CN116590355B - 一种利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法 - Google Patents
一种利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法 Download PDFInfo
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Abstract
本发明公开了一种利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,属于生物医药领域。该方法以大肠杆菌为模板,克隆出葡萄糖脱氢酶基因Ecgdh;构建重组表达葡萄糖脱氢酶基因Ecgdh体系,表达出葡萄糖脱氢酶;再以麦芽糖为底物,以NADH为电子供体,经葡萄糖脱氢酶催化制备麦芽糖酸;所述的葡萄糖脱氢酶基因Ecgdh的核苷酸序列如SEQ ID NO.1所示。本发明获得的葡萄糖脱氢酶,与以PQQ为辅因子的醌蛋白葡萄糖脱氢酶相比大大提高,相较于其他醌蛋白葡萄糖脱氢酶对于麦芽糖具有更强的底物偏好性,适合大规模的制备麦芽糖酸,为麦芽糖酸的合成提供了新的方式。
Description
技术领域
本发明属于生物医药技术领域,涉及一种依赖于NADH的葡萄糖脱氢酶基因的挖掘、表达及催化麦芽糖生成麦芽糖酸的方法,具体涉及一种利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法。
背景技术
麦芽糖酸又称为第四代果酸,是麦芽糖氧化的产物,易溶于水,不易溶于醇。麦芽糖酸属于植物来源的,具有超温和、不刺激的优点,广泛用于化妆品中,用于改善皮肤的水合度,提升皮肤紧致性和弹性,减少皱纹等;在临床中,麦芽糖酸能辅助多种皮肤炎症的治疗,减缓刺激性药物对皮肤引起的不适症状;麦芽糖酸是一种天然抗氧化剂,用做食物和饲料保鲜剂。
目前研究中,常利用醌蛋白葡萄糖脱氢酶(PQQGDH)在辅因子吡咯喹啉醌(PQQ)作用下催化麦芽糖生成麦芽糖酸。以PQQ为辅因子的醌蛋白GDH广泛存在于革兰氏阴性致病菌中,如醋酸钙不动杆菌、产气克雷伯氏杆菌等,它们自身能生成PQQ;其他菌种如大肠杆菌、鲁氏不动杆菌等,自身不能合成PQQ,只能通过在培养基中添加外源PQQ后才能形成有活性的全酶。而PQQ的产量低,价格昂贵。因此,挖掘不依赖于PQQ的葡萄糖脱氢酶基因对于麦芽糖酸的大规模制备尤为重要。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供一种利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,对于麦芽糖具有更强的底物偏好性,适合大规模的制备麦芽糖酸。
为了解决上述技术问题,本发明所采用的技术方案如下:
一种利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,以大肠杆菌为模板,克隆出葡萄糖脱氢酶基因Ecgdh;构建重组表达葡萄糖脱氢酶基因Ecgdh体系,表达出葡萄糖脱氢酶;再以麦芽糖为底物,以NADH为电子供体,经葡萄糖脱氢酶催化制备麦芽糖酸;所述的葡萄糖脱氢酶基因Ecgdh的核苷酸序列如SEQ ID NO.1所示。
所述的葡萄糖脱氢酶基因Ecgdh的表达蛋白,其氨基酸序列如SEQ ID NO.2所示。
所述重组表达葡萄糖脱氢酶基因Ecgdh体系为重组大肠杆菌表达体系或重组毕赤酵母表达体系。
所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,葡萄糖脱氢酶催化反应体系为:1-200mM Tris-HCl溶液中,添加有0.1-50mM NADH、0.1-100μM葡萄糖脱氢酶和10-50g/L麦芽糖;葡萄糖脱氢酶催化反应温度为20-50℃;反应pH为5-9,反应时间为5-24h。
所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,葡萄糖脱氢酶催化反应温度为37℃;反应pH为7;反应时间为5h。
所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,构建重组表达葡萄糖脱氢酶基因Ecgdh体系的具体过程为:
将克隆得到的葡萄糖脱氢酶基因Ecgdh,经Gibbson组装,同源重组整合到pET29a(+)的Nde I和Xho I位点,构建重组表达载体pET29a(+)-Ecgdh;将重组表达载体转化E.coli BL21获得葡萄糖脱氢酶的大肠杆菌重组表达菌株E.coli BL21pET29a(+)-Ecgdh。
所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,表达出葡萄糖脱氢酶的过程为:平板上挑取重组大肠杆菌表达菌株E.coli BL21 pET29a(+)-Ecgdh单菌落,接种于3mL LB培养基中,37℃,200rpm培养过夜;按1%(v/v)的比例转接50mL TB培养基中,37℃,培养至OD600nm为0.6-0.8,加入终浓度为0.1mM的IPTG,25℃,诱导5h后8000rpm,4℃离心5min,收集菌体;
用预冷的Tris-HCl(pH 7.0)清洗、重悬菌体至50mL,冰浴超声,条件为:400W,工作2s,间歇3s,200个循环至菌液澄清,14000rpm,4℃离心20min,收集上清,即为粗酶液;
用Buffer A平衡Ni-NTA Agarose Fast Flow柱子后,将葡萄糖脱氢酶粗酶液透过0.22μm滤膜后,上清液以3mL/min的速度通过Ni-NTA Agarose Fast Flow柱子,结合10min后,Buffer B洗脱收集酶活性部分,浓缩,冷冻干燥,获得纯酶。
所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,构建重组表达葡萄糖脱氢酶基因Ecgdh体系的具体过程为:
将克隆得到的葡萄糖脱氢酶基因Ecgdh,经Gibbson组装,同源重组整合到pPIC9K的EcoR I和Not I位点,构建出重组表达载体pPIC9K-Ecgdh;将重组表达载体转化毕赤酵母得到转化子P.patoris GS115pPIC9K-Ecgdh;
所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,表达出葡萄糖脱氢酶的过程为:从平板上挑取毕赤酵母转化子P.patoris GS115pPIC9K-Ecgdh单菌落,接种于3mL YPD培养基中,30℃,200rpm培养过夜;按1%(v/v)的比例转接50mL BMMY培养基中,20℃,200rpm诱导5d;将上述培养物于8000rpm,4℃离心5min收集上清,即为葡萄糖脱氢酶粗酶液;
用Buffer A平衡Ni-NTA Agarose Fast Flow柱子后,将葡萄糖脱氢酶粗酶液透过0.22μm滤膜后,上清液以3mL/min的速度通过Ni-NTA Agarose Fast Flow柱子,结合10min后,Buffer B洗脱收集酶活性部分,浓缩,冷冻干燥,获得纯酶。
有益效果:相比于现有技术,本发明的技术优势为:
1)本申请基于公共数据库NCBI,挖掘葡萄糖脱氢酶基因序列,通过微生物细胞重组表达葡萄糖脱氢酶基因,获得葡萄糖脱氢酶,在此基础上,催化麦芽糖合成麦芽糖酸。
2)本申请获得的葡萄糖脱氢酶,相较于以PQQ为辅因子的醌蛋白葡萄糖脱氢酶对于麦芽糖具有更强的底物偏好性,适合大规模的制备麦芽糖酸。
附图说明
图1为葡萄糖脱氢酶基因的克隆及其鉴定结果图;
图2为重组大肠杆菌细胞工厂的构建合成葡萄糖脱氢酶示意图;图中,A为重组大肠杆菌E.coli BL21 pET29a(+)-Ecgdh的构建示意图;B为SDS-PAGE分析大肠杆菌细胞工厂合成葡萄糖脱氢酶情况,其中泳道M表示marker,泳道1为重组大肠杆菌E.coli BL21pET29a(㈡全细胞样品,泳道2为重组大肠杆菌E.coli BL21 pET29a(+)胞内上清样品,泳道3为重组大肠杆菌E.coli BL21pET29a(+)-Ecgdh全细胞样品,泳道4为重组大肠杆菌E.coliBL21 pET29a(+)-Ecgdh胞内上清样品,泳道5为Ecgdh蛋白纯化样品;
图3是HPLC分析麦芽糖酸标品色谱图;
图4是添加NADH辅因子的葡萄糖脱氢酶催化麦芽糖的产物HPLC色谱图;
图5是未添加NADH辅因子的葡萄糖脱氢酶催化麦芽糖的产物HPLC色谱图。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1依赖于NADH的葡萄糖脱氢酶基因的克隆根据大肠杆菌来源的葡萄糖脱氢酶(GenBank:BAB96699.1)基因序列设计引物FI/R1(ATGTTGATCCCATTTCTGTT,CGATTCCAGTCGCAGTAATT),利用高保真PCR聚合酶Prime Star,以E.coli DH5α为模板扩增大肠杆菌葡萄糖脱氢酶基因片段。经片段回收后,利用平末端连接试剂盒Blunting KinationLigation Kit(TaKaRa),按照试剂盒使用说明进行载体pUC19和片段的连接,最后42℃、90s转化E.coli JM109获得备选基因的重组克隆载体,并经过测序鉴定待测基因的核苷酸序列(SEQ ID NO.1),与GenBank:BAB96699.1的基因序列一致,电泳鉴定结果如图1所示,命名为葡萄糖脱氢酶基因Ecgdh。
实施例2利用微生物细胞制备葡萄糖脱氢酶
1、利用重组大肠杆菌制备葡萄糖脱氢酶
将克隆得到的葡萄糖脱氢酶基因Ecgdh,经Gibbson组装,同源重组整合到pET29a(+)的Nde I和Xho I位点,构建重组表达载体pET29a(+)-Ecgdh。利用上述重组表达载体转化E.coli BL21获得葡萄糖脱氢酶的大肠杆菌重组表达菌株E.coli BL21 pET29a(+)-Ecgdh,以备后续葡萄糖脱氢酶的制备(图2A)。
平板上挑取重组大肠杆菌表达菌株E.coli BL21pET29a(+)-Ecgdh单菌落,接种于3mL LB培养基(加入终浓度为50mg/L的卡那霉素)中,37℃,200rpm培养过夜。按1%(v/v)的比例转接50mL TB培养基(加入终浓度为50mg/L的卡那霉素)中,37℃,培养至OD600nm为0.6-0.8,加入终浓度为0.1mM的IPTG,25℃,诱导5h后8000rpm,4℃离心5min,收集菌体。用预冷的Tris-HCl(pH 7.0)清洗、重悬菌体至50mL,冰浴超声,条件为:400W,工作2s,间歇3s,200个循环至菌液澄清,14000rpm,4℃离心20min,收集上清,即为粗酶液(图2B)。
2、重组毕赤酵母细胞工厂的构建及葡萄糖脱氢酶的制备
将克隆得到的葡萄糖脱氢酶基因Ecgdh,经Gibbson组装,同源重组整合到pPIC9K的EcoR I和Not I位点,构建出重组表达载体pPIC9K-Ecgdh。以上述重组表达载体转化毕赤酵母得到转化子P.patoris GS115 pPIC9K-Ecgdh,以备后续利用毕赤酵母细胞工厂制备葡萄糖脱氢酶。
从平板上挑取毕赤酵母转化子P.patoris GS115 pPIC9K-Ecgdh单菌落,接种于3mL YPD培养基中,30℃,200rpm培养过夜。按1%(v/v)的比例转接50mL BMMY培养基(加入终浓度为0.5g/L的甲醇)中,20℃,200rpm诱导5d。将上述培养物于8000rpm,4℃离心5min收集上清,即为葡萄糖脱氢酶粗酶液。
3、葡萄糖脱氢酶的纯化
用Buffer A(20mM Tris-HC1,0.5M NaCl,20mM咪唑,pH 7.4)平衡Ni-NTA AgaroseFast Flow柱子后,将葡萄糖脱氢酶粗酶液1、2分别透过0.22μm滤膜后,上清液以3mL/min的速度通过Ni-NTA Agarose Fast Flow柱子,结合10min后,Buffer B(20mM Tris-HCl,0.5MNaCl,500mM咪唑,pH 7.4)洗脱收集酶活性部分,浓缩,冷冻干燥。
实施例3麦芽糖酸的制备
本申请以麦芽糖为原料,反应液包括20mM Tris-HCl(pH7.0),3mM NADH(以不添加该组分为对照样)、3μM葡萄糖脱氢酶(实施例2制备)、100mM麦芽糖,37℃,反应5h后70℃加热5min终止反应,制得葡萄糖脱氢酶的催化产物,即麦芽糖酸。
采用高效液相色谱测定麦芽糖酸的产量。所用HPLC-MS的高效液相色谱条件为色谱柱:Agilent AdvanceBio MS Spent Media 2.1×150mm;流动相:0.2%磷酸水溶液(A),乙腈溶液(B);检测波长:210nm;流速:0.25mL/min。洗脱程序为时间(min)0:泵10%+泵B90%,时间(min)2:泵A10%+泵B90%,时间(min)4:泵A 60%+泵B 40%,时间(min)6:泵A10%+泵B 90%。其对应的所用质谱条件:离子源:ESI;雾化器流速:1.5L/min;干燥气流速:5L/min;离子源温度:200℃;传输线温度:250℃。
所用MALDI-TOF-MS的质谱条件为:在阴离子模式下,一级质谱每张谱图累加800次,二级质谱累加1200次,碰撞诱导解离条件为1000eV碰撞能加空气碰撞(1kV,Gas on),源内电压8kV,碰撞池电压7kV。所用数据库搜索条件:串联质谱数据信噪比设为4,质谱质粒误差设为0.2Da,通过Mascot软件分析,并利用NCBI数据库检索或者利用Data Explorer提供的离子碎片计算器(Ion fragmentation calculator)和Protein-Prospector MS-Productprogram辅助进行人工解析,鉴定麦芽糖酸的结构。
结果表明,在发酵体系中添加NADH辅因子,重组大肠杆菌或酿酒酵母合成的麦芽糖酸的出峰时间与标样一致(图3、图4),为4.5min,而未添加NADH辅因子的,不能制备出麦芽糖酸(图5),由此表明重组大肠杆菌和毕赤酵母成功合成了麦芽糖酸,经过5h催化反应后,麦芽糖酸的产量为20g/L。
Claims (8)
1. 一种利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,其特征在于,以大肠杆菌为模板,克隆出葡萄糖脱氢酶基因Ecgdh;构建重组表达葡萄糖脱氢酶基因Ecgdh体系,表达出葡萄糖脱氢酶;再以麦芽糖为底物,以NADH为电子供体,经葡萄糖脱氢酶催化制备麦芽糖酸;所述的葡萄糖脱氢酶基因Ecgdh的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,其特征在于,所述重组表达葡萄糖脱氢酶基因Ecgdh体系为重组大肠杆菌表达体系或重组毕赤酵母表达体系。
3. 根据权利要求1所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,其特征在于,葡萄糖脱氢酶催化反应体系为:1-200 mM Tris-HCl溶液中,添加有0.1-50 mM NADH、0.1-100 μM葡萄糖脱氢酶和10-50 g/L麦芽糖;葡萄糖脱氢酶催化反应温度为20-50℃;反应pH为5-9,反应时间为5-24 h。
4.根据权利要求1所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,其特征在于,葡萄糖脱氢酶催化反应温度为37℃;反应pH为7;反应时间为5h。
5.根据权利要求1所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,其特征在于,构建重组表达葡萄糖脱氢酶基因Ecgdh体系的具体过程为:
将克隆得到的葡萄糖脱氢酶基因Ecgdh,经Gibbson组装,同源重组整合到pET29a (+)的Nde I和Xho I位点,构建重组表达载体pET29a(+)-Ecgdh;将重组表达载体转化E. coliBL21获得葡萄糖脱氢酶的大肠杆菌重组表达菌株E. coli BL21 pET29a (+)-Ecgdh。
6. 根据权利要求5所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,其特征在于,表达出葡萄糖脱氢酶的过程为:平板上挑取重组大肠杆菌表达菌株E. coli BL21pET29a (+)-Ecgdh单菌落,接种于3 mL LB培养基中,37℃,200 rpm培养过夜;按1%的比例转接50 mL TB培养基中,37℃,培养至OD 600 nm为0.6-0.8,加入终浓度为0.1 mM的IPTG,25℃,诱导5 h后8000 rpm,4℃离心5 min,收集菌体;
用预冷的Tris-HCl pH7.0清洗、重悬菌体至50 mL,冰浴超声,条件为:400 W,工作2 s,间歇3 s,200个循环至菌液澄清,14000 rpm,4oC离心20 min,收集上清,即为粗酶液;
用Buffer A平衡Ni-NTA Agarose Fast Flow柱子后,将葡萄糖脱氢酶粗酶液透过0.22μm滤膜后,上清液以3 mL/min的速度通过Ni-NTA Agarose Fast Flow 柱子,结合10 min后,Buffer B洗脱收集酶活性部分,浓缩,冷冻干燥,获得纯酶;
其中,Buffer A的组成为:20mM Tris-HC1,0.5M NaCl,20mM咪唑,pH7.4;Buffer B的组成为:20mM Tris-HCl,0.5M NaCl,500mM咪唑,pH 7.4。
7.根据权利要求1所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,其特征在于,构建重组表达葡萄糖脱氢酶基因Ecgdh体系的具体过程为:
将克隆得到的葡萄糖脱氢酶基因Ecgdh,经Gibbson组装,同源重组整合到 pPIC9K的EcoR I和Not I位点,构建出重组表达载体pPIC9K-Ecgdh;将重组表达载体转化毕赤酵母得到转化子P. patoris GS115 pPIC9K-Ecgdh。
8. 根据权利要求7所述的利用葡萄糖脱氢酶催化麦芽糖合成麦芽糖酸的方法,其特征在于,表达出葡萄糖脱氢酶的过程为:从平板上挑取毕赤酵母转化子P. patoris GS115pPIC9K-Ecgdh单菌落,接种于3 mL YPD培养基中,30℃,200 rpm培养过夜;按1% v/v的比例转接50 mL BMMY培养基中,20℃,200 rpm诱导5 d;将上述培养物于8000 rpm,4℃离心5min收集上清,即为葡萄糖脱氢酶粗酶液;
用Buffer A平衡Ni-NTA Agarose Fast Flow柱子后,将葡萄糖脱氢酶粗酶液透过0.22μm滤膜后,上清液以3 mL/min的速度通过Ni-NTA Agarose Fast Flow 柱子,结合10 min后,Buffer B洗脱收集酶活性部分,浓缩,冷冻干燥,获得纯酶;
其中,Buffer A的组成为:20mM Tris-HC1,0.5M NaCl,20mM咪唑,pH7.4;Buffer B的组成为:20mM Tris-HCl,0.5M NaCl,500mM咪唑,pH 7.4。
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