CN114196640B - 一种源于新型贝类的l-肌肽合成酶atpgd及其应用 - Google Patents
一种源于新型贝类的l-肌肽合成酶atpgd及其应用 Download PDFInfo
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- CN114196640B CN114196640B CN202111498661.8A CN202111498661A CN114196640B CN 114196640 B CN114196640 B CN 114196640B CN 202111498661 A CN202111498661 A CN 202111498661A CN 114196640 B CN114196640 B CN 114196640B
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Abstract
本发明公开了一种源于新型贝类的L‑肌肽合成酶ATPGD及其应用,属于海洋生物技术领域。本发明首次从鳞砗磲中发现ATPgrasp结构域的蛋白ATPGD,其氨基酸序列如SEQ IDNO.2所示,能够将β‑丙氨酸和L‑组氨酸催化合成为L‑肌肽,为L‑肌肽的生物合成提供一种全新的酶制剂。本发明还公开了利用鳞砗磲ATPGD高效合成L‑肌肽的方法。
Description
技术领域
本发明属于海洋生物技术领域,具体涉及一种源于新型贝类的L-肌肽合成酶ATPGD及其应用。
背景技术
L-肌肽(β-丙氨酰-L-组氨酸)及其类似物(高肌肽、鹅肌肽),是广泛存在于哺乳动物的大脑、肌肉和其他重要组织中的天然活性二肽。该二肽具有良好的抗氧化活性,在清除自由基,减少脂质过氧化等方面作用显著,因此被大量应用于化妆品、食品保藏、细胞氧化相关疾病如高血压、心脏病、白内障、肿瘤等的辅助治疗等方面。目前,肌肽的生产主要依靠化学合成,但该路线相对复杂且容易造成环境污染,于是条件温和且环保的生物酶催化法成为近年来该二肽合成的探索方向之一。
鳞砗磲(Tridacna squamosa)是一种分布于印度、太平洋海域的大型贝类,属于双壳纲,帘蛤目,鸟蛤科,砗磲亚科。
发明内容
本发明的第一个目的是提供一种新型贝类合成具有抗氧化作用的L-肌肽合成酶ATPGD,其氨基酸序列如SEQ ID NO.2所示或与SEQ ID NO.2所示的序列有95%以上的同源性;
本发明第二个目的是提供编码上述L-肌肽合成酶ATPGD的编码基因。
优选地,所述的编码基因,其核苷酸序列如SEQ ID NO.1所示,或与SEQ ID NO.1所示的序列有95%以上的同源性。
本发明第三个目的是提供一种包含L-肌肽合成酶ATPGD的编码基因的表达载体。
优选地,所述的表达载体为pGEX4T-1。
本发明第四个目的是提供一种包含上述表达载体的宿主细胞。
优选地,所述的宿主细胞为大肠杆菌BL21(DE3)。
本发明第五个目的是提供所述的肌肽合成酶ATPGD、编码基因、表达载体、宿主细胞在制备酶制剂或合成L-肌肽中的应用。
本发明第六个目的是提供一种高效合成L-肌肽的方法,是将上述L-肌肽合成酶ATPGD的编码基因在宿主细胞中进行表达,生物合成L-肌肽合成酶ATPGD;将L-肌肽合成酶ATPGD与β-丙氨酸、L-组氨酸以及ATP混合,酶催化合成L-肌肽。
优选地,所述的宿主细胞为大肠杆菌BL21(DE3)。
优选地,将上述L-肌肽合成酶ATPGD的编码基因在宿主细胞中进行表达是将所述的编码基因转入表达载体如pGEX4T-1中,然后转化进入宿主细胞中进行表达。
本发明的第七个目的是提供上述合成的L-肌肽在制备抗氧化产品中的应用;进一步地,可应用于化妆品、食品保藏、细胞氧化相关疾病如高血压、心脏病、白内障、肿瘤等的辅助治疗等方面。
与现有技术相比,本发明有益效果如下:
本发明首次从鳞砗磲中发现ATPgrasp结构域的蛋白ATPGD,能够将β-丙氨酸和L-组氨酸催化合成为L-肌肽。本发明中的ATPGD能够合成具有抗氧化作用的L-肌肽,为L-肌肽的生物合成提供一种全新的酶制剂。
附图说明
图1为鳞砗磲ATPGD催化L-肌肽生成的模式图。
图2为含有鳞砗磲ATPGD重组蛋白的反应体系在60min和120min时肌肽的产量。
图3为含有鳞砗磲ATPGD重组蛋白的反应体系产生肌肽分子的结构质谱鉴定结果。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
下列实施例中未注明具体实验条件和方法,所采用的技术手段通常为本领域技术人员所熟知的常规手段。
实施例1ATPGD全长基因的获得
1、RNA提取
总RNA的提取使用Trizol(Invitorgen)提取,具体步骤如下:(1)将50mg鳞砗磲鳃组织放入1ml Trizol溶液的离心管中,充分研磨,室温放置10min,(2)加入200μL氯仿,剧烈震荡40s,室温放置5min;(3)4℃,12,000×g离心10min,吸取上清转移至新管;(4)加入0.5ml的异丙醇,混匀,室温静置10min;(5)4℃,12,000×g离心10min,弃上清;(6)1ml 75%乙醇清洗两次沉淀,4℃,12,000×g离心10min;(7)弃上清,室温放置2-3min,加入100μLDEPC水溶解RNA,-80℃保存备用。以上步骤中所有容器、枪头均经过无RNAase处理。
2.反转录和ATPGD全长基因的获得
取约1μg上述提取的RNA,使用PrimeScriptTM 1st Strand cDNA Synthesis Kit(TaKaRa)进行反转录反应获得cDNA,-80℃保存。获取ATPGD全长基因,其中,ATPGD全长基因的核苷酸序列如下:
atgacgagtttccgtgaaaggttcgaggaccacgccgggtctataacttcggcgaatgcagacgttgatttgtcgactttggcaggattcgataaggagcagactgaggaccccttcgtaaaagattactattattccctacaacacacgctgtcagagacgggccaccccaaaacgatagaccggacctgcgttccgagaactacacataatgacgaacacgtgactgtcgccatcttagcttcgccacgtgatgtcgtaggattattgttggaaggtggcaagcaagcacccgatacattccatcttatcttatcaacaacctggttgtctaaaaaggacgagggaggacactattgctccagtgggctatgggtgacaaaagcaataacttttcacaaaaatggacagtcctatattgatgtgtttaaaccactccgaaaagtgacatactttatggattttttcactgaaaatatcacaggaggacagagaaatgacggggagaaatttgagaatgatttagactgtctaatgagcagttccgtcaagttatctgagcttattgacaataaagtttggacacgatgtatcatggcggatgttgaaatggcgtatccaatcaccctggcttttatgtaccagcccagttatagtgtacaaagcaatcacagatatataacactcgtgaggctggaaacaaaagaaagcggagaaattcacaaaagagtccgagagtttctggacagttactcgatgaaagaagtaaccaaggaggtttatcaaaatccattacagttcttatacgttatatcccgtggcgtacccctcctgcatagcaacgcattacaatattacgccaagcgcgagaaatgtaaggtcgtggtcaagctttctggtccgcaaagtagcggcagtgctggctactacaataagacagacaagaatgctatcaccgatgctgttacagggttactagaggatatacaaccaggatctgcagttctggtcgaggaatttatcgagggcttaactccatttgacaggagtgcatcttgttgtgctacacctaaaccttggggcgctgacagaaacttctccttcagagtaagagtcacggtgtgtcgagattttaatgataagcctgtaacggtgcaggtaagctgtgggatgcaaccacggtctcgactaaccaacgggaacaacactgtcccgcagtcgctgaatagcacactgttagcctacaatgtgtttgaaaaggaacagagagatgaggctgagaaaagaataagggaacagtgcgaggctgttatgtccaagatcatggaacgggaaaaatatttaacagtggaacagcgaggttgtctacgagcgcaaacagatgtgattgactgtcctgcaaaagacgaagtagaggattttatgtatttcgacttttcgaaccatctccttgacaaagaccacgcagtgaacatcagtactatgcttagcgacgggaagttacaaatcgacggttgtctaaccttctttgaagattgtgggccgctggcagctttattacgagaaaaactggacttgtcgaaatcgggaattagtcatgaatcggcgatgaatgccaagaaaaaaagccagacgtttgaaatactgcgctcaaggaaggacgacacgccccattggccaaggtcttatctgtattcgtccgcctcatacagaataacggacaaggaaacattggttactgcaactaaagacatcaaattcccggccattgtgaaactggaacatggctcaagctctgtcggtgtaaagcttgtacagagtaatgaagaactaatgcaagagtacgagcagatcaggacgaccttgacccgagagacagactttcccggtataggtttgggctttgacaacacgttattggtgatggactatcataacgggtccgaacacgatgttgacgttgtcatctttaacagacaattgattgctgccttcatttcagataatggaccaactcgggaacctgtttatatagagacttgtgcgcttatgccatcctgtttaccgacagataagcaagcccagcttgtggcagctgcgtatcagtgttgtgtagaactcggtttagataatgggactttcaacgtagaactaaagatgactgcaggtgggccaaaacttctcgaagtaaatgctaggatgggaggattttatctacgggactgggtgaaacgtttgtatggcgtcgacttaatgcttgtttctatcatgattgcctgtgggatcaaaccattcattccaaaatttgattcaagatcccacattatgggaaccatgttggtgccttcattacatagtcatatgctcgacaatgacgtatataaagtgaagctggagtcactgattaaatcagaggaggtgatatggaaccagttccttaaaactgcaaagtcagactcagaaaagtttgagaaaccgtttgcaaaccttgccgttcaagcacaaactattacagaagctcggaacaaacttgtagctgtgtgtaaagatttaggtattcacaaggagagttatgatgtggaatattttaccaaagaattttttttgaaaaaaaaaaaaaaaaaagaatctgtcgcagatctacatatacatgtagatctaatggtaactttctatgatgatgacttgttggagccttcttcatctcatacgactgattag(SEQ IDNO.1),其编码的蛋白的氨基酸序列如SEQ ID NO.2所示。
实施例2重组蛋白的原核表达
1.原核表达载体构建
(1)设计一对涵盖鳞砗磲ATPGD ORF的引物(F:5'-ATGACGAGTTTCCGTGAAAGGTTCG-3';R:5'-CTAATCAGTCGTATGAGATGAAGAAGGCTC-3');(2)使用这对引物进行PCR扩增(反应体系:10倍稀释的鳃组织cDNA 0.5μl,引物(10mM)各1μl,Premix TaKaRaEx Taq(TaKaRa)12.5μl,ddH2O 10μl。反应程序:95℃3min;95℃15s,60℃15s,72℃3min;72℃5min)。PCR产物通过1.5%琼脂糖凝胶检测符合预期大小后,使用HiPure Gel Pure DNA Mini Kit(Magen)回收纯化,测序验证无误后产物-20℃保存。(3)以PCR产物为模板,使用引物对(5'-CCGCGTGGATCCCCGGAATTCATGACGAGTTTCCGTGAAAGGT-3';5'-GTCACGATGCGGCCGCTCGAGCTAATCAGTCGTATGAGATGAAGAAGG-3')再次进行扩增添加重组接头(反应体系:模板0.5μl,引物(10mM)各1μl,Premix TaKaRa Ex Taq(TaK-aRa)12.5μl,ddH2O 10μl。反应程序:95℃3min;95℃15s,60℃15s,72℃3min;72℃5min),pGEX4T-1载体用EcoRI和Xho-I进行双酶切,并回收纯化PCR产物和酶切产物;MonCloneTM Single Assembly Cloning Mix(Monad)连接PCR产物和酶切产物;(4)将连接产物转化至大肠杆菌DH5α中,转化菌在含有氨苄西林(Amp,终浓度100μg/L)的LB平板37℃过夜培养,挑取阳性克隆,在LB液体培养基中37℃培养过夜,测序验证载体正确无误后,扩大培养,使用HiPure Plasmid/BAC EF Mini Kits(Magen)提取质粒,由此得到含有鳞砗磲ATPGD基因的pGEX4T-1载体。
2.重组蛋白的原核表达
(1)将获得的含有鳞砗磲ATPGD基因的pGEX4T-1载体,转化至大肠杆菌BL21(DE3);(2)挑选单克隆在含有氨苄西林(Amp,终浓度100μg/L)的LB液体培养基中进行培养至OD600=0.6,加入IPTG至终浓度为0.1mM,16℃,180rmp的条件下诱导表达;(3)16h后收集菌液,4℃,12,000×g离心10min,收集菌体;获得包含鳞砗磲ATPGD基因的pGEX4T-1载体的细菌。
3.重组蛋白的纯化
用含有1mM PMSF的PBS重悬步骤2获得的细菌,超声破碎;使用GST SefinoseTMResin(BBI Life sciences)纯化重组蛋白(即鳞砗磲ATPGD重组蛋白),在12%SDS-PAGE胶分析纯化产物,分光光度法测定浓度。
实施例3L-肌肽的合成
使用实施例2获得的包含约15μg鳞砗磲ATPGD重组蛋白的0.2ml溶液(含有20mMGSH的PBS作为溶剂)与1ml含有0.5mM的β-丙氨酸、0.5mM的L-组氨酸以及0.5mM的MgATP的PBS底物溶液37℃孵育60min和120min;其中对照组是将含有15μg的BSA蛋白的0.2ml溶液(含有20mM GSH的PBS作为溶剂)与1ml含有0.5mM的β-丙氨酸、0.5mM的L-组氨酸以及0.5mM的MgATP的PBS底物溶液进行混合。每组设置3个生物学重复,后加入200μl的30%(v/v)HCl终止反应,3kDa超滤膜过滤后,去除蛋白等大分子。使用HPLC对包含肌肽的滤液进行浓度测定,发现含有鳞砗磲ATPGD重组蛋白的反应体系,在60min和120min反应时间内可以分别产生76.8μM和167.2μM的肌肽(图2),质谱鉴定结果肌肽分子结构相一致(图3)。
序列表
<110> 中国科学院南海海洋研究所
<120> 一种源于新型贝类的L-肌肽合成酶ATPGD及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2721
<212> DNA
<213> 鳞砗磲(Tridacna squamosa)
<400> 1
atgacgagtt tccgtgaaag gttcgaggac cacgccgggt ctataacttc ggcgaatgca 60
gacgttgatt tgtcgacttt ggcaggattc gataaggagc agactgagga ccccttcgta 120
aaagattact attattccct acaacacacg ctgtcagaga cgggccaccc caaaacgata 180
gaccggacct gcgttccgag aactacacat aatgacgaac acgtgactgt cgccatctta 240
gcttcgccac gtgatgtcgt aggattattg ttggaaggtg gcaagcaagc acccgataca 300
ttccatctta tcttatcaac aacctggttg tctaaaaagg acgagggagg acactattgc 360
tccagtgggc tatgggtgac aaaagcaata acttttcaca aaaatggaca gtcctatatt 420
gatgtgttta aaccactccg aaaagtgaca tactttatgg attttttcac tgaaaatatc 480
acaggaggac agagaaatga cggggagaaa tttgagaatg atttagactg tctaatgagc 540
agttccgtca agttatctga gcttattgac aataaagttt ggacacgatg tatcatggcg 600
gatgttgaaa tggcgtatcc aatcaccctg gcttttatgt accagcccag ttatagtgta 660
caaagcaatc acagatatat aacactcgtg aggctggaaa caaaagaaag cggagaaatt 720
cacaaaagag tccgagagtt tctggacagt tactcgatga aagaagtaac caaggaggtt 780
tatcaaaatc cattacagtt cttatacgtt atatcccgtg gcgtacccct cctgcatagc 840
aacgcattac aatattacgc caagcgcgag aaatgtaagg tcgtggtcaa gctttctggt 900
ccgcaaagta gcggcagtgc tggctactac aataagacag acaagaatgc tatcaccgat 960
gctgttacag ggttactaga ggatatacaa ccaggatctg cagttctggt cgaggaattt 1020
atcgagggct taactccatt tgacaggagt gcatcttgtt gtgctacacc taaaccttgg 1080
ggcgctgaca gaaacttctc cttcagagta agagtcacgg tgtgtcgaga ttttaatgat 1140
aagcctgtaa cggtgcaggt aagctgtggg atgcaaccac ggtctcgact aaccaacggg 1200
aacaacactg tcccgcagtc gctgaatagc acactgttag cctacaatgt gtttgaaaag 1260
gaacagagag atgaggctga gaaaagaata agggaacagt gcgaggctgt tatgtccaag 1320
atcatggaac gggaaaaata tttaacagtg gaacagcgag gttgtctacg agcgcaaaca 1380
gatgtgattg actgtcctgc aaaagacgaa gtagaggatt ttatgtattt cgacttttcg 1440
aaccatctcc ttgacaaaga ccacgcagtg aacatcagta ctatgcttag cgacgggaag 1500
ttacaaatcg acggttgtct aaccttcttt gaagattgtg ggccgctggc agctttatta 1560
cgagaaaaac tggacttgtc gaaatcggga attagtcatg aatcggcgat gaatgccaag 1620
aaaaaaagcc agacgtttga aatactgcgc tcaaggaagg acgacacgcc ccattggcca 1680
aggtcttatc tgtattcgtc cgcctcatac agaataacgg acaaggaaac attggttact 1740
gcaactaaag acatcaaatt cccggccatt gtgaaactgg aacatggctc aagctctgtc 1800
ggtgtaaagc ttgtacagag taatgaagaa ctaatgcaag agtacgagca gatcaggacg 1860
accttgaccc gagagacaga ctttcccggt ataggtttgg gctttgacaa cacgttattg 1920
gtgatggact atcataacgg gtccgaacac gatgttgacg ttgtcatctt taacagacaa 1980
ttgattgctg ccttcatttc agataatgga ccaactcggg aacctgttta tatagagact 2040
tgtgcgctta tgccatcctg tttaccgaca gataagcaag cccagcttgt ggcagctgcg 2100
tatcagtgtt gtgtagaact cggtttagat aatgggactt tcaacgtaga actaaagatg 2160
actgcaggtg ggccaaaact tctcgaagta aatgctagga tgggaggatt ttatctacgg 2220
gactgggtga aacgtttgta tggcgtcgac ttaatgcttg tttctatcat gattgcctgt 2280
gggatcaaac cattcattcc aaaatttgat tcaagatccc acattatggg aaccatgttg 2340
gtgccttcat tacatagtca tatgctcgac aatgacgtat ataaagtgaa gctggagtca 2400
ctgattaaat cagaggaggt gatatggaac cagttcctta aaactgcaaa gtcagactca 2460
gaaaagtttg agaaaccgtt tgcaaacctt gccgttcaag cacaaactat tacagaagct 2520
cggaacaaac ttgtagctgt gtgtaaagat ttaggtattc acaaggagag ttatgatgtg 2580
gaatatttta ccaaagaatt ttttttgaaa aaaaaaaaaa aaaaagaatc tgtcgcagat 2640
ctacatatac atgtagatct aatggtaact ttctatgatg atgacttgtt ggagccttct 2700
tcatctcata cgactgatta g 2721
<210> 2
<211> 906
<212> PRT
<213> 鳞砗磲(Tridacna squamosa)
<400> 2
Met Thr Ser Phe Arg Glu Arg Phe Glu Asp His Ala Gly Ser Ile Thr
1 5 10 15
Ser Ala Asn Ala Asp Val Asp Leu Ser Thr Leu Ala Gly Phe Asp Lys
20 25 30
Glu Gln Thr Glu Asp Pro Phe Val Lys Asp Tyr Tyr Tyr Ser Leu Gln
35 40 45
His Thr Leu Ser Glu Thr Gly His Pro Lys Thr Ile Asp Arg Thr Cys
50 55 60
Val Pro Arg Thr Thr His Asn Asp Glu His Val Thr Val Ala Ile Leu
65 70 75 80
Ala Ser Pro Arg Asp Val Val Gly Leu Leu Leu Glu Gly Gly Lys Gln
85 90 95
Ala Pro Asp Thr Phe His Leu Ile Leu Ser Thr Thr Trp Leu Ser Lys
100 105 110
Lys Asp Glu Gly Gly His Tyr Cys Ser Ser Gly Leu Trp Val Thr Lys
115 120 125
Ala Ile Thr Phe His Lys Asn Gly Gln Ser Tyr Ile Asp Val Phe Lys
130 135 140
Pro Leu Arg Lys Val Thr Tyr Phe Met Asp Phe Phe Thr Glu Asn Ile
145 150 155 160
Thr Gly Gly Gln Arg Asn Asp Gly Glu Lys Phe Glu Asn Asp Leu Asp
165 170 175
Cys Leu Met Ser Ser Ser Val Lys Leu Ser Glu Leu Ile Asp Asn Lys
180 185 190
Val Trp Thr Arg Cys Ile Met Ala Asp Val Glu Met Ala Tyr Pro Ile
195 200 205
Thr Leu Ala Phe Met Tyr Gln Pro Ser Tyr Ser Val Gln Ser Asn His
210 215 220
Arg Tyr Ile Thr Leu Val Arg Leu Glu Thr Lys Glu Ser Gly Glu Ile
225 230 235 240
His Lys Arg Val Arg Glu Phe Leu Asp Ser Tyr Ser Met Lys Glu Val
245 250 255
Thr Lys Glu Val Tyr Gln Asn Pro Leu Gln Phe Leu Tyr Val Ile Ser
260 265 270
Arg Gly Val Pro Leu Leu His Ser Asn Ala Leu Gln Tyr Tyr Ala Lys
275 280 285
Arg Glu Lys Cys Lys Val Val Val Lys Leu Ser Gly Pro Gln Ser Ser
290 295 300
Gly Ser Ala Gly Tyr Tyr Asn Lys Thr Asp Lys Asn Ala Ile Thr Asp
305 310 315 320
Ala Val Thr Gly Leu Leu Glu Asp Ile Gln Pro Gly Ser Ala Val Leu
325 330 335
Val Glu Glu Phe Ile Glu Gly Leu Thr Pro Phe Asp Arg Ser Ala Ser
340 345 350
Cys Cys Ala Thr Pro Lys Pro Trp Gly Ala Asp Arg Asn Phe Ser Phe
355 360 365
Arg Val Arg Val Thr Val Cys Arg Asp Phe Asn Asp Lys Pro Val Thr
370 375 380
Val Gln Val Ser Cys Gly Met Gln Pro Arg Ser Arg Leu Thr Asn Gly
385 390 395 400
Asn Asn Thr Val Pro Gln Ser Leu Asn Ser Thr Leu Leu Ala Tyr Asn
405 410 415
Val Phe Glu Lys Glu Gln Arg Asp Glu Ala Glu Lys Arg Ile Arg Glu
420 425 430
Gln Cys Glu Ala Val Met Ser Lys Ile Met Glu Arg Glu Lys Tyr Leu
435 440 445
Thr Val Glu Gln Arg Gly Cys Leu Arg Ala Gln Thr Asp Val Ile Asp
450 455 460
Cys Pro Ala Lys Asp Glu Val Glu Asp Phe Met Tyr Phe Asp Phe Ser
465 470 475 480
Asn His Leu Leu Asp Lys Asp His Ala Val Asn Ile Ser Thr Met Leu
485 490 495
Ser Asp Gly Lys Leu Gln Ile Asp Gly Cys Leu Thr Phe Phe Glu Asp
500 505 510
Cys Gly Pro Leu Ala Ala Leu Leu Arg Glu Lys Leu Asp Leu Ser Lys
515 520 525
Ser Gly Ile Ser His Glu Ser Ala Met Asn Ala Lys Lys Lys Ser Gln
530 535 540
Thr Phe Glu Ile Leu Arg Ser Arg Lys Asp Asp Thr Pro His Trp Pro
545 550 555 560
Arg Ser Tyr Leu Tyr Ser Ser Ala Ser Tyr Arg Ile Thr Asp Lys Glu
565 570 575
Thr Leu Val Thr Ala Thr Lys Asp Ile Lys Phe Pro Ala Ile Val Lys
580 585 590
Leu Glu His Gly Ser Ser Ser Val Gly Val Lys Leu Val Gln Ser Asn
595 600 605
Glu Glu Leu Met Gln Glu Tyr Glu Gln Ile Arg Thr Thr Leu Thr Arg
610 615 620
Glu Thr Asp Phe Pro Gly Ile Gly Leu Gly Phe Asp Asn Thr Leu Leu
625 630 635 640
Val Met Asp Tyr His Asn Gly Ser Glu His Asp Val Asp Val Val Ile
645 650 655
Phe Asn Arg Gln Leu Ile Ala Ala Phe Ile Ser Asp Asn Gly Pro Thr
660 665 670
Arg Glu Pro Val Tyr Ile Glu Thr Cys Ala Leu Met Pro Ser Cys Leu
675 680 685
Pro Thr Asp Lys Gln Ala Gln Leu Val Ala Ala Ala Tyr Gln Cys Cys
690 695 700
Val Glu Leu Gly Leu Asp Asn Gly Thr Phe Asn Val Glu Leu Lys Met
705 710 715 720
Thr Ala Gly Gly Pro Lys Leu Leu Glu Val Asn Ala Arg Met Gly Gly
725 730 735
Phe Tyr Leu Arg Asp Trp Val Lys Arg Leu Tyr Gly Val Asp Leu Met
740 745 750
Leu Val Ser Ile Met Ile Ala Cys Gly Ile Lys Pro Phe Ile Pro Lys
755 760 765
Phe Asp Ser Arg Ser His Ile Met Gly Thr Met Leu Val Pro Ser Leu
770 775 780
His Ser His Met Leu Asp Asn Asp Val Tyr Lys Val Lys Leu Glu Ser
785 790 795 800
Leu Ile Lys Ser Glu Glu Val Ile Trp Asn Gln Phe Leu Lys Thr Ala
805 810 815
Lys Ser Asp Ser Glu Lys Phe Glu Lys Pro Phe Ala Asn Leu Ala Val
820 825 830
Gln Ala Gln Thr Ile Thr Glu Ala Arg Asn Lys Leu Val Ala Val Cys
835 840 845
Lys Asp Leu Gly Ile His Lys Glu Ser Tyr Asp Val Glu Tyr Phe Thr
850 855 860
Lys Glu Phe Phe Leu Lys Lys Lys Lys Lys Lys Glu Ser Val Ala Asp
865 870 875 880
Leu His Ile His Val Asp Leu Met Val Thr Phe Tyr Asp Asp Asp Leu
885 890 895
Leu Glu Pro Ser Ser Ser His Thr Thr Asp
900 905
Claims (8)
1.一种L-肌肽合成酶ATPGD,其特征在于,其氨基酸序列如SEQ ID NO.2所示。
2.一种编码权利要求1所述L-肌肽合成酶ATPGD的基因。
3.根据权利要求2所述的基因,其特征在于,其核苷酸序列如SEQ ID NO.1所示。
4.一种表达载体,其特征在于,其含有权利要求2或3所述的基因。
5.权利要求1所述的L-肌肽合成酶ATPGD、权利要求2或3所述的基因、权利要求4所述的表达载体在制备酶制剂或合成L-肌肽中的应用。
6.一种合成L-肌肽的方法,其特征在于,将权利要求2或3所述的基因在宿主细胞中进行表达,生物合成L-肌肽合成酶ATPGD;将L-肌肽合成酶ATPGD与β-丙氨酸、L-组氨酸以及ATP混合,酶催化合成L-肌肽。
7.根据权利要求6所述的方法,其特征在于,所述的宿主细胞为大肠杆菌BL21(DE3)。
8.根据权利要求6所述的方法,其特征在于,将权利要求2或3所述的基因在宿主细胞中进行表达是将权利要求2或3所述的基因转入表达载体中,然后转化进入宿主细胞中进行表达。
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