CN108103044A - 一种酯酶WDEst17及其编码基因和应用 - Google Patents
一种酯酶WDEst17及其编码基因和应用 Download PDFInfo
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- CN108103044A CN108103044A CN201810106976.5A CN201810106976A CN108103044A CN 108103044 A CN108103044 A CN 108103044A CN 201810106976 A CN201810106976 A CN 201810106976A CN 108103044 A CN108103044 A CN 108103044A
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- Prior art keywords
- wdest17
- esterase
- gene
- ethyl
- hydroxybutanoate
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Abstract
本发明公开了一种酯酶WDEst17及其编码基因和应用。本发明从囊孢菌Dactylosporangium aurantiacum subsp.Hamdenensis NRRL18085中克隆到了一个酯酶基因WDEst17,其核苷酸序列如SEQ ID NO.1所示,全长为750bp,其编码的酯酶WDEst17的氨基酸序列如SEQ ID NO.2所示,共包含249个氨基酸。利用酯酶WDEst17制备得到了光学纯度大于99%的(R)‑3‑羟基丁酸乙酯。酯酶WDEst17具有较高的酶活和立体选择性等优点,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WDEst17及其编码基因和应用。
背景技术:
3-羟基丁酸乙酯的两种手性对映体是合成多种手性药物和化工产品的重要中间体。例如,(S)-3-羟基丁酸乙酯是昆虫信息素和手性香茅醇的重要手性中间体;而光学纯的(R)-3-羟基丁酸乙酯(R-EHB)是合成β-内酰胺酶抑制剂、β-内酰胺类抗生素和其他一些精细化工产品的重要中间体。因此,3-羟基丁酸乙酯等β-羟基酯的合成已经成为化工产业手性合成领域的研究热点。
生物酶具有立体位点区域和底物的高度专一性,利用酶促反应催化具有反应条件温和、位点选择性强、副反应少、光学纯度高以及环境污染小等优点。酯酶(esterase)作为一种绿色的生物催化剂,不仅具有良好的有机溶剂和表面活性剂耐受性、较高的立体选择性,而且反应还不需要价格昂贵的辅酶和辅因子参与,已被广泛应用于手性药物和手性化工产品的合成。因此开发具有光学选择性的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WDEst17及其编码基因和应用。
本发明从一株囊孢菌Dactylosporangium aurantiacum subsp.HamdenensisNRRL18085基因组中开发了一种新的酯酶WDEst17及其编码基因酯酶基因WDEst17,构建了含有酯酶基因WDEst17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WDEst17,其可应用于制备(R)-3-羟基丁酸乙酯。
本发明的第一个目的是提供一种酯酶WDEst17,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WDEst17的酯酶基因WDEst17。
优选,所述的酯酶基因WDEst17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WDEst17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WDEst17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WDEst17在制备(R)-3-羟基丁酸乙酯中的应用。
优选,所述的应用为酯酶WDEst17在拆分(±)-3-羟基丁酸乙酯得到(R)-3-羟基丁酸乙酯中的应用。
进一步优选,所述的应用为取酯酶WDEst17于pH为6.0~9.0的缓冲液中,再加入(±)-3-羟基丁酸乙酯,进行反应,得到(R)-3-羟基丁酸乙酯。
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。
本发明的酯酶基因WDEst17来自一株囊孢菌Dactylosporangium aurantiacumsubsp.Hamdenensis NRRL18085,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,从基因组测序的囊孢菌Dactylosporangium aurantiacumsubsp.Hamdenensis NRRL18085中筛选得到酯酶基因WDEst17,全长为750bp(从起始密码子到终止密码子),编码249个氨基酸。通过克隆编码成熟的酯酶WDEst17的酯酶基因WDEst17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WDEst17。
酯酶WDEst17作为催化剂拆分(±)-3-羟基丁酸乙酯,可得到光学纯度为99%的(R)-3-羟基丁酸乙酯。酯酶WDEst17作为绿色的生物催化剂,具有较高的酶活和立体选择性,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WDEst17的蛋白表达纯化情况。其中,M为蛋白Marker,1为未经IPTG诱导的含有pET-28a(+)-WDEst17的大肠杆菌BL21(DE3),2为经IPTG诱导的含有pET-28a(+)-WDEst17的大肠杆菌BL21(DE3),3为镍柱穿透液,4为Ni柱纯化后得到的酯酶WDEst17,5为经过脱盐柱后的酯酶WDEst17。
图2是酯酶WDEst17的底物特异性。
图3是酯酶WDEst17水解反应的最适pH。
图4是酯酶WDEst17水解反应的最适反应温度和温度稳定性。
图5是不同浓度NaCl对酯酶WDEst17酶活性影响。
图6是酯酶WDEst17拆分(±)-3-羟基丁酸乙酯反应GC图;A为样品(±)-3-羟基丁酸乙酯气相图,B为酯酶WDEst17拆分(±)-3-羟基丁酸乙酯反应3.5h后的气相图,其中S代表(S)-3-羟基丁酸乙酯,R代表(R)-3-羟基丁酸乙酯。
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WDEst17来自一株囊孢菌Dactylosporangium aurantiacumsubsp.Hamdenensis NRRL18085,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WDEst17引物设计及开放阅读框边界确定
提取囊孢菌Dactylosporangium aurantiacum subsp.Hamdenensis NRRL18085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WDEst17的开放阅读框,其核苷酸序列如SEQ ID NO.1所示,全长为750bp(从起始密码子到终止密码子),其编码的酯酶WDEst17的氨基酸序列如SEQ IDNO.2所示,共249个氨基酸,该基因是一个全新的酯酶基因。根据分析得到的酯酶基因WDEst17序列,设计引物如下:正向引物:5′-CATGGATCCGTGAGCATCCCGCAGACA-3′,下划线部分为BamH I酶切位点;反向引物:5′-CACAAGCTTTCAGACGCCACGGAGTGCG-3′,下划线部分为Hind III酶切位点。
实施例2:酯酶基因WDEst17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′-CATGGATCCGTGAGCATCCCGCAGACA-3′,反向引物:5′-CACAAGCTTTCAGACGCCACGGAGTGCG-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,囊孢菌Dactylosporangium aurantiacum subsp.HamdenensisNRRL18085的总DNA作为DNA模板,建立如表1所示反应体系:
表1PCR反应体系
使用以下PCR扩增程序扩增酯酶基因WDEst17:a.94℃变性3min;b.94℃变性30s,55~65℃退火0.5~1min,72℃延伸1min,进行20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,置于凝胶成像系统中观察。回收768bp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:BamHI 2μL,Hind III 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BamH I和Hind III按以下体系双酶切,酶切时间1-2h。酶切体系为:BamH I 2μL,Hind III 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20℃,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WDEst17核苷酸序列进行比对,确认是将酯酶基因WDEst17(其核苷酸序列如SEQ IDNO.1所示)插入到pET-28a(+)质粒中,结果完全正确后确认得到带有酯酶基因WDEst17的pET-28a(+)质粒(命名为pET-28a(+)-WDEst17),可用于进行下一步试验。
实施例3:酯酶基因WDEst17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,220rpm;
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),得到原培养物;
3、将培养好的摇瓶在冰水中迅速冷却至0℃,将原培养物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原培养物回收的细胞用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(+)-WDEst17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(+)-WDEst17的大肠杆菌BL21(DE3)。
实施例4:酯酶WDEst17的表达和纯化
4.1蛋白诱导
含有pET-28a(+)-WDEst17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20个小时。350mL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,pH 7.5)PBS缓冲液重悬菌体,超声400w,超4s,停6s,破碎10min,4℃,10000rpm离心20min,收集上清。
4.2酯酶WDEst17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WDEst17(图1),纯化的蛋白大小约29.8kD,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。
4.3酯酶WDEst17酶活测定
酯酶WDEst17活力测定采用对硝基苯酚酯,具体方法如下:(1)配制10mM的对硝基苯酚酯溶液;(2)在1mL反应体系中加入940μL Tris-HCl buffer(50mM,pH 8.0),40μL乙醇,10μL对硝基苯酚酯溶液(10mM),10μL浓度为0.5mg/mL酯酶WDEst17纯酶液(即步骤4.2制备得到的纯化的酯酶WDEst17);(3)于35℃条件下,4min后,410nm测定吸光度。
酶活单位定义:1min内水解对硝基苯酚酯,释放1μmol对硝基苯酚所需的酶量定义为一个酶活单位。
实施例5:酯酶WDEst17的酶学性质
5.1水解不同长度的对硝基苯酚酯
按照4.3的测定条件,比较酯酶WDEst17作用不同长度的对硝基苯酚酯(C2-C8),结果如图2,说明酯酶WDEst17对长链对硝基苯酚酯基本上没有水解活性,而对于短链对硝基苯酚酯的作用效果较好,最佳的底物是C2,即对硝基苯酚乙酸酯。
5.2最适pH和pH稳定性
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4.3中测定条件所述的缓冲液(Tris/HCl buffer)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WDEst17的酶活力的影响(底物为对硝基苯酚乙酸酯),结果说明酯酶WDEst17酶活在Tris/HCl pH为8.5时活性最高(图3),pH高于8.5、小于8.5活性都会急剧下降。
5.3最适温度和温度稳定性
在pH 8.5、50mM Tris/HCl作为缓冲溶液,按4.3中的反应混合液(反应混合液包括940μL pH 8.5、50mM Tris/HCl缓冲液,40μL乙醇,10μL对硝基苯酚酯溶液(10mM);以对硝基苯酚乙酸酯作为底物)置于不同的温度(25~60℃)下处理1h后,加入等量的酯酶WDEst17(均加入10μL浓度为0.5mg/mL酯酶WDEst17纯酶液),在各自的温度下反应4min,405nm测定酶活。结果说明,酯酶WDEst17最适反应温度在40℃(图4)。
将酯酶WDEst17在25~60℃条件下处理40min后,于30℃,pH 8.5、50mM Tris/HCl的缓冲溶液中,按4.3测定方法(以对硝基苯酚乙酸酯作为底物)测定酯酶WDEst17酶活。结果说明,酯酶WDEst17在30℃以下的稳定性最好,随着温度升高,稳定性逐渐降低,60℃处理40min后酶活残余约40%(图4)。
5.4NaCl浓度对WDEst17酶活性的影响
将酯酶WDEst17分别加入到含有不同浓度的NaCl的pH8.5、50mM Tris/HCl的缓冲溶液中,按4.3测定方法(以对硝基苯酚乙酸酯作为底物,pH 8.5、50mM Tris/HCl作为缓冲溶液)在35℃下反应4min,405nm测定酶活。结果说明,在NaCl浓度不超过4M条件下,WDEst17的酶活随着NaCl浓度的增加而提高,当NaCl浓度为4M时,酯酶WDEst17的相对酶活达到165.42%(图5)。以上结果说明,酯酶WDEst17是一个对钠盐具有非常好的耐受性的酯酶。
5.5金属离子抑制
以50mM pH8.5的Tris/HCl为溶剂配制不同金属离子溶液,每种金属离子浓度均为1mM,将酯酶WDEst17在各种金属离子溶液中4℃下处理12h;以不加金属离子的50mM、pH8.5的Tris/HCl溶液为对照(Control)。再按照4.3中的测定方法(以对硝基苯酚乙酸酯作为底物,pH 8.5、50mM Tris/HCl作为缓冲溶液)测定酶活,结果见表3,大部分已测得金属离子对酯酶WDEst17的水解活性都产生了不同程度的抑制,尤其是Cu2+和Zn2+,酶活分别为60.45±6.89%、46.98±6.59%。
表3金属离子对酯酶WDEst17酶活力的影响
5.6有机溶剂和表面活性剂对酯酶WDEst17酶活性的影响
将酯酶WDEst17分别加入到表4中的有机溶剂和表面活性剂(以pH 8.5、50mMTris/HCl为溶剂配制)中处理12h(对照为蒸馏水,其他溶液的浓度为体积分数),然后按照4.3的测定方法(以对硝基苯酚乙酸酯作为底物,pH 8.5、50mM Tris/HCl作为缓冲溶液)测定酶活。结果表明正癸烷、Tween-20和Tween-80能极大地促进酯酶WDEst17酶活,最高达到137.37±1.77%。
表4有机溶剂和表面活性剂对酯酶WDEst17酶活性的影响
实施例6:酯酶WDEst17在拆分(±)-3-羟基丁酸乙酯中的应用
本法采用在水相中拆分(±)-3-羟基丁酸乙酯。
在优化的条件下,即在0.5mL 50mM、pH8.5的Tris/HCl缓冲溶液中,加入终浓度3.5×103U/L的酯酶WDEst17,于35℃,200rpm条件下,拆分5mM的(±)-3-羟基丁酸乙酯,在3.5h内可得到光学纯度为99%的(R)-3-羟基丁酸乙酯(图6)。
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WDEst17及其编码基因和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 750
<212> DNA
<213> 囊孢菌 NRRL18085(Dactylosporangium aurantiacum subsp. HamdenensisNRRL18085)
<400> 1
gtgagcatcc cgcagacaac cgcgccgtgg ttcgaccagc ggttccggtc ggtcaccgcg 60
acccacagcc tctactgctt cccgttcgcg ggcggctccg ccacctacta cgccgcctgg 120
gagaagtact tcaccgggcg gatcgagctg gtgccggtgc agctgccggg gcgcggcgcc 180
cggatggcgg agcagcccgt cagtgacctg gccggcctcg ccgacgacct cgccgaggtg 240
atcgacggcg agccgaccga gaccgtgctc ttcgggcaca gcatgggcgc catcctcgcc 300
ttcgaggtcg cccgccggct caagaccgcc gggcgtcccg tgcggcacct gttcgtgacg 360
ggccggcccg cgccgccgat cgtgcgcccg cgggagccgg tcagcgacct gccgcgggcc 420
gagttcatcg agatgctgcg cgattacggc gccgccgaca acgcggtctt cgagcacgac 480
gacctgctgg acctgctgat gccgatgctc cgggccgact tctcgatgat cgagcgctac 540
cggatggcgc ccgggccgcg gctgtcgtgc ccggtcaccg cctggtgcgg cgacagcgac 600
ccgggcgtgc cgccgtcggc gatggccccg tggggcgagc agacgtcggg ggcgttcgcg 660
ctctcggtcc tgcccggcgg gcacttcttc ctcaccgagc accacgcccg gatcgtgcgg 720
gaggtccacg ccgcactccg tggcgtctga 750
<210> 2
<211> 249
<212> PRT
<213> 囊孢菌 NRRL18085(Dactylosporangium aurantiacum subsp. HamdenensisNRRL18085)
<400> 2
Val Ser Ile Pro Gln Thr Thr Ala Pro Trp Phe Asp Gln Arg Phe Arg
1 5 10 15
Ser Val Thr Ala Thr His Ser Leu Tyr Cys Phe Pro Phe Ala Gly Gly
20 25 30
Ser Ala Thr Tyr Tyr Ala Ala Trp Glu Lys Tyr Phe Thr Gly Arg Ile
35 40 45
Glu Leu Val Pro Val Gln Leu Pro Gly Arg Gly Ala Arg Met Ala Glu
50 55 60
Gln Pro Val Ser Asp Leu Ala Gly Leu Ala Asp Asp Leu Ala Glu Val
65 70 75 80
Ile Asp Gly Glu Pro Thr Glu Thr Val Leu Phe Gly His Ser Met Gly
85 90 95
Ala Ile Leu Ala Phe Glu Val Ala Arg Arg Leu Lys Thr Ala Gly Arg
100 105 110
Pro Val Arg His Leu Phe Val Thr Gly Arg Pro Ala Pro Pro Ile Val
115 120 125
Arg Pro Arg Glu Pro Val Ser Asp Leu Pro Arg Ala Glu Phe Ile Glu
130 135 140
Met Leu Arg Asp Tyr Gly Ala Ala Asp Asn Ala Val Phe Glu His Asp
145 150 155 160
Asp Leu Leu Asp Leu Leu Met Pro Met Leu Arg Ala Asp Phe Ser Met
165 170 175
Ile Glu Arg Tyr Arg Met Ala Pro Gly Pro Arg Leu Ser Cys Pro Val
180 185 190
Thr Ala Trp Cys Gly Asp Ser Asp Pro Gly Val Pro Pro Ser Ala Met
195 200 205
Ala Pro Trp Gly Glu Gln Thr Ser Gly Ala Phe Ala Leu Ser Val Leu
210 215 220
Pro Gly Gly His Phe Phe Leu Thr Glu His His Ala Arg Ile Val Arg
225 230 235 240
Glu Val His Ala Ala Leu Arg Gly Val
245
Claims (9)
1.一种酯酶WDEst17,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WDEst17的酯酶基因WDEst17。
3.根据权利要求2所述的酯酶基因WDEst17,其特征在于,所述的酯酶基因WDEst17的核苷酸序列如SEQ ID NO.1所示。
4.一种含有权利要求2所述的酯酶基因WDEst17的重组表达载体。
5.一种含有权利要求2所述的酯酶基因WDEst17的基因工程菌。
6.权利要求1所述的酯酶WDEst17在制备(R)-3-羟基丁酸乙酯中的应用。
7.根据权利要求6所述的应用,其特征在于,所述的应用为酯酶WDEst17在拆分(±)-3-羟基丁酸乙酯制备得到(R)-3-羟基丁酸乙酯中的应用。
8.根据权利要求7所述的应用,其特征在于,所述的应用为取酯酶WDEst17于pH为6.0~9.0的缓冲液中,再加入(±)-3-羟基丁酸乙酯,进行反应,得到(R)-3-羟基丁酸乙酯。
9.根据权利要求8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。
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CN106085985A (zh) * | 2016-07-27 | 2016-11-09 | 中国科学院南海海洋研究所 | 一种酯酶WDEst9及其编码基因和应用 |
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CN106085985A (zh) * | 2016-07-27 | 2016-11-09 | 中国科学院南海海洋研究所 | 一种酯酶WDEst9及其编码基因和应用 |
Non-Patent Citations (3)
Title |
---|
WANG,Y.等: "Dactylosporangium aurantiacum subsp. hamdenensis WDEst17 gene, complete cds,GenBank: KY321752.1", 《GENBANK》 * |
WANG,Y.等: "WDEst17 [Dactylosporangium aurantiacum subsp. hamdenensis],GenBank: ATO93963.1", 《GENBANK》 * |
YILONG WANG 等: "Functional characterization of salt‐tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)‐3‐hydroxybutyrate", 《CHIRALITY》 * |
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