CN108396016A - 一种酯酶phe21及其编码基因和在手性乙酸仲丁酯制备中的应用 - Google Patents
一种酯酶phe21及其编码基因和在手性乙酸仲丁酯制备中的应用 Download PDFInfo
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- CN108396016A CN108396016A CN201810114144.8A CN201810114144A CN108396016A CN 108396016 A CN108396016 A CN 108396016A CN 201810114144 A CN201810114144 A CN 201810114144A CN 108396016 A CN108396016 A CN 108396016A
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- Prior art keywords
- phe21
- esterase
- sec
- butyl acetate
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- DCKVNWZUADLDEH-RXMQYKEDSA-N [(2r)-butan-2-yl] acetate Chemical compound CC[C@@H](C)OC(C)=O DCKVNWZUADLDEH-RXMQYKEDSA-N 0.000 claims abstract description 11
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Abstract
本发明公开了一种酯酶PHE21及其编码基因和在手性乙酸仲丁酯制备中的应用。本发明从海洋假单胞菌(Pseudomonadaceae oryzihabitans HUP022)中克隆到了一个酯酶基因PHE21,其核苷酸序列如SEQ ID NO.1所示,全长为966bp,其编码的酯酶PHE21的氨基酸序列如SEQ ID NO.2所示,共包含321个氨基酸。利用重组表达的酯酶PHE21作为催化剂,一方面可以直接催化水解反应制备光学纯度大于98%的(S)‑乙酸仲丁酯,另一方面还可以催化转酯反应制备光学纯度大于64%的(R)‑乙酸仲丁酯。酯酶PHE21在生物医药和精细化工领域具有非常大的应用前景。
Description
技术领域:
本发明属于生物化工和生物技术领域,具体涉及一种酯酶PHE21及其编码基因和在手性乙酸仲丁酯制备中的应用。
背景技术:
手性化合物是一类由相同原子组成,立体结构互为镜像的一类化合物,不同对映体往往会表现出截然不同的生理活性和毒性。如苯并***烷的2个对映体都有镇痛作用,但(-)的异构体服用后会成瘾,而(+)的异构体则不会;R型的“反应停”是孕妇用镇痛药和止痛药,而S型的“反应停”对胎儿则有致畸作用;抗菌药氧氟沙星的右旋体能损害肝、肾功能,但左旋的氧氟沙星药效很高,且毒性极小,其在各国上市后,深受人们的欢迎。所以在合成手性药物的过程中,前体原料的光学纯度是非常重要的环节。
手性仲丁醇及其酯类衍生物是重要的手性化学品和拆分剂,在工业上有很大需求,广泛用于香料工业、医药工业、洗涤剂工业和精细化工工业。如手性丁醇及其酯作为一种环保型溶剂,不仅是合成手性化合物特别是一些抗结核药(如乙胺丁醇)和一些抗血压药(如拉贝洛尔)的重要关键中间体,而且还广泛应用于硝基漆、丙烯酸漆等化工产品的溶剂和农药乳油剂等农药产业。
手性化合物的合成主要有:(1)化学法,即利用对映体的物理和化学性质的差异进行分离。通常有盐析法、包结法、以及组合拆分。其缺点是要求对映体之间的性质差异要大,适用的化合物不多。(2)色谱拆分,这种方法是利用填料对手性对映体吸附性质的差异来实现,其缺点是设备昂贵,普及性较差。(3)合成法,通过设计反应来进行手性化合物的化学合成。这种方法缺点在于反应过程通常比较剧烈,耗费能量,而且反应中使用大量有毒的有机溶剂。(4)生物酶法,生物脂肪酶/酯酶具有立***点区域和底物的高度专一性,而且反应条件温和、副反应少、光学纯度高以及环境污染小等优点,因此开发具有光学选择性的脂肪酶/酯酶具有重要的意义。
发明内容:
本发明的针对现有技术中的不足,提供一种新的酯酶PHE21及其编码基因和在手性乙酸仲丁酯制备中的应用。
本发明从一株海洋假单胞菌(Pseudomonadaceae oryzihabitans HUP022)中开发了一种新的酯酶PHE21及其编码基因PHE21,构建了含有PHE21的重组表达载体和基因工程菌,培养基因工程菌后获得酯酶PHE21,其可应用于制备手性乙酸仲丁酯。
本发明的第一个目的是提供一种酯酶PHE21,其氨基酸序列如SEQ ID NO.2所示。
本发明的第二个目的是提供一种编码所述的酯酶PHE21的酯酶基因PHE21。
优选,所述的酯酶基因PHE21的核苷酸序列如SEQ ID NO.1所示。
本发明还提供一种含有所述的酯酶基因PHE21的重组表达载体。所述的表达载体,优选pET28a(+)载体。
本发明还提供一种含有所述的酯酶基因PHE21的基因工程菌。所述的基因工程菌,优选大肠杆菌BL21(DE3)。
本发明的第三个目的是提供所述的酯酶PHE21在制备(S)-乙酸仲丁酯或(R)-乙酸仲丁酯中的应用。
优选,所述的应用为酯酶PHE21在拆分(±)-乙酸仲丁酯制备得到(S)-乙酸仲丁酯中的应用。
进一步优选,所述的应用为:取酯酶PHE21于pH为6.0~9.0的缓冲液中,再加入(±)-乙酸仲丁酯,进行反应,得到(S)-乙酸仲丁酯。
所述的缓冲液,优选为柠檬酸-柠檬酸钠缓冲液、磷酸缓冲液、Tris-HCl缓冲液和甘氨酸-NaOH缓冲液中的一种。
优选,所述的应用为酯酶PHE21在催化(±)-仲丁醇的转酯反应制备得到(R)-乙酸仲丁酯中的应用。
进一步优选,所述的应用为:取酯酶PHE21于反应介质中,再加入(±)-仲丁醇和酰基供体,进行反应,得到(R)-乙酸仲丁酯。
所述的反应介质,优选为1,4-二氧六环、四氢呋喃、叔丁醇、异丙醇、二氯甲烷、甲苯、环己烷、正己烷、正庚烷和异辛烷中的一种。
所述的酰基供体,优选乙酰乙烯酯、乙酰异丙烯酯和乙酸乙酯中的一种。
本发明的酯酶基因PHE21来自深海样品中筛选得到的一株海洋假单胞菌(Pseudomonadaceae oryzihabitans HUP022),保存在中国科学院南海海洋研究所实验室。本发明用生物信息学分析的方法,从基因组测序的假单胞菌(Pseudomonadaceaeoryzihabitans HUP022)中筛选得到酯酶基因PHE21,全长为966bp(从起始密码子到终止密码子),编码321个氨基酸。通过克隆编码成熟的酯酶PHE21的酯酶基因PHE21并将其连接表达载体pET-28a(+)后转化大肠杆菌BL21(DE3),培养并诱导表达后,得到了重组表达的酯酶PHE21。酯酶PHE21可用于制备手性乙酸仲丁酯,利用重组表达的酯酶PHE21作为催化剂,一方面可以直接催化水解反应制备光学纯度大于98%的(S)-乙酸仲丁酯,另一方面还可以催化转酯反应制备光学纯度大于64%的(R)-乙酸仲丁酯。酯酶PHE21在生物化工和生物医药等领域具有非常大的应用价值。
附图说明:
图1是酯酶基因PHE21的NCBI blast对比结果。
图2是pH对酶动力学水解拆分乙酸仲丁酯的影响。
图3是温度对酶动力学水解拆分乙酸仲丁酯的影响。
图4是底物浓度对酶动力学水解拆分乙酸仲丁酯的影响。
图5是酶浓度对酶动力学水解拆分乙酸仲丁酯的影响。
图6是反应时间对酶动力学水解拆分乙酸仲丁酯的影响。
图7是酯酶PHE21拆分(±)-乙酸仲丁酯反应GC图;A为样品(±)-乙酸仲丁酯气相图,B为酯酶PHE21拆分(±)-乙酸仲丁酯反应3h后的气相图,其中S代表(S)-乙酸仲丁酯,R代表(R)-乙酸仲丁酯。
图8是反应时间对转酯反应的影响。
图9是酯酶PHE21催化(±)-仲丁醇转酯反应GC图;A为样品(±)-乙酸仲丁酯气相图,B为酯酶PHE21催化(±)-仲丁醇转酯反应4h后的气相图,其中S代表(S)-乙酸仲丁酯,R代表(R)-乙酸仲丁酯。
具体实施方式:
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
下列实例中未具体注明的实验方法,均可按照常规方法进行,或按照所用产品生产厂商的使用说明。下述实施例中所用的材料、试剂等,如无特殊说明,均可通过商业途径得到。
本发明的酯酶基因PHE21来自深海样品中筛选得到的一株海洋假单胞菌(Pseudomonadaceae oryzihabitans HUP022),该菌保存在中国科学院南海海洋研究所实验室。
实施例1:酯酶基因PHE21引物设计及开放阅读框边界确定
提取假单胞菌(Pseudomonadaceae oryzihabitans HUP022)的基因组DNA,经测序验证无误后,利用生物信息学手段对基因组进行注释,分析其中的酯酶基因,确定了其中酯酶基因PHE21的开放阅读框,其核苷酸序列如SEQ ID NO.1所示,全长为966bp(从起始密码子到终止密码子),其编码的酯酶PHE21的氨基酸序列如SEQ ID NO.2所示,共321个氨基酸;该基因是一个全新的酯酶基因,与其它酯酶基因序列的最大相似度为96%(图1)。根据分析得到的酯酶基因PHE21序列,设计引物如下:正向引物:5′-CACGAATTCATGCCCGACGTCTTCGCGCG-3′,下划线部分为EcoRI酶切位点;反向引物:5′-CCGCTCGAGTTAGGCGGCCTGCTGCAGATGC-3′,下划线部分为Xho I酶切位点。
实施例2:酯酶基因PHE21的克隆及载体构建
2.1PCR扩增
将实施例1设计的引物(正向引物:5′-CACGAATTCATGCCCGACGTCTTCGCGCG-3′,反向引物:5′-CCGCTCGAGTTAGGCGGCCTGCTGCAGATGC-3′)送至上海生物工程有限公司合成引物,合成的引物使用TE稀释到10μM,假单胞菌(Pseudomonadaceae oryzihabitans HUP022)的总DNA作为DNA模板,建立如表1所示反应体系:
表1 PCR反应体系
使用以下PCR扩增程序扩增酯酶基因PHE21:a.94℃变性3min;b.94℃变性30s,55~65℃退火0.5min,72℃延伸1min,进行30个循环;c.72℃延伸10min,冷却到10℃。
将PCR产物在1%琼脂糖凝胶中,120V电压下电泳20min,置于凝胶成像***中观察。回收984bp左右的条带。PCR产物按照胶回收试剂盒的方法进行回收,使用20μL无菌水洗脱,得到纯化回收的PCR产物。
2.2酶切
将纯化回收的PCR产物使用以下体系进行双酶切,酶切时间1h。酶切体系为:EcoRI2μL,XhoI 2μL,DNA<0.3μg,灭菌的双蒸水加至30μL。酶切后纯化回收得到经过双酶切的PCR产物。
质粒pET-28a(+)的双酶切:挑取含有质粒pET-28a(+)的大肠杆菌DH5α单菌落,过夜培养。使用质粒提取试剂盒提取质粒,用BamHI和XhoI按以下体系双酶切,酶切时间1h。酶切体系为:EcoRI 2μL,XhoI 2μL,质粒DNA<1μg,灭菌的双蒸水加至20μL。酶切后纯化回收得到经过双酶切的pET-28a(+)载体。
上述双酶切使用的限制性内切酶为Thermo公司生产的快速内切酶,酶切后的纯化回收使用核酸纯化回收试剂盒(Magen,Hipure Gel Pure DNA Micro Kit),质粒提取试剂盒为上海捷瑞生物工程有限公司的质粒小量制备试剂盒,操作方法按其使用说明书。
2.3连接
将经过双酶切的PCR产物和pET-28a(+)载体按照3:1的摩尔比例进行连接。连接使用的T4连接酶购自北京全式金生物技术有限公司,连接使用的酶量为5U/5μL连接体系,连接温度为25℃,连接时间30min。由此得到连接产物。
2.4转化及筛选
取5μL连接产物于50μL大肠杆菌DH5a感受态细胞中,冰浴30min,后于42℃水浴锅热激90s,冰浴2min后加入500μL LB液体培养基,37℃200rpm转速下,孵育培养1h。取一定量的菌液涂布于含100μg/mL卡那霉素的LB平板,培养20h后挑选单菌落。单菌落于5mL LB培养基中过夜培养后提取质粒,进行双酶切验证,酶切片段与基因大小相同的即为阳性克隆。
2.5基因核苷酸序列测定
将筛选的正确阳性克隆送至上海美吉生物医药有限公司进行测序,测序结果与酯酶基因PHE21核苷酸序列进行比对,确认是将酯酶基因PHE21(其核苷酸序列如SEQ ID NO.1所示)***到pET-28a(+)质粒中,结果完全正确后确认得到带有酯酶基因PHE21的pET-28a(+)质粒(命名为pET-28a(+)-PHE21),可用于进行下一步试验。
实施例3:酯酶基因PHE21在大肠杆菌BL21(DE3)中的高效表达
3.1大肠杆菌BL21(DE3)感受态细胞制备
1、将少量大肠杆菌BL21(DE3)菌种接入5mL LB试管液中,37℃过夜摇培,250rpm;
2、按1%体积比的接种量将过夜摇培后的大肠杆菌BL21(DE3)菌液接种到300mlLB摇瓶中,37℃摇培3-4h(≥300rpm);
3、将培养好的摇瓶在冰水中迅速冷却至0℃,分装至冰预冷的离心管(50mL),冰置数分钟;
4、4℃,4000rpm离心10min回收细胞,去上清;
5、用冰预冷的10mL 0.1M的CaCl2重悬细胞,4℃,4000rpm离心10min回收细胞;
6、重复5,用10mL 0.1M的CaCl2重悬细胞,冰浴1h以上;
7、4℃,4000rpm离心10min回收细胞;
8、50mL原培养物用2mL含体积分数15%DMSO的0.1M CaCl2来重悬,分装于1.5mL离心管,100μL每管,-80℃保藏。由此得到大肠杆菌BL21(DE3)感受态细胞。
3.2转化
取实施例2中得到的pET-28a(+)-PHE21质粒0.5~1μL与50μL大肠杆菌BL21(DE3)感受态细胞混合,冰浴30min,于42℃水浴锅热激45s,冰浴2min后加入500μL LB液体培养基,37℃200rpm培养1h。培养物离心后涂布50μg/mL的卡那霉素LB平板,培养过15h后挑选单菌。由此得到含有pET-28a(+)-PHE21的大肠杆菌BL21(DE3)。
实施例4:酯酶PHE21的表达和纯化
4.1蛋白诱导
含有pET-28a(+)-PHE21的大肠杆菌BL21(DE3)在LB培养基中37℃培养至OD600为0.5左右,加IPTG至终浓度0.2mM,20℃培养16个小时。300mL菌液4000rpm,4℃离心10min,收集菌体,用PBS缓冲液洗涤菌体2次,4000rpm,10min收集菌体。用30mL(50mM,pH7.4)Tris-HCl缓冲液重悬菌体,超声400w,超5s,停5s,破碎10min,4℃,10000rmp离心10min,收集上清。将收集的上清用冻干机冻干得到酯酶PHE21粗酶粉,放于-80℃冰箱保存。4.2酯酶PHE21的纯化
用镍离子亲和层析柱对步骤4.1中收集的上清进行纯化得纯化的酯酶PHE21(即酯酶PHE21纯酶液,浓度为10mg/mL),纯化的蛋白大小约37kD,符合理论预期。具体实施方案如下:使用10mM的咪唑洗脱5个柱体积,30mM咪唑洗脱30个柱体积,最后使用100~1000mM咪唑洗脱5个柱体积,收集中间3.5mL。用脱盐柱SephadexG25进行脱盐,具体操作方法参照GE公司的操作手册进行。
实施例5:酯酶PHE21在拆分(±)-乙酸仲丁酯中的应用
5.1酶动力学水解拆分乙酸仲丁酯获得光学纯的(S)-乙酸仲丁酯
酶动力学水解拆分乙酸仲丁酯获得光学纯的(S)-乙酸仲丁酯受到助溶剂种类和比例、反应温度、转速等等诸多条件的影响。本发明对上述参数进行了优化,反应混合液为:在500μL50mM,pH7.5的Na2HPO4/NaH2PO4缓冲溶液中,加入终浓度0.01g/L的酯酶PHE21(即加入1mL/L的步骤4.2制得的酯酶PHE21纯酶液),0.02M的(±)-乙酸仲丁酯,于37℃,200rpm条件下反应120min。
具体分析条件为:采用福立气相色谱仪,配有手性柱(30m×0.25mm Cyclosil Bchirl column)和氢离子火焰检测器。仪器分析条件设置为:注射器温度220℃,检测器温度250℃,载气为N2,流速1.2mL/min,采用梯度升温进行分析:80℃保持1min,15℃/min,120℃保持1min,10℃/min到220℃,保持1min。
5.2pH对酶动力学水解拆分乙酸仲丁酯的影响
配制不同的缓冲溶液,这些缓冲溶液具有不同的pH,如表2所示,其浓度均为50mM:
表2 不同缓冲体系的pH
将5.1中测定条件所述的缓冲液(Na2HPO4/NaH2PO4buffer)按照表2中的缓冲溶液分别进行替换,测定不同pH的缓冲溶液对酯酶PHE21水解拆分乙酸仲丁酯的影响(图2),结果说明,在pH为8的缓冲溶液中,酯酶PHE21对底物(±)-乙酸仲丁酯的拆分效果最好。(S)-乙酸仲丁酯的对映体过量值和转化率分别为70%和41.2%。
5.3温度对酶动力学水解拆分乙酸仲丁酯的影响
在pH8.0,50mM Na2HPO4/NaH2PO4作为缓冲溶液,按5.1中的反应混合液置于不同的温度(25~50℃)下进行水解拆分乙酸仲丁酯的实验,在不同的反应温度条件下,(S)-乙酸仲丁酯的对映体过量值和转化率如图3所示。结果说明,在反应温度为35℃时,(S)-乙酸仲丁酯的对映体过量值最高,为80%。当反应温度高于40℃或者低于30℃时,(S)-乙酸仲丁酯的对映体过量值都会下降,说明温度对酯酶PHE21的光学选择性具有很大的影响。
5.4有机溶剂和表面活性剂对酶动力学水解拆分乙酸仲丁酯的影响
在水解拆分乙酸仲丁酯的实验中,按5.1中的反应混合液将酯酶PHE21和底物(±)-乙酸仲丁酯置于pH8.0,50mM Na2HPO4/NaH2PO4缓冲溶液中,在温度为35℃的条件下,加入11种不同的有机溶剂和5种不同的表面活性剂进行水解拆分反应,结果如表3所示。结果表明,实验中的11种不同的有机溶剂和5种表面活性剂会对酯酶PHE21的立体选择性产生不同程度的抑制,说明有机溶剂和表面活性剂会对酯酶PHE21的立体选择性产生较大的影响。
表3 有机溶剂和表面活性剂对酶动力学水解拆分乙酸仲丁酯的影响
5.5底物浓度对酶动力学水解拆分乙酸仲丁酯的影响
在最适反应温度(35℃)和最适反应pH(8.0)条件下,按5.1中的反应混合液分别在反应体系中加入不同浓度的底物(±)-乙酸仲丁酯,测试底物浓度对酯酶PHE21水解拆分乙酸仲丁酯的影响,反应结果如图4所示。结果说明,在底物浓度为0.04M时,酯酶PHE21水解(±)-乙酸仲丁酯的效果最好,产物(S)-乙酸仲丁酯的对映体过量值>91%,转化率为48.9%,底物浓度越高,拆分效果越差。
5.6酶浓度对酶动力学水解拆分乙酸仲丁酯的影响
在最适反应温度(35℃)和最适反应pH(8.0)条件下,按5.1中的反应混合液中加入最适浓度的底物(±)-乙酸仲丁酯(0.04M),分别在反应体系中加入不同浓度的纯酶液,测试酯酶PHE21水解拆分(±)-乙酸仲丁酯(0.04M)的最适酶浓度,反应结果如图5所示。结果说明,在酶浓度为0.016g/L时,水解拆分(±)-乙酸仲丁酯(0.04M)的效果已达到最好,产物(S)-乙酸仲丁酯的对映体过量值>92%,转化率为49.7%,当酶浓度大于0.03g/L时,产物(S)-乙酸仲丁酯的对映体过量值和转化率基本不变。
5.7反应时间对酶动力学水解拆分乙酸仲丁酯的影响
在最适反应条件下(反应温度35℃,pH为8.0,50mM Na2HPO4/NaH2PO4作为缓冲溶液,底物(±)-乙酸仲丁酯浓度为0.04M,酯酶PHE21浓度为0.016g/L),按5.1中的反应条件下分别反应不同时间(40~200min),结果如图6所示。结果表明,反应180min后,产物(S)-乙酸仲丁酯的对映体过量值已达到最高,对映体过量值>98%,产率为83.6%,当反应时间大于180min后,产物(S)-乙酸仲丁酯的对映体过量值和转化率基本不变。在24h内可得到大于98%光学纯度的(S)-乙酸仲丁酯(图7)。
实施例6:酯酶PHE21在催化(±)-仲丁醇转酯反应制备(R)-乙酸仲丁酯中的应用
在优化的条件下,即在3mL反应体系中,加入1.7×106U/L的酯酶PHE21粗酶粉,30mM的乙酸乙烯酯和10mM的(±)-仲丁醇,其余为环己烷,于45℃,200rpm条件下进行转酯反应,结果如图8所示,当反应时间为4小时时,反应产物(R)-乙酸仲丁酯的对映体过量值和转化率达到最优,分别为64%和43%。在24h内可得到光学纯度大于64%的(R)-乙酸仲丁酯(图9)。
具体分析条件为:采用福立气相色谱仪,配有手性柱(30m×0.25mm Cyclosil Bchirl column)和氢离子火焰检测器。仪器分析条件设置为:注射器温度220℃,检测器温度250℃,载气为N2,流速1.2mL/min,采用梯度升温进行分析:80℃保持1min,15℃/min,120℃保持1min,10℃/min到220℃,保持1min。
序列表
<110> 中国科学院南海海洋研究所
<120> 一种酯酶PHE21及其编码基因和在手性乙酸仲丁酯制备中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 966
<212> DNA
<213> 海洋假单胞菌(Pseudomonadaceae oryzihabitans HUP022)
<400> 1
atgcccgacg tcttcgcgcg cctcactccc gaggtggccg gcctgctggc gcagctggcc 60
gcccacccgc agccgccgct ggacagcctc accccggcgc aggcgcgcgc cgcctatgtg 120
cagctgaatc gtgccctcgg cctgccgcca gcgccggtga agcgggtgga ggcgctcagc 180
gctccggggc cgctggggcc catcccgctg cgcctctacc gcccctatgc ggaagacgat 240
cgaccgcgcc cgctggtgat ctatctgcac ggcggtggct gggtgatcgg cgacctggac 300
agccatgatt cgctctgccg gcagatcgcg atcggaaccg gctatgcggt gctggcggtg 360
cactatgccc tcgcccccga gcatccggca ccggcgagcc ccgacgatgt gctggccgcc 420
ctgcactggc tggccgaggc cggcagcgcc ttccacctcg atctcgaccg catcgccgtg 480
gccggcgaca gcgccggtgg cgggctggcg gccatggccg cattgtacct gcgcgaagga 540
ccgctcaggc tcaaggccca ggtgctgatc taccctggcg tcgacaacac gcccgaggcc 600
tggaatcacc cctcgcggat cgagaacgcc caggtcccac cgctgacccg gccaatgatg 660
gattactttt cggggatgta tctccaggac gtggacaccc gcgacccgcg cgtctcgccg 720
ctttatgccg cctcccacgc cgacctgccg ccggcgctga tcttcggcgc cgaatgcgac 780
gccctgcgcg acgatgcccg cctctatgcc caggcgctga tcggcgccgg caacgccgtg 840
gagtaccacg agttgccggg catgatccat ggcttcatcg agatgctcgg ggtgctgccc 900
agcgtgcgct ggaccctggc gcggatgaat gcctttctgc gcgagcatct gcagcaggcc 960
gcctga 966
<210> 2
<211> 321
<212> PRT
<213> 海洋假单胞菌(Pseudomonadaceae oryzihabitans HUP022)
<400> 2
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Pro Ala Pro Ala Ser Pro Asp Asp Val Leu Ala Ala Leu His Trp Leu
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Ala Gly Asp Ser Ala Gly Gly Gly Leu Ala Ala Met Ala Ala Leu Tyr
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Gly Val Asp Asn Thr Pro Glu Ala Trp Asn His Pro Ser Arg Ile Glu
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Asn Ala Gln Val Pro Pro Leu Thr Arg Pro Met Met Asp Tyr Phe Ser
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Gly Met Tyr Leu Gln Asp Val Asp Thr Arg Asp Pro Arg Val Ser Pro
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Leu Tyr Ala Ala Ser His Ala Asp Leu Pro Pro Ala Leu Ile Phe Gly
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Ala Glu Cys Asp Ala Leu Arg Asp Asp Ala Arg Leu Tyr Ala Gln Ala
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Leu Ile Gly Ala Gly Asn Ala Val Glu Tyr His Glu Leu Pro Gly Met
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Ala
Claims (10)
1.一种酯酶PHE21,其特征在于,其氨基酸序列如SEQ ID NO.2所示。
2.一种编码权利要求1所述的酯酶PHE21的酯酶基因PHE21。
3.根据权利要求2所述的酯酶基因PHE21,其特征在于,所述的酯酶基因PHE21的核苷酸序列如SEQ ID NO.1所示。
4.权利要求1所述的酯酶PHE21在制备(S)-乙酸仲丁酯或(R)-乙酸仲丁酯中的应用。
5.根据权利要求4所述的应用,其特征在于,所述的应用为酯酶PHE21在拆分(±)-乙酸仲丁酯制备得到(S)-乙酸仲丁酯中的应用。
6.根据权利要求5所述的应用,其特征在于,所述的应用为:取酯酶PHE21于pH为6.0-9.0的缓冲液中,再加入(±)-乙酸仲丁酯,进行反应,得到(S)-乙酸仲丁酯。
7.根据权利要求6所述的应用,其特征在于,所述的缓冲液为柠檬酸-柠檬酸钠缓冲液、磷酸缓冲液、Tris-HCl缓冲液和甘氨酸-NaOH缓冲液中的一种。
8.根据权利要求4所述的应用,其特征在于,所述的应用为酯酶PHE21在催化(±)-仲丁醇的转酯反应制备得到(R)-乙酸仲丁酯中的应用。
9.根据权利要求8所述的应用,其特征在于,所述的应用为:取酯酶PHE21于反应介质中,再加入(±)-仲丁醇和酰基供体,进行反应,得到(R)-乙酸仲丁酯。
10.根据权利要求9所述的应用,其特征在于,所述的反应介质为1,4-二氧六环、四氢呋喃、叔丁醇、异丙醇、二氯甲烷、甲苯、环己烷、正己烷、正庚烷和异辛烷中的一种;所述的酰基供体为乙酰乙烯酯、乙酰异丙烯酯和乙酸乙酯中的一种。
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