CN109652470B - 一种脂肪酶在拆分(r,s)-扁桃酸甲酯中的应用 - Google Patents
一种脂肪酶在拆分(r,s)-扁桃酸甲酯中的应用 Download PDFInfo
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- CN109652470B CN109652470B CN201811639784.7A CN201811639784A CN109652470B CN 109652470 B CN109652470 B CN 109652470B CN 201811639784 A CN201811639784 A CN 201811639784A CN 109652470 B CN109652470 B CN 109652470B
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Abstract
本发明提供了一种脂肪酶及其编码基因,含有该编码基因的载体、工程菌及其应用。所述脂肪酶的氨基酸序列如SEQ ID NO.1所示,其编码基因如SEQ ID NO.2所示。本发明脂肪酶基因可与表达载体连接构建得到含该基因的胞内表达重组质粒,再转化至大肠杆菌菌株中,获得重组大肠杆菌;该重组大肠杆菌经细胞破碎,分离纯化获得重组脂肪酶;该重组脂肪酶具有催化拆分(R,S)‑扁桃酸甲酯的能力,可以生成(R)‑扁桃酸甲酯。
Description
(一)技术领域
本发明属于生物催化技术领域,涉及一种脂肪酶及其编码基因,含有该编码基因的载体、工程菌,及通过该脂酶立体选择性催化水解拆分(R,S)-扁桃酸甲酯对映体合成单一构型的(R)-扁桃酸甲酯对映体的应用。
(二)背景技术
手性是自然界的本质属性之一,构成生命有机体的分子都具有手性的特征。具有手性中心的药物的单一对映体可能具有不同的药理作用。外消旋扁桃酸、单一对映体扁桃酸及他们的衍生物都是重要的医药原料和医药中间体。(R)-扁桃酸及(S)-扁桃酸是重要的拆分介质,R-扁桃酸是制备多种手性药物的重要中间体,如用于半合成青霉素和头孢菌素等抗生素、血管扩张剂、杀菌剂、镇痉剂、减肥药物、抗肿瘤药物等的合成。R-扁桃酸的国际市场需求逐年增长,市场潜力巨大,加快R-扁桃酸的研究开发具有重要的意义。制备单一对映体扁桃酸的方法有:化学拆分法和生物酶法。化学拆分难度大,费用高,生物酶法涉及脂肪酶和脱氢酶,且生物酶法的反应条件温和,副作用少,对环境污染少,效率高。所以,本实验对(R,S)-扁桃酸甲酯对映体进行有效的手性拆分的研究具有重要的意义。
脂肪酶(Lipase,EC 3.1.1.3)全称为三酰基甘油酰基水解酶(Triacylglycerolacylhydrolase),是目前研究与工业化应用最多的一类水解酶。脂肪酶能催化酯水解、酯合成、醇解、酸解、酯酯交换和氨解反应。脂肪酶广泛存在于自然界的各种生物体,特别是在微生物和动植物组织中。动物脂肪酶主要存在于胰脏等器官组织,比如猪肝酯酶和猪胰脂肪酶,但由于动物脂肪酶活性较低、酶提取纯化成本较高和原料来源有限等因素,限制了其在工业中的应用。微生物脂肪酶来源丰富,种类繁多,不受季节气候等因素的影响,微生物生长产酶周期较短,而且具有耐有机溶剂、底物专一性强、催化选择性高和催化活性高等特点,因而微生物来源的脂肪酶有较高的工业化应用价值。目前市场上大部分商品化脂肪酶都通过细菌、真菌和酵母等微生物培养发酵获得。丹麦Novo Nordisk公司、日本Amano公司和美国Genencor公司等主要酶制造商开发了多种的商品化酶制剂。这些酶制剂公司利用分子改造技术对微生物脂肪酶进行改造,提高酶活力、稳定性和立体选择性等酶学性质,以满足不同工业领域应用的需求。因而构建脂肪酶基因工程菌从而大量生产重组脂肪酶具有十分重要意义。
(三)发明内容
本发明目的是提供一种立体选择性脂肪酶、编码基因、含有该基因的重组载体、该重组载体转化得到的重组基因工程菌,及其在生物法拆分(R,S)-扁桃酸甲酯中的应用。
本发明采用的技术方案是:
本发明提供一种脂肪酶在拆分(R,S)-扁桃酸甲酯中的应用(如图1所示),所述脂肪酶氨基酸序列如SEQ ID NO.1所示,编码基因序列为SEQ ID NO.2所示,具体的应用方法为:以含立体选择性脂肪酶编码基因的工程菌经发酵培养获得的湿菌体冻干后的粗酶为催化剂,以(R,S)-扁桃酸甲酯为底物,以pH 7.0缓冲液为反应介质,在20-45℃、600-800rpm条件下进行拆分反应,反应完全后,将反应液分离纯化,获得(R)-扁桃酸甲酯。所述催化剂用量以缓冲液体积计为10g/L,所述底物终浓度以缓冲液体积计为5-20g/L。
进一步,优选反应时间为20-120min,更优选反应条件为35℃、800rpm反应60min。
进一步,所述缓冲液为pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液。
进一步,所述催化剂按如下方法制备:将含立体选择性脂肪酶编码基因的工程菌(优选大肠杆菌BL21)接种在LB培养基中,37℃培养OD600至为0.4-0.6,加IPTG至终浓度0.02mM,30℃培养10-12h,菌液8000rpm,4℃离心10min,收集菌体,再用PBS缓冲液洗涤菌体2次,8000rpm,4℃离心10min,收集菌体,冻干,得到脂肪酶粗酶粉;LB培养基组成:胰蛋白胨10g/L、酵母粉5g/L、NaCl 5g/L,溶剂为去离子水,pH值自然。
进一步,本发明所述反应液分离纯化的方法为:反应结束后,反应液用4mM HCl进行酸化至pH 2.0,然后用等体积乙酸乙酯萃取,分液漏斗分离出有机相,再经纯水洗涤两次,饱和NaCl洗涤两次,干燥,旋蒸,得到最终产物(R)-扁桃酸甲酯。
本发明提供一种立体选择性脂肪酶,其氨基酸序列如SEQ ID NO.1所示:
MTTTKIETRHGVMAVDDSATDGFPVVFIHGNSASRSIFRHQVDADWAGDYRMITLDLLGHGDSEDASDPEAAYTQNGYADAIVDVLTELGVEGAVVVGWSLGGHIALELIAKFPGLTGIVITGTPPANPETLGDAFKAGDGLSIGGTEVITAQEAELYARMTAVPFEQAALDAAIRTDGRARKIMFDAFGAGRESDQKALVATASVPLAVIDGAEDPFVNTSYVASLTFANLWEGTYHVIPGVGHAAFWEAPEVYNPLLQRFLGSFAQ。
由于氨基酸序列的特殊性,任何含有SEQ ID NO.1所示氨基酸序列的多肽的片段或其变体,如其保守性变体、生物活性片段或衍生物,只要该多肽的片段或多肽变体与前述氨基酸序列同源性在90%以上、且具有相同的酶活性,均属于本发明保护范围之列。具体的,所述改变可包括氨基酸序列中氨基酸的缺失、插入或替换;其中,对于变体的保守性改变,所替换的氨基酸具有与原氨基酸相似的结构或化学性质,如用亮氨酸替换异亮氨酸,变体也可具有非保守性改变,如用色氨酸替换甘氨酸。
本发明所述蛋白的片段、衍生物或类似物是指基本上保持本发明所述的蛋白酶相同的生物学功能或活性的蛋白,可以是下列情形:(Ⅰ)一个或多个氨基酸残基被保守或非保守氨基酸残基(优选的是保守氨基酸残基)取代,并且取代的氨基酸可以是也可以不是由遗传密码子编码的;(Ⅱ)一个或多个氨基酸残基上的某个基团被其它基团取代;(Ⅲ)成熟蛋白与另一种化合物(比如延长蛋白半衰期的化合物,例如聚乙二醇)融合;(Ⅳ)附加的氨基酸序列融合进成熟的蛋白而形成的蛋白序列(如用来纯化此蛋白的序列或蛋白原序列)。
所述蛋白可以是重组蛋白、天然蛋白或合成蛋白,可以是纯天然纯化的产物,或是化学合成的产物,或使用重组技术从原核或真核宿主(例如:细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主,本发明的蛋白可以是糖基化的。本发明的蛋白还可以包括或不包括起始的甲硫氨酸残基。
本发明所述立体选择性脂肪酶的编码基因,具体的,所述大肠杆菌密码子优化后的编码基因核苷酸序列如SEQ ID NO.2所示:
CCATGGGCATGACAACCACCAAGATTGAGACCCGTCATGGTGTGATGGCCGTGGATGATAGCGCAACCGATGGCTTTCCGGTGGTGTTTATTCATGGTAATAGCGCCAGCCGCAGCATCTTTCGTCATCAAGTTGATGCCGACTGGGCCGGCGATTATCGCATGATCACTTTAGATCTGCTGGGTCATGGTGATAGCGAAGACGCAAGTGATCCGGAAGCCGCATATACCCAGAATGGCTACGCCGATGCCATTGTGGATGTGCTGACCGAACTGGGTGTGGAAGGTGCCGTGGTGGTTGGCTGGAGTCTGGGTGGCCACATCGCACTGGAACTGATCGCCAAATTTCCGGGTTTAACCGGCATTGTGATTACCGGCACCCCGCCGGCAAATCCGGAAACTTTAGGTGATGCCTTTAAAGCCGGCGATGGTTTAAGCATTGGTGGTACCGAAGTGATCACCGCCCAAGAAGCCGAGCTGTATGCACGCATGACAGCAGTGCCTTTTGAGCAAGCTGCTTTAGATGCAGCCATTCGTACCGATGGCCGTGCCCGCAAAATCATGTTTGATGCCTTCGGCGCTGGTCGCGAAAGCGATCAGAAAGCTTTAGTTGCAACAGCCAGCGTGCCGCTGGCCGTTATTGATGGCGCCGAAGATCCGTTTGTGAACACCAGCTACGTTGCCTCTTTAACCTTCGCAAATCTGTGGGAGGGCACCTATCATGTGATTCCGGGTGTGGGCCATGCCGCATTTTGGGAAGCCCCGGAGGTGTACAATCCGCTGCTGCAACGCTTTCTGGGCAGCTTTGCCCAGCTCGAG
由于核苷酸序列的特殊性,任何SEQ ID NO.2所示多核苷酸的变体,只要其与该多核苷酸具有70%以上同源性、且具有相同的功能,均属于本发明保护范围之列。所述多核苷酸的变体是指一种具有一个或多个核苷酸改变的多核苷酸序列。此多核苷酸的变体可以是天然发生的等位变异体或非天然发生的变异体,包括取代变异体、缺失变异体和插入变异体。如本领域所知的,等位变异体是一个多核苷酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,但不会从实质上改变其编码的氨基酸的功能。
另外,可与SEQ ID NO:2所示多核苷酸序列杂交的多核苷酸(至少具有50%同源性,优选具有70%同源性),也在本发明保护范围之列,特别是在严格条件下可与本发明所述核苷酸序列杂交的多核苷酸。所述“严格条件”是指:(1)在较低离子强度和较高温度下的杂交和洗脱,如0.2SSC,0.1%SDS,60℃;或(2)杂交时加用变性剂,如50%(v/v)甲酰胺,0.1%小牛血清,0.1%Ficoll,42℃;或(3)仅在两条序列之间的同源性至少在95%以上,更好是97%以上时才发生杂交。并且,可杂交的多核苷酸编码的蛋白与SEQ ID NO:1所示的蛋白有相同的生物学功能和活性。
本发明还涉及含有所述编码基因的重组载体,以及利用所述重组载体转化得到的重组基因工程菌。
本发明的有益效果主要体现在:本发明提供了一种脂肪酶基因核苷酸序列;该脂肪酶基因可与表达载体连接构建得到含该基因的胞内表达重组质粒,再转化至大肠杆菌菌株中,获得重组大肠杆菌,再利用重组大肠杆菌或重组脂肪酶为生物催化剂催化拆分(R,S)-扁桃酸甲酯,可以生成(R)-扁桃酸甲酯,对映体过量值>99%和转化率达到50.6%,产物(R)-扁桃酸甲酯的收率达到98.4%。本发明生物催化的手性合成反应具有条件温和、效率高,高度的化学选择性、区域选择性以及对映体选择性等优点,而且生物催化过程具有无毒、无污染和能耗低等特点,是一种环境友好的合成方法。
(四)附图说明
图1为重组脂肪酶对映选择性水解拆分(R,S)-扁桃酸甲酯的反应示意图;
图2为扁桃酸甲酯标样气相色谱图;
图3为脂肪酶催化(R,S)-扁桃酸甲酯水解反应1h的气相色谱图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此,本领域的普通技术人员根据这些实施方式所做出的方法上的变换均包含在本发明的保护范围内。
实施例1酶催化拆分(R,S)-扁桃酸甲酯的脂肪酶筛选
称取0.01g多种现有具有水解活性的冻干酯酶菌粉或10uL的菌液于2mL EP管中,加入1mL或者990μL PB(pH 7.0、0.2mM)作为反应溶剂,然后加入0.01g底物(R,S)-扁桃酸甲酯,以不加菌体为空白对照,置于35℃,800rpm恒温混匀仪中反应60min。反应结束后,反应液经2mM HCl酸化,再加入1mL乙酸乙酯,涡旋振荡器振荡2min,充分萃取,离心(1200rpm,3min),得到有机相。取700μL乙酸乙酯层通过气相色谱检测菌体的立体选择性及酶催化水解活性,筛选得到底物的对映体过量值>99%和转化率达到50.6%的微生物菌株。最终选择源自红串红球菌(Rhodococcus erythropolis)的GenBank:WP_047269443.1的蛋白质序列,通过大肠杆菌表达系统密码子优化后获得基因序列SEQ ID NO.2所示核苷酸序列,编码蛋白氨基酸序列为SEQ ID NO.1所示。将该片段连接到pET28b载体上获得克隆载体pET28b-lip并将其转化于大肠杆菌Escherichia coli BL21中,获得重组大肠杆菌,记为大肠杆菌BL21(F2)。对重组质粒测序,并利用软件对测序结果进行分析,该序列含有一个长为804bp的开放阅读框(SEQ ID NO.2)。
具体气相分析条件:采用Agilent6890气相色谱仪,BGB-174手性毛细管色谱柱(30.0m×0.25mm×0.25um),FID检测器;检测条件为柱温从初始温度100℃(恒温保持3min)升温至200℃(恒温保持2min),升温速率5℃/min,进样温度250℃,检测器温度250℃,空气流量和氢气流量分别为300mL/min和40mL/min。载气为高纯N2,柱头压93.5Kpa;尾吹气流量25.0mL·min-1;分流比50:1,进样体积为1uL。气相结果所示,(R)-扁桃酸甲酯和(S)-扁桃酸甲酯的保留时间分别为14.4min和14.7min(如图2所示)。
实施例2脂肪酶蛋白诱导条件
将实施例1获得的大肠杆菌BL21(F2)接种在LB培养基中,37℃培养OD600至为0.5(大概培养2h),加IPTG至终浓度0.02mM,30℃培养10-12h。300mL菌液8000rpm,4℃离心10min,收集菌体,再用PBS缓冲液洗涤菌体2次,8000rpm,10min收集菌体。将收集的菌体用冻干机冻干得到脂肪酶F2粗酶粉,放于4℃冰箱保存。LB培养基组成:胰蛋白胨10g/L、酵母粉5g/L、NaCl 5g/L,溶剂为水,pH自然。
实施例3不同脂肪酶催化(R,S)-扁桃酸甲酯的水解拆分效果比较
在pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液1mL中,加入终浓度10g/L的不同脂肪酶(实施例2制备的脂肪酶F2粗酶粉、Novozym 435、Lipozyme TL IM、Lipozyme RM IM、PSIM),0.02g(R,S)-扁桃酸甲酯,于恒温混匀仪35℃,800rpm条件下反应60min,取反应液按实施例1方法检测(R)-扁桃酸甲酯的对映体过量值和转化率,结果见表1所示。结果显示,在35℃反应1h后,脂肪酶435对(R,S)-扁桃酸甲酯两种构型都进行水解,脂肪酶TL IM、RM IM和PSIM对底物没有水解拆分活性,则以上四种商品脂肪酶对底物(R,S)-扁桃酸甲酯都没有对映体选择性,而当脂肪酶F2催化反应时,产物(R)-扁桃酸甲酯的ee值>99%,转化率为50.6%,气相色谱图3所示。
表1不同脂肪酶催化(R,S)-扁桃酸甲酯的水解拆分效果比较
实施例4反应时间对酶动力学水解拆分(R,S)-扁桃酸甲酯的影响
在pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液1mL中,加入终浓度10g/L的实施例2制备的脂肪酶F2粗酶粉,0.02g(R,S)-扁桃酸甲酯,于恒温混匀仪35℃,800rpm条件下反应不同时间(20min~120min),取反应液按实施例1方法检测(R)-扁桃酸甲酯的对映体过量值和转化率,结果见表2所示。
结果表明,反应60min后,产物(R)-扁桃酸甲酯的对映体过量值已达到最高,对映体过量值>99%,转化率为50.6%,当反应时间大于60min后,产物(R)-扁桃酸甲酯的对映体过量值基本不变,而转化率在增大。
表2反应时间对酶催化反应的影响
实施例5反应温度对酶动力学水解拆分(R,S)-扁桃酸甲酯的影响
在pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液1mL中,加入终浓度10g/L的实施例2制备的脂肪酶F2粗酶粉,0.02g(R,S)-扁桃酸甲酯,于恒温混匀仪不同温度(20-45℃),800rpm条件下反应不同时间60min,取反应液按实施例1方法检测(R)-扁桃酸甲酯的对映体过量值和转化率,结果见表3所示。
结果说明,在反应温度为35℃时,(R)-扁桃酸甲酯的对映体过量值最高,其ee值>99%。当反应温度高于35℃或者低于35℃时,(R)-扁桃酸甲酯的对映体过量值都会下降,说明温度对脂肪酶F2的光学选择性具有很大的影响。
表3反应温度对反应的影响
实施例6产物(R)-扁桃酸甲酯的分离提取
在50mL圆底烧瓶中加入20mL pH 7.0,0.2mM Na2HPO4/NaH2PO4缓冲溶液,称取0.2g实施例2制备的脂肪酶F2,再加入终浓度5g/L的(R,S)-扁桃酸甲酯,用50mM NaOH进行流加滴定反应,控制反应pH维持在7.0,在磁力搅拌器35℃,600rpm条件下反应3h。反应结束后得到的反应液,用4mM HCl进行酸化至pH 2.0,然后用1:1体积的乙酸乙酯萃取,分液漏斗分离出有机相,再经纯水洗涤两次,饱和NaCl洗涤两次,干燥,旋蒸,得到最终产物,并称重,产物(R)-扁桃酸甲酯的收率达到98.4%。
序列表
<110> 浙江工业大学
<120> 一种脂肪酶在拆分(R,S)-扁桃酸甲酯中的应用
<160> 2
<170> SIPOSequenceListing 1.0
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tgctgcaacg ctttctgggc agctttgccc agctcgag 818
Claims (9)
1.一种脂肪酶在拆分(R,S)-扁桃酸甲酯中的应用,其特征在于所述脂肪酶的氨基酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的应用,其特征在于所述脂肪酶编码基因的核苷酸序列如SEQ IDNO.2所示。
3.如权利要求1所述的应用,其特征在于所述应用的方法为:以含脂肪酶编码基因的工程菌经发酵培养获得的湿菌体冻干后的粗酶为催化剂,以(R,S)-扁桃酸甲酯为底物,以pH7.0缓冲液为反应介质,在20-45℃、800rpm条件下进行拆分反应,反应完全后,将反应液分离纯化,获得(R)-扁桃酸甲酯。
4.如权利要求3所述的应用,其特征在于所述催化剂用量以缓冲液体积计为10g/L,所述底物终浓度以缓冲液体积计为5-20g/L。
5.如权利要求3所述的应用,其特征在于反应时间为20-120min。
6.如权利要求3所述的应用,其特征在于所述反应条件为35℃、800rpm反应60min。
7.如权利要求3所述的应用,其特征在于所述缓冲液为pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液。
8.如权利要求3所述的应用,其特征在于所述催化剂按如下方法制备:将含脂肪酶编码基因的工程菌接种在LB培养基中,37℃培养OD600至为0.4-0.6,加IPTG至终浓度0.02mM,30℃培养10-12h,菌液8000rpm,4℃离心10min,收集菌体,再用PBS缓冲液洗涤菌体2次,8000rpm,4℃离心10min,收集菌体,冻干,得到脂肪酶粗酶粉。
9.如权利要求3所述的应用,其特征在于所述反应液分离纯化的方法为:反应结束后,反应液用4mM HCl进行酸化至pH 2.0,然后用等体积乙酸乙酯萃取,分液漏斗分离出有机相,再经纯水洗涤两次,饱和NaCl洗涤两次,干燥,旋蒸,得到最终产物(R)-扁桃酸甲酯。
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