻糖的GDP-甘露糖脱水酶(SeGMD)和异构还原酶(SeGER)也来源Salmonella enterica。从数据库中搜索获得所述蔗糖合酶SlSUS(NP_001234655.2)、CDP-泰威糖2-差向异构酶SeC2E(DW4091412.1)、GDP-甘露糖脱水酶SeGMD(ACN45760.1)和异构还原酶SeGER(WP_001041701.1)基因序列,经合成后分别克隆到pET30a质粒中,分别为,将SlSUS的编码序列为平端连接到pET30a的多克隆位点得到pET30a-SlSUS,将SeC2E的编码序列连接到pET30a的Nde I/Xho I位点之间(替换原位点之间的序列)得到pET30a-SeC2E,将SeGMD的编码序列连接到pET30a的Kpn I/Xho I位点之间得到pET30a-SeGMD,将SeGER的编码序列连接到pET30a的Kpn I/Xho I位点之间得到pET30a-SeGER。分别将上述四种重组质粒(pET30a-SlSUS、pET30a-SeC2E、pET30a-SeGMD、pET30a-SeGER)转化到大肠杆菌E.coli BL21中,具体过程如下:(1)基因合成返回的重组质粒干粉进行离心,加水稀释至1ng/μL;
(2)取1μL稀释后的重组质粒移入100μL感受态细胞中;
(3)静置25min的间隙调好水浴锅温度,迅速放入42℃水浴中热激1min,拿出快速插回冰中,静置2min的破坏以减少E.coli;
(4)取200μL已灭菌的LB液体培养基,加入到热激后的每管感受态细胞中于37℃,800rpm震荡培养50min,使其复苏;
(5)将震荡培养好的菌液均匀加入并涂布到含有0.1mg/mL卡那霉素的LB固体培养基中。并将平板倒置放于37℃恒温培养箱中培养12-16h,直至菌斑长成单菌落合适挑取的状态。
2、重组质粒在大肠杆菌中的异源表达
(1)挑取转入E.coli BL21的单菌落接种至5mL LB液体培养基中,加入50mg/mL的卡那霉素5μL,置于37℃,250rpm的恒温摇床过夜培养;
(2)将5mL过夜培养的菌液转移至400mL经高温灭菌的新鲜LB液体培养基中进行扩大培养。当发酵液的OD600达到0.5-0.8之间时向其中加入IPTG溶液,使IPTG终浓度为1mmol/L。在18℃,250rpm条件下过夜诱导表达;
(3)分别取IPTG诱导前后的发酵液各取1mL,12000rpm离心5min,弃上清液,菌体沉淀保存于-20℃作为SDS-PAGE分析的样品。
3、重组载体镍的亲和层析柱纯化
(1)将IPTG诱导后的菌液转移至收菌离心瓶中,4℃下4000rpm离心15min,弃上清;
(2)将诱导后的菌体中加入10mL Lysis Buffer(细胞裂解液),充分吸打使融解使其没有沉淀颗粒。将混合液转移到50mL离心管中加入100μL 100mM PMSF(苯甲基磺酰氟),在超声破碎仪的作用下超声20min,促使细胞破碎,释放目的蛋白。超声破碎条件:1.5s破碎/2.5s间歇,15min,功率200W;
(3)破碎后的细胞悬液再次于4℃下进行12000rpm离心15min,取上清至冰上备用。细胞裂解液配制:称取Tris 6.06g,氯化钠5.84g溶于800mL超纯水中,加入10mL Triton X-100,搅拌均匀后,盐酸调节pH至8.0,定容至1L,4℃保存。半衰期短,在即将进行破碎时再加入准备各种镍亲和层析所用缓冲液,各种试剂配制如下:①Washing buffer洗脱缓冲液:称取Tris 6.06g、氯化钠2.92g于800mL超纯水中溶解,用稀HCl调pH至8.0,最后用超纯水定容至1L,至4℃冰箱保存;②蛋白洗脱液配制:称取Tris 3.03g,氯化钠1.46g,咪唑17.02g溶于400mL超纯水中,用盐酸调节pH至8.0,定容至500mL,室温保存。
4、镍柱纯化
含有6×His-tag的重组蛋白采用Ni2+亲和层析的方法进行纯化,纯化步骤如下:(1)将纯化柱固定在铁架台上,用约5-10倍柱体积(1个柱体积约5mL)的washing buffer缓冲液冲洗平衡蛋白层析柱;
(2)将预处理中经细胞破碎后离心得到的上清液上样至预先平衡好的层析柱中,控制上样速度,延长酶流经层析柱的时间,上样后用约10-20柱体积的washing buffer冲洗层析柱,以去除杂质蛋白及其他物质的非特异性结合,直至层析柱洗脱液中蛋白浓度A280不再降低(在0.1以下);
(3)用蛋白洗脱液洗脱重组蛋白,同时收集层析柱洗脱液15管左右,每管1mL,测定收集到的各管洗脱液的A280值,留取A280值相对较高的洗脱液加入20%-25%体积的甘油,以100μL每管进行分装,液氮冷冻后置于-80℃超低温冰箱,备用;
(4)待洗脱下来的溶液A280不再降低,再用蛋白结合液再次平衡层析柱,然后将层析柱保存在20%乙醇中。
纯化过程中要各取适量的破碎离心后的上清液和沉淀置于-20℃冰箱作为SDS-PAGE分析的样品。
5、重组质粒表达产物的SDS-PAGE分析
配制SDS-PAGE所需缓冲液、染色液和脱色液备用,所用溶液配方如下:SDS-PAGE电泳缓冲液:称取Tris 3.05g,甘氨酸31.3g溶于800mL超纯水中,并加入10%SDS溶液50mL,加入超纯水定容至1L;5×loading buffer:称取DTT 0.39g,SDS 0.5g,溴酚蓝0.025g,溶于2.5mL甘油和2.5mL 0.25M Tris/HCl(pH 6.8)中,分装于1.5mL EP管中,4℃保存;考马斯亮蓝R-250染色液(1L):考马斯亮蓝R-2502g,400mL甲醇,100mL冰醋酸,500mL水;脱色液(1L):100mL冰醋酸,450mL乙醇和450mL水混合均匀;清洁玻璃板及SDS-PAGE凝胶制备装置,进行组装验漏。根据蛋白质质量配制不同比分的分离胶,不同分子质量的蛋白需要制备不同配比的蛋白胶,其中10%浓度的分离胶适用于检测分子质量为30-90kDa的蛋白,12%浓度的分离胶适用于检测分子质量为20-80kDa的蛋白。将SDS-PAGE凝胶灌入制胶装置中,分离胶上可用水液封防止不均匀,凝结后再灌入浓缩胶。静置30-60min后可在分离胶与水层之间出现肉眼可见的分界线时,倒掉分离胶的上面水,制备分离胶并加入夹板中,迅速插入合适大小的梳子。将整块SDS-PAGE凝胶静置30min左右,将其放入电泳槽中浸泡几分钟慢慢拔出梳子。
将诱导前和诱导后留置样品分别加入100和400μL水吸打混匀,各取20μL,裂解上清取2μL配18μL水,沉淀取适量浓度配水至20μL,春花的蛋白根据其OD值(OD×体积=20)配相应提及的水至20μL,所有样品加入5μL的5×loading buffer,95℃灭活10分钟使蛋白彻底变性。向SDS-PAGE凝胶孔位中依次加入蛋白Marker、诱导前、诱导后、裂解上清、纯化酶的样品。然后以电压120伏运行电泳,直至蓝色指示条带恰好跑出分离胶为止。取出凝胶盘用考马斯亮蓝R-250染色液对电泳凝胶进行染色,染色约30分钟。倒掉考马斯亮蓝,用脱色液进行脱色,约2小时换一次直至条带清晰可见。对凝胶扫描并观察结果、结果如图1;图1结果显示这4个条带指示的分子质量与理论值相符(SlSUS、SeC2E、SeGMD和SeGER论值分别为96.6kDa、37.9kDa、42.3kDa和36.2kDa。
实施例2:4种重组酶纯化后脱盐
纯化后的酶溶液中含有大量的咪唑,所以用PD-10脱盐柱对蛋白酶进行脱盐处理,详细步骤如下:
(1)将一次性脱盐柱PD-10柱内原有溶液流干;
(2)配置pH为8,浓度为10mM的Tris缓冲液并用缓冲液冲洗5倍柱体积的量(此步骤为了防止使用去离子水冲洗蛋白产生沉淀);
(3)吸取2.5ml经镍亲和层析柱纯化后的混合液加入脱盐柱中使其自由洗脱;
(4)用3ml缓冲液冲洗柱身,根据OD260吸光值收集目的蛋白。
(5)纯化后的SlSUS、SeC2E、SeGMD和SeGER的蛋白浓度分别为2.0±0.9mg/mL;22±1.5mg/mL;25±1.2mg/mL和38±2.6mg/mL。
实施例3:SlSUS酶促反应产物的TLC检测
由于蔗糖合酶催化蔗糖的分解成葡萄糖和果糖,并将葡萄糖和GDP反应生成GDP-葡萄糖。SlSUS理论催化原理表明最适核苷酸是尿苷二磷酸(UDP),对鸟苷二磷酸(GDP)亲和力最弱。反应体系包括:5μL 50mM Tris-Cl(pH6.0)(南京寿德有限公司),5μL 50mM蔗糖,2.5μL 5mM GDP(Sigma公司),1μL 2mM MgCl2,20μL SlSUS(2.0mg/mL),6.5μL超纯水。反应在37℃下进行,反应结束后取少量混合物进行离心稀释。各取1微升滴在板子下方吹干置于层析缸内。TLC展开剂配方:正丁醇(N-butanol)/乙醇(Ethanol)/水=5/3/2,(v/v/v),展开剂混匀后提前加入层析缸中使其充分饱和。由于GDP本身具有共轭双键在紫外下可见,如图2所示,用水替代SUS的空白对照显示只有GDP,没有多余的物质产生,而加入SlSUS的反应体系b有新的物质产生。蔗糖和果糖在紫外下不可见,只能进行地衣酚染色。将TLC板子进行地衣酚染色吹干后,图2A显示,加入SlSUS的反应体系成功将蔗糖分解生成果糖,空白对照反应体系中没有果糖生成,结合紫外下检测(图2B)显示SlSUS具有分解蔗糖并利用GDP产生GDP-葡萄糖的能力。
实施例4:SeC2E酶促反应产物的UPLC检测
C2E理论催化原理表明对是胞苷二磷酸葡萄糖(CDP-Glc)的亲和力最大,而对鸟苷二磷酸(GDP)活化的葡萄糖亲和力最小。反应体系为:3μL SlSUS反应后混合物上清或2mMGDP-甘露糖(Sigma公司),5μL 50mM Tris-Cl(pH7.5)(南京寿德有限公司),1μL 2mMMgCl2,3μL 2mM NAD+(上海如吉生物科技发展有限公司),18μL SeC2E(22mg/mL)。反应在45℃下过夜,反应结束后取少量混合物进行离心稀释。吸50μL打入进样瓶依据上述SeC2E酶活性测定方法进行检测。图3显示,当GDP-甘露糖作为底物时,反应体系加入了SeC2E后,底物GDP-甘露糖(GDP-Man)的峰明显下降,且在3min处有一个新的峰出现。而空白对照的反应体系GDP-甘露糖峰值不变无新的峰产生。当用SlSUS反应过夜后的混合物进行检测时,则在相同出峰位置出现强烈的峰,推测在GDP-甘露糖后面的峰是GDP-葡萄糖(GDP-Glc)。以该混合物浓缩后为底物,继续添加SeC2E并在相同最适反应条件下过夜反应,用UPLC检测结果显示,GDP-葡萄糖峰有明显降低,产物GDP-甘露糖峰出现。说明SeC2E对GDP-葡萄糖和GDP-甘露糖都具有催化活性,可以偶联SlSUS产生GDP-甘露糖。
实施例5:SeGMD和SeGER酶促反应产物的UPLC检测
GDP-甘露糖先由SeGMD催化生成GDP-4-脱氢-6-脱氧-甘露糖,然后GDP-4-脱氢-6-脱氧-甘露糖再由SeGER酶在辅助因子NADPH的参与下催化形成GDP-L-岩藻糖(GDP-L-Fuc)。为检测SeGMD和SeGER酶的活性,在反应体系中加入岩藻糖基转移酶HpFucT和pNP-乳糖,将SeGMD和SeGER反应产物GDP-L-Fuc转化为pNP-岩藻糖基乳糖,然后用UPLC检测pNP-岩藻糖基乳糖的生成。
将来源于Helicobacterpylori(幽门螺杆菌)的岩藻糖基转移酶HpFucT(序列号:WP_000487428.1)基因合成后克隆到pET30a的Nde I/Xho I位点,按实施例1和实施例2的方法转化大肠杆菌,并表达纯化,获得HpFucT用于偶联酶促反应产物分析。
SeGMD和SeGER反应体系为:4μl 2mM GDP-甘露糖(Sigma公司),5μl 50mM Tris(pH7.5)(南京寿德有限公司),4μl 2mM NADPH(上海如吉生物科技发展有限公司),2μl 2mMpNP-lactose(苏州卡博森斯生物公司),6μl SeGMD,4μl SeGER,15μl HpFucT,在37F反应过夜。反应完成后95后下加热10min终止反应并离心,取上清适当稀释后吸50μL打入进样瓶进行检测。UPLC结果如图2-6结果显示:对比没有加入SeGMD、SeGER和HpFucT酶的空白对照,只检测出pNP-乳糖底物峰没有产生新峰,而加入SeGMD、SeGER和HpFucT酶的反应体系,在pNP-乳糖出峰位置的前0.3min产生一个新的峰。样品经过进MS用ESI电离子进行检测(Shimadzu公司),模式设置为采用正离子scan扫描模式,m/z范围设置在200-1000之间。分离结果图4和图5显示和UPLC结果相同,测得靠近6min的底物峰中含有质荷比为486[M+Na]+最多,正好对应pNP-乳糖的质荷比加Na+的分子量463+23。新出现的峰中含有质荷比为632[M+Na]+做多,对应pNP-岩藻糖基乳糖的质荷比加Na+的分子量609+23,证明SeGMD和SeGER有活性,可成功将GDP-甘露糖转化为GDP-岩藻糖,随后GDP-岩藻糖在HpFucT酶的催化下与pNP-乳糖结合生成pNP-岩藻糖基乳糖,因此证明GMD和GER均具有活性,可催化合成GDP-岩藻糖。
实施例6:多酶级联催化合成GDP-岩藻糖
多酶级联催化GDP-岩藻糖的85μL反应体系包括:5μL 500mM Tris-HCl(pH7.5)(南京寿德有限公司),5μL 100mM GDP(Sigma公司),5μL 1M蔗糖,1μL 100mM MgCl2(Sigma公司),加入14μL SlSUS(2.0mg/mL)在37℃下反应12小时,然后加入4μL 20mM NAD+(上海如吉生物科技发展有限公司)和20μL SeC2E(22mg/mL)在45℃下反应12小时,最后再添加4μL20mM NADPH(上海如吉生物科技发展有限公司),6μL SeGMD(25mg/mL)和4μL SeGER(38mg/mL),在25反应12小时。如图6所示,经HpFucT酶偶联反应测定,GDP-岩藻糖的最佳转化率可达20%。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
Claims (10)
1.生物酶组合物在制备GDP-岩藻糖中的应用,所述生物酶组合物包括蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶;
所述蔗糖合酶来源于番茄(Solanum locopersicum),氨基酸序列的序列号为NP_001234655;
所述CDP-泰威糖2-差向异构酶来源于肠道沙门氏菌(Salmonella enterica),氨基酸序列的序列号为DW4091412.1;
所述GDP-甘露糖脱水酶来源于肠道沙门氏菌(Salmonella enterica),氨基酸序列的序列号为ACN45760.1;
所述异构还原酶来源于肠道沙门氏菌(Salmonella enterica),氨基酸序列的序列号为WP_001041701.1。
2.与生物酶组合物相关的生物材料在制GDP-岩藻糖中的应用,其特征在于,所述生物材料为如下任一所示:
A1)编码权利要求1所述的蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶或者异构还原酶的核酸分子,
A2)含有所述核酸分子的表达盒,
A3)含有所述核酸分子的重组载体,
A4)含有所述核酸分子的重组菌,
A5)含有所述核酸分子的重组细胞。
3.根据权利要求2所述的应用,其特征在于,所述重组菌为向所述宿主菌中导入能够表达述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和/或异构还原酶的核酸分子,经诱导培养后获得表达所述述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和/或异构还原酶的重组菌;所述重组细胞为向所述宿主细胞中导入能够表达述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和/或异构还原酶的核酸分子,经诱导培养后获得表达所述述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和/或异构还原酶的重组细胞。
4.权利要求1-3任一中所述的生物酶组合物或相关生物材料。
5.一种多级多酶级联催化合成GDP-岩藻糖的方法,其特征在于,包括如下步骤:
1)以鸟苷二磷酸和蔗糖为底物,经权利要求1中所述的蔗糖合酶催化反应生成GDP-葡萄糖;
2)以GDP-葡萄糖为底物,经权利要求1中所述的CDP-泰威糖2-差向异构酶催化反应生成GDP-甘露糖;
3)以GDP-甘露糖为底物,经权利要求1中所述的DP-甘露糖脱水酶和异构还原酶催化反应生成GDP-岩藻糖。
6.根据权利要求5所述的方法,其特征在于,所述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶均是以粗酶液、粗酶粉、纯酶或全细胞的形式发生催化作用的。
7.根据权利要求5所述的方法,其特征在于,所述催化反应为经蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶共表达的全细胞催化反应。
8.根据权利要求6所述的方法,其特征在于,所述粗酶液、粗酶粉和纯酶均按照包括如下步骤的方法制备得到:在宿主细胞中表达所述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和/或异构还原酶,得到重组细胞;裂解所述重组细胞获得所述粗酶液、粗酶粉或纯酶。
9.根据权利要求6所述的方法,其特征在于,所述全细胞均按照包括如下步骤的方法制备得到:在宿主细胞中表达所述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶,得到的重组细胞即为所述全细胞。
10.根据权利要求9所述的方法,其特征在于,所述重组细胞是按照包括如下步骤的方法制备获得的:向所述宿主细胞中导入能够表达述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的核酸分子,经诱导培养后获得表达所述述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的重组细胞;更进一步,所述“能够表达述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶和异构还原酶的核酸分子”是通过重组载体的形式导入到所述宿主细胞中的;所述重组载体为携带有述蔗糖合酶、CDP-泰威糖2-差向异构酶、GDP-甘露糖脱水酶或异构还原酶的编码基因的细菌质粒、噬菌体、酵母质粒或逆转录病毒包装质粒;和/或所述宿主细胞为原核细胞或低等真核细胞;具体的,所述原核细胞为细菌;所述低等真核细胞为酵母细胞;更加具体的,所述细菌为大肠杆菌。
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