CN104704124A - 制备癸二酸的方法 - Google Patents
制备癸二酸的方法 Download PDFInfo
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- CN104704124A CN104704124A CN201380046581.6A CN201380046581A CN104704124A CN 104704124 A CN104704124 A CN 104704124A CN 201380046581 A CN201380046581 A CN 201380046581A CN 104704124 A CN104704124 A CN 104704124A
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Abstract
制备癸二酸的方法,该方法包括在第一步骤(i)中,通过油酸水合酶的催化将亚油酸与水反应以形成10-羟基-12-十八烯酸,在第二步骤(ii)中,热解10-羟基-12-十八烯酸生成1-辛烯和10-氧代-癸酸,然后在第三步骤(iii)中,将10-氧代-癸酸氧化成癸二酸。
Description
本发明涉及新的制备癸二酸的方法。本发明尤其涉及癸二酸的化学-酶制备,其中用亚油酸作为原料,将其羟基化成10-羟基-12-十八烯酸并进一步转化成癸二酸。
现有技术
癸二酸目前从蓖麻油通过蓖麻油酸(12-羟基-9-顺式-十八烯酸)在高压和高温下的碱性裂解来制备。
癸二酸及其衍生物是可生物降解的聚合物、增塑剂、润滑剂、液压机液体、蜡烛和化妆品的重要成分。
关于不饱和脂肪酸的微生物氧化的概述记载于以下出版物中:Hou C.T.(1995)Adv.Appl.Microbiol.,41,1-23。
Schroepfer G.J.等人记载了通过假单胞菌制剂将亚油酸以57%的收率酶水合成10-羟基-12-十八烯酸((1970)J.Biol.Chem.,245,3798-3801)。
在US 4,582,804中,Litchfield&Pierce公开了玫瑰红红球(Rhodococcus rhodochrous)的细胞催化亚油酸水合生成10-羟基-12-十八烯酸,收率22%。
Hou报道了通过黄杆菌(Flavobacterium)DS5酶体系将亚油酸以55%的收率水合生成10-羟基-12-十八烯酸(Hou C.T.(1994)J.Am.Oil Chem.Soc.,71,975-978)。
利用来自绵羊瘤胃的粪肠球菌(Enterococcus faecalis)菌株也显示了相同的转化,收率为22%(Hudson J.A.等人(1998)FEMS Microbiology Letters,169,277-282)。
Demir等人的报道描述了将亚油酸化学-酶转化成顺式-9,反式-11-十八二烯酸(CLA),这是一种具有抗癌、降低脂肪和抑制高血压性能的化合物。将亚油酸通过植物乳杆菌(Lactobacillus plantarum)转化成10-羟基-12-十八烯酸,然后用碘在微波照射下处理以高收率制得CLA(Demir A.S.等人(2010)J.Agric.Food Chem.,58,1646-1652)。
尽管许多报道描述了使用完整的微生物或细胞提取物来水合不饱和脂肪酸,但直到2009年才对酶进行了详细地表征。Bevers等人首次描述了油酸水合酶(EC 4.2.1.53)从脑膜炎败血伊丽莎白菌(Elizabethkingia meningoseptica)的分离、在大肠杆菌中的重组表达和表征(Bevers L.E.等人(2009)J.Bacteriol.,191,5010-5012)。
WO 2008/119735记载了通过利用来自酿脓链球菌(Streptococcus pyogenes)的水合酶生产羟基脂肪酸的方法。
最近的报道表明,来自嗜麦芽糖寡养单胞菌(Stenotrophomonas maltophilia)和来自纺锤形赖氨酸芽孢杆菌(Lysinibacillus fusiformis)的油酸水合酶能够水合亚油酸,尽管与油酸相比具有降低的比活性(Young-Chul Joo等人(2012)J.Biotechnol.,158,17-23和Bi-Na Kim等人(2011)Appl.Microbiol.Biotechnol.,在线发表)。
目的
由于对癸二酸的需求日益增加,因此,本发明的目的是提供新的合成癸二酸的路线,其使用除蓖麻油酸之外的易于获得的离析物作为原料。
发明的主题
按照权利要求,该目的通过如下制备癸二酸的方法来实现:在第一步骤(i)中通过油酸水合酶催化将亚油酸与水反应以形成10-羟基-12-十八烯酸,在第二步骤(ii)中,将10-羟基-12-十八烯酸热解生成1-辛烯和10-氧代-癸酸,然后在第三步骤(iii)中将10-氧代-癸酸氧化成癸二酸。
步骤(i)
本发明的方法起始于将亚油酸转化成10-羟基-12-十八烯酸。对于本发明方法的步骤(i),可使用化学纯的亚油酸以及含有亚油酸作为主要成分的底物,优选亚油酸占底物的60重量%以上、更优选70重量%或80重量%以上。
该底物可从具有高亚油酸含量的甘油酯形式的油类通过水解甘油酯并回收游离酸形式的亚油酸或其盐来制备。该油类是例如红花油(78%亚油酸)、葡萄子油(73%亚油酸)、婴粟子油(70%亚油酸),或者更优选葵花油(68%亚油酸)。
如果亚油酸从复合油例如葵花油制备,则脂肪酸制剂除了亚油酸之外还可含有其它当进行本发明的反应步骤(i)时可以存在的脂肪酸。这些其它的脂肪酸可在后来的反应步骤中、优选在步骤(ii)之后从反应中除去。
关于适合步骤(i)的酶,在以上所述的现有技术中有多种油酸水合酶,例如来自有机体假单胞菌属、红球菌属、黄杆菌属、肠球菌属、赖氨酸芽孢杆菌属、乳酸菌属、寡养单胞菌属、伊丽莎白菌的那些。
本领域已知这些酶可将亚油酸转化成10-羟基-12-十八烯酸。除了这些酶之外,本领域技术人员通过利用油酸转化成10-羟基硬脂酸的模型反应或从亚油酸生成10-羟基-12-十八烯酸的靶向反应筛选微生物可以容易地发现其它油酸水合酶。该反应可在试管试验中进行,因此可在短时间内同时筛选成千上万的微生物。
由于某些油酸水合酶的序列是已知的,因此也可以在电脑中从微生物基因组中筛选其它油酸水合酶并将阳性群用于将油酸转化成10-羟基硬脂酸或将亚油酸转化成10-羟基-12-十八烯酸的靶向反应的测试。
另一种方式是对已知油酸水合酶的基因工程,以通过比较已知油酸水合酶的序列找到保守区或同源区并找到定向基因突变的起点来获得具有提高的活性或更好的温度或溶剂耐受性的酶。
优选的酶是油酸水合酶EC 4.2.1.53。该类酶的代表是来自脑膜炎败血伊丽莎白菌的酶(Bevers等人(2009)J.Bacteriol.191,5010-5012)。核苷酸序列和相应的氨基酸序列以SEQ ID NO:1和2公开。
优选的酶是具有SEQ ID NO:2或所述多肽序列的片段(其中所述的片段足以使蛋白具有油酸水合酶的酶活性)的酶,或是具有包含编码油酸水合酶并且与编码SEQ ID NO:2的核苷酸序列的互补链在严格条件下杂交的核酸序列或包含所述核苷酸序列的片段(其中所述的片段足以编码具有油酸水合酶的酶活性的蛋白)的酶。
本发明进一步涉及具有油酸水合酶的酶活性以及SEQ ID NO:2描述的氨基酸序列或与SEQ ID NO:2所描述的氨基酸序列至少75%或80%、优选至少85%、90%或95%、更优选至少95%或97%、最优选至少98%或99%相同的氨基酸序列的酶。
为了提高酶的溶解度和表达水平,可将该酶用N-(或C)-末端融合伙伴(蛋白质或多肽)重组表达。用作提高溶解度和/或表达水平的融合伙伴的常用蛋白或标记是:麦芽糖结合蛋白、硫氧还蛋白、绿色荧光蛋白、谷胱甘肽-S-转移酶、二硫化物氧化还原酶/异构酶、T7tag、SET tag、Nus A、Mistic和SUMO。
在本发明的上下文中,术语“在严格条件下杂交”是指杂交在体外在足以保证特异性杂交的严格条件下进行。严格的体外杂交条件对于本领域技术人员来说是已知的,并且可参见文献(例如Sambrook and Rus-sell(2001)Molecular Cloning:A Laboratory Manual,第三版,Cold Spring Har-bour Laboratory Press,Cold Spring Harbour,NY)。术语“特异性杂 交”是指,如果靶序列是复杂混合物例如DNA或RNA分子的一部分的话,一种分子在严格条件下会优先与特定核酸序列、即靶序列结合,但不会与其它序列结合或至少以相当低的程度结合。
严格条件取决于特定的情况。较长的序列在较高的温度下特异性杂交。通常,选择严格条件以使杂交温度比特定序列在规定的离子强度和规定的pH值下的熔点(Tm)低约5℃。Tm是其中50%与靶序列互补的分子与靶序列以平衡状态杂交的温度(在规定的pH值、规定的离子强度和规定的核酸浓度下)。通常,对于短分子来说(即,例如10至50个核苷酸),严格条件是其中的盐浓度至少为约0.01至1.0M钠离子浓度(或者是另一种盐的浓度)、pH为7.0至8.3、温度至少是30℃。此外,严格条件还包括加入可使杂合体不稳定的活性剂例如甲酰胺。在本文所用的严格条件下的杂交中,至少60%彼此同源的核苷酸序列通常保持相互杂交。优选按照如下方式选择严格条件:至少约65%、优选至少约70%、特别优选至少约75%或更多彼此同源的序列通常保持相互杂交。优选的非限制性的严格杂交条件的实例是在6x氯化钠/柠檬酸钠(SSC)中在约45℃下杂交,然后在0.2x SSC、0.1%SDS中在50至65℃下进行一次或多次洗涤步骤。例如,在取决于核酸类型的标准杂交条件下,温度范围通常是42℃至58℃,在含水缓冲液中在0.1至5x SSC的浓度下进行(pH 7.2)。
如果在上述缓冲液中存在有机溶剂例如50%甲酰胺,则标准条件下的温度约为42℃。优选用于DNA:DNA杂交的杂交条件是例如0.1x SSC和20℃至45℃,优选30℃至45℃。优选用于DNA:RNA杂交的杂交条件是例如0.1x SSC和30℃至55℃,优选45℃至55℃。以上所述的杂交温度是例如用长度约为100个碱基对且G/C含量为50%的核酸在不存在甲酰胺的情况下确定的。本领域技术人员知晓如何利用上述内容或者按照如下教科书确定所需的杂交条件:Current Protocols in Molecular Biology,John Wiley&Sons,N.Y.(1989),Hames und Higgins(publisher)1985,Nucleic Acids Hybridization:A Practical Approach,IRL Press at Oxford University Press,Oxford;Brown(出版商)1991,Essential Molecular Biology:A Practical Approach,IRL Press at Oxford University Press,Oxford。
酶水合的立体特异性对于本发明方法来说不是关键的。因此,步骤(i)中制备的10(R)或10(S)-羟基-12-十八烯酸或混合物(外消旋体)均可用于下面的步骤(ii)中。
亚油酸生成10-羟基-12-十八烯酸的酶转化可以在含有亚油酸和水的反应介质中进行。如果将亚油酸以游离酸(油相)的形式使用,则含酶的水溶液或缓冲水溶液形成第二液相(水相)。两种液相应该彻底混合形成乳液以便快速反应。
然而,步骤(ii)还可利用固定化酶进行,该固定化酶易于从反应介质中除去并可重复利用。通常将酶通过不同的方法例如吸附、共价结合、膜包裹、凝胶包裹和交联来固定。用于固定的载体材料的性能应当最优化以避免酶失活。常用载体可以是有机(天然和非天然)或无机材料。无机材料通常具有良好的耐压性能,而有机材料表现出良好的化学稳定性。无机载体通常是基于硅-或铝氧化物或其混合物的多孔材料。天然的有机载体是例如多糖例如纤维素、淀粉、葡聚糖、琼脂或壳多糖。蛋白质例如胶原蛋白、明胶或白蛋白也可使用。合成的有机载体包括聚(甲基)丙烯酸酯、聚丙烯酰胺、乙烯基-和烯丙基聚合物、聚酯、聚酰胺。
步骤(i)可在2-相体系中进行,其中将酶制剂(水相)加入到含亚油酸的有机相中。水相/亚油酸相的比率可以在宽范围内变化。
反应可在或不存在其它溶剂的条件下进行。关于溶剂的选择,本领域技术人员可根据产物收率、反应速率、所形成的悬浮液的易处理性以及溶剂的成本来确定。
有利的溶剂是可与亚油酸混合的溶剂,并且是化学惰性的,即,不会与酶反应或抑制酶的活性。
在生物催化过程中常用的有机溶剂是:己烷、庚烷、十二烷、十六烷、乙醚、异丙醚、丁醚、四氢呋喃、二恶烷、甲苯、二甲基亚砜、丙酮、2-戊酮、2-庚酮。步骤(i)的反应温度取决于所用酶的热稳定性,并且通常是 10至50℃,优选20至40℃。然而,如果使用热稳定性高的酶,则反应温度可在50℃以上。
将形成的10-羟基-12-十八烯酸通过常规方法例如结晶或萃取从反应介质中回收。
对于本发明的反应的下一步骤、即步骤(ii),将10-羟基-12-十八烯酸以游离酸或以酯的形式使用,优选低级酯例如10-羟基-12-十八烯酸的甲酯或乙酯。
如果在反应中使用10-羟基-12-十八烯酸的酯,则可在步骤(ii)的热解之前,将从步骤(i)回收的10-羟基-12-十八烯酸通过化学或酶的方法酯化。用于酯化的优选方法是通过脂肪酶的酶转化。
步骤(ii)
对于该步骤(ii)的描述,术语“10-羟基-12-十八烯酸”是指游离酸10-羟基-12-十八烯酸或10-羟基-12-十八烯酸的酯,例如10-羟基-12-十八烯酸的甲酯或乙酯。
10-羟基-12-十八烯酸热解生成10-氧代-癸酸是逆烯(retro-ene)型反应。为了选择逆烯重排以及抑制竞争的脱水反应,最好是快速蒸发10-羟基-12-十八烯酸。
该反应可在约400至高达800℃的温度下进行,优选500至600℃。最佳的温度范围取决于底物的停留时间以及底物的性质。如果使用10-羟基-12-十八烯酸甲酯,在微型反应器内采用600℃的温度以及1-2秒的停留时间可以得到最好的结果。如果用游离酸10-羟基-12-十八烯酸代替酯,在较低的温度例如575℃可检测到10-羟基-12-十八烯酸的完全转化。然而,就逆烯反应对脱水反应的选择性而言,游离酸10-羟基-12-十八烯酸的选择性小于10-羟基-12-十八烯酸甲酯的选择性。详细的比较可参见工作实施例。
反应可在小型或微型反应器中进行,例如直径为0.1至3mm的毛细管。关键点是在小型或微型反应器内具有高加热速率和快速的蒸发,停留时间<10秒,优选<1秒。为了保持这些特性,本领域技术人员已知的小型或微 型结构的装置是适当的。反应可利用或不利用溶剂进行。如果使用溶剂,则可加入溶剂最多至99%(w/w)。所用的溶剂在热解过程中所用的温度和条件下不应该反应或分解。优选的溶剂选自适当的醚类例如THF或二恶烷。THF是用于步骤(ii)的最优选的溶剂。
还可将水加入到反应混合物中。
取决于原料10-羟基-12-十八烯酸,步骤(ii)中形成的10-氧代-癸酸可以是游离酸10-氧代-癸酸或10-氧代-癸酸的相应酯。对于该步骤(ii)的描述,术语“10-氧代-癸酸”是指10-氧代-癸酸的游离酸以及酯。
将10-氧代-癸酸与逆烯重排的第二产物(1-辛烯)通过常规方法例如蒸馏或萃取相分离。在使用不纯的亚油酸例如葵花油水解产物作为原料的情况下,未转化的脂肪酸(例如硬脂酸或10-羟基硬脂酸)可通过常规方法例如蒸馏、结晶或萃取除去。
对于下一步骤的10-氧代-癸酸氧化成癸二酸,由10-氧代-癸酸、1-辛烯和可能的其它脂肪酸组成的步骤(ii)的产物混合物通常可直接使用而无需纯化,或者可将10-氧代-癸酸通过上述方法纯化。甲酯或游离的脂肪酸均可使用。
步骤(iii)
对于步骤(iii),可将回收的10-氧代-癸酸以游离酸或酯的形式使用。
将10-氧代-癸酸中的醛官能团氧化成二碳酸癸二酸可按照已知的方法进行,例如通过利用温和的氧化剂例如氧气或空气在最高至100℃以及最高至7巴的条件下、不利用催化剂或通过氧化还原金属例如Cu、Fe、Co、Mn等均质催化将氧代-醛氧化成氧代碳酸(Industrial Organic Chemistry,Wiley-VCH,H.-J.Arpe(出版商),2007,pp.149)。
根据步骤(i)中用作亚油酸源的原料纯度的不同以及癸二酸的应用的不同,在本发明的方法中可能需要额外的纯化和回收步骤,这是本领域技术人员熟知的。当在步骤(iii)中使用10-氧代-癸酸的酯时,酯的水解将产生游离的癸二酸。
工作实施例
实施例1
油酸水合酶的表达和表征
利用为大肠杆菌而优化的密码子来合成编码来自脑膜炎败血伊丽莎白菌的油酸水合酶的基因。下面的制备方法改编自Bevers等人(Bevers L.E.等人(2009)J.Bacteriol.,191,5010-5012)。
对于重组酶的制备,将基因克隆到pBAD(HisA)载体(Invitrogen)内,其可诱导阿拉伯糖的表达。将E.coli TOP10 One Shot(Invitrogen)用pBAD(HisA)-OH转化并铺到LB-Agar-Amp板上(过夜,37℃)。将单一菌落接种入2xYT-Amp并在37℃下继续培养5小时。
蛋白表达的诱导通过将5mL该培养液加入到补充有0.2%阿拉伯糖的500mL 2xYT-Amp中并在37℃下继续孵育18小时来实现。诱导后通过离心分离收集细胞(20’,4000rpm,4℃)并重新悬浮在20mM Tris-HCl(pH 8),50mM NaCl和1mM CaCl2中。
将细胞悬浮液超声处理(3’,15”开/关循环,80%振幅,在4℃下),然后将澄清上清液用于该报告中所述的大多数生物催化转化(通常是40mg/mL总蛋白;≈13%油酸水合酶,基于Agilent 2100 Bioanalyzer)。将该酶进一步通过Ni-亲和色谱纯化(His-tag纯化)。在这种情况下,将诱导的细胞重新悬浮于20mM Tris-HCl(pH8),50mM NaCl,1mM CaCl2和10mM咪唑。纯化过程中的洗涤缓冲液含有20mM咪唑,蛋白质洗脱用500mM咪唑在相同缓冲液中来实现。将含有油酸水合酶的级分收集并用20mM Tris-HCl(pH 8),50mM NaCl和1mM CaCl2透析。将酶原液(5.8mg/mL)保存在4℃下。
所表达的蛋白质的身份通过N-端蛋白测序来确认。
实施例2
油酸转化成10-羟基-硬脂酸(10-HSA)
第一步,将实施例1的重组制备的油酸水合酶用其作用于油酸(OA)以生成10-羟基硬脂酸(10-HSA)的野生型活性来表征。将表达油酸水合酶的细菌按照实施例1所述的方法超声处理并将200μL澄清上清液(5mg/mL总蛋白含量,≈600μg油酸水合酶)加入到含10mM油酸的20mM Tris-HCl(pH 8),50mM NaCl和1mM CaCl2的乳液中(终体积2mL)。
作为阴性对照,利用超声处理的不表达油酸水合酶的E.coli TOP10的上清液(5.6mg/mL总蛋白含量,无油酸水合酶)进行相同的反应。将反应混合物在搅拌下在室温下孵育过夜。通过加入50μL 3M HCl停止反应(最终的pH 1-2)。
此时,将4mL MTBE加入到反应混合物中以萃取有机底物(OA)和产物(10-HSA)。通过将500μL三甲基氢氧化硫(TMSH;0.1M的甲醇溶液)加入到100μL产物溶液中(30’,在100℃下)将反应产物衍生化并通过GC分析。
图1显示了该酶转化的GC分析。
图1(A)将油酸与E.coli TOP10在室温下孵育过夜。油酸的保留时间:8.161min。(B)将油酸与表达油酸水合酶的E.coli TOP10在室温下孵育过夜。10-HAS的保留时间:9.340min。
正如所预期的那样,TOP10 E.coli细胞中表达的油酸水合酶能够将油酸几乎完全转化(>95%)成10-HAS。超声处理的不含油酸水合酶的E.coli TOP10细胞不能转化油酸。
实施例3
亚油酸转化成10-羟基-12-十八烯酸(10-HOA)
将油酸水合酶在E.coli TOP10(10L培养液)中按照实施例1进行表达。通过将细胞团重新悬浮于100mL 20mM Tris-HCl(pH 8),50mM NaCl,1mM CaCl2中、然后按照前面所述的方法超声处理完成细胞的溶解。总蛋白浓度是26mg/mL(13%油酸水合酶)。
将上清液(300-400mg油酸水合酶)加入到含有900mL 20mM Tris-HCl(pH 8),50mM NaCl,1mM CaCl2和14.4g亚油酸(≈50mM)的溶液中。将反应混合物在室温下搅拌72小时。反应完成后,通过加入3M HCl将pH调节至1.5。然后将产物用1L MTBE萃取并用100g硅藻土535过滤。通过蒸馏除去MTBE后,以高收率得到反应产物10-HOA(13.6g,收率:89%)。通过将400μL N-三甲基甲硅烷基咪唑(TSIM)加入到100μL产物溶液(30’,在100℃下)中来制备GC和GC-MS分析的样品。
图2(A)10-HOA的GC分析:10-HOA的保留时间:9.971min。(B)反应产物的GC-MS分析,表明10-HOA的常规裂解方式。
实施例4
10-羟基-12-十八烯酸(10-HOA)在微型反应器中的热解
将微型反应器用于热解反应。示意性的实验装置如图3所示。将进料用Bischoff HPLC泵打入反应器内。反应器是浸没在可加热至最高800℃的固体铜块内的内径为1/16”的钢管。在经过冷却器(室温下的铝块)后,产物混合物被收集在烧瓶中。采用该装置,可以使停留时间<1秒。此外,整个混合物被非常快速地加热至所需温度。
图3显示了在微型反应器内热解10-羟基-12-十八烯酸(10-HOA)的示意性实验装置。
a.10-HOA甲酯(Me-10-HOA)的热解
在第一个微型反应器实验中,选择10-羟基-12-十八烯酸的甲酯作为反应物,以易于处理(即,较好的蒸发性能)。将MTBE和THF用作溶剂。在MTBE的情况下,观察到大量二甲基化的10-氧代-癸酸(与甲醇形成缩醛),得出的结论是溶剂在反应过程中分解。因此,选择THF用于进一步的实验(未观察到分解产物)。对不同的温度和停留时间进行评价,并对加入水的影响(几乎等摩尔量的水溶于反应物溶液中)进行评估。结果如表2所述。
从表2可以看出,温度对10-羟基-12-十八烯酸甲酯的转化率具有最大的影响,而停留时间的变化仅有较小的影响(停留时间的更大变化因反应器 装置的限制而不可行,但可能对转化率和选择性有较大的影响)。在500℃下,仅有约25%的10-羟基-12-十八烯酸甲酯被转化(条目1和2),在550℃下,其已达到约40%(条目3-6),在600℃下可实现全部转化(条目7和8)。加入水似乎对选择性具有有益的效果。最佳结果在600℃、停留时间τ=1.3秒得到,10-羟基-12-十八烯酸甲酯被完全转化,朝向逆烯产物的选择性约为75%,朝向脱水产物(亚油酸甲酯和异构体,条目8)的选择性仅为6.5%。
表2:在微型反应器内10-羟基-12-十八烯酸甲酯热解的结果(通过甲硅烷基化样品的GC分析)。
b.10-羟基十八-12-烯酸(10-羟基-12-十八烯酸)的热解
由于使用10-羟基-12-十八烯酸甲酯作为反应物意味着在从亚油酸到癸二酸的整个反应路线中多了一个步骤,因此用获得了成功的微型反应器设置用游离酸10-羟基-12-十八烯酸(10wt%THF乳液的形式)进行了试验。与10-羟基-12-十八烯酸甲酯的热解相比,在较低的温度下(575℃,表3中的条目5)就已获得10-羟基-12-十八烯酸的完全转化。在10-羟基-12-十八烯酸甲酯的情况下,停留时间不会对反应有较大程度的影响。然而,对10-氧代-癸酸的选择性明显降低(48对74%,比较表2中的条目8)。观察到更多的副反应形式的脱水作用,这可能是由于游离脂肪酸的蒸发行为与其甲酯相比较差而导致的。
表3:在微型反应器内10-羟基-12-十八烯酸热解的结果(通过甲硅烷基化样品的GC分析)。
实施例5
10-氧代-癸酸氧化成癸二酸
10-氧代-癸酸氧化成癸二酸可按照H.-J.Arpe所述的方法进行(Industrial Organic Chemistry,Wiley-VCH,2007,pp.149):将醛用温和氧化剂例如空气或纯氧在液相中在最高100℃且最高7巴下、未催化或通过氧化还原活性金属例如Cu、Fe、Co、Mn均质催化氧化。
Claims (9)
1.制备癸二酸的方法,该方法包括
在第一步骤(i)中,通过油酸水合酶的催化将亚油酸与水反应以形成10-羟基-12-十八烯酸,
在第二步骤(ii)中,将10-羟基-12-十八烯酸热解生成1-辛烯和10-氧代-癸酸,然后
在第三步骤(iii)中,将10-氧代-癸酸氧化成癸二酸。
2.根据权利要求1所述的方法,其中油酸水合酶是具有SEQ ID NO:2的多肽。
3.根据权利要求1所述的方法,其中在步骤(i)之后将10-羟基-12-十八烯酸酯化成10-羟基-12-十八烯酸酯,随后在步骤(ii)中将10-羟基-12-十八烯酸酯热解。
4.根据权利要求3所述的方法,其中10-羟基-12-十八烯酸酯是甲酯。
5.根据权利要求1所述的方法,其中步骤(ii)中的热解在400至800℃的温度下进行,更优选500至600℃。
6.根据权利要求1所述的方法,其中热解在微型结构装置中进行。
7.根据权利要求1所述的方法,其中步骤(ii)中的热解在作为溶剂的THF中进行。
8.根据权利要求1所述的方法,其中步骤(iii)中的氧化通过空气进行。
9.根据权利要求1所述的方法,其中的氧化步骤(iii)通过氧化还原活性金属催化。
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- 2013-09-03 IN IN1320DEN2015 patent/IN2015DN01320A/en unknown
- 2013-09-03 RU RU2015112327A patent/RU2673343C2/ru active
- 2013-09-03 CA CA2883141A patent/CA2883141C/en active Active
- 2013-09-03 BR BR112015004662-2A patent/BR112015004662B1/pt active IP Right Grant
- 2013-09-03 US US14/423,448 patent/US9834798B2/en active Active
- 2013-09-03 ES ES13758836.4T patent/ES2692950T3/es active Active
- 2013-09-03 EP EP13758836.4A patent/EP2893026B1/en active Active
- 2013-09-03 JP JP2015530364A patent/JP6174149B2/ja active Active
- 2013-09-03 WO PCT/EP2013/068144 patent/WO2014037328A1/en active Application Filing
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN112226428A (zh) * | 2020-10-29 | 2021-01-15 | 华东理工大学 | 油酸水合酶突变体及其在制备10-羟基硬脂酸中的应用 |
CN112226428B (zh) * | 2020-10-29 | 2022-11-08 | 华东理工大学 | 油酸水合酶突变体及其在制备10-羟基硬脂酸中的应用 |
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ES2692950T3 (es) | 2018-12-05 |
EP2706117A1 (en) | 2014-03-12 |
WO2014037328A1 (en) | 2014-03-13 |
JP2015529075A (ja) | 2015-10-05 |
CN104704124B (zh) | 2018-08-10 |
CA2883141C (en) | 2022-03-29 |
EP2893026B1 (en) | 2018-08-01 |
BR112015004662A2 (pt) | 2017-12-12 |
US9834798B2 (en) | 2017-12-05 |
CA2883141A1 (en) | 2014-03-13 |
RU2015112327A (ru) | 2016-10-27 |
RU2673343C2 (ru) | 2018-11-26 |
EP2893026A1 (en) | 2015-07-15 |
IN2015DN01320A (zh) | 2015-07-03 |
BR112015004662B1 (pt) | 2022-02-15 |
JP6174149B2 (ja) | 2017-08-02 |
US20150259713A1 (en) | 2015-09-17 |
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