CN1216061A - 具有磷脂酶活性的蛋白质 - Google Patents
具有磷脂酶活性的蛋白质 Download PDFInfo
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- CN1216061A CN1216061A CN199898800009A CN98800009A CN1216061A CN 1216061 A CN1216061 A CN 1216061A CN 199898800009 A CN199898800009 A CN 199898800009A CN 98800009 A CN98800009 A CN 98800009A CN 1216061 A CN1216061 A CN 1216061A
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- aspergillus
- albumen
- phospholipase activity
- ala
- lysophospholipase
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Abstract
本发明涉及具有磷脂酶活性的蛋白质,其特征在于它其具有曲霉属溶血磷脂酶的成熟序列或由它衍生的序列,并且它可以在至少一个位点被酶切,其中在酶切时所述限制片段可选地或者通过至少一个在还原条件下可酶切的键连接或者至少一个所述未连接限制片段具有磷脂酶活性,本发明还涉及通过在适当培养基中发酵培养适当转化的产生溶血磷脂酶的宿主生物体并从该无细胞培养滤液中分离具有磷脂酶活性的蛋白而生产该蛋白的方法,其中在酸性至微弱碱性范围进行发酵。
Description
描述
本发明涉及具有磷脂酶活性的蛋白质，它具有曲霉属溶血磷脂酶的成熟序列或由它衍生的序列，它可以在至少一个位点被酶切，其中所述限制片段可选地或者通过至少一个在还原条件下可酶切的键连接或者至少一个所述未连接限制片段具有磷脂酶活性。本发明另外涉及生产该蛋白质的方法以及该蛋白质用于植物油脱胶和做为焙烤辅助剂的用途。
当使食用油脱胶时，用磷脂酶以低成本和环保的方式将不能水合的磷脂变为水溶性，因此可以温和地从该食用油中除去。欧洲专利申请0513709(Rhm/Lurgi)第一次提出了有效的酶促脱胶工艺。在该工艺中，将先前用水脱胶的一种食用油用磷脂酶的水溶液乳化，产生小于10μm的小滴。水解(pH3-6，温度50-70℃)后，分离水相。Lurgi将此酶促脱胶工艺做为“EnzyMax工艺”引入食用油工业。DE 4339556描述该工业的一个变化，包括通过加入表面活性剂或加溶剂将该酶从含有胶的用过的水相中溶解出来而再使用该酶，并将它做为大致无胶的含酶溶液序列使用。
可能仅用微生物生产用于在大工业规模上运行该工艺所需的足够数量的酶。因此需要有一允许生产无限量该磷脂酶的微生物源。DE-OS 19527275.9(Rhm/Lurgi)，日期为1995年7月26日，声明已在黑曲霉中发现合适的磷脂酶。该磷脂酶将卵磷脂酶切为溶血卵磷脂，但也能够将溶血卵磷脂进一步酶切产生磷脂酰胆碱。来自曲霉属的仅能酶切溶血卵磷脂的纯溶血磷脂酶在脱胶工艺中无效。这也适用于非酰基酶切的磷脂酶C和D。
也可以将磷脂酶做为焙烤辅助剂使用以改进面团加工。
本发明的目标是提供提高纯度的低成本磷脂酶。应当可以通过转化的宿主生物大量生产该磷脂酶。使用该酶，应该可以制备特别适于水解磷脂并因此用于澄清淀粉水解产物，并用于制备焙烤辅助剂的制剂。
按照本发明、通过具有磷脂酶活性的蛋白质达到这个目标，该蛋白的特征在于它具有曲霉属溶血磷脂酶的成熟序列或由它衍生的序列，它可以在至少一个位点被酶切，其中在酶切时，所述限制片段或者通过至少一个在还原条件下可酶切的键连接或者至少一个所述未连接限制片段具有磷脂酶活性。还可以通过具有磷脂酶活性的蛋白质达到这个目标，该蛋白的特征在于它被抗来自臭曲霉RH 3046的纯化磷脂酶的抗体所识别。
已意外地发现按照DE-OS 19620649.9用可从曲霉属分离的脱氧核糖核酸(DNA)转化的微生物不仅编码溶血磷脂酶，而且在一定培养条件下，继续加工产生磷脂酶。该磷脂酶因此具有与该溶血磷脂酶相同的一级结构，但具有不同的二级和三级结构并因此具有不同的生理性质。相应的序列示于DE-OS 19620649.9的SEQ ID no.1。已从黑曲霉分离出另一个磷脂酶编码序列，仅6％的其氨基酸与来自泡盛曲霉的同源序列不同。来自黑曲霉的磷脂酶和来自臭曲霉的溶血磷脂酶都由270个氨基酸组成，并且分子量都为36000Da(参看SEQ ID no.1+2)。在得到该转化微生物方面请查阅DE-OS 19620649.9的说明书。在现有技术中从未描述过来自曲霉属的磷脂酶。Nakaya等的论文(Eur.J.Biochem.1990,193(1)31-38)描述了具有与磷脂酶A2类似序列的蛋白质。
使用蛋白化学方法可以从溶血磷脂酶分离磷脂酶并得到提高纯度的磷脂酶。比较该纯化的磷脂酶与溶血磷脂酶发现以下差别：
在还原条件下用SDS凝胶电泳测定来自泡盛曲霉的磷脂酶和溶血磷脂酶的分子量，磷脂酶为约30000 Da，溶血磷脂酶为约36000Da，而在非还原条件下测定时，这两种酶的分子量相同，约为36000Da。在还原条件下，该磷脂酶分解为二条链，较大的那条链(30000Da)保留在电泳凝胶中。由于方法学的原因，在同一电泳凝胶中不能检测到约6000Da大小的片段，但由磷脂酶由二条肽链组成的发现可以推测出。该蛋白质的测序结果证实了这个观点。
来自臭曲霉的磷脂酶的蛋白测序发现与该溶血磷脂酶序列有很高的同源性，但也有差异。在磷脂酶中，发现比例为1∶1的二个NH2末端基团，而在溶血磷脂酶中只发现一个。该磷脂酶的二个NH2末端基团中的一个属于6000Da的肽，而另一个则是该30000Da蛋白质的NH2末端基团。该较小的肽对应于该成熟溶血磷脂酶蛋白(参看DE-OS 19620649.9中的序列ID no.1)的1-44氨基酸，该30000Da蛋白质的序列对应于该溶血磷脂酶(参看DE-OS 19620649.9中的序列ID no.1)的45-270氨基酸。
该发现清楚地表明来自臭曲霉的磷脂酶可以通过加工该溶血磷脂酶而得到，其中该加工是在细胞内还是在细胞外进行和怎样进行尚待澄清。磷脂酶与溶血磷脂酶的关系示于图1。
另外，磷脂酶与溶血磷脂酶的区别在于它们的等电点、最适pH和最适温度，并在温度稳定性方面区别明显。这些参数在下表中做了比较。表1.来自臭曲霉的磷脂酶和溶血磷脂酶的性质比较
磷脂酶 溶血磷脂酶分子量(SDS凝胶，还原) 30000Da 36000Da分子量(SDS凝胶，非还 36000Da 36000Da原)等电点 pH4.3 pH4.2最适温度 50℃ 55℃最适pH pH3-4 pH4.5pH稳定性(60℃1小时) pH3.5 pH4.5
＞75％残留活性 10％残留活性
发酵条件对从该微生物得到磷脂酶而不是溶血磷脂酶是必要的。有必要在酸性至微弱碱性培养基中培养以形成该磷脂酶。适于该目的的合适pH值范围是2-9，优选为3-8。在这些条件下，优先形成磷脂酶。使用下列方法：
首先以达到最简单地生产该磷脂酶为目标选择合适的宿主。尽管可以考虑许多霉菌种做为可能的宿主，例如嗜热属腐质霉属、Thermomyces和毛霉属、根霉属、犁头霉属、毛壳属、木霉属、梭孢壳属、青霉属和头孢属，但最好使用曲霉属的种。一旦用按照本发明的质粒转化后，可以分离那些转化体，转化体与所述宿主相比，可以形成大量的磷脂酶。该转化宿主生物最好是米曲霉、酱油曲霉、黑曲霉、泡盛曲霉、臭曲霉、椭圆曲霉、棘孢曲霉、碳黑曲霉或海枣曲霉种的曲霉菌株或绿色木霉、Trichoderma longibrachiatum或Trichoderma reesei种的木霉属菌株。
将用选择质粒(最好是pAN7-1、p3SR2或pSTA10)和表达质粒(最好是pKC3、pKC9、pKC12或pKCN2)共转化宿主菌株产生的转化体在该宿主菌株的常规营养溶液中培养，该溶液含有至少一种可代谢碳源(例如玉米粉、淀粉、淀粉糊精)和至少一种可代谢有机氮源(例如玉米浸出液、酵母自溶物、大豆粉、大豆蛋白质或大豆胨，该有机氮源可单独存在或与无机氮源(例如铵盐或硝酸盐)组合存在，将该溶液灭菌后，将其pH值调节至酸性至微弱碱性。可以通过加入特异促进磷脂酶形成的物质补充该营养溶液。这类物质存在于大豆磷脂中，但它们也存在于其它种类的物质中，例如聚氧乙烯醚。当用该转化体的分生孢子或营养菌丝体接种该已灭菌的营养溶液后，该转化体在通气的条件下在20-60℃，优选25-45℃的温度下生长，并产生按照本发明的磷脂酶。通过加入酸或碱在培养中校正该培养物的pH值，使之保持在酸性至微弱碱性范围，优选为pH3-8。在培养48-120小时后，通过分离不溶营养溶液残留物和生物量(一般用过滤)并用常规方法(例如超滤)浓缩该滤液回收该磷脂酶。可以将该浓缩液(保留液)用以植物油脱胶或用于处理磷脂。另外，可以将该磷脂酶用于改进食料的流变学性质。
下列微生物已按照布达佩斯特条约的条款储存于德意志微生物保藏中心(DSM)Mascheroder Weg 1B,38124 Braunschweig，德国：
米曲霉RH 3745：保藏号为DSM 11283(保藏日期：1996年11月11日)
椭圆曲霉RH 3886：保藏号为DSM 11284(保藏日期：1996年11月11日)
臭曲霉RH 3046：保藏号为DSM 10652(保藏日期：1996年4月24日)
大肠杆菌DH5α pKC3：保藏号为DSM 10653(保藏日期：1996年4月24日)
大肠杆菌DH5αpKC9：保藏号为DSM 10654(保藏日期：1996年4月24日)
大肠杆菌DH5αpKC12：保藏号为DSM 10655 (保藏日期：1996年4月24日)
大肠杆菌pKCN2：保藏号为DSM 11352(保藏日期：1996年12月23日)
黑曲霉RH3909：保藏号为DSM 11353(保藏日期：1996年12月23日)
下列实施例和图更详细地说明本发明。
图1展示来自曲霉属的溶血磷脂酶基因的加工和该磷脂酶的分离。
图2展示质粒pKCN2的结构。
实施例
实施例1表达载体pKCN2的结构来自黑曲霉NRRL3的溶血磷脂酶基因的分离
采用Hynes,M.J.等(1983),Mol.Cell.Biol.3,1430-1439描述的方法分离黑曲霉NRRL3的染色体DNA。
用Sau3AI部分水解得到的高分子量DNA并用蔗糖密度梯度离心进行分级分离。将大小为9-20kb的DNA分子插入用BamHⅠ/EcoRⅠ水解的EMBL3-DNA中并进行体外包装。
将来自质粒pKCl/36的HindⅢ/SalⅠ-cDNA片段用做杂交探针以鉴定在λEMBL3基因文库中的染色体溶血磷脂酶基因。质粒pKCl/36含有从臭曲霉RH 3046分离得到的该溶血磷脂酶cDNA。
在杂交和重复选择后，鉴定出二个阳性克隆。制备1号克隆的噬菌体DNA并用BamHⅠ消化。在Southern杂交后，在约9kb处展示一个阳性信号。将该BamHⅠ片段克隆入pUC18并将得到的含有该完整染色体溶血磷脂酶基因的质粒命名为pKCN。表达载体pKCN2的结构
将质粒pKCN2中的该溶血磷脂酶基因置于米曲霉α淀粉酶启动子和构巢曲霉trpC终止子的控制之下。
用PCR方法从该质粒pKCN分离该溶血磷脂酶基因。使用具有下列序列的二个寡核苷酸引物：KC29:5＇-GGA ATT CAC CTG CTA ACC ATG TTC TCT GGA CGG TTT GGA GTG-3＇
BspMⅠ Met(SEQ ID no.3)KC43:5＇-CG GGATCC AAG CTA TAG CAG ACA CTC TGA AAT TG-3＇
BamHⅠ AMB(SEQ ID no.4)
将50mM KCl、1.5mM MgCl2、0.2mM dNTP(dATP、dCTP、dGTP、dTTP各0.2mM)、KC29和KC43各50pmol、1ng pKCN做为模板和2.5 U Tag聚合酶一同混合于0.1ml 20mM tris/HCl,pH8.4的反应体积内进行聚合酶链式反应。将该混合物经过20个循环(94℃ 40秒；40℃ 1分钟；72℃ 1分钟)。反应完成时，将该扩增片段纯化，并用BspMⅠ和BamHⅠ水解并插入用NcoⅠ/BamHⅠ酶切的质粒pKE2中。该质粒pKE2含有米曲霉α淀粉酶启动子和构巢曲霉trpC终止子。
通过限制分析和后续测序证实了质粒pKCN2的结构。
实施例2曲霉属和Trichoderma reesei菌株的转化方法
从二周龄的欲转化真菌菌株的陪氏平皿培养物中，借助在约10ml0.85％NaCl中的匙状小竹板(spatula)通过浮集法制备孢子悬浮液。在四个各含有100ml Czapek-Dox基本培养基(Oxoid)和0.1％酵母膏的1升摇瓶中各接种1ml孢子悬浮液，并在轨道式摇床上以120转/分钟在28℃培养约16小时。将这四个摇瓶中的菌丝体收集于滤纸上并用约50ml MP缓冲液(10mM磷酸盐缓冲液中含1.2M MgSO4,pH5.8)清洗。将缓冲液排干后，将该湿润的菌丝体称重。一般得到约3-5g湿菌丝体。
在每克湿菌丝体中加入5ml MP缓冲液、120μl Novozym溶液(在6ml MP缓冲液中有1g Novozym234(Novo Nordisk))和25μl β-葡糖苷酸酶(Sigma)。将该菌丝体悬浮液置于冰水中5分钟。然后加入60μl牛血清白蛋白溶液(在4ml MP缓冲液中有0.2g牛血清白蛋白，过滤灭菌)，将该混合物于30℃温和振荡温育。用显微镜目视监测原生质体的形成。一旦不再观察到原生质体形成的进一步增加时，终止温育该混合物以收获所述原生质体。这一般在3-5小时后发生。
将该原生质体悬浮液通过用MP缓冲液饱和的玻璃棉滤器以除去任何仍然存在的粗菌丝体组分并转移至微量离心管中。将一层600mM山梨醇、100mM tris/HCl、pH7.0导入该微量离心管的上半部。将该微量离心管于2500g离心10分钟。将原生质体从夹层取出并悬浮于1M山梨醇、10mM tris/HCl,pH7.5。然后通过于1500g离心用STC缓冲液(1M山梨醇、10 mM tris/HCl,pH7.5、10mM CaCl2)洗涤二次，最终悬浮于1ml STC缓冲液中。
通过在25μl 10mM tris/HCl,pH8.0中混合300μl原生质体悬浮液、约10μg做为选择质粒的p3SR2和10μg表达该LPL的特定质粒，并将该混合物于0℃温育10分钟来转化米曲霉。将另外25μl的同样的质粒混合物与400μl PEG溶液(在10mM tris/HCl,pH7.5、50mM CaCl2中有60％聚乙二醇6000(Fluka))组合，极小心地混匀并于室温温育5分钟。加入另外600μl PEG溶液，混匀并将该混合物于室温温育另外20分钟。将该混合物于45℃与约9ml乙酰胺软琼脂(含有10mM做为唯一氮源的乙酰胺、1M蔗糖、0.6％(重量)琼脂的基本培养基)混合并分配至四个含有同样培养基但具有1.5％(重量)琼脂(Oxoid)且有15mM CsCl的陪氏平皿中。将这些平皿于28℃培养。6-10天后，将快速生长的菌落(转化体)再接种至不含蔗糖的乙酰胺培养基，通过单孢子菌落方法纯化二次，并最终转移至完全培养基，例如马铃薯-右旋糖-琼脂。
也可以用质粒p3SR2转化黑曲霉、泡盛曲霉、日本曲霉或臭曲霉种的菌株。然而，最好用质粒pAN7-1进行转化。按照与上述质粒p3SR2相似的方式进行原生质体制备和加入质粒DNA。然而，将全部转化混合物而不是乙酰胺软琼脂加入100ml Czapek-Dox基本培养基(Oxoid)并小心混合，该培养基含有100μg潮霉素B/ml、1.5％(重量)琼脂(Oxoid)和1M蔗糖并已冷却至约45℃。然后将该混合物以10ml一份置于陪氏平皿中，在每个这种平皿中已加入了含有1.5％(重量)琼脂(Oxoid)但不含潮霉素和蔗糖的10ml Czapek-Dox基本培养基(Oxoid)做为固体底层。一旦上部琼脂层固化，将所述陪氏平皿于30-37℃培养。约3-10天后可以传代培养抗潮霉素B的转化体，并且为了测试抗性，将转化体转移至含有50μg潮霉素B/ml和1.5％(重量)琼脂(Oxoid)的Czapek-Dox基本培养基(Oxoid)。
使用第三个选择原理转化酱油曲霉或海枣曲霉，因为这些种的菌株都代谢乙酰胺并对潮霉素B有抗性。通过在含氯酸盐的营养培养基(Cove,D.J.(1976)Heredity 36,191-203)上选择，分离具有缺陷型硝酸盐还原酶(niaD)基因的突变体(即用硝酸盐做为唯一氮源时不生长)。该缺陷被质粒pSTA10的转化所抵消(Unkles,S.E等(1989)Mol.GenGenet.218,99-104)，该质粒含有该硝酸盐还原酶基因的全部信息，使得该转化体可以在硝酸盐做为唯一氮源时生长，而未转化细胞的生长则受阻止。
该选择方法同样适用于其它曲霉属种以及酱油曲霉；然而，与利用潮霉素B抗性或乙酰胺代谢相比，制备所述niaD突变体需要额外的努力。
实施例3分泌PL的转化体的生产黑曲霉、泡盛曲霉、臭曲霉、碳黑曲霉和椭圆曲霉的转化体
按照实施例1描述的方法制备这些曲霉属种的原生质体，并用质粒pAN7-1和质粒pKC3、pKC9、pKCl2或pKCN2中的一个共转化。按上述方法将所述转化混合液平板接种于潮霉素上再生所述原生质体，从所述再生平板上分离所述转化体，纯化并用下列营养溶液在振荡测试中测试它门的PL生产
麦芽糖糊精 3.75％
玉米浸出液 3.0％
KH2PO4 1.0％
K2HPO4 0.7％
Triton X-100 0.10％
以上均溶于自来水中，于121℃灭菌30分钟。
为此目的，从所述振荡培养物中过滤出生物质，并测定该培养过滤液中磷脂酶活性(PLU)。转化体由于磷脂酶活性显著提高而区别于该宿主菌株。米曲霉和棘孢曲霉的转化体
按照实施例2所述对这些种进行原生质体制备和转化。用质粒p3SR2和质粒pKC3、pKC9、pKC12或pKCN2中的一个共转化所述原生质体。按上述方法将所述转化混合液平板接种于含有乙酰胺做为唯一氮源的营养培养基上再生所述原生质体，从所述再生平板上分离所述转化体，纯化并用下列营养溶液在振荡测试中测试它们的PL生产
麦芽糖糊精 3.75％
玉米浸出液 3.0％
(NH4)2PO4 0.5％
Triton X-100 0.10％
以上均溶于自来水中，于121℃灭菌30分钟。酱油曲霉和海枣曲霉的转化体
将按照Cove(1976)制备的酱油曲霉RH 3782和海枣曲霉RH3828的突变体菌株酱油曲霉RH3782 niaD22和海枣曲霉RH 3828niaD在按照以下制备的营养溶液中培养
葡萄糖(Merck) 2％
麦芽膏(Oxoid) 0.5％
细菌蛋白胨 0.025％
(Bacto-peptone)(Difco)
去离子水
调节pH至5.0；于121℃灭菌30分钟。
用实施例1所述方法从该菌丝体得到原生质体。用做为选择质粒的pSTA10和质粒pKC3、pKC9或pKCl2中的一个共转化所述原生质体。将该转化混合液与由以下组成的9ml软琼脂(渗透压稳定)混合再生所述原生质体
0.1M磷酸钠缓冲液pH 6.0 15ml
1M蔗糖(Merck) 10.28g
Millipore水补至 29.1ml
琼脂(Oxoid) 0.18g(=0.6％)
于121℃灭菌30分钟，然后无菌加入
盐溶液(7.14.2) 0.6ml
1M NaNO3溶液 0.3ml并且将该混合物均分于具有相同组成但用1％琼脂制备的四个琼脂平板上。于37℃培养6-10天后，从所述琼脂平板分离所述转化体，并通过平板接种于硝酸盐/蔗糖琼脂上纯化。通过选择和在含有硝酸盐做为唯一氮源的营养培养基上后续纯化得到许多转化体，用下列营养溶液测试在振荡烧瓶中PL生产
麦芽糖糊精 3.75％
玉米浸出液 3.0％
KH2PO4 1.0％
K2HPO4 0.7％
Triton X-100 1.0％
以上均溶于自来水中，于121℃灭菌30分钟。
除了不产生或仅产生少量PL的转化体和未转化宿主菌株之外，还发现了在该培养物滤液中表现出显著增加的PL活性的转化体。这些菌株命名为共转化体，适于该酶的生产。表2比较了转化体和未转化宿主的典型的PL形成。表2：宿主菌株和转化体PL形成的比较菌株或转化体 相对PL活性米曲霉RH 3745 100米曲霉RH 3745 p3SR pKC9 3000-4000米曲霉RH 3745 p3SR pKCN2 2000-2500酱油曲霉RH 3782 niaD22 100酱油曲霉RH 3782 niaD22 pSTA10 pKC9 500-700臭曲霉RH 3046 100臭曲霉RH 3046 pAN7-1 pKC9 1000-1500海枣曲霉RH 3828 niaD 100海枣曲霉RH 3828 niaD pSTA10 pKC9 400-600椭圆曲霉 100椭圆曲霉pAN7-1 pKCl2 800-900异形曲霉niaD 100异形曲霉niaD pSTA10 pKC9 900-1000碳黑曲霉 100碳黑曲霉pAN7-1 pKC9 400-600菌株或转化体 相对PL活性棘孢曲霉 100棘孢曲霉p3SR2 pKC9 900-1200黑曲霉 100黑曲霉pAN7-1 pKCl2 700-1000泡盛曲霉 100泡盛曲霉pAN7-1 pKCl2 600-800
实施例4从臭曲霉纯化PL
将2080ml的臭曲霉RH 3788的培养保留液用3520ml蒸馏水稀释以降低电导率，并用160ml NaOH调至pH7.0。该样品的体积为5760ml，电导率为7.8mS/em。
在另一步骤中，在DEAE-Fractogel(Merck)上进行离子交换层析。为此目的，分四份(每份1440ml)将5760ml的该酶溶液导入DEAE-Fractogel柱(高278mm，直径100mm)。将该柱用缓冲液A(20mM自Na2HPO4/K2PO4制备的磷酸盐缓冲液，pH7.0+15mM NaCl)冲洗。用缓中液A至缓冲液B(缓冲液A+1M NaCl)的连续梯度洗脱。以70ml/min的洗脱流速进行洗脱，收集350ml的分部收集液。
测试该分部收集液的PL的存在。通过测定PL活性达到这点。这里，一个PL单位定义为在pH3.5和40℃下卵磷脂水溶液中导致水解速率为1μM/分钟的酶量。
按以下测定PL活性：
底物：1g Epikuron 200(来自Lucas Meyer的磷脂酰胆碱)+100g蒸馏水+5ml 0.32M CaCl2溶液，并用Ultra Turrax匀浆。
分析：将10ml底物与10ml 1％ Triton X-100溶液(Fluka)和5ml0.0033M柠檬酸一水合物溶液混合，并于40℃保持10分钟；将pH保持在3.4-3.5，加入0.1ml酶溶液并将该混合物于40℃温育10分钟。测试混合物中的该酶浓度应不超过2.5U/g。该反应时间完成时，将该混合物用0.01M KOH返滴定至pH10.0，其中快速加入前5ml，然后降低滴定速率以防止过量滴定。
将该酶加热至95℃ 15分钟并失活得到空白值(BV)。冷却后，对主值(MV)进行稀释并对主值继续进行该程序。计算：
在1995年7月26日的专利申请195 27 274.9中，用卵磷脂酶单位LU/g表述磷脂酶。一个卵磷脂酶单位是于40℃、pH8于1分钟内从蛋黄释放1μM脂肪酸的酶量。1LU/g,pH8对应于108PLU/g,pH3.5。
PL在约0.11M NaCl下开始洗脱。将四轮含PL的分部收集液合并(8950ml)，并使用Amicon CH2A浓缩器(中空纤维柱(cartridge)MW10,000)浓缩至2570ml。将该样品与782ml的3M硫酸铵溶液一起搅拌(样品现含有0.7M硫酸铵)。
在下一步骤中，将3352ml样品导入苯基Sepharose 6快速流动低置换柱(Pharmacia，柱高215mm，直径100mm)。用缓冲液C(20mM磷酸盐缓冲液，pH7.0+0.5M硫酸铵)重新洗涤该柱，并用从缓冲液C至缓冲液D(20mM磷酸盐缓冲液，pH7.0)的连续降低梯度洗脱。合并含有PL的分部收集液(790ml)并用浓缩器(如上述)浓缩，对缓冲液D透析；得到150ml样品。
在另一步骤中，将5个30ml份的该样品导入Mono Q(Pharmacia,6.3ml)阴离子交换层析柱。用缓冲液D冲洗该柱并用从缓冲液D至缓冲液B的连续梯度进行洗脱，PL于约200mM NaCl时开始洗脱。
合并含有PL的分部收集液并通过PD-10柱(Pharmacia)对缓冲液E(20mM磷酸盐缓冲液，pH7.1)透析。该样品体积为24ml。通过Moho P HR5/20(层析聚焦)最终纯化PL
将该24ml(见上述)导入Mono P柱(柱高200mm，直径5mm)。用缓冲液E重新洗涤该柱。用缓冲液F(Phramacia的Polybuffer 74，用蒸馏水1∶10稀释并用1M HCl调节至pH4.0)洗脱该样品。当13倍柱体积的缓冲液F流过该柱后，PL被洗脱。
在SDS凝胶电泳中，该纯化蛋白展示一个分子量为约31000道尔顿的一致的条带。等电点为约pH4.3。以这种方式纯化的蛋白用于测序。以这种方式分离的磷脂酶用于在兔子中得到抗体。按照Harlowe和Lane(参见Ed Harlowe和Lane,Antibodies，冷泉港实验室，1988)描述的标准方法进行免疫。可以将得到的抗血清直接用于Western印记分析(也由Harlowe和David Lane描述)，其中它特异性地标记该磷脂酶条带。
实施例5豆油脱胶
将具有残留磷酸酯含量为160ppm的200g湿脱胶豆油在圆底烧瓶中加热至40℃。加入含有20mg柠檬酸和100单位磷脂酶的10g水。该酶源自含有该磷脂酶结构的黑曲霉转化体发酵。于pH3.5测定活性。为此目的，将含有0.5ml 0.32M CaCl2的10ml 1％磷脂酰胆碱(得自Lucas Meyer的Epikuron 200)与10ml Triton X-100溶液和5ml 0.0033M柠檬酸一水合物混合，并于40℃保持10分钟。加入0.1ml相应稀释的酶溶于，于40℃温育10分钟。用0.01M KOH溶液将该混合物滴定至pH10。减去该空白值(将酶溶液于该混合物中加热至95℃15分钟)，按照实施例3进行计算。
通过外部离心泵将该圆底烧瓶的内容物剧烈分散，该烧瓶内容物每分钟约通过该泵一次。水相的颗粒大小低于1μ。每隔二小时取样并测定其磷含量。得到以下值：时间(小时)0 2 4 6 8磷含量 160 24 12 7 3(ppm)
在那些使用所述方法、但却未加入该酶制剂而加入了相应量的乳清蛋白(即非酶市售蛋白)或溶血磷脂酶(来自Enzyme Biosystems,USA的G-Zyme，每200ml豆油1000溶血磷脂酶单位)的试验中，未能将该磷含量减少至低于80ppm。
实施例6面团质量的改进
用按照本发明的磷脂酶进行以下焙烤试验。用以下制备面团：100重量份面粉、2重量份盐、3重量份面包酵母、58-60重量份水和40-50ppm维生素C(相对于面团重量)，在螺旋捏合机(制造厂：Kemper)中于低水平1处理2分钟、于高水平2处理6分钟。在捏合开始前加入所述酶和其它添加剂。面团温度为23-25℃。静置20分钟后，将该面团分成350g的部分以制备开放焙烤的白面包、成形、于32℃和80％相对湿度发酵70或90分钟，并于230℃焙烤32分钟。表3展示了各种酶添加剂的面包体积。该焙烤结果显示加入磷脂酶改进了该面包的焙烤体积和面包屑结构。从延长的发酵时间(90分钟)的优良焙烤结果可以明显看出该面团的稳定化作用。
表3：焙烤试验
| 添加剂/100g面粉 | 焙烤体积，70分钟发酵 | ％ | 90分钟发酵 | ％ | 多孔结构 |
| 无添加剂 | 1000ccm | 100 | 1050ccm | 100 | 不规则 |
| 真菌淀粉酶，10000 SKB | 1050ccm | 105 | 1130ccm | 107 | 不规则 |
| 真菌淀粉酶，10000 SKB+磷脂酶，2500 PLU | 1100ccm | 110 | 1225ccm | 117 | 不规则 |
| 真菌淀粉酶，50000 SKB | 1225ccm | 122 | 1275ccm | 121 | 不规则 |
| 真菌淀粉酶，50000 SKB+磷脂酶，12500 PLU | 1275ccm | 128 | 1365ccm | 130 | 规则 |
| 真菌木聚糖酶，12000 UXYL | 1325ccm | 133 | 1375ccm | 131 | 规则 |
| 真菌木聚糖酶，1200 UXYL+磷脂酶，12500 PLU | 1375ccm | 138 | 1475ccm | 140 | 规则 |
顺序表(1)一般信息：(ⅰ)申请人
(A)名称：Roehm GmbH
(B)街道：Kirschenallee
(C)城市：Darmstadt
(D)国家：德国
(F)邮编：64293(ⅱ)发明名称：具有磷脂酶活性的蛋白(ⅲ)顺序数：4(ⅳ)计算机阅读形式：
(A)数据媒体：软盘
(B)计
算机：IBMPC兼容机
(C)操作系统：PC-DOS/MS-DOS
(D)软件：PatentIn Release #1.0，版本#1.30(EPO)(2)SEQ ID NO:1的信息：(ⅰ)顺序特征：
(A)长度：1368碱基对
(B)类型：核苷酸
(C)链性：双链
(D)拓扑学：线性(ⅱ)分子类型：基因组DNA(ⅲ)假设：否(ⅳ)反义：否(ⅸ)特征：
(A)名称/关键词：内含子
(B)位置：222...275(ⅸ)特征：
(A)名称/关键词：内含子
(B)位置：442...486(ⅸ)特征：
(A)名称/关键词：内含子
(B)位置：824...874(ⅸ)特征：
(A)名称/关键词：CDS
(B)位置：连接(140...221,276...441,487...823,
875...1180)(ⅸ)特征：
(A)名称/关键词：成熟肽
(B)位置：221...1180(ⅸ)特征：
(A)名称/关键词：信号肽
(B)位置：140...220(ⅹⅰ)顺序描述：SEQ ID NO:1: ATGGGGAATT GGGGTGGGTA ATATGATACA GGTATAAAAG GGGGCTCGGA GGTGCAGTTG 6GATAGAAGCA TTGTGTGTGC ATTGCAGCAG TCCGTTGGTC TCACGTCTCT GGTTGCCTCG 12ATTGTATATA TACTGCAGG ATG TTC TCT GGA CGG TTT GGA GTG CTT TTG ACA 17
Met Phe Ser Gly Arg Phe Gly Val Leu Leu Thr
-27 -25 -20GCG CTT GCT GCG CTG TGT GCT GCG GCA CCG ACA CCA CTT GAT GTG CGG A 22Ala Leu Ala Ala Leu Cys Ala Ala Ala Pro Thr Pro Leu Asp Val Arg
-15 -10 -5GTAGGTGTGC CTGATTTGAA GTGGCTGGAT AGCACTGATG AAGGTTTTGA.ATAG GT 27
Ser
1GTC TCG ACT TCC ACG TTG GAT GAG CTG CAA TTG TTC TCG CAA TGG TCT 32Val Ser Thr Ser Thr Leu Asp Glu Leu Gln Leu Phe Ser Gln Trp Ser
5 10 15GCC GCA GCT TAT TGC TCG AAC AAT ATC GAC TCG GAC GAC TCT AAC GTG 37Ala Ala Ala Tyr Cys Ser Asn Asn Ile Asp Ser Asp Asp Ser Asn Val
20 25 30ACA TGC ACG GCC GAC GCC TGT CCA TCA GTC GAG GAG GCG AGC ACC AAG 42Thr Cys Thr Ala Asp Ala Cys Pro Ser Val Glu Glu Ala Ser Thr Lys
35 40 45ATG CTG CTG GAG TTT GAC CT GTATGTTGCT CCAGTGAAAT GGATAGAACA 47Met Leu Leu Glu Phe Asp Leu50 55CAGCTGATTG AATAG G ACA AAT AAC TTT GGA GGC ACA GCC GGT TTC CTG 52
Thr Asn Asn Phe Gly Gly Thr Ala Gly Phe Leu
60 65GCC GCG GAC AAC ACC AAC AAG CGG CTC GTG GTC GCC TTC CGA GGC AGT 56Ala Ala Asp Ash Thr Asn Lys Arg Leu Val Val Ala Phe Arg Gly Ser
70 75 80AGC ACC ATC AAG AAC TGG ATT GCT GAT CTC GAC TTC ATC CTG CAA GAT 61Ser Thr Ile Lys Asn Trp Ile Ala Asp Leu Asp Phe Ile Leu Gln Asp
85 90 95AAC GAT GAC CTC TGT ACT GGC TGC AAG GTT CAC ACT GGA TTC TGG AAG 66Asn Asp Asp Leu Cys Thr Gly Cys Lys Val His Thr Gly Phe Trp Lys100 105 110 115GCA TGG GAA GCC GCT GCA GAC AAT CTG ACG AGC AAG ATC AAG TCC GCG 71Ala Trp Glu Ala Ala Ala Asp Asn Leu Thr Ser Lys Ile Lys Ser Ala
120 125 130ATG AGC ACG TAT TCG GGC TAT ACC CTC TAC TTC ACC GGG CAC AGC TTG 76CMet Ser Thr Tyr Ser Gly Tyr Thr Leu Tyr Phe Thr Gly His Ser Leu
135 140 145GGC GGC GCA TTG GCT ACA CTG GGA GCA ACG GTC TTG CGA AAT GAC GGT 808Gly Gly Ala Leu Ala Thr Leu Gly Ala Thr Val Leu Arg Asn Asp Gly
150 155 160TAT AGC GTT GAA CTG GTGAGTGCTT CAGAGGGTGA TCATTAAACA GCCGGTTCTG 863Tyr Ser Val Glu Leu
165ACAGTCAATA G TAC ACC TAT GGA TGT CCT CGA GTC GGA AAC TAT GCG CTG 913
Tyr Thr Tyr Gly Cys Pro Arg Val Gly Asn Tyr Ala Leu
170 175 180GCC GAG CAC ATC ACC AGC CAG GGA TCT GGA GCG AAC TTC CCT GTT ACA 961Ala Glu His Ile Thr Ser Gln Gly Ser Gly Ala Asn Phe Pro Val Thr
185 190 195CAC TTG AAC GAC ATC GTC CCC CGG GTG CCA CCC ATG GAC TTT GGA TTC 1009His Leu Asn Asp Ile Val Pro Arg Val Pro Pro Met Asp Phe Gly Phe
200 205 210AGC CAG CCA AGT CCA GAA TAC TGG ATC ACC AGT GGC ACC GGA GCC AGT 1057Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr Ser Gly Thr Gly Ala Ser
215 220 225GTC ACG GCG TCG GAT ATT GAA CTC ATC GAG GGA ATC AAT TCG ACG GCG 1105Val Thr Ala Ser Asp Ile Glu Leu Ile Glu Gly Ile Ash Ser Thr Ala230 235 240 245GGG AAT GCA GGC GAA GCA ACG GTG GAC GTT TTG GCT CAC TTG TGG TAC 1153Gly Asn Ala Gly Glu Ala Thr Val Asp Val Leu Ala His Leu Trp Tyr
250 255 260TTT TTC GCA ATT TCA GAG TGT CTG CTA TAGCTTGGAC AGTCCGATGA 1200Phe Phe Ala Ile Ser Glu Cys Leu Leu
265 270AATAAGTGCG GAGAGAAAGT GTAAATAGTA ATTAAGTATA TATCAGGCAG AGAAGCAGTG 1260GTGGTCAGAG AAGAAAGAGT GAGTCCCATT ACGTAGCAGA TAACCACGTG TGGAGGCGCT 1320GTTCCTCCAC TTGCAGTTGC GGCCATCAAT CATATTCTTC TCCTTACT 1368(2)SEQ ID NO:2的信息：(ⅰ)顺序特征：
(A)长度：297个氨基酸
(B)类型：氨基酸
(D)拓扑学：线性(ⅱ)分子类型：蛋白质(ⅸ)顺序描述：SEQ ID NO:2:Met Phe Ser Gly Arg Phe Gly Val Leu Leu Thr Ala Leu Ala Ala Leu-27 -25 -20 -15Cys Ala Ala Ala Pro Thr Pro Leu Asp Val Arg Ser Val Ser Thr Ser
-10 -5 l 5Thr Leu Asp Glu Leu Gln Leu Phe Ser Gln Trp Ser Ala Ala Ala Tyr
10 15 20Cys Ser Asn Asn Ile Asp Ser Asp Asp Ser Asn Val Thr Cys Thr Ala
25 30 35Asp Ala Cys Pro Ser Val Glu Glu Ala Ser Thr Lys Met Leu Leu Glu
40 45 50Phe Asp Leu Thr Asn Asn Phe Gly Gly Thr Ala Gly Phe Leu Ala Ala
55 60 65Asp Asn Thr Asn Lys Arg Leu Val Val Ala Phe Arg Gly Ser Ser Thr70 75 80 85Ile Lys Asn Trp Ile Ala Asp Leu Asp Phe Ile Leu Gln Asp Asn Asp
90 95 100Asp Leu Cys Thr Gly Cys Lys Val His Thr Gly Phe Trp Lys Ala Trp
105 110 115Glu Ala Ala Ala Asp Asn Leu Thr Ser Lys Ile Lys Ser Ala Met Ser
120 125 130Thr Tyr Ser Gly Tyr Thr Leu Tyr Phe Thr Gly His Ser Leu Gly Gly
135 140 145Ala Leu Ala Thr Leu Gly Ala Thr Val Leu Arg Asn Asp Gly Tyr Ser150 155 160 165Val Glu Leu Tyr Thr Tyr Gly Cys Pro Arg Val Gly Asn Tyr Ala Leu
170 175 180Ala Glu His Ile Thr Ser Gln Gly Ser Gly Ala Asn Phe Pro Val Thr
185 190 195His Leu Asn Asp Ile Val Pro Arg Val Pro Pro Met Asp Phe Gly Phe
200 205 210Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr Ser Gly Thr Gly Ala Ser
215 220 225Val Thr Ala Ser Asp Ile Glu Leu Ile Glu Gly Ile Asn Ser Thr Ala230 235 240 245Gly Asn Ala Gly Glu Ala Thr Val Asp Val Leu Ala His Leu Trp Tyr
250 255 260Phe Phe Ala Ile Ser Glu Cys Leu Leu
265 270(2)SEQ ID NO:3的信息：(ⅰ)顺序特征：
(A)长度：42个碱基对
(B)类型：核苷酸
(C)链性：双链
(D)拓扑学：线性(ⅱ)分子类型：基因组DNA(ⅲ)假设：否(ⅳ)反义：否(ⅹⅰ)顺序描述：SEQ ID NO:3:GGAATTCACC TGCTAACCAT GTTCTCTGGA CGGTTTGGAG TG 42(2)SEQ ID NO:4的信息：(ⅰ)顺序特征：
(A）长度：34个碱基对
(B)类型：核苷酸
(C)链性：单链
(D)拓扑学：线性
(ⅱ)分子类型：基因组DNA
(ⅲ)假设：否
(ⅳ)反义：否
(ⅹⅰ)顺序描述：SEQ ID NO:4:CGGGATCCAA GCTATAGCAG ACACTCTGAA ATTG
Claims (13)
1．具有磷脂酶活性的蛋白质，其特征在于具有曲霉属溶血磷脂酶的成熟序列或由它衍生的序列，它可以在至少一个位点被酶切，其中在酶切时所述限制片段可选地或者通过至少一个在还原条件下可酶切的键连接或者至少一个所述未连接限制片段具有磷脂酶活性。
2．按照权利要求1的蛋白，其特征在于它在至少一个位点被酶切，并且所述限制片段或者通过至少一个在还原条件下可酶切的键连接或者至少一个所述未连接限制片段具有磷脂酶活性。
3．按照权利要求1或2之一的蛋白，其特征在于具有磷脂酶活性的序列与曲霉属溶血磷脂酶的类似序列有至少80％的同源性。
4．按照权利要求1、2或3之一的蛋白，其特征在于它具有臭曲霉溶血磷脂酶的成熟序列或由它衍生的序列，并且该酶切位点位于该氨基酸序列的44和45位之间。
5．按照权利要求1-4之一的蛋白，其特征在于它可以从曲霉属培养物分离。
6．按照权利要求5的蛋白，其特征在于它可以从臭曲霉、黑曲霉或米曲霉培养物分离。
7．具有磷脂酶活性的蛋白，其特征在于它由抗从臭曲霉RH 3046纯化的磷脂酶的抗体所识别。
8．按照权利要求1具有磷脂酶活性的蛋白的生产方法，即通过在适当培养基中发酵培养适当转化的产生溶血磷脂酶的宿主生物体并从该无细胞培养滤液中分离具有磷脂酶活性的蛋白，其特征在于在酸性至微弱碱性范围进行发酵。
9．按照权利要求8的方法，其特征在于在pH值为2-9、最好为3-8下进行发酵。
10．按照权利要求8或9的方法，其特征在于用曲霉属菌株或木霉属菌株做为该转化宿主生物体。
11．按照权利要求10的方法，其特征在于用臭曲霉或米曲霉做为该转化宿主生物体。
12．按照权利要求1-7之一的蛋白使植物油脱胶的用途。
13．按照权利要求1-7之一的蛋白做为焙烤辅助剂的用途。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19701348.1 | 1997-01-16 | ||
| DE19701348A DE19701348A1 (de) | 1997-01-16 | 1997-01-16 | Protein mit Phospholipaseaktivität |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1216061A true CN1216061A (zh) | 1999-05-05 |
| CN1152954C CN1152954C (zh) | 2004-06-09 |
Family
ID=7817548
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB988000091A Expired - Fee Related CN1152954C (zh) | 1997-01-16 | 1998-01-08 | 具有磷脂酶活性的蛋白质 |
Country Status (16)
| Country | Link |
|---|---|
| US (1) | US6140094A (zh) |
| EP (1) | EP0904357B1 (zh) |
| CN (1) | CN1152954C (zh) |
| AR (1) | AR010099A1 (zh) |
| AT (1) | ATE348151T1 (zh) |
| AU (1) | AU6208098A (zh) |
| BR (1) | BR9805893A (zh) |
| CA (1) | CA2243476C (zh) |
| DE (2) | DE19701348A1 (zh) |
| DK (1) | DK0904357T3 (zh) |
| ES (1) | ES2275299T3 (zh) |
| HU (1) | HU224831B1 (zh) |
| MY (1) | MY117580A (zh) |
| PT (1) | PT904357E (zh) |
| TW (1) | TW565610B (zh) |
| WO (1) | WO1998031790A1 (zh) |
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| CN1328375C (zh) * | 2003-03-04 | 2007-07-25 | 独立行政法人制品评价技术基盘机构 | 来自于曲霉菌的磷脂酶a2 |
| CN100404672C (zh) * | 2003-04-28 | 2008-07-23 | 诺维信公司 | 磷脂酶和其生产方法 |
| CN100545261C (zh) * | 2002-05-21 | 2009-09-30 | Dsmip资产有限公司 | 新的磷脂酶及其用途 |
| CN102388311A (zh) * | 2009-02-10 | 2012-03-21 | 阿特罗瓦西公司 | 用于定量测定样品中磷脂酶a1或a2活性的酶测定法 |
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| DE69604491T3 (de) | 1995-06-07 | 2008-09-25 | Danisco A/S | Methode zur verbesserung der eigenschaften von mehlteig, sowie zusammensetzung zur teigverbesserung und verbesserte nahrungsmittel |
| US6936289B2 (en) | 1995-06-07 | 2005-08-30 | Danisco A/S | Method of improving the properties of a flour dough, a flour dough improving composition and improved food products |
| DE19620649A1 (de) * | 1996-05-22 | 1997-11-27 | Roehm Gmbh | Rekombinant hergestellte Lysophospholipase aus Aspergillus |
| EP1433852B1 (en) | 1997-04-09 | 2006-06-28 | Danisco A/S | Use of lipase for improving doughs and baked products |
| ES2188143T5 (es) † | 1998-04-20 | 2008-11-01 | Novozymes A/S | Preparacion de masa y de productos horneados. |
| EP1098988B9 (en) | 1998-07-21 | 2007-10-24 | Danisco A/S | Foodstuff |
| AU2011204998B2 (en) * | 1998-11-27 | 2013-02-14 | Novozymes A/S | Lipolytic enzyme variants |
| CA2353379C (en) | 1998-11-27 | 2011-01-04 | Novozymes A/S | Lipolytic enzyme variants |
| US7312062B2 (en) * | 1998-11-27 | 2007-12-25 | Novozymes A/S | Lipolytic enzyme variants |
| ATE494366T1 (de) | 1999-10-14 | 2011-01-15 | Novozymes As | Lysophospholipase aus aspergillus |
| US20020192792A1 (en) | 2000-04-28 | 2002-12-19 | Palle Schneider | Laccase mutants |
| EP1352057B1 (en) | 2001-01-10 | 2009-03-25 | Novozymes A/S | Lipolytic enzyme variant |
| CN1526013A (zh) * | 2001-02-23 | 2004-09-01 | 诺维信公司 | 脂解酶基因 |
| ATE362315T1 (de) | 2001-05-18 | 2007-06-15 | Danisco | Verfahren zur herstellung von teig mit einem enzym |
| AU2003203139B2 (en) | 2002-01-16 | 2007-11-08 | Novozymes A/S | Lipolytic enzyme variants and method for their production |
| US6773731B2 (en) * | 2002-10-18 | 2004-08-10 | James S. Campbell | Liquid egg yolk product comprising lysophospholipoprotein |
| US20050196766A1 (en) * | 2003-12-24 | 2005-09-08 | Soe Jorn B. | Proteins |
| MXPA05007653A (es) | 2003-01-17 | 2005-09-30 | Danisco | Metodo. |
| US7955814B2 (en) | 2003-01-17 | 2011-06-07 | Danisco A/S | Method |
| US7906307B2 (en) | 2003-12-24 | 2011-03-15 | Danisco A/S | Variant lipid acyltransferases and methods of making |
| GB0716126D0 (en) | 2007-08-17 | 2007-09-26 | Danisco | Process |
| US7718408B2 (en) | 2003-12-24 | 2010-05-18 | Danisco A/S | Method |
| GB0405637D0 (en) | 2004-03-12 | 2004-04-21 | Danisco | Protein |
| EP2267107A3 (en) | 2004-07-16 | 2011-05-25 | Danisco A/S | Lipolytic Enzyme. Uses Thereof In The Food Industry |
| US20070148311A1 (en) * | 2005-12-22 | 2007-06-28 | Bunge Oils, Inc. | Phytosterol esterification product and method of make same |
| DE102006046857A1 (de) | 2006-10-02 | 2008-04-03 | Ab Enzymes Gmbh | Klonierung, Expression und Verwendung saurer Lysophospholipasen |
| DE102006046719A1 (de) | 2006-10-02 | 2008-04-03 | Ab Enzymes Gmbh | Klonierung, Expression und Verwendung saurer Phospholipasen |
| AU2007344910B2 (en) | 2007-01-25 | 2013-03-28 | Dupont Nutrition Biosciences Aps | Production of a lipid acyltransferase from transformed Bacillus licheniformis cells |
| CN101589142B (zh) * | 2007-01-30 | 2012-06-06 | 三菱化学食品株式会社 | 甘油糖脂脂肪酶 |
| US8460905B2 (en) * | 2007-09-11 | 2013-06-11 | Bunge Oils, Inc. | Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases with reduced reaction time |
| US8956853B2 (en) * | 2007-01-30 | 2015-02-17 | Bunge Oils, Inc. | Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases |
| US8241876B2 (en) | 2008-01-07 | 2012-08-14 | Bunge Oils, Inc. | Generation of triacylglycerols from gums |
| DE102009051013A1 (de) | 2009-10-28 | 2011-06-09 | Ab Enzymes Gmbh | Klonierung, Expression und Verwendung saurer Phospholipasen |
| CA2788148C (en) | 2010-03-12 | 2018-03-13 | Dupont Nutrition Biosciences Aps | An enzymatic process for refining a plant oil |
| AR085251A1 (es) | 2011-02-23 | 2013-09-18 | Danisco | Proceso para tratar aceite vegetal |
| WO2013104660A1 (en) | 2012-01-13 | 2013-07-18 | Dupont Nutrition Biosciences Aps | Process for treating a plant oil comprising hydrolysing chlorophyll or a chlorophyll derivative and involving partial caustic neutralisation |
| DK2840900T3 (da) | 2012-04-25 | 2019-09-09 | Novozymes As | Fremgangsmåde til bagning |
| RU2763469C2 (ru) | 2017-02-20 | 2021-12-29 | Новозимс А/С | Липолитический фермент для применения в хлебопечении |
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| ES2180541T5 (es) * | 1992-06-16 | 2008-12-01 | Sankyo Lifetech Company Limited | Nueva fosfolipasa a1, procedimiento para su preparacion y uso de la misma. |
| BR9506861A (pt) * | 1994-02-22 | 1997-09-23 | Novo Nordisk As | Processo para preparar e produzir uma variante de uma enzima lipolítica originária variante de enzima liplítica construção de dna vetor célula hospedeira aditivo detergente e composição detergente |
| DE19527274A1 (de) * | 1995-07-26 | 1997-01-30 | Metallgesellschaft Ag | Enzymatisches Verfahren zur Entschleimung von pflanzlichen Ölen mit Aspergillus-Phospholipase |
| DE19620649A1 (de) * | 1996-05-22 | 1997-11-27 | Roehm Gmbh | Rekombinant hergestellte Lysophospholipase aus Aspergillus |
-
1997
- 1997-01-16 DE DE19701348A patent/DE19701348A1/de not_active Ceased
- 1997-12-08 MY MYPI97005899A patent/MY117580A/en unknown
-
1998
- 1998-01-02 AR ARP980100020A patent/AR010099A1/es not_active Application Discontinuation
- 1998-01-08 AT AT98904046T patent/ATE348151T1/de active
- 1998-01-08 BR BR9805893A patent/BR9805893A/pt not_active Application Discontinuation
- 1998-01-08 PT PT98904046T patent/PT904357E/pt unknown
- 1998-01-08 CA CA002243476A patent/CA2243476C/en not_active Expired - Fee Related
- 1998-01-08 US US09/142,469 patent/US6140094A/en not_active Expired - Lifetime
- 1998-01-08 AU AU62080/98A patent/AU6208098A/en not_active Abandoned
- 1998-01-08 ES ES98904046T patent/ES2275299T3/es not_active Expired - Lifetime
- 1998-01-08 HU HU9901640A patent/HU224831B1/hu not_active IP Right Cessation
- 1998-01-08 CN CNB988000091A patent/CN1152954C/zh not_active Expired - Fee Related
- 1998-01-08 DE DE59813840T patent/DE59813840D1/de not_active Expired - Lifetime
- 1998-01-08 WO PCT/EP1998/000081 patent/WO1998031790A1/de active IP Right Grant
- 1998-01-08 DK DK98904046T patent/DK0904357T3/da active
- 1998-01-08 EP EP98904046A patent/EP0904357B1/de not_active Expired - Lifetime
- 1998-07-18 TW TW087111741A patent/TW565610B/zh not_active IP Right Cessation
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100545261C (zh) * | 2002-05-21 | 2009-09-30 | Dsmip资产有限公司 | 新的磷脂酶及其用途 |
| CN1328375C (zh) * | 2003-03-04 | 2007-07-25 | 独立行政法人制品评价技术基盘机构 | 来自于曲霉菌的磷脂酶a2 |
| CN100404672C (zh) * | 2003-04-28 | 2008-07-23 | 诺维信公司 | 磷脂酶和其生产方法 |
| CN102388311A (zh) * | 2009-02-10 | 2012-03-21 | 阿特罗瓦西公司 | 用于定量测定样品中磷脂酶a1或a2活性的酶测定法 |
Also Published As
| Publication number | Publication date |
|---|---|
| ATE348151T1 (de) | 2007-01-15 |
| CA2243476C (en) | 2009-01-06 |
| CN1152954C (zh) | 2004-06-09 |
| TW565610B (en) | 2003-12-11 |
| PT904357E (pt) | 2007-03-30 |
| EP0904357A1 (de) | 1999-03-31 |
| DE59813840D1 (de) | 2007-01-25 |
| CA2243476A1 (en) | 1998-07-23 |
| HUP9901640A2 (hu) | 1999-08-30 |
| HU224831B1 (en) | 2006-02-28 |
| AR010099A1 (es) | 2000-05-17 |
| ES2275299T3 (es) | 2007-06-01 |
| US6140094A (en) | 2000-10-31 |
| HK1019074A1 (zh) | 2000-01-21 |
| MY117580A (en) | 2004-07-31 |
| BR9805893A (pt) | 1999-08-24 |
| DK0904357T3 (da) | 2007-04-10 |
| AU6208098A (en) | 1998-08-07 |
| DE19701348A1 (de) | 1998-07-23 |
| WO1998031790A1 (de) | 1998-07-23 |
| HUP9901640A3 (en) | 2001-09-28 |
| EP0904357B1 (de) | 2006-12-13 |
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