CN102186967B - 包含消泡剂的疏水蛋白溶液 - Google Patents
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Abstract
包含至少300mg/l疏水蛋白和至少0.3mg/l消泡剂的水溶液,其中消泡剂/疏水蛋白重量比低于0.2,优选低于0.15,更优选低于0.1。
Description
发明的技术领域
本发明涉及一种包含消泡剂的疏水蛋白溶液。特别地,它涉及一种通过发酵方法获得的包含消泡剂的疏水蛋白溶液。
发明背景
发泡是需氧深层发酵中的常见问题。发泡由于将气体鼓入(sparging)发酵培养基而引起,将气体鼓入发酵培养基的目的是为正在培养的需氧有机体(例如细菌、酵母、真菌、藻类、细胞培养物)的生长提供氧气。若发酵培养基含有诸如蛋白质、多糖或脂肪酸之类的表面活性组分,则当鼓入的气泡脱离液体时,可在培养基的表面上形成泡沫。发泡引起许多问题,包括不合需要地将产品、营养素和细胞带(stripping)入泡沫,并且可能使工艺保持变得困难。控制发泡的一种已知方法是使用消泡剂,其中的几类是常用的:基于硅氧烷的消泡剂(例如聚二甲基硅氧烷)、聚亚烷基二醇(例如聚丙二醇)、脂肪酸、聚酯和天然油(例如亚麻籽油、大豆油)。消泡剂代替气泡表面上的发泡组分,导致泡沫因气泡聚结而破灭。在发酵开始和/或期间加入消泡剂。
当发酵产品有意用于食品、个人用品或药品时,非常希望通过生产有机体将所述产品分泌到发酵培养基中(即,细胞外而非细胞内制备)。这样不需要通过物理或化学方法破坏细胞,以便释放供回收的产品。通过保持细胞完整,可容易地将细胞材料与产品分离,以致产品不含细胞内材料和遗传材料,所述材料通常被视为不合需要的污染物。若生产有机体已经过基因修饰,这样做可能尤其重要。然而,细胞外制备疏水蛋白可加强发酵罐中的发泡程度。使用消泡剂在疏水蛋白的细胞外制备中产生特殊问题,其原因有二:第一,由于疏水蛋白本身促进了发酵罐中的发泡,因此所需消泡剂的量增加。第二,由于消泡剂与疏水蛋白一起存在将削弱疏水蛋白的功能(functionality),因此必须基本上去除消泡剂。
Bailey等,Appl.Microbiol.Biotechnol.58(2002)p721-727公开了通过里氏木霉(Trichodermareesei)转化株的发酵来制备疏水蛋白HFBI和HFBII。使用消泡剂(StruktolJ633)来防止发泡,并使用双水相萃取提纯疏水蛋白。
目前已经发现,疏水蛋白溶液中可以存在某种水平的消泡剂,而疏水蛋白保留它的至少部分功能。因此,存在一种包含消泡剂的疏水蛋白溶液,从而简化它的生产过程并导致显著的节约是可能的。
发明简述
本发明的目的是提供一种包含至少300mg/l疏水蛋白和至少0.3mg/l消泡剂的水溶液,其中消泡剂/疏水蛋白重量比低于0.2,优选低于0.15,更优选低于0.1。
优选地,该水溶液包含至少0.5mg/l消泡剂。
还优选,疏水蛋白是Ⅱ类疏水蛋白,最优选是来自里氏木霉(Trichodermareesei)的HFBI或HFBⅡ。
还优选,消泡剂具有浊点。
优选对该水溶液进行浓缩,以便消泡剂/疏水蛋白比例保持低于0.2,但是它的疏水蛋白含量高于1g/l,优选10g/l,甚至更优选100g/l。
发明详述
除非另有定义,本文使用的所有技术和科学术语均具有本领域(例如细胞培养、分子遗传学、核酸化学、杂交技术和生物化学领域)普通技术人员通常所理解的相同含义。用于分子和生物化学方法的标准技术可见于Sambrook等,MolecularCloning:ALaboratoryManual,第3版,(2001)ColdSpringHarborLaboratoryPress,ColdSpringHarbor,N.Y.,以及Ausubel等,ShortProtocolsinMolecularBiology(1999)第4版,JohnWiley&Sons.Inc.(完整版的名称为CurrentProtocolsinMolecularBiology)中。
疏水蛋白
可通过培养丝状真菌如丝状菌(例如,木霉),担子菌和子囊菌获得疏水蛋白。特别优选的宿主是食品等级的有机体,如栗疫病菌,其分泌一种称为cryparin的疏水蛋白(MacCabe和VanAlfen,1999,App.Environ.Microbiol65:5431-5435)。类似地,可从枯草芽孢杆菌(Bacillussubtilis)获得表面活性肽,可从例如铜绿假单胞菌(Pseudomanasaeruginosa)、红平红球菌(Rhodococcuserythropolis)、分枝杆菌属物种(Mycobacteriumspecies)和球拟酵母(Torulopsisbombicola)获得糖脂(Desai和Banat,MicrobiologyandMolecularBiologyReviews,Mar.1997,p47-64)。
我们以前曾在EP1623631中发现,疏水蛋白允许产生具有优异的抗歧化(disproportionation)和抗聚结稳定性的水性泡沫。由于疏水蛋白是非常有效的发泡剂,因此它们在发酵培养基中的存在对泡沫控制形成一个特别挑战。
疏水蛋白是一类充分定义的蛋白(Wessels,1997,Adv.Microb.Physio.38:1-45;Wosten,2001,AnnuRev.Microbiol.55:625-646),其能够在疏水/亲水界面处自组装,并具有以下保守序列:
Xn-C-X5-9-C-C-X11-39-C-X8-23-C-X5-9-C-C-X6-18-C-Xm,(SEQIDNo.1)
其中X代表任何氨基酸,n和m独立地代表整数。典型地,疏水蛋白具有至多125个氨基酸的长度。该保守序列中的半胱氨酸残基(C)是二硫桥键(bridge)的一部分。在本发明的上下文中,术语“疏水蛋白”具有更宽的含义,包括仍然显示在疏水-亲水界面自组装产生蛋白质膜的特性的功能上等价的蛋白质,例如包含以下序列或其部分,仍然显示在疏水-亲水界面自组装产生蛋白质膜的特性的蛋白质:
Xn-C-X1-50-C-X0-5-C-X1-100-C-X1-100-C-X1-50-C-X0-5-C-X1-50-C-Xm(SEQIDNo.2)。
根据本发明的定义,可通过将蛋白质吸附到特氟隆上,并使用圆二色性,以确定二级结构(一般是α-螺旋)的存在来检测自组装(DeVocht等人,1998,Biophys.J.74:2059-68)。
可以通过在蛋白质溶液中孵育特氟隆片,然后用水或缓冲剂洗涤至少3次来确定膜的形成(Wosten等人,1994,Embo.J.13:5848-54)。可通过如本领域充分建立的任何合适的方法例如用荧光标记物标记,或者通过使用荧光抗体来显现蛋白质膜。m和n的值典型为0-2000,但更通常地,m和n的总和小于100或200。在本发明的上下文中,疏水蛋白的定义包括疏水蛋白与另一种多肽的融合蛋白,以及疏水蛋白与其他分子如多糖的缀合物。
至今所鉴定的疏水蛋白一般归类为I类或II类。这两种类型已在真菌中鉴定为分泌性蛋白质,它们在疏水界面处自组装成两亲膜。I类疏水蛋白的组装体(Assemblage)一般相对不可溶解,而II类疏水蛋白的那些组装体容易溶解在各种溶剂中。优选地,所述疏水蛋白溶于水,这意味着,其至少0.1%,优选至少0.5%可溶于水。至少0.1%可溶于水是指使99.9mL水中的0.1g疏水蛋白在20℃下经受30000g离心30分钟时,不发生疏水蛋白沉淀。
在丝状细菌如放线菌(Actinomycetesp.)和链霉菌(Streptomycessp.)中也鉴定了疏水蛋白样蛋白质(例如“chaplins”)(WO01/74864;Talbot,2003,Curr.Biol,13:R696-R698)。与真菌疏水蛋白形成对比,这些细菌蛋白可能只形成至多一个二硫桥键,因为它们可能只有两个半胱氨酸残基。此类蛋白质是具有SEQIDNo.1和2中所示共有序列的疏水蛋白的功能性等价体的一个实例,并包括在本发明的范围内。
已经从超过16个真菌物种中克隆了多于34种编码疏水蛋白的基因(参见例如WO96/41882,其给出了双孢蘑菇(Agaricusbisporus)中鉴定的疏水蛋白的序列;以及Wosten,2001,AnnuRev.Microbiol.55:625-646)。就本发明而言,与天然存在的疏水蛋白在氨基酸水平上具有至少80%的同一性的疏水蛋白也包括在术语“疏水蛋白”内。
消泡剂
术语“消泡剂”既包括通常在发生发泡前加入的消泡剂,也包括通常在一旦已经形成泡沫就加入的那些消泡剂(有时称作“去泡剂(defoamers)”)。用于本发明的消泡剂的定义见“泡沫和它在发酵系统中的缓解”-BethJunker-BiotechnologyProgress,2007,23,768-784。
适用于本发明的一组特定消泡剂是显示浊点的那些消泡剂。浊点是当发生相分离(即,消泡剂分子形成散射光的聚集体)时,消泡剂水溶液变得明显浑浊的温度,如J.Eastoe在T.Cosgrove编著的ColloidScience:Principles,MethodsandApplications(BlackwellPublishing,2005)中SurfactantAggregationandAdsorptionatInterfaces第63页所述。
显示浊点的消泡剂的例子包括基于聚亚烷基二醇(PAG)的化合物,例如环氧乙烷/环氧丙烷嵌段共聚物、基于环氧乙烷/环氧丙烷嵌段共聚物的多元醇、以及环氧乙烷和环氧丙烷的聚醚;以及基于脂肪酸酯的化合物。
浊点取决于表面活性剂的组成和化学结构。例如,对于聚氧乙烯(PEO)非离子型表面活性剂,对于给定的疏水基团,浊点随着EO含量的增加而升高。优选地,消泡剂的浊点在0℃与90℃之间,更优选在5℃与60℃之间。
优选地,消泡剂包含至少一种非离子型表面活性剂/聚合物,如聚醚、聚亚烷基二醇、环氧乙烷/环氧丙烷嵌段共聚物、基于环氧乙烷/环氧丙烷嵌段共聚物的多元醇、基于聚丙二醇的聚醚分散体或烷氧基化脂肪酸酯。基于PAG的消泡剂(例如可得自希伦赛勒赫公司(SchillandSeilacher)的StruktolJ647)、基于EO/PO嵌段共聚物的多元醇(例如可得自希伦赛勒赫公司的StruktolJ647)及其他非离子型表面活性剂消泡剂在消灭泡沫方面特别有效,即使存在强有力的发泡剂如疏水蛋白时也是如此。
可以使用消泡剂的混合物,在该情况下,这种混合物的浊点定义为单独组分中的最高浊点。
显示浊点的一些常见的市售消泡剂示于表1中。
表1
消泡剂测定方法
使用用于非离子型表面活性剂的LangeLCK433水测试试剂盒测定滤液中消泡剂的浓度。这运用非离子型表面活性剂(如J647)与指示剂TBPE(四溴酚酞乙酯)形成复合物的原理,可以在二氯甲烷中提取该复合物,并进行光度测定以确定其浓度。首先,绘制校准曲线。通过吸取3.00gStruktolJ647的等分试样,并用MilliQ水于15℃稀释到1L,来制备StruktolJ647的0.3%(w/v)溶液。从该溶液吸取等分试样,并用MilliQ水稀释到给定浓度:6,15,30,60,150和300mg/L。MilliQ水用作空白样品。每个浓度的0.2ml样品被添加到含有TBPE和二氯甲烷的试剂盒测试试管中。轻轻混合试管2分钟,并停止30分钟。然后根据测试试剂盒的说明书,在LangeDR2800分光光度计中于605nm处进行测定。图2显示了得到的校准曲线图。
滤液然后用MilliQ水稀释1/10。在分光光度计中如前所述对0.2ml样品进行测定,从校准曲线图对每种滤液中的消泡剂浓度进行读数。滤液中剩余的消泡剂的含量(%)如下计算:
(滤液中测定的浓度)/(已知的起始浓度)x100%。
使用LangeLCK333水测试试剂盒,绘制合适浓度范围内的校准曲线,通过类似的技术,可以测定降至0.2mg/L(2x10-5%w/v)的消泡剂浓度。在这种情况下,添加待测定的2ml等分试样样品到测试试剂盒中,而不是0.2ml。
发酵方法与消泡剂的去除
通过在生物反应器(例如工业发酵罐)内,在液体发酵培养基中培养宿主细胞,进行发酵以生产发泡剂。选择培养基的组成(例如营养素、碳源等)、温度和pH,为培养物的生长和/或发泡剂的制备提供适当的条件。将空气或富氧空气正常鼓入培养基中,为培养物的呼吸提供氧气。
可将消泡剂包括在初始培养基组合物中和/或在发酵期间按需加入。通常的实践是采用泡沫检测方法,例如电导探针,其自动启动消泡剂的加入。在本发明中,消泡剂的存在终浓度优选为0.1-20g/L,更优选为1-10g/L。
在步骤i)期间,即,在发酵期间,发酵罐温度可高于或低于消泡剂的浊点。优选地,发酵罐温度高于消泡剂的浊点,因为消泡剂在高于其浊点的温度下对引起气泡聚结和泡沫崩塌最有效。一般对发酵罐温度进行选择,以达到用于生长宿主细胞和/或制备的最适宜条件。
在发酵结束时,必须基本上去除消泡剂,以确保发泡剂的功能不受削弱。通过确保发酵培养基的温度高于消泡剂的浊点,以使消泡剂相分离,实现消泡剂的去除。可通过任何合适的方法将相分离的消泡剂从发酵培养基中去除,所述方法例如:
-过滤,例如死端过滤或压滤器
-膜(交叉流)过滤,例如微滤或超滤
-离心
-吸附,使用例如活性炭、二氧化硅或硅藻土作为吸附剂。
若发酵培养基的温度高于所述浊点至少10℃,优选高于所述浊点至少20℃,最优选高于所述浊点至少30℃,则去除更多消泡剂。发酵培养基的温度优选低于90℃,更优选低于75℃。在一个优选的实施方案中,消泡剂的浊点为20-30℃,发酵培养基的温度为40-60℃。
用于分离消泡剂的优选方法是膜过滤。一般认为,在高于消泡剂的浊点的温度下对含消泡剂的发酵液进行膜过滤,导致由沉淀的消泡剂引起的膜污垢,导致低的渗透通量以及后续的处理困难:参见例如Yamagiwa等,J.Chem.Eng.Japan,26(1993)p13-18和WO01/014521。因此,先前认为膜过滤应在低于浊点的温度下进行。然而,在高于消泡剂的浊点约25℃的温度下进行超滤和微滤操作时,获得可接受的通量。
为了确保发泡剂产品不含细胞内材料和遗传材料(其通常视为不合需要的污染物),必须从发酵培养基中去除细胞。在一个优选的实施方案中,在去除沉淀的消泡剂的同时,将细胞从培养基中分离,例如在高于浊点的温度下所进行的微滤步骤中分离。
在另选的实施方式中,可在去除消泡剂之前,在单独的步骤中,在低于浊点的温度下从培养基中去除细胞——例如通过过滤(例如死端过滤或压滤器)、膜/交叉流过滤(例如微滤或超滤)或离心。在该实施方案中,提纯和/或浓缩步骤(例如通过超滤)可在去除细胞之后但在分离消泡剂之前进行(再次在低于浊点的温度下)。然后将培养基加热至高于浊点的温度,以致可如已经描述的那样将消泡剂去除。
一旦已从发酵培养基中去除消泡剂和细胞,就可根据需要,例如通过超滤进一步提纯和浓缩发泡剂产品。若发泡剂是疏水蛋白,可通过例如WO01/57076中所描述的程序将其从发酵培养基中提纯,所述程序涉及将疏水蛋白吸附到一表面上,然后使该表面与表面活性剂如Tween20接触,以将疏水蛋白从表面上洗脱。还参见Collen等,2002,BiochimBiophysActa.1569:139-50;Calonje等,2002,Can.J.Microbiol.48:1030-4;Askolin等,2001,ApplMicrobiolBiotechnol.57:124-30;和DeVries等,1999,EurJBiochem.262:377-85。
现将参考以下实施例进一步描述本发明,所述实施例仅仅是说明性的而非限制性的。
实施例1:从包含发泡剂的发酵液中去除消泡剂
对酿酒酵母(Saccharomycescerevisiae)遗传修饰菌株进行分批补料式发酵。通过掺入编码来自真菌里氏木霉(Trichodermareesei)的疏水蛋白HFBII(一种发泡剂)的基因,对所述菌株修饰,以致在发酵过程中实现疏水蛋白的细胞外表达。基本如vandeLaarT等在BiotechnolBioeng.96(3):483-94(1997)中所述进行发酵,使用葡萄糖作为碳源,并在300L发酵容器中将该工艺过程按比例放大(scaling)到总体积为150L。使用消泡剂StruktolJ647来控制发酵过程中的发泡(而非vandeLaarT等所使用的StruktolJ673)。
发酵结束时,在15℃(即,低于消泡剂J647的浊点)下微滤发酵液,以去除酵母细胞。在具有孔径为0.1μm的Kerasep陶瓷膜的中试装置(pilotscaleplant)上进行微滤,使用去离子水进行两份体积的渗滤(twovolumesofdiafiltration)。然后,再次在15℃下,对液体进行超滤,以部分提纯HFBII。通过1kDSynder缠绕式(spiralwound)聚合物膜,在0.9巴的跨膜压力下进行超滤,和进行四份体积的渗滤。
超滤步骤之后,发酵液中消泡剂的浓度经测量为0.196g/L。HFBII的浓度通过如下所述的高效液相色谱(HPLC)测量为0.320g/L。分析之前,用60%乙醇水溶液稀释样品,使其浓度约为200μg/ml。在30℃下,在VydacProteinC4柱(250x4.6mm)上进行HPLC分离。通过214nm处的UV检测测量疏水蛋白,通过与得自VTT生物技术公司(VTTBiotechnology)(埃斯普(Espoo),芬兰)的已知HFBII浓度的样品作比较,计算其浓度。
将不含细胞的发酵液加热至50℃,在该温度下保持30分钟,并且过滤(0.2μm孔径),以去除消泡剂。如前所述测量滤液中消泡剂和HFBII的剩余量,表2中给出结果(阶段1)。然后将来自该第一阶段的滤液再加热至50℃,在该温度下保持额外30分钟,并如前所述过滤。测量所得滤液中HFBII和消泡剂的浓度,也在表2中给出结果(阶段2)。
表2
阶段1 | 阶段2 | |
滤液中HFB II的量(g/L) | 0.32 | 0.30 |
剩余的初始HFB II浓度% | 100% | 93.75% |
滤液中消泡剂的量(g/L) | 0.05 | .028 |
剩余的初始消泡剂浓度% | 25.5% | 14.3% |
消泡剂∶疏水蛋白的质量比 | 0.156 | 0.093 |
发现得到的疏水蛋白溶液具有令人满意的发泡特性。
适当时,以上各单独部分中提到的本发明各种特征和实施方式经过必要的修改后,应用于其他部分。因此,适当时,一个部分中指明的特征可与其他部分中指明的特征相组合。
在以上说明书中提到的所有出版物均引入这里作为参考。不背离本发明范围的本发明所述方法的各种修改和变化对本领域技术人员来说是显而易见的。尽管已结合具体的优选实施方案描述了本发明,但应理解,所要求保护的本发明不应不适当地受限于此类具体的实施方案。实际上,对于相关领域的技术人员显而易见的用于实施本发明的所述方式的各种修改有意包括在所附权利要求书的范围之内。
Claims (10)
1.包含至少300mg/lII类疏水蛋白和至少0.3mg/l的具有浊点的消泡剂的水溶液,其中该消泡剂/疏水蛋白重量比低于0.2,且其中该浊点为20-30℃。
2.根据权利要求2的溶液,其中该疏水蛋白是来自里氏木霉的HFBI或HFBII。
3.根据权利要求1或2的溶液,其中该消泡剂包括聚亚烷基二醇。
4.根据权利要求1的溶液,其中该消泡剂/疏水蛋白重量比低于0.1。
5.根据权利要求1或2的溶液,其包含至少1g/l的所述疏水蛋白。
6.根据权利要求1或2的溶液,其包含至少100g/l的所述疏水蛋白。
7.用于制备根据权利要求1的溶液的方法,该溶液包含至少300mg/l的II类疏水蛋白和至少0.3mg/l的具有20-30℃的浊点的消泡剂,包括步骤:
a)在含有营养素和氧气、含有浓度为0.1-20g/l的具有浊点的消泡剂的液体发酵培养基中培养产生II类疏水蛋白的宿主细胞;
b)在高于消泡剂的浊点的温度下,通过过滤或膜(交叉流)过滤或离心或吸附将消泡剂从发酵培养基中分离,以制备包含II类疏水蛋白的溶液且其中该消泡剂/疏水蛋白重量比低于0.2。
8.根据权利要求7的方法,其中步骤b)中的温度高于该消泡剂的浊点至少10℃。
9.根据权利要求7的方法,其中该溶液包含至少1g/l的所述疏水蛋白。
10.根据权利要求7的方法,其中该溶液包含至少100g/l的所述疏水蛋白。
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Also Published As
Publication number | Publication date |
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CA2740326A1 (en) | 2010-04-22 |
CN102186967A (zh) | 2011-09-14 |
JP2012505645A (ja) | 2012-03-08 |
ZA201102685B (en) | 2012-06-27 |
CA2740326C (en) | 2017-06-13 |
US20100099844A1 (en) | 2010-04-22 |
BRPI0919788A2 (pt) | 2016-09-06 |
MX2011004080A (es) | 2011-07-28 |
DK2346987T3 (en) | 2016-04-25 |
WO2010043520A1 (en) | 2010-04-22 |
AU2009304092B2 (en) | 2013-09-05 |
AU2009304092A1 (en) | 2010-04-22 |
US9115349B2 (en) | 2015-08-25 |
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