CN114540318B - 具有催化乙醇醛合成乙醇酸功能的酶及其应用 - Google Patents
具有催化乙醇醛合成乙醇酸功能的酶及其应用 Download PDFInfo
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- CN114540318B CN114540318B CN202111282888.9A CN202111282888A CN114540318B CN 114540318 B CN114540318 B CN 114540318B CN 202111282888 A CN202111282888 A CN 202111282888A CN 114540318 B CN114540318 B CN 114540318B
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- aldh
- glycolaldehyde
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Abstract
本发明涉及具有催化乙醇醛合成乙醇酸功能的酶—乙醛脱氢酶ALDH‑BL21和ALDH‑11300。本发明通过定点突变得到酶活提高的突变体蛋白,还提供了以该功能的酶为催化剂以乙醇醛为底物合成乙醇酸及以甲醛为原料,将该功能的酶与乙醇醛合成酶GLAS级联以甲醛为底物合成乙醇酸的应用。与现有技术相比,本发明所提供的具有催化乙醇醛合成乙醇酸功能的酶可以快速合成大量的乙醇酸,并实现了多酶将甲醛转化为乙醇酸,甲醛可以通过CO2的多酶催化获得,该发明为CO2合成高附加值化合物途径提供了新思路,为实现碳中和开辟新的生物化学途径,具有潜在的应用前景。
Description
技术领域
本发明属于酶的定向进化改造和生物催化应用技术领域,涉及具有催化乙醇醛合成乙醇酸功能的酶及其应用。
背景技术
乙醇酸又称羟基乙酸、甘醇酸,是一种重要的有机合成中间体和化工产品,广泛应用于有机合成、清洗、电镀、纺织、皮革、灭菌等行业。此外,乙醇酸的聚合物具有生物可降解性,解决了传统塑料制品存在的难以降解的问题,因此也可被广泛应用于医学、包装及其他许多领域。
传统的乙醇酸制备方法主要分为(1)以甲醛和CO为原料,在强酸催化下通过甲醛的羰基化合成乙醇酸;(2)以氯乙酸为原料,在碳酸钡或碳酸钙为催化剂的作用下水解制备乙醇酸;(3)以乙二醛为原料,在碱性条件下氧化乙二醛合成乙醇酸等。中国专利CN102584566A公开一种以乙二醛为底物,在氢氧化钾、相转移催化剂的作用下经歧化反应合成得到羟基乙酸钾,在经酸化得到乙醇酸的乙醇酸制备方法。中国专利CN105085227A提供了使用水解反应器水解乙醇酸甲酯然后经甲醇精制塔分离再经浓缩塔浓缩得到乙醇酸的方法。上述方法均是利用化学方法制备乙醇酸,毒性大,条件苛刻,合成周期长且产品纯度低。
目前,通过酶法合成乙醇酸的方法几乎没有报道。以CO2为原料,通过多酶合成乙醇酸的途径也几乎没有报道。开发一个绿色高效的乙醇酸合成途径是一直以来需要解决而由尚未解决的难题。
发明内容
本发明所要解决的技术问题是针对制备乙醇酸的方法条件苛刻,对环境污染大,酶法合成乙醇酸方法几乎没有报道等问题,提供了具有催化乙醇醛合成乙醇酸功能的酶-乙醛脱氢酶ALDH-BL21和ALDH-11300,并通过定点突变得到酶活提高的突变体蛋白;本发明还提供了以该功能的酶为催化剂以乙醇醛为底物合成乙醇酸及以甲醛为原料,将该功能的酶与乙醇醛合成酶GLAS级联以甲醛为底物合成乙醇酸的应用。
为此,本发明第一方面提供了一种具有催化乙醇醛合成乙醇酸功能的酶,其为乙醛脱氢酶ALDH-BL21和/或乙醛脱氢酶ALDH-11300。
根据本发明的一些实施方式,所述乙醛脱氢酶ALDH-BL21为以下ALDH-BL21蛋白质中的任一种:
以下蛋白质中的任一种:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质;
(A3)与(A1)或(A2)所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(A4)在(A1)-(A3)中任一种蛋白质的N端和/或C端连接标签后得到的融合蛋白。
本发明中,氨基酸序列如SEQ ID No.1所示的乙醛脱氢酶ALDH-BL21为野生乙醛脱氢酶ALDH-BL21,其基因ydcW来源于大肠杆菌BL21(Escherichia coli BL21),该基因的GenBank登录号:CAQ31929.1,在不改变ALDH-BL21氨基酸序列的前提下,将该基因ydcW的密码子替换为大肠杆菌偏好(高频使用)的密码子,经密码子优化后,得到优化后ydcW基因序列,具有大肠杆菌偏爱密码子,其核苷酸序列如SEQ ID No.3所示,亦即,经密码子优化的编码氨基酸序列如SEQ ID No.1所示的ALDH-BL21蛋白(野生乙醛脱氢酶ALDH-BL21)的基因ydcW的核苷酸序列如SEQ ID No.3所示。
根据本发明:所述(A2)中,所述“将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质”为将SEQ ID No.1所示的氨基酸序列中的氨基酸残基进行一个或多个突变后得到的蛋白质;
在本发明的一些实施例中,所述(A2)中,所述“将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质”为所述ALDH-BL21蛋白突变体,其与SEQ ID No.1相比,在或仅在如下任一个位置或多个位置存在突变:氨基酸序列为SEQ ID No.1的乙醛脱氢酶ALDH-BL21氨基酸序列从N端到C端的第379位,第438位,第439位,第440位,第442位,第452位,第456位,第460位,第463位。
在本发明的一些进一步的实施例中,所述(A2)中,所述“将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质”与SEQ ID No.1相比,存在或仅存在如下任一个或多个突变:V379I,L438T,V439D,S440A,M442A,Y452D,M456G,G460A,D463A。
根据本发明的另一些实施方式,所述乙醛脱氢酶ALDH-11300为以下ALDH-11300蛋白质中的任一种:
(B1)氨基酸序列为SEQ ID No.2的蛋白质;
(B2)将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质;
(B3)与(B1)或(B2)所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(B4)在(B1)-(B3)中任一种蛋白质的N端和/或C端连接标签后得到的融合蛋白。
本发明中,氨基酸序列如SEQ ID No.2所示的乙醛脱氢酶ALDH-11300为野生乙醛脱氢酶ALDH-11300,其基因为aldh-11300来源于中度嗜热菌(Deinococcusgeothermalis)DSM 11300菌株,该基因为aldh-11300的GenBank登录号:ABF45418.1,在不改变ALDH-11300氨基酸序列的前提下,将该aldh-11300基因的密码子替换为大肠杆菌偏好(高频使用)的密码子,经密码子优化后,得到优化后aldh-11300基因序列,具有大肠杆菌偏爱密码子,其核苷酸序列为SEQ ID No.4,亦即,经密码子优化的编码氨基酸序列如SEQ ID No.2所示的ALDH-11300蛋白(野生乙醛脱氢酶ALDH-11300)的基因aldh-11300的核苷酸序列如SEQ IDNo.4所示。
根据本发明,所述(B2)中,所述“将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质”为将SEQ ID No.2所示的氨基酸序列中的氨基酸残基进行一个或多个突变后得到的蛋白质;
在本发明的一些实施例中,所述(B2)中,所述“将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质”为所述ALDH-11300蛋白突变体,其与SEQ ID No.2相比,在或仅在如下任一个位置或多个位置存在突变:氨基酸序列为SEQ ID No.2的乙醛脱氢酶ALDH-11300氨基酸序列从N端到C端的375位,第377位,第402位,第447位。
在本发明的一些进一步的实施例中,所述(B2)中,所述“将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质”为与SEQ ID No.2相比,存在或仅存在如下任一个或多个突变:E375D,L377V,Y402F,R447Q。
本发明第二方面提供了一种具有催化乙醇醛合成乙醇酸功能的酶的相关生物材料,为能够表达所述乙ALDH-BL21蛋白或ALDH-BL21突变体,或者ALDH-11300蛋白或ALDH-11300蛋白突变体的核酸分子,或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
所述核酸分子可以是DNA,如cDNA、基因组DNA或重组DNA;所述核酸分子也可以是RNA,如mRNA等。
所述重组载体可为重组表达载体,也可为重组克隆载体。
所述表达盒可由能够启动所述核酸分子转录的启动子,所述核酸分子,以及转录终止序列组成。
根据本发明的一些实施方式,编码所述ALDH-BL21蛋白质的核酸分子具体地为以下任一所示:
(C1)编码区包括如SEQ ID NO.3所示的经过密码子优化的核苷酸序列的DNA分子;
(C2)核苷酸序列如SEQ ID NO.3所示的经过密码子优化的核苷酸序列的DNA分子;
(C3)与(C1)或(C2)中所述的核苷酸序列具有75%或75%以上同一性,且编码本发明第一方面中的所述的ALDH-BL21蛋白质的DNA分子;
(C4)在严格条件下与(C1)或(C2)中所述的核苷酸序列杂交,且编码本发明第一方面中的所述的ALDH-BL21蛋白质的DNA分子。
在本发明的一些进一步具体的实施例中,所述核酸分子为所述ALDH-BL21蛋白突变体的编码基因,具体如下:与SEQ ID NO.3相比,存在或仅存在如下任一个或多个突变:GTG 1135-1137 ATT;CTG1312-1314 ACC;GTG 1315-1317 GAT;AGC 1318-1320 GCG;ATG1324-1326 GCG;TAT 1354-1356 AAC;ATG 1366-1368 GGC;GGC 1378-1380 GCG;GAT 1387-1389 GCG。
根据本发明的一些实施方式,编码所述ALDH-11300蛋白质的核酸分子具体地为以下任一所示:
(D2)核苷酸序列如SEQ ID NO.4所示的经过密码子优化的核苷酸序列的DNA分子;
(D3)与(D1)或(D2)中所述的核苷酸序列具有75%或75%以上同一性,且编码本发明第一方面中的所述的ALDH-11300蛋白质的DNA分子;
(D4)在严格条件下与(D1)或(D2)中所述的核苷酸序列杂交,且编码本发明第一方面中的所述的ALDH-11300蛋白质的DNA分子。
在本发明的一些进一步具体的实施例中,所述核酸分子为所述ALDH-11300蛋白突变体的编码基因,具体如下:与SEQ ID NO.4相比,存在或仅存在如下任一个或多个突变:GAA 1123-1125 GAT;CTG 1129-1131 GTG;TAT 1204-1206 TTT;CGC 1339-1341 CAG。
本发明第三方面提供了一种以乙醇醛为底物合成乙醇酸的方法,其包括利用具有催化乙醇醛合成乙醇酸功能的酶或相关生物材料,以乙醇醛为底物反应产生乙醇酸;
所述具有催化乙醇醛合成乙醇酸功能的酶为如本发明第一方面所述所述的酶;所述具有催化乙醇醛合成乙醇酸功能的酶的相关生物材料为本发明第二方面所述的相关生物材料;
优选地,所述合成乙醇酸的反应温度为4-80℃。
本发明第四方面提供了一种以甲醛为底物多酶级联合成乙醇酸的方法,其特征在于,包括以下步骤:(1)利用乙醇醛合成酶GALS将甲醛转化为乙醇醛;(2)利用具有催化乙醇醛合成乙醇酸功能的酶或相关生物材料将乙醇醛转化为乙醇酸;
所述具有催化乙醇醛合成乙醇酸功能的酶为如本发明第一方面所述所述的酶;所述具有催化乙醇醛合成乙醇酸功能的酶的相关生物材料为本发明第二方面所述的相关生物材料;
优选地,所述合成乙醇酸的反应温度为4-80℃。
本发明所述用语“TPP”是指焦磷酸硫胺素,“NAD+”是指氧化型辅酶Ⅰ。
本发明中所述用语“酶活力”(enzymeactivity)也称酶活性,是指酶催化一定化学反应的能力;本发明中酶活力以比活力表示,是指每g酶蛋白所具有的酶活力,单位为U/g。
本发明中所述“野生型”是指由野生菌发酵培养获得的酶蛋白,以及氨基酸或碱基序列未进行改变所翻译表达得到的酶蛋白。
本发明中所述用语“蛋白”与“蛋白质”可以互相使用。
本发明中所述用语“野生”与“野生型”可以互相使用。
在本发明中,对于氨基酸取代,使用下述命名法:原始氨基酸,位置,取代氨基酸。对于碱基取代,使用下述命名法:原始碱基,位置,取代碱基。
本发明中的检测方法及仪器:
(1)采用10mL规格镍琼脂糖凝胶FF亲和层析柱填料预装柱(中国bersee)进行蛋白纯化。
(2)采用HPLC U3000型高效液相色谱仪(Thermo Fisher Scientific)测定产物。
(3)采用870型酶标仪(Thermo Fisher Scientific)进行纯化后蛋白质浓度测定。
本发明提供了催化乙醇醛合成乙醇酸的酶,并通过定点突变得到酶活提高的突变体蛋白,其中,乙醛脱氢酶ALDH-BL21的Y452D突变体的酶活与野生型相比提高了17.62%,乙醛脱氢酶ALDH-11300的E375D突变体的酶活与野生型相比提高了22.92%;本发明还提供了以该功能的酶为催化剂以乙醇醛为底物合成乙醇酸及以甲醛为原料,将该功能的酶与乙醇醛合成酶GLAS级联以甲醛为底物合成乙醇酸的应用。与现有技术相比,本发明所提供的具有催化乙醇醛合成乙醇酸功能的酶可以快速合成大量的乙醇酸,并实现了多酶将甲醛转化为乙醇酸,甲醛可以通过CO2的多酶催化获得,该发明为CO2合成高附加值化合物途径提供了新思路,为实现碳中和开辟新的生物化学途径,具有潜在的应用前景。
附图说明
下面结合附图来对本发明作进一步详细说明:
图1为pET-22b-ALDH-BL21质粒图谱。
图2为pET-22b-ALDH-11300质粒图谱。
图3为ALDH-BL21野生型及其1号和2号突变体蛋白的SDS凝胶电泳图。
图4为ALDH-11300野生型及其1号和2号突变体蛋白的SDS凝胶电泳图。
图5示出不同ALDH-BL21及其突变体催化乙醇醛生成乙醇酸的结果。
图6示出不同ALDH-11300及其突变体催化乙醇醛生成乙醇酸的结果。
图7示出酶催化反应体系的HPLC结果,其中产物乙醇酸出峰时间为11.7min左右。
具体实施方式
为使本发明容易理解,下面将结合附图和实施例详细说明本发明。但在详细描述本发明前,应当理解本发明不限于描述的具体实施方式。还应当理解,本文中使用的术语仅为了描述具体实施方式,而并不表示限制性的。
除非另有定义,本文中使用的所有术语与本发明所属领域的普通技术人员的通常理解具有相同的意义。虽然与本文中描述的方法和材料类似或等同的任何方法和材料也可以在本发明的实施或测试中使用,但是现在描述了优选的方法和材料。
实施例
以下通过具体实施例对于本发明进行具体说明。下文所述实验方法,如无特殊说明均为实验室常规方法。下文所述实验材料,如无特别说明均可由商业渠道获得。
以下实施例中涉及到的培养基配方如下:
LB液体培养基:蛋白胨1%、酵母提取物0.5%、NaCl 1%;
LB固体培养基:琼脂1.5%、蛋白胨1%、酵母提取物0.5%、NaCl 1%;
培养基中的单位为%(W/V)。
实施例1:酶蛋白的获得
本发明中,氨基酸序列如SEQ ID No.1所示的乙醛脱氢酶ALDH-BL21的基因ydcW来源于大肠杆菌(Escherichia coli BL21),该基因的GenBank登录号:CAQ31929.1,在不改变ALDH-BL21氨基酸序列的前提下,将该基因ydcW的密码子替换为大肠杆菌偏好(高频使用)的密码子,经密码子优化后,得到优化后ydcW基因序列,具有大肠杆菌偏爱密码子,其核苷酸序列如SEQ ID No.3所示。
本发明中,氨基酸序列如SEQ ID No.2所示的乙醛脱氢酶ALDH-11300的基因为aldh-11300来源于中度嗜热菌(Deinococcusgeothermalis)DSM 11300菌株,该基因为aldh-11300的GenBank登录号:ABF45418.1,在不改变ALDH-11300氨基酸序列的前提下,将该aldh-11300基因的密码子替换为大肠杆菌偏好(高频使用)的密码子,经密码子优化后,得到优化后aldh-11300基因序列,具有大肠杆菌偏爱密码子,其核苷酸序列为SEQ IDNo.4。
实施例2:表达载体的构建
将SEQ ID No.3所示的优化后ydcW基因替换pET-22b载体(Novagen,Amp,见图1)酶切位点NdeI和XhoI之间的DNA片段,得到重组质粒,命名为pET-22b-ALDH-BL21。
将SEQ ID No.4所示的优化后aldh-11300基因替换pET-22b载体(Novagen,Amp,见图2)酶切位点NdeI和XhoI之间的DNA片段,得到重组质粒,命名为pET-22b-ALDH-11300。
实施例3:野生型酶蛋白的表达
(1)将大肠杆菌表达型重组质粒pET-22b-ALDH-BL21和pET-22b-ALDH-11300分别转入E.coli BL21(DE3)(TransGen)中,获得重组菌。采用氨苄抗性平板进行阳性克隆筛选(Amp,100mg/mL),37℃过夜培养;
(2)将挑单克隆至4mL LB液体培养基中(Amp,100mg/mL),在37℃、180rpm的恒温培养箱中培养12-16h;
(3)将步骤(2)培养的菌液接菌到50mL LB液体培养基中(Amp,100mg/mL),在37℃、150rpm的恒温培养箱中培养至OD600为0.6-0.8后,加入IPTG诱导剂(终浓度为0.4mM),在30℃、150rpm的恒温培养箱中诱导培养24h;
(4)培养结束,4℃、12000rpm离心10min收集菌体,加入10mL含有10mM咪唑和100mMNaCl的PBS溶液(0.05M,pH=7.5)重悬菌体,超声破碎15min;
(5)细胞破碎后4℃、12000rpm离心20min,所得上清为实验所需粗酶液;
(6)粗酶液过镍琼脂糖凝胶FF亲和层析柱调料预装柱纯化得到纯化酶1;使用30KDa的超滤膜超滤纯化溶液1得到最终纯化酶液,使用SDS凝胶电泳验证蛋白表达正确(图3和图4);
(7)使用G250考马斯亮蓝与酶液进行反应,然后使用870型酶标仪(Thermo FisherScientific)检测595nm的吸光度,从而计算蛋白浓度。
实施例4:ALDH-BL21突变体的制备
对重组质粒pET-22b-ALDH-BL21进行定点突变。定点突变采用Fast site-directMutagenesis System kit(Transgene,FM111-02)试剂盒方法。
ALDH-BL21蛋白的具体突变形式共涉及9种,如表1所示。
表1 ALDH-BL21突变体蛋白和基因突变位点
注:蛋白取代的编号是从SEQ ID No.1所示氨基酸序列的N端为起始的;基因取代的编号是从SEQ ID No.3所示核苷酸序列的5’端为起始的。表中,对于氨基酸取代,使用下述命名法:原始氨基酸,位置(即在SEQ ID No.1中的位置),取代氨基酸。相应地,在SEQ IDNo.1的第452位用天冬氨酸取代原有的酪氨酸命名为“Y452D”。对于碱基取代,使用下述命名法:原始碱基,位置(即在SEQ ID No.3中的位置),取代碱基。相应的,在SEQ ID No.3的第1354-1356位用TAT取代原有的AAC命名为“TAT 1354-1356AAC”。
实施例5:ALDH-11300突变体的制备
对重组质粒pET-22b-ALDH-11300进行定点突变。定点突变采用Fast site-directMutagenesis System kit(Transgene,FM111-02)试剂盒方法。
ALDH-11300蛋白的具体突变形式共涉及4种,如表2所示。
表2 ALDH-11300突变体蛋白和基因突变位点
注:蛋白取代的编号是从SEQ ID No.2所示氨基酸序列的N端为起始的;基因取代的编号是从SEQ ID No.4所示核苷酸序列的5’端为起始的。表中,对于氨基酸取代,使用下述命名法:原始氨基酸,位置(即在SEQ ID No.2中的位置),取代氨基酸。相应地,在SEQ IDNo.2的第402位用苯丙氨酸取代原有的天冬氨酸命名为“Y402F”。对于碱基取代,使用下述命名法:原始碱基,位置(即在SEQ ID No.4中的位置),取代碱基。相应的,在SEQ ID No.4的第1204-1206位用TAT取代原有的TTT命名为“TAT 1204-1206TTT”。
实施例6:酶活测定
反应体系:20mM乙醇醛,20mM NAD+,上述野生型蛋白或突变体蛋白100μg,反应体系总体积为1mL,缓冲液为0.05M,pH=8.0的PBS。30~50℃中反应3~6h后,利用HPLC检测产物乙醇酸的生成量计算酶活。
HPLC检测乙醇酸方法:带有紫外检测器的U3000高效液相色谱仪,HPX-87H有机酸柱子(BIO-RAD,300×7.8mm),65℃,0.6mL/min,检测波长210nm,流动相为5mM硫酸。
1个酶活力单位定义为:在特定条件下,在1min内转化1μmol底物(甲醛)生成产物(乙醇酸)的产量的酶量,故比酶活的计算公式为:
U/g=(C乙醇酸(μmol)/(时间(min)))/蛋白质量(g)
野生型ALDH-BL21及其突变体蛋白在30℃条件下反应6h的比活力如表3所示(图5):
表3野生型ALDH-BL21及其突变体蛋白的比活力
突变体 | 氨基酸突变位点 | 比活力(U/g) |
ALDH-BL21 | -- | 168.74 |
M1 | V379I | 134.95 |
M2 | L438T | 129.20 |
M3 | V439D | 47.63 |
M4 | S440A | 176.16 |
M5 | M442A | 33.95 |
M6 | Y452D | 198.38 |
M7 | M456G | 74.73 |
M8 | G460A | 93.95 |
M9 | D463A | 67.04 |
野生型ALDH-11300及其突变体蛋白在45℃条件下反应6h的比活力如表4所示(图6):
表4野生型ALDH-11300及其突变体蛋白的比活力
突变体 | 氨基酸突变位点 | 比活力(U/g) |
ALDH-11300 | -- | 376.14 |
M1 | E375D | 462.36 |
M2 | L377V | 332.32 |
M3 | Y402F | 336.89 |
M4 | R447Q | 399.01 |
使用HPLC检测产物乙醇酸的生成,HPLC结果如图7所示,乙醇酸的出峰时间为11.7min左右。
实施例7:催化乙醇醛合成乙醇酸的应用
反应体系:底物乙醇醛,辅酶NAD+,上述野生型蛋白或突变体蛋白,反应体系总体积为1mL,缓冲液为0.05M,pH=8.0的PBS。4~80℃中反应后,利用HPLC检测产物乙醇酸的生成量计算酶活。
反应方程式如式(1)所示。
在反应体系中加入100mM乙醇醛,100mM NAD+,1mg野生型ALDH-11300蛋白,反应体系总体积为1mL,缓冲液为0.05M,pH=8.0的PBS,40℃反应6h后,检测得到5.84g/L乙醇酸。
实施例8:催化甲醛合成乙醇酸的应用
反应体系:底物甲醛,无水硫酸镁,TPP,NAD+,上述野生型蛋白或突变体蛋白,乙醇醛合成酶GLAS,反应体系总体积为1mL,缓冲液为0.05M,pH=8.0的PBS。4~80℃中反应后,利用HPLC检测产物乙醇酸的生成量计算酶活。
反应方程式如式(2)所示。
在反应体系中加入50mM甲醛,25mM NAD+,2mM无水硫酸镁,0.1mM TPP,2mg乙醇醛合成酶GLAS,1mg野生型ALDH-11300蛋白,反应体系总体积为1mL,缓冲液为0.05M,pH=8.0的PBS,35℃反应6h后,检测得到0.65g/L乙醇酸。
乙醇醛合成酶基因由文献(该文献的DOI号:10.1038/s41467-019-09095)获得(基于该文献由华大基因合成获得)。
应当注意的是,以上所述的实施例仅用于解释本发明,并不构成对本发明的任何限制。通过参照典型实施例对本发明进行了描述,但应当理解为其中所用的词语为描述性和解释性词汇,而不是限定性词汇。可以按规定在本发明权利要求的范围内对本发明做出修改,以及在不背离本发明的范围和精神内对本发明进行修订。尽管其中描述的本发明涉及特定的方法、材料和实施例,但是并不意味着本发明限于其中公开的特定例,相反,本发明可扩展至其他所有具有相同功能的方法和应用。
序列表
<110> 北京化工大学
<120> 具有催化乙醇醛合成乙醇酸功能的酶及其应用
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aaacgcaccc acatggaact gggcggcaaa gcgccggtga ttgtgtttga tgatgcggat 780
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Claims (6)
1.一种具有催化乙醇醛合成乙醇酸功能的酶,其为乙醛脱氢酶ALDH-11300的如SEQ IDNo.2所示的氨基酸序列经过E375D突变或R447Q突变且具有相同功能的ALDH-11300蛋白突变体。
2.一种具有催化乙醇醛合成乙醇酸功能的酶的相关生物材料,其为能够表达ALDH-11300蛋白的经密码子优化的编码野生乙醛脱氢酶ALDH-11300的基因ALDH-11300或能够表达ALDH-11300蛋白突变体的核酸分子,或含有能够表达ALDH-11300蛋白的经密码子优化的编码野生乙醛脱氢酶ALDH-11300的基因ALDH-11300或能够表达ALDH-11300蛋白突变体的核酸分子的表达盒、重组载体、重组菌或转基因细胞系;
其中,所述野生乙醛脱氢酶ALDH-11300的氨基酸序列如SEQ ID No.2所示;
所述经密码子优化的编码野生乙醛脱氢酶ALDH-11300的基因ALDH-11300的核苷酸序列如SEQ ID No.4所示;
所述ALDH-11300蛋白突变体与SEQ ID No.2相比,存在E375D突变或R447Q突变。
3.一种以乙醇醛为底物合成乙醇酸的方法,其包括利用具有催化乙醇醛合成乙醇酸功能的酶或相关生物材料,以乙醇醛为底物反应产生乙醇酸;
所述具有催化乙醇醛合成乙醇酸功能的酶为如权利要求1所述的酶;所述具有催化乙醇醛合成乙醇酸功能的酶的相关生物材料为权利要求2所述的相关生物材料。
4.如权利要求3所述的方法,其特征在于,所述合成乙醇酸的反应温度为4-80℃。
5.一种以甲醛为底物多酶级联合成乙醇酸的方法,其特征在于,包括以下步骤:(1)利用乙醇醛合成酶GALS将甲醛转化为乙醇醛;(2)利用具有催化乙醇醛合成乙醇酸功能的酶或相关生物材料将乙醇醛转化为乙醇酸;
所述具有催化乙醇醛合成乙醇酸功能的酶为如权利要求1所述的酶;所述具有催化乙醇醛合成乙醇酸功能的酶的相关生物材料为权利要求2所述的相关生物材料。
6.如权利要求5所述的方法,其特征在于,所述合成乙醇酸的反应温度为4-80℃。
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CN103403172A (zh) * | 2010-11-08 | 2013-11-20 | 德意诺夫 | 酶及其用途 |
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CN112226398A (zh) * | 2020-10-30 | 2021-01-15 | 江南大学 | 一种高效生产戊二酸的重组大肠杆菌及其构建方法 |
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CN103403172A (zh) * | 2010-11-08 | 2013-11-20 | 德意诺夫 | 酶及其用途 |
WO2019063507A1 (en) * | 2017-09-26 | 2019-04-04 | Dsm Ip Assets B.V. | IMPROVED PROCESS FOR THE PRODUCTION OF ETHANOL |
CN112226398A (zh) * | 2020-10-30 | 2021-01-15 | 江南大学 | 一种高效生产戊二酸的重组大肠杆菌及其构建方法 |
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