CN116042732A - 一种黄素单加氧酶制备酚类化合物的方法 - Google Patents
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Abstract
本发明公开了一种黄素单加氧酶制备酚类化合物方法,是采用4‑羟基苯乙酸‑3‑单加氧酶与NAD(P)H‑黄素氧化还原酶共表达作为反应催化剂,以单酚类化合物、NADH和FAD为底物,O2为氧化剂,反应得到酚类化合物。本发明创新性地利用4‑羟基苯乙酸‑3‑单加氧酶与NAD(P)H‑黄素氧化还原酶共表达双酶级联体系,提高了4‑羟基苯乙酸‑3‑单加氧酶的活力,高效绿色的合成酚类化合物,具有反应条件温和、工艺步骤简单、成本低且合成效率高等优点﹐为酚类化合物的绿色生产提供了一种新方法。
Description
技术领域
本发明属于酶的基因工程技术领域,具体涉及一种酚类化合物的合成制备方法。
背景技术
橄榄和橄榄油中的橄榄多酚对人体有许多健康益处,羟基酪醇是橄榄多酚最主要的成分,主要以酯类的形式存在油橄榄的果实和树叶中。羟基酪醇的健康功效非常广泛,被认为是最强大的抗氧化剂之一,同时具有多种生物和药理活性。羟基酪醇不仅可以预防心血管疾病、调节代谢综合征、抗炎、抗肿瘤、预防和调节呼吸系统疾病,表现出明显的抗菌效果,同时还具有保护神经和皮肤的作用,并且对骨骼的形成和维持产生重要影响。羟基酪醇无明显毒性反应,其在医药、食品和化妆品行业的应用前景近年受到广泛关注。
目前,羟基酪醇的制备方法有直接提取法、化学合成法以及生物酶法制备。由于橄榄和橄榄油的商业需求,大多数天然羟基酪醇在橄榄废弃物(如橄榄废水、半固体副产品或橄榄叶等)中通过有机溶剂、酸或酶直接提取得到。然而,从橄榄废弃物中提取羟基酪醇耗时长且效率低,化学合成法反应过程复杂且试剂难以完全分离,环境污染较大,因此酶法制备羟基酪醇成为人们研究的热点,具有广阔的应用前景。
酶法制备羟基酪醇以酪醇为底物,通过单加氧酶将酪醇羟基化得到酚类化合物羟基酪醇,该方法具有较高的催化效率并且对环境更友好。黄素单加氧酶(Flavoproteinmonooxygenases,FPMOs)属于氧化还原酶类,能够在温和条件下活化分子氧,从而将底物氧化,是一类被广泛应用的单加氧酶。4-羟基苯乙酸-3-单加氧酶B(4-hydroxyphenylacetate3-monooxygenaseB;HpaB)是一种来源于大肠杆菌的黄素单加氧酶,具有广泛的底物特异性,可以催化黄素腺嘌呤二核苷酸递氢体(FADH2)、氧分子与底物酚类化合物发生反应,可用于将酪醇羟基化得到羟基酪醇。然而体外生物系统的构建经常出现依赖于辅助因子的反应中需要昂贵的辅助因子的问题,限制了酶的实际应用,因此开发FADH2高效生成系统成为催化反应过程优化的重要一部分。
NAD(P)H-黄素氧化还原酶(NAD(P)H-flavinoxidoreductase;HpaC)以NADH作为电子供体催化游离黄素的还原,进行黄素间氢离子的传递,该酶不含任何辅基,在氧化还原前同时容纳还原的吡啶核苷酸和黄素。以还原型辅酶Ⅰ(NADH)和黄素腺嘌呤二核苷酸(FAD)为底物,生成NAD+和FADH2,上述HpaB则利用FADH2催化酚类化合物的羟基化。酶体外级联反应已成为一种环境友好和经济的有价值化学品生产的替代方法,将HpaB和HpaC进行级联共表达进行以黄素、单酚为底物合成双酚的反应,可以防止没有底物时FADH2的浪费氧化,节约成本的同时可以绿色无污染的高效进行羟基酪醇的酶法合成,在工业应用中具有重要意义。
因此,我们建立了4-羟基苯乙酸-3-单加氧酶HpaB和NAD(P)H-黄素氧化还原酶HpaC双酶级联体系制备羟基酪醇的合成路径。
发明内容
提供了一种黄素单加氧酶制备酚类化合物的方法。
发明目的:本发明所要解决的技术问题是针对现有条件确定酚类化合物的合成路径,将HpaB进行表达,提供了一种黄素单加氧酶制备酚类化合物方法。本发明另一个目的是提供一种双酶级联体系,将HpaC进行表达,实现了黄素分子间氢的转移,高效获得FADH2,节约了成本。将HpaB与HpaC共表达,提高了HpaB的催化活性,实现了羟基酪醇的高效合成。
技术方案:为了实现以上目的,本发明采取的技术方案为:一种酚类化合物,它是由黄素单加氧酶与黄素氧化还原酶共表达制备得到,包括以下步骤:
(1)HpaB的表达
以大肠杆菌编码基因hpaB为模板,克隆至表达载体,再经宿主进行表达。
(2)HpaB的活力测定
以酪醇为底物,通过高效液相色谱法测定羟基酪醇转化率来评价HpaB酶活力。50μL反应包含20mM磷酸缓冲液(pH7.0)、200μM酪醇、10μMFADH2以及HpaB;反应在30℃下孵育5min。
(3)HpaB与HpaC共表达
所述共表达是将HpaB编码基因和HpaC基因通过linker连接后克隆至表达载体,再经宿主进行表达。
(4)酚类化合物的酶法合成
以酪醇、NADH、FAD为底物,O2为氧化剂,上述表达蛋白为催化剂进行酚类化合物的酶法制备。
经上述操作后即制得酚类化合物。
作为优选方案,所述共表达是将4-羟基苯乙酸-3-单加氧酶编码基因和NAD(P)H-黄素氧化还原酶编码基因经linker连接后克隆至表达载体,再经宿主进行表达;
作为优选方案,所述4-羟基苯乙酸-3-单加氧酶HpaB,来源于大肠杆菌BL21(EscherichiacoliBL21),GenBank编号为:CAD6019151.1;
作为优选方案,所述NAD(P)H-黄素氧化还原酶HpaC,来源于大肠杆菌BL21(EscherichiacoliBL21),GenBank编号为:CAD6019161.1;
作为优选方案,所述酶来源包括但不限于大肠杆菌;
作为优选方案,所述表达载体为pET-28a质粒;
作为优选方案,所述linker包括但不限于:GSGGTG、SSRGRTSG、GGSGGSGGSGGSGGS、GGGGSGGGGS;
作为优选方案,所述linker为GGGGSGGGGS;
作为优选方案,所述宿主包括但不限于:大肠杆菌BL21、枯草芽孢杆菌;
本发明提供的技术方案之一,是由上述方法制备的黄素单加氧酶和黄素氧化还原酶共表达产物-连接蛋白HpaB-HpaC;所述连接蛋白HpaB-HpaC较单独表达HpaB酶活力提高了7%左右;
本发明提供的技术方案之二,是上述连接蛋白HpaB-HpaC的应用,特别是在制备酚类化合物中的应用,更特别的是在制备羟基酪醇中的应用,所述应用为以连接蛋白HpaB-HpaC催化底物酪醇、NADH和FAD合成羟基酪醇;
进一步地,反应条件为30℃水浴6h。
有益效果:
本发明将黄素单加氧酶和黄素氧化还原酶使用linker连接进行共表达后获得的连接蛋白HpaB-HpaC,相比于原始HpaB单体,连接蛋白的HpaB活力提高了7%左右,使用该连接蛋白以酪醇为底物,使用NADH和FAD实现了黄素分子间氢的转移,高效的获得了FADH2,以FADH2为氢供体,实现了羟基酪醇的酶法合成。
附图说明
图1为本发明合成路径示意图。
图2为本发明hpaB、hpaB-hpaC基因电泳图,其中(1)为hpaB基因,(2)为hpaB-hpaC基因。
具体实施方式
下面结合实施例对本发明的技术内容做进一步说明,但本发明不只限于这些实施例,不能以下述实施例来限定本发明的保护范围。
本发明实施例所用培养基如下:
LB培养基:酵母提取物5.0g/L,胰蛋白胨10.0g/L,NaCl 10.0g/L,其余为水。
上述培养基的固态培养基添加2%琼脂。
本发明及实施例所用溶液如下:
20mM磷酸缓冲液:38mL 0.2M NaH2PO4,62mL 0.2M Na2HPO4,pH7.0;
Wash Buffer(mM):咪唑100,Tris-HCl 20,二硫苏糖醇1,NaCl 200;
Elution Buffer(mM):咪唑500,Tris-HCl 20,二硫苏糖醇1,NaCl 200;
Lysis Buffer(mM):咪唑20,Tris-HCl 20,二硫苏糖醇1,NaCl 200。
以下将通过具体实施例对本发明作进一步解释说明。
实施例1:4-羟基苯乙酸-3-单加氧酶编码基因hapB和NAD(P)H-黄素氧化还原酶编码基因hpaC的获得
1.4-羟基苯乙酸-3-单加氧酶编码基因hapB和NAD(P)H-黄素氧化还原酶编码基因hpaC来自实验室保存的大肠杆菌BL21(Escherichia coli BL21)菌株,利用美国OMEGA公司的Bacterial DNA Kit D3350-02按照说明书提取基因组。
(1)菌株活化:用接种环从甘油管中蘸取大肠杆菌菌液,接种于LB固体培养基平板,三区划线,37℃恒温培养12h;
(2)转接:从培养菌体的平板上挑取边缘整齐、表面光滑的单菌落接种于5mL液体LB培养基中,220r/min、37℃条件下培养12h;
(3)收集菌体:取适量培养菌液分装于1.5mL EP管,12000r/min离心2min,弃上清;
(4)加入250μL ddH2O重悬菌体,并加入50μL的50mg/mL溶菌酶,37℃水浴10min;
(5)加入100μL BTL Buffer和20μL蛋白酶K,旋涡振荡;
(6)55℃水浴40-50min,每隔20-30min,振荡混匀;
(7)加入5μL RNA酶,颠倒混匀数次,室温放置5min;
(8)12000rpm离心2min,去掉未消化部分,将上清部分转移至新的1.5mL EP管;
(9)加入220μL BDL Buffer,振荡混匀,65℃水浴10min;
(10)加入220μL无水乙醇,吹吸混匀;
(11)转移至吸附柱,静置1min,12000rpm离心1min,弃滤液;
(12)加入500μL HBC Buffer,12000rpm,离心1min,弃滤液;
(13)加入700μL DNA Wash Buffer,12000rpm,离心1min,弃滤液;
(14)加入500μL DNA Wash Buffer,12000rpm,离心1min,弃滤液;
(15)12000rpm,离心2min,55℃金属浴10min,晾干;
(16)加入40μL ddH2O洗脱并于-20℃保存基因组。
2.4-羟基苯乙酸-3-单加氧酶编码基因hapB扩增
4-羟基苯乙酸-3-单加氧酶编码基因hapB的引物(上游引物F1、下游引物R1)序列如下:F1:CATGCCATGGGCATGAAACCGGAAGATTTTC(划线部分为NcoI酶切位点)R1:CCGCTCGAGTTTCAGCAGTTTATCCAGC(划线部分为XhoI酶切位点)
PCR扩增的反应体系为50μL,其组成为:
Prime STAR Max | 25μL |
上游引物F1(20μmol/L) | 2μL |
下游引物R1(20μmol/L) | 2μL |
基因组 | 2μL |
<![CDATA[ddH<sub>2</sub>O]]> | 19μL |
总体积 | 50μL |
注:上述所需试剂来自宝生物工程有限公司Takara。
扩增程序的设置为:预变性:97℃30s;变性:97℃10s;退火:53℃45s;延伸:72℃20s;上述反应30个循环;延伸:72℃10min。
将PCR产物进行琼脂糖凝胶电泳,大肠杆菌编码基因hapB的条带约1500bp(见图2),再由DNA切胶回收试剂盒回收PCR产物,使用限制性内切酶NcoI和XhoI对pET-28a质粒进行双酶切,用小量DNA回收试剂盒回收得到线性化质粒pET-28a。使用连接酶将hapB的PCR产物与线性化质粒pET-28a在37℃连接1h。将连接产物转化至大肠杆菌BL21感受态细胞,涂布于含硫酸卡那霉素抗性的固体培养基平板上,置于37℃培养箱培养12h。将平板长出的转化子转接试管并提质粒,提出质粒送于金唯智测序,测序结果显示hapB已成功构建至pET-28a质粒上。
实施例2:共表达重组菌株的构建
以重组质粒pET-28a-hapB为模板,设计引物(上游引物F2、下游引物R2),引物序列为F2:TAAGAAGGAGATATACCATGGCATGAAACCGGAAGATTTTCR2:GCTACCACCACCACCGCTACCACCACC ACCCTGCCAATCGCATCC(划线部分为linker)
进行PCR扩增带有完整linker的4-羟基苯乙酸-3-单加氧酶编码基因hapB片段,扩增的反应体系为:
上游引物F2 | 2.0μL |
下游引物R2 | 2.0μL |
pET-28a-hapB | 2.0μL |
Primer Star Max酶 | 25μL |
<![CDATA[ddH<sub>2</sub>O]]> | 19μL |
扩增程序为:98℃预变性30s;98℃变性10s,53℃退火20s,72℃延伸20s,反应30个循环;72℃延伸10min。
以提取的pET-28a-hapC为模板,设计引物(上游引物F3、下游引物R3),引物序列为:F3:GGTGGTGGTGGTAGCGGTGGTGGTGGTAGCATGCAGCTGGATGAACAG(划线部分为linker)
R3:GTGGTGGTGGTGGTGCTCGAGAATCGCCGCT TCCAT
进行PCR扩增带有完整linker的NAD(P)H-黄素氧化还原酶编码基因hpaC片段,其扩增的反应体系为:
上游引物F3 | 2.0μL |
下游引物R3 | 2.0μL |
pET-28a-hapC | 2.0μL |
Primer Star Max酶 | 25μL |
<![CDATA[ddH<sub>2</sub>O]]> | 19μL |
扩增程序为:98℃预变性30s;98℃变性10s,53℃退火20s,72℃延伸8s,反应30个循环;72℃延伸10min。
使用限制性内切酶NcoI和XhoI对pET-28a质粒进行双酶切,用小量DNA回收试剂盒回收得到线性化质粒pET-28a。使用同源重组酶将两段PCR产物与线性化质粒pET-28a在50℃水浴连接15min。将连接产物转化至大肠杆菌BL21感受态细胞,涂布于含硫酸卡那霉素抗性的固体培养基平板上,置于37℃培养箱培养12h。将平板长出的转化子转接试管并提质粒,提出质粒送于金唯智测序,测序结果显示hapB-linker-hapC片段已成功构建至pET-28a质粒上,得到重组质粒pET-28a-hapB-hapC以及重组菌株BL21(pET-28a-hapB-hapC)。
实施例3:HpaB和HpaB-HpaC在大肠杆菌中的表达及制备
1.平板三区划线活化重组菌株;
2.挑取单菌落接种于接种于加入卡那霉素抗性的5mL LB液体试管中,37℃,220r/min振荡培养12h;
3.以2%的接种量转接于含有卡那霉素抗性的250mL LB液体培养基中,37℃,220r/min振荡培养2-3h使菌液OD600达到0.6-0.8;
4.加入IPTG诱导剂进行诱导(终浓度为0.5mmol/L),在110r/min,16℃的摇床中振荡培养16-20h;
5.将上述菌液用500mL离心杯于4℃,8000r/min离心10min获得菌体沉淀;
6.用40mL pH7.0的Lysis Buffer将收集的菌体震荡重悬,超声破碎并离心得到破碎上清;
7.将上述破碎上清经无菌滤膜后与Ni2+树脂混合,于0-4℃,以100r/min在磁力搅拌器上使蛋白与Ni2+树脂结合50-70min,将与Ni2+树脂结合后的蛋白溶液倒入到纯化层析柱中,Wash Buffer洗脱杂蛋白;
8.用孔径大小为10kDa的超滤管对含有目的蛋白Elution Buffer洗脱液在2500g离心力下超滤,当超滤管内洗脱液离心至1.5mL左右时,将收集管中滤出液倒掉,向超滤管中加入10mL的Tris-HCl(pH7.0、50mM),重复两次后,超滤管中的上清剩余1mL左右时,转移至新EP管中,制备得到纯的HpaB和HpaB-HpaC,4℃保存备用。
实施例4:HpaB酶活力的测定
HpaB活性通过高效液相色谱(HPLC)方法分析酪醇转化为羟基酪醇的能力表示。反应通常在20mM磷酸缓冲液(pH7.0)进行,底物包含10mM FADH2、1mM酪醇、1mL HpaB或HpaB-HpaC。反应条件为30℃,水浴6h。HPLC分析采用C18柱(150×4.6mm)与LC-10Avp系统(日本岛津)。流动相包括乙腈(溶剂A)和水(溶剂B)(两者都包括含1%甲酸),柱温30℃,流速为0.4mL/min。HPLC程序如下:5min10%-15%B(v/v),5-15min15%-40%B,20-22min40%-60%B,22-25min10%B。在280nm处测定羟基酪醇产量,HpaB酶活力定义为在pH为7.0,温度为30℃条件下,每分钟生成1mM羟基酪醇所需要的酶量为一个酶活力单位,以U表示。上述测定得到HpaB酶活力为27U/mL,HpaB-HpaC表现出来的HpaB活力为29U/mL,相比原始HpaB酶活力提高了7.4%。
实施例5:羟基酪醇的制备
实验在30℃下进行,总体积为500μL,20mMTris-HCl(pH7.5),包含20mM氯化钠,1mM酪醇,10μMHpaB-HpaC,2μMFAD和2μMNADH。轻轻地搅拌一下混合物。用5%三氯乙酸(w/v)分别在反应1h、2h、3h、4h、5h、6h、7h时停止反应,30,000g离心10min,样品与甲醇混合,7500g离心10min,用0.22μm尼龙过滤器过滤用HPLC分析羟基酪醇。
采用高效液相色谱(HPLC)检测羟基酪醇。测定条件具体为:使用Agilent126(高效液相色谱仪,色谱柱为C18柱(4.6mmID×250mm,5um),流动相为80%(v/v)的甲酸(0.1%,w/v)和20%(v/v)的纯甲醇,流速1mL/min,柱温30℃,进样量10.0uL,检测波长为280nm,洗脱25min。结果表明制备羟基酪醇在6h达到最高,转化率为88%。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对专利范围的限制。应当指出的是,对于本领域技术人员来说,在不脱离本专利构思的前提下,上述各实施方式还可以做出若干变形、组合和改进,而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
本发明中使用到如下序列:
1.HpaB氨基酸序列
MKPEDFRASTQRPLTGEEYLKSLQDGREIYIYGERVKDVTTHPAFRNAAASVAQLYDALHKPEMQDSLC
WNTDTGSGGYTHKFFRVAKSADDLRQQRDAIAEWSRLSYGWMGRTPDYKAAFGCALGANPGFYGQFE
QNARNWYTRIQETGLYFNHAIVNPPIDRHLPTDKVKDVYIKLEKETDAGIIVSGAKVVATNSALTHYNMV
GFGSAQVMGENPDFALMFVAPMDADGVKLISRASYEMVAGATGSPYDYPLSSRFDENDAILVMDNVLIP
WENVLIYRDFDRCRRWTMEGGFARMYPLQACVRLAVKLDFITALLKKSLECTGTLEFRGVQADLGEVVA
WRNTFWALSDSMCSEATPWVNGAYLPDHAALQTYRVLAPMAYAKIKNIIERNVTSGLIYLPSSARDLNNP
QIDQYLAKYVRGSNGMDHVQRIKILKLMWDAIGSEFGGRHELYEINYSGSQDEIRLQCLRQAQSSGNMD
KMMAMVDRCLSEYDQNGWTVPHLHNNDDINMLDKLLK
2.HpaB碱基序列
ATGAAACCGGAAGATTTTCGCGCGAGCACGCAGCGCCCGCTGACCGGCGAAGAATATCTGAAAAGCC
TGCAAGATGGCCGCGAAATTTATATTTATGGCGAACGCGTGAAAGATGTTACCACCCATCCGGCGTTTC
GCAACGCGGCCGCGAGCGTGGCGCAGCTGTATGATGCGCTGCATAAACCGGAAATGCAAGATAGCCT
GTGCTGGAACACCGATACCGGCAGCGGCGGCTATACCCATAAATTTTTTCGCGTGGCGAAAAGCGCG
GATGATCTGCGTCAGCAGCGCGATGCGATTGCGGAATGGAGCCGCCTGAGCTATGGCTGGATGGGCC
GCACCCCGGATTATAAAGCGGCGTTTGGCTGCGCGCTGGGCGCGAACCCGGGCTTTTATGGTCAGTTT
GAACAGAACGCGCGCAACTGGTATACCCGCATTCAAGAAACCGGCCTGTATTTTAACCATGCGATTGT
GAACCCGCCGATTGATCGCCATCTGCCGACCGATAAAGTGAAAGATGTGTATATTAAACTGGAAAAAG
AAACCGATGCGGGCATTATTGTGAGCGGCGCGAAAGTGGTGGCGACCAACAGCGCGCTGACCCATTA
TAACATGGTGGGCTTTGGCAGCGCGCAAGTGATGGGCGAAAACCCGGATTTTGCGCTGATGTTTGTTG
CCCCGATGGATGCGGATGGCGTGAAACTGATTAGCCGCGCGAGCTATGAAATGGTGGCGGGCGCGAC
CGGCAGCCCGTATGATTATCCGCTGAGCAGCCGCTTTGATGAAAACGATGCGATTCTGGTGATGGATA
ACGTGTTAATTCCGTGGGAAAACGTTCTGATTTATCGCGATTTTGATCGCTGCCGCCGCTGGACGATGG
AAGGCGGCTTTGCGCGCATGTATCCGCTGCAAGCGTGCGTGCGCCTGGCGGTGAAACTGGATTTTATT
ACCGCGCTGCTGAAAAAAAGCCTGGAATGCACCGGCACCCTGGAATTTCGCGGCGTGCAAGCGGATC
TGGGCGAAGTGGTGGCGTGGCGCAACACCTTTTGGGCGCTGAGCGATAGCATGTGCAGCGAAGCGA
CCCCGTGGGTGAACGGCGCGTATCTGCCGGATCATGCGGCGCTGCAGACCTATCGCGTGTTAGCGCCG
ATGGCCTATGCGAAAATTAAAAACATTATTGAACGCAACGTGACGAGCGGCCTGATTTATCTGCCGAG
CAGCGCGCGCGATCTGAACAACCCGCAGATTGATCAGTATCTGGCGAAATATGTGCGCGGCAGCAAC
GGCATGGATCATGTGCAGCGCATTAAAATTCTGAAACTGATGTGGGATGCGATTGGCAGCGAATTTGG
CGGCCGCCATGAACTGTATGAAATTAACTATAGCGGCAGCCAAGATGAAATTCGCCTGCAGTGCCTGC
GCCAAGCGCAGAGCAGCGGCAACATGGATAAAATGATGGCGATGGTGGATCGCTGCCTGAGCGAATA
TGATCAGAACGGCTGGACCGTGCCGCATCTGCATAACAACGATGATATTAACATGCTGGATAAACTGC
TGAAA
3.HpaC氨基酸序列
MQLDEQRLRFRDAMASLSAAVNIITTEGDTGQCGITATAVCSVTDTPPSLMVCINANSAMNPVFQGNGKL
CVNVLNHEQELMARHFAGMTGMAMEERFSLSCWQKGPLAQPVLKGSLASLEGEIRDVQAIGTHLVYLV
EIKNIILSAEGHGLIYFKRRFHPVMLEMEAAI
4.HpaC碱基序列
ATGCAGCTGGATGAACAGCGCCTGCGCTTTCGCGATGCGATGGCGAGCCTGAGCGCGGCGGTGAACA
TTATTACCACCGAAGGCGATACCGGTCAGTGCGGCATTACCGCGACCGCGGTGTGCAGCGTGACCGAT
ACCCCGCCGAGCCTGATGGTGTGCATTAACGCGAACAGCGCGATGAACCCGGTGTTTCAAGGCAACG
GCAAACTGTGCGTGAACGTGCTGAACCATGAACAAGAACTGATGGCGCGCCATTTTGCGGGCATGAC
CGGCATGGCGATGGAAGAACGCTTTAGCCTGAGCTGCTGGCAGAAAGGCCCGCTGGCGCAGCCGGT
GCTGAAAGGCAGCCTGGCGAGCCTGGAAGGCGAAATTCGCGATGTGCAAGCGATTGGCACCCATCTG
GTGTATCTGGTGGAAATTAAAAACATTATTCTGAGCGCGGAAGGTCATGGCCTGATTTATTTTAAACGC
CGCTTTCATCCGGTGATGCTGGAAATGGAAGCGGCGATT
5.HpaB-HpaC氨基酸序列
MKPEDFRASTQRPLTGEEYLKSLQDGREIYIYGERVKDVTTHPAFRNAAASVAQLYDALHKPEMQDSLC
WNTDTGSGGYTHKFFRVAKSADDLRQQRDAIAEWSRLSYGWMGRTPDYKAAFGCALGANPGFYGQFE
QNARNWYTRIQETGLYFNHAIVNPPIDRHLPTDKVKDVYIKLEKETDAGIIVSGAKVVATNSALTHYNMV
GFGSAQVMGENPDFALMFVAPMDADGVKLISRASYEMVAGATGSPYDYPLSSRFDENDAILVMDNVLIP
WENVLIYRDFDRCRRWTMEGGFARMYPLQACVRLAVKLDFITALLKKSLECTGTLEFRGVQADLGEVVA
WRNTFWALSDSMCSEATPWVNGAYLPDHAALQTYRVLAPMAYAKIKNIIERNVTSGLIYLPSSARDLNNP
QIDQYLAKYVRGSNGMDHVQRIKILKLMWDAIGSEFGGRHELYEINYSGSQDEIRLQCLRQAQSSGNMD
KMMAMVDRCLSEYDQNGWTVPHLHNNDDINMLDKLLKGGGGSGGGGSMQLDEQRLRFRDAMASLSA
AVNIITTEGDTGQCGITATAVCSVTDTPPSLMVCINANSAMNPVFQGNGKLCVNVLNHEQELMARHFAG
MTGMAMEERFSLSCWQKGPLAQPVLKGSLASLEGEIRDVQAIGTHLVYLVEIKNIILSAEGHGLIYFKRRF
HPVMLEMEAAI
6.HpaB-HpaC碱基序列
ATGAAACCGGAAGATTTTCGCGCGAGCACGCAGCGCCCGCTGACCGGCGAAGAATATCTGAAAAGCC
TGCAAGATGGCCGCGAAATTTATATTTATGGCGAACGCGTGAAAGATGTTACCACCCATCCGGCGTTTC
GCAACGCGGCCGCGAGCGTGGCGCAGCTGTATGATGCGCTGCATAAACCGGAAATGCAAGATAGCCT
GTGCTGGAACACCGATACCGGCAGCGGCGGCTATACCCATAAATTTTTTCGCGTGGCGAAAAGCGCG
GATGATCTGCGTCAGCAGCGCGATGCGATTGCGGAATGGAGCCGCCTGAGCTATGGCTGGATGGGCC
GCACCCCGGATTATAAAGCGGCGTTTGGCTGCGCGCTGGGCGCGAACCCGGGCTTTTATGGTCAGTTT
GAACAGAACGCGCGCAACTGGTATACCCGCATTCAAGAAACCGGCCTGTATTTTAACCATGCGATTGT
GAACCCGCCGATTGATCGCCATCTGCCGACCGATAAAGTGAAAGATGTGTATATTAAACTGGAAAAAG
AAACCGATGCGGGCATTATTGTGAGCGGCGCGAAAGTGGTGGCGACCAACAGCGCGCTGACCCATTA
TAACATGGTGGGCTTTGGCAGCGCGCAAGTGATGGGCGAAAACCCGGATTTTGCGCTGATGTTTGTTG
CCCCGATGGATGCGGATGGCGTGAAACTGATTAGCCGCGCGAGCTATGAAATGGTGGCGGGCGCGAC
CGGCAGCCCGTATGATTATCCGCTGAGCAGCCGCTTTGATGAAAACGATGCGATTCTGGTGATGGATA
ACGTGTTAATTCCGTGGGAAAACGTTCTGATTTATCGCGATTTTGATCGCTGCCGCCGCTGGACGATGG
AAGGCGGCTTTGCGCGCATGTATCCGCTGCAAGCGTGCGTGCGCCTGGCGGTGAAACTGGATTTTATT
ACCGCGCTGCTGAAAAAAAGCCTGGAATGCACCGGCACCCTGGAATTTCGCGGCGTGCAAGCGGATC
TGGGCGAAGTGGTGGCGTGGCGCAACACCTTTTGGGCGCTGAGCGATAGCATGTGCAGCGAAGCGA
CCCCGTGGGTGAACGGCGCGTATCTGCCGGATCATGCGGCGCTGCAGACCTATCGCGTGTTAGCGCCG
ATGGCCTATGCGAAAATTAAAAACATTATTGAACGCAACGTGACGAGCGGCCTGATTTATCTGCCGAG
CAGCGCGCGCGATCTGAACAACCCGCAGATTGATCAGTATCTGGCGAAATATGTGCGCGGCAGCAAC
GGCATGGATCATGTGCAGCGCATTAAAATTCTGAAACTGATGTGGGATGCGATTGGCAGCGAATTTGG
CGGCCGCCATGAACTGTATGAAATTAACTATAGCGGCAGCCAAGATGAAATTCGCCTGCAGTGCCTGC
GCCAAGCGCAGAGCAGCGGCAACATGGATAAAATGATGGCGATGGTGGATCGCTGCCTGAGCGAATA
TGATCAGAACGGCTGGACCGTGCCGCATCTGCATAACAACGATGATATTAACATGCTGGATAAACTGC
TGAAAGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCATGCAGCTGGATGAACAGCGCCTGCGCTTTCG
CGATGCGATGGCGAGCCTGAGCGCGGCGGTGAACATTATTACCACCGAAGGCGATACCGGTCAGTGC
GGCATTACCGCGACCGCGGTGTGCAGCGTGACCGATACCCCGCCGAGCCTGATGGTGTGCATTAACGC
GAACAGCGCGATGAACCCGGTGTTTCAAGGCAACGGCAAACTGTGCGTGAACGTGCTGAACCATGA
ACAAGAACTGATGGCGCGCCATTTTGCGGGCATGACCGGCATGGCGATGGAAGAACGCTTTAGCCTG
AGCTGCTGGCAGAAAGGCCCGCTGGCGCAGCCGGTGCTGAAAGGCAGCCTGGCGAGCCTGGAAGGC
GAAATTCGCGATGTGCAAGCGATTGGCACCCATCTGGTGTATCTGGTGGAAATTAAAAACATTATTCTG
AGCGCGGAAGGTCATGGCCTGATTTATTTTAAACGCCGCTTTCATCCGGTGATGCTGGAAATGGAAGC
GGCGATT
Claims (10)
1.一种黄素单加氧酶制备酚类化合物的方法,其特征在于,所述方法是4-羟基苯乙酸-3-单加氧酶和NAD(P)H-黄素氧化还原酶共表达催化制备酚类化合物。
2.如权利要求1所述的一种黄素单加氧酶制备酚类化合物的方法,所述方法是4-羟基苯乙酸-3-单加氧酶和NAD(P)H-黄素氧化还原酶共表达获得的。
3.如权利要求2所述的一种黄素单加氧酶制备酚类化合物的方法,其特征在于,所述共表达是将4-羟基苯乙酸-3-单加氧酶编码基因和NAD(P)H-黄素氧化还原酶编码基因经linker连接后克隆至表达载体,在经宿主进行表达。
4.如权利要求3所述的一种黄素单加氧酶制备酚类化合物的方法,其特征在于,所述4-羟基苯乙酸-3-单加氧酶,GenBank编号为:CAD6019151.1,但不限于此来源的4-羟基苯乙酸-3-单加氧酶基因。
5.如权利要求3所述的一种黄素单加氧酶制备酚类化合物的方法,其特征在于,所述NAD(P)H-黄素氧化还原酶,GenBank编号为:CAD6019161.1,但不限于此来源的NAD(P)H-黄素氧化还原酶基因。
6.如权利要求3所述的一种黄素单加氧酶制备酚类化合物的方法,其特征在于,所述表达载体为pET-28a质粒,包括但不限于pET-28a质粒、pET-22b质粒;所述宿主包括但不限于:大肠杆菌BL21、枯草芽孢杆菌。
7.如权利要求3所述的一种黄素单加氧酶制备酚类化合物的方法,其特征在于,所述linker包括但不限于:GSGGTG、SSRGRTSG、GGSGGSGGSGGSGGS、GGGGSGGGGS。
8.如权利要求3所述的一种黄素单加氧酶制备酚类化合物的方法,其特征在于,将4-羟基苯乙酸-3-单加氧酶的编码基因hpaB与NAD(P)H-黄素氧化还原酶的编码基因hpaC经linker(GGGGSGGGGS)连接后,克隆至pET-28a表达载体,再在大肠杆菌BL21中表达实现。
9.由权利要求1-8任意一项所述方法制备的4-羟基苯乙酸-3-单加氧酶和NAD(P)H-黄素氧化还原酶共表达产物-连接蛋白HpaB-HpaC。
10.由权利要求9所述HpaB、HpaC和连接蛋白HpaB-HpaC在制备酚类化合物中的应用。
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US20100047887A1 (en) * | 2006-11-27 | 2010-02-25 | Jihane Achkar | Method for preparing hydroxytyrosol |
CN112126610A (zh) * | 2019-06-25 | 2020-12-25 | 枫杨生物研发(南京)有限公司 | 一种用于生产羟基酪醇的工程菌 |
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