CN101103118B - 使用两种脂解酶的混合物从甘油三酯和醇产生脂肪酸烷基酯的方法 - Google Patents
使用两种脂解酶的混合物从甘油三酯和醇产生脂肪酸烷基酯的方法 Download PDFInfo
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Abstract
用于产生脂肪酸烷基酯的方法,其中将包含甘油三酯和醇的溶液与第一脂解酶和第二脂解酶接触,所述第一脂解酶对游离脂肪酸的活性相对高于对甘油三酯的活性,并且所述第二脂解酶对甘油三酯的活性相对高于对游离脂肪酸的活性。
Description
发明领域
本发明涉及从甘油三酯产生脂肪酸烷基酯的方法,其通过使用一种促成甘油三酯转化成脂肪酸烷基酯的第一脂解酶,和另一种促成游离脂肪酸转化成脂肪酸烷基酯的第二脂解酶。
背景技术
生物柴油通常被归类为油脂的单烷基酯,近来由于其有益于环境而吸引了人们更多的注意。尽管目前人们成功地用化学方法生产生物柴油(使用例如NaOH和/或甲醇钠作为催化剂),但仍存在几个限制其发展的相关问题,例如高含量的游离脂肪酸所致的油的预加工问题、从酯和甘油相去除化学催化剂的问题和在甘油回收期间去除无机盐的问题。
人们主要通过使用脂解酶作为催化剂来防止化学催化剂导致的不利因素,并且近几年来对于在转酯作用中使用固定化或未固定化的脂肪酶以产生生物柴油发生了兴趣。
真菌酯酶可以用于酯的酶促生产中,其中它们可以取代催化剂如无机酸(例如硫酸、氯化氢和氯磺酸),I、II、III和IV族金属的两性氢氧化物等。酶用于酯合成的用途在现有技术中已经有所描述,尤其是根据EnzymeNomenclature (Recommendations of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology,1992或之后的)归类于EC 3.1.1羧酸酯水解酶的酶。
WO 88/02775公开了源自Candida antarctica的脂肪酶A和B。该文件称C.antarctica脂肪酶B(CALB)对于酯的合成更有效。
角质酶(cutinase)是能够底物角质的脂解酶。已知角质酶来自多种真菌(P.E.Kolattukudy in“Lipases”,Ed.B.Borgstr m and H.L.Brockman,Elsevier1984,471-504)。源自Humicola insolens的一种角质酶的氨基酸序列已被公布(US 5,827,719)。
许多研究人员已报导了在有机溶剂的存在下能够达到烷基酯的高产量,但是由于有机溶剂的毒性和可燃性,在无溶剂介质中由脂肪酶催化的醇解是更理想的。已证明在不含有机溶剂的含水体系中发生由脂肪酶催化的甲醇分解作用。在这种体系中,对甲醇敏感性较小的脂肪酶是有利的(Kaieda et al.J.Biosci.Bioeng.2001,91:12-15)。众所周知,过多的短链醇例如甲醇可能严重地导致脂肪酶失活。然而,为了将油完全转化成其相应的甲酯,需要至少三摩尔当量的甲醇。Du等人(Biotechnol.Appl.Biochem.2003,38:103-106)比较性地研究了不连续分批和连续分批操作过程中油/甲醇的摩尔比率的影响。
为了避免脂肪酶失活,通过在整个反应中逐步添加甲醇来保持低的甲醇浓度(Shimada et al.J Mol.Catalysis Enzymatic,2002,17:133-142;Xu et al.2004,Biocat.Biotransform.22:45-48)。
Boutur等人(J.Biotechnol.1995,42:23-33)报道了源自Candida deformans的脂肪酶,其既能催化甘油三酯(TG)的醇解,又能催化游离脂肪酸(FFA)的酯化作用,但不是在相同的反应条件下。在Boutur等人描述的条件下,酶只催化酯化作用。
为了实现更经济地生产用于生物柴油的脂肪酸乙酯,人们需要将油脂更快地转化成它们相应的甲酯,并在所述转化中实现更高的产量。
发明概述
本发明涉及产生脂肪酸烷基酯例如脂肪酸甲酯(FAME)和脂肪酸乙酯的方法。这些酯也被称为生物柴油,因为人们将它们用作矿物柴油的添加剂,以获得无硫、十六烷数较高、且部分地基于可再生资源的燃料。
本发明的方法包括含有醇、甘油三酯和/或游离脂肪酸的溶液,将该溶液与特异性不同的第一脂解酶和第二脂解酶接触,其中所述脂解酶催化甘油三酯或游离脂肪酸或二者的混合物转化成脂肪酸烷基酯。使用下面的实施例1和2中所述的方法测定第一和第二脂解酶活性。
第一脂解酶定义为对TG的活性/对FFA的活性的比率在0.2以下的脂解酶。第二脂解酶定义为对TG的活性/对FFA的活性的比率在0.5以上的脂解酶。
根据本发明将第一脂解酶和第二脂解酶相组合,对于甘油三酯及甘油三酯和游离脂肪酸的组合向脂肪酸烷基酯的转化产生协同作用,从而在较短的时间内获得较高的转化百分比。
此外,本发明涉及使用如上所述的第一和第二脂解酶产生脂肪酸烷基酯的分批方法或连续、分阶段方法,其中醇是连续或分步添加的,并且其中酶循环利用或只用一次。
发明详述
本发明涉及生产脂肪酸烷基酯的方法。本发明所述方法包括溶液,其包含醇和底物,该底物包含甘油三酯和/或游离脂肪酸。将所述溶液与特异性不同的第一脂解酶和第二脂解酶接触,其中所述脂解酶催化甘油三酯或游离脂肪酸或二者的混合物转化成脂肪酸烷基酯。
底物根据本发明用于产生脂肪酸烷基酯的适合底物是种类广泛的植物油脂;菜籽油和大豆油是最常使用的,但其他作物例如芥菜(mustard)、向日葵(sunflower)、油菜(canola)、椰子(coconut)、大麻(hemp)、棕榈油(palm oil)和甚至藻类(algae)也显示了应用前景。该底物可以是未经加工品质的或进一步加工的(精炼的、脱色的并且脱臭的)。也可以使用动物脂肪,包括牛羊油脂(tallow)、熟猪油(lard)、家禽、鱼油(marine oil)以及废植物和动物油脂,其通常称为黄色油脂(yellow grease)和褐色润滑脂(brown grease)。合适的油脂可以是纯甘油三酯或甘油三酯和游离脂肪酸的混合物,其通常见于废植物油和动物脂肪。底物也可以从植物油脱臭器馏出物(deodorizer distillate)获得。底物中脂肪酸的类型包含那些作为植物和动物油脂中的甘油酯天然存在的脂肪酸。这些脂肪酸包括,仅举数例:油酸、亚油酸、亚麻酸、棕榈酸(palmetic acid)和月桂酸。未加工的植物油中的次要成分通常是磷脂、游离脂肪酸和偏甘油酯,即单酸甘油酯和二酯酸甘油酯。当用于本文时,短语“脂肪酸残基”指游离的脂肪酸或酯化的脂肪酸,如甘油三酯、二酯酸甘油酯、单酸甘油酯或脂肪酸烷基酯中的脂肪酸。
生物柴油脂肪酸烷基酯,例如脂肪酸甲酯(FAME)和脂肪酸乙酯,也称为生物柴油,因为它们被用作化石柴油的添加剂。生物柴油构成基于石油的柴油燃料的日益重要的添加剂或替代品,因为其产自可再生资源。
醇 用在本发明所述方法中的醇优选地是具有1-5个碳原子(C1-C5)的低级醇。优选的醇是甲醇和乙醇。
脂解酶所述脂解酶对甘油三酯和游离脂肪酸的活性的测定分别如实施例1和实施例2所述。
根据本发明,第一脂解酶定义为:对甘油三酯的活性(其作为甘油三酯到脂肪酸烷基酯的转化率被测量)与对FFA的活性(其作为FFA到脂肪酸烷基酯的转化率被测量)的比率在0.2以下的脂解酶。第二脂解酶定义为:对甘油三酯的活性(其作为甘油三酯到脂肪酸烷基酯的转化率被测量)与对FFA的活性(其作为FFA到脂肪酸烷基酯的转化率被测量)的比率在0.5以上的脂解酶。
因此,本发明涉及用于生产脂肪酸烷基酯的方法,其特征在于将包含甘油三酯和醇的溶液与第一脂解酶和第二脂解酶接触,其中所述第一脂解酶对甘油三酯的活性与对FFA的活性的比率在0.2以下,所述第二脂解酶对甘油三酯的活性与对FFA的活性的比率在0.5以上。
第一脂解酶对甘油三酯的活性与对FFA的活性的比率优选为0.01-0.2,更优选0.01-0.1,更优选0.125-0.05,更优选0.015-0.025,更优选0.02-0.024。第二脂解酶对甘油三酯的活性与对FFA的活性的比率优选为0.5-20,更优选0.6-10,更优选0.7-5,更优选0.8-1.5。
如上所述,脂解酶对甘油三酯和游离脂肪酸的活性的测定分别如实施例1和实施例2中所述。以下,计算了受试脂解酶对甘油三酯(缩写为TG)的活性比对游离脂肪酸(缩写为FFA)的活性的比率,其中对甘油三酯的活性在实施例1中测定,对游离脂肪酸的活性在实施例2中测定:
CALB:TG/FFA=0.55/26.41=0.021
H.insolens角质酶:TG/FFA=12.13/10=1.213
细毛嗜热霉脂肪酶:TG/FFA=13.22/16.25=0.814.
根据本发明结合第一脂解酶和第二脂解酶,对甘油三酯和/或游离脂肪酸向脂肪酸烷基酯的转化产生了协同作用,从而在较短的时间内获得了较高的转化百分比。
在本发明所述方法的优选实施方案中,本发明的第一脂解酶是如WO88/02775中公开的源自CandidaAntarctica(CALB)的脂肪酶B,而第二脂解酶是WO 00/60063中示例的细毛嗜热霉(Thermomyces lanuginosus)(曾为Humicola lanuginosus)脂肪酶变体和WO 01/92502的实施例2中公开的Humicola insolens角质酶变体之一,在下文中分别称为细毛嗜热霉脂肪酶和H.insolens角质酶。在第二个优选的实施方案中,第一脂解酶包括Hyphozymasp.脂肪酶和近平滑念珠菌(Candida parapsilosis)脂肪酶,而本发明的第二脂解酶包括如WO 88/02775中公开的C.antarctica脂肪酶A和来自Humicolalanuginosus(EP 258 068)、皱落念珠菌(Candida rugosa)、洋葱假单胞菌(Pseudomonas cepacia)、白地霉(Geotricum candidum)、曼赫根毛霉(Rhizomucormiehei)、隐球菌属菌种(Crytococcus spp.)S-2和近平滑念珠菌的脂肪酶。
在第三个实施方案中,第一脂解酶与CALB、Hyphozyma sp.脂肪酶或近平滑念珠菌脂肪酶同源,而第二脂解酶与以下酶同源:细毛嗜热霉脂肪酶、H.insolens角质酶或任何来自Humicola lanuginosus(EP 258068)、皱落念珠菌、洋葱假单胞菌、白地霉、曼赫根毛霉、隐球菌属菌种S-2和近平滑念珠菌的脂肪酶。
优选地,根据本发明方法的第一脂解酶与CALB具有60%同一性,而第二脂解酶与细毛嗜热霉脂肪酶、H.insolens角质酶具有60%同一性。更优选地,第一脂解酶与CALB的同一性是70%,甚至更优选地第一脂解酶与CALB的同一性是75%、80%、85%、88%、90%、92%、94%、95%、96%、97%、98%或甚至99%。类似地,第二脂解酶与细毛嗜热霉脂肪酶和H.insolens角质酶的同一性优选是70%,更优选地,第二脂解酶与细毛嗜热霉脂肪酶或H.insolens角质酶的同一性是75%、80%、85%、88%、90%、92%、94%、95%、96%、97%、98%或甚至99%。
可将该酶作为冻干粉末、固定化或在水溶液中的形式应用。
对于本发明而言,同一性程度可根据Needleman,S.B.and Wunsch,C.D.,(1970),Journal of Molecular Biology,48,443-45所述的方法合适地测定,多肽序列的比较使用下列设置:GAP产生罚分3.0和GAP延伸罚分0.1。测定可以利用已知的计算机程序实现,例如GCG程序包(Program Manual for theWisconsin Package,Version 8,August 1994,Genetics Computer Group,575Science Drive,Madison,Wisconsin,USA 53711)中提供的GAP。
可以根据Needleman(如上)所述的方法,使用相同的参数来对两个给定序列进行比对。这可以利用GAP程序(如上)来实现。
此外,本发明涉及使用如上所述的第一和第二脂解酶产生脂肪酸烷基酯 的分批方法和/或连续、分阶段的方法,其中连续或分步地添加醇,并且其中将酶循环利用或只用一次。如果酶在水相中,可通过油水分离器(decanter)、沉降器或通过离心将该相与脂肪相分离。在连续方法中,可以用逆流方式(counter currently)对分别为油和水的两相进行处理。Kosugi,Y;Tanaka,H.andTomizuka,(1990),Biotechnology and Bioengineering,vol.36,617-622描述了一种用固定化的脂肪酶水解植物油的连续逆流方法。
脂肪酸烷基酯制备概述
将包含甘油三酯的底物与醇,优选甲醇或乙醇混合,并在往复式振荡水浴(reciprocal water shaking bath)(200rpm)上加热至30-60℃,优选50℃。优选添加水,混合该溶液,并且进一步加热至所需的温度。添加酶,将溶液剧烈混合,并置于处于所需温度的往复式振荡水浴上,优选50℃和200rpm以进行反应。可以使用高剪切混合器来混合反应混合物的各相,高剪切混合器例如来自Silverson或IKA Labortechnik的类型,如在植物油的酶促脱胶中使用的高剪切混合器(Clausen,K.(2001),European Journal of Lipid Science andTechnology,vol.103,333-340)。
[甲醇]/[脂肪酸残基]的摩尔比率应该是至少0.1,最大10,优选0.3-5,更优选0.4-2。可将醇逐渐地分步加入反应。水可单独地添加或包含在酶的水溶液中添加。反应混合物中水的终浓度可以是0-50%(w/w),优选5-40%,更优选5-30%。底物包含1-99%(w/w)的甘油三酯,优选70-95%。此外,底物可以包含总计0.01-95%(w/w)的游离脂肪酸,优选0.01-30%。并且,可以存在单酸甘油酯和二脂酸甘油酯以及磷脂。
可以通过在一段确定的反应时间后从反应混合物抽取样品来监测该反应过程。将所述样品于14000rpm离心14分钟。上层由不溶于水相的脂肪物质组成,对其用1H NMR(使用CDCl3作为溶剂)加以分析。反应结束之后,通过倾析(decanting)或离心去除甘油相。
克隆编码脂解酶的DNA序列
编码亲本脂解酶的DNA序列可以使用本领域熟知的各种方法从任何产生所述脂解酶的细胞或微生物分离。首先,应该使用来自产生待研究脂解酶的生物体的染色体DNA或信使RNA建立基因组DNA和/或cDNA文库。然 后,如果该脂解酶的氨基酸序列是已知的,可以合成标记的寡核苷酸探针,并使用该探针从由所述生物体制备的基因组文库鉴定出编码脂解酶的克隆。或者,可以使用含有其它已知脂解酶基因的同源序列的标记寡核苷酸探针作为探针,使用较低严紧度的杂交和洗涤条件,来鉴定编码磷脂酶的克隆。
另一种鉴定编码脂解酶的克隆的方法涉及将基因组DNA片段插入表达载体例如质粒中,用所得基因组DNA文库转化角质酶阴性的细菌,随后将转化的细菌涂布(plate)到含有脂解酶底物(即甘油三酯)的琼脂上,从而使表达该脂解酶的克隆得以被鉴定。
或者,编码该酶的DNA序列可以通过已建立的标准方法合成制备,例如由S.L.Beaucage and M.H.Caruthers,(1981),Tetrahedron Letters 22,p.1859-1869描述的亚磷酰胺(phosphoroamidite)方法,或由Matthes et al.,(1984),EMBO J.3,p.801-805描述的方法。在亚磷酰胺方法中,合成寡核苷酸,例如在自动DNA合成仪中合成;纯化;退火;连接并且克隆到合适的载体中。
最后,该DNA序列可以是基因组和合成混合来源,合成和cDNA混合来源,或基因组和cDNA混合来源,通过根据标准技术连接合成来源、基因组来源或cDNA来源的片段而制备(这些片段对应于整个DNA序列的不同部分,视情况而定)。也可以使用特异性引物通过聚合酶链式反应(PCR)来制备该DNA序列,例如US 4,683,202或R.K.Saiki et al.,(1988),Science 239,1988,pp.487-491中所述。
表达载体
携带编码本发明脂解酶的DNA序列的重组表达载体可以是任何可以方便地接受重组DNA方法处理的载体,并且载体的选择通常取决于要导入该载体的宿主细胞。所述载体可以是这样的载体:其被引入到宿主细胞中时,整合到宿主细胞基因组中,并且与其整合进入的染色体一起复制。合适的表达载体的实例包括pMT838。
本发明所述的表达载体也可以包含合适的转录终止子,在真核细胞中还可包含聚腺苷酸化序列,它们与编码本发明脂解酶的DNA序列可操作地连接。终止序列和聚腺苷酸化序列可以适当地来自与启动子相同的来源。
载体可以进一步包含使该载体能够在所讨论的宿主细胞中复制的DNA序列。这种序列的实例是以下质粒的复制起点:pUC19、pACYC177、pUB110、 pE194、pAMB1和pIJ702。
载体还可以包含选择标记,例如其产物补足宿主细胞中缺陷的基因,例如来自枯草芽孢杆菌(B.subtilis)或地衣芽孢杆菌(B.licheniformis)的dal基因,或赋予抗生素抗性的基因,该抗生素抗性例如氨苄青霉素、卡那霉素、氯霉素或四环素的抗性。此外,该载体可以包含曲霉菌属选择性标记例如amdS、argB、niaD和sC,导致潮霉素抗性的标记,或所述选择可以通过共转化完成,例如WO 91/17243中所述。
分别用于连接编码角质酶变体的本发明DNA构建体、启动子、终止子和其它元件,并将它们插入到含有复制必需的信息的合适载体中的方法,对本领域的技术人员而言是熟知的(参见,例如,Sambrook et al.,Molecular Cloning:A Laboratory Manual,2nd Ed.,Cold Spring Harbor,1989)。
启动子
在载体中,DNA序列应当可操作地连接至合适的启动子序列。该启动子可以是在所选择的宿主细胞中表现出转录活性的任何DNA序列,并且可以源自编码与该宿主细胞同源或异源的蛋白质的基因。
用于指导编码本发明脂解酶DNA序列转录,特别是在细菌宿主中转录的合适启动子的实例,是大肠杆菌lac操纵子的启动子、天蓝色链霉菌(Streptomyces coelicolor)琼脂酶基因dagA启动子、地衣芽孢杆菌α-淀粉酶基因(amyL)的启动子、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)产麦芽糖淀粉酶基因(amyM)的启动子、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶(amyQ)的启动子、枯草芽孢杆菌xylA和xylB基因的启动子等。对于在真菌宿主中的转录,有用启动子的实例是那些下列来源的启动子:编码米曲霉(A.oryzae)TAKA淀粉酶的基因、源自酿酒酵母(S.cerevisiae)的TPI(丙糖磷酸异构酶)启动子(Alber et al.(1982),J.Mol.Appl.Genet 1,p.419-434)、曼赫根毛霉天冬氨酸蛋白酶、黑曲霉(A.niger)中性α-淀粉酶、黑曲霉酸稳定α-淀粉酶、黑曲霉葡糖淀粉酶、曼赫根毛霉脂肪酶、米曲霉碱性蛋白酶、米曲霉丙糖磷酸异构酶或构巢曲霉(A.nidulans)乙酰胺酶。
宿主细胞
包含如上定义的本发明DNA构建体或表达载体的本发明细胞可有利地 作为宿主细胞用于重组产生本发明的脂解酶。可以用编码脂解酶的本发明的DNA构建体转化所述细胞,方便地,通过将该DNA构建体(一个或多个拷贝)整合到宿主染色体中。通常认为这种整合是有利的,因为该DNA序列更有可能稳定地保持在所述细胞中。所述DNA构建体向宿主染色体中的整合可以根据常规方法进行,例如通过同源或异源重组。或者,可使用如上所述与不同类型的宿主细胞相关的表达载体转化所述细胞。
本发明所述细胞可以是高等生物体的细胞,例如哺乳动物或昆虫的细胞,尤其是微生物细胞,例如细菌或真菌(包括酵母)细胞。
合适细菌是实例是革兰氏阳性细菌例如枯草芽孢杆菌、地衣芽孢杆菌、迟缓芽孢杆菌(Bacillus lentus)、短芽孢杆菌(Bacillus brevis)、嗜热脂肪芽孢杆菌、嗜碱芽孢杆菌(Bacillus alkalophilus)、解淀粉芽孢杆菌、凝结芽孢杆菌(Bacillus coagulans)、环状芽孢杆菌(Bacillus circulans)、灿烂芽孢杆菌(Bacilluslautus)、巨大芽孢杆菌(Bacillus megaterium)、苏云金芽孢杆菌(Bacillusthuringiensis),或浅青紫链霉菌(Streptomyces lividans)或鼠灰链霉菌(Streptomyces murinus),或革兰氏阴性菌例如大肠杆菌。细菌转化可以例如通过原生质体转化或使用感受态细胞,以本身已知的方式来实现。
酵母生物体可以优选地选自酵母属(Saccharomyces)或裂殖酵母(Schizosaccharomyces)属的物种,例如酿酒酵母(Saccharomyces cerevisiae)。
宿主细胞还可以是丝状真菌,例如属于曲霉菌属物种的菌株,具体地是米曲霉或黑曲霉,或镰孢属(Fusarium)的菌株,例如下列菌种的菌株:尖孢镰孢(Fusarium oxysporum)、禾本科镰孢(Fusarium graminearum)(在完全状态被命名为玉蜀黍赤霉(Gibberella zeae),曾命名为Sphaeria zeae,与Gibberellaroseum和Gibberella roseum f.sp.cerealis同义)或Fusarium sulphureum(在完全状态被命名为Gibberella puricaris,与Fusarium trichothecioides、杆孢状镰孢(Fusarium bactridioides)、接骨木镰孢(Fusarium sambucinum)、粉红镰孢(Fusarium roseum)和粉红镰孢禾本科变种(Fusarium roseum var.graminearum)同义)、Fusarium cerealis(与Fusarium crokkwellense 同义)、或Fusariumvenenatum。
在本发明的具体实施方案中,所述宿主细胞是蛋白酶缺陷的或蛋白酶负型菌株。其可以是例如蛋白酶缺陷菌株米曲霉JaL 125,该菌株缺失名为“alp”的碱性蛋白酶基因。该菌株在WO 97/35956(Novo Nordisk)中有所描述。
丝状真菌细胞可以通过涉及下述内容的方法,以本身已知的方式来加以转化:形成原生质体,转化该原生质体,然后再生细胞壁。使用曲霉属作为宿主微生物在EP 238023(Novo NordiskA/S)中有所描述,本文中将其内容通过引用并入。
通过培养转化体产生脂解酶
本发明涉及,特别地,产生本发明的脂解酶的方法,该方法包括在有益于所述脂解酶产生的条件下培养宿主细胞,和从该细胞和/或培养基中回收所述脂解酶。
用于培养该细胞的培养基可以是适于培育所述宿主细胞并获得本发明所述脂解酶的表达的任何常规培养基。合适的培养基可以从供应商获得或可以根据公开的配方制备(例如,如美国典型培养物保藏中心(American TypeCulture Collection)的目录中所述)。
从宿主细胞分泌的脂解酶可以方便地通过公知的方法从培养基中回收,包括通过离心或过滤从培养基分离细胞,以及用盐例如硫酸铵从培养基中沉淀蛋白质性成分,接着使用色谱方法例如离子交换色谱、亲和色谱等。
材料和方法
对三丁精(tributyrin)的脂肪酶活性(LU)
通过用阿拉伯树胶作为乳化剂乳化三丁精(甘油三丁酸酯)来制备脂解酶的底物。在30℃pH 7的水解三丁精,然后进行pH恒定的滴定实验(pH-stattitration experiment)。一个脂肪酶活性单位(1LU)等于在标准条件下每分钟能够释放1μmol丁酸的酶的量。
脂肪酸烷基酯的制备
将8.00克底物与甲醇混合(0.500ml=>0.395克)。使用以下类型的底物:
实施例1)100%色拉油(精炼、脱色并且脱臭的大豆油,RBD SBO);
实施例2)100%油酸;
实施例3)油酸于RBD SBO中的20%w/w的混合物
将底物-甲醇混合物在往复式振荡水浴(200rpm)上加热至50℃。添加去离子水(体积取决于所添加的酶的体积;水的总量:4.00ml,其包括添加酶带来 的水),其相当于总混合物的32w/w%。将所述混合物加热至50℃。随后将酶加入到该混合物中,剧烈地混合10秒,并置于50℃200rpm的往复式振荡水浴上。可以使用高剪切混合器混合反应混合物的各个相,所述高剪切混合器例如来自Silverson Ltd.UK或IKAKunkel的类型。
三小时反应时间后从反应混合物中抽取样品,并且在14000rpm离心14分钟。上层由在水相中不溶的脂肪物质组成,通过1H NMR(使用CDCl3作为溶剂)Varian 400MHz频谱仪(Varian Inc.CA,USA)对其进行分析。根据来自脂肪酸甲酯的甲基信号-COOCH 3(3.70ppm)和来自脂肪酸残基的甲基信号CH 3CH2-(1.0-0.9ppm)的比率来确定脂肪酸残基到脂肪酸甲酯的转化率。
酶剂量基于总计0.4mg蛋白质/8.00克底物。为了测试两种酶组合的协同作用;向8克底物添加每种酶各0.2mg,与0.4mg/8克底物剂量的每种单独的酶进行比较。为了将蛋白质的量与酶活性关联起来,可以应用标准酶活性测定,在这里使用如上所述的LU测定(对三丁精的脂肪酶活性)。测试了下列酶制剂:
1.细毛嗜热霉脂肪酶(TLL,比活性7000LU/mg蛋白质)
2.C.antarctica脂肪酶B(CALB,比活性500LU/mg蛋白质)
3.H.insolens角质酶(角质酶,比活性1800LU/mg蛋白质)
用于单酶实验的酶剂量和添加的水体积:
1.TLL:0.700ml的4000LU/ml酶溶液+3.30ml水
2.CALB:1.680ml的119LU/ml酶溶液+2.32ml水
3.角质酶:0.450ml的1600LU/ml酶溶液+3.55ml水
用于组合酶实验的酶剂量和添加的水体积:
1.TLL+CALB:(0.350ml的4000LU/ml TLL溶液+0.840ml的119LU/ml CALB溶液+2.810ml的水)
2.角质酶+CALB:(0.225ml的1600LU/ml角质酶溶液+0.840ml的119LU/ml CALB溶液+2.935ml的水)。
实施例
实施例1从甘油三酯制备脂肪酸烷基酯
根据上述的通用方法,使用精炼、脱色并脱臭的大豆油(RBD SBO,色拉油)作为底物。
使用不同脂解酶经过3小时反应时间之后脂肪酸残基转化为FAME的转化率(%)示于表1,而用CALB和TLL的组合达到的转化率(%)示于表2。四个同样实验的以%计的变异系数(Coefficient of Variation)(CV%)确定为2.2%。
CALB | 0.55 |
角质酶 | 12.13 |
TLL | 13.22 |
表1.单酶,脂肪酸残基转化为FAME的%转化率
CALB+TLL | 16.21 |
表2.酶组合,脂肪酸残基转化为FAME的%转化率
实施例2从油酸制备脂肪酸烷基酯
根据上述的通用方法使用油酸作为底物。使用不同脂解酶经过3小时反应时间之后,脂肪酸残基转化为FAME的转化率(%)示于表3。
CALB | 26.41 |
角质酶 | 10 |
TLL | 16.25 |
表3.单酶,脂肪酸残基转化为FAME的%转化率
实施例3从含有游离脂肪酸的甘油三酯制备脂肪酸烷基酯
根据上述的通用方法,使用RBD SBO中20%w/w油酸的混合物作为底物。使用不同脂解酶和所述酶的组合,经过3小时反应时间之后,脂肪酸残基转化为FAME的转化率(%)示于表4和5。
CALB | 16.58 |
角质酶 | 11.22 |
TLL | 14.09 |
表4.单酶,脂肪酸残基转化为FAME的%转化率
CALB+角质酶 | 18.82 |
CALB+TLL | 20 |
表5.酶组合,脂肪酸残基转化为FAME的%转化率
Claims (15)
1.用于产生脂肪酸烷基酯的方法,其中通过将包含底物和醇的溶液与第一脂解酶和第二脂解酶接触来制造反应混合物,所述底物包含甘油三酯和游离脂肪酸,所述醇是具有1-5个碳原子的低级醇,所述第一脂解酶对甘油三酯的活性与对FFA的活性的比率在0.2以下,所述第二脂解酶对甘油三酯的活性与对FFA的活性的比率在0.5以上。
2.根据权利要求1的方法,其中所述底物包含0.01-95%以重量计的游离脂肪酸。
3.根据权利要求1或2的方法,其中所述甘油三酯源自一种或多种植物油原料或者源自一种或多种动物脂肪。
4.根据权利要求3的方法,其中所述植物油原料选自菜籽油、大豆油、芥子油、向日葵子油、油菜油、椰子油、大麻油、棕榈油和妥尔油(tall oil)。
5.根据权利要求3的方法,其中所述动物脂肪选自牛油、熟猪油、家禽油和鱼油。
6.根据权利要求1或2的方法,其中所述醇和脂肪酸残基的摩尔比率为0.4-2。
7.根据权利要求1或2的方法,其中所述醇是甲醇或乙醇。
8.权利要求1或2的方法,其中所述反应混合物进一步包含多达50%(w/w)的水。
9.根据权利要求1或2的方法,其中所述第一脂解酶对甘油三酯的活性与对FFA的活性的比率为0.01-0.2,所述第二脂解酶对甘油三酯的活性与对FFA的活性的比率为0.5-20。
10.根据权利要求1或2的方法,其中所述第一脂解酶是来自CandidaAntarctica的脂肪酶B、Hyphozyma sp.脂肪酶或近平滑念珠菌脂肪酶。
11.根据权利要求1或2的方法,其中所述第二脂解酶为选自下组的脂解酶:细毛嗜热霉脂肪酶、H.insolens角质酶、C.antarctica脂肪酶A、来自皱落念珠菌、洋葱假单胞菌、白地霉、曼赫根毛霉、隐球菌属菌种S-2和近平滑念珠菌的脂肪酶。
12.根据权利要求1或2的方法,其中所述方法是以分批方式进行。
13.根据权利要求1或2的方法,其中所述方法是以连续方式进行。
14.根据权利要求1或2的方法,其中使用高剪切混合器混合所述反应混合物中的溶液相。
15.根据权利要求1或2的方法,其中所述方法是以逆流方式进行。
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US9040263B2 (en) | 2010-07-28 | 2015-05-26 | Butamax Advanced Biofuels Llc | Production of alcohol esters and in situ product removal during alcohol fermentation |
CN103429747A (zh) | 2011-01-21 | 2013-12-04 | 诺维信公司 | 产生脂肪酸烃基酯 |
EP2670854A1 (en) | 2011-02-04 | 2013-12-11 | Novozymes A/S | Fatty acid esterification process |
US10815506B2 (en) | 2014-05-28 | 2020-10-27 | Novozymes A/S | Production of fatty acid alkyl esters with caustic treatment |
CN105062697B (zh) * | 2015-08-24 | 2021-06-15 | 华南农业大学 | 一种利用预处理提高餐厨油脂酶法制备生物柴油产量的方法 |
CN106480114B (zh) * | 2015-08-25 | 2021-10-08 | 丰益(上海)生物技术研发中心有限公司 | 制备生物柴油的方法 |
EP3359679A1 (en) | 2015-10-09 | 2018-08-15 | Novozymes A/S | Enzymatic or non-enzymatic biodiesel polishing process |
WO2023209070A1 (en) * | 2022-04-29 | 2023-11-02 | Novozymes A/S | Production of fatty acid alkyl esters |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0407959A2 (en) * | 1989-07-11 | 1991-01-16 | Lion Corporation | Process for producing polyol fatty acid monoesters |
US5713965A (en) * | 1996-04-12 | 1998-02-03 | The United States Of America As Represented By The Secretary Of Agriculture | Production of biodiesel, lubricants and fuel and lubricant additives |
CN1441848A (zh) * | 2000-07-13 | 2003-09-10 | 日本水产株式会社 | 利用脂肪酶的甘油酯的制造方法 |
Family Cites Families (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4056442A (en) * | 1976-06-01 | 1977-11-01 | The Dow Chemical Company | Lipase composition for glycerol ester determination |
JPS60234588A (ja) * | 1984-05-07 | 1985-11-21 | Asahi Denka Kogyo Kk | 長鎖高度不飽和脂肪酸アルコ−ルエステルの製造法 |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
DK122686D0 (da) | 1986-03-17 | 1986-03-17 | Novo Industri As | Fremstilling af proteiner |
DE3750450T2 (de) | 1986-08-29 | 1995-01-05 | Novo Industri As | Enzymhaltiger Reinigungsmittelzusatz. |
ATE117018T1 (de) | 1986-10-17 | 1995-01-15 | Novo Nordisk As | Positionsmässig nicht spezifische lipase von candida-arten; verfahren für ihre herstellung und ihre verwendung. |
US5288619A (en) * | 1989-12-18 | 1994-02-22 | Kraft General Foods, Inc. | Enzymatic method for preparing transesterified oils |
KR100237148B1 (ko) | 1990-05-09 | 2000-01-15 | 한센 핀 베네드 | 엔도글루칸아제 효소를 함유하는 셀룰라제 제조물 |
US5173965A (en) | 1991-09-30 | 1992-12-29 | Panner Donna J | Releasably attached two section gown |
BR9509525A (pt) | 1994-10-26 | 1995-10-26 | Novo Nordisk As | Construção de dna vetor de expressão recombinante célula processo para produzir a enzima que exibe atividade lipolítica enzima que exibe atividade lipolítica preparação de enzima aditivo de detergente e composição de detergente |
WO1997035956A1 (en) | 1996-03-27 | 1997-10-02 | Novo Nordisk A/S | Alkaline protease deficient filamentous fungi |
US6939702B1 (en) | 1999-03-31 | 2005-09-06 | Novozymes A/S | Lipase variant |
MXPA02011911A (es) | 2000-06-02 | 2003-05-27 | Novozymes As | Variantes de cutinasa. |
CN1238469C (zh) * | 2004-01-16 | 2006-01-25 | 清华大学 | 有机介质反应体系中脂肪酶转化油脂生产生物柴油新工艺 |
US7473539B2 (en) * | 2004-09-20 | 2009-01-06 | Sunho Biodiesel Corporation | Methods for producing alkyl esters |
-
2006
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- 2006-01-10 JP JP2007549795A patent/JP5133701B2/ja not_active Expired - Fee Related
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- 2006-01-10 WO PCT/DK2006/000016 patent/WO2006072256A2/en active Application Filing
- 2006-01-10 CN CN2006800019976A patent/CN101103118B/zh active Active
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- 2006-01-10 US US11/813,121 patent/US20080113419A1/en not_active Abandoned
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0407959A2 (en) * | 1989-07-11 | 1991-01-16 | Lion Corporation | Process for producing polyol fatty acid monoesters |
US5713965A (en) * | 1996-04-12 | 1998-02-03 | The United States Of America As Represented By The Secretary Of Agriculture | Production of biodiesel, lubricants and fuel and lubricant additives |
CN1441848A (zh) * | 2000-07-13 | 2003-09-10 | 日本水产株式会社 | 利用脂肪酶的甘油酯的制造方法 |
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BRPI0606389A2 (pt) | 2011-08-23 |
CA2593282A1 (en) | 2006-07-13 |
ATE503019T1 (de) | 2011-04-15 |
EP1838860B1 (en) | 2011-03-23 |
US20080113419A1 (en) | 2008-05-15 |
US9593352B2 (en) | 2017-03-14 |
EP1838860A2 (en) | 2007-10-03 |
ES2363094T3 (es) | 2011-07-20 |
JP2008526217A (ja) | 2008-07-24 |
BRPI0606389B1 (pt) | 2018-02-14 |
WO2006072256A3 (en) | 2007-02-08 |
DE602006020837D1 (de) | 2011-05-05 |
CA2593282C (en) | 2016-03-15 |
US20110201066A1 (en) | 2011-08-18 |
WO2006072256A2 (en) | 2006-07-13 |
CN101103118A (zh) | 2008-01-09 |
JP5133701B2 (ja) | 2013-01-30 |
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