CN113930457A - 一种双酶偶联合成(s)-香茅醇的方法 - Google Patents
一种双酶偶联合成(s)-香茅醇的方法 Download PDFInfo
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- CN113930457A CN113930457A CN202111142193.0A CN202111142193A CN113930457A CN 113930457 A CN113930457 A CN 113930457A CN 202111142193 A CN202111142193 A CN 202111142193A CN 113930457 A CN113930457 A CN 113930457A
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- ysadh
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- citronellol
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- alcohol dehydrogenase
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Abstract
本发明公开了供了一种双酶偶联合成(S)‑香茅醇的方法,以含醇脱氢酶YsADH基因的工程菌和含老黄酶NemR‑PS基因的工程菌各自经发酵培养获得的湿菌体混合后作为催化剂,以橙花醇为底物,以NADP+为辅酶,以pH 6‑9缓冲液为反应介质构成反应体系,在25‑55℃、0‑900rpm条件下反应完全后,反应液分离纯化,最终获得(S)‑香茅醇。当以100mM橙花醇为底物时,经12h反应后产物(S)‑香茅醇的转化率高达99.74%,产物e.e.值>99%。与现有技术相比,所建立的(S)‑香茅醇合成方法绿色高效,原子经济性高,具有优越的位点选择性、化学选择性和对映体选择性。
Description
(一)技术领域
本发明属于生物催化领域,涉及一种以橙花醇为底物基于辅酶自循环的(S)-香茅醇合成方法。
(二)背景技术
香茅醇具有玫瑰香气,是香水的重要配料。(S)-香茅醇的香型不同于(R)-香茅醇,可满足高端香水的配制要求。(S)-香茅醇具有多种生物活性,可以抑制伤寒杆菌及金黄色葡萄杆菌活性。(S)-香茅醇是合成手性化学品的重要中间体,可用于合成全顺式的玫瑰醚、(S)-胡薄荷酮和3(S)-甲基-庚酸等。
(S)-香茅醇的化学合成通常以橙花醇、香茅醛、柠檬醛等作为底物,利用化学加氢得到(S)-香茅醇。香茅醛和柠檬醛既有C=C键,也有C=O键,它们的还原在化学选择性和对映体选择性上仍是挑战。橙花醇含有两个C=C键,它的还原合成(S)-香茅醇需要满足严格的位点选择性和对映体选择性。与化学法相比,生物法还原橙花醇、香茅醛、柠檬醛等生成(S)-香茅醇,具有优异的位点选择性、化学选择性和对映体选择性,并且条件温和、绿色环保。然而,由于缺乏高活力的专用酶,目前生物法还达不到实际应用的要求。此外,生物法还原橙花醇、香茅醛或柠檬醛需要配置辅酶循环体系,为了驱动辅酶循环,往往需要添加过量的辅底物,因而降低了原子经济性。
为了提高(S)-香茅醇的合成效率,生物催化剂选用了高活力的醇脱氢酶YsADH 和老黄酶NemR-PS。为了提高生物催化合成(S)-香茅醇的原子经济性,反应设计融合了辅酶自循环体系:醇脱氢酶YsADH利用NADP+催化橙花醇脱氢生成橙花醛和 NADPH,而老黄酶NemR-PS利用NADPH还原橙花醛生成(S)-香茅醛,该过程辅酶自循环,不需要辅底物驱动;生成的(S)-香茅醛由醇脱氢酶YsADH进一步还原成光学纯的(S)-香茅醇,所建立的方法具有严格的位点选择性、化学选择性以及对映体选择性。与专利文献(申请号:2020112165477;一种双酶偶联合成(R)-香茅醇的方法) 相比,老黄酶NemR-PS替代老黄酶OYE2y-HG,与醇脱氢酶YsADH相偶联催化不同的底物(橙花醇)转化为不同的产物(S)-香茅醇,适应底物浓度达100mM,反应更为高效。与专利文献(申请号:2021108203589;一种三酶共表达重组菌及其在(S)- 香茅醇合成中的应用)相比,老黄酶NemR-PS与醇脱氢酶YsADH二酶偶联替代老黄酶NemR-PS、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6三酶偶联,利用不同的底物获得了相同的产物(S)-香茅醇;反应不需要辅底物葡萄糖,提高了反应的原子经济性。
(三)发明内容
本发明目的是提供一种双酶偶联合成(S)-香茅醇的方法,以橙花醇为底物基于辅酶自循环的(S)-香茅醇合成方法,醇脱氢酶YsADH利用NADP+催化橙花醇脱氢生成橙花醛和NADPH,而老黄酶NemR-PS利用NADPH还原橙花醛生成(S)-香茅醛,该过程辅酶自循环,不需要辅底物驱动;(S)-香茅醛在醇脱氢酶YsADH催化下进一步还原生成光学纯的(S)-香茅醇。该方法原子经济性高,具有优越的位点选择性、化学选择性和对映体选择性。
本发明采用的技术方案是:
本发明提供一种双酶偶联合成(S)-香茅醇的方法,所述方法为:以含醇脱氢酶YsADH基因的工程菌和含老黄酶NemR-PS基因的工程菌各自经发酵培养获得的湿菌体混合后作为催化剂,以橙花醇为底物,以NADP+为辅酶,以pH 6-9缓冲液为反应介质构成反应体系,在25-55℃、0-900rpm条件下反应完全后,反应液分离纯化,最终获得(S)-香茅醇。
进一步,所述醇脱氢酶YsADH基因来源于约克氏菌(Yokenella sp.WZY002),氨基酸序列为SEQ ID NO.1所示,核苷酸序列为SEQ ID NO.2所示。所述约克氏菌 (Yokenellasp.WZY002)保藏于中国典型培养物保藏中心,地址:中国武汉,武汉大学,邮编:430072,保藏编号:CCTCC No:M2013099,保藏日期:2013年3月22 日,已在专利申请CN201310188883.9中公开。
进一步,所述含醇脱氢酶YsADH基因的工程菌是将醇脱氢酶YsADH基因导入大肠杆菌构建获得的;具体为:将SEQ ID NO.2所示醇脱氢酶YsADH编码基因插入 pET28b载体的Nco I和Hind III限制性酶切位点,得到重组载体pET28b-YsADH;将重组载体pET28b-YsADH导入宿主细胞E.coli BL21(DE3),得到重组基因工程菌E. coli BL21(DE3)/pET28b-YsADH。
进一步,所述老黄酶NemR-PS基因来源于斯氏普罗威登斯菌(Providenciastuartii; Department of Infectious Diseases,National Institute of HealthDr.Ricardo Jorge,Lisbon, Portugal),其氨基酸序列如SEQ ID NO.3所示,核苷酸序列如SEQ ID NO.4所示。
进一步,所述含老黄酶NemR-PS基因的工程菌是将老黄酶NemR-PS基因导入大肠杆菌构建获得的;具体为:将SEQ ID NO.4所示老黄酶NemR-PS编码基因插入 pET28b载体的Nco I和Xho I限制性酶切位点,得到重组载体pET28b-NemR-PS;将重组载体pET28b-NemR-PS导入宿主细胞E.coli BL21(DE3),得到基因工程菌E.coli BL21(DE3)/pET28b-NemR-PS。
进一步,所述含醇脱氢酶YsADH基因的工程菌的湿菌体与含老黄酶NemR-PS 基因的工程菌的湿菌体以质量比0.25-4:1混合,优选为2:1。所述催化剂加入量以湿菌体总量计为100-200g/L,优选120g/L。
进一步,底物橙花醇用乙醇为溶剂预先配成1M橙花醇溶液,再根据反应体系中底物终浓度酌量添加,所述底物橙花醇加入终浓度为10-100mM,优选为100mM;所述辅酶NADP+加入终浓度为0-1.0mM,优选为0.6mM。
进一步,所述反应时间为2-12h,优选反应条件为40℃、600rpm下反应12h。
进一步,所述缓冲液优选为pH 7.5的50mM Tris-HCl缓冲液。
进一步,所述含醇脱氢酶YsADH基因工程菌的湿菌体按如下方法制备:将含醇脱氢酶YsADH基因的工程菌(优选E.coli BL21(DE3)/pET28b-YsADH)接种于含有终浓度100μg/mL卡那霉素的LB液体培养基中,在37℃和200rpm下培养过夜,然后以体积浓度2%的接种量转接于含有100μg/mL卡那霉素的LB液体培养基中,在 37℃和200rpm培养至菌体浓度OD600至0.6~0.8,向培养物中加入终浓度0.2mM的 IPTG,在21℃下诱导培养12h,获得诱导培养液;再将诱导培养液于4℃和8000rpm 下离心10min,弃去上清液;然后用pH 8.0、50mMTris-HCl缓冲液重悬菌体,于4℃和8000rpm下离心10min,弃去上清液,收集湿菌体。
进一步,所述含老黄酶NemR-PS基因工程菌的湿菌体按如下方法制备:将含老黄酶NemR-PS基因的工程菌(优选E.coli BL21(DE3)/pET28b-NemR-PS)接种于含有终浓度100μg/mL卡那霉素的LB液体培养基中,在37℃和200rpm下培养过夜,以体积浓度2%的接种量转接于终浓度100μg/mL卡那霉素的LB液体培养基中,在37℃和200rpm培养至菌体浓度OD600至0.6~0.8,向培养物中加入终浓度0.2mM的IPTG,在24℃下诱导培养12h,获得诱导培养液;再将诱导培养液于4℃和8000rpm下离心10min,弃去上清液;然后用pH 8.0、50mM Tris-HCl缓冲液重悬菌体,于4℃和 8000rpm下离心10min,弃去上清液,收集湿菌体。
进一步,所述反应液分离纯化方法为:反应液在12000rpm下离心2min,取上清,加入4倍反应液体积的乙酸乙酯,在200rpm和30℃条件下萃取1h,萃取结束后,在12000rpm下离心1min,取上层有机相,经减压蒸馏去除乙酸乙酯,最终获得(S)-香茅醇。
与现有技术相比,本发明的有益效果主要体现在:本发明提供了一种以橙花醇为底物基于辅酶自循环的(S)-香茅醇合成方法,在辅酶自循环中,醇脱氢酶YsADH利用NADP+催化橙花醇脱氢生成橙花醛和NADPH,而老黄酶NemR-PS利用NADPH 还原橙花醛生成(S)-香茅醛,该过程辅酶循环不需要辅底物驱动(图1)。当以100mM 橙花醇为底物时,经12h反应后产物(S)-香茅醇的得率高达99.74%,产物e.e.值>99%。与现有技术相比,所建立的(S)-香茅醇合成方法绿色高效,原子经济性高,具有优越的位点选择性、化学选择性和对映体选择性。
(四)附图说明
图1为以橙花醇为底物基于辅酶自循环的(S)-香茅醇合成方法示意图。
图2为实施例1工程菌诱导前后上清液的SDS-PAGE胶图;从左到右,泳道M, Blueplus II protein marker;泳道1,未经诱导基因工程菌E.coli BL21(DE3)/pET28b-YsADH;泳道2,诱导后的基因工程菌E.coli BL21(DE3)/pET28b-YsADH,加粗条带对应为YsADH,其分子量大小为37kDa;泳道3,未经诱导基因工程菌E.coli BL21(DE3)/pET28b-NemR-PS;泳道4,诱导后的基因工程菌E.coli BL21(DE3)/pET28b-NemR-PS,加粗条带对应为NemR-PS,分子量大小为39kDa。
图3为BCA法测蛋白质浓度的标准曲线。
图4为气相色谱图;a,标样(S)-香茅醛(20.128min)和(R)-香茅醛(21.098min);b,标样(S)-香茅醛(20.128min),橙花醇(27.208min),香茅醇(27.480min),香叶醇(28.419min),橙花醛(29.890min)和香叶醛(28.842min);c,(S)-香茅醇(95.481min)和(R)-香茅醇(96.249min);d,反应液中萃取获得的(S)-香茅醇(95.481min)。
图5为以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的最适催化温度。
图6为以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的最适催化pH。
图7为以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的最适辅酶NADP+添加量。
图8为以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的最适搅拌速度。
图9为以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的醇脱氢酶YsADH和老黄酶NemR-PS最适质量比。
图10为基于辅酶自循环合成(S)-香茅醇时不同底物浓度下的反应进程。
图11为底物橙花醇(a)和产物香茅醇(b)的气相-质谱联用(GC-MS)谱图。
图12为产物香茅醇的1H(a)和13C(b)的核磁共振(NMR)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:
LB液体培养基组成(1L):胰化蛋白胨10g,酵母提取物5g,NaCl 10g。用1 M的NaOH调节pH值到7.0~7.5,定容至1L。在121℃高压灭菌20min,4℃储存。
实施例1:表达醇脱氢酶YsADH、老黄酶NemR-PS的湿菌体、粗酶液制备
1、醇脱氢酶YsADH基因工程菌的构建
人工合成已公开的来源于约克氏菌(Yokenella sp.WZY002;保藏于中国典型培养物保藏中心,地址:中国武汉,武汉大学,邮编:430072,保藏编号:CCTCC No: M2013099,保藏日期:2013年3月22日,已在专利申请CN201310188883.9中公开) 的醇脱氢酶YsADH编码基因,其氨基酸序列和核苷酸序列分别如SEQ ID NO.1和 SEQ ID NO.2所示。
基因工程菌E.coli BL21(DE3)/pET-28b-YsADH的构建:将SEQ ID NO.2所示醇脱氢酶YsADH编码基因插入pET28b载体的Nco I和Hind III限制性酶切位点,得到重组载体pET28b-YsADH;将重组载体pET-28b-YsADH导入宿主细胞E.coli BL21(DE3),得到基因工程菌E.coli BL21(DE3)/pET28b-YsADH。
2、老黄酶NemR-PS基因工程菌的构建
人工合成已公开的来源于斯氏普罗威登斯菌(Providencia stuartii;Department of Infectious Diseases,National Institute of Health Dr.RicardoJorge,Lisbon,Portugal)的老黄酶NemR-PS的编码基因,其氨基酸序列和核苷酸序列分别如SEQ ID NO.3和 SEQ ID NO.4所示。
基因工程菌E.coli BL21(DE3)/pET28b-NemR-PS的构建:将SEQ ID NO.4所示老黄酶NemR-PS编码基因插入插入pET28b载体的Nco I和Xho I限制性酶切位点,得到重组载体pET28b-NemR-PS;将重组载体pET-28b-NemR-PS导入宿主细胞E.coli BL21(DE3),得到基因工程菌E.coli BL21(DE3)/pET28b-NemR-PS。
3、表达醇脱氢酶YsADH与老黄酶NemR-PS的湿菌体制备
湿菌体E.coli BL21(DE3)/pET28b-YsADH按如下方法制备:将基因工程菌E.coliBL21(DE3)/pET28b-YsADH接种于含有终浓度100μg/mL卡那霉素的LB液体培养基中,在37℃、200rpm下培养过夜,然后以体积浓度2%的接种量转接于含有100μg/mL 卡那霉素的LB液体培养基中,在37℃、200rpm培养至菌体浓度OD600至0.6~0.8,向培养物中加入终浓度0.2mM的IPTG,在21℃下诱导培养12h,获得诱导培养液。同样条件下,以不添加IPTG的培养液为未诱导对照培养液。再将诱导培养液于4℃、 8000rpm下离心10min,弃去上清液;然后用pH 8.0、50mM Tris-HCl缓冲液重悬菌体,于4℃和8000rpm下离心10min,弃去上清液,收集湿菌体作为生物催化剂,于 -80℃条件下保存备用。
湿菌体E.coli BL21(DE3)/pET28b-NemR-PS按如下方法制备:将基因工程菌E.coli BL21(DE3)/pET28b-NemR-PS接种于含有终浓度100μg/mL卡那霉素的LB液体培养基中,在37℃、200rpm下培养过夜,然后以体积浓度2%的接种量转接于含有100 μg/mL卡那霉素的LB液体培养基中,在37℃、200rpm培养至菌体浓度OD600至0.6~ 0.8,向培养物中加入终浓度0.2mM的IPTG,在24℃下诱导培养12h,获得诱导培养液。同样条件下,以不添加IPTG的培养液为未诱导对照培养液。再将诱导培养液于4℃、8000rpm下离心10min,弃去上清液;然后用pH 8.0、50mM Tris-HCl缓冲液重悬菌体,于4℃和8000rpm下离心10min,弃去上清液,收集湿菌体作为生物催化剂,于-80℃条件下保存备用。
SDS-PAGE检测样品的制备:取未诱导对照培养液和诱导培养液各1mL,在12000rpm离心1min,弃去上清,留取菌体。菌体随后各加入100μL超纯水,将菌重悬成菌悬液。之后,各取20μL菌悬液,加入4μL 6x Protein Loading Buffer混匀后,煮沸 10min。煮沸结束后,12000rpm离心1min,各取15μL上清液用于SDS-PAGE检测,蛋白质Marker为BluePlusProtein Marker(14-120kDa)。如图2所示,SDS-PAGE检测表明,醇脱氢酶YsADH与老黄酶NemR-PS均在大肠杆菌中成功表达。
4、醇脱氢酶YsADH、老黄酶NemR-PS的粗酶液制备
将每1g步骤3离心收集的湿菌体中加入20mL的50mM Tris-HCl缓冲液(pH 8.0),用玻璃棒搅拌成菌悬液,在冰浴(0℃)条件下超声破碎10min,超声工作1s,间歇2s,超声功率250W。经超声破碎后的菌悬液在8000rpm、4℃下离心10min,所得到的上清液即为粗酶液,放在4℃保存备用。
5、醇脱氢酶YsADH和老黄酶NemR-PS的体积比活力测定
醇脱氢酶YsADH的酶活力采用分光光度计的单因素动力学方法测定NADPH在340nm处吸光值的变化来计算酶活。酶活检测体系为:20mM香叶醇,0.4mM NADP+, 50μL粗酶液,加50mM Tris-HCl(pH 8.0)补足1mL。酶活力单位(U)定义:在45℃下,每分钟还原1μmol的NADP+所需的酶量。每次做三组平行实验,计算平均值和标准误差。醇脱氢酶(ADH)的体积酶活力计算如公式1。
老黄酶NemR-PS的酶活力采用分光光度计的单因素动力学方法测定NADPH在340nm处吸光值的变化来计算酶活。酶活检测体系为:20mM柠檬醛,0.4mM NADPH,50μL粗酶液,加入KH2PO4缓冲液(pH 7.0)补足1mL。酶活力单位(U) 定义:在30℃下,每分钟消耗1μmolNADPH所需的酶量。每次做三组平行实验,计算平均值和标准误差。老黄酶体积酶活力计算如公式1。
D:稀释倍数,为1;A1:样品吸光度;A2:空白对照吸光度;Vt:反应总体系,为1mL。
e:摩尔吸光系数,为常数6220;Vs:酶液体积,为0.05mL;d:比色皿直径,为1cm。
经酶活力测定,醇脱氢酶YsADH粗酶液体积比酶活为5.07U/mL;老黄酶 NemR-PS体积比酶活为1.50U/mL。
6、醇脱氢酶YsADH和老黄酶NemR-PS粗酶液的蛋白浓度测定
根据BCA法蛋白质浓度测定试剂盒绘制蛋白浓度标准曲线,以蛋白含量为横坐标,吸光值为纵坐标,绘制标准曲线,如图3所示,测得的线性关系公式为y= 0.4478x+0.20925,其中y为562nm处的吸光度值,x为BSA溶液蛋白浓度(mg/mL),标准偏差为R2=0.99056。
在利用BCA法蛋白质浓度测定试剂盒测量醇脱氢酶YsADH与老黄酶NemR-PS 时,每次做三组平行实验,计算平均值和标准误差。经测定,醇脱氢酶YsADH粗酶液的蛋白质浓度为7.57mg/mL;老黄酶NemR-PS粗酶液的蛋白质浓度为8.82mg/mL。
7、比酶活
体积酶活与蛋白质浓度的比值,即可得出相应的比酶活。经计算醇脱氢酶YsADH粗酶液的比酶活为0.67U/mg,老黄酶NemR-PS粗酶液的比酶活为0.17U/mg。
实施例2:以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的初始反应体系构建
实施例1步骤3制备的醇脱氢酶YsADH湿菌体与老黄酶NemR-PS湿菌体按一定比例于缓冲液(50mM Tris-HCl)中混合为催化剂,以橙花醇为底物,加入辅酶 NADP+,构成10mL的反应总体系。底物橙花醇用乙醇为溶剂预先配成1M溶液,再根据反应体系中底物终浓度酌量添加。
初始未进行条件优化的反应体系如下:底物橙花醇终浓度60mM、辅酶NADP+终浓度0.4mM,湿菌体终浓度120g/L(其中醇脱氢酶YsADH湿菌体与老黄酶 NemR-PS湿菌体按质量比1:1的比例混合),加入pH 8.0的50mM Tris-HCl缓冲液中,在pH 8.0、200rpm和30℃下反应4h。同样条件下,以不加催化剂为空白对照。
反应结束后取300μL反应液于12000rpm离心3min,取200μL上清加入800μL 乙酸乙酯萃取30min。萃取结束后,在12000rpm下离心1min,取上层有机相600μL 加入0.8g无水硫酸钠,然后在12000rpm下再离心1min,取100μL上清液,补加乙酸乙酯至终体积0.5mL,利用气相色谱法检测样品中各成分的含量。每次做三组平行实验,计算平均值和标准误差。
气相色谱条件如下:气相色谱仪,Agilent 6890N;手性色谱柱,BGB-174(柱长30m,柱内径250μm,固定液涂层厚0.25μm);检测器,FID,250℃;载气,N2;载气流量,2.27mL/min;分流比:1:19;进样量:1.0μL;进样口温度:250℃。
底物、可能的中间产物和产物升温程序:90℃保持25min,以20℃/min升温至 160℃,保持2min,随后以20℃/min升温至180℃,保持3min,共34.50min。结果如图4中a和b所示,底物(橙花醇、香叶醇)、可能的中间产物((S)-香茅醛,(R)- 香茅醛,橙花醛,香叶醛)和产物(香茅醇)的保留时间分别如下:(S)-香茅醛,20.128 min;(R)-香茅醛,21.098min;橙花醇,27.208min;香茅醇,27.480min;香叶醇, 28.419min;橙花醛,29.890min;香叶醛,28.842min。
(S)-香茅醇和(R)-香茅醇的分析采用特定的升温程序:75℃保持30min,以 0.4℃/min升温至120℃,保持10min,随后以20℃/min升温至180℃,保持3min,共158.50min。如图4中c所示,(S)-香茅醇和(R)-香茅醇的保留时间分别为95.481min 和96.249min。在所有的后续反应中,反应液中萃取获得的产物构型均如图4中d所示,为光学纯(S)-香茅醇。
实施例3:以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的最适温度
选取25℃、30℃、35℃、40℃、45℃、50℃和55℃,其他操作及反应条件同实施例2。每次做三组平行实验,计算平均值和标准误差,结果如图5所示。在25℃时, (S)-香茅醇的转化率为43.75%;当催化温度为40℃时,转化率已达到72.39%。当温度升高至45和50℃时,转化率降至65.78%和55.27%。综上,最佳反应温度为40℃。
实施例4:以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的最适催化pH
将反应体系的pH设为6.0、6.5、7.0、7.5、8.0、8.5和9.0,反应温度为40℃。其他操作及反应条件同实施例2。每次做三组平行实验,计算平均值和标准误差。结果如图6所示,当pH为6.0时,(S)-香茅醇的转化率为42.95%。当pH偏中性或弱碱性时,转化率也较高,当pH为7.5时,转化率高达67.38%。当pH 8.0和pH 9.0时,转化率分别为55.94%和40.39%。由此可见,最适的反应pH为7.5。
实施例5:以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的最适辅酶NADP+添加量
反应温度为40℃、pH为7.5。其他操作及反应条件同实施例2。其中辅酶NADP+终浓度为0-1.0mM(选取0、0.2、0.4、0.6、0.8和1mM)下反应。每次做三组平行实验,计算平均值和标准误差。如图7所示,当NADP+不添加时,(S)-香茅醇的转化率为15.37%,NADP+浓度在0-1.0mM之间时,NADP+的增加有利于反应的进行,0.6 mM的浓度时,转化率为34.45%。当NADP+的浓度大于0.6mM时,转化率增加幅度不大。因此,反应中使用0.6mM NADP+较为经济。
实施例6:以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的最适搅拌速度
选取转速为取300、400、500、600、700、800和900rpm。反应温度为40℃, pH为7.5,辅酶NADP+终浓度为0.6mM。其他操作及反应条件同实施例2。每次做三组平行实验,计算平均值和标准误差。如图8所示,当反应的转速为300rpm时, (S)-香茅醇的转化率为53.2%。当反应转速为600rpm时,转化率提高到72.1%,当转速高于600rpm时,转化率迅速下降,当转速到900rpm时,转化率降至38.82%。综上,反应的最佳转速为600rpm。
实施例7:以橙花醇为底物基于辅酶自循环合成(S)-香茅醇的醇脱氢酶YsADH和老黄酶NemR-PS最适质量比
选取醇脱氢酶YsADH与老黄酶NemR-PS的湿菌体质量比为4:1、3:1、2:1、1:1、 1:2和1:3和1:4。反应温度为40℃,pH为7.5,辅酶NADP+终浓度为0.6mM,反应转速为600rpm。其他操作及反应条件同实施例2。每次做三组平行实验,计算平均值和标准误差,结果如图9所示。如图9所示,醇脱氢酶YsADH与老黄酶NemR-PS 的比例为4:1时,(S)-香茅醇的转化率为80.64%。当醇脱氢酶YsADH与老黄酶 NemR-PS的配比时4:1减少至3:1、2:1时,产物的转化率也由80.64%升高至86.22%,随后醇脱氢酶YsADH与老黄酶NemR-PS的配比调整为1:1、1:2、1:3和1:4时,产物的转化率由75.35%降至45.39%。因此,催化体系的最适醇脱氢酶YsADH与老黄酶NemR-PS的湿菌体质量比为2:1,即在10mL的体系中,醇脱氢酶YsADH与老黄酶NemR-PS按0.8g:0.4g的比例混合加入。
实施例8:基于辅酶自循环合成(S)-香茅醇时不同底物浓度下的反应进程及反应产物的鉴定
反应进程:同实施例2,底物终浓度分别为20mM、40mM、60mM、80mM和 100mM,湿菌体终浓度120g/L,醇脱氢酶YsADH与老黄酶NemR-PS的湿菌体质量比为2:1(0.8g:0.4g),辅酶NADP+浓度为0.6mM,pH 7.5的50mM Tris-HCl缓冲液。反应温度为40℃,转速为600rpm,反应体系为10mL。每隔一个小时取一个样,反应时长为12h。如图10所示,2h内20mM橙花醇转化完全;4h内40mM 橙花醇转化完全;6h内60mM橙花醇转化完全;8h内80mM橙花醇转化完全;而100mM橙花醇经12h反应后,(S)-香茅醇的转化率为99.74%,反应液中检测不到中间产物橙花醛和(S)-香茅醛。经气相色谱-质谱联用分析,反应液中的底物和产物分别确认为橙花醇和香茅醇(图11中a和b)。气相色谱-质谱联用仪型号为Agilent 7890A/5975C,气相色谱条件如实施例2(不需要检测器FID,其相关条件除外),质谱检测条件如下:辅助加热器温度,250℃;MS四极杆温度,150℃;离子源温度, 230℃;质谱扫描范围,30-500amu;发射电流,200μA;电子能量,70eV。
产物鉴定:底物终浓度为100mM,湿菌体终浓度120g/L,醇脱氢酶YsADH与老黄酶NemR-PS的湿菌体质量比为2:1(0.8g:0.4g),辅酶NADP+浓度为0.6mM, pH 7.5的50mMTris-HCl缓冲液,反应体系为10mL,转速为600rpm,反应温度为 40℃,反应时长为12h。反应结束后,反应液在12000rpm下离心2min,取上清,加入4倍反应液体积的乙酸乙酯,在200rpm和30℃条件下萃取1h,萃取结束后,在12000rpm下离心1min,取上层有机相。由于反应转化率>99%,几乎没有底物和中间产物残留,上层有机相经减压蒸馏去除乙酸乙酯,直接获得产物。产物经手性气相色谱分析,为光学纯的(S)-香茅醇。产物经进一步的1H和13C核磁共振分析,所得的结构信息也与香茅醇相符(图12中a和b)。产物1H NMR(600MHz,溶剂氘代氯仿)的化学位移信息:δ5.08(dddd,J=8.6,5.8,2.7,1.3Hz,1H),3.71–3.60(m,2H), 1.99–1.90(m,2H),1.66(d,J=1.6Hz,3H),1.62–1.58(m,3H),1.58–1.52(m,2H), 1.40–1.29(m,2H),1.16(dddd,J=13.4,9.5,7.7,5.8Hz,1H),0.89(d,J=6.6Hz,3H)。产物13C NMR(151MHz,溶剂氘代氯仿)的化学位移信息:δ131.23,124.70,61.11,39.87, 37.21,29.17,25.70,25.45,19.51,17.62。
序列表
<110> 浙江工业大学
<120> 一种双酶偶联合成(R)-香茅醇的方法
<160> 4
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 约克氏菌(Yokenella sp. )
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Asp Asn Glu Trp Gly Phe Ser Gln Tyr Pro Leu Val Ala Gly His Glu
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Arg Ala Asp Trp Gln Trp Val Ile Pro Leu Pro Glu Ser Ile Asp Ile
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Ile Gly Gly Leu Gly His Ile Ala Ile Lys Leu Leu His Ala Met Gly
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<213> 约克氏菌(Yokenella sp. )
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gccgggcatg aagtgattgg ccgcgtggcg gcgctcggca gtgcggcgca ggaaaaaggg 240
gtgaaagttg gtcagcgcgt gggcgtaggc tggacggcgc gcagctgtgg gcattgcgat 300
gcatgtatca gcggtaatca gattaactgc ctggaaggcg ccgtagccac cattctcaac 360
cgtggcggtt ttgccgagaa actgcgggca gactggcagt gggtgatccc gcttccggag 420
agcatcgata ttgagtcggc aggtcctctg ttatgcggcg gtattacggt ttttaaacct 480
ctgctgatgc accacatcac cgcgaccagt cgcgtggggg tgatcggcat cggcggtctt 540
gggcacattg ccattaaact gttgcacgca atgggctgtg aagtgaccgc attcagctcg 600
aatccgtcga aagaacagga agtgctggca atgggggcgg ataaagtcgt gaacagtcgc 660
gatccagacg cgttaaatgc gctggcaggc cagtttgatc tcattatcaa caccgttaat 720
gtcgacctcg actggcagcc ctactttgaa gcgctggcct atggcggcca tttccacacc 780
gtcggcgcag tgatgaagcc gctgccggtt ccggcgttta cattgattgc tggcgatcgc 840
agcatctccg gctcagcaac cggtacgccc tatgagctgc gcaaattgat gaagtttgcc 900
gggcgcagca aggtctcgcc gacgacagag ctgttcccaa tgtcgcaaat caacgaagcc 960
atccagcacg ttcgcgacgg caaagcgcgt taccgcgtgg tactgcaagc cgacttttga 1020
<210> 3
<211> 365
<212> PRT
<213> 斯氏普罗威登斯菌 (Providencia stuartii)
<400> 3
Met Ser Gln Lys Lys Leu Phe Thr Pro Leu Lys Val Gly Thr Leu Thr
1 5 10 15
Ala Pro Asn Arg Ile Phe Met Ala Pro Leu Thr Arg Leu Arg Ser Ile
20 25 30
Glu Pro Gly Asp Ile Pro Thr Pro Leu Met Gly Glu Tyr Tyr Arg Gln
35 40 45
Arg Ala Thr Ala Gly Leu Ile Ile Ser Glu Ala Thr Gln Ile Ser Ala
50 55 60
Gln Ser Lys Gly Tyr Ala Gly Ala Pro Gly Leu His Ser Ala Glu Gln
65 70 75 80
Ile Ala Ala Trp Lys Lys Ile Thr Ser Thr Val His Glu Ala Gly Gly
85 90 95
Arg Ile Ala Val Gln Leu Trp His Thr Gly Arg Ile Ser His Val Ser
100 105 110
Leu Gln Pro Asn Gly Leu Ala Pro Val Ala Pro Ser Ala Ile Ser Ala
115 120 125
Gly Thr Arg Thr Ser Leu Arg Asp Glu Asn Gly Arg Ala Ile Arg Val
130 135 140
Asp Thr Ser Met Pro Arg Ala Leu Glu Thr Glu Glu Ile Pro Ala Ile
145 150 155 160
Val Asn Asp Phe Arg Gln Ala Val Ala Asn Ala Arg Glu Ala Gly Phe
165 170 175
Asp Met Ala Glu Leu His Ala Ala His Gly Tyr Leu Leu His Gln Phe
180 185 190
Leu Ser Pro Ser Ala Asn His Arg Thr Asp Gln Tyr Gly Gly Thr Arg
195 200 205
Glu Asn Arg Ala Arg Phe Leu Leu Asp Val Val Asp Ala Val Cys Ala
210 215 220
Glu Trp Gly Ser Glu His Ile Gly Ile Arg Ile Ser Pro Ile Gly Thr
225 230 235 240
Phe Gln Asn Thr Asp Asn Gly Pro Asn Glu Val Asp Asp Ala Leu Tyr
245 250 255
Leu Ile Glu Glu Leu Asp Lys Arg His Ile Ala Tyr Leu His Leu Ser
260 265 270
Glu Pro Asp Trp Ala Gly Gly Gln Pro Tyr Thr Asp Asp Phe Arg Gln
275 280 285
Lys Val Arg Glu Arg Phe His Gly Val Ile Ile Gly Ala Gly Ala Tyr
290 295 300
Thr Thr Glu Lys Ala Glu Asn Leu Ile Glu Lys Gly Leu Ile Asp Ala
305 310 315 320
Val Ala Phe Gly Arg Asp Phe Ile Ala Asn Pro Asp Leu Val Val Arg
325 330 335
Leu Lys Asn Lys Ala Ala Leu Asn Pro Gln Arg Pro Glu Ser Phe Tyr
340 345 350
Gly Gly Gly Ala Glu Gly Tyr Thr Asp Tyr Pro Ser Leu
355 360 365
<210> 4
<211> 1095
<212> DNA
<213> 斯氏普罗威登斯菌 (Providencia stuartii)
<400> 4
atgagccaga aaaagctgtt tacgccgctg aaagttggta ccctgaccgc accgaatcgt 60
atttttatgg cgccgctgac ccgcctgcgt agcattgaac cgggcgatat tccgacgccg 120
ctgatgggcg aatattatcg ccagcgtgcc accgcaggcc tgattattag cgaagcaacc 180
cagattagtg cacagtcaaa aggttatgca ggtgcaccag gtctgcatag cgcagaacag 240
attgcagcat ggaaaaagat tacctcaacc gttcatgaag caggtggtcg tattgcagta 300
cagctgtggc atacgggtcg tattagtcat gttagcctgc agccgaacgg tctggcaccg 360
gttgcaccgt cagcaatttc agccggcaca cgtaccagtc tgcgtgatga aaatggtcgt 420
gccattcgtg tagataccag catgccgcgt gcactggaaa ccgaagaaat tccggcaatt 480
gttaatgatt ttcgccaggc agttgcaaat gcccgtgaag ctggttttga tatggctgaa 540
ctgcatgcag cacatggtta tctgctgcat cagtttctga gcccgtcagc aaatcatcgt 600
accgatcagt atggcggtac ccgtgaaaat cgtgcacgtt ttctgctgga tgttgttgat 660
gcagtttgtg cagaatgggg tagcgaacat attggtattc gtattagtcc gattggtacc 720
tttcagaata cagataatgg tcctaatgaa gttgatgatg ctctgtatct gattgaagaa 780
ctggataaac gtcatattgc atatctgcat ctgagcgaac cggattgggc aggtggtcag 840
ccatataccg atgattttcg tcagaaagtt cgtgaacgtt ttcatggtgt tattattggt 900
gcaggcgcat atacgacaga aaaagcagaa aatctgattg aaaaaggtct gattgatgca 960
gttgcctttg gtcgtgattt tattgcgaat ccggatctgg ttgtgcgtct gaaaaataaa 1020
gcagcactga acccacagcg tccggaaagc ttttatggtg gtggtgccga aggttatacc 1080
gattatccga gcctg 1095
Claims (10)
1.一种双酶偶联合成(S)-香茅醇的方法,其特征在于所述方法为:以含醇脱氢酶YsADH基因的工程菌和含老黄酶NemR-PS基因的工程菌各自经发酵培养获得的湿菌体混合后作为催化剂,以橙花醇为底物,以NADP+为辅酶,以pH 6-9缓冲液为反应介质构成反应体系,在25-55℃、0-900rpm条件下反应完全后,反应液分离纯化,最终获得(S)-香茅醇。
2.如权利要求1所述的方法,其特征在于所述醇脱氢酶YsADH基因核苷酸序列为SEQ IDNO.2所示;老黄酶NemR-PS基因核苷酸序列为SEQ ID NO.4所示。
3.如权利要求1所述的方法,其特征在于所述含醇脱氢酶YsADH基因的工程菌按如下方法构建:将醇脱氢酶YsADH编码基因插入pET28b载体的Nco I和Hind III限制性酶切位点,得到重组载体pET28b-YsADH;将重组载体pET28b-YsADH导入宿主细胞E.coli BL21(DE3),得到基因工程菌E.coli BL21(DE3)/pET28b-YsADH。
4.如权利要求1所述的方法,其特征在于所述含老黄酶NemR-PS基因的工程菌按如下方法构建:将老黄酶NemR-PS编码基因插入pET28b载体的Nco I和Xho I限制性酶切位点,得到重组载体pET28b-NemR-PS;将重组载体pET28b-NemR-PS导入宿主细胞E.coli BL21(DE3),得到基因工程菌E.coli BL21(DE3)/pET28b-NemR-PS。
5.如权利要求1所述的方法,其特征在于所述含醇脱氢酶YsADH基因工程菌的湿菌体与含老黄酶NemR-PS基因工程菌的湿菌体以质量比0.25-4:1混合,所述催化剂加入量以湿菌体总量计为120g/L。
6.如权利要求1所述的方法,其特征在于所述底物以1M橙花醇乙醇溶液形式加入,所述橙花醇加入终浓度为10-100mM;所述辅酶加入终浓度0-1mM。
7.如权利要求1所述的方法,其特征在于所述缓冲液为pH 7.5的50mM Tris-HCl缓冲液。
8.如权利要求1所述的方法,其特征在于所述含醇脱氢酶YsADH基因工程菌的湿菌体按如下方法制备:将含醇脱氢酶YsADH基因的工程菌接种于含有终浓度100μg/mL卡那霉素的LB液体培养基中,在37℃和200rpm下培养过夜,然后以体积浓度2%的接种量转接于含有100μg/mL卡那霉素的LB液体培养基中,在37℃和200rpm培养至菌体浓度OD600至0.6~0.8,向培养物中加入终浓度0.2mM的IPTG,在21℃下诱导培养12h,获得诱导培养液;再将诱导培养液于4℃和8000rpm下离心10min,弃去上清液;然后用pH 8.0、50mM Tris-HCl缓冲液重悬菌体,于4℃和8000rpm下离心10min,弃去上清液,收集湿菌体。
9.如权利要求1所述的方法,其特征在于所述含老黄酶NemR-PS基因工程菌的湿菌体按如下方法制备:将含老黄酶NemR-PS基因的工程菌接种于含有终浓度100μg/mL卡那霉素的LB液体培养基中,在37℃和200rpm下培养过夜,以体积浓度2%的接种量转接于终浓度100μg/mL卡那霉素的LB液体培养基中,在37℃和200rpm培养至菌体浓度OD600至0.6~0.8,向培养物中加入终浓度0.2mM的IPTG,在24℃下诱导培养12h,获得诱导培养液;再将诱导培养液于4℃和8000rpm下离心10min,弃去上清液;然后用pH 8.0、50mM Tris-HCl缓冲液重悬菌体,于4℃和8000rpm下离心10min,弃去上清液,收集湿菌体。
10.如权利要求1所述的方法,其特征在于所述反应液分离纯化方法为:反应液在12000rpm下离心2min,取上清,加入4倍反应液体积的乙酸乙酯,在200rpm和30℃条件下萃取1h,萃取结束后,在12000rpm下离心1min,取上层有机相,经减压蒸馏去除乙酸乙酯,最终获得(S)-香茅醇。
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