CN109576239A - 耐热磷酸化酶及其应用 - Google Patents
耐热磷酸化酶及其应用 Download PDFInfo
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- CN109576239A CN109576239A CN201811541294.3A CN201811541294A CN109576239A CN 109576239 A CN109576239 A CN 109576239A CN 201811541294 A CN201811541294 A CN 201811541294A CN 109576239 A CN109576239 A CN 109576239A
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- glycosylglycerol
- sucrose
- preculture
- resisting
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- 229930006000 Sucrose Natural products 0.000 claims description 39
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Abstract
本发明涉及耐热磷酸化酶及其用途。该耐热磷酸化酶具有SEQ ID NO:2所示的氨基酸序列。该耐热磷酸化酶可以有效用于甘油葡糖苷的催化合成,酶活力高,制备甘油葡糖苷时酶用量较小,生产成本较低,且甘油葡糖苷的产量较高。
Description
技术领域
本发明涉及生物技术领域,具体地,本发明涉及耐热磷酸化酶及其应用,更具体地,本发明涉及耐热蔗糖磷酸化酶、分离的核酸、构建体、微生物、全细胞催化剂以及甘油葡糖苷的制备方法。
背景技术
甘油葡糖苷是一种由甘油分子与葡萄糖分子以糖苷键结合的物质。它是一种天然细胞激活剂,常用作化妆品原料,具有以下功能:能保护细胞膜和细胞结构不变性、不受损伤;具有优异的锁水能力,在极度干燥的环境中细胞中依然有水存;分子量小,易吸收。目前制备甘油葡糖苷的方法主要包括化学合成和酶催化合成法。
2000年,日本Takenaka F.等鉴定了日本清酒中3种不同构型的甘油葡糖苷,并通过化学合成的方法实现这3种物质的化学合成(Biosci Biotechnol Biochem,2000,64:378-385)。然而,化学合成方法具有高污染、高能耗、制备工艺复杂等缺点。
专利WO2008034158公开了一种利用蔗糖磷酸化酶(SPase)催化蔗糖和甘油转化为甘油葡糖苷的酶催化合成方法,在0.3M蔗糖、2.0M甘油、20U/mL酶、pH 7.0、7.5h反应条件下,甘油葡糖苷产物浓度约0.29M,即产量约73g/L。然而,该酶催化合成方法成本高、产量较低,难以在工业生产中广泛应用。
因此,甘油葡糖苷的制备方法还需进一步深入研究。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的:
发明人对专利WO2008034158进行深入研究后发现,首先,该专利中所用的SPase酶为商业化酶制剂或纯的酶,商业化酶制剂价格十分昂贵,生产成本较高,而纯的酶需要经过复杂的分离纯化工艺方可得到,生产工艺过于复杂。其次,SPase酶活力不高,制备甘油葡糖苷时酶的用量较大,增大了生产成本。另外,该专利生产甘油葡糖苷时产量较低。综合上述几种因素,该专利方法生产成本较高,产量较低,难以在工业生产中广泛应用。
基于上述问题,发明人经过大量的实验探索,构建了耐热重组蔗糖磷酸化酶(TSPase)的大肠杆菌工程菌株,并将其发酵所得的菌体作为生物催化剂进行甘油葡糖苷的制备。该制备方法直接采用含TSPase酶的菌体细胞作为生物催化剂,避免了购买昂贵的商业用酶制剂或者进行复杂的菌体破壁、浓缩处理,生产成本更低,制备工艺更加简便,且甘油葡糖苷的产量更高。另外,TSPase酶的活力高,制备甘油葡糖苷时催化剂用量较少,降低了生产成本。
为此,在本发明的第一方面,本发明提出了一种耐热磷酸化酶。根据本发明的实施例,所述耐热磷酸化酶具有SEQ ID NO:2所示的氨基酸序列。根据本发明实施例的耐热磷酸化酶可以有效用于甘油葡糖苷的催化合成,酶活力高,制备甘油葡糖苷时用量较小,生产成本较低,且甘油葡糖苷的产量较高。
在本发明的第二方面,本发明提出了一种分离的核酸。根据本发明的实施例,所述分离的核酸具有SEQ ID NO:1所示的核苷酸序列。过表达根据本发明实施例的核酸可以获得前面所述的耐热磷酸化酶,进而有效用于甘油葡糖苷的催化合成,且生产成本低,产量高。
在本发明的第三方面,本发明提出了一种构建体。根据本发明的实施例,所述构建体携带上述分离的核酸。根据本发明实施例的构建体转染受体细胞后获得的微生物可以有效表达前面所述的耐热磷酸化酶,进而用于甘油葡糖苷的催化合成,且生产成本低,产量高。在一些实施例中,所述构建体的载体为pET-28A,由此,所述构建体的转染效果好。
在本发明的第四方面,本发明提出了一种微生物。根据本发明的实施例,所述微生物携带上述分离的核酸或上述任一项所述的构建体。发明人发现,根据本发明实施例的微生物可直接用于甘油葡糖苷的催化合成,且生产成本低,产量高。根据本发明的实施例,所述微生物为大肠杆菌。在一些实施例中,所述微生物为大肠杆菌BL21。
在本发明的第五方面,本发明提出了一种全细胞催化剂。根据本发明的实施例,所述全细胞催化剂包括上述任一项所述的微生物。发明人发现,采用所述全细胞催化剂进行甘油葡糖苷的催化合成可以避免购买昂贵的商业用酶制剂或者进行复杂的菌体破壁、浓缩处理,生产成本低,且甘油葡糖苷的产量高。
根据本发明的实施例,上述全细胞生物催化剂还可进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述全细胞催化剂是通过如下方式制备得到的:在适合微生物增殖的条件下,将上述任一项所述的微生物进行预培养和发酵处理;将发酵处理产物进行离心处理,以便获得离心沉淀物,所述离心沉淀物构成所述全细胞催化剂。发明人发现,通过所述方式制备得到的全细胞催化剂可以直接用于甘油葡糖苷的催化合成,无需购买昂贵的商业用酶制剂或者进行复杂的菌体破壁、浓缩处理,生产成本更低,制备工艺更加简便,且甘油葡糖苷的产量更高。
根据本发明的实施例,所述预培养和发酵处理是在温度28~35℃、转速为200~250rpm的条件下进行的。根据本发明的实施例,所述预培养处理的时间为15~25小时。根据本发明的实施例,所述预培养处理后,菌液的OD600nm为20~25。根据本发明的实施例,所述发酵处理的时间为45~55小时。发明人发现,所述预培养和发酵处理可以使菌体得到高效扩增,前面所述的耐热磷酸化酶在菌体中的高效表达所获得的全细胞生物催化剂可高效用于甘油葡糖苷的催化合成。
根据本发明的实施例,在预培养处理中,培养基包括:1.5~2.5%的酵母浸粉、0.3~0.8%的蛋白胨、0.05~0.15%的氯化钠、0.003~0.008%的七水硫酸镁、0.03~0.08%的磷酸二氢钾、0.10~0.20%的磷酸氢二钠、0.20~0.30%的蔗糖、0.001~0.003%的氯化钙、0.001~0.003%的7水硫酸锌、0.001~0.003%的七水硫酸亚铁以及余量的水。发明人发现,所述微生物在上述培养基中进行预培养,可有效提高菌体的活力和密度,使得菌体在短时间内达到发酵培养所需的接种密度。
根据本发明的实施例,在发酵处理中,培养基包括:2.5~3.5%的酵母浸粉、0.20~0.30%的蛋白胨、0.30~0.80%的氯化钠、0.005~0.015%的七水硫酸镁、0.20~0.30%的磷酸二氢钾、0.50~0.60%的磷酸氢二钠、0.005~0.015%的氯化钙、0.30~0.40%的蔗糖、0.20~0.30%的甘油、0.10~0.15%的乳糖以及余量的水。发明人发现,预培养后的菌体在上述培养基中进行发酵培养,可使得菌体得到快速扩增,且保持高活力。
根据本发明的实施例,所述预培养处理后、发酵处理前,进一步包括将预培养处理产物接种在发酵培养基中。根据本发明的实施例,接种比为0.5~1.5%。需要说明的是,所述接种比为体积比。
在本发明的第六方面,本发明提出了一种甘油葡糖苷的制备方法。根据本发明的实施例,所述方法包括:将蔗糖以及甘油在上述任一项所述的全细胞催化剂的催化条件下进行脱水缩合反应,以便获得所述甘油葡糖苷。发明人发现,所述制备方法直接采用含TSPase酶的菌体细胞作为生物催化剂,避免了购买昂贵的商业用酶制剂或者进行复杂的菌体破壁、浓缩处理,生产成本低,制备工艺简便,且甘油葡糖苷的产量高。
根据本发明的实施例,上述方法还可进一步包括如下附加技术特征至少之一:
根据本发明的实施例,所述脱水缩合反应是在温度为50~60℃,如在51、52、53、54、55、56、57、58或59℃的恒温条件下进行缩合反应18~28小时,如20、22、24或26小时。需要说明的是,所述恒温条件是指在50~60℃之间任一温度下进行恒温反应。发明人发现,若所述脱水缩合反应的温度过高,耐热磷酸化酶已失活或活性降低,无法有效催化合成甘油葡糖苷或甘油葡糖苷的产量显著降低;若所述脱水缩合反应的温度过低,耐热磷酸化酶活性降低,同时细胞内其他具有水解副反应的杂蛋白并未失活,导致甘油葡糖苷的产量和纯度均显著降低。由此,所述脱水缩合反应在上述条件下进行时,耐热磷酸化酶的活性较高,同时细胞内其他具有水解副反应的杂蛋白均已失活或活性较低,进而甘油葡糖苷的产量和纯度更高。
根据本发明的实施例,所述脱水缩合反应是在柠檬酸三钠缓冲液存在的条件下进行的。在一些实施例中,所述柠檬酸三钠缓冲液的pH为4.0~6.5,如4.2、4.4、4.6、4.8、5.0、5.2、5.4、5.6、5.8、6.0、6.2或6.4。发明人发现,若所述pH过大或过小,甘油葡糖苷的产量均显著降低。由此,所述脱水缩合反应在上述条件下进行时,甘油葡糖苷的产量更高。
根据本发明的实施例,所述全细胞催化剂的浓度为50~150g/L,如70、100、130g/L;所述蔗糖的浓度为60~300g/L,如100、150、200、250g/L;所述甘油的浓度为20~150g/L,如50、80、100、130g/L;所述柠檬酸缓冲液的浓度为20~60mmol/L,如30、40、50mmol/L。发明人发现,所述全细胞催化剂、蔗糖、甘油、柠檬酸缓冲液的浓度在上述范围时,甘油葡糖苷的产量更高。
在本发明的第七方面,本发明提出了上述耐热蔗糖磷酸化酶、上述分离的核酸、上述任一项所述的构建体、上述任一项所述的微生物或上述任一项所述的全细胞催化剂在制备甘油葡糖苷中的用途。
具体实施方式
下面详细描述本发明的实施例,所述实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
本发明基于目前所报道的采用蔗糖磷酸化酶制备甘油葡糖苷的不成熟现况,构建耐热蔗糖磷酸化酶(TSPase)高表达工程菌株,采用该重组工程菌株的完整细胞生物催化制备甘油葡糖苷。具体为:采用基因重组技术,将来自Thermoanaerobacteriumthermosaccharolyticum的耐热蔗糖磷酸化酶编码基因通过pet28a(+)质粒导入大肠杆菌BL21(DE3)中,构建重组蔗糖磷酸化酶的工程菌株E.coli BL21/pET-TSpase。并以发酵所得菌体全细胞为生物催化剂,在柠檬酸三钠缓冲液体系中,以蔗糖和甘油为原料制备甘油葡糖苷。
利用本发明构建的耐热蔗糖磷酸化酶进行催化反应具有以下优势:
一是耐热蔗糖磷酸化酶的酶活高,不需购买昂贵的商业用酶制剂或者进行菌体破壁、浓缩处理,可以直接使用发酵液或菌体作为生物催化剂,成本更低,制备工艺更加简便。
二是耐热蔗糖磷酸化酶在50-60℃的条件下反应,在该温度条件下,细胞内其他具有水解副反应的杂蛋白均已失活,反应产物单一,产品纯度高。
三是采用耐热蔗糖磷酸化酶进行催化反应,在温度较高的条件下,反应向正向移动,转化率和产量较高,且利用蔗糖为糖基供体,价廉易得,总生产成本低。
本发明的目的是提供一种耐热重组蔗糖磷酸化酶的大肠杆菌工程菌株制备甘油葡糖苷的方法,反应步骤如下:
1、将源自Thermoanaerobacterium thermosaccharolyticum的耐热蔗糖磷酸化酶编码基因进行克隆表达,构建重组耐热蔗糖磷酸化酶的大肠杆菌工程菌株,具体如下:
1)蔗糖磷酸化酶克隆菌株的构建,包括以下步骤:
从Thermoanaerobacterium thermosaccharolyticum提取基因组作为模板设计一对引物,然后进行PCR、琼脂凝胶分离纯化回收获得目标基因DNA片段。然后将上述PCR产物和质粒pET-28A使用内切酶BamHI和XhoI进行双酶切并使用DNA纯化试剂盒纯化,再用T4连接酶将PCR产物和质粒pET-28A连接。连接产物转入到大肠杆菌感受态细胞BL21(DE3)中,得到工程菌E.coli BL21/pET-TSpase。
本发明构建的耐热蔗糖磷酸化酶的核苷酸序列如:SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:2所示。
ATGGCTCTGAAAAATAAAGTGCAACTGATTACCTACCCGGATAGCCTGGGCGGTGACCTGAAAACCCTGAACGATGTGCTAGAAAAATACTTCAGCGACGTTTTTGGCGGTGTCCATATTCTGCCGCCGTTCCCGAGCTCTGGTGATCGTGGTTTTGCGCCGATTACCTACTCTGAAATCGAACCGAAATTCGGCACGTGGTACGATATTAAGAAAATGGCCGAAAACTTCGACATACTGCTAGATCTAATGGTCAATCACGTGAGTCGTCGCTCCATTTACTTTCAGGATTTTCTGAAGAAAGGCCGCAAAAGTGAATATGCGGATATGTTTATTACCCTGGACAAACTGTGGAAAGATGGCAAACCGGTGAAAGGTGATATCGAAAAAATGTTCCTGCGTCGCACCCTGCCGTACTCCACGTTTAAAATTGAAGAAACCGGCGAAGAAGAAAAAGTTTGGACCACGTTCGGTAAAACGGATCCGTCAGAACAGATCGACCTGGATGTGAACTCGCATCTGGCAAAAGAATTTCTGCTGGGCGTTTTCAAAACCTTCTCAAACTTCGGTGTTAATATAGTCAGACTAGATGCTGTGGGCTATGTTATTAAGAAAATTGGCACGTCGTGCTTTTTCGTGGAACCGGAAATTTACGAATTTCTGGATTGGATCAAAGGCCAGGCGGCCAGTTATGGTATAGAACTACTGCTAGAAGTCCACTCCCAGTTCGAAGTGCAATATAAACTGGCCGAACGTGGCTTTCTGATTTACGACTTCATCCTGCCGTTTACCGTTCTGTATACGCTGATCAACAAAAGTAACGAAATGCTGTACGATTACCTGAAAAACCGCCCGATTAATCAGTTTACCATGCTGGACTGCCATGATGGCATTCCGGTCAAACCGGACCTGGATGGTCTGATCGACACCAAGAAAGCGAAAGAAGTGGTTGATATTTGTGTTCAGCGTGGCGCCAACCTGAGCCTGATCTATGGTGATAAATACAAATCTGAAGACGGCTTCGATGTGCACCAAATCGGTTGCACCTATTACAGCGCGCTGAATTGTGATGACGATGCGTATCTGGCAGCTCGCGCCATTCAGTTTTTCACGCCGGGCATCCCGCAAGTCTATTACGTGGGCCTGCTGGCAGGTGTTAATGATTTTGAAGCTGTCAAACGTACCAAAGAAGGCCGTGAAATTAACCGCCATAATTACGGTCTGAAAGAAATCGAAGAATCTGTGCAGAAGAAAGCGGTTCAACGCCTGCTGAAACTGATTCGTTTCCGCAACGAATATGAAGCTTTCAATGGTGAATTTATGGTGCAGGACTGCCAAAAAGATGAAATTCGTCTGACCTGGAAGAAAGATGATAAACGCTGTAGCCTGTTTATCGATCTGAAAACCTACAAAACGACGATTGACTACATTAACGAAAACGGCGAAGAAG TGAAATACCT GGTG(SEQ ID NO:1)。
MALKNKVQLITYPDSLGGDLKTLNDVLEKYFSDVFGGVHILPPFPSSGDRGFAPITYSEIEPKFGTWYDIKKMAENFDILLDLMVNHVSRRSIYFQDFLKKGRKSEYADMFITLDKLWKDGKPVKGDIEKMFLRRTLPYSTFKIEETGEEEKVWTTFGKTDPSEQIDLDVNSHLAKEFLLGVFKTFSNFGVNIVRLDAVGYVIKKIGTSCFFVEPEIYEFLDWIKGQAASYGIELLLEVHSQFEVQYKLAERGFLIYDFILPFTVLYTLINKSNEMLYDYLKNRPINQFTMLDCHDGIPVKPDLDGLIDTKKAKEVVDICVQRGANLSLIYGDKYKSEDGFDVHQIGCTYYSALNCDDDAYLAARAIQFFTPGIPQVYYVGLLAGVNDFEAVKRTKEGREINRHNYGLKEIEESVQKKAVQRLLKLIRFRNEYEAFNGEFMVQDCQKDEIRLTWKKDDKRCSLFIDLKTYKTTIDYINENGEEVKYLV(SEQ ID NO:2)。
2)重组菌株的诱导表达
将耐热蔗糖磷酸化酶的重组大肠杆菌E.coli BL21/pET-TSpase接种于含卡那霉素的LB培养基中进行种子培养,然后转接入诱导培养基进行蔗糖磷酸化酶发酵生产,获得含重组耐热蔗糖磷酸化酶的全细胞催化剂。
3)甘油葡糖苷的制备
将含重组蔗糖磷酸化酶的全细胞催化剂与缓冲液进行混合,然后加入蔗糖和甘油原料,再调节反应体系pH值,最后在一定转化温度下进行生物催化合成甘油葡糖苷。
下面将结合具体实施例对本发明进行进一步的解释说明。
实施例1
1、耐热蔗糖磷酸化酶大肠杆菌重组菌构建
从Thermoanaerobacterium thermosaccharolyticum(ATCC 7956)提取基因组作为模板设计一对引物:
TSpase(+):CGCGGATCCATGGCTCTGAAAAATAAAGTGCAACTG(SEQ ID NO:3),下划线为酶切位点BamHI
TSpase(-):CCGCTCGAGCACCAGGTATTTCACTTCTTCGCCGT(SEQ ID NO:4),下划线为酶切位点XhoI。
然后进行PCR反应,反应条件:94℃预变性2分钟;94℃变性30秒,55℃退火30秒,72℃延伸1.5分钟,30个循环;72℃保温10分钟。
PCR反应完毕后,将经凝胶回收、验证和纯化PCR反应后的产物以及质粒pET-28A使用内切酶BamHI和XhoI进行双酶切并使用DNA纯化试剂盒纯化,再用T4连接酶将PCR产物和质粒pET-28A连接。连接产物转入到大肠杆菌感受态细胞BL21(DE3)中,得到工程菌E.coliBL21/pET-TSpase。
2、重组菌株的诱导表达
将耐热蔗糖磷酸化酶的重组大肠杆菌E.coli BL21/pET-TSpase接种于装有50mL种子培养基的500mL三角瓶中。然后在温度32℃、转速220rpm条件下振荡培养20小时至菌液OD600nm达到20~25后,按接种比为1%的比例接入装有100mL发酵培养基的500mL三角瓶中进行发酵生产蔗糖磷酸化酶。然后在发酵温度为32℃、转速220rpm条件下振荡培养48小时,之后通过离心机4000转/10min离心收集含蔗糖磷酸化酶的菌体浆状物,该浆状物即可作为全细胞生物催化剂。
种子培养基组成为:酵母浸粉2.0%、蛋白胨0.5%、氯化钠0.1%、七水硫酸镁0.005%、磷酸二氢钾0.05%、磷酸氢二钠0.15%、蔗糖0.25%、氯化钙0.002%、7水硫酸锌0.002%、七水硫酸亚铁0.002%,余量为水。其中,各比例为重量百分比。
发酵培养基组成为:酵母浸粉3.0%、蛋白胨0.25%、氯化钠0.5%、七水硫酸镁0.01%、磷酸二氢钾0.25%、磷酸氢二钠0.54%、氯化钙0.01%、蔗糖0.35%、甘油0.25%、乳糖0.15%,余量为水。其中,各比例为重量百分比。
3、甘油葡糖苷的制备
称取上述步骤2中含重组耐热蔗糖磷酸化酶的菌体浆状物100g/L,然后加入蔗糖200g/L,甘油106g/L,pH 6.5的柠檬酸缓冲液50mM,在50℃恒温环境中转化(脱水缩合反应)24小时,取样测定转化液中甘油葡糖苷的产量。测定方法采用离子色谱法(IC),其检测条件如下:ICS-3000(DIONEX)离子色谱仪,内径为4×250mm的A1色谱柱,流动相为800mM/L NaOH,流速为0.4mL/min。
结果:甘油葡糖苷产量高达138.1g/L,基于底物蔗糖的转化率为93%。
实施例2
将按照实施例1步骤1的方法制备的耐热蔗糖磷酸化酶的重组大肠杆菌E.coliBL21/pET-TSpase接种于装有50mL种子培养基的500mL三角瓶中。然后在温度28℃、转速200rpm条件下振荡培养25小时至菌液OD600nm达到20后,按接种比为1.5%的比例接入装有100mL发酵培养基的500mL三角瓶中进行发酵生产蔗糖磷酸化酶。然后在发酵温度为28℃、转速200rpm条件下振荡培养55小时,之后通过离心机4000转/10min离心收集含蔗糖磷酸化酶的菌体浆状物,该浆状物即可作为全细胞生物催化剂。
种子培养基组成为:酵母浸粉1.5%、蛋白胨0.3%、氯化钠0.05%、七水硫酸镁0.003%、磷酸二氢钾0.03%、磷酸氢二钠0.10%、蔗糖0.20%、氯化钙0.001%、7水硫酸锌0.001%、七水硫酸亚铁0.001%,余量为水。其中,各比例为重量百分比。
发酵培养基组成为:酵母浸粉2.5%、蛋白胨0.2%、氯化钠0.3%、七水硫酸镁0.005%、磷酸二氢钾0.2%、磷酸氢二钠0.5%、氯化钙0.005%、蔗糖0.3%、甘油0.2%、乳糖0.1%,余量为水。其中,各比例为重量百分比。
甘油葡糖苷的制备步骤同实施例1。
结果:甘油葡糖苷产量高达134.0g/L,基于底物蔗糖的转化率为90.2%。
实施例3
将按照实施例1步骤1的方法制备的耐热蔗糖磷酸化酶的重组大肠杆菌E.coliBL21/pET-TSpase接种于装有50mL种子培养基的500mL三角瓶中。然后在温度35℃、转速250rpm条件下振荡培养15小时至菌液OD600nm达到25后,按接种比为0.5%的比例接入装有100mL发酵培养基的500mL三角瓶中进行发酵生产蔗糖磷酸化酶。然后在发酵温度为35℃、转速200rpm条件下振荡培养45小时,之后通过离心机4000转/10min离心收集含蔗糖磷酸化酶的菌体浆状物,该浆状物即可作为全细胞生物催化剂。
种子培养基组成为:酵母浸粉2.5%、蛋白胨0.8%、氯化钠0.15%、七水硫酸镁0.008%、磷酸二氢钾0.08%、磷酸氢二钠0.20%、蔗糖0.30%、氯化钙0.003%、7水硫酸锌0.003%、七水硫酸亚铁0.003%,余量为水。其中,各比例为重量百分比。
发酵培养基组成为:酵母浸粉3.5%、蛋白胨0.3%、氯化钠0.8%、七水硫酸镁0.008%、磷酸二氢钾0.3%、磷酸氢二钠0.6%、氯化钙0.015%、蔗糖0.4%、甘油0.3%、乳糖0.15%,余量为水。其中,各比例为重量百分比
甘油葡糖苷的制备步骤同实施例1。
结果:甘油葡糖苷产量高达136.0g/L,基于底物蔗糖的转化率为91.5%。
实施例4
称实施例1步骤2中含重组耐热蔗糖磷酸化酶的菌体浆状物50g/L,然后加入蔗糖60g/L,甘油20g/L,pH 4.0的柠檬酸缓冲液20mM,转化18小时,其余步骤与实施例1中步骤3所述的甘油葡糖苷的制备条件相同。转化终止取样测定转化液中甘油葡糖苷的产量。
结果:甘油葡糖苷产量高达41.9g/L,基于底物蔗糖的转化率为94.1%。
实施例5
称实施例1步骤2中含重组耐热蔗糖磷酸化酶的菌体浆状物150g/L,然后加入蔗糖300g/L,甘油150g/L,pH 5.0的柠檬酸缓冲液60mM,在55℃恒温环境中转化28小时,其余步骤与实施例1中步骤3所述的甘油葡糖苷的制备条件相同。转化终止取样测定转化液中甘油葡糖苷的产量。
结果:甘油葡糖苷产量高达189.8g/L,基于底物蔗糖的转化率为85.2%。
实施例6
称实施例1步骤2中含重组耐热蔗糖磷酸化酶的菌体浆状物125g/L,然后加入蔗糖250g/L,甘油125g/L,pH 6.5的柠檬酸缓冲液50mM,在60℃恒温环境中转化28小时,其余步骤与实施例1中步骤3所述的甘油葡糖苷的制备条件相同。转化终止取样测定转化液中甘油葡糖苷的产量。
结果:甘油葡糖苷产量高达167.3g/L,基于底物蔗糖的转化率为90.1%。
实施例7
称实施例1步骤2中含重组耐热蔗糖磷酸化酶的菌体浆状物75g/L,然后加入蔗糖150g/L,甘油100g/L,pH 6.0的柠檬酸缓冲液40mM,在50℃恒温环境中转化24小时,其余步骤与实施例1中步骤3所述的甘油葡糖苷的制备条件相同。转化终止取样测定转化液中甘油葡糖苷的产量。
结果:甘油葡糖苷产量高达101.4g/L,基于底物蔗糖的转化率为91%。
对比例1
与实施例1的区别仅在于步骤3中转化温度(即缩合反应的温度)为70℃,其余步骤与实施例1相同。
结果:甘油葡糖苷产量仅为38.0g/L,基于底物蔗糖的转化率仅为25.6%。
结论:转化温度对于实现本发明的技术效果具有重要影响。
对比例2
与实施例1的区别仅在于步骤3中转化温度(即缩合反应的温度)为40℃,其余步骤与实施例1相同。
结果:甘油葡糖苷产量仅为66.8g/L,基于底物蔗糖的转化率仅为45%。
结论:转化温度对于实现本发明的技术效果具有重要影响。
对比例3
与实施例1的区别仅在于步骤3中柠檬酸三钠缓冲液的pH值为8.0,其余步骤与实施例1相同。
结果:甘油葡糖苷产量仅为56.8g/L,基于底物蔗糖的转化率仅为38.2%。
结论:pH值对于实现本发明的技术效果具有重要影响。
对比例4
与实施例1的区别仅在于步骤3中柠檬酸三钠缓冲液的pH值为3.5,其余步骤与实施例1相同。
结果:甘油葡糖苷产量仅为22.5g/L,基于底物蔗糖的转化率仅为15.1%。
结论:pH值对于实现本发明的技术效果具有重要影响。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
SEQUENCE LISTING
<110> 清华大学
<120> 耐热磷酸化酶及其应用
<130> PIDC3185617
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1464
<212> DNA
<213> Artificial
<220>
<223> 耐热蔗糖磷酸化酶的核苷酸序列
<400> 1
atggctctga aaaataaagt gcaactgatt acctacccgg atagcctggg cggtgacctg 60
aaaaccctga acgatgtgct agaaaaatac ttcagcgacg tttttggcgg tgtccatatt 120
ctgccgccgt tcccgagctc tggtgatcgt ggttttgcgc cgattaccta ctctgaaatc 180
gaaccgaaat tcggcacgtg gtacgatatt aagaaaatgg ccgaaaactt cgacatactg 240
ctagatctaa tggtcaatca cgtgagtcgt cgctccattt actttcagga ttttctgaag 300
aaaggccgca aaagtgaata tgcggatatg tttattaccc tggacaaact gtggaaagat 360
ggcaaaccgg tgaaaggtga tatcgaaaaa atgttcctgc gtcgcaccct gccgtactcc 420
acgtttaaaa ttgaagaaac cggcgaagaa gaaaaagttt ggaccacgtt cggtaaaacg 480
gatccgtcag aacagatcga cctggatgtg aactcgcatc tggcaaaaga atttctgctg 540
ggcgttttca aaaccttctc aaacttcggt gttaatatag tcagactaga tgctgtgggc 600
tatgttatta agaaaattgg cacgtcgtgc tttttcgtgg aaccggaaat ttacgaattt 660
ctggattgga tcaaaggcca ggcggccagt tatggtatag aactactgct agaagtccac 720
tcccagttcg aagtgcaata taaactggcc gaacgtggct ttctgattta cgacttcatc 780
ctgccgttta ccgttctgta tacgctgatc aacaaaagta acgaaatgct gtacgattac 840
ctgaaaaacc gcccgattaa tcagtttacc atgctggact gccatgatgg cattccggtc 900
aaaccggacc tggatggtct gatcgacacc aagaaagcga aagaagtggt tgatatttgt 960
gttcagcgtg gcgccaacct gagcctgatc tatggtgata aatacaaatc tgaagacggc 1020
ttcgatgtgc accaaatcgg ttgcacctat tacagcgcgc tgaattgtga tgacgatgcg 1080
tatctggcag ctcgcgccat tcagtttttc acgccgggca tcccgcaagt ctattacgtg 1140
ggcctgctgg caggtgttaa tgattttgaa gctgtcaaac gtaccaaaga aggccgtgaa 1200
attaaccgcc ataattacgg tctgaaagaa atcgaagaat ctgtgcagaa gaaagcggtt 1260
caacgcctgc tgaaactgat tcgtttccgc aacgaatatg aagctttcaa tggtgaattt 1320
atggtgcagg actgccaaaa agatgaaatt cgtctgacct ggaagaaaga tgataaacgc 1380
tgtagcctgt ttatcgatct gaaaacctac aaaacgacga ttgactacat taacgaaaac 1440
ggcgaagaag tgaaatacct ggtg 1464
<210> 2
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<223> 耐热蔗糖磷酸化酶的核苷酸序列
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Asp Arg Gly Phe Ala Pro Ile Thr Tyr Ser Glu Ile Glu Pro Lys Phe
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Gly Thr Trp Tyr Asp Ile Lys Lys Met Ala Glu Asn Phe Asp Ile Leu
65 70 75 80
Leu Asp Leu Met Val Asn His Val Ser Arg Arg Ser Ile Tyr Phe Gln
85 90 95
Asp Phe Leu Lys Lys Gly Arg Lys Ser Glu Tyr Ala Asp Met Phe Ile
100 105 110
Thr Leu Asp Lys Leu Trp Lys Asp Gly Lys Pro Val Lys Gly Asp Ile
115 120 125
Glu Lys Met Phe Leu Arg Arg Thr Leu Pro Tyr Ser Thr Phe Lys Ile
130 135 140
Glu Glu Thr Gly Glu Glu Glu Lys Val Trp Thr Thr Phe Gly Lys Thr
145 150 155 160
Asp Pro Ser Glu Gln Ile Asp Leu Asp Val Asn Ser His Leu Ala Lys
165 170 175
Glu Phe Leu Leu Gly Val Phe Lys Thr Phe Ser Asn Phe Gly Val Asn
180 185 190
Ile Val Arg Leu Asp Ala Val Gly Tyr Val Ile Lys Lys Ile Gly Thr
195 200 205
Ser Cys Phe Phe Val Glu Pro Glu Ile Tyr Glu Phe Leu Asp Trp Ile
210 215 220
Lys Gly Gln Ala Ala Ser Tyr Gly Ile Glu Leu Leu Leu Glu Val His
225 230 235 240
Ser Gln Phe Glu Val Gln Tyr Lys Leu Ala Glu Arg Gly Phe Leu Ile
245 250 255
Tyr Asp Phe Ile Leu Pro Phe Thr Val Leu Tyr Thr Leu Ile Asn Lys
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Ser Asn Glu Met Leu Tyr Asp Tyr Leu Lys Asn Arg Pro Ile Asn Gln
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Phe Thr Met Leu Asp Cys His Asp Gly Ile Pro Val Lys Pro Asp Leu
290 295 300
Asp Gly Leu Ile Asp Thr Lys Lys Ala Lys Glu Val Val Asp Ile Cys
305 310 315 320
Val Gln Arg Gly Ala Asn Leu Ser Leu Ile Tyr Gly Asp Lys Tyr Lys
325 330 335
Ser Glu Asp Gly Phe Asp Val His Gln Ile Gly Cys Thr Tyr Tyr Ser
340 345 350
Ala Leu Asn Cys Asp Asp Asp Ala Tyr Leu Ala Ala Arg Ala Ile Gln
355 360 365
Phe Phe Thr Pro Gly Ile Pro Gln Val Tyr Tyr Val Gly Leu Leu Ala
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Gly Val Asn Asp Phe Glu Ala Val Lys Arg Thr Lys Glu Gly Arg Glu
385 390 395 400
Ile Asn Arg His Asn Tyr Gly Leu Lys Glu Ile Glu Glu Ser Val Gln
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Lys Lys Ala Val Gln Arg Leu Leu Lys Leu Ile Arg Phe Arg Asn Glu
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Tyr Glu Ala Phe Asn Gly Glu Phe Met Val Gln Asp Cys Gln Lys Asp
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Glu Ile Arg Leu Thr Trp Lys Lys Asp Asp Lys Arg Cys Ser Leu Phe
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cgcggatcca tggctctgaa aaataaagtg caactg 36
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<213> Artificial
<220>
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Claims (10)
1.一种耐热磷酸化酶,其特征在于,具有SEQ ID NO:2所示的氨基酸序列。
2.一种分离的核酸,其特征在于,具有SEQ ID NO:1所示的核苷酸序列。
3.一种构建体,其特征在于,携带权利要求2所述的分离的核酸。
4.根据权利要求3所述的构建体,其特征在于,所述构建体的载体为pET-28A。
5.一种微生物,其特征在于,所述微生物携带权利要求2所述的分离的核酸或权利要求3或4所述构建体。
6.根据权利要求5所述的微生物,其特征在于,所述微生物为大肠杆菌;任选地,所述微生物为大肠杆菌BL21。
7.一种全细胞催化剂,其特征在于,包括权利要求5或6所述的微生物;
任选地,所述全细胞催化剂是通过如下方式制备得到的:
在适合微生物增殖的条件下,将权利要求5~6任一项所述的微生物进行预培养和发酵处理,
将发酵处理产物进行离心处理,以便获得离心沉淀物,所述离心沉淀物构成所述全细胞催化剂;
任选地,所述预培养和发酵处理是在温度28~35℃、转速为200~250rpm的条件下进行的;
任选地,所述预培养处理的时间为15~25小时;
任选地,所述预培养处理后,菌液的OD600nm为20~25;
任选地,所述发酵处理的时间为45~55小时;
任选地,在预培养处理中,培养基包括:1.5~2.5%的酵母浸粉、0.3~0.8%的蛋白胨、0.05~0.15%的氯化钠、0.003~0.008%的七水硫酸镁、0.03~0.08%的磷酸二氢钾、0.10~0.20%的磷酸氢二钠、0.20~0.30%的蔗糖、0.001~0.003%的氯化钙、0.001~0.003%的7水硫酸锌、0.001~0.003%的七水硫酸亚铁以及余量的水;
任选地,在发酵处理中,培养基包括:2.5~3.5%的酵母浸粉、0.20~0.30%的蛋白胨、0.30~0.80%的氯化钠、0.005~0.015%的七水硫酸镁、0.20~0.30%的磷酸二氢钾、0.50~0.60%的磷酸氢二钠、0.005~0.015%的氯化钙、0.30~0.40%的蔗糖、0.20~0.30%的甘油、0.10~0.15%的乳糖以及余量的水;
任选地,所述预培养处理后、发酵处理前,进一步包括将预培养处理产物接种在发酵培养基中;
任选地,接种比为0.5~1.5%。
8.一种甘油葡糖苷的制备方法,其特征在于,包括:
将蔗糖以及甘油在权利要求7所述的全细胞催化剂的催化条件下进行脱水缩合反应,以便获得所述甘油葡糖苷。
9.根据权利要求8所述的方法,其特征在于,所述脱水缩合反应是在温度为50~60℃的条件下反应18~28小时;
任选地,所述脱水缩合反应是在柠檬酸三钠缓冲液存在的条件下进行的,优选地,所述柠檬酸三钠缓冲液的pH为4.0~6.5;
任选地,所述全细胞催化剂的浓度为50~150g/L,所述蔗糖的浓度为60~300g/L,所述甘油的浓度为20~150g/L,所述柠檬酸缓冲液的浓度为20~60mmol/L。
10.权利要求1所述的耐热蔗糖磷酸化酶、权利要求2所述的分离的核酸、权利要求3~4任一项所述的构建体、权利要求5~6任一项所述的微生物或权利要求7所述的全细胞催化剂在制备甘油葡糖苷中的用途。
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CN115851648A (zh) * | 2021-09-24 | 2023-03-28 | 中国科学院天津工业生物技术研究所 | 一种热稳定性及催化活性提高的甘油葡萄糖苷磷酸化酶突变体及其应用 |
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