CN101035894A - 用脂肪酶水解酯键 - Google Patents
用脂肪酶水解酯键 Download PDFInfo
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- CN101035894A CN101035894A CNA2005800344056A CN200580034405A CN101035894A CN 101035894 A CN101035894 A CN 101035894A CN A2005800344056 A CNA2005800344056 A CN A2005800344056A CN 200580034405 A CN200580034405 A CN 200580034405A CN 101035894 A CN101035894 A CN 101035894A
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- China
- Prior art keywords
- lipase
- seq
- glu
- gly
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明人已鉴定了厌氧嗜热细菌中具有脂肪酶活性的多肽。因此,本发明提供水解底物中的酯键的方法,包括用特定脂肪酶(具有脂肪酶活性的多肽)处理所述底物。本发明还提供用于所述方法的脂肪酶和编码所述脂肪酶的多核苷酸。
Description
发明所属领域
本发明涉及用脂肪酶处理而水解底物中的酯键的方法。还涉及用于所述方法的脂肪酶和编码所述脂肪酶的多核苷酸。
发明背景
菌株热硫化氢热厌氧杆菌(Thermoanaerobacterthermohydrosulfuricus)DSM 7021、布氏嗜热厌氧杆菌(Thermoanaerobacterbrockii)subsp.brockii DSM 1457和Caldanaerobacter subterraneus subsp.tengcongensis DSM 15242是公众可获得的。
Thermoanaerobacter tengcongensis的全部基因组序列已经公开(Bao etal.,Genome Res.12,689-700,2002(GenBank AE008691))。SWALL:Q8R921显示258个氨基酸的序列,描述为″hydrolases of the alpha/beta superfamily″(AAM25001.1)。
发明概述
发明人已鉴定了厌氧嗜热细菌中具有脂肪酶活性的多肽。
因此,本发明提供水解底物中的酯键的方法,包括用脂肪酶(具有脂肪酶活性的多肽)处理所述底物。本发明还提供用于所述方法的脂肪酶和编码所述脂肪酶的多核苷酸。
所述多肽可以具有序列SEQ ID NO:2或4,或者与其中之一具有高度同一性(identity),或者可以通过取代、缺失、和/或插入一个或多个氨基酸而衍生于其中之一。
所述多核苷酸可以具有序列SEQ ID NO:1或3,或者与其中之一具有高度同一性或者可以与其杂交,或者可以是存在于菌株DSM 7021、DSM1457、或DSM 15242中的基因组的一部分,其可以用引物对LipCtTb-ForLipCtTb-Rev(SEQ ID NO:5-6)或用引物对LipTtg-for和LipTtg-rev(SEQ IDNO:7-8)扩增。
发明详述
基因组DNA来源
编码脂肪酶的DNA序列可以从Caldanaerobacter、热厌氧杆菌属(Thermoanaerobacter)、热厌氧菌属(Thermoanaerobium)或梭菌属(Clostridium)的厌氧嗜热菌株分离。因此,序列表中所示DNA序列和多肽从以下所述生物分离。如以下所指出的,从两种生物获得了相同的序列。
系统分类 | 基原异名 | 保藏号 | DNA序列 | 多肽序列 |
热硫化氢热厌氧杆菌 | 热硫化氢梭状芽孢杆菌(Clostridiumthermohydrosulfuricum) | DSM 7021 | SEQ IDNO:1 | SEQ IDNO:2 |
布氏嗜热厌氧杆菌subsp.brockii | 布氏嗜热厌氧细菌(Thermoanaerobiumbrockii) | DSM 1457 | SEQ IDNO:1 | SEQ IDNO:2 |
Caldanaerobactersubterraneus subsp.tengcongensis | Thermoanaerobactertengcongensis | DSM15242 | SEQ IDNO:3 | SEQ IDNO:4 |
所述菌株商业上可由DSMZ-Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH,Mascheroder Weg 1b,38124 Braunschweig,德国获得。
序列同一性
本发明的多肽和多核苷酸与SEQ ID NO:1-4中任一个可以具有70%以上、80%以上、90%以上或95%以上的同一性。可以如US 6162628所述完成两个序列的比对和氨基酸或核苷酸同一性的计算。
脂肪酶特性
脂肪酶对大量酯,特别是水不溶性底物包括三酰基甘油(甘油三酯)和对硝基苯基棕榈酸酯是活性的。脂肪酶对甘油三酯中的2-位酯键显示异乎寻常的偏好。
脂肪酶是来自消旋酯的S-对映体选择性(anantioselective)、构成性(S)-醇。
工业用途
脂肪酶可以用作去垢剂的添加剂,如WO 2002062973所述。
脂肪酶可用于从甘油三酯和甘油生产甘油二酯。
脂肪酶可通过水解消旋酯混合物,用于对映体选择性酯水解。一个例子是(S)-(-)-3-丁炔-2-醇,其可用作药物中间体。
可通过将脂肪酶添加到生面团用于烘烤制备基于生面团的产品,特别是焙烤产品,如WO 9826057、WO 0032758、WO 2003100044、WO2004064537或丹麦专利申请PA 2003 01762中所述。
脂肪酶可用于甘油三酯的酯交换,如WO 9522606或WO 9933964所述。
脂肪酶可用于酯合成例如生物柴油(biodiesel)的生产。
脂肪酶可用于聚合反应例如二酸和二醇的缩合。
实施例
实施例1:完整脂肪酶基因的扩增
用如下所示引物扩增来自热硫化氢热厌氧杆菌DSM7021和布氏嗜热厌氧菌subsp.brockii DSM 1457的完整脂肪酶基因:
名称 | SEQ ID | 序列 | 长度 | Tm | %GC |
LipCtTb-For | SEQ ID NO:5 | 5’-ATGCAAAAGGCTGTTGAAATTAC-3’ | 23 | 55.3℃ | 34.8% |
LipCtTb-Rev | SEQ ID NO:6 | 5’-TTATCCCTTTAACAATTCCTTTTTG-3’ | 25 | 54.8℃ | 28.0% |
用以下所示引物扩增来自Caldanaerobacter subterraneus subsp.tengcongensis DSM 15242的完整脂肪酶基因:
名称 | SEQ ID | 序列 | 长度 | Tm | %GC |
Lip Ttg-for | SEQ ID NO:7 | 5’-ATGCAGAAGGCTGTAGAGTTTAC-3’ | 23 | 58.9℃ | 43.5% |
Lip Ttg-rev | SEQ ID NO:8 | 5’-TTATCCCTTTAATTCTCTTTCAAAG-3’ | 25 | 54.8℃ | 28.0% |
实施例2:从热硫化氢热厌氧杆菌生产脂肪酶
在含有20ml相应液体培养基的50ml瓶子中,将菌株DSM 7021于65℃培养在旋转震荡器(160rpm)上32h。
基础培养基包括(每升):NaCl,3.0g;KH2PO4,2.5g;NaH2PO4,0.8g;MgSO4×7H2O,0.1g;CaCl2×2H2O,0.05;FeCl3×6H2O,0.01g;(NH4)2SO4,1.5g;SrCl2×6H2O,0.03g;H3BO3,0.03g;Na2WO4,0.03g;酵母提取物,1.5g;蛋白胨,1.5g;痕量元素溶液141,1ml,维生素溶液141,1ml;刃天青,0.001g;NaHCO3,1.0g;半胱氨酸,0.3g;pH 7.2。刚要接种前,将1mg Na2S×9H2O注射在20ml容量的瓶子中。此外,将0.05g Na2S2O3添加到带有培养基的瓶子中。
将所述菌株培养在上述复合培养基上,发现在含有作为碳源和能量来源的0.5%葡萄糖的培养基中,在没有脂肪酶诱导剂的情况下合成胞外脂肪酶。酶的生产与生长并行,在65℃和pH 7.2下生长32h后达到其最大值(12U/l)。发现大约89%的酶分泌到培养液中。在典型脂肪酶诱导剂橄榄油和Tween 80存在下,脂肪酶活性没有增加。
所述生物被鉴定为以对硝基苯基棕榈酸酯还有橄榄油为底物的脂肪酶生产菌。
实施例3:从热硫化氢热厌氧杆菌纯化脂肪酶
实施例1的培养肉汤中的胞外脂肪酶通过三步程序纯化。第一步为疏水性相互作用层析。脂肪酶在1-0M KCl梯度内没有从Phenyl-Sepharose柱上解除吸附,但在10-12%二甲亚砜下洗脱,从大批其它蛋白质良好地分离。
疏水性相互作用层析后获得的脂肪酶溶液上样在所使用的hydroxilapatite柱上,这是第二个纯化步骤。脂肪酶在接近磷酸钠缓冲液梯度(220至250mM范围)中点洗脱。活性级分进一步加到凝胶过滤柱。最终的凝胶过滤产生三个峰值。酶活性存在于第二个主峰中。脂肪酶相对于粗提物纯化大约133.5倍,产率为10.2%。纯化脂肪酶的特异活性为12.3U/mg。
在通常的还原性条件下对热预处理的纯化脂肪酶进行SDS-PAG电泳产生一个相对分子量大约34.2kDa的蛋白质条带。不存在(absence)去垢剂时,脂肪酶在天然条件下通过天然PAG电泳迁移,显示69kDa的单一条带,这与凝胶过滤测定的68.5kDa分子量一致。
测定了所述条带在天然PAG电泳后的活性,α-乙酸萘酯(naphtyl acetate)活性与考马斯亮蓝R-250染色的蛋白质条带相符。SDS-PAG电泳后的纯化脂肪酶的解脂活性能够通过用Triton X-100去除SDS恢复。这表明所述酶也是活性单体。在未经Triton X-100处理冲洗的条件下检测了所有脂肪酶活性。
实施例4:从热硫化氢梭状芽孢杆菌、布氏嗜热厌氧杆菌和Thermoanaerobacter tengcongensis克隆脂肪酶
菌株、质粒和培养基
采用标准技术用E.coli TOP-10(Invitrogen)或TunerTM(DE3)pLacI(Novagen)实施细菌克隆试验。E.coli TOP-10与适于蓝/白测试(blue/white assays)的克隆载体pCR 2.1-TOPO(Invitrogen)组合使用。E.coli TunerTM(DE3)pLacI与包含T7启动子的载体pETBlue-1(Novagen)组合使用,以克隆和表达脂肪酶基因。Lura-Betani培养基用于E.coli细胞。以下述浓度添加抗生素:羧苄青霉素,50μg/ml;四环素,15μg/ml;氯霉素,34μg/ml;卡那霉素,50μg/ml。
N末端氨基酸序列分析
测定了来自热硫化氢梭状芽孢杆菌和布氏嗜热厌氧杆菌的脂肪酶N末端氨基酸序列的达17个氨基酸残基。所述N末端氨基酸序列如SEQ ID NO:2的残基1-17所示,是100%一致的。来自热硫化氢梭状芽孢杆菌和布氏嗜热厌氧菌subsp.brockii的脂肪酶N末端序列与来自Thermoanaerobactertengcongensis(菌株MB4T,Genbank登录号AE008691)的水解酶N末端序列(AAM25001.1)对比显示88%的同源性。
数据库检索和计算分析
脂肪酶基因序列利用Entrez检索系统在the National Center forBiotechnology Information(NCBI)获得。用BLASTP于NCBI获得与脂肪酶基因序列具有同源性的区域。脂肪酶基因与Thermoanaerobacter tengcongensis水解酶基因的比对利用CLUSTALW在eBioinformatics进行。
实施例5:从热硫化氢热厌氧杆菌和布氏嗜热厌氧杆菌PCR扩增脂肪酶并在pCR 2.1-TOPO载体中克隆
脂肪酶基因片段的PCR扩增
利用QIAGEN Genomic DNA Kit从热硫化氢热厌氧杆菌和布氏嗜热厌氧杆菌菌株提取DNA,以从细菌分离基因组DNA。染色体DNA用作模板,利用所有可能的引物(表1)组合扩增脂肪酶片段。依照下述条件利用Biometra热循环仪(T 3000型热循环仪)实施PCR反应:模板DNA添加在由1×PCR缓冲液、3mM MgCl2、0.2mM dNTPs、和0.15U μl-1Taq聚合酶组成的缓冲液中,至终浓度1.5ng μl-1。以终浓度3pmol μl-1添加正向和反向引物。实施如下的二十五个热循环:Seq1(94℃,20s)、Seq2(55℃,40s)、Seq3(72℃,1min)。
用于PCR筛选的寡核苷酸:
SEQ ID | 名称 | 序列 | 功能 |
NO:9NO:10NO:11NO:12NO:13NO:14NO:15NO:16NO:17NO:18NO:19NO:20 | LF/NT/CTTLF/NT/ATTLF/OAH/CATF/CRI/TTCF/CRI/GGAF/CRI/GCGR/CRI/TCCR/CRI/AAAR/CRI/TCTR/CRII/CAAR/CRII/AAGR/CTI/TTT | CTTAAGGGGGATGTTGCATCTTCATTAAGGGGGGTACTGCATCTGCATGGGTTTACCGGAAATAAAGTGGTTCAGGCGAAAGCGACGGAGGGAACAGGTGAAAGTGATGGAGAATTGCGGTGAAAGTGATGGAGACTTTTCCGTCGCTTTCGCCTGAACAAATTCTCCATCACTTTCACCTGTTCCTCTCCATCACTTTCACCGCTGTCCTCCCATGCTGAGTCCCAATCCTCCCATGCTGAAGCCAAGTTTTGTATGGTCCGCTCCTTCTAT | 正向正向正向正向正向正向反向反向反向反向反向反向 |
脂肪酶基因片段的克隆
采用标准克隆技术(TA Cloning Kit,Invitrogen)将选择的PCR扩增物连接到载体pCR2.1-TOPO中,并在感受态TOP-10 E.coli细胞中转化。按照传统蓝/白筛选选择阳性克隆。用NucleoSpin Plasmid Kit(Macherey-Nagel)分离质粒。
用于鉴定与脂肪酶有同源性的序列的PCR筛选
在NCBI利用BLASTN分析所述序列。用引物F/CRI/GCG和R/CRII/CAA(SEQ ID NO:14和18),来自热硫化氢梭状芽孢杆菌的gDNA用作模板,扩增与来自Thermoanaerobacter tengcongensis的水解酶α/β超家族核苷酸序列有84%同一性的142-bp片段:
TGCGGTGAAAGTGATGGAGACTTTAGTGAAATGACATTTAGCAGTGAATTGGAAGATGCAAGACAAATTTTAAAGTTTGTGAAAGAGCAACCTACGACTGACCCTGAGAGAATAGGACTACTTGGGACTCAGCATGGGAGGA(SEQ ID NO:21)
用引物F/CRI/GCG和R/CRII/AAG(SEQ ID NO:14和19),来自布氏嗜热厌氧杆菌的gDNA用作模板,扩增与来自Thermoanaerobacter tengcongensis的水解酶α/β超家族核苷酸序列有81%同一性的141-bp片段:
TGCGGTGAAAGTGATGGAGACTTTAGTGAAATGACATTTAGCAGTGAATTGGAAGATGCAAGACAAATTTTAAAGTTTGTGAAAGAGCAACCTACGACTGACCCTGAGAGAATAGGACTACTTGGCTTCAGCATGGGAGGA(SEQ ID NO:22)
实施例6:反向PCR
用来自热硫化氢热厌氧杆菌和布氏嗜热厌氧杆菌sp.brockii的DNA实施反向PCR。
用限制酶消化gDNA
允许扩增已知序列边界外的DNA片段的反向PCR技术用于完成脂肪酶基因。在1×RE-缓冲液B中,每个反应分别用20U的限制酶BamHI和HindIII将基因组DNA(~1.4μg)消化为小片段。所述消化反应于37℃在300μl总体积中进行24h。限制性反应液用1/10体积3M NaOAc和2.5体积的纯乙醇于-20℃沉淀2h,4℃下,13000rpm离心30min。丸状沉淀在室温下风干20min,然后重悬在100μl ddH2O中。
DNA片段的自连接
将0.5μl(200U)T4连接酶(MBI,BioLabs)、30μl T4连接酶-缓冲液(MBI)和10mM ATP添加到消化过的DNA。所述连接反应于4℃实施48h。连接反应液用1/10体积3M NaOAc和2.5体积的纯乙醇于-20℃沉淀2h,4℃下,13000rpm离心30min。丸状沉淀在室温下风干20min,然后重悬在100μl ddH2O中。
用构建的引物实施反向PCR
环状DNA片段用作模板,用所有可能的引物(表2)组合扩增脂肪酶片段。依照下述条件利用Biometra热循环仪(T 3000型热循环仪)实施PCR反应:模板环状DNA片段添加在由1×PCR缓冲液、3mM MgCl2、0.2mM dNTPs、和0.15U μl-1Taq聚合酶组成的缓冲液中,至终浓度~1.35ng μl-1。以终浓度3pmol μl-1添加正向和反向引物。如下进行三十个热循环:Seq1(94℃,20s)、Seq2(55℃,45s)、Seq3(72℃,2min)。
表2.用于反向PCR的寡核苷酸
订正了表2的最后一栏。
SEQ ID | 名称 | 序列 | 长度 | Tm | %GC |
23 | 1F_Inv2CT | GACATTTAGCAGTGAATTGGAAGATGC | 27 | 62℃ | 41% |
24 | 2F_Inv2CT | TTTGTGAAAGAGCCTACGACTGACC | 25 | 63℃ | 48% |
25 | 3R_Inv2CT | GCACTTTACCCTTAACATCATCAGGC | 26 | 63℃ | 46% |
26 | 4R_Inv2CT | GACTCTACTTTATTGCCTGTAAAACCG | 27 | 62℃ | 41% |
程序ContigExpressTM(Vector NTI,InforMax,Inc.,North Bethesda,Maryland针对Mac OS用户开发的软件包)用于分析序列,并完成脂肪酶基因。
实施例7:从热硫化氢梭状芽孢杆菌、布氏嗜热厌氧杆菌和Thermoanaerobacter tengcongensis表达脂肪酶
AccepTor Vector Kit(Novagen)用于pETBlue-1载体中T7lac启动子控制下IPTG可诱导的脂肪酶基因表达。设计所述试剂盒用于利用Taq DNA聚合酶简化所产生的PCR产物的克隆,其在其反应产物上留下单一的3’-dA突出端(overhang)。线性化pETBlue-1载体包含单一的3’-dU突出端,其适合于这些产物的直接连接,无需中间反应。转化后,细菌复制质粒时,dU残基被dT残基替换。
NovaBlue宿主用于初始克隆并验证pETBlue-1载体中的构建体,然后将所述重组质粒转化到Tuner(DE3)pLacI菌株中,用于在E.coli中表达。
插入物的制备
PCR扩增脂肪酶基因。如以上实施例1所述,染色体DNA用作模板,利用构建的引物(对于来自热硫化氢梭状芽孢杆菌和布氏嗜热厌氧杆菌的脂肪酶基因为SEQ ID NO:5-6;对于来自Thermoanaerobacter tengcongensis的脂肪酶基因为SEQ ID NO:7-8)扩增完整的脂肪酶基因。依照下述条件实施PCR反应:将模板DNA添加在由1×PCR-缓冲液、3mM MgCl2、0.2mMdNTPs组成的缓冲液中,至终浓度~1.5ng μl-1。热启动后添加0.2U μl-1的Hifi-聚合酶。以终浓度3pmol μl-1添加正向和反向引物。如下实施二十五个热循环:Seq1(94℃,15s)、Seq2(50℃,30s)、Seq3(68℃,1min 20s)。用NucleoSpinExtraction Kit(Macherey Nagel)纯化PCR产物。
连接
在10μl的总体积中将50ng μl-1pETBlue-1载体与~50ng扩增产物连接。反应物于16℃保温1h。
NovaBlue SinglesTM感受态细胞的转化
为了进行转化,将1μl的连接反应物直接添加到NovaBlue Singles感受态细胞。在42℃水浴中用“热休克”法转化刚好30秒。通过蓝/白筛选选择羧苄青霉素抗性标记的阳性克隆。
用pETBlue-1重组体转化TunerTM(DE3)pLacI感受态细胞
将从蓝/白筛选宿主NovaBlue鉴定分离的pETBlue-1重组体转化到Tuner(DE3)pLacI表达宿主中,用于基于IPTG的诱导。该菌株携带T7 RNA聚合酶基因的染色体拷贝,设计用于在pETBlue-1载体中T7lac启动子的控制下实施可用IPTG诱导的目标基因表达。将~1ng μl-1的pETBlue-1重组体质粒直接添加到感受态细胞。在42℃水浴中用“热休克”法转化刚好30秒。
生长和诱导
制备了3ml(DE3)pLacI表达宿主菌株中pETBlue-1重组体的发酵剂培养物。生长LB培养基包含羧苄青霉素,50μg ml-1;氯霉素,34μg ml-1和1%葡萄糖。将100ml用发酵剂培养物接种的培养基孵育至OD600为1.0。然后添加1mM IPTG。培养物于37℃振动孵育4h,以充分诱导。
实施例8:热硫化氢热厌氧杆菌脂肪酶在枯草芽孢杆菌(Bacillussubtilis)中的表达。
线性整合载体系统用于所述基因的表达克隆。所述线性整合构建体是PCR融合产物,由两个带有强启动子和氯霉素抗性标记的枯草芽孢杆菌同源染色体区域之间的基因融合而成。所述融合物通过SOE PCR(Horton,R.M.,Hunt,H.D.,Ho,S.N.,Pullen,J.K.and Pease,L.R.(1989)Engineering hybridgenes without the use of restriction enzymes,gene splicing by overlap extensionGene 77:61-68)制得。SOE PCR法也描述于专利申请WO 2003095658)。所述基因在三重启动子系统(如WO 99/43835所述)的控制下表达,所述三重启动子系统由来自地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因(amyL)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶基因(amyQ)、和包括稳定序列的苏云金芽孢杆菌(Bacillus thuringiensis)cryIIIA启动子组成。编码氯霉素乙酰基-转移酶的基因用作标记(Described in eg.Diderichsen,B.;Poulsen,G.B.;Joergensen,S.T.;A useful cloning vector for Bacillus subtilis.Plasmid 30:312(1993))。在芽孢杆菌染色体上,最终的基因构建体通过同源重组整合到果胶酸裂合酶位点中。
用QIAmp Tissue Kit(Qiagen,Hilden,德国)分离热硫化氢热厌氧杆菌的染色体DNA。PCR扩增前3个片段:用特异性引物oth296(SEQ ID NO.:27)和oth297(SEQ ID NO.:28)扩增来自热硫化氢热厌氧杆菌的基因组DNA上的基因片段。从菌株iMB1361(专利申请WO 2003095658中所述)的基因组DNA,用引物260558(SEQ ID NO.:29)和iMB1361Uni1(SEQ ID NO.:30)扩增上游侧翼片段,用引物260559(SEQ ID NO.:31)和DwC 1361(SEQ IDNO.:32)扩增下游侧翼片段。
用校对聚合酶(Proof Start Polymerase(Qiagen))扩增所述基因片段。用High Fidelity PCR System”(Boehringer Mannheim,德国)扩增两个侧翼DNA片段。依照标准程序(按照厂商的推荐)完成PCR反应。PCR条件如下:94℃2min,然后10个循环的(94℃15秒、50℃45秒、68℃4min),接下来20个循环的(94℃15秒、50℃45秒、68℃4min(每个循环+20秒延伸)),68℃10min一个循环结束。
将所得的3个片段等摩尔比混合,在以下条件下实施新的PCR反应:最初94℃2min.,然后10个循环的(94℃15秒、50℃45秒、68℃5min.),10个循环的(94℃15秒、50℃45秒、68℃8min.),15个循环的(94℃15秒、50℃45秒、68℃8min.,每个循环额外加20秒)。第一个循环后,添加两种末端引物260558(SEQ ID NO.:29)和260559(SEQ ID NO.:31)(每种20pMol)。将2μl的PCR产物转化到枯草芽孢杆菌中,在含氯霉素(6μg/ml培养基)的LB平板上选择转化体。选择包含所述构建体的克隆用于在液体培养基中发酵,所述构建体不含导致氨基酸改变的突变。
发酵、纯化和活性测试
所述克隆在从-80℃储存物获得的含6micro g/ml氯霉素的LB琼脂板上划线,37℃培养过夜。将菌落转移到500ml振动烧瓶中添加有6micro g/ml氯霉素的100ml LB或PS-1培养基。培养物于30℃ 275rpm振动1或3天。用已在实施例3中描述的方法离心细胞,从上清纯化酶。如实施例9中所述测定活性。
实施例9:脂肪酶的特性
温度的影响
纯化的SEQ ID NO:2和4的脂肪酶均在75℃(10分钟反应)显示出最佳活性,在85-90℃以上活性很小。
pH的影响
纯化的SEQ ID NO:2的脂肪酶于pH 8.0显示最佳活性,于pH 6.5-9.0显示>80%的活性,低于pH 6.0和高于pH 10.0几乎没有活性。SEQ ID NO:4的脂肪酶于pH 7.0显示最佳活性,于pH 6.5-9.0显示>60%的活性,低于pH6.0和高于pH 11.0几乎没有活性。
金属离子的影响
在存在达10mM的以下金属离子时,脂肪酶活性几乎没有改变:Na+、K+、Ca2+、Cu2+、Ag+、Mg2+、Mn2+、Sr2+、Rb+、Co2+、Ni2+和Al3+。以下离子降低所述活性:Zn2+、Fe2+、Fe3+和Cr3+。
去垢剂成分的影响
30℃下与以重量计达10%的多种化合物一起孵育1.5小时后测试脂肪酶的活性。与CHAPS(3-[(3-胆酰胺丙基)二甲铵基]-1-丙磺酸)、PVA(聚乙烯醇)和EDTA(乙二胺四乙酸)一起孵育后,脂肪酶保留>75%的活性。与Tween-20和Tween-80或Triton X-100一起孵育降低所述活性。SDS引起完全的酶抑制。
溶剂的影响
浓度以体积计达50%的下述溶剂对SEQ ID NO:2的脂肪酶的活性几乎没有影响:叔丁醇、乙醇、乙腈、异丙醇、吡啶、DMSO、丙酮、二甲基甲酰胺和甲醇。
抑制剂的影响
浓度达10mM的下述化合物对SEQ ID NO:2和4的脂肪酶的活性几乎没有影响:b-巯基乙醇、尿素、pHMB、盐酸胍、DTT和碘代-醋酸酯。0.1-1mM的PMSF和Pefablock同时灭活两种脂肪酶。
实施例10:来自热硫化氢热厌氧杆菌的脂肪酶的底物特异性
对pNP-酯的底物特异性
通过与作为底物的1mM(pH 8.0)的多种pNP-酯于70℃反应10min,测试SEQ ID NO:2的脂肪酶。获得了与SEQ ID NO:4的脂肪酶类似的结果。
pNP-酯 相对活性
pNP-醋酸酯(C2:0) 9
pNP-丁酸酯(C4:0) 57
pNP-己酸酯(C6:0) 81
pNP-辛酸酯(C8:0) 90
pNP-癸酸酯(C10:0) 100
pNP-月桂酸酯(C12:0) 84
pNP-豆蔻酸酯(C14:0) 68
pNP-棕榈酸酯(C16:0) 32
pNP-硬脂酸酯(C18:0) 8
发现所述脂肪酶与链长C6-C14的底物一起具有高活性。
对甘油三酯的底物特异性
通过与作为底物的10mM(pH 8.0)的多种甘油三酯于70℃反应25h,测试SEQ ID NO:2的脂肪酶。获得了与SEQ ID NO:4的脂肪酶类似的结果。
甘油三酯 相对活性
三乙酸甘油酯(C2:0) 5
三丁酸甘油酯(C4:0) 10
三己酸甘油酯(C6:0) 74
三辛酸甘油酯(C8:0) 100
三癸酸甘油酯(C10:0) 15
三月桂酸甘油酯(C12:0) 11
三肉豆蔻酸甘油酯(C14:0) 8
三棕榈酸甘油酯(C16:0) 22
三硬脂酸甘油酯(C18:0) 9
三油酸甘油酯(C18:1) 12
橄榄油 10
发现脂肪酶对C6和C8具有良好的活性,但对其它链长的活性低。
实施例11:醇解
测试了由SEQ ID NO:2的脂肪酶催化的多种三酰甘油酯的醇解。所述脂肪酶醇解了所有底物。三硬脂酸甘油酯作为底物时观察到最高产率(转化率67%)。对于其它底物,转化率高于40%。所述脂肪酶以最高速率催化从三酰甘油酯合成1,3-二酰甘油酯和1-和3-单酰甘油酯。没有产生sn2-单酸甘油酯。所述酶显示出对2-位酯键异乎寻常的偏好。所述蛋白质的2-位特异性随酯键的长度而增强。
实施例12:对映体选择性
发现SEQ ID NO:2的脂肪酶对以下四种底物是活性的:1-苯基-1-乙基-醋酸酯、1-苯基-2-丙基-醋酸酯、丁炔醇醋酸酯(butynol acetate)和丁炔醇丁酸酯,并且对后两种是相对S-对映体选择性的。用这两种底物显示了所述脂肪酶的(S)-偏好和可接受的E-值(分别为16.7和9.2)。形成(S)-醇。与丁炔醇醋酸酯相比,脂肪酶对丁炔醇丁酸酯更具对映体选择性。在24h的反应时间后,随着时间推移,对于所有四种底物转化率均提高,达到20-30%以上。在40h的反应时间后,随着时间推移,对照转化率,所述酶针对两种底物的对映体选择性均降低,对于丁炔醇丁酸酯,从16.7降到8.06,对于丁炔醇醋酸酯,从9.15降到2.65。脂肪酶显示出对(S)-对映体更高的偏好,但随着时间推移,其区分对映体的能力下降。对于其它两种底物,脂肪酶的对映体选择性(E≥1)随时间推移是恒定的。
实施例13:位置特异性
对单酸甘油酯(MG)的位置特异性
发现与单棕榈酰甘油分子中的2-位酯键相比,SEQ ID NO:2的脂肪酶对1-位酯键的水解较弱(小2倍(less than 2 fold))。所述酶显示出对2-位酯键异乎寻常的偏好。
对甘油三酯(TG)的位置特异性
针对下述甘油三酯(TG、三酰甘油酯):三月桂酸甘油酯(C12)、三豆蔻酸甘油酯(C14)、三棕榈酸甘油酯(C16)、三硬脂酸甘油酯(C18)、三油酸甘油酯(C18:1),测试了来自热硫化氢热厌氧杆菌的脂肪酶(SEQ ID NO:2)的位置特异性。将每种TG(3mmol)溶解在有机溶剂(2ml丙酮)中,并于65℃、400rpm预平衡过15min。添加干燥的乙醇(3mmol),并将反应混合物于65℃、400rpm孵育15min。添加脂肪酶(以TG重量为基础10%)以启动反应。在4-ml带螺丝帽的管形瓶中进行反应,并用磁搅拌器混合反应混合物(400rpm)。周期性地取出等份量的反应混合物(20μl),并用氯仿(80μl)稀释为合适的稀释液,接下来通过Iatroscan分析以测定酰基甘油成分(composition)。
反应期间利用Iatroscan分析法(Iatroscan,Iatron Laboratories,Inc.,东京,日本)通过TLC/FID定量测定反应介质的甘油酯成分变化。分析前扫描空白色谱棒(chromarod)。用硼酸(3%)处理色谱棒并干燥5min后,将0.1ml的反应介质(在氯仿中稀释为合适的稀释液)点样到色谱棒上,所点的样在苯∶氯仿∶醋酸(50∶30∶0.5,以体积计)混合物中显影10cm,35min。干燥后,将色谱棒置于110℃烘箱中5min,在160ml/min的氢气流速和2.0l/min的风速下进行扫描,产生色谱。
以下以剩余底物(TG)的%和下述产物的%给出由来自热硫化氢梭状芽孢杆菌的脂肪酶于65℃催化反应7h后的三酰甘油酯醇解结果:脂肪酸(FA)、甘油二酯(DG,1,3-DG自1,2-和2,3-DG分离)、单酸甘油酯(MG,2-MG自1-和3-MG分离)。
TG | FA | DG1,3- | DG1,2-2,3- | MG2- | MG1-3- | |
三月桂酸甘油酯(C12) | 58 | 2 | 12 | 6 | - | 22 |
三豆蔻酸甘油酯(C14) | 66 | 1 | 12 | 2 | - | 19 |
三棕榈酸甘油酯(C16) | 65 | 3 | 13 | 1 | - | 18 |
三硬脂酸甘油酯(C18) | 33 | 10 | 48 | - | - | 9 |
三油酸甘油酯(C18:1) | 63 | 2 | 18 | - | - | 17 |
结果显示所有底物都被所述脂肪酶醇解。三硬脂酸甘油酯作为底物时观察到最高产率(转化率67%)。对于其它底物,转化率高于40%。所述脂肪酶以最高速率催化从三酰甘油酯形成1,3-二酰甘油酯与1-和3-单酰甘油酯。没有产生sn2-单酸甘油酯。所述酶显示出对2-位酯键异乎寻常的偏好。所述蛋白质的2-位特异性随酯键的长度增强。
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<210>23
<211>27
<212>DNA
<213>人工的
<220>
<223>1F_Inv2CT
<400>23
gacatttagc agtgaattgg aagatgc 27
<210>24
<211>25
<212>DNA
<213>人工的
<220>
<223>2F_Inv2CT
<400>24
tttgtgaaag agcctacgac tgacc 25
<210>25
<211>26
<212>DNA
<213>人工的
<220>
<223>3R_Inv2CT
<400>25
gcactttacc cttaacatca tcaggc 26
<210>26
<211>27
<212>DNA
<213>人工的
<220>
<223>4R_Inv2CT
<400>26
gactctactt tattgcctgt aaaaccg 27
<210>27
<211>46
<212>DNA
<213>人工的
<220>
<223>引物oth296
<400>27
tgaaaaaaag gagaggataa agaatgcaaa aggctgttga aattac 46
<210>28
<211>46
<212>DNA
<213>人工的
<220>
<223>引物oth297
<400>28
ggagcggatt gaacatgcga ttatcccttt aacaattcct ttttga 46
<210>29
<211>21
<212>DNA
<213>人工的
<220>
<223>260558
<400>29
gagtatcgcc agtaaggggc g 21
<210>30
<211>29
<212>DNA
<213>人工的
<220>
<223>iMB1361Uni1
<400>30
tctttatcct ctcctttttt tcagagctc 29
<210>31
<211>23
<212>DNA
<213>人工的
<220>
<223>260559
<400>31
gcagccctaa aatcgcataa agc 23
<210>32
<211>23
<212>DNA
<213>人工的
<220>
<223>DwC 1361
<400>32
taatcgcatg ttcaatccgc tcc 23
Claims (6)
1.水解底物中酯键的方法,其包括用脂肪酶处理所述底物,所述脂肪酶:
a)具有与SEQ ID NO:2或4具有至少70%同一性的氨基酸序列;
b)由中等严紧条件下与SEQ ID NO:1或3的互补链杂交的核酸序列所编码;
c)具有能够自SEQ ID NO:2或4通过取代、缺失、和/或插入一个或多个氨基酸而获得的氨基酸序列;或者
d)由存在于菌株DSM 7021、DSM 1457、或DSM 15242中的基因组的脂肪酶编码部分编码,并且能够用SEQ ID NO:5和6或用SEQ ID NO:7和8扩增。
2.权利要求1的方法,其中所述酯键是二级醇酯键,特别是甘油三酯2-位的键。
3.脂肪酶,其
a)具有与SEQ ID NO:2具有至少90%同一性的氨基酸序列;
b)由中等严紧条件下与核酸序列SEQ ID NO:1的互补链杂交的核酸序列所编码;
c)具有能够自SEQ ID NO:2通过取代、缺失、和/或插入一个或多个氨基酸而获得的氨基酸序列;或者
d)由存在于菌株DSM 7021或DSM 1457中基因组的脂肪酶编码部分所编码,并且能够用SEQ ID NO:5和6扩增。
4.编码前述权利要求的脂肪酶的多核苷酸。
5.包含选自以下序列的多核苷酸:
a)编码脂肪酶并与之具有至少90%同一性的多核苷酸;
b)编码脂肪酶并在高严紧条件下与核苷酸SEQ ID NO:1的互补链杂交的多核苷酸;
c)存在于菌株DSM 7021或DSM 1457中的基因组的脂肪酶编码部分,其能够用SEQ ID NO:5和6扩增,或者
d)a)、b)或c)的多核苷酸的互补链。
6.生产多肽的方法,包括:
a)获得编码多肽的多核苷酸,所述多肽具有来自厌氧细菌的脂肪酶的活性;
b)用所述多核苷酸转化芽孢杆菌细胞;
c)在有助于生产所述多肽的条件下培养转化的细胞;和
d)回收所述多肽。
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US (1) | US7972831B2 (zh) |
EP (1) | EP1805302B1 (zh) |
CN (2) | CN102212509A (zh) |
WO (1) | WO2006037334A1 (zh) |
Cited By (1)
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CN102325891A (zh) * | 2008-08-29 | 2012-01-18 | 邦奇油类公司 | 水解酶、编码它们的核酸及制备和使用它们的方法 |
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US7919683B2 (en) | 2006-11-13 | 2011-04-05 | Pioneer Hi-Bred International, Inc. | Cloning and sequencing of the ferulate esterase gene from Lactobacillus buchneri LN4017 |
EP1964512A3 (en) * | 2007-02-28 | 2008-10-29 | Sysmex Corporation | Method of measuring skin conductance, method of analyzing component concentration, skin conductive measuring apparatus, and component concentration analyzer |
CA2699406C (en) | 2007-09-12 | 2019-09-03 | Martek Biosciences Corporation | Biological oils and production and uses thereof |
WO2011082190A1 (en) | 2009-12-28 | 2011-07-07 | Martek Biosciences Corporation | Recombinant thraustochytrids that grow on sucrose, and compositions, methods of making, and uses thereof |
CN107760644A (zh) * | 2017-12-06 | 2018-03-06 | 慎东 | 一种饲用大肠杆菌高密度发酵的培养基及其应用 |
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FR2731015B1 (fr) | 1995-02-24 | 1997-05-30 | Sci Sartone | Procede d'enrichissement enzymatique d'huiles d'origine marine et les triglycerides d'acides gras polyinsatures ainsi obtenus |
JP4175868B2 (ja) | 2002-11-13 | 2008-11-05 | 株式会社ニフコ | ホールプラグ |
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2005
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- 2005-10-05 CN CN2011100810414A patent/CN102212509A/zh active Pending
- 2005-10-05 US US11/574,996 patent/US7972831B2/en not_active Expired - Fee Related
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102325891A (zh) * | 2008-08-29 | 2012-01-18 | 邦奇油类公司 | 水解酶、编码它们的核酸及制备和使用它们的方法 |
CN102325891B (zh) * | 2008-08-29 | 2015-01-07 | 邦奇油类公司 | 水解酶、编码它们的核酸及制备和使用它们的方法 |
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EP1805302A1 (en) | 2007-07-11 |
US20090029410A1 (en) | 2009-01-29 |
WO2006037334A1 (en) | 2006-04-13 |
US7972831B2 (en) | 2011-07-05 |
EP1805302B1 (en) | 2012-08-15 |
CN102212509A (zh) | 2011-10-12 |
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