CN114015735B - 一种蔗糖磷酸化酶和葡萄糖异构酶级联催化合成黑曲霉二糖的方法 - Google Patents
一种蔗糖磷酸化酶和葡萄糖异构酶级联催化合成黑曲霉二糖的方法 Download PDFInfo
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- CN114015735B CN114015735B CN202111413918.5A CN202111413918A CN114015735B CN 114015735 B CN114015735 B CN 114015735B CN 202111413918 A CN202111413918 A CN 202111413918A CN 114015735 B CN114015735 B CN 114015735B
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Abstract
本发明公开了一种蔗糖磷酸化酶和葡萄糖异构酶级联催化合成黑曲霉二糖的方法，属于酶工程领域。本发明以来源于青春双歧杆菌蔗糖磷酸化酶(BaSP)和葡萄糖异构酶(GI)为研究对象，利用BaSP水解蔗糖产生的葡萄糖启动转糖苷反应，通过GI将副产物果糖转化为转糖苷受体葡萄糖，促进整个反应向转糖苷方向进行，抑制水解作用，最大程度利用副产物，并降低辅底物葡萄糖的添加。在单一底物蔗糖的条件下，级联反应的黑曲霉二糖产量约为BaSP单酶反应的192％，同时果糖产量降低为BaSP单酶反应的80％。本发明构建的酶级联反应对于促进黑曲霉二糖绿色、高效合成具有重要意义。
Description
技术领域
本发明涉及一种蔗糖磷酸化酶和葡萄糖异构酶级联催化合成黑曲霉二糖的方法，属于酶工程领域。
背景技术
黑曲霉二糖(2-O-α-D-glucopyranosyl-D-glucose,nigerose)是由两个葡萄糖通过α-1,3糖苷键键合的二糖，不易被机体消化，具有抗龋齿和促进双歧杆菌增殖的作用。同时，黑曲霉二糖及其衍生的低聚糖是一种低热量甜味剂，其良好的风味可以赋予食品丰富而醇厚的口感，例如:一、可以作为高甜度甜味剂的风味改良剂，降低高甜度甜味剂具有的独特异味、苦味和刺激味，解除高甜度甜味剂在食品种类、用量及使用方法等方面的限制；二、可以作为含盐食品的风味改良剂，消除“盐过度”，降低食盐用量而不影响其香味；三、可以作为食品煮崩防止剂，有效预防食品在加热或加压处理过程中煮崩，并改善口感。此外，黑曲霉二糖可以用作生物相容性纳米载体，持续输送药物。综上而言，黑曲霉二糖作为一种功能性食品引起了人们的广泛关注。
在自然界中，黑曲霉二糖通常作为多糖的组成单元出现，如nigeran,elucinan,pseudonigeran,isolichenin等。这些多糖是丝状真菌(青霉菌或曲霉菌)细胞壁的组成成分。此外，也可以从啤酒、蜂蜜、清酒、甘薯淀粉水解物中分离出黑曲霉二糖，但由于产物含量很低，导致分离纯化非常困难，难以大规模生产。
目前，黑曲霉二糖的制备方法有化学降解法和酶合成法。
化学降解法是以酸或乙酰化试剂降解含有α-1,3糖苷键连接的多糖。葡聚糖在酸水解过程中，α-1,3糖苷键不稳定；而在乙酰解过程中，α-1,2糖苷键和α-1,3糖苷键较稳定。故乙酰解优于酸水解。但该法存在操作步骤繁琐，反应时间长，产量低，使用浓硫酸、氯仿等危险化学品等问题，逐渐被酶合成法所替代。
酶合成法由于催化条件温和且生产更为安全成为当前生产黑曲霉二糖的研究热点。当前用于酶法制备黑曲霉二糖的酶有α-葡萄糖苷酶(α-glucosidase)、黑曲霉二糖磷酸化酶(nigerose phosphorylase)、蔗糖磷酸化酶(sucrose phosphorylase)。它们的来源以真菌和肠道微生物为主。α-葡萄糖苷酶使低聚麦芽糖水解，将释放的葡萄糖以α-1,3糖苷键或α-1,4糖苷键转移至葡萄糖基部分，因此在制备黑曲霉二糖过程中会产生大量含有α-1,4糖苷键的低聚糖，影响后续的分离纯化。黑曲霉二糖磷酸化酶通过反向磷酸化，以D-葡萄糖和β-葡萄糖-1-磷酸为底物合成黑曲霉二糖，由于使用了昂贵的β-葡萄糖-1-磷酸，导致生产成本的提高，且反应后期黑曲霉二糖会逐渐转化成曲二糖，影响黑曲霉二糖的产量。蔗糖磷酸化酶已经进行了许多详细研究，包括阐明反应机理、结晶、热稳定性、固定化。其可以通过水解蔗糖，将葡萄糖基部分转移至葡萄糖的C-3位置，形成黑曲霉二糖，成为新型生产黑曲霉二糖的方法。目前，有报道通过对青春双歧杆菌来源的蔗糖磷酸化酶(BaSP)的理性设计，提高转糖基效率来生产黑曲霉二糖。然而，利用BaSP制备黑曲霉二糖的方法需要添加辅底物葡萄糖，且反应会产生副产物果糖，给分离纯化带来一定困难，限制了其工业化生产进程。
发明内容
为了解决上述问题，本发明提供一种蔗糖磷酸酶和葡萄糖异构酶级联催化生产黑曲霉二糖的方法。以BaSP为应用实例，将BaSP的基因与pET-30a(+)连接，导入到E.coliBL21中表达，以蔗糖和葡萄糖为底物，通过其转糖苷作用生成黑曲霉二糖。二是从10种GI中筛选出比酶活和果糖转化率最高的一株。三是级联反应初始阶段，利用BaSP水解蔗糖产生的葡萄糖启动转糖苷反应，其后通过GI将副产物果糖转化为转糖苷受体葡萄糖，促进整个反应向转糖苷方向进行，抑制水解作用，最大程度利用副产物，并降低辅底物葡萄糖的添加(图1)。本发明将蔗糖磷酸酶与葡萄糖异构酶联用，对于促进黑曲霉二糖绿色、高效合成具有重要意义。
本发明的第一个目的是提供一种生产黑曲霉二糖的方法，所述方法为以蔗糖为反应底物，利用蔗糖磷酸化酶和葡萄糖异构酶级联催化蔗糖生成黑曲霉二糖。
在本发明的一种实施方式中，将蔗糖磷酸化酶和葡萄糖异构酶以一定比例添加至含有蔗糖的反应体系中进行催化反应；
或将表达蔗糖磷酸化酶的全细胞和表达葡萄糖异构酶的全细胞以一定比例添加至含有蔗糖的反应体系中进行催化反应；
或将共表达蔗糖磷酸化酶和葡萄糖异构酶的全细胞添加至含有蔗糖的反应体系中进行催化反应。
在本发明的一种实施方式中，还可以添加少量或不添加葡萄糖作为辅底物。
在本发明的一种实施方式中，葡萄糖的添加量为0～10mM。
在本发明的一种实施方式中，所述反应的缓冲体系包括40～60mM MOPS-NaOHbuffer。
在本发明的一种实施方式中，所述反应条件为50～54℃，pH 5.0～8.0，时间90～120h。
在一种实施方式中，所述蔗糖磷酸化酶的氨基酸序列如SEQ ID NO.2所示。
在一种实施方式中，所述葡萄糖异构酶的氨基酸序列的NCBI登录号为P12851、P12070、Q9FKK7、A5CPC1、Q9ZAI3、Q9RFM4、P24300、P09033、P26997、Q5GUF2。
本发明的第二个目的是提供一种组合酶制剂，所述酶制剂包括蔗糖磷酸化酶和葡萄糖异构酶。
在一种实施方式中，所述蔗糖磷酸化酶的氨基酸序列如SEQ ID NO.2所示。
在一种实施方式中，所述葡萄糖异构酶的氨基酸序列的NCBI登录号为P12851、P12070、Q9FKK7、A5CPC1、Q9ZAI3、Q9RFM4、P24300、P09033、P26997、Q5GUF2。
本发明还提供所述方法或所述组合酶制剂在制备含有黑曲霉二糖的产品中的应用。
有益效果：
1、本发明通过蔗糖磷酸化酶BaSP和葡萄糖异构酶GI的级联反应的应用实例，降低辅底物葡萄糖的添加，促进反应向转糖苷方向进行，抑制水解作用，大幅降低生产成本。最大程度利用副产物果糖，降低副产物果糖的累积，利于后续黑曲霉二糖的分离纯化。
2、相较于BaSP单酶催化合成黑曲霉二糖，本发明的方法在不添加辅底物葡萄糖的条件下，酶级联反应的黑曲霉二糖产量约为BaSP单酶反应的192％，同时果糖产量降低为BaSP单酶反应的80％。在添加少量辅底物葡萄糖(10mM)的条件下，酶级联反应的黑曲霉二糖产量约为蔗糖磷酸化酶单酶反应的158％，同时果糖产量降低为BaSP单酶反应的80％。
3、利用本发明的方法生产黑曲霉二糖，终产物中不含有曲二糖，进一步利于后续产物分离提纯。
附图说明
图1级联反应机理；
图2 BaSP 27℃表达情况；
图3 BaSP反应液相结果；
图4 7种葡萄糖异构酶的表达情况，a)粗酶液；b)细胞碎片；c)葡萄糖异构酶CMGI、SVGI和SLGI的16℃表达情况；d)SKGI的表达情况；
图5葡萄糖异构酶的表达情况，a)SKGI的详细纯化情况。M.低分子质量标准蛋白；1.粗酶；2.流穿液；3.Binding buffer透过液；4.Binding buffer+100mM咪唑洗脱；5.Binding buffer+150mM咪唑洗脱；6.Binding buffer+500mM咪唑；b)6种葡萄糖异构酶的纯化后的纯度和浓度比较。7.AMGI；8.TTGI；9.XOGI；10.ASGI；11.SKGI；12.CMGI；
图6咔唑-硫酸显色法检测果糖浓度标准曲线；
图7 XOGI和ASGI的最适反应温度；
图8葡萄糖异构酶的比活力比较；
图9蔗糖磷酸化酶和葡萄糖异构酶级联反应。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市售商品或者可以通过已知方法制备。
PrimerStar Mix DNA聚合酶(Takara)、DNA上样缓冲液(Takara)、SanPrep柱式PCR产物纯化试剂盒、SanPrep胶回收试剂盒及SanPrep柱式质粒DNA小量抽提试剂盒(SangonBiotech,Shanghai)；LE琼脂糖(核酸电泳)、4S Green Plus无毒核酸染料(SangonBiotech,Shanghai)；蛋白上样缓冲液(5x)(碧云天)、低分子质量预混蛋白Marker(Takara)；DNA 15000 ladder、DNA 5000 ladder和DNA 10000 ladder购买于上海宝生物有限公司；镍柱购自Novagen，DEAE Sepharose Fast Flow购自GE Healthcare，透析袋购自北京索莱宝科技有限公司(中国)；胰蛋白胨和酵母提取物购买于英国Oxoid公司，琼脂粉和氯化钠购自国药集团，硫酸卡那霉素购自百灵威科技。
下述实施例中涉及到的培养基：
LB(Luria-Bertani)液体培养基配置(1L)：NaCl 10g，胰蛋白胨10g，酵母提取物5g，121℃，20min高压蒸汽灭菌后备用；
LB(Luria-Bertani)固体培养基配置(1L)：NaCl 10g，胰蛋白胨10g，酵母提取物5g，琼脂粉15g，高压蒸汽灭菌(121℃，20min)后倒入15mm*90mm无菌培养皿，为无抗生素LB固体培养基，加抗生素则为对应的抗生素选择性LB固体培养基，凝固后放于4℃冰箱备用。
下述实施例中涉及到的方法：
葡萄糖异构酶的结构异构活性测定：在pH 7.0，最适温度的反应条件下，以1.5M的葡萄糖为底物，添加0.4mg/mL纯化后的酶液，以及0.15mM CoSO4和1.5mM MgSO4，反应0、5、10、20、30、45、60、90、120min，反应液稀释后通过半胱氨酸-咔唑显色法检测酶活性。
实施例1：青春双歧杆菌蔗糖磷酸化酶突变体克隆、表达、纯化及酶反应
(1)蔗糖磷酸化酶突变体全基因合成
在NCBI获取青春双歧杆菌(NC_008618.1 Bifidobacterium adolescentis ATCC15703)来源的蔗糖磷酸化酶的氨基酸序列(如SEQ ID NO.1所示)。以氨基酸序列如SEQ IDNO.1所示的蔗糖磷酸化酶为亲本酶进行定点突变，将氨基酸序列的第135位Arg突变为Tyr，第342位Asp突变为Gly，第344位Tyr突变为Gln，第345位Gln突变为Phe，并根据大肠杆菌的偏好性进行密码子优化于上海生工生物工程股份有限公司合成基因序列。并通过限制性内切酶位点NdeI和XhoI插入到商品化载体PUC57-Kan，得到含有目的基因BaSP的质粒PUC57-BaSP。
(2)构建重组质粒pET-30a(+)-BaSP
将步骤(1)制备的PUC57-BaSP与pET-30a(+)分别进行NdeI和XhoII双酶切，通过DNA胶回收试剂盒回收目的基因片段与pET-30a(+)片段，并使用T4连接酶连接，获得重组DNA。
通过热激法将重组DNA导入至感受态E.coli Top10菌株，并涂布于含有kana的LB琼脂平板，37℃倒置培养16h。挑取平板上的单菌落进行菌落PCR，使用琼脂糖核酸电泳检测条带，挑选条带大小正确的单菌落进行测序。将测序成功的重组质粒命名为pET-30a(+)-BaSP。
菌落PCR：反应体系参照表1，总体积为50μL：
表1 PCR反应体系
反应程序：PCR反应扩增条件参照表2：
表2 PCR反应扩增条件
(3)酶的表达与纯化
1)步骤(2)中测序成功的重组质粒pET-30a(+)-BaSP转入E.coli BL21(DE3)感受态细胞中，转化液涂平板，37℃过夜培养；然后挑取单菌落过夜培养获得活化菌液，将活化菌液以1％(v/v)的接种量接种于LB培养基中，37℃培养至OD600值0.6-0.8，加入浓度为1M的IPTG，27℃诱导18h，获得发酵液。
2)将步骤1)中的发酵液离心，将收集的菌体用Lysis Buffer(60mM Na2HPO4，250mMNaCl，11mM imidazole，pH＝8)悬浮混匀，放置冰浴中，超声破碎的条件为210W功率，工作5s，间隔9s，超声破碎15min，得到粗酶液，将粗酶液用0.22μm过滤膜过滤，备用；
3)将步骤2)中过滤过的粗酶液通过Ni离子亲和层析柱，先用Washing Buffer(60mM Na2HPO4，250mM NaCl，11mM imidazole，pH＝8)清洗蛋白，最后用Elution Buffer(60mM Na2HPO4，250mM NaCl，230mM imidazole，pH＝8)洗脱，收集含有目的蛋白的洗脱液；对含有目的蛋白的洗脱液进行透析，透析液为浓度20mM MOPS-NaOH(pH 7.0)的缓冲液，共透析3次，每次6个小时，透析结束后，收集透析袋中的纯酶液于EP管中，4℃保存备用。采用SDS-PAGE蛋白电泳检验蛋白纯化效率及纯度。
如图2所示，上清液，沉淀，100mM洗脱液分别稀释20倍上样；流穿液，250mM洗脱液，500mM洗脱液原浓度上样。目标蛋白分子量为57.6kDa，100mM洗脱液相应位置处出现较宽蛋白条带。证明100mM洗脱液即可将大量目标蛋白洗脱下来。
(4)青春双歧杆菌蔗糖磷酸化酶合成黑曲霉二糖及检测
1)在50mM MOPS-NaOH buffer(pH 7.0)的缓冲体系中，以100mM蔗糖和100mM葡萄糖为底物，添加1mg/mL纯化酶液，52℃反应96h，沸水浴10min停止反应
2)反应液5000rpm离心2min，对蔗糖、葡萄糖、果糖、黑曲霉二糖进行HPLC检测。利用示差折光检测仪分离和检测糖的浓度；其中色谱柱为Asahipak NH2P-50G 4A预装柱和NH2P-50 4E色谱分离柱，流动相为乙腈、水(75:25)的混合液。
3)如图3所示，底物蔗糖反应完全，大部分水解生成葡萄糖和果糖，少部分通过转糖基作用将葡萄糖基部分转移至底物葡萄糖上，生成黑曲霉二糖。黑曲霉二糖产量较低，转化率为12.3％。
实施例2：葡萄糖异构酶的筛选
(1)构建葡萄糖异构酶表达载体
从UniProt数据库(https://www.uniprot.org/)中搜索葡萄糖异构酶的EC号(EC:5.3.1.5)，下载不同来源的约300条葡萄糖异构酶的基因序列，通过Multiple SequenceAlignment by CLUSTALW网页(https://www.genome.jp/tools-bin/clustalw)进行序列比对，同时参考用于工业化生产的葡萄糖异构酶的酶学信息和活力数据，综合挑选10条活力较高且序列相似性约为95％、90％、80％、75％、70％、65％、50％、25％、20％不等的葡萄糖异构酶基因进行基因合成。基因信息见表3；基因的序列优化和合成由生工生物工程(上海)股份有限公司完成；密码子优化选择对象为Escherichia coli；克隆载体为pET-22b，获得一系列葡糖糖异构酶的重组质粒pET-22b-AMGI、pET-22b-ASGI、pET-22b-ATGI、pET-22b-CMGI、pET-22b-SKGI、pET-22b-SLGI、pET-22b-SRGI、pET-22b-SVGI、pET-22b-TTGI、pET-22b-XOGI。
表3葡萄糖异构酶基因的基本信息
(2)葡萄糖异构酶表达条件的优化
将步骤(1)中构建的重组质粒分别转化到感受态E.coli BL21(DE3)中，转化液涂平板，37℃过夜培养。挑取单菌落接种到6mL含有氨苄青霉素(100μg/mL)的LB培养基中，在200rpm和37℃下过夜培养获得活化菌液，将活化菌液以1％(v/v)的接种量接种到含100μg/mL氨苄青霉素的500mL LB培养基中，37℃培养至OD600达到约0.6-0.8时，加入0.6mM IPTG，并在28℃，200rpm下孵育24h，获得菌液。取1.5mL菌液，4℃、10000rpm离心5min，弃去上清液，菌体保存在-20℃冰箱。
(3)葡萄糖异构酶的表达情况
将步骤(2)中的菌体用150μL含10mM咪唑的50mM磷酸钠盐缓冲液(pH 7.0)重悬，超声破碎释放蛋白，超声破碎的条件为50W功率、工作5s、暂停5s，共4次，菌体全程置于冰上。4℃、10000rpm离心15min，将上清液和沉淀分开保存。采用SDS-PAGE蛋白电泳检验不同来源的葡萄糖异构酶的表达情况。
SDS-PAGE样品制备如下：取20μL上清液，加入5μL蛋白上样缓冲液(5x)，混匀；沉淀用48μL去离子水重悬，加入12μL蛋白上样缓冲液(5x)，混匀。沸水浴10min，冷却后室温下离心。
SDS-PAGE凝胶制备方法如下：有数据显示12％聚丙烯酰胺凝胶的有效分离范围是12-60kDa，鉴于葡萄糖异构酶的分子量在42-53kDa之间，因此选择在12聚丙烯酰胺凝胶中进行SDS-PAGE。
从图4a和图4b的结果来看，AMGI、TTGI、ASGI和XOGI能够表达可溶性蛋白；CMGI、SLGI、SVGI表达的蛋白会形成包涵体；ATGI、SRGI不能表达。因此，需要调整CMGI、SLGI、SVGI的表达条件，使之表达出可溶性蛋白。
(4)葡萄糖异构酶的纯化
将步骤(1)中构建的重组质粒分别转化到感受态E.coli BL21(DE3)中，转化液涂平板，37℃过夜培养，挑取单菌落分别接种到6mL含有氨苄青霉素(100μg/mL)的LB培养基中，并在200rpm和37℃下过夜培养获得活化菌液，将活化菌液以1％(v/v)的接种量接种到含100μg/mL氨苄青霉素的500mL LB培养基中，37℃培养至OD600达到0.6-0.8时，加入0.6mMIPTG，并在28或16℃，200rpm下孵育20h，获得发酵液。将发酵液进行离心，6000rpm、4℃下离心15min收获细胞，离子水洗去多余培养基，在6000rpm、4℃下离心5min收获细胞，-20℃保存。
将细胞悬浮在50mL Binding buffer(含有10mM咪唑和200mM NaCl的50mM Na2HPO4缓冲液(pH 7.0))中，超声破碎的条件为210W功率，工作5s，暂停9s，超声破碎15min。8000rpm、4℃，离心15min，收集上清，获得粗酶液，4℃保存备用。
先用5倍柱体积的Binding buffer平衡镍螯合树脂(5mL)，随后将粗酶液加载到树脂上，依次用含50-500mM咪唑的缓冲液A进行洗脱，收集含有目的蛋白的洗脱液，通过SDS-PAGE分析酶纯度。用100mM磷酸钾缓冲液(pH 7.5)透析洗脱液，获得纯酶液。以牛血清白蛋白为标准，通过Bradford法测定纯酶浓度。
纯化结果如图5所示，其中，5种酶(AMGI、TTGI、ASGI、XOGI、SKGI)的过表达情况较好，酶的纯度和浓度都较高；CMGI的过表达会形成较多包涵体，纯化后浓度较低，但纯度较好；SLGI纯化后立即析出失活，因此认为该酶在表达过程中，折叠错误而无法生成可溶性蛋白，后续选取6种葡萄糖异构酶(AMGI、TTGI、ASGI、XOGI、CMGI、SKGI)的纯酶用于后续检测。
(5)酶活力检测
酶活通过果糖的生成量确定，以D-果糖为标品绘制标准曲线。在15mL试管里加入1.0mL果糖溶液(0、5、10、15、20、25、30μg/mL)，分别加入2.80mL的浓硫酸(75％，w/v)，振荡器混匀，立刻置于46℃的恒温水浴中保温5min。加入0.2mL半胱氨酸盐酸盐和色氨酸混显色剂(0.08％的色氨酸和2.5％半胱氨酸盐酸盐)并混匀，继续保温30min。冷却至室温，取混合物200μL置于96孔板中，用酶标仪扫描OD518nm处的吸光度值。按上述程序制备3组平行的样品，标准曲线如图6所示。
标准反应混合物(1mL)由90mM PBS缓冲液(pH 7.0)、1.5M乳糖、0.15mM CoSO4、1.5mM MgSO4和0.5mg/mL酶组成。酶促反应在70℃进行，每0、5、10、20、30、45、60、90、120min，取100μL，煮沸3min。反应液稀释1000倍，检测果糖的生成量。
选择XOGI和ASGI测试最适反应温度，选择30℃、40℃、50℃、60℃和70℃进行检测，结果如图7所示，XOGI在70℃下1h失活，其最适反应温度为60℃，而ASGI的最适反应温度是70℃，综上，选择XOGI外的其他酶在70℃条件下进行酶促反应。
6种不同来源的葡萄糖异构酶的比活性和反应2h的果糖转化率如表4和图8所示，其中TTGI、AMGI、XOGI、ASGI和CMGI的比酶活分别为21.5、12.0、8.9、7.1和5.4U/mg，反应2h的果糖转化率分别为50.3％、50.1％、36.3％、25.3％、24.0％和15.4％。比酶活最高的是SKGI，为37.0U/mg，同时它的果糖转化率也最高，2h的转化率达到81.3％。
表4葡萄糖异构酶的表达量和比酶活。
实施例3：蔗糖磷酸化酶和葡萄糖异构酶级联反应
在52℃，50mM MOPS-NaOH buffer(pH 7.0)的缓冲体系中，以100mM蔗糖和0、10mM葡萄糖为底物，分别添加1mg/mL实施例1中获得的BaSP纯酶和1mg/mL实施例2中获得的SKGI纯酶，反应96h，沸水浴10min停止反应。反应液5000rpm离心2min，对黑曲霉二糖(目标产物)、果糖(副产物)进行HPLC检测。利用示差折光检测仪分离和检测糖的浓度；其中色谱柱为Asahipak NH2P-50G 4A预装柱和NH2P-50 4E色谱分离柱，流动相为乙腈、水(75:25)的混合液。
如图9所示，不添加辅底物葡萄糖的条件下，酶级联反应的黑曲霉二糖产量约为BaSP单酶反应的192％，同时果糖产量降低为BaSP单酶反应的80％。在添加少量辅底物葡萄糖的条件下，酶级联反应的黑曲霉二糖产量约为蔗糖磷酸化酶单酶反应的158％，同时果糖产量降低为BaSP单酶反应的80％。
实施例4：蔗糖磷酸化酶和葡萄糖异构酶级联反应
在52℃，50mM MOPS-NaOH buffer(pH 7.0)的缓冲体系中，以100mM蔗糖为底物，分别添加1mg/mL实施例1中获得的BaSP纯酶和1mg/mL实施例2中获得的TTGI纯酶，反应96h，沸水浴10min停止反应。反应液5000rpm离心2min，对黑曲霉二糖(目标产物)、果糖(副产物)进行HPLC检测。利用示差折光检测仪分离和检测糖的浓度；其中色谱柱为AsahipakNH2P-50G 4A预装柱和NH2P-50 4E色谱分离柱，流动相为乙腈、水(75:25)的混合液。
实施例5：蔗糖磷酸化酶和葡萄糖异构酶级联反应
在52℃，50mM MOPS-NaOH buffer(pH 7.0)的缓冲体系中，以100mM蔗糖为底物，分别添加1mg/mL实施例1中获得的BaSP纯酶和1mg/mL实施例2中获得的AMGI纯酶，反应96h，沸水浴10min停止反应。反应液5000rpm离心2min，对黑曲霉二糖(目标产物)、果糖(副产物)进行HPLC检测。利用示差折光检测仪分离和检测糖的浓度；其中色谱柱为AsahipakNH2P-50G 4A预装柱和NH2P-50 4E色谱分离柱，流动相为乙腈、水(75:25)的混合液。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种蔗糖磷酸化酶和葡萄糖异构酶级联催化合成黑曲霉二糖的方法
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Claims (8)
1.一种生产黑曲霉二糖的方法，其特征在于，所述方法为以蔗糖为反应底物，利用蔗糖磷酸化酶和葡萄糖异构酶级联催化蔗糖生成黑曲霉二糖，所述蔗糖磷酸化酶的氨基酸序列如SEQ ID NO.2所示，所述葡萄糖异构酶的氨基酸序列的NCBI登录号为P12851、P12070、A5CPC1、Q9ZAI3、P26997、Q5GUF2。
2.根据权利要求1所述的方法，其特征在于，将蔗糖磷酸化酶和葡萄糖异构酶以一定比例添加至含有蔗糖的反应体系中进行催化反应；
或将表达蔗糖磷酸化酶的全细胞和表达葡萄糖异构酶的全细胞以一定比例添加至含有蔗糖的反应体系中进行催化反应；
或将共表达蔗糖磷酸化酶和葡萄糖异构酶的全细胞添加至含有蔗糖的反应体系中进行催化反应。
3.根据权利要求1或2所述的方法，其特征在于，还可以添加少量或不添加葡萄糖作为辅底物。
4.根据权利要求3所述的方法，其特征在于，葡萄糖的添加量为0～10mM。
5.根据权利要求1或2所述的方法，其特征在于，所述反应的缓冲体系包括40～60mMMOPS-NaOH buffer。
6.根据权利要求1或2所述的方法，其特征在于，所述反应条件为50～54℃，pH 5.0～8.0，时间90～120h。
7.一种组合酶制剂，其特征在于，所述酶制剂包括蔗糖磷酸化酶和葡萄糖异构酶，所述蔗糖磷酸化酶的氨基酸序列如SEQ ID NO.2所示，所述葡萄糖异构酶的氨基酸序列的NCBI登录号为P12851、P12070、A5CPC1、Q9ZAI3、P26997、Q5GUF2。
8.权利要求1～6任一所述方法或权利要求7所述组合酶制剂在制备含有黑曲霉二糖的产品中的应用。
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| CN109423485A (zh) * | 2017-08-25 | 2019-03-05 | 中国科学院微生物研究所 | 蔗糖磷酸化酶突变体及其应用 |
| CN112980762A (zh) * | 2021-03-05 | 2021-06-18 | 江南大学 | 一种黑曲霉二糖磷酸化酶及其在黑曲霉二糖制备中的应用 |
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| CN109423485A (zh) * | 2017-08-25 | 2019-03-05 | 中国科学院微生物研究所 | 蔗糖磷酸化酶突变体及其应用 |
| CN112980762A (zh) * | 2021-03-05 | 2021-06-18 | 江南大学 | 一种黑曲霉二糖磷酸化酶及其在黑曲霉二糖制备中的应用 |
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| One Pot Enzymatic Production of Nigerose from Common Sugar Resources Employing Nigerose Phosphorylase;Takanori Nihira等;J. Appl. Glycosci.;第61卷;75-80 * |
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