CN116622676A - 黑曲霉脂肪酶、编码基因及应用 - Google Patents
黑曲霉脂肪酶、编码基因及应用 Download PDFInfo
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- CN116622676A CN116622676A CN202310607080.6A CN202310607080A CN116622676A CN 116622676 A CN116622676 A CN 116622676A CN 202310607080 A CN202310607080 A CN 202310607080A CN 116622676 A CN116622676 A CN 116622676A
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- aspergillus niger
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Abstract
本发明属于生物技术领域,具体涉及黑曲霉脂肪酶、编码基因及应用。针对在水相体系中酶促酯化反应研究较少的问题,本发明提供一种黑曲霉脂肪酶、编码基因及应用。其黑曲霉脂肪酶的氨基酸序列如SEQ ID No.1或SEQ ID No.2所示。本发明的脂肪酶在含有乙醇、丁酸、戊酸、己酸、庚酸和辛酸的水相体系中能合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯。本发明的脂肪酶用于白酒酿造的水相体系中催化合成重要风味酯己酸乙酯和辛酸乙酯,具有较好的工业化应用前景。
Description
技术领域
本发明属于生物技术领域,具体涉及黑曲霉脂肪酶、编码基因及应用。
背景技术
众所周知,传统白酒中含有数千种风味化合物,其决定了白酒的品质,这些风味物质主要包括酯类、醇类、酸类和其它挥发物。其中,短链脂肪酸酯是白酒重要的香气物质,乙酸乙酯是清香型白酒主体香,而己酸乙酯已被鉴定为浓香型白酒中重要特征香气物质。己酸乙酯赋予浓香型白酒一种地窖的香味,类似于菠萝、泥巴、酸菜和植物灰的混合气味,含量的高低是决定浓香型白酒品质的重要参数。然而,在传统白酒酿造过程中产酯生香较慢,发酵周期长,粮耗高、出酒率低。因此,探究高效的己酸乙酯等短链脂肪酸酯的合成对于缩短传统白酒发酵周期以及提高白酒品质具有重要作用和应用价值。
白酒中的酯来源于原料、自发的化学酯化和微生物合成酯这三个主要途径,其中,微生物合成和基于酯化酶的酶促反应是白酒中短链脂肪酸酯的主要来源,尤其对于浓香型白酒而言,酒醅中的乙醇和窖泥中的己酸通过酶促反应是己酸乙酯最为主要的来源。酯化反应主要由羧酸水解酶家族的酶催化,其中报道最多的酶是脂肪酶、酯酶和角质酶。据文献报道,有机相体系适合大部分的酶促酯化反应,有少部分的酶促酯化反应发生在水相。然而,在白酒生产中,原料中有53-58%的水分,可视为水相体系。白酒发酵过程中的某些微生物所具有的酶可以在水相体系中进行酯合成反应,但是关于白酒酯化酶的挖掘及其在水相条件下合成酯的催化机理研究报道很少。因此,脂肪酶在水相体系中催化合成酯的发现及其催化性能的分析,不仅有助于丰富对白酒酯化酶催化机制的了解,也为白酒风味绿色生物制造和工艺优化提供了新的素材和思路。
发明内容
针对在水相体系中酶促酯化反应研究较少的问题,本发明提供一种在水相体系中合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯的脂肪酶、编码基因及应用,其来源于黑曲霉(Aspergillus niger)。
本发明提供黑曲霉脂肪酶,分别定义为黑曲霉脂肪酶ANI_1_236084、黑曲霉脂肪酶ANI_1_266144,其对照组黑曲霉脂肪酶定义为ANI_1_58064。
本发明首先提供黑曲霉脂肪酶,其氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
黑曲霉脂肪酶ANI_1_236084氨基酸序列SEQ ID NO.1:
MFLRREFGAVAALSVLAHAAPAPAPMQRRDISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDSSSYGDPTGFIAVDPTNELIVLSFRGSSDLSNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPELLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTAGNDGTLLDSTTAHRWYTIYISECS。
黑曲霉脂肪酶ANI_1_266144氨基酸序列SEQ ID NO.2:
MFSGRFGVLLTALAALSAAAPTPLDVRSVSTSTLDELQLFSQWSAAAYCSNNIDSDDSNVTCTADACPSVEEASTKMLLEFDLTNNFGGTAGFLAADNTNKRLVVAFRGSSTIKNWIADLDFILQDNDDLCTGCKVHTGFWKAWEAAADNLTSKIKSAMSTYSGYTLYFTGHSLGGALATLGATVLRNDGYSVELYTYGCPRVGNYALAEHITSQGSGANFRVTHLNDIVPRLPPMDFGFSQPSPEYWITSGTGASVTASDIELIEGINSTAGNAGEATVDVLAHLWYFFAISECLL。
本发明还提供了编码所述黑曲霉脂肪酶的编码基因,分别如SEQ ID NO.3或SEQID NO.4所示。
黑曲霉脂肪酶ANI_1_236084的编码基因SEQ ID NO.3:
ATGTTTCTCCGCAGGGAATTTGGGGCTGTTGCAGCCCTATCTGTGCTGGCCCATGCTGCTCCCGCACCTGCTCCGATGCAGCGTAGAGACATCTCCTCTACCGTCTTGGACAATATCGACCTCTTCGCCCAATACAGTGCAGCAGCTTACTGCTCCTCCAACATCGAGTCCACCGGCACGACTCTGACCTGCGACGTAGGCAATTGCCCTCTCGTCGAGGCAGCCGGTGCCACGACCATCGATGAGTTTGACGACAGCAGCAGCTACGGCGACCCGACGGGGTTCATCGCCGTTGACCCGACGAACGAGTTAATCGTTCTGTCTTTCCGGGGCAGTTCCGACCTCTCGAACTGGATTGCCGACCTAGACTTCGGCCTCACATCCGTAAGCAGCATCTGTGATGGCTGTGAGATGCACAAGGGCTTCTACGAGGCCTGGGAAGTCATTGCCGACACCATCACATCCAAGGTGGAGGCCGCCGTCTCCAGCTATCCGGACTACACCCTCGTGTTCACCGGACACAGCTACGGCGCTGCATTGGCGGCTGTCGCGGCCACCGTGCTCCGCAACGCCGGATACACTCTTGACCTGTACAACTTCGGCCAGCCCCGTATTGGCAACCTCGCCTTAGCCGACTACATCACCGACCAAAACATGGGCAGCAACTACCGCGTCACGCACACCGATGACATCGTGCCTAAGCTGCCTCCGGAGCTGCTGGGCTACCACCACTTCAGTCCGGAGTACTGGATCACCAGCGGCAATGATGTGACGGTGACAACGTCGGACGTCACCGAGGTCGTGGGGGTGGATTCGACGGCTGGGAATGACGGCACGCTGCTTGACAGTACGACTGCCCATCGGTGGTACACGATCTACATTAGTGAATGCTCGTAG。
黑曲霉脂肪酶ANI_1_266144的编码基因SEQ ID NO.4:
ATGTTCTCTGGACGGTTTGGAGTGCTTTTGACGGCGCTCGCTGCGCTGAGTGCTGCGGCACCGACACCACTTGATGTGCGGAGTGTCTCGACTTCCACGTTGGATGAGCTGCAATTGTTCTCGCAATGGTCTGCCGCAGCTTATTGCTCGAACAATATCGACTCGGACGACTCTAACGTGACATGCACGGCCGACGCCTGTCCATCAGTCGAGGAGGCGAGCACCAAGATGCTGCTGGAGTTTGACCTGACAAATAACTTTGGAGGCACAGCCGGTTTCCTGGCCGCGGACAACACCAACAAGCGGCTCGTGGTCGCCTTCCGAGGCAGTAGCACCATCAAGAACTGGATTGCTGATCTCGACTTCATCCTGCAAGATAACGATGACCTCTGTACTGGCTGCAAGGTTCACACTGGATTCTGGAAGGCATGGGAAGCCGCTGCAGACAATCTGACGAGCAAGATCAAGTCCGCGATGAGCACGTATTCGGGCTATACCCTCTACTTCACCGGGCACAGCTTGGGCGGCGCATTGGCTACACTGGGAGCAACGGTCTTGCGAAATGACGGTTATAGCGTTGAACTGTACACCTATGGATGTCCTCGAGTCGGAAACTATGCGCTGGCCGAGCACATCACCAGCCAGGGATCTGGAGCGAACTTCCGCGTTACACACTTGAACGACATCGTCCCCCGGTTGCCACCCATGGACTTTGGATTCAGCCAGCCAAGTCCAGAATACTGGATCACCAGTGGCACCGGAGCCAGTGTCACGGCGTCGGATATTGAACTCATCGAGGGAATCAATTCGACGGCGGGGAATGCAGGCGAAGCAACGGTGGACGTTTTGGCTCACTTGTGGTACTTTTTCGCAATTTCAGAGTGTCTGCTATAG。
本发明还提供了上述黑曲霉脂肪酶在水相体系中催化合成酯类物质的应用。
本发明还提供了上述黑曲霉脂肪酶在制备含酯类物质的酒用酯香液或白酒中的应用。
其中,所述酯类物质包括:丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯或辛酸乙酯中至少一种。
其中,所述应用中,黑曲霉脂肪酶是通过构建含有所述黑曲霉脂肪酶编码基因的大肠杆菌重组质粒,将重组质粒转入大肠杆菌中,经过诱导表达获得。
优选地,所述大肠杆菌为pET32(a)表达载体。
其中,所述应用中,黑曲霉脂肪酶在水相体系中催化合成酯类物质的反应条件为:pH为4.0,温度为30~40℃。
优选地,所述应用中,脂肪酶ANI_1_266144催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯的含量分别为51.26mg/L、80.58mg/L、207.15mg/L、487.3mg/L和794.94mg/L。
脂肪酶ANI_1_236084催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯的含量分别为44.35mg/L、45.96mg/L、118.86mg/L、427.57mg/L和615.2mg/L。
有益效果:本发明利于基因工程技术,获得黑曲霉脂肪酶,定义为黑曲霉脂肪酶ANI_1_236084、黑曲霉脂肪酶ANI_1_266144。本发明的脂肪酶在含有乙醇、丁酸、戊酸、己酸、庚酸和辛酸的水相体系中能合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯。本发明的脂肪酶ANI_1_236084、ANI_1_266144用于白酒酿造的水相体系中催化合成重要风味酯己酸乙酯和辛酸乙酯,具有较好的工业化应用前景。
附图说明
图1是实施例1脂肪酶ANI_1_266144、ANI_1_236084和ANI_1_58064编码基因PCR扩增结果。M:DNA分子量标准,泳道1:黑曲霉脂肪酶ANI_1_266144核酸条带;泳道2:黑曲霉脂肪酶ANI_1_236084核酸条带;泳道3:黑曲霉脂肪酶ANI_1_58064核酸条带;
图2是实施例2和3所表达的脂肪酶ANI_1_266144、ANI_1_236084和ANI_1_58064的粗酶液及纯酶液;a、b、c图分别为ANI_1_266144、ANI_1_236084和ANI_1_58064;
图3是实施例4脂肪酶ANI_1_266144、ANI_1_236084和ANI_1_58064在水相中催化合成风味酯的含量测定;
图4是实施例5脂肪酶ANI_1_266144(a)和ANI_1_236084(b)最适反应pH的确定图;
图5是实施例6脂肪酶ANI_1_266144(a)和ANI_1_236084(b)最适反应温度的确定图。
具体实施方式
本发明的目的在于提供一种具有在水相体系中合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯的脂肪酶ANI_1_236084、ANI_1_266144,均来源于黑曲霉。
本发明的脂肪酶ANI_1_236084、ANI_1_266144的氨基酸序列分别如SEQ ID No.1或SEQ ID No.2所示。
本发明编码脂肪酶ANI_1_236084、ANI_1_266144的基因序列分别如SEQ ID No.3或SEQ ID No.4所示。
本发明还提供了重组载体,其含有上述脂肪酶ANI_1_236084、ANI_1_266144的编码基因;其中,所述重组载体选自昆虫杆状病毒表达载体、哺乳动物细胞表达载体、大肠杆菌表达载体、酵母表达载体中至少一种。在本发明的一种优选实施方式中,所述重组载体是在基础质粒pET32(a)的多克隆位点插入SEQ ID NO.3或SEQ ID NO.4所示的核苷酸序列。
本发明还提供了重组细胞,其含有上述重组载体。其中,所述重组细胞采用昆虫细胞、哺乳动物细胞、大肠杆菌、酵母中至少一种。在本发明的一种优选实施方式中,本发明提供了含有所述重组细胞的重组菌,其是通过将上述重组载体导入宿主细胞所得到,其中所述宿主细胞是E.coli BL21(DE3)。
本发明还提供了脂肪酶ANI_1_266144、ANI_1_236084在水相体系中合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯中的应用。
其中,所述脂肪酶在水相体系中合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯的方法包括以下步骤:
1)以脂肪酶对应的编码基因为模板,用引物对进行PCR扩增,得到目的基因片段;
2)将所述目的基因片段插入质粒表达载体中,得到重组载体;
3)将所述重组载体转化到原核表达系统中,得到重组细胞;
4)将所述重组细胞进行目的蛋白的诱导表达,收集菌体,破碎,收集上清液;
5)将所述上清液进行蛋白纯化,超滤浓缩,得到纯酶液;
6)将纯酶液与酸和醇混合,催化反应,萃取,即得。
在本发明的一种实施方式中,所涉及的脂肪酶在水相体系中合成风味酯的最优反应体系为包含100μL所述的超滤浓缩后纯酶液,终浓度为1M乙醇,终浓度为10mM的丁酸、戊酸、己酸、庚酸或辛酸中的一种,用50mM的柠檬酸缓冲液补至终体积为1mL。
本发明提供另一种黑曲霉对照组,为黑曲霉脂肪酶ANI_1_58064,其氨基酸序列SEQ ID NO.5:
MYIPSVLLLAASLFHGATALPTPGSTPIPPSQDPWYSAPEGFEEADPGAILRVRPAPGNLTVVVGNASAAYNILYRTTDSQYKPSWAVTTLLVPPVAASAAVNQSVLLSHQIAYDSFDVNASPSYAMYTSPPSDIILALQRGWFVNVPDYEGPNASFTAGVQSGHATLDSVRSVLASGFGLNEDAQYALWGYSGGALASEWAAELQMQYAPELNIAGLAVGGLTPNVTSVMDTVTSTISAGLIPAAALGLSSQHPETYEFILSQLKTTGPYNRTGFLAAKDLTLSEAEVFYAFQNIFDYFVNGSATFQAEVVQKALNQDGYMGYHGFPQMPVLAYKAIHDEISPIQDTDRVIKRYCGLGLNILYERNTIGGHSAEQVNGNARAWNWLTSIFDGTYAQQYKTEGCTIRNVTLNTTSSVY。
黑曲霉脂肪酶ANI_1_58064的编码基因SEQ ID NO.6:
ATGTATATCCCCTCGGTGCTGCTTCTGGCCGCGAGCCTGTTCCATGGCGCAACGGCGCTGCCCACGCCCGGCTCCACGCCCATCCCGCCCAGCCAGGATCCCTGGTACAGTGCGCCCGAGGGCTTCGAGGAGGCTGATCCCGGTGCCATCCTGCGCGTGCGGCCCGCGCCCGGCAACTTGACCGTGGTAGTGGGCAATGCGTCGGCGGCCTACAACATCCTCTACCGCACTACAGACAGTCAGTACAAGCCCTCCTGGGCTGTGACCACCCTGCTGGTGCCCCCCGTGGCCGCCTCCGCCGCCGTCAACCAGAGTGTCCTGCTCTCCCACCAGATCGCCTACGATTCGTTCGACGTCAATGCCAGTCCCAGCTACGCCATGTACACCAGCCCGCCCTCCGATATTATCCTCGCCCTGCAGCGCGGCTGGTTCGTTAACGTCCCCGATTACGAGGGCCCCAATGCCTCTTTCACCGCCGGTGTGCAGTCCGGCCATGCCACCCTCGACTCGGTCCGCAGCGTGCTCGCCTCCGGATTCGGCCTGAACGAGGACGCCCAGTACGCTCTGTGGGGTTACTCTGGCGGTGCCTTGGCCAGCGAATGGGCTGCTGAACTGCAGATGCAATACGCTCCCGAGTTGAACATTGCCGGTCTGGCCGTGGGTGGTCTCACTCCCAATGTTACCAGCGTCATGGACACGGTGACCTCGACCATCAGTGCGGGACTCATCCCCGCCGCCGCCCTGGGTCTGTCGAGCCAGCACCCCGAGACCTACGAGTTCATCCTCAGCCAGCTCAAGACGACGGGACCCTACAACCGCACAGGATTCCTAGCCGCCAAGGACCTGACCCTGTCCGAGGCGGAGGTCTTCTACGCCTTCCAGAACATCTTCGATTACTTTGTCAACGGATCGGCCACGTTCCAGGCGGAGGTGGTGCAGAAGGCGCTGAACCAGGACGGATACATGGGCTACCATGGGTTCCCGCAGATGCCGGTGCTCGCGTACAAGGCTATTCACGATGAGATCAGTCCCATCCAGGATACGGATCGCGTGATCAAGCGCTACTGTGGTCTGGGATTGAACATCTTGTATGAGCGGAACACCATCGGTGGCCACTCGGCAGAGCAGGTGAATGGCAACGCCAGGGCGTGGAACTGGTTGACGAGCATTTTCGACGGAACGTATGCGCAGCAGTACAAGACCGAGGGGTGCACGATCCGCAATGTCACTCTGAACACGACTTCCTCCGTTTATTAG。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,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。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
下述实施例所用的引物合成及测序工作均由苏州金唯智生物科技有限公司完成。所用的蛋白表达载体为pET32(a)。所用的蛋白表达细胞为E.coli BL21(DE3)。所用的萃取剂为正己烷。
实施例1脂肪酶ANI_1_266144、ANI_1_236084和ANI_1_58064编码基因的克隆
1、总RNA的提取
总RNA的提取参照Trizol抽提法,具体步骤如下:
(1)将黑曲霉CBS513.88接种到PDA培养基(索莱宝,P8931)中,利用布氏漏斗抽滤获得培养好的黑曲霉菌丝体,菌丝体立即放置于液氮中保存并保藏于-80℃冰箱中。
(2)在2.0mL去核酸酶螺帽管中加入1mL RNA提取液(Trizol),备用。
(3)将-80℃冰箱中保藏的菌丝置于用液氮预冷的研钵中,并迅速研磨至粉末,其间不停的添加液氮使菌丝处于冷冻状态。
(4)待液氮将要挥发完时,取50mg菌丝粉末置于Trizol中,并在涡旋振荡器上以最大转速保持30s,进行两次。
(5)放置摇床中,室温条件下轻摇5min(转速50rpm左右即可),以除去核小体。
(6)加入0.2mL氯仿,置于涡旋振荡器上最大转速保持15s。
(7)室温轻摇2min后,4℃,12000rpm,离心15min,取上清液,转移至去核酸酶EP管中。
(8)加入0.5mL的异丙醇,充分混匀后室温条件下轻摇10min,以使RNA充分沉淀。
(9)4℃,12000rpm,离心10min,弃上清。
(10)1mL 75%酒精(DEPC处理水配制)洗涤EP管,4℃,7500rpm,离心5min,弃上清。
(11)置于室温约10min,以除去乙醇。
(12)添加100μL去核酸酶无菌水,室温溶解,如不溶可在60℃水浴锅中放置10min。
(13)琼脂糖凝胶电泳观察总RNA的完整性。
2、RNA的纯化
总RNA的纯化参见QIAGEN RNA纯化试剂盒内的说明书,具体步骤体如下:
(1)将总RNA用去核酸酶无菌水调整至100μL,加入350μL裂解液(RLT),混匀。
(2)加入250μL无水乙醇,混匀后,将所有液体置于RNeasy Mini Spin Column中,10000rpm,离心15s,弃去滤液。
(3)加入350μL洗涤缓冲液(RW1),后转移至纯化柱中,10000rpm,离心15s,弃去滤液。
(4)将10μL含有DNaseI酶的溶液加至70μL DNA消化缓冲液(RDD)中,混匀后加至柱膜上,置于室温15min。
(5)加入350μL洗涤缓冲液RW1至纯化柱中,10000rpm,离心15s,弃去滤液。
(6)加500μL漂洗缓冲液RPE至纯化柱中,10000rpm,离心15s,弃去滤液。
(7)将步骤6重复一次。
(8)将纯化柱置于一个去核酸酶1.5mL EP管中,加入50μL去核酸酶无菌水至柱膜上,10000rpm,离心1min,洗脱RNA。
(9)琼脂糖凝胶电泳检查RNA的完整性并测定其浓度。
(10)立即将RNA反转录为cDNA或将RNA进行分装后保存于-80℃,避免RNA的反复冻融。
3、反转录
本实验采用PrimeScriptTM RT reagent Kit with gDNA Eraser试剂盒进行RNA的反转录,具体操作步骤如下:
(1)去除基因组DNA反应
按照表1中的体系,在冰上配置反应混合溶液,为保证准确性应先按反应数+2的量配制反应液,分装到反应管中,最后加入RNA样品,42℃水浴锅中反应2min。
(2)反转录反应
按照表2,在冰上配置反应混合溶液,为保证准确性应先按反应数+2的量配制反应液,然后再分装10μL到(1)反应管中,轻柔混匀后立即放入37℃水浴锅反应15min,而后在85℃水浴锅中反应5s,获得cDNA溶液,于-20℃冷冻保存。
表1基因组DNA去除反应体系
试剂 | 使用量 |
5×gDNA Eraser Buffer | 2μL |
gDNA Eraser | 1μL |
Total RNA | 1-2μL |
RNase Free ddH2O | up to 10μL |
表2RNA逆转录体系
试剂 | 使用量 |
步骤(1)的反应液 | 10μL |
Prime Script RT Enzyme Mix I | 1μL |
RT Primer Mix | 1μL |
5×Prime Script Buffer | 4μL |
RNase Free ddH2O | 4μL |
Total | 20μL |
4、PCR扩增脂肪酶ANI_1_266144、ANI_1_236084和ANI_1_58064编码基因
通过PCR扩增脂肪酶ANI_1_266144、ANI_1_236084和ANI_1_58064编码基因。
用于扩增ANI_1_266144的引物对为正向引物SEQ ID No.7:
5’-CTGATATCGGATCCGAATTCGCACCGACACCACTTGATGT-3’;
反向引物SEQ ID No.8:
5’-GTGGTGGTGGTGGTGGTGCTCGAGCTATAGCAGACACTCTGAAATTGCGAAAA-3’
用于扩增ANI_1_236084的引物对为正向引物SEQ ID No.9:
5’-CTGATATCGGATCCGAATTCGCTCCCGCACCTGCTC-3’;
反向引物SEQ ID No.10:
5’-GTGGTGGTGGTGGTGGTGCTCGAGCTACGAGCATTCACTAATGTAGATCGTG-3’
用于扩增ANI_1_58064的引物对为正向引物SEQ ID No.11:
5’-CTGATATCGGATCCGAATTCCTGCCCACGCCCGGCTCC-3’;
反向引物SEQ ID No.12:
5’-GTGGTGGTGGTGGTGGTGCTCGAGCTAATAAACGGAGGAAGTCGTGTTCAG-3’
PCR反应体系如表3所示:
表3本实施例中所用的PCR扩增体系
反应体系 | 加样量 |
ddH2O | 补足50μL |
10×PCR Buffer | 5.0μL |
dNTP(0.2mmol/L) | 4μL |
上、下游引物(10m mol/L) | 各1.5μL |
模板:酵母总DNA | 1.0μL |
LA-Taq DNA聚合酶 | 0.25μL |
表4PCR反应程序(LA Taq酶)
反应程序 | 温度 | 时间 |
预变性 | 95℃ | 5min |
变性 | 94℃ | 45s |
退火 | 58℃ | 45s |
延伸 | 72℃ | 1.5min |
终延伸 | 72℃ | 10min |
保温 | 4℃ | ---- |
编码基因扩增结果通过凝胶电泳检测,结果如图1所示。
实施例2重组表达载体的构建
1、质粒线性化
载体pET32(a)用EcoRⅠ、XhoⅠ双酶切。双酶切具体反应体系如表5所示,酶切条件为37℃反应30min。随后对酶切产物进行DNA凝胶回收即得到线性化的载体DNA。
表5双酶切反应体系
a若为Plasmid DNA,则添加1μg,若为PCR product,则添加0.2μg,具体的XμL,则需根据DNA浓度计算。
2、连接转化
采用VazymeClonExpress II One Step Cloning Kit将目的片段及线性化载体进行同源重组,具体反应体系的配制见6,反应条件:PCR仪中37℃孵育30min。随后转化E.coliDH5α。
表6重组反应体系
组分 | 20μL |
LinearizedVectorsa | XμL |
Insertsa | YμL |
5×CEⅡBuffer | 4μL |
ExnaseⅡ | 2μL |
ddH2O | Add to 20μL |
aX/Y根据公式:最佳载体质量=[0.02×碱基对数]ng(0.03pmol);最佳插入质量=[0.04×碱基对数]ng(0.06pmol)计算载体和插入片段用量。
3、重组菌株的构建及诱导表达
将重组质粒转化至宿主E.coli.BL21(DE3)感受态细胞,从转化平板上挑单菌落于5mL含抗生素的液体LB培养基(胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L)中37℃,200rpm过夜培养,种子液按1%(V/V)转接至50mL含抗生素的液体LB培养基中16℃培养至OD600为0.6~0.8左右,加入IPTG(异丙基-β-D-硫代半乳糖苷)至终浓度0.1mM,诱导20h,以不加IPTG为对照组。诱导结束分别离心(4℃,6000rpm,10min)收集菌体,用适量的100mMTris-HCl(pH8.0)缓冲液重悬菌体,随后将菌悬液置于冰水混合物中用超声波破碎仪破碎细胞,破碎功率选择30%,工作程序为超声2s,停顿3s,破碎20~30min,具体破碎时间根据蛋白本身和菌体量决定。将破碎后的菌液于4℃,8000rpm离心20min,将含有可溶胞内蛋白的上清液倒入无菌离心管中。
实施例3脂肪酶ANI_1_266144、ANI_1_236084和ANI_1_58064的纯化和超滤浓缩
1、镍柱纯化蛋白
将实施例2中的上清液选择可以与His-tag螯合的的Ni柱进行亲和层析,纯化结果如图2所示。具体步骤如下:
(1)用10mL去离子水洗掉柱子里的乙醇。
(2)加入10倍柱体积的结合缓冲液平衡镍柱。
(3)将粗酶液与镍柱中的镍与磁力搅拌器上混合40min,收集流出液体为穿出样品,未结合在柱上的蛋白会在此时穿出。
(4)加入10倍柱体积结合缓冲液再次平衡镍柱,一些结合能力弱的杂蛋白会在此时被洗脱下来。
(5)用100mM咪唑洗脱并收集流出液。
(6)最后用10倍柱体积的洗脱缓冲液彻底洗脱Ni柱。
(7)最后加入5~10倍柱体积的20%的乙醇封柱子,于4℃保存。
2、重组蛋白的超滤浓缩
已纯化好的蛋白倒入预冷的超滤管中,最高转速4000rpm,用置换缓冲液(20mMTris-HCl,pH 8.0),反复置换使蛋白液最终的咪唑浓度和盐浓度降至20mM左右。获得脂肪酶ANI_1_266144、ANI_1_236084和ANI_1_580644的纯酶液。
实施例4脂肪酶ANI_1_266144、ANI_1_236084和ANI_1_58064在水相中催化合成风味酯的检测
反应体系为1mL,进行水相酯合成的检测,体系中包含100uL的纯酶液(蛋白浓度为0.5mg/mL),终浓度10mM的丁酸、戊酸、己酸、庚酸或辛酸中的一种,终浓度为1M的乙醇,最后加入柠檬酸缓冲液中(50mM,pH2.0),以不加酶液的反应体系为对照组。上述混合反应液在30℃以150rpm的转速搅拌24h。此后向反应混合物中加入等体积的正己烷,剧烈涡旋30s,10000rpm离心5min,将上层即正己烷层过滤后进行气相色谱分析。脂肪酶ANI_1_266144催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯的含量分别为51.26mg/L、80.58mg/L、207.15mg/L、487.3mg/L和794.94mg/L;脂肪酶ANI_1_236084催化合成丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯的含量分别为44.35mg/L、45.96mg/L、118.86mg/L、427.57mg/L和615.2mg/L;脂肪酶ANI_1_58064与对照组无差异。
实施例5pH对脂肪酶ANI_1_266144和脂肪酶ANI_1_236084的影响
选择pH为2、3、4、5和6的柠檬酸钠缓冲液,终浓度为10mM的己酸,其它反应条件与实施例4一致,以最高酶活为100%,其它为相对酶活。结果证实本发明脂肪酶ANI_1_236084与脂肪酶ANI_1_266144的最适pH为4.0,如图4所示。
实施例6温度对脂肪酶ANI_1_266144和脂肪酶ANI_1_236084的影响
选择反应温度为20℃、30℃、40℃和50℃,其他反应条件同实施例4,以最高酶活为100%,其它为相对酶活。结果证实本发明脂肪酶ANI_1_236084与脂肪酶ANI_1_266144的最适反应温度为30℃,但在温度为40℃的反应条件下,脂肪酶ANI_1_236084有较好的活性,表现出更宽泛的适用温度范围,如图5所示。
需要说明的是,本说明书中描述的具体特征、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以及不同实施例的特征进行结合和组合。
Claims (7)
1.黑曲霉(Aspergillus niger)脂肪酶,其特征在于:其氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.根据权利要求1所述的黑曲霉脂肪酶,其特征在于:编码所述黑曲霉脂肪酶的编码基因如SEQ ID NO.3或SEQ ID NO.4所示。
3.权利要求1或2所述的黑曲霉脂肪酶在水相体系中催化合成酯类物质的应用。
4.权利要求1或2所述的黑曲霉脂肪酶在制备含酯类物质的酒用酯香液或白酒中的应用。
5.根据权利要求3或4所述的应用,其特征在于:所述酯类物质包括:丁酸乙酯、戊酸乙酯、己酸乙酯、庚酸乙酯和辛酸乙酯中至少一种。
6.根据权利要求3~5任一项所述应用,其特征在于:所述黑曲霉脂肪酶是通过构建含有所述黑曲霉脂肪酶编码基因的大肠杆菌重组质粒,将重组质粒转入大肠杆菌中,经过诱导表达获得。
7.根据权利要求3~5任一项所述应用,其特征在于:所述黑曲霉脂肪酶在水相体系中催化合成酯类物质的反应条件为:pH为4.0,温度为30~40℃。
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