CN100424170C - 脂肪酶、其基因、产生该酶的亚罗解脂酵母及其应用 - Google Patents
脂肪酶、其基因、产生该酶的亚罗解脂酵母及其应用 Download PDFInfo
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- CN100424170C CN100424170C CNB2005101126385A CN200510112638A CN100424170C CN 100424170 C CN100424170 C CN 100424170C CN B2005101126385 A CNB2005101126385 A CN B2005101126385A CN 200510112638 A CN200510112638 A CN 200510112638A CN 100424170 C CN100424170 C CN 100424170C
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Abstract
本发明涉及一种用于制备一种脂肪酶,具体是氨基酸序列为SEQ ID NO:1或其保守突变序列的脂肪酶,还涉及编码该脂肪酶的基因、产生该脂肪酶的亚罗解脂酵母(Yarrowia lipolytica)以及利用该脂肪酶合成酯的方法。
Description
技术领域
本发明涉及一种用于制备一种脂肪酶、编码该脂肪酶的基因、产生该脂肪酶的亚罗解脂酵母(Yarrowia liolytica)以及利用该脂肪酶合成酯类的方法。
背景技术
酯类物质是食品、饮料、医药、生物能源等许多工业领域常用的化学物质,但长期以来,工业上脂肪酸酯均是通过化学法来生产。即用无机催化剂如浓硫酸、苯磺酸等在高温高压下催化酸醇的酯化反应来完成。虽然产率较高,但也存在许多难以克服的缺点。如:高温高压反应能耗高、副反应多,产物色泽深、分离和精制困难等,而且酸对设备腐蚀严重,不利于生产。随着生物技术的发展,特别是酶工程的研究,为生物催化剂合成脂肪酸酯提供了新的方法。与传统的化学法相比,酶法合成具有反应条件温和,特异性强,副反应少,产物品质高等特点。特别是非水相中脂肪酶促反应,拓宽了酶的应用范围和领域,成为近年来酶学研究的热点。
“微生物脂肪酶催化酯的合成”CN 1223300A(1999)报道了以脂肪醇、一元脂肪酸为底物,游离的脂肪酶为催化剂,在15℃~40℃条件下,于有机溶剂中催化脂肪酸酯的合成。该方法所用脂肪酶是用华根霉生产,反应为分批操作,在搅拌罐中进行。10~36hr底物转化率可达95%。反应结束后,过滤分离脂肪酶和反应液,减压蒸馏得到产物酯。该方法采用游离脂肪酶,因此反应批次不高,不利于产物的分离纯化。
Leite等人(1998)研究了固定化米黑毛霉脂肪酶催化的酯化反应。以异辛烷为溶剂通过渗透蒸发膜移走反应中生成的水。Bufeiend等人(1999)中空纤维膜反应器,以辛烷为溶剂,通过将底物循环反应合成长链脂肪酸酯。膜反应器催化效率高,能选择性移走反应中生成的水,但膜反应器也存在造价高,操作复杂、膜易堵塞等问题。
亚罗解脂酵母(Y.lipolytica)属于非常规酵母,FDA认证为安全级酵母(GRAS),该菌株被大规模用于生产柠檬酸、单细胞蛋白,工业生产中积累了大量的经验。亚罗解脂酵母分泌几种胞外蛋白包括酸性和碱性蛋白酶、RNA酶、酯酶、几种脂肪酶等(Barth and Gaillardin,1997)。1976年,Ota等人报道Y.lipolytica生产一个胞外脂肪酶及两个与细胞结合(Cell-bound)的脂肪酶。1996年,Ota等人纯化出39kDa的胞外脂肪酶并测定了N-端氨基酸序列。1997年,Destain等人报道Y.lipolytica在500L发酵罐中大规模生产脂肪酶,最高酶活达到1000U/ml发酵液,该方法虽然进行了大规模生产但是单位体积发酵液的酶活不高。2000年,Fignede等人采用基因工程的方法来提高胞外脂肪酶Lip2的生产,在15L发酵罐中,优化条件下,发酵酶活达到了10000U/ml发酵液。该方法虽然单位发酵液的酶活比较高,但是生产规模小,只在15L的发酵罐中进行,而且发酵的控制条件比较严格,难于进行放大。目前,关于Y.lipolytica胞外脂肪酶的应用报道很少,Sandoval等人报道用Y.lipolytica脂肪酶拆分手性酸,得到的产物的光学纯度大大提高。关于Y.lipolytica脂肪酶的其它应用未见报道。
发明内容
本发明为了克服现有技术中的不足,提供一种新型的脂肪酶、编码该脂肪酶的基因、高产该脂肪酶的菌株和使用该脂肪酶合成酯的方法。
在一个方面中,本发明提供了一种脂肪酶,其氨基酸序列为SEQ ID NO:1或其保守突变序列。SEQ ID NO:1由301个氨基酸残基组成,其中164-168氨基酸(GHSLG)为脂肪酶的保守五肽序列。与Lip2基因(AJ012632)编码的氨基酸序列相比,其中第179位氨基酸由甘氨酸(G)突变成天冬氨酸(D)。本文中所述的术语“保守突变”指由另一生物学相似残基替代氨基酸残基。保守突变的例子包括将亲水残基如异亮氨酸、缬氨酸、亮氨酸或蛋氨酸替代为另一种,将极性残基替代为另一种,例如将精氨酸替代为赖氨酸、将谷氨酸替代为天冬氨酸或谷氨酰胺替代为天冬酰胺等。
在另一个方面中,本发明提供了一种编码上述脂肪酶的基因,更具体地提供了核苷酸序列为SEQ ID NO:2的编码所述脂肪酶的基因。该基因由906个碱基组成。与Lip2基因的核苷酸序列相比,其中第536位核苷酸由g突变为a。
可以理解的是,由于氨基酸密码子的兼并性,本发明中编码脂肪酶的基因除了SEQ ID:2所示的序列外,还包括编码上述脂肪酶氨基酸序列的其他核酸分子。
在另一个方面中,本发明提供了一种产生上述脂肪酶的亚罗解脂酵母(Yarrowialipolytica),其已于2005年9月30日在中国微生物菌种保藏管理委员会普通微生物中心(地址:北京市海淀区中关村北一条13号中国科学院微生物研究所,邮编:100080)提交生物保藏,保藏编号为CGMCC No.1470。
在又一个方面中,本发明提供了一种利用上述脂肪酶合成酯的方法,其包括:
(a)获得权利要求1所述的脂肪酶;
(b)使用步骤(a)中所得脂肪酶合成酯。
本发明技术人员通过阅读本说明书,可以从上述的亚罗解脂酵母提取得到所述的脂肪酶,也可以通过本领域中公知的基因重组技术在本领域中公知的合适基因表达系统中容易地获得,还可以通过本领域技术人员公知的肽合成方法进行化学合成。
在本发明的一个实施方案中,步骤(b)中通过固定化脂肪酶合成酯。
在一个优选的实施方案中,其中所述的固定化脂肪酶包含上述脂肪酶、载体和共固定剂。固定化载体先用一定组成和配比的共固定剂活化。将固定化载体和共固定剂按1∶1~1∶3(W∶V)比例混合,干燥。脂肪酶用去离子水溶解,按1000-30000单位/克载体的比例与活化后的固定化载体混合,晾干待用。
在另一个优选的实施方案中,所述载体包括固体颗粒,如硅胶、硅藻土;和膜状纺织品,包括天然纤维织物如棉布和化学纤维织物如涤纶,尤其是膜状纺织品固定化酶,其具有表面积大、吸附性强、价格便宜、稳定性好以及以重复利用的特点。
在另一个优选的实施方案中,所述共固定剂选自(包括)高分子化合物、表面活性剂、蛋白质、无机盐中的至少一种。
在一个进一步的优选实施方案中,所述共固定剂选自PEG6000、椰子油、吐温80、明胶、卵磷脂和硫酸镁中的至少一种,共固定剂的总浓度为0.5%-2%,各组分的质量比为明胶∶卵磷脂∶PEG6000∶吐温80∶硫酸镁∶椰子油=5∶1∶1∶2∶1∶1。
在本发明的一个实施方案中,酯的合成通过脂肪酸和醇的酯化反应进行,优选将脂肪酸和醇以3∶1~1∶5的摩尔比和脂肪酶混合,于20-50℃反应9-50小时。具体而言,将底物脂肪酸和醇按摩尔比3∶1~1∶5的比例加入含有有机溶剂的酶反应器中。加入固定化酶开始反应。在20-50℃的条件下,搅拌反应9-50小时,转化率可以达到56-97%,在反应过程中加入分子筛或变色硅胶除去反应中生成的水,可增加反应的转化率。反应结束后,固定化酶可以直接分离,用少量的有机溶剂洗涤后可再次使用。反应液过滤后在真空度0.05-0.08MPa条件下减压蒸馏,蒸发出反应媒体有机溶剂,得到产物酯。蒸发出的溶剂经过冷凝后可回收重复使用。
本发明在有机溶剂中完成的酯化反应方程式为:
酶活的定义为:40°条件下,pH8.0的磷酸缓冲溶液中水解橄榄油,每分钟释放出1μmol脂肪酸的酶量为一个酶活单位。
其中所述的脂肪酸选自C8~C18一元脂肪酸和二元酸的至少一种,优选为辛酸、癸酸、棕榈酸、硬脂酸、油酸、亚油酸、亚麻酸、己二酸、壬二酸、癸二酸中的至少一种;醇选自C1~C18的伯醇、C3~C5的仲醇以及甘油、维生素C中至少一种,优选为甲醇、丙醇、丁醇、己醇、异辛醇、癸醇、十二醇、十八醇、异丙醇、异丁醇、甘油和维生素C中的至少一种。
反应所用的有机溶剂为丙酮、正己烷、环己烷、石油醚、正庚烷或正辛烷等,对于某些底物可直接采用无溶剂体系。
在本发明的另一个实施方案中,其中步骤(b)中酯的合成通过底物醇或者酸和酯的转酯化反应进行,优选将底物醇或者底物酸和底物酯以摩尔比3∶1~1∶7与有机溶剂和固定化脂肪酶混合,于20-50°反应9-50小时。具体而言,将底物酸或者醇和酯按摩尔比3∶1~1∶7的比例加入含有有机溶剂的酶反应器中。加入固定化酶开始反应。在20-50°的条件下,搅拌反应9-50小时,转化率可以达到80-93%。反应结束后,固定化酶可以直接分离,用少量的有机溶剂洗涤后可再次使用。反应液通过分离纯化得到产物酯。蒸发出的溶剂经过冷凝后可回收重复使用。
本发明在有机溶剂中完成的酯化反应方程式为:
或
其中所述底物酸选自棕榈酸、油酸中的至少一种;底物醇选自甲醇、乙醇中的至少一种;底物酯选自维生素A醋酸酯、豆油、红花油、亚麻油、玉米油、棕榈油、色拉油中的至少一种。
反应所用的有机溶剂选自丙酮、正己烷、环己烷、石油醚、正庚烷中的至少一种,对于某些底物可直接采用无溶剂体系。
因此,本发明的一个优点是通过一系列的诱变方法得到一株高产脂肪酶的菌株,该菌株于500L发酵罐中,优化的条件下,发酵脂肪酶活力可达到8000U/ml发酵液,为生产提供了廉价的酶催化剂。发酵液经过离子交换和疏水层析纯化后,脂肪酶比酶活可达到18600U/mg。
本发明的另一个优点是本发明生产的脂肪酶有广泛的底物适应性,作用的底物包括一元、二元脂肪酸,一元的伯醇,甘油,维生素A、C等。将酶固定在载体上,提高了酶的稳定性和使用批次,并且有利于连续化生产,大大简化了后续的分离纯化步骤,由于该发明采用生物催化剂,反应中使用的溶剂可以回收重复利用,因此不会环境造成污染,为酯类的工业化生产提供了非常有前景的绿色工艺路线。
附图说明
图1说明复合诱变系谱I,其中UV为紫外线,n为快中子,γ为γ射线,ME为磁场;
图2说明复合诱变系谱II,其中n为快中子,γ为γ射线,ME为磁场;
图3说明不同的氮源a、b(a为脱脂豆粕、b为全脂豆粕)(4%)对脂肪酶产量的影响;
图4说明温度对脂肪酶产量的影响;
图5说明pH对脂肪酶产量的影响;
图6说明500L发酵罐中发酵时酶产量随时间的变化。
具体实施方式
下面通过具体的实施方案描述本发明的亚罗解脂酵母菌的获得以及利用该菌株中的脂肪酶合成酯的方法。除非特别指明,本发明中所用的实验方法均为本领域技术人员所公知的方法。此外,实施例应理解为说明性的,而不是要限制本发明的范围,本发明的实质和范围仅由权利要求书限定。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对这些实施方案中的菌种诱变条件、分离纯化条件、底物类型、反应条件进行的各种改变或改动也属于本发明的保护范围。
实施例1:亚罗解脂酵母菌种的获得
本菌种是经过进行紫外线、亚硝基胍诱变后再进行快中子、γ射线和磁场复合处理相结合得到的脂肪酶高产育种。简言之,紫外诱变时间分别为1分钟、1.5分钟、2分钟,快中子诱变剂量分别为5千拉特、10千拉特、15千拉特,γ射线诱变剂量分别为5万伦琴、10万伦琴、15万伦琴,如图1和2所示。图1说明复合诱变系谱I,其中UV为紫外线,n为快中子,γ为γ射线,ME为磁场;图2说明复合诱变系谱II,其中n为快中子,γ为γ射线,ME为磁场。经过上述一系列诱变过程,最终得到了酶活为2246U/mL的高产脂肪酶菌株。
麦芽汁斜面培养基(麦芽汁0.3%,酵母粉0.3%,牛肉蛋白胨0.5%,葡萄糖1%,琼脂2%)上、26℃下培养48hr后出现白色菌落,72hr后菌落长大,呈淡黄色,较肥厚,边缘整齐,略带油脂光泽,96hr后菌落匍匐展开,变得单薄,并且表面出现皱褶。
实施例2:发酵条件的优化
比较了不同的氮源a、b(a为脱脂豆粕、b为全脂豆粕)(4%)对脂肪酶产量的影响,结果见图3。从图3的曲线可以看出,以b作为氮源的培养基脂肪酶产量很低,远小于以a作为氮源。
选择有利于产脂肪酶的油脂c、d、e(c为豆油、d为菜籽油、e为橄榄油)作为碳源(2.5%)进行研究,结果比较见下表1:
表1
培养时间(HR) | 24 | 48 | 72 | 96 |
c | 440 | 960 | 1520 | 1760 |
d | 360 | 720 | 1340 | 1480 |
e | 320 | 640 | 880 | 1080 |
从表中可见,在比较的三种碳源中,c的脂肪酶产量最高。
在恒温摇床中,不同温度下培养96小时,结果见图4。从图4可知,产脂肪酶的温度较窄,以28℃~30℃最适,超过30℃,脂肪酶产量明显减少,34℃基本不产脂肪酶。
培养基在接种前用1N NaOH和1N HCl将pH调节到一定值,28℃振荡培养96小时,结果见下表2。从表2可见,发酵初始pH6~7最利于生产脂肪酶,而发酵液的自然pH在5.7左右。
初始pH | 4 | 5 | 6 | 7 | 8 | 9 | 自然 |
终pH | 4 | 4.8 | 5.6 | 6.4 | 6.7 | 7.2 | 5.7 |
酶活(U/mL) | 400 | 1460 | 1680 | 1600 | 1040 | 700 | 1620 |
在获得较好的培养基配方(4%脱脂豆粕、2.5%豆油、0.1%K2HPO4、0.1%司盘(SPAN)60)后,我们在30L发酵罐中进行了初步的实验以研究其发酵过程(发酵温度为26℃,搅拌转速为350rpm,pH5.7,通气量为0.5vvm)的变化规律和脂肪酶的产量,结果见图5。从图5可以看出,两批有代表性的发酵的脂肪酶活力增长和pH变化趋势基本一致,发酵40小时左右开始产酶,80小时左右进入产酶旺盛期,130小时左右脂肪酶活力达到最高峰,之后开始下降;发酵过程中pH保持在4~5之间,波动不大,在酶活达到最高峰后回升到5.4。综上所述,发酵时间应当为130小时左右,脂肪酶产量可达5300U/mL。
实施例3:发酵和脂肪酶的提取
随后在500L发酵罐中进行了中试放大实验,培养基组分为4%豆粕、2.5%豆油、0.1%K2HPO4、0.1%司盘(SPAN)60、03%泡敌;发酵温度为26℃,搅拌转速为200rpm,pH5.7,通气量为0.35vvm。发酵酶活达到了8000U/mL,结果如图6。
脂肪酶的提取通过本领域技术人员公知的方法进行。简言之,将发酵液4000rpm离心10min,取上清液,加入4%聚乙二醇6000絮凝浓缩,自然沉降分层,取下层液。向下层液中加入3倍体积的丙酮,得到沉淀,干燥后得到粗酶产品。经上述操作后得到的粗酶产品酶活为5×104u/g,收率为65%,外观为灰黄色粉末。
实施例4 酯化脂肪酶基因的克隆
亚罗解脂酵母发酵液经过离心分离得到上清,上清液用3倍体积的冷丙酮沉淀,得到的沉淀物冷丙酮洗涤3次,室温干燥得到粗酶粉。此酶粉用25mM的磷酸缓冲液(pH7.5)溶解,离心得到上清液。此上清液经过Q-sepharose FF层析柱后收集脂肪酶组分,该组分加入硫酸铵至终浓度为0.8M,然后经过Butyl sepharose FF层析柱,收集脂肪酶组分,得到纯化的脂肪酶。此脂肪酶组分在SDS-聚丙烯酰胺凝胶电泳上得到单一条带,此条带转印到PVDF膜上进行蛋白质N端测序,得到氨基酸序列为VYTSTETSHIDQESY。
此序列同亚罗解脂酵母基因组的蛋白序列比较,结果发现此序列与Lip2基因(AJ012632)编码的氨基酸序列匹配。分析发现此序列含有保守的五肽序列GHSLG(第194-198氨基酸),N-端1-30氨基酸为该脂肪酶的信号肽序列。根据lip2脂肪酶的基因序列(SEQNO.1)设计如下引物:
引物1:5’-ACGAGAATTCGTGTACACCTCTACCGAGACC-3’
引物2:5’-AATAAAGCTTGATACCACAGACACCCTCGGT-3’
根据该引物,以亚罗解脂酵母基因组为模板,PCR扩增得到DNA序列,经过测序得到SEQ ID NO:2的基因序列,该序列与Lip2基因同源,编码该脂肪酶的成熟肽,只是第536位核苷酸由g突变为a,导致所编码的氨基酸序列中第179位氨基酸由甘氨酸(G)突变成天冬氨酸(D)。
实施例5 脂肪酶的固定化
将棉布和共固定剂按质量体积比为1∶1的比例混合,室温晾干得到活化载体。共固定剂为PEG6000、椰子油、吐温80、明胶、卵磷脂和硫酸镁混合物,其质量比为明胶∶卵磷脂∶PEG6000:吐温80∶硫酸镁∶椰子油=5∶1∶1∶2∶1∶1。亚罗解脂酵母脂肪酶水溶液按8000U/克活化载体的比例与活化载体混合,浸泡,室温晾干得到固定化脂肪酶。
实施例6
将硅藻土和共固定剂按质量体积比为1∶1.5的比例混合,室温晾干得到活化载体。共固定剂为PEG6000、椰子油、吐温80、明胶、卵磷脂和硫酸镁混合物,其质量比为明胶∶卵磷脂∶PEG6000∶吐温80∶硫酸镁∶椰子油=5∶1∶1∶2∶1∶1。亚罗解脂酵母脂肪酶水溶液按8000IU/克活化载体的比例与活化载体混合,浸泡,室温晾干得到固定化脂肪酶。
实施例7
将涤纶和共固定剂按质量体积比为1∶3的比例混合,室温晾干得到活化载体。共固定剂为PEG6000、椰子油、吐温80、明胶、卵磷脂和硫酸镁混合物,其质量比为明胶∶卵磷脂∶PEG6000∶吐温80∶硫酸镁∶椰子油=5∶1∶1∶2∶1∶1。亚罗解脂酵母脂肪酶水溶液按8000IU/克活化载体的比例与活化载体混合,浸泡,室温晾干得到固定化脂肪酶。
实施例8
0.085g实施例5中制备的固定化脂肪酶(16000u/g,)加入含有0.85mol/L浓度油酸和甲醇的5ml正己烷中,40℃条件下,反应16hr后,反应的转化率达95%。经过在-0.05Mpa压力下减压蒸馏得到产物油酸甲酯。
实施例9
0.12g实施例5中制备的固定化酶(9000U/g)加入到含1g棕榈酸(0.54mol/L)和0.67g 2-乙基己醇(0.7mol/L)的5mL石油醚中。40℃条件下,振荡反应(190r/min),22hr反应转化率达96.7%。半衰期达150hr。
实施例10
0.05g实施例5中制备的脂肪酶(16000u/g)加入含有0.7mol/L浓度硬脂酸和丁醇的5ml的环己烷中,40℃条件下,反应24hr后,反应的转化率达97%.经过在-0.05Mpa压力下减压蒸馏得到产物硬脂酸丁酯。
实施例11
0.12g实施例5中制备的固定化酶(9000U/g)加入到含1g硬脂酸和0.62g 2-乙基己醇(0.65mol/L)的5mL正庚烷中。45℃条件下,振荡反应(190r/min),50hr反应转化率达95%。
实施例12
20g实施例5中制备的固定化酶(9000U/g)加入含138g棕榈酸(0.54mol)和92g异辛醇(0.706mol)的690mL的石油醚中,40℃条件下搅拌反应。固定化酶固定在不锈钢丝网上置于反应器内,反应24hr转化率可达95%。反应液经碱液洗涤、过滤后在0.05~0.08的Mpa真空度下蒸发出有机溶剂,得到产品棕榈酸异辛脂。产品色泽浅,酸值为0.205mgKOH/g。
实施例13
0.2g实施例5中制备的固定化酶(9000U/g)加入含0.2g癸酸和0.147g异丁醇的20ml正己烷中,50℃条件下,振荡反应(190r/min),24hr反应转化率达91%。
实施例14
0.2g实施例6中制备的固定化酶(9000U/g)加入含0.2g辛酸和0.135g异丙醇的20ml正己烷中,35℃条件下,振荡反应(190r/min),20hr反应转化率达93%。
实施例15
0.3g实施例5中制备的固定化酶加入含0.2g癸二酸和0.147g异丁醇的10ml正己烷中,40℃条件下,振荡反应,9hr反应转化率达91%。
实施例16
0.12g实施例5中制备的固定化酶加入含0.2g癸二酸和0.535g十八醇的20ml石油醚中,30℃条件下,振荡反应,12hr反应转化率达89%。
实施例17
0.3g实施例7中制备的固定化酶加入含0.2g癸二酸和0.2575g辛醇的10ml正己烷中,50℃条件下,振荡反应,18hr反应转化率达88%。
实施例18
0.25g实施例6中制备的固定化酶加入含0.2g己二酸和0.3886g十二醇的20ml正辛烷中,20℃条件下,振荡反应,48hr反应转化率达89%。
实施例19
0.3g固定化酶加入含0.2g己二酸和0.203g庚醇的20ml正己烷中,37℃条件下,振荡反应,24hr反应转化率达92%。
实施例20
0.2g实施例5中制备的固定化酶加入0.2g癸二酸和2g己醇,无溶剂条件下,40℃下反应20h,转化率达94%。
实施例21
0.16g实施例5中制备的固定化酶加入0.2g己二酸和2g辛醇,无溶剂条件下,35℃下反应50h,转化率达90%。
实施例22
0.50g实施例5中制备的固定化酶加入0.0956g维生素C和1.051g棕榈酸的10mL丙酮溶剂,40℃,反应72小时,转化率为65.3%。
实施例23
0.60g实施例7中制备的固定化酶加入0.0956g维生素C和0.956g月桂酸的10mL丙酮溶剂,37℃,反应72小时,转化率为56.5%。
实施例24
0.40g固定化酶加入0.0956g维生素C和1.203g豆蔻酸的10mL丙酮溶剂,40℃,反应72小时,转化率为58.2%。
实施例25
0.50g实施例5中制备的固定化酶加入0.0956g维生素C和1.000g亚油酸的10mL丙酮溶剂,35℃,反应72小时,转化率为61.6%。
实施例26
0.65g实施例5中制备的固定化酶加入0.0956g维生素C和1.203g亚麻酸的10mL丙酮溶剂,40℃,反应72小时,转化率为63.6%。
实施例27
0.3g实施例5中制备的固定化酶加入含0.100g(0.3mmol)维生素A醋酸酯和0.234g(0.9mmol)棕榈酸的10ml正己烷中,35℃下反应24h转化率达84%。
实施例28
0.4g实施例5中制备的固定化酶加入含0.100g(0.3mmol)维生素A醋酸酯和0.254g(0.9mmol)油酸的20ml正己烷中,40℃下反应24h转化率达80%。
实施例29
0.3g实施例5中制备的固定化酶加入含2.00g色拉油和0.25g甲醇的10ml石油醚中,40℃下反应30h转化率达93%。
实施例30
0.3g实施例5中制备的固定化酶加入含2.00g豆油和0.27g甲醇的10ml正庚烷中,35℃下反应40h转化率达92%。
实施例31
0.4g实施例5中制备的固定化酶加入含2.00g红花油和0.30g甲醇的10ml石油醚中,40℃下反应50h转化率达93%。
实施例32
0.35g实施例5中制备的固定化酶加入含2.00g亚麻油和0.28g甲醇的10ml正己烷中,30℃下反应50h转化率达90%。
实施例33
0.3g实施例5中制备的固定化酶加入含2.00g玉米油和0.26g甲醇的10ml石油醚中,40℃下反应30h转化率达92%。
实施例34
0.3g实施例6中制备的固定化酶加入含2.00g棕榈油和0.29g甲醇的10ml环己烷中,30℃下反应40h转化率达89%。
实施例35
0.3g实施例5中制备的固定化酶加入含2.00g色拉油和0.36g乙醇的10ml石油醚中,40℃下反应30h转化率达91%。
实施例36
0.4g实施例5中制备的固定化酶加入含0.110g甘油和0.190g癸酸的20ml正己烷中,37℃下反应24h转化率达到96%。
实施例37
0.4g实施例5中制备的固定化酶加入含0.110g甘油和0.178g辛酸的20ml正己烷中,40℃下反应24h转化率达到92%。
序列表
<110>北京化工大学
<120>脂肪酶、其基因、产生该酶的亚罗解脂酵母及其应用
<160>2
<170>Patentln version 3.2
<210>1
<211>301
<212>PRT
<213>亚罗解脂酵母
<220>
<221>mise_特征
<222>(179)
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gacccccgtc tcatctttga tgtttctggt tacctcgctg ttgatcatgc ctccaagcag 240
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cagcaggtca atgtgattgg aaaccatctg cagtacttcg tcaccgaggg tgtctgtggt 900
atc 903
Claims (17)
1. 一种产生氨基酸序列为SEQ ID NO:1的脂肪酶的亚罗解脂酵母(Yarrowialipolytica),其保藏编号为CGMCC No.1470。
2. 一种利用脂肪酶合成酯的方法,其包括:
(a)使用权利要求1所述的亚罗解脂酵母提取脂肪酶;
(b)使用步骤(a)中所得脂肪酶合成酯。
3. 根据权利要求2的方法,其中步骤(b)中通过固定化脂肪酶合成酯。
4. 根据权利要求3的方法,其中所述的固定化脂肪酶包含氨基酸序列为SEQ ID NO:1的脂肪酶、载体和共固定剂,其中,载体与共固定剂的质量体积比为1∶1~1∶3,脂肪酶与载体的比例为1000-30000单位/克载体。
5. 根据权利要求4的方法,其中所述载体为固体颗粒和膜状纺织品。
6. 根据权利要求5的方法,其中所述固体颗粒为硅胶、硅藻土,所述膜状纺织品为天然纤维织物和化学纤维织物。
7. 根据权利要求6的方法,其中所述天然纤维织物为棉布,所述化学纤维织物为涤纶。
8. 根据权利要求4的方法,其中所述共固定剂选自高分子化合物、表面活性剂、蛋白质、无机盐中的至少一种。
9. 根据权利要求8的方法,其中所述共固定剂选自PEG6000、椰子油、吐温80、明胶、卵磷脂和硫酸镁中的至少一种。
10. 根据权利要求2的方法,其中步骤(b)中所述酯的合成通过脂肪酸和醇的酯化反应进行。
11. 根据权利要求10的方法,其中脂肪酸和醇以3∶1~1∶5的摩尔比加入,再和脂肪酶混合,于20-50℃反应9-50小时。
12. 根据权利要求11的方法,其中所述的脂肪酸选自C8~C18一元脂肪酸和二元酸的至少一种,醇选自C1~C18的伯醇、C3~C5的仲醇以及甘油、维生素C中的至少一种。
13. 根据权利要求12的方法,其中所述的脂肪酸选自辛酸、癸酸、棕榈酸、硬脂酸、油酸、亚油酸、亚麻酸、己二酸、壬二酸、癸二酸中的至少一种;所述醇选自甲醇、丙醇、丁醇、己醇、异辛醇、异丙醇、异丁醇、癸醇、十二醇、十八醇、甘油、维生素C中的至少一种。
14. 根据权利要求2的方法,其中步骤(b)中酯的合成通过底物醇或者酸和酯的转酯化反应进行。
15. 根据权利要求14的方法,其特征在于底物醇或者酸和底物酯以摩尔比3∶1~1∶7加入,再与固定化脂肪酶混合,于20-50℃反应9-50小时。
16. 根据权利要求15的方法,其中所述底物酸选自棕榈酸、油酸中的至少一种;底物醇选自甲醇、乙醇中的至少一种;底物酯选自维生素A醋酸酯、豆油、红花油、亚麻油、玉米油、棕榈油、色拉油中的至少一种。
17. 根据权利要求10-16中任一项的方法,其中所述反应使用有机溶剂或不用溶剂,所述有机溶剂选自丙酮、正己烷、环己烷、石油醚、正庚烷或正辛烷中的至少一种。
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EP06021211A EP1775344B1 (en) | 2005-10-11 | 2006-10-10 | Lipase, its gene, the strain and the application of this lipase |
DK06021211.5T DK1775344T3 (da) | 2005-10-11 | 2006-10-10 | Lipase, dens gen, stammen og anvendelsen af denne lipase |
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CN101104861B (zh) * | 2007-04-28 | 2012-01-18 | 北京化工大学 | 生物催化制备s-布洛芬及s-布洛芬酯的方法 |
MY144876A (en) * | 2007-11-23 | 2011-11-30 | Univ Putra Malaysia | A method for producing adipate ester |
CA2823222C (en) | 2010-06-18 | 2019-07-16 | Butamax(Tm) Advanced Biofuels Llc | Extraction solvents derived from oil for alcohol removal in extractive fermentation |
US9040263B2 (en) | 2010-07-28 | 2015-05-26 | Butamax Advanced Biofuels Llc | Production of alcohol esters and in situ product removal during alcohol fermentation |
US8765425B2 (en) | 2011-03-23 | 2014-07-01 | Butamax Advanced Biofuels Llc | In situ expression of lipase for enzymatic production of alcohol esters during fermentation |
US8759044B2 (en) | 2011-03-23 | 2014-06-24 | Butamax Advanced Biofuels Llc | In situ expression of lipase for enzymatic production of alcohol esters during fermentation |
CN102212572B (zh) * | 2011-04-28 | 2013-08-14 | 浙江大学 | 酵母展示脂肪酶催化合成l-抗坏血酸油酸酯的方法 |
CN106929502B (zh) * | 2015-12-30 | 2021-12-21 | 丰益(上海)生物技术研发中心有限公司 | 固定化脂肪酶颗粒 |
CN105936922B (zh) * | 2016-06-02 | 2020-03-17 | 北京化工大学 | 一种利用33℃棕榈油酶促酯交换制备类可可脂的方法 |
CN107779444A (zh) * | 2016-08-31 | 2018-03-09 | 北京超纳生物质化工技术研究院 | 用于制备低碳链甘油三酯的催化剂及利用该催化剂制备低碳链甘油三酯的方法 |
CN109971663B (zh) * | 2019-04-28 | 2020-06-09 | 江南大学 | 一株耐热亚罗解脂酵母及其应用 |
CA3166033A1 (en) * | 2019-12-30 | 2021-07-08 | Dsm Ip Assets B.V. | Lipase-modified strain |
CN112608954B (zh) * | 2020-11-27 | 2023-03-10 | 安徽泰格生物技术股份有限公司 | 一种维生素c棕榈酸酯的制备方法 |
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