CN107119035B - 苯丙氨酸变位酶、编码基因、重组载体、宿主细胞、多重pcr引物及它们的应用 - Google Patents
苯丙氨酸变位酶、编码基因、重组载体、宿主细胞、多重pcr引物及它们的应用 Download PDFInfo
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- CN107119035B CN107119035B CN201710251330.1A CN201710251330A CN107119035B CN 107119035 B CN107119035 B CN 107119035B CN 201710251330 A CN201710251330 A CN 201710251330A CN 107119035 B CN107119035 B CN 107119035B
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- phenylalanine
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- mutase
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- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 title claims abstract description 69
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 239000013598 vector Substances 0.000 title claims abstract description 20
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- 150000001413 amino acids Chemical group 0.000 claims abstract description 10
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- 125000003729 nucleotide group Chemical group 0.000 claims description 4
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Abstract
本发明涉及酶工程领域,具体地,公开了一种苯丙氨酸变位酶、编码基因、重组载体、宿主细胞、多重PCR引物及它们的应用。所述苯丙氨酸变位酶的氨基酸序列如SEQ ID NO:1所示。本发明通过对现有的苯丙氨酸变位酶进行定点突变,使得突变后的苯丙氨酸变位酶具有更好的催化活性,同时能够降低α‑苯丙氨酸的生成能力。并通过建立表达载体,构建重组菌,实现高效表达,获得满足工业化生产要求的苯丙氨酸变位酶。
Description
技术领域
本发明涉及酶工程领域,具体地,涉及苯丙氨酸变位酶、编码基因、重组载体、宿主细胞、多重PCR引物及它们的应用。
背景技术
β-苯丙氨酸是β-内酯和内酰胺的前体,是天然产物萜类、生物碱及功能性多肽的重要结构单元,广泛应用于食品、医药、化妆品、饲料等工业领域,需求逐年提高。生产β-苯丙氨酸有化学拆分、不对称化学合成、酶转化四种方法,其中不对称化学合成因其工艺相对简单,是目前最主要的生产方法。但不对称化学合成需要昂贵的手性试剂、高温高压的条件、强酸强碱的环境,而且存在着环境污染的问题。随着β-苯丙氨酸需求量的不断增加,开发绿色的生产技术具有十分明显的经济和社会效益。与不对称化学合成方法相比,酶转化法制备β-苯丙氨酸因其具有工艺简单、生产条件温和、立体选择性高、生产过程绿色无污染等优点,因此越来越受到重视。目前酶法合成主要采用青霉素酰化酶、转氨酶等,但上述酶的使用要经多步酶催化反应,而且还需要添加外源性辅因子,如金属离子、核黄素、磷酸吡哆醛等,导致反应过程控制复杂,产率低,难实现大规模工业化生产。
苯丙氨酸变位酶(PAM,EC 5.4.3.10)可在碱性条件下一步催化3-苯基丙烯酸加氨合成β-芳香基丙氨酸,但同时能形成副产物α-苯丙氨酸,难以除去,导致产物β-芳香基丙氨酸纯度不高。
因此,提供一种能够对野生型的苯丙氨酸变位酶进行定点突变的分子改造,制备突变体,用于合成β-芳香基丙氨酸,实现提高转化率,满足工业化生产β-芳香基丙氨酸的要求的苯丙氨酸变位酶、编码基因、重组载体、宿主细胞、多重PCR引物及它们的应用是本发明亟需解决的问题。
发明内容
针对上述现有技术,本发明的目的在于克服现有技术中生产β-苯丙氨酸或生产条件要求高,或使用的原料价格高,或容易产生大量的副产物等问题,从而提供一种能够对野生型的苯丙氨酸变位酶进行定点突变的分子改造,制备突变体,用于合成β-芳香基丙氨酸,实现提高转化率,满足工业化生产β-芳香基丙氨酸的要求的苯丙氨酸变位酶、编码基因、重组载体、宿主细胞、多重PCR引物及它们的应用。
为了实现上述目的,本发明提供了一种苯丙氨酸变位酶,其中,所述苯丙氨酸变位酶的氨基酸序列如SEQ ID NO:1所示。
本发明还提供了一种编码基因,其中,所述编码基因能够编码上述所述的苯丙氨酸变位酶。
本发明还提供了一种重组载体,其中,所述重组载体含有上述所述的编码基因。
本发明还提供了一种宿主细胞,其中,所述宿主细胞含有根据上述所述的编码基因,或者含有根据上述所述的重组载体。
本发明还提供了一种多重PCR引物,其中,所述多重PCR引物用于制备如上述所述的编码基因。
本发明还提供了上述所述的苯丙氨酸变位酶、上述所述的编码基因、上述所述的重组载体、上述所述的宿主细胞在制备β-苯丙氨酸中的应用。
通过上述技术方案,本发明通过对现有的苯丙氨酸变位酶进行定点突变,使得突变后的苯丙氨酸变位酶具有更好的催化活性,同时能够降低α-苯丙氨酸的生成能力。并通过建立表达载体,构建重组菌,实现高效表达,获得满足工业化生产要求的苯丙氨酸变位酶。
本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本发明的进一步理解,并且构成说明书的一部分,与下面的具体实施方式一起用于解释本发明,但并不构成对本发明的限制。在附图中:
图1是α-苯丙氨酸和β-苯丙氨酸标准样的高效液相色谱图;
图2是天然的苯丙氨酸变位酶催化3-苯基丙烯酸后的合成产物的高效液相色谱图;
图3是本发明的苯丙氨酸变位酶催化3-苯基丙烯酸后的合成产物的高效液相色谱图;
图4是SDS-PAGE(SDS-聚丙烯酰胺凝胶)电泳图;其中,泳道M为分子量标记,泳道a来自硫酸铵沉淀浓缩后的样品,泳道b来自实施例3中步骤4)最终得到的如SEQ ID NO:1所示的苯丙氨酸变位酶。
附图标记说明
1 α-苯丙氨酸 2 β-苯丙氨酸
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
本发明提供了一种苯丙氨酸变位酶,其中,所述苯丙氨酸变位酶的氨基酸序列如SEQ ID NO:1。
本发明通过对现有的苯丙氨酸变位酶进行定点突变,使得突变后的苯丙氨酸变位酶具有更好的催化活性,同时能够降低α-苯丙氨酸的生成能力。并通过建立表达载体,构建重组菌,实现高效表达,获得满足工业化生产要求的苯丙氨酸变位酶。当然,这里的苯丙氨酸变位酶的来源没有特别的限定,只要能够得到上述氨基酸序列的苯丙氨酸变位酶即可。例如,可以为人工进行合成,也可以为通过该氨基酸序列获得其编码基因,再由相对应的生物表达得到该苯丙氨酸变位酶。
本发明还提供了一种编码基因,其中,所述编码基因能够编码上述所述的苯丙氨酸变位酶。众所周知,遗传密码子具有简并性,本领域技术人员可以根据常规技术手段获得核苷酸序列不同,且能够编码上述苯丙氨酸变位酶的编码基因。
例如,在本发明的一种优选的实施方式中,所述编码基因的核苷酸序列可以进一步选择为如SEQ ID NO:2所示。
本发明还提供了一种重组载体,其中,所述重组载体含有根据上述所述的编码基因。
本发明还提供了一种宿主细胞,其中,所述宿主细胞含有根据上述所述的编码基因,或者含有根据上述所述的重组载体。
当然,这里的宿主细胞可以为本领域常规使用的细胞类型,例如,可以为原核细胞,进一步地,可以选择为大肠杆菌。
本发明还提供了一种多重PCR引物,其中,所述多重PCR引物用于制备上述所述的编码基因。
同样地,这里的多重PCR引物可以根据编码基因的不同,由本领域技术人员根据常规技术手段予以选择和获得。
进一步优选的实施方式中,所述多重PCR引物包括如SEQ ID NO:4和SEQ ID NO:5所示的引物对1,如SEQ ID NO:6和SEQ ID NO:7所示的引物对2,以及如SEQ ID NO:8和SEQID NO:9所示的引物对3。
本发明还提供了上述所述的苯丙氨酸变位酶、上述编码基因、上述重组载体、上述宿主细胞在制备β-苯丙氨酸中的应用。
以下将通过实施例对本发明进行详细描述。以下实施例中,所述引物均由上海生工生物工程技术有限公司合成且引物浓度为100pmol/L,各原料为常规市售品。
实施例1
将紫杉(Taxus chinensis)中的苯丙氨酸变位酶的基因序列(具体见NCBI公布的收录编号为AY724735.1的序列),提交给上海生工生物工程技术有限公司合成该酶的基因(并命名为pam),将上述该基因(pam)与克隆载体pUC载体连接,获得克隆载体pUC-pam。
将克隆载体pUC-pam和空表达载体pET28a分别用限制性内切酶EcoRⅠ和NdeⅠ进行双酶切,割胶回收目的基因片段和pET28a载体,然后将双酶切产物于16℃下过夜连接,将连接产物转入大肠杆菌E.coli JM109中,涂布含有50μg/mL的卡那霉素的LB平板上,于37℃培养,挑选单菌落,采用菌落PCR和质粒双酶切进行鉴定阳性克隆,得到重组质粒并命名为pET-28-pam;
以上述制得的重组质粒pET-28-pam为模板,利用PCR技术引物引起核苷酸突变(其突变引物对分别如SEQ ID NO:4和SEQ ID NO:5所示的引物对1,如SEQ ID NO:6和SEQ IDNO:7所示的引物对2,以及如SEQ ID NO:8和SEQ ID NO:9所示的引物对3所示),获得突变质粒pET-28-pam/Cys107His/Gln319Met/Asp458Phe。
PCR扩增体系为:
DNA聚合酶:0.5μL
10×buffer:5μL
DNA模板(即得到的重组质粒pET-28-pam):1μL
双蒸水:38.5μL
dNTP:3μL
引物:各1μL
PCR扩增条件为:94℃预变性1min;94℃变性1min,56℃退火30s,72℃延伸7min,共25个循环。
将上述PCR扩增产物采用DNA纯化试剂盒进行纯化回收。
实施例2
将上述纯化回收的PCR扩增产物采用DpnI限制性内切酶于37℃下酶切消化1h。
消化反应体系为:
DpnI限制性内切酶:0.5μL
10×buffer:1μL
纯化回收后的PCR扩增产物:8μL
双蒸水:0.5μL
将上述消化后的产物采用DNA纯化试剂盒进行纯化回收。
实施例3
取实施例2中消化后的回收的产物在42℃的条件下热击60s,转化至大肠杆菌E.coli JM109感受态细胞。而后涂布于含Kan抗性(10mg/L)的固体LB平板上,于37℃条件下培养8h。然后挑取单菌落,接入含有50mg/L的Kan的LB液体培养基中培养,提取质粒,进行酶切和PCR验证。选择阳性克隆质粒送至上海生工测序。测序正确的质粒在42℃的条件下60s后转化大肠杆菌E.coli BL21感受态细胞,而后涂布于含Kan抗性(10mg/L)的LB平板上,于37℃条件下培养8h,挑选阳性转化子,即为能够产生如SEQ ID NO:1所示的苯丙氨酸变位酶的宿主细菌,该酶的编码基因如SEQ ID NO:2所示。
实施例4
1)将实施例3中制得的宿主细菌接入LB液体培养基中于37℃条件下培养8h,获得种子液。将种子液转接至新鲜的LB培养基中于37℃条件下培养至OD600达0.6时,添加终浓度0.5mmol/L的IPTG(异丙基硫代-β-D-半乳糖苷),并在26℃下进行诱导24h,从而获得大量的含有如SEQ ID NO:1所示的苯丙氨酸变位酶的游离细胞。
2)采用超声破碎方法对步骤1)中制得的游离细胞进行破碎(功率250W,超声1s,间歇3s,共15min)后冷冻离心,收集上层清液,制备获得无细胞抽提液,向获得的无细胞抽提液中加入硫酸铵至硫酸铵饱和度为55%,而后置于4℃的条件下静置使如SEQ ID NO:1所示的苯丙氨酸变位酶充分沉淀,低温离心收集该沉淀。将上述沉淀溶解在浓度为25mmol/L,且pH为8.7的Tris盐酸缓冲液中并且于4℃条件下透析;
3)将上述透析后的如SEQ ID NO:1所示的苯丙氨酸变位酶蛋白液加入到预先用浓度为25mmol/L,且pH为8.7的Tris盐酸缓冲液平衡的阴离子交换柱Resource Q中。用25mmol/L,且pH为8.7的Tris盐酸缓冲液(其中,该Tris盐酸缓冲液中还含有浓度不大于0.5mol/L的氯化钠)进行线性梯度洗脱,洗脱时的流速为1mL/min,检测波长为280nm,收集有活性的如SEQ ID NO:1所示的苯丙氨酸变位酶。
4)将步骤3)中收集到的全部活性部分用硫酸铵(饱和度为55%)浓缩并用少量pH为8.7的Tris盐酸缓冲液溶解,将浓缩后的酶液加入到预先用pH为8.7,浓度为25mmol/L的Tris盐酸缓冲液平衡好的Superdex 7510/300GL层析柱中,用含0.5mol/L氯化钠的pH为8.7,浓度为25mmol/L的Tris盐酸缓冲液进行洗脱,流速为0.4mL/min,检测波长为280nm,收集有活性的部分,得到如SEQ ID NO:1所示的苯丙氨酸变位酶,同时进行SDS-PAGE检测该苯丙氨酸变位酶的纯度,检测后得到的电泳图如图4所示。
5)测定纯蛋白的酶活性,酶活性定义是指样品在3-苯基丙烯酸为0.1mmol/L,反应体系pH为8.7,25mmol的Tris盐酸缓冲液,反应温度为30℃的条件下每分钟生成1mmol的β-苯丙氨酸需要的酶量为1个酶单位(U),步骤4)得到的洗脱后的如SEQ ID NO:1所示的苯丙氨酸变位酶的酶活性为4.2U/mg,而来源于紫杉(Taxus chinensis)的天然苯丙氨酸变位酶(其氨基酸序列如SEQ ID No:3所示)的酶活性为2.6U/mg。
测试例
向步骤4)中最终获得的含有如SEQ ID NO:1所示的苯丙氨酸变位酶的溶液(其中,该溶液中含有洗脱时的pH为8.7,浓度为25mmol/L的Tris盐酸缓冲液)中添加100μmol的3-苯基丙烯酸,在温度为30℃的条件下转化合成β-苯丙氨酸,利用高效液相色谱仪监测反应进程,结果表明,3-苯基丙烯酸12h以内即能转化完全,高效液相色谱仪检测反应液中的α-苯丙氨酸含量,本发明中的苯丙氨酸变位酶催化与野生型的酶催化相比,α-苯丙氨酸含量下降了80%,含量仅仅为6.8%(图1-3所示)。
通过上述可以看出,本发明通过将对来源于紫杉(Taxus chinensis)的天然苯丙氨酸变位酶的氨基酸序列(如SEQ ID No:3所示)进行改造,将天然的苯丙氨酸变位酶氨基酸序列的458位天冬酰胺(Asp),107位的半胱氨酸(Cys)和319位的谷氨酰胺(Gln)分别突变为苯丙氨酸(Phe),组氨酸(His)和甲硫氨酸(Met),获得本发明中的苯丙氨酸变位酶,提高了其催化活力,本发明中的苯丙氨酸变位酶的酶活达到4.2U/mg,相比天然酶的酶活(2.6U/mg)提高了1.6倍,并且使副产物α-苯丙氨酸形成能力显著降低。结果表明,以3-苯基丙烯酸为底物,副产物α-苯丙氨酸生成量下降了80%,含量仅仅为6.8%(如图1-图3所示)。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
SEQUENCE LISTING
<110> 安徽工程大学
<120> 苯丙氨酸变位酶、编码基因、重组载体、宿主细胞、多重PCR引物及它们的
应用
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<223> The sequence is synthesized.
<400> 2
atggggtttg ccgtggaatc gcgttctcac gtaaaggata tattggggct gatcaacgcg 60
ttcaacgagg tgaagaaaat tacagtagac ggtacgaccc ccatcacggt ggcccatgtc 120
gcggcgctgg cccggaggca tgacgtgaag gttgcgttgg aggcggagca atgcagagcc 180
cgtgtggaaa cctgctcttc gtgggtgcag cgcaaggcgg aagacggcgc cgacatatac 240
ggcgtcacca cgggcttcgg cgcgtgctcg agccggagga ccaaccggct gagcgagctg 300
caggagtcgc tcatacgcca cctgctcgcg ggggtgttta ctaaaggatg cgctccctcc 360
gtcgacgagc tccccgcgac cgccacccgc agcgccatgc tgctccgcct taatagtttt 420
acctatggat gttccggcat ccggtgggag gtcatggaag cgctggaaaa gcttctcaac 480
agcaatgtct ctcctaaagt gcctctccgg ggttctgtga gcgcttcggg agacctcatc 540
ccgctcgcct acattgcagg gctcctgatc gggaagccta gcgtaatcgc tcgcataggc 600
gacgatgtcg aggtccctgc gcccgaggcg ttgagcaggg tggggcttcg gccattcaag 660
ctccaggcca aagaagggct ggcgctcgtc aacggcacct ccttcgccac cgcggtcgcc 720
tccaccgtca tgtacgacgc caatgttctg ttgctgctcg tcgaaacgct ttgcggaatg 780
ttctgcgagg tgatctttgg aagggaggag ttcgcgcatc cgctgatcca taaagtgaag 840
ccgcacccgg gccagatcga atcggcggag ctgctcgagt ggctgctgcg gtcgagcccg 900
tttcaggagc tgtcgaggga gtattacagt attgataagc tgaagaaacc gaaaatggat 960
cgctatgctc tgaggtcgag cccgcagtgg ttggctcctc tggtgcagac aatcagagac 1020
gccaccacta cagtggagac ggaggtcaat tccgccaatg ataaccccat cattgaccac 1080
gccaatgaca gggctctcca tggtgcgaat ttccagggca gcgccgtcgg tttctacatg 1140
gactacgtgc gcatcgcagt agccgggctg gggaaactct tgttcgctca gttcacggag 1200
ctgatgatcg aatattacag caacggccta ccggggaacc tctccctggg gccggacctg 1260
agcgtggact acggcctcaa ggggctcgac atcgccatgg ccgcctacag ctccgagctt 1320
cagtacctgg cgaatcccgt gaccacacac gtgcacagcg cggaacagca ctttcaggac 1380
atcaactctc tggcgctcat ctccgcccgc aagacggagg aggcgttgga tatcttaaag 1440
ctcatgatcg cctcgcattt aacagcaatg tgccaggcgg tggaccttcg gcagctcgaa 1500
gaagccctag taaaagtcgt ggagaatgtc gtttccaccc ttgcagacga atgcggcctc 1560
cctaacgaca caaaggcgag gcttttatat gtagccaaag cggtgcctgt ttacacatac 1620
ctggaatccc cctgcgaccc cacgcttccc ctcttgttag gcctgaaaca gtcctgtttc 1680
gataccattc tggctctcca caaaaaagac ggcattgaga cggacacctt ggtcgatcgg 1740
ctcgccgagt tcgagaagcg gctgtccgac cgcctggaaa acgagatgac ggcagtgagg 1800
gttttgtacg aaaagaaagg gcataaaacg gcagacaaca acgacgccct cgtgagaatc 1860
cagggttcca aattccttcc tttttacaga tttgttcggg aagagctcga cacaggtgtg 1920
atgagtgcga gaagagagca gacgccgcaa gaggacgtgc agaaagtgtt cgatgcaatt 1980
gccgacggca gaattacggt gcctctactg cactgcctgc aagggtttct cggccaacca 2040
aatgggtgcg ccaacggcgt ctag 2064
<210> 3
<211> 687
<212> PRT
<213> Taxus chinensis
<400> 3
Met Gly Phe Ala Val Glu Ser Arg Ser His Val Lys Asp Ile Leu Gly
1 5 10 15
Leu Ile Asn Ala Phe Asn Glu Val Lys Lys Ile Thr Val Asp Gly Thr
20 25 30
Thr Pro Ile Thr Val Ala His Val Ala Ala Leu Ala Arg Arg His Asp
35 40 45
Val Lys Val Ala Leu Glu Ala Glu Gln Cys Arg Ala Arg Val Glu Thr
50 55 60
Cys Ser Ser Trp Val Gln Arg Lys Ala Glu Asp Gly Ala Asp Ile Tyr
65 70 75 80
Gly Val Thr Thr Gly Phe Gly Ala Cys Ser Ser Arg Arg Thr Asn Arg
85 90 95
Leu Ser Glu Leu Gln Glu Ser Leu Ile Arg Cys Leu Leu Ala Gly Val
100 105 110
Phe Thr Lys Gly Cys Ala Pro Ser Val Asp Glu Leu Pro Ala Thr Ala
115 120 125
Thr Arg Ser Ala Met Leu Leu Arg Leu Asn Ser Phe Thr Tyr Gly Cys
130 135 140
Ser Gly Ile Arg Trp Glu Val Met Glu Ala Leu Glu Lys Leu Leu Asn
145 150 155 160
Ser Asn Val Ser Pro Lys Val Pro Leu Arg Gly Ser Val Ser Ala Ser
165 170 175
Gly Asp Leu Ile Pro Leu Ala Tyr Ile Ala Gly Leu Leu Ile Gly Lys
180 185 190
Pro Ser Val Ile Ala Arg Ile Gly Asp Asp Val Glu Val Pro Ala Pro
195 200 205
Glu Ala Leu Ser Arg Val Gly Leu Arg Pro Phe Lys Leu Gln Ala Lys
210 215 220
Glu Gly Leu Ala Leu Val Asn Gly Thr Ser Phe Ala Thr Ala Val Ala
225 230 235 240
Ser Thr Val Met Tyr Asp Ala Asn Val Leu Leu Leu Leu Val Glu Thr
245 250 255
Leu Cys Gly Met Phe Cys Glu Val Ile Phe Gly Arg Glu Glu Phe Ala
260 265 270
His Pro Leu Ile His Lys Val Lys Pro His Pro Gly Gln Ile Glu Ser
275 280 285
Ala Glu Leu Leu Glu Trp Leu Leu Arg Ser Ser Pro Phe Gln Glu Leu
290 295 300
Ser Arg Glu Tyr Tyr Ser Ile Asp Lys Leu Lys Lys Pro Lys Gln Asp
305 310 315 320
Arg Tyr Ala Leu Arg Ser Ser Pro Gln Trp Leu Ala Pro Leu Val Gln
325 330 335
Thr Ile Arg Asp Ala Thr Thr Thr Val Glu Thr Glu Val Asn Ser Ala
340 345 350
Asn Asp Asn Pro Ile Ile Asp His Ala Asn Asp Arg Ala Leu His Gly
355 360 365
Ala Asn Phe Gln Gly Ser Ala Val Gly Phe Tyr Met Asp Tyr Val Arg
370 375 380
Ile Ala Val Ala Gly Leu Gly Lys Leu Leu Phe Ala Gln Phe Thr Glu
385 390 395 400
Leu Met Ile Glu Tyr Tyr Ser Asn Gly Leu Pro Gly Asn Leu Ser Leu
405 410 415
Gly Pro Asp Leu Ser Val Asp Tyr Gly Leu Lys Gly Leu Asp Ile Ala
420 425 430
Met Ala Ala Tyr Ser Ser Glu Leu Gln Tyr Leu Ala Asn Pro Val Thr
435 440 445
Thr His Val His Ser Ala Glu Gln His Asp Gln Asp Ile Asn Ser Leu
450 455 460
Ala Leu Ile Ser Ala Arg Lys Thr Glu Glu Ala Leu Asp Ile Leu Lys
465 470 475 480
Leu Met Ile Ala Ser His Leu Thr Ala Met Cys Gln Ala Val Asp Leu
485 490 495
Arg Gln Leu Glu Glu Ala Leu Val Lys Val Val Glu Asn Val Val Ser
500 505 510
Thr Leu Ala Asp Glu Cys Gly Leu Pro Asn Asp Thr Lys Ala Arg Leu
515 520 525
Leu Tyr Val Ala Lys Ala Val Pro Val Tyr Thr Tyr Leu Glu Ser Pro
530 535 540
Cys Asp Pro Thr Leu Pro Leu Leu Leu Gly Leu Lys Gln Ser Cys Phe
545 550 555 560
Asp Thr Ile Leu Ala Leu His Lys Lys Asp Gly Ile Glu Thr Asp Thr
565 570 575
Leu Val Asp Arg Leu Ala Glu Phe Glu Lys Arg Leu Ser Asp Arg Leu
580 585 590
Glu Asn Glu Met Thr Ala Val Arg Val Leu Tyr Glu Lys Lys Gly His
595 600 605
Lys Thr Ala Asp Asn Asn Asp Ala Leu Val Arg Ile Gln Gly Ser Lys
610 615 620
Phe Leu Pro Phe Tyr Arg Phe Val Arg Glu Glu Leu Asp Thr Gly Val
625 630 635 640
Met Ser Ala Arg Arg Glu Gln Thr Pro Gln Glu Asp Val Gln Lys Val
645 650 655
Phe Asp Ala Ile Ala Asp Gly Arg Ile Thr Val Pro Leu Leu His Cys
660 665 670
Leu Gln Gly Phe Leu Gly Gln Pro Asn Gly Cys Ala Asn Gly Val
675 680 685
<210> 4
<211> 36
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 4
tcgctcatac gccacctgct cgcgggggtg tttact 36
<210> 5
<211> 36
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 5
ccccgcgagc aggtggcgta tgagcgactc ctgcag 36
<210> 6
<211> 36
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 6
aagaaaccga aaatggatcg ctatgctctg aggtcg 36
<210> 7
<211> 36
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 7
agcatagcga tccattttcg gtttcttcag cttatc 36
<210> 8
<211> 36
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 8
gcggaacagc actttcagga catcaactct ctggcg 36
<210> 9
<211> 36
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 9
gttgatgtcc tgaaagtgct gttccgcgct gtgcac 36
Claims (6)
1.一种苯丙氨酸变位酶,其特征在于,所述苯丙氨酸变位酶的氨基酸序列如SEQ IDNO:1所示。
2.一种编码基因,其特征在于,所述编码基因能够编码权利要求1所述的苯丙氨酸变位酶。
3.根据权利要求2所述的编码基因,其中,所述编码基因的核苷酸序列如SEQ ID NO:2所示。
4.一种重组载体,其特征在于,所述重组载体含有根据权利要求2或3所述的编码基因。
5.一种宿主细胞,其特征在于,所述宿主细胞含有根据权利要求2或3所述的编码基因,或者含有根据权利要求4所述的重组载体。
6.权利要求1所述的苯丙氨酸变位酶、权利要求2或3所述的编码基因、权利要求4所述的重组载体、权利要求5所述的宿主细胞在制备β-苯丙氨酸中的应用。
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Non-Patent Citations (4)
Title |
---|
Mechanism-inspired engineering of phenylalanine aminomutase for enhanced β-regioselective asymmetric amination of cinnamates.;WU Bian等;《Angewandte Chemie International Edition》;20121231;第51卷(第2期);482-486 * |
Mechanistic,mutational,and structural evaluation of a Taxus phenylalanine aminomutase.;LEI FENG等;《Biochemistry》;20111231;第50卷(第14期);2919-2930 * |
Phenylalanime Aminomutase-Catalyzed Addition of Ammonia to Subsituted Cinnamic Acids:a Route to Enantiopure α-and β-Amino Acids.;SZYMANSKI W等;《Journal of Organic Chemistry》;20091231;第74卷(第23期);9152-9157 * |
产β-苯丙氨酸变位酶基因工程菌的构建及其发酵条件优化;葛飞等;《基础研究》;20170131;第33卷(第1期);16-21 * |
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