CN1129956A - 羧酸与叔醇的酯化方法 - Google Patents
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Abstract
使用南极洲假丝醇母(Candida antarctica)脂肪酶A或具有就叔醇酯酶来说底物活性类似于南极洲假丝酵母脂肪酶A的脂肪酶种,在低水状态下酶促制备醇部分在酯键周围有立体位阻的酯类,即衍生于叔醇的酯类。
Description
本发明涉及使用脂肪酶酶促制备羧酸酯的方法。
已知(例如从WO-A-8802775.(NOVO Industri A/s)中已知)在酯水解、酯合成和酯交换(酸解、醇解、酯交换)反应中使用脂肪酶,但所有这些反应都局限于利用伯醇和仲醇。
法国专利说明书FR-A-2,617,501(Soc.Nat.Elf Aquitaine)中甚至提出以类似叔丁醇或叔戊醇的叔醇作为溶剂,在其中进行酶促反应,这表明预期此溶剂和待用酶处理的底物之间不发生任何反应。S.Okumura等人在Biochim.Biophys.Acta 575,156-165(1979)中以实验证实了此观点,并在其中研究了经微生物脂肪酶的酯合成。黑色曲霉(Aspergillus niger)、德列马根霉(Rhizopus delemar)、白地霉(Geotrichum Candidum)或圆弧青霉(Penicillum cyclopium)的脂肪酶中无一能合成叔醇、酚或糖醇的酯类(参见表I)。
A.Zaks和A.M.Klibanov发现猪胰脂肪酶在99%的有机介质中可催化三丁酸甘油酯和多种伯醇或仲醇之间的醇解反应(Science224(15 June 1984)1249-1251)。已发现在叔醇的醇解反应中,干的脂肪酶完全没有活性,但是表I中已显示经湿的猪胰脂肪酶(酶中有占重量3.6%的水)催化的三丁酸甘油酯和叔丁醇之间醇解反应的初速度是每100mg脂肪酶22μmol/h,然而表I中未显示出产物的任何产量。最后I.L.Gatfield描述了(Lebensm.-Wiss.u.-Technol.19(1986)87-88)在米赫毛霉(Mucor miehei)(ex Rapi-dase,Seclin,France)脂肪酶的影响下,在非水非均相系统中酯的酶促合成。已发现伯和仲脂肪醇可有效酯化,而叔醇却不行。表1中叔丁醇显示出与油酸(酸和醇摩尔数相等;使用3wt%的米赫毛霉酶;在室温下在密闭容器中磁力搅拌3天)之间酯化反应的程度为14%。本申请的申请人重复了此实验却未生成任何油酸叔丁基酯,然而可假定测出的14%的酯化反应程度实际上与叔丁醇中存在的杂质(特别是仲丁醇)相关,因为所使用的检测技术是基于滴定,它不能把各个酯类区分开来。
这篇文章中缺少的任何酸值和酯化值不允许任何进一步的推断,但在他们的德国专利申请DE-A-3,108,927(此文章中提及)中,发明人从酸值和酯化值中计算出了转化水平,这在此类反应中是很平常的,但尽管在此专利申请中也使用了米赫毛霉脂肪酶(酯酶30,000;商标,ex Rapidase,Seclin,France),可此发明清楚地局限于伯醇和仲醇,它清楚地声明在此酶促反应中叔醇无反应性。
因此直至现在人们仍认为在从醇和酸酶促合成酯的过程中,不可能合成其醇部分在酯键周围有立体位阻的酯,即衍生于叔醇的酯。然而这种酶促酯合成的可用性是有价值的,特别是在“化学纯的”酯的合成中,其中不存在任何痕量的催化剂或副产物,也没有形成颜色的危险。
本发明提供了这种制备有立体位阻的酯类的酶促方法。已发现某些脂肪酶,即那些对其醇部分在酯键周围有立体位阻的酯具有水解活性的脂肪酶,能够利用醇和酸组分来制备这种酯。
因此,本发明涉及使用脂肪酶在低水状态下酶促制备羧酸酯的方法,它事实上有如下特征:使用脂肪酶来制备其醇部分在酯键周围有立体位阻的酯类,此脂肪酶选自对相同酯类具有水解活性的脂肪酶种。优选的脂肪酶是南极洲假丝酵母(Candida antarctica)脂肪酶A。已知南极洲假丝酵母脂肪酶含有两种组分:脂肪酶A和B,它们可通过例如凝胶过滤从脂肪酶中产生。也可通过重组DNA技术制备它们。脂肪酶A比脂肪酶B具有更好的热稳定性,但是已发现脂肪酶B不能合成本发明的有立体位阻的酯类。也可使用就叔醇酯来说底物活性类似于南极洲假丝酵母脂肪酶A的脂肪酶种。
使用发明所用特定脂肪酶的突变体和变异体也被认为在本发明的范围之内。已发现适用于本发明的其它脂肪酶是来自皱摺假丝酵母(Candida rugosa)、白地霉和Candida cylindracae的脂肪酶。
优选地,根据本发明所使用的特定脂肪酶可通过本来已知的任何方法,例如K.Mosbach(编辑)在Methods in Enzymology.Volume44,“Immobilized enzymes”.Academic Press.New York,1976中描述的方法固定到不溶性的无机或有机载体上。
特别优选的是如欧洲专利说明书EP-B-0,232,933(AKZO N.V)和EP-B-0,322,213和EP-A-0,424,130(both Unilever NV)中所述的固定化方法。通常这些方法包括从水溶液中将脂肪酶吸附到疏水颗粒、多孔固体上,它们选自疏水聚合物,如链烯同聚物或共聚物、聚苯乙烯、聚丙烯酸酯、聚酰胺或者疏水无机材料,如硅烷化过的二氧化硅、玻璃或陶瓷。预先用极性、与水互溶的有机溶剂例如乙醇处理固体材料,在所述溶剂中固体材料是不溶性的,但是溶剂不会使脂肪酶失活。适当材料的例子是Accurel EP100(商标、ex AKZO、荷兰)和硅烷化过的二氧化硅Grace 6 1500 MP(商标、多孔二氧化硅,ex W.RGrace,德国)。另外也可如欧洲专利说明书EP-B-140,542(NovoNordisk A/S)中所述将脂肪酶固定在离子交换树脂上。从水溶液中将脂肪酶吸附到弱的阴离子交换树脂(例如Duolite ES-568(商标、ex Rohm and Haas,法国))上。
此方法可连续进行或分批进行。优选地,应通过蒸馏、全蒸发或吸附到分子筛上不断除去反应所形成的水。进行反应的温度在约20℃至约90℃之间变化,优选从50℃至80℃。
可以改变醇和羧酸各自的用量,但优选使用大致等摩尔量的醇和羧酸。按照本发明的方法中的反应可通过从系统中除去水来驱动,例如通过共-蒸馏,加入干燥剂(如分子筛)、冷凝或冷却表面技术或全蒸发以除去水。
基于需酯化的醇和酸的总重量,酶的用量可达到重量的30%,但优选使用占重量1%至20%的酶。
按照本发明的方法可在不添加水时进行,但是对那些需要水的酶而言,水的存在量(基于起始反应混合物的总量)至多占重量的5%,优选至多占重量的2.5%。可被酯化的酸是具有2至54个碳原子的饱和或不饱和、直链或支链的一元羧酸,例如乙酸、癸酸、月桂酸、硬脂酸、异硬脂酸、肉豆蔻酸和不饱和的或多不饱和的脂肪酸,如油酸、亚油酸等等。也可使用多元羧酸如壬二酸和聚合酸(通过聚合具有8至24个碳原子的不饱和的脂肪酸得到如三聚物酸、二聚物酸或氢化二聚物酸。
按照本发明,可使用的叔醇可具有4至54个碳原子,如叔丁醇,叔戊醇、格尔伯特醇、萜烯醇等等。最好侧链所含碳原子数不超过2个。所述叔醇优选地是一元醇。
在酯混合物的制备中,可使用一元羧酸的多种混合物。
下述实施例将有助于进一步阐明本发明。
实施例I
在容器中将油酸(纯度为90%,171.55g)与叔醇(纯度超过99%,45.0g)和4.3g蒸馏水搅拌混合。将25g固定化的南极洲假丝酵母脂肪酶A(Code SP433,ex Novo Industri、丹麦)(固定在多孔聚丙烯珠上,Accurel EP100 ex AKZO)加入其中,将反应混合物的温度升高至60℃并维持117小时。大约每隔24小时对混合物施以真空处理,典型地为20毫巴持续1小时以通过与叔丁醇共蒸馏除去水。每次真空除水后再在反应混合物中加入45g叔丁醇。
反应结束后,使用300g氧化铝(基本活性(basic activity)2)通过柱纯化以除去游离的脂肪酸,60-80石油醚用作洗脱剂。这样可生成66g无水产物,通过GC分析显示出它是叔丁基油酸酯,它具有按油酸计算的32%的转化率。终产物含有极少量的游离酸(酸值=0.15)。
实施例II
将油酸(纯度超过99%,828mg)与叔戊醇(纯度超过99%,258mg)和23mg蒸馏水混合,加入62.5mg如实施例I的固定化南极洲假丝酵母脂肪酶A(Code SP433,ex NovoIndustri、丹麦)。将反应混合物的温度升至60℃,22小时后,通过对样品进行GC分析测定反应所形成的叔戊基油酸酯的量。除去70.4mg,使用60-80石油醚作为洗脱剂从氧化铝柱(基本活性2)上洗下混合物以除去游离脂肪酸,使用2.5mg二十碳烷作为GC分析的内标,所得终产物的产率按油酸计算为14.8%,通过GCMS证实产物本身。
实施例III
按与实施例II所述相同的方法,使用780mg里哪醇(纯度为97%)和1427mg油酸(纯度为99%)和44mg水制备里哪基油酸酯。加入125mg固定化的南极洲假丝酵母脂肪酶A(Code SP433ex Novo Industri、丹麦),将反应混合物的温度升高至60℃,所得终产物的产率按油酸计算产率为3.0%,通过GCMS证实产物本身。实施例IV
在此实施例中研究了大量固定化的脂肪酶(固定在如实施例I所述的大孔聚丙烯上(Accurel,如EP-B-322,213中所述)形成有立体位阻的酯类的适宜性。
在磁力搅拌容器中将油酸(纯度超过99%,548mg)和叔丁醇(纯度超过99%,144mg)和14mg蒸馏水混合,加入40mg脂肪酶催化剂(见表1)。
将反应混合物的温度升至40℃,持续期为64小时。
反应结束后,使用氧化铝(基本活性2)通过柱纯化除去游离的脂肪酸;60-80石油醚用作洗脱剂。使用2.5mg二十碳烷作为内标通过对样品进行GC分析测定反应所形成的叔丁基油酸酯的量。
表1
固定化的脂肪酶 | 理论荷载 | %产率 |
对照(无脂肪酶) | 0 | |
雪白根霉 | 12.5 KLU/G | 0 |
节杆菌属 | 37.1 KLU/G | 0 |
德列马根霉 | 38.4 KLU/G | 0 |
腐质霉属 | 2.23 KLU/G | 0 |
嗜热假单胞菌 | 56.0 KLU/G | 0 |
Pseudomonas P. | 3.8 KLU/G | 0 |
日本根霉 | 40.1 KLU/G | 0 |
皱摺假丝酵母 | 10.7 KLU/G | 0.6 |
白地霉 | 4.1 KLU/G | 0 |
白地霉 | 26.1 KLU/G | 0 |
猪胰脂肪酶 | 193.4 KLU/G | 0 |
生物脂肪酶 | 4.83 KLU/G | 0 |
角质酶 | 22.0 KLU/G | 0 |
Chromobacter viscosium | 43.7 KLU/G | 0 |
Pseudomonas glumae | 71.3 KLU/G | 0 |
南极洲假丝酵母A | 27.0 KLU/G | 5.6 |
从这些实验中可明显看出仅有少量特定的脂肪酶可用于按照本发明的方法。
比较实施例A
在磁力搅拌容器中将油酸(纯度超过99%,858mg)与叔丁醇(纯度超过99%,225mg)和22mg蒸馏水混合,加入62.5mg固定化的脂肪酶催化剂(天然米赫毛霉,Code SP 282,ex NovoIndustri A/S、丹麦),在另一分开的混合物中加入62.5mg固定化的脂肪酶,它是经克隆的米赫毛霉(Code SP 392,ex Novo IndustriA/S、丹麦)。
将反应混合物的温度升至40℃并维持20小时,反应期结束后,以60-80石油醚为洗脱剂,使用氧化铝(基本活性2)通过柱纯化除去游离的脂肪酸。使用2.5mg二十碳烷作为内标,通过对样品进行GC分析测定反应所形成的叔丁基油酸酯的量。
表2
固定化的脂肪酶 | 偏码 | %产率 |
天然米赫毛霉 | SP 282 | 0 |
经克隆的米赫毛霉 | SP 392 | 0 |
比较实施例B
使用猪胰脂肪酶进行如Zaks和Klibanov(Science 224、P1249-1251,1984)所述的比较实验,另外还将南极洲假丝酵母脂肪酶A和两个其它的醇裂解酯交换反应进行了比较。
将甘油三丁酰甘油酯(纯度大于99%,0.975g)与叔丁醇(纯度大于99%,0.2392g)和8.5mg(占重量的0.7%)蒸馏水混合,加入125mg猪胰脂肪酶(PPL/EP 100 ST623),将反应混合物的温度升至40℃,持续24小时。
按相同的方法,使用猪胰脂肪酶研究了两个其它的醇裂解反应。
将甲基油酸酯(纯度大于99%,0.859g)与叔丁醇(纯度大于99%,0.2146g)和7.5mg(占重量的0.7%)蒸馏水混合,加入125mg适当的猪胰脂肪酶并在40℃下温育24小时。
按与前述实施例相同的方法将类似的甲基油酸酯与正丁醇混合以作为一系列对照反应,通过对样品进行GC分析测定产物的形成,结果概括于下列表3中
表3
脂肪酶 | 底物 | %产量 |
猪胰脂肪酶 | 叔丁醇+三丁酸甘油酯 | 0 |
猪胰脂肪酶 | 叔丁醇+甲基油酸酯 | 0 |
猪胰脂肪酶 | 正丁醇+甲基油酸酯 | 29.6 |
这些实验清楚地表明当叔醇存在时Zaks和Klibanov的结果呈阴性。
Claims (12)
1.一种在低水状态下使用脂肪酶酶促制备羧酸酯的方法,其特征在于使用脂肪酶制备其中醇部分在酯键周围有立体位阻的酯类,所述脂肪酶选自对相同酯类具有水解活性的脂肪酶种。
2.按照权利要求1的方法,其特征在于所述脂肪酶是南极洲假丝酵母(Candida antarctica)脂肪酶A。
3.按照权利要求1的方法,其特征在于所述脂肪酶是就叔醇酯来说底物活性类似于南极洲假丝酵母脂肪酶A的脂肪酶种。
4.按照权利要求1的方法,其特征在于所述有立体位阻的酯的醇部分选自具有4至54个碳原子的叔醇及其混合物。
5.按照权利要求1的方法,其特征在于所述叔醇的侧链所含的碳原子数不超过2个。
6.按照权利要求1的方法,其特征在于所述有立体位阻的酯的醇部分是叔萜烯醇。
7.按照权利要求1的方法,其特征在于所述有立体位阻的酯的醇部分是一元叔醇。
8.按照权利要求1的方法,其特征在于所述有立体位阻的酯的酸组分选自具有2至54个碳原子的饱和或不饱和、直链或支链的一元羧酸,多元羧酸及其混合物。
9.按照权利要求1的方法,其特征在于存在的水的量按起始反应混合物的总量计算至多占其重量的5%。
10.按照权利要求1的方法,其特征在于存在的水的量按起始反应混合物的总量计算至多占其重量的2.5%。
11.按照权利要求1的方法,其特征在于所述脂肪酶被固定在不溶性的无机或有机载体上。
12.按照权利要求1的方法,其特征在于反应可通过从系统中除去水来驱动。
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CN1102960C (zh) * | 1999-07-30 | 2003-03-12 | 无锡轻工大学 | 一种甘油酯类生物表面活性剂的制备方法 |
CN115595340A (zh) * | 2022-11-28 | 2023-01-13 | 凯莱英生命科学技术(天津)有限公司(Cn) | 合成亚烷基二元酸单酯的方法 |
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GB9607458D0 (en) * | 1996-04-10 | 1996-06-12 | Zeneca Ltd | Enzymatic process for stereoselective preparation of therapeutic amides |
WO2001053511A1 (en) * | 2000-01-21 | 2001-07-26 | Novozymes A/S | Process for preparing esters |
KR100985423B1 (ko) | 2002-07-02 | 2010-10-05 | 카오카부시키가이샤 | 고정화 효소의 제조방법 |
US6924129B2 (en) * | 2002-10-23 | 2005-08-02 | Polytechnic University | Enzyme-catalyzed esterification of pendant carboxylic acid groups |
DE10251144A1 (de) * | 2002-10-31 | 2004-05-19 | Röhm GmbH & Co. KG | Makroporöses Kunststoffperlenmaterial |
EP2899280A1 (en) | 2014-01-28 | 2015-07-29 | Ernst-Moritz-Arndt-Universität Greifswald | Process for the enzymatic production of oligo-/polyesters |
CN104293848A (zh) * | 2014-10-13 | 2015-01-21 | 青岛科技大学 | 一种生产(1s,3r) 亚环己基二氧基庚烯醇的方法 |
US20210330597A1 (en) * | 2020-04-27 | 2021-10-28 | New Jersey Institute Of Technology | Nanoparticle Depot For Controlled And Sustained Gene Delivery |
CN114426993B (zh) * | 2022-02-18 | 2024-01-26 | 山东省农业科学院 | 从高油酸葵花籽油中获取1,3-甘油二酯的方法 |
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CN1102960C (zh) * | 1999-07-30 | 2003-03-12 | 无锡轻工大学 | 一种甘油酯类生物表面活性剂的制备方法 |
CN115595340A (zh) * | 2022-11-28 | 2023-01-13 | 凯莱英生命科学技术(天津)有限公司(Cn) | 合成亚烷基二元酸单酯的方法 |
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