CN111849935A - 川桑咖啡酸氧甲基转移酶comt3及其应用 - Google Patents
川桑咖啡酸氧甲基转移酶comt3及其应用 Download PDFInfo
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- CN111849935A CN111849935A CN201910342241.7A CN201910342241A CN111849935A CN 111849935 A CN111849935 A CN 111849935A CN 201910342241 A CN201910342241 A CN 201910342241A CN 111849935 A CN111849935 A CN 111849935A
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- Prior art keywords
- comt3
- acid oxygen
- caffeic acid
- oxygen methyltransferase
- hydroxytryptamine
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Abstract
本发明公开了川桑咖啡酸氧甲基转移酶COMT3及其应用,咖啡酸氧甲基转移酶COMT由全长为1125bp的COMT3基因编码374个氨基酸,PI=4.98,将川桑咖啡酸氧甲基转移酶基因COMT3克隆后构建原核表达系统,重组表达后进行酶活测定,测得具有较高的咖啡酸氧甲基转移酶活性,因此川桑咖啡酸氧甲基转移酶基因COMT3可以作为工程菌的目的基因,川桑N‑乙酰‑5羟色胺转移酶SNAT5也可以作为体外催化5‑羟色胺转化为5‑甲氧基‑色胺或N‑乙酰‑5‑羟色胺转化为褪黑素,具有很好的应用前景。
Description
技术领域
本发明涉及生物技术领域,具体涉及川桑咖啡酸氧甲基转移酶COMT3,还涉及川桑咖啡酸氧甲基转移酶COMT3的应用。
背景技术
褪黑素,学名N-乙酰基-5-甲氧基-色胺,是一种吲哚胺,色氨酸的代谢产物。目前已知它广泛存在于人体、动物体及植物体内,生理作用非常广泛。由于其首先在人体的松果体内被发现,因此又被称为松果体素。随后的研究发现,其广泛存在于人体内的各个部位,含量极少,只有pg(1×10-12g)/mL水平。目前已知的褪黑素的生理功能主要有调节生物体的昼夜节律,缓解睡眠障碍;抗氧化,褪黑素本身及其代谢产物不仅有强大的自由基清除能力,而且能够诱导生物体相关抗氧化酶活性增强,增强免疫作用,抗肿瘤,抗衰老,特别是对阿尔茨海默病有明显效果等优点。
长期以来褪黑素被认为是动物体内专有的,自从1995年在植物中检测到褪黑素之后,人们陆续在许多高等植物中也发现褪黑素存在。在动物中,褪黑素的生物合成途径已经非常清楚。在植物中褪黑素的合成途径包括3条:(1)色氨酸脱羧酶将色氨酸转化成色胺;色氨酸羟化酶将色胺转化为5-羟色胺;N-乙酰-5羟色胺转移酶将5-羟色胺转化为N-乙酰-5-羟色胺;N-乙酰-5-羟色胺氧甲基转移酶或者咖啡酸氧甲基转移酶将N-乙酰-5羟色胺转化成褪黑素。(2)色氨酸脱羧酶将色氨酸转化成色胺;N-乙酰-5羟色胺转移酶将色胺转化为N-乙酰-5羟色胺;N-乙酰色胺氧甲基转移酶将N-乙酰-5羟色胺转化成褪黑素。(3)色氨酸脱羧酶将色氨酸转化成色胺;色氨酸羟化酶将色胺转化为5-羟色胺;N-乙酰-5-羟色胺氧甲基转移酶或者咖啡酸氧甲基转移酶将5-羟色胺转化成5-甲氧基-色胺;N-乙酰色胺转移酶将5-甲氧基-色胺转化成褪黑素。
咖啡酸氧甲基转移酶(COMT3)可以将5-羟色胺转化为5-甲氧基-色胺和将N-乙酰-5-羟色胺转化为褪黑素,因此咖啡酸氧甲基转移酶COMT3是褪黑素合成中的重要酶促步骤。川桑中含有褪黑素,但是其含量极低,并不能大量提取获得。因此,研究褪黑素合成途径的咖啡酸氧甲基转移酶COMT3及其基因,有利于体外合成途径的建立,为褪黑素规模化生产点奠定基础。
发明内容
有鉴于此,本发明采用分子生物学技术,克隆出褪黑素合成相关酶咖啡酸氧甲基转移酶基因COMT3的核苷酸序列、分析该基因的核苷酸和翻译的氨基酸序列,然后采用大肠杆菌原核表达系统进行褪黑素合成相关酶咖啡酸氧甲基转移酶COMT3的功能验证。用发酵的产物纯化、得到相关的色氨酸脱羧酶直接体外合成5-甲氧基-色胺和褪黑素。即本发明的目的之一在于提供一种川桑咖啡酸氧甲基转移酶COMT3;本发明的目的之二在于提供川桑咖啡酸氧甲基转移酶基因COMT3;本发明的目的之三在于提供所述川桑咖啡酸氧甲基转移酶COMT3在制备5-羟色胺转化为5-甲氧基-色胺或N-乙酰-5-羟色胺转化为褪黑素的催化剂中的应用;本发明的目的之四在于提供所述川桑咖啡酸氧甲基转移酶基因COMT3在原核生物中重建褪黑素合成途径中的应用;本发明的目的之五在于提供制备重组咖啡酸氧甲基转移酶COMT3的方法;本发明的目的之六在于提供由所述的方法制得的重组川桑咖啡酸氧甲基转移酶COMT3;本发明的目的之七在于提供所述重组川桑咖啡酸氧甲基转移酶COMT3在制备5-羟色胺转化为5-甲氧基-色胺或N-乙酰-5-羟色胺转化为褪黑素的催化剂中的应用。
为达到上述目的,本发明提供如下技术方案:
1、一种川桑咖啡酸氧甲基转移酶COMT3,所述川桑咖啡酸氧甲基转移酶COMT3的氨基酸序列如SEQ ID NO.4所示。
优选的,编码川桑咖啡酸氧甲基转移酶COMT3的核苷酸序列如SEQ ID NO.3所示。
2、一种川桑咖啡酸氧甲基转移酶基因COMT3,所述川桑咖啡酸氧甲基转移酶基因COMT3的核苷酸序列如SEQ ID NO.3所示。
3、所述川桑咖啡酸氧甲基转移酶COMT3在作为5-羟色胺转化为5-甲氧基-色胺或N-乙酰-5-羟色胺转化为褪黑素的催化剂中的应用。咖啡酸氧甲基转移酶COMT3可以在体外催化合成,也可以在宿主细胞内催化反应,宿主细胞可以为原核细胞,也可以为真核细胞。
4、所述川桑咖啡酸氧甲基转移酶基因COMT3在宿主细胞中重建褪黑素合成途径中的应用,所述COMT3催化5-羟色胺转化为5-甲氧基-色胺或N-乙酰-5-羟色胺转化为褪黑素的关键酶基因。
5、一种制备重组咖啡酸氧甲基转移酶COMT3的方法,所述将如SEQ ID NO.3所示的川桑咖啡酸氧甲基转移酶基因COMT3连入Pcold-tf质粒KpnⅠ和EcoRI酶切位点,获得重组表达载体Pcold-tf-COMT3,再将获得的重组表达载体转化表达菌株B21(DE3),在28℃、IPTG终浓度为1mM条件下诱导表达,提取,纯化,获得重组川桑咖啡酸氧甲基转移酶COMT3。
优选的,所述提取的方法是收集菌体,在功率为200W条件下超声破碎,超声总时间15min,每超声1s,暂停3s;然后在4℃、15000g条件下离心20min,收集上清。
优选的,所述纯化是使用镍柱纯化,用含咪唑浓度为100mM的洗脱液进行洗脱。
6、由所述的方法制得的重组川桑咖啡酸氧甲基转移酶COMT3。制得的重组酶当底物为5-羟色胺时候,其表达产物的酶促反应最适底物浓度为0.05μM,最适温度为37℃,最适pH为酸性(pH=5.4),最适反应时间为10分钟;当底物为N-乙酰-5-羟色胺时,其表达产物的酶促反应最适底物浓度为0.5μm,最适温度为55℃,最适pH为碱性(PH=7.8),最适反应时间为30分钟。
7、所述重组川桑咖啡酸氧甲基转移酶COMT3在作为5-羟色胺转化为5-甲氧基-色胺或N-乙酰-5-羟色胺转化为褪黑素的催化剂中的应用。
本发明的有益效果在于:本发明公开了川桑咖啡酸氧甲基转移酶COMT3及其基因,将川桑咖啡酸氧甲基转移酶COMT3基因进行原核表达和酶活性的测定,测得其具有较高的咖啡酸氧甲基转移酶的活性,其DNA碱基序列和氨基酸序列均与已报道的咖啡酸氧甲基转移酶基因序列有差异。因此,我们认为这是新的咖啡酸氧甲基转移酶基因,其原核表达的酶活性高,作为工程菌的目的基因,制备重组蛋白作为催化剂体外合成褪黑素前体或褪黑素,褪黑素前体最后经不同的途径合成褪黑素,作为抗肿瘤,抗衰老,特别是对阿尔茨海默病候选药物,具有很好的应用前景。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为目的基因扩增电泳图。
图2为Pcold-tf-MnCOMT3诱导上清经镍柱纯化后咪唑洗脱SDS-PAGE电泳图(1:蛋白质Marker;2:Pcold-tf空载(+IPTG);3:重组子(-IPTG);4:重组子(+IPTG);5:上清经最适浓度咪唑浓度洗脱液)。
图3为(5-羟色胺)在咖啡酸氧甲基转移酶的催化下生成物经UPLC-MS/MS鉴定的质谱图(A:5-甲氧基-色胺);B:生成物(COMT3))。
图4为5-羟色胺在咖啡酸氧甲基转移酶的催化下生成物经UPLC-MS/MS鉴定的质谱图(A:褪黑素标品;B:生成物(COMT3))。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好的理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
本发明中使用5-甲氧基-色胺(Sigma,市售价1050元/100mg)和褪黑素(Sigma,市售价2599元/100mg)作为标品对照,检验咖啡酸氧甲基转移酶基因所表达的酶活性。
实施例1、川桑咖啡酸氧甲基转移酶基因COMT3克隆
根据Morus.COMT3datebase上已报道的Morus.COMT3咖啡酸氧甲基转移酶基因(Morus013036)设计扩增咖啡酸氧甲基转移酶基因COMT3的引物。COMT3上游引物为:5'-ggggtaccatggcatccccattggagct-3'(SEQ ID NO.1),COMT3下游引物为:5'-cggaattcttaaagaactccataaccc-3'(SEQ ID NO.2)。以川桑的cDNA为模版,SEQ ID NO.1和SEQ ID NO.2所示序列为引物进行PCR扩增,扩增获得COMT3基因全长,扩增产物进行琼脂糖凝胶电泳,结果如图1所示。回收目的基因然后与pMD19-T载体连接,获得pMD19-COMT3载体,然后将pMD19-COMT3载体转入E.coli.Trans1-T1感受态细胞,获得的阳性克隆送往华大基因公司测序;结果显示,COMT3基因的核苷酸序列如SEQ ID NO.3所示,其编码的氨基酸序列如SEQ ID NO.4所示。
实施例2、川桑咖啡酸氧甲基转移酶基因COMT3重组载体构建及原核表达
将实施例克隆得到的目的基因COMT3与Pcold-tf空质粒分别用KpnⅠ和EcoRI进行双酶切,回收产物以T4DNA连接酶将其连接,获得重组质粒Pcold-tf-COMT3,将重组质粒转化入大肠杆菌DH5ɑ感受态细胞,经鉴定正确的Pcold-tf-COMT3送往华大基因公司测序,测序结果与第一次测序得到的序列一致从而获得了正确的序列。
提取Pcold-tf-COMT3质粒,将其转入表达菌株B21(DE3),在1.5ml的离心管加入450μl含有Amp抗性的LB培养基,按照1:100扩大培养到试管中,28℃,220rpm摇床培养至OD600=0.6,取1ml菌液保存了作为诱导前阳性对照,加入IPTG进行诱导,IPTG中浓度为1mM,28℃下诱导8h,诱导后各取1ml菌液保存作为诱导后总蛋白,剩余菌液4℃,5000rpm离心10min收菌,弃上清,菌体用PBS洗两次;超声破碎,超声功率为200W,超声1s,暂停3s,超声总时间15min;4℃,15000×g,20min,取上清于新的离心管中。将之前保存的诱导前和诱导后的菌液10000rpm离心1min沉淀用PBS重悬,加入适量5×Loading buffer。取破碎离心后的上清加入适量5×Loading buffer。将加入的样品沸水浴,之后12000rpm离心2min,吸取上清进行检测目的蛋白的表达情况。
将含Pcold-tf-COMT3质粒的菌株诱导8小时的上清进行镍柱纯化,分别用含咪唑浓度为:100mM,200mM,250mM,300mM的洗脱液进行洗脱,SDS-PAGE检测其咪唑洗脱浓度,结果为100mM的咪唑可以将大量的目的蛋白洗脱下来(图2)。
实施例3、重组咖啡酸氧甲基转移酶COMT3浓度及活性
取Pcold-tf-COMT3质粒的菌株诱导8小时的上清经镍柱纯化,后采用UPLC测定酶促反应的产物的物质量来表示酶活力,酶的活力单位定义为每分钟生成1nmol的物质的量为一个比活力即1U诱导诱导菌液摇了8小时之后,5ml上清经纯化后,用10ml咪唑浓度为100mM洗脱,液洗脱后,洗脱液里面所含的咖啡酸氧甲基转移酶的浓度为0.089mg/ml;
以浓度为0.2μM的5-羟色胺为底物,在温度为37℃、pH为5.4条件下以酶活力0.0831U的咖啡酸氧甲基转移酶反应20min,然后用UPLC-MS/MS鉴定,结果如图3所示,结果显示,生成5-甲氧基-色胺总量为1.66nmol(317ng)。
以浓度为0.5μM的N-乙酰-5-羟色胺为底物,在温度为45℃、pH为7.8条件下以酶活力2.122U的咖啡酸氧甲基转移酶反应30min,然后用UPLC-MS/MS鉴定,结果如图4所示。结果显示,生成褪黑素总量为42.46nmol(9893.2ng)。
上述结果表明,咖啡酸氧甲基转移酶COMT3经原核表达后具有咖啡酸氧甲基转移酶活性,可以直接作为体外合成褪黑素的催化剂,也可以构建原核生物中重建褪黑素合成途径以大规模合成褪黑素。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 西南大学
<120> 川桑咖啡酸氧甲基转移酶COMT3及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggggtaccat ggcatcccca ttggagct 28
<210> 2
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cggaattctt aaagaactcc ataaccc 27
<210> 3
<211> 1125
<212> DNA
<213> 川桑(Morus alba)
<400> 3
atggcatccc cattggagct gggaaacgat cgaaaattcc ttgacagtga gggcagaaat 60
aaagaagaag aagacaaaga aaacttctcc tatgccatgc agcttgtgtt ttccaccgtg 120
ctgtccatgt ctttgcaaac tgcaattgag ctcggagttt tcgacatcat agcgaaagcc 180
ggtgaaggcg ctaagctttc gccggcggag attacggcgc agatgaccac cgacaaccct 240
gaggcaccca tcatgcttga ccgcatactt agggtgctgg caagccactc catattgaga 300
tgttcggtgg ttggtgatgt taacagtgag tcggattttc aaaggttgta cagtcttggt 360
cctgtggcca agtattttgc gactaatgaa gatggtgttt ctttgggacc cttgatggcg 420
ttgatacaag acaagatctt cttggatagc tggccccaac taaaagaggc agttcttaag 480
ggaggaattg catttaacag agtctatgga acacacgcct tcgagtatcc aggcttggac 540
ccaagattta atcatgtttt caacaaagca atgtacaatc aaactactat tgttattaag 600
aacatcctca aattttacaa aggttttaag gaccttgcaa aattggtaga tgttggtggc 660
ggtctaggag tgacccttaa cctaatcact tccaaatacc cacatattaa gggtattaac 720
tttgacttgc cacatgttat agaacacgcg ccttcttatc caggtgttga acacgtgggt 780
ggagatatgt ttgaaaaggt tcctaccggg gatgcaattt ttatgaagtg gattctgcat 840
gattggagtg atgaacactg cttaaagcta ttgaaaaact gttacaaagc cactccagaa 900
aatggaaagg tcatagttgt ggaatcagtc cttccagtcg aggcagaaac taataccgct 960
gtgaaaagca cctcccaact tgatgtgtta atgatgactc aaaatccagg aggaaaggaa 1020
aggagcacgc aacaattcct ggccttggca actgatgcag gatttagggg cattaaattt 1080
gaatatttta tttgtaactt ttgggttatg gagttcttta agtag 1125
<210> 4
<211> 374
<212> PRT
<213> 川桑(Morus alba)
<400> 4
Met Ala Ser Pro Leu Glu Leu Gly Asn Asp Arg Lys Phe Leu Asp Ser
1 5 10 15
Glu Gly Arg Asn Lys Glu Glu Glu Asp Lys Glu Asn Phe Ser Tyr Ala
20 25 30
Met Gln Leu Val Phe Ser Thr Val Leu Ser Met Ser Leu Gln Thr Ala
35 40 45
Ile Glu Leu Gly Val Phe Asp Ile Ile Ala Lys Ala Gly Glu Gly Ala
50 55 60
Lys Leu Ser Pro Ala Glu Ile Thr Ala Gln Met Thr Thr Asp Asn Pro
65 70 75 80
Glu Ala Pro Ile Met Leu Asp Arg Ile Leu Arg Val Leu Ala Ser His
85 90 95
Ser Ile Leu Arg Cys Ser Val Val Gly Asp Val Asn Ser Glu Ser Asp
100 105 110
Phe Gln Arg Leu Tyr Ser Leu Gly Pro Val Ala Lys Tyr Phe Ala Thr
115 120 125
Asn Glu Asp Gly Val Ser Leu Gly Pro Leu Met Ala Leu Ile Gln Asp
130 135 140
Lys Ile Phe Leu Asp Ser Trp Pro Gln Leu Lys Glu Ala Val Leu Lys
145 150 155 160
Gly Gly Ile Ala Phe Asn Arg Val Tyr Gly Thr His Ala Phe Glu Tyr
165 170 175
Pro Gly Leu Asp Pro Arg Phe Asn His Val Phe Asn Lys Ala Met Tyr
180 185 190
Asn Gln Thr Thr Ile Val Ile Lys Asn Ile Leu Lys Phe Tyr Lys Gly
195 200 205
Phe Lys Asp Leu Ala Lys Leu Val Asp Val Gly Gly Gly Leu Gly Val
210 215 220
Thr Leu Asn Leu Ile Thr Ser Lys Tyr Pro His Ile Lys Gly Ile Asn
225 230 235 240
Phe Asp Leu Pro His Val Ile Glu His Ala Pro Ser Tyr Pro Gly Val
245 250 255
Glu His Val Gly Gly Asp Met Phe Glu Lys Val Pro Thr Gly Asp Ala
260 265 270
Ile Phe Met Lys Trp Ile Leu His Asp Trp Ser Asp Glu His Cys Leu
275 280 285
Lys Leu Leu Lys Asn Cys Tyr Lys Ala Thr Pro Glu Asn Gly Lys Val
290 295 300
Ile Val Val Glu Ser Val Leu Pro Val Glu Ala Glu Thr Asn Thr Ala
305 310 315 320
Val Lys Ser Thr Ser Gln Leu Asp Val Leu Met Met Thr Gln Asn Pro
325 330 335
Gly Gly Lys Glu Arg Ser Thr Gln Gln Phe Leu Ala Leu Ala Thr Asp
340 345 350
Ala Gly Phe Arg Gly Ile Lys Phe Glu Tyr Phe Ile Cys Asn Phe Trp
355 360 365
Val Met Glu Phe Phe Lys
370
Claims (10)
1.一种川桑咖啡酸氧甲基转移酶COMT3,其特征在于:所述川桑咖啡酸氧甲基转移酶COMT3的氨基酸序列如SEQ ID NO.4所示。
2.根据权利要求1所述川桑咖啡酸氧甲基转移酶COMT3,其特征在于:编码川桑咖啡酸氧甲基转移酶COMT3的核苷酸序列如SEQ ID NO.3所示。
3.一种川桑咖啡酸氧甲基转移酶基因COMT3,其特征在于:所述川桑咖啡酸氧甲基转移酶基因COMT3的核苷酸序列如SEQ ID NO.3所示。
4.权利要求1所述川桑咖啡酸氧甲基转移酶COMT3在作为5-羟色胺转化为5-甲氧基-色胺或N-乙酰-5-羟色胺转化为褪黑素的催化剂中的应用。
5.权利要求3所述川桑咖啡酸氧甲基转移酶基因COMT3在宿主细胞中重建褪黑素合成途径中的应用,其特征在于:所述COMT3催化5-羟色胺转化为5-甲氧基-色胺或N-乙酰-5-羟色胺转化为褪黑素的关键酶基因。
6.一种制备重组咖啡酸氧甲基转移酶COMT3的方法,其特征在于:所述将如SEQ IDNO.3所示的川桑咖啡酸氧甲基转移酶基因COMT3连入Pcold-tf质粒KpnⅠ和EcoRI酶切位点,获得重组表达载体Pcold-tf-COMT3,再将获得的重组表达载体转化表达菌株B21(DE3),在28℃、IPTG终浓度为1mM条件下诱导表达,提取,纯化,获得重组川桑咖啡酸氧甲基转移酶COMT3。
7.根据权利要求6所述制备重组川桑咖啡酸氧甲基转移酶COMT3的方法,其特征在于:所述提取的方法是收集菌体,在功率为200W条件下超声破碎,超声总时间15min,每超声1s,暂停3s;然后在4℃、15000g条件下离心20min,收集上清。
8.根据权利要求6所述制备重组川桑咖啡酸氧甲基转移酶COMT3的方法,其特征在于:所述纯化是使用镍柱纯化,用含咪唑浓度为100mM的洗脱液进行洗脱。
9.由权利要求6~8任一项所述的方法制得的重组川桑咖啡酸氧甲基转移酶COMT3。
10.权利要求9所述重组川桑咖啡酸氧甲基转移酶COMT3在作为5-羟色胺转化为5-甲氧基-色胺或N-乙酰-5-羟色胺转化为褪黑素的催化剂中的应用。
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CN114958794A (zh) * | 2022-06-14 | 2022-08-30 | 南京工业大学 | 一种苯乙醇胺-N-甲基转移酶hPNMT54及其克隆表达与应用 |
CN117683794A (zh) * | 2024-02-04 | 2024-03-12 | 湖南工程学院 | 一种促进人参中褪黑素和人参皂苷合成的PgCOMT2基因及其应用 |
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CN113528411A (zh) * | 2021-06-29 | 2021-10-22 | 河北维达康生物科技有限公司 | 生产n-乙酰基-5-甲氧基色胺的基因工程菌、构建方法及应用 |
CN114958794A (zh) * | 2022-06-14 | 2022-08-30 | 南京工业大学 | 一种苯乙醇胺-N-甲基转移酶hPNMT54及其克隆表达与应用 |
CN114958794B (zh) * | 2022-06-14 | 2023-06-02 | 南京工业大学 | 一种苯乙醇胺-N-甲基转移酶hPNMT54及其克隆表达与应用 |
CN117683794A (zh) * | 2024-02-04 | 2024-03-12 | 湖南工程学院 | 一种促进人参中褪黑素和人参皂苷合成的PgCOMT2基因及其应用 |
CN117683794B (zh) * | 2024-02-04 | 2024-04-26 | 湖南工程学院 | 一种促进人参中褪黑素和人参皂苷合成的PgCOMT2基因及其应用 |
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