CN107794263B - 一种提高黑色素产率的基因及其应用 - Google Patents
一种提高黑色素产率的基因及其应用 Download PDFInfo
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- CN107794263B CN107794263B CN201710915508.8A CN201710915508A CN107794263B CN 107794263 B CN107794263 B CN 107794263B CN 201710915508 A CN201710915508 A CN 201710915508A CN 107794263 B CN107794263 B CN 107794263B
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Abstract
本发明公开了一种提高黑色素产率的基因，该基因是将基因Cjhppd的编码蛋白的氨基酸序列的第280位的Tyr突变成Phe，命名为Cjhppd Y280F，其核苷酸序列如SEQ ID NO:1所示；同时公开了该基因在生产黑色素中的应用。本发明提供的基因Cjhppd Y280F能够显著提高脓黑素的产量，将其用于生产脓黑素具有方法简单、生产成本低，生产周期短、产量高、可以控制等优点，且生产的脓黑素为天然黑色素，不需要动、植物原材料，其与人工合成黑色素相比具有安全、无毒等优势。本发明生产的黑色素可以作为原料用于制备保健食品、日用化妆品、防晒膏、防晒霜、黑发剂以及生物菌肥的菌体保护和酶制剂等。
Description
技术领域
本发明涉及基因工程技术领域，具体地说是一种提高黑色素产率的基因及其应用。
背景技术
黑色素（Melanin）是一类分子结构未知的高分子量黑色色素类物质，广泛存在于动物、植物和微生物体中，其结构复杂多样，能提高生物生存、竞争的能力。黑色素是一种生物大分子，难溶于水，不溶于大部分有机溶剂和酸液。黑色素的结构非常复杂，到目前为止，行业内的研究人员仍然对其缺乏全面、清楚的认识。据研究表明，黑色素的基础结构是一些共价交联的吲哚环，而且不同生物体产生的黑色素的结构也各不相同。一般来讲，黑色素分为四种类型，分别是真黑素（eumelanin）、棕黑素（phaeomelanin）、异黑素（allomelanin）和脓黑素（pyomelanin）。
随着研究的深入，人们发现天然黑色素有着非常强大的功能，其具有抗氧化、防止衰老、防止紫外线辐射等，因此，人们可以利用天然黑色素制备各种功能性日用化妆品，如防晒膏、防晒霜、染发剂等，也可以将其作为酒类、饮料类、婴儿保健品及大众食品的添加剂研制出各种保健食品。另外，人们研究发现天然黑色素还与某些疾病，衰老，肤色等有关，如近年来，人们发现一些可溶性黑色素在体外对艾滋病病毒有显著的抑制作用，因此，天然黑色素将来有可能成为一种新的抗艾滋病药物。此外，天然黑色素还可以作为一种生物半导体材料应用。
由于天然色素与人工合成色素相比具有安全、无毒、营养价值高等优点，这就促使人们更多地获得天然色素。目前，天然色素主要是从动物、植物和微生物中提取。其中从动植物中提取的色素成本高，应用受到一定局限，而利用微生物生产天然色素，不仅可以克服动植物为原料生产天然色素的很多缺点，并且易于工业化。目前，人们已发现多种微生物可以用来生产色素，如将类胡萝卜素在大肠杆菌、产朊假丝酵母、酿酒酵母和运动发酵单孢菌中进行生产；如Minra等将欧氏杆菌中控制番茄红素合成的基因转入产蛋白假丝酵母，得到番茄红素和八氢番茄红素；如阮丽芳等将嗜麦芽假单胞菌中产黑色素 mel 基因克隆到穿梭载体p HT3101中，将构建的重组质粒 p HTAM 转入苏云金芽胞杆菌受体菌株 BMB171中，得到重组菌株 RSA，mel 基因得到成功表达；如从Streptomyces avermitilis 克隆得到的一段基因HPDs 在大肠杆菌中表达，并在发酵液中发现产生一种由尿黑酸氧化聚合而产生的色素；又如Steven等将海洋细菌Shewanella colwellian D的hppd基因克隆到大肠杆菌中发酵产生脓黑素；Keon从Mycosphaerella graminicola 中分离出 hppd 基因并克隆到大肠杆菌中，在发酵液中得到脓黑素；也如姚红宝在硕士论文中公开了将黄连 hppd 基因在大肠杆菌中进行原核表达，重组菌的表达产物 hppd催化对羟苯基丙酮酸氧化形成尿黑酸，尿黑酸暴露在氧气中时发生氧化聚合形成一种褐色色素-脓黑素。虽然现有研究已经通过转入特定基因的工程菌来生产黑色素，但是，我们发现这些研究中的生产黑色素的产量相对偏低。
发明内容
本发明的目的是提供一种提高黑色素产率的基因及其应用，为生产黑色素提供了一种高效的生产途径。
本发明的目的是通过以下技术方案实现的：一种提高黑色素产率的基因，该基因是将基因Cjhppd的编码蛋白的氨基酸序列的第280位的Tyr突变成Phe，命名为Cjhppd Y280F，基因Cjhppd Y280F的核苷酸序列如SEQ ID NO : 1所示。
其中基因Cjhppd的核苷酸序列如SEQ ID NO :3所示；所述基因Cjhppd的编码蛋白的氨基酸序列如SEQ ID NO :4所示。
一种核苷酸序列如SEQ ID NO : 1所示的提高黑色素产率的基因所编码蛋白，其氨基酸序列如SEQ ID NO : 2所示。
一种含有核苷酸序列如SEQ ID NO : 1所示的提高黑色素产率的基因的质粒，所述质粒为pET21d-Cjhppd Y280F；所述质粒中的基因处于T7表达框架的调控之下。
一种含有核苷酸序列如SEQ ID NO : 1所示的提高黑色素产率的基因的工程菌株，即将质粒pET21d-Cjhppd Y280F转入到大肠杆菌BL21（DE3）感受态细胞得到的重组菌株。
本发明还公开了如SEQ ID NO : 1所示的提高黑色素产率的基因在生产黑色素中的应用，其具体的应用方法是：将基因Cjhppd Y280F连接到质粒载体上，导入大肠杆菌菌株中，获得能够产生黑色素的重组菌，活化培养，诱导表达使其产生黑色素，分离提取，获得黑色素。
所述质粒载体为pET21d，所述大肠杆菌菌株为BL21（DE3）。
所述诱导表达是指将重组菌活化后，按体积比为1:50的比例接种于装有50 mL的LB液体发酵培养基（附加100μg/mL氨苄青霉素）的250 mL三角瓶中，220 r/min转速，37℃下振荡培养；当培养液菌浓度达到A600≈0.6时，加入IPTG至终浓度为1 mmol/L，培养温度调整到30℃，诱导重组hppd酶蛋白表达，继续培养重组菌产生色素，48 h后停止发酵。
本发明通过分子生物学方法将基因Cjhppd的编码蛋白的氨基酸序列中280位由原来的酪氨酸（Tyr, Y）突变成苯丙氨酸（Phe, F），将突变后的基因Cjhppd Y280F连接到质粒上并转入大肠杆菌内，获得了与基因Cjhppd相似的的表达载体和重组菌，通过诱导表达产生黑色素，特别地，在研究过程中意外发现突变后的基因Cjhppd Y280F较突变前能够显著提高黑色素的产量；而且突变后的基因Cjhppd Y280F产生的黑色素与突变前基因Cjhppd产生的黑色素属于相同的脓黑素，且同样在光照和温度变化条件下以及外加氧化剂和还原剂的条件下保持稳定的特性；且添加物蔗糖、葡萄糖不影响色素的稳定性；对细菌具有紫外线保护作用，并且具有抗氧化及可能的酶蛋白活性保护作用。
将本发明提供的基因Cjhppd Y280F应用于生产脓黑素，具有方法简单、生产成本低，生产周期短、产量高、可以控制等优点，且生产的脓黑素为天然黑色素，不需要动、植物原材料，与人工合成黑色素相比有无毒、安全等优势。本发明生产的黑色素可以用于保健食品工业、日用化妆品工业，制备防晒膏、防晒霜和黑发剂洗护工业以及生物菌肥的菌体保护和酶制剂等领域。
附图说明
图1为实施例1和对比例1得到的重组菌株所生产的黑色素的傅里叶红外光谱图。
图2为实施例4制作的黑色素的质量浓度与OD值关系的标准曲线。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。实施例中所用的试验材料、试剂等，如无特殊说明，均可从商业途径得到。
以下实施例所使用的部分培养基、母液配方如下：
LB液体基本培养基：NaCl 10 g/L、蛋白胨10 g/L、酵母粉5 g/L，pH＝7.0。若为固体培养需在LB液体基本培养基中再加琼脂15 g/L即可。
LB液体发酵培养基：LB液体基本培养基+1 g/L的L-酪氨酸。
氨苄青霉素母液（100 mg/mL）：溶解1 g氨苄青霉素钠盐于足量的灭菌水中，最后定容至10 mL，用0.22 μm滤膜过滤除菌。
实施例1
1、突变：采用北京全式金生物技术公司Fast Mutagenesis System进行，以重组质粒pET21d-Cjhppd为目标质粒，设计突变位点，将基因Cjhppd的编码蛋白的氨基酸序列的第280位的Tyr突变成Phe，即选择Y280位点突变成F，相应的密码子由TAT突变成TTT。其中已公开基因Cjhppd的核苷酸序列如SEQ ID NO:3所示，其编码氨基酸序列如SEQ ID NO:4所示；突变后基因Cjhppd-Y280F的核苷酸序列如SEQ ID NO:1所示，其编码氨基酸序列如SEQ IDNO:2所示。
2、设计点突变引物：引物序列(5'—3')为：
Cjhppd Y280F Fw：GCCAGATACAAACTTTTTTGGAACATAA；
Cjhppd Y280F Rv：AAAGTTTGTATCTGGCTCTTTCTCTTTG。
3、PCR体系及条件：
PCR体系如下：
Template（模板） 质粒 0.2 μl
Forward Primer（正向引物）（10 μM） 1 μl
Reverse Primer（反向引物）（10 μM） 1 μl
TransStartFastPfuFly DNA Polymerase（TransStartFastPfuFly DNA聚合酶） 1μl
5×TransStart FastPfuFly Buffer（TransStartFastPfuFly 缓冲液） 10 μl
dNTPs（脱氧核糖核苷三磷酸）（2.5 mM each） 5 μl
ddH2O to final volume（加超纯水至终体积） 50 μl
PCR条件如下：
采用质量比浓度为1.5%琼脂糖凝胶电泳检测PCR产物；
加1 μl DMT酶于PCR产物中，轻混，37℃孵育1 h。
4、转化
（1）加入5 μl DMT酶消化产物于50 μl DMT感受态细胞中（在感受态细胞刚刚解冻时加入产物），轻弹混匀，冰浴30 min；
（2）42 ℃准确热激45 sec，立即置于冰上2 min；
（3）加500 μL 平衡至室温的SOC，250 rpm，37℃培养1 h；
（4）在4000 rpm离心2 min，弃掉部分上清，保留100 μL，轻吹悬浮菌体，取全部菌液涂板（Amp+ 终浓度为100 μg/mL），37℃培养过夜；
5、挑单菌落摇菌后送测序公司测序（挑取3个克隆），同时加甘油保存菌种。
6、比对测序结果，取位点突变正确核苷酸序列如SEQ ID NO : 1所示、编码氨基酸序列如SEQ ID NO : 2所示的菌株进行摇菌，提取相应的突变菌株对应的质粒。
7、取点突变的质粒及原始质粒转入大肠杆菌BL21（DE3）感受态细胞。
（1）加入1 μL质粒于50 μL的大肠杆菌BL21（DE3）感受态细胞中（在感受态细胞刚刚解冻时加入产物），轻弹混匀，冰浴30 min；
（2）42℃热激45 sec，立即置于冰上2 min；
（3）加500 μL 平衡至室温的SOC，250 rpm，37℃培养1 h；
（4）在4000 rpm离心2 min，弃掉部分上清，保留100 μl，轻吹悬浮菌体，取全部菌液涂板（Amp+ 终浓度为100 μg/mL），37℃培养过夜；挑取单菌落摇菌后，加甘油保存菌种。
通过以上步骤对目标质粒pET21d-Cjhppd进行点突变，并将点突变质粒pET21d-Cjhppd Y280F转化到克隆感受态细胞中，对测序正确的单克隆进行质粒提取，并将质粒pET21d-Cjhppd Y280F转入表达大肠杆菌菌株BL21（DE3）感受态细胞中，得重组菌。
实施例2
1、重组菌的活化培养：将转质粒pET21d-Cjhppd Y280F的重组大肠杆菌菌株BL21(DE3)进行活化培养，在超净工作台下，用灭菌牙签分别挑取-80℃冰箱保存的实施例1获得的重组菌，接种在含5mL LB液体发酵培养基（附加100μg/mL氨苄青霉素）的50mL三角瓶中，过夜培养活化。
2、重组菌诱导表达及色素产生
将重组菌活化后，按体积比为1:50的比例接种于装有50mL LB液体发酵培养基（附加100μg/mL氨苄青霉素）的250 mL三角瓶中，220 r/min转速下振荡培养。当培养液菌浓度达到A600≈0.6时，加入IPTG至终浓度为1 mmol/L，诱导重组hppd酶蛋白表达，继续培养重组菌产生色素，48 h后停止发酵，测定发酵液中色素产量。
3、色素的分离提取
重组菌培养液经一层滤纸和两层神奇滤布（Miracloth）过滤去除菌体，用6mol/L盐酸调整滤液pH至2，静置4h，8000r/min离心20min，收集沉淀得色素粗提物。粗提物溶解于用5mol/L氢氧化钠调整pH值为12的超纯水中，充分混匀后，8000r/min，离心20min，取上清调整pH值至2，沉淀色素，8000r/min，离心20min，收集沉淀，再重复以上步骤两次；之后，二次无水乙醇、一次丙酮依次洗涤沉淀，8000r/min，离心20min，直到上清液为无色；最后将沉淀60℃烘干至恒重，即为色素提取物-脓黑素。
对比例1
以质粒pET21d-Cjhppd为目标质粒，基因Cjhppd的核苷酸序列如SEQ ID NO : 3所示，其编码氨基酸序列如SEQ ID NO : 4所示，将其转入表达大肠杆菌BL21（DE3）感受态细胞中，得重组菌，采用如实施例2相同的具体步骤进行菌株活化培养、诱导表达及色素产生、测定发酵液中色素的产量，色素的分离提取，得到脓黑素。
实施例3 产生的脓黑素的红外光谱（IR）
对实施例2和对比例1得到的脓黑素用KBr压片600-4000cm-1扫描，其结果如图1所示，图中显示，两种菌株产生的脓黑素红外光谱相同，说明为同种黑色素。
实施例4 色素产量测定
1、色素标准曲线测定
采用分光光度法测定色素的吸收光谱及吸收峰，选择进行测量色素的波长。
将实施例3中提取到的色素50mg先用1mL浓度为1 mol/L的NaOH溶液溶解，然后加水定容至10mL，并调pH至7.0 （本实验所用色素溶液均按此方法配制），配制成5 mg/mL的色素溶液母液备用。稀释一定倍数使质量浓度分别为5mg/mL、4mg/mL、3mg/mL、2mg/mL、1mg/mL、0.5mg/mL，进行紫外可见光谱扫描，并测定400 nm处的吸光值，以色素质量浓度为横坐标，测定的OD400为纵坐标，制作标准曲线如图2所示。
2、发酵液中色素的测定
将实施例2和对比例1中的发酵液收集，同等条件下，测定400 nm处的吸光值，减去相同条件下空载体菌体发酵液的在400 nm处的吸光值，计算出重组菌发酵液中色素的OD400。再将OD400代入制备的标准曲线计算出重组菌发酵液中色素的产量，其测定结果见表1。这里的空载体指的是在表达菌体BL21（DE3）中含有空载体pET21d的菌体。
表1 实施例2和对比例1的脓黑素产量结果（mg/mL)
SEQUENCE LISTING
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gatgctacaa acactgctcg cagattctca tggggtctcg gtatgccaat ggtcgcaaaa 180
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gcaaatggcg cgaaaccttc gtcctctccg attaaactcg atgaaggcgt agttctttcg 480
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agttgtccat ttttacctgg gtttgaagaa gtgggtgaag tttcgtcatc tcgaggacta 600
gattttggta ttagaaggtt agaccatgca gtaggaaatg taccaaattt ggcagaagca 660
ataggatatt tgaaagagtt taccgggttt catgagtttg ctgagttcac agcagaagat 720
gtaggaacca ccgaaagcgg gttgaactcc atagttttag ctagtaatga tgaaatggta 780
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ttggaacata atgaaggtgc tggtgttcag catttggctt tggttagtga agatatattt 900
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aggcttggat gcatgttaaa ggatgaagaa gggaaaacat atcagaaggc aggatgtgga 1200
ggatttggga agggtaactt ctctgagctc ttcaaatcta ttgaagaata tgagaagact 1260
ttggaggcca aagctaatgt tgttgctgca tag 1293
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Met Val Pro Ser Thr Ala Ser Asn Leu Lys Leu Val Gly His Thr Asn
1 5 10 15
Phe Val His Asn Asn Pro Lys Ser Asp Lys Phe His Val Lys Lys Phe
20 25 30
His His Ile Glu Phe Trp Ser Thr Asp Ala Thr Asn Thr Ala Arg Arg
35 40 45
Phe Ser Trp Gly Leu Gly Met Pro Met Val Ala Lys Ser Asp Leu Ser
50 55 60
Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Arg Ser Gly Glu Leu
65 70 75 80
Asn Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Ile Ala Gly Asn Thr
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Leu Thr His Thr Ala Ser Ile Pro Thr Tyr Ser His Asn Leu Ala Arg
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Leu Phe Ala Ser Thr His Gly Leu Ala Val Arg Ala Ile Ala Ile Glu
115 120 125
Val Gln Asp Ala Glu Leu Ala Tyr Asn Ile Ser Val Ala Asn Gly Ala
130 135 140
Lys Pro Ser Ser Ser Pro Ile Lys Leu Asp Glu Gly Val Val Leu Ser
145 150 155 160
Glu Ile Gln Leu Tyr Gly Asp Val Val Leu Arg Tyr Leu Ser Phe Lys
165 170 175
Asn Thr Asn Gln Ser Cys Pro Phe Leu Pro Gly Phe Glu Glu Val Gly
180 185 190
Glu Val Ser Ser Ser Arg Gly Leu Asp Phe Gly Ile Arg Arg Leu Asp
195 200 205
His Ala Val Gly Asn Val Pro Asn Leu Ala Glu Ala Ile Gly Tyr Leu
210 215 220
Lys Glu Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala Glu Asp
225 230 235 240
Val Gly Thr Thr Glu Ser Gly Leu Asn Ser Ile Val Leu Ala Ser Asn
245 250 255
Asp Glu Met Val Leu Leu Pro Met Asn Glu Pro Val Tyr Gly Thr Lys
260 265 270
Arg Lys Ser Gln Ile Gln Thr Phe Leu Glu His Asn Glu Gly Ala Gly
275 280 285
Val Gln His Leu Ala Leu Val Ser Glu Asp Ile Phe Thr Thr Leu Arg
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Glu Met Arg Arg Arg Ser Gly Val Gly Gly Phe Glu Phe Met Pro Ser
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Pro Pro Pro Thr Tyr Tyr Lys Asn Leu Lys Asn Arg Ala Gly Asp Val
325 330 335
Leu Ser Asp Glu Gln Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val
340 345 350
Asp Arg Asp Ala Gln Gly Thr Leu Leu Gln Ile Phe Thr Lys Pro Val
355 360 365
Gly Asp Arg Pro Thr Ile Phe Val Glu Ile Ile Gln Arg Leu Gly Cys
370 375 380
Met Leu Lys Asp Glu Glu Gly Lys Thr Tyr Gln Lys Ala Gly Cys Gly
385 390 395 400
Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu
405 410 415
Tyr Glu Lys Thr Leu Glu Ala Lys Ala Asn Val Val Ala Ala
420 425 430
<210> 3
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atggttccca gcacagcctc caatttgaag ctagtcggcc acacaaattt tgtacacaat 60
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gatgctacaa acactgctcg cagattctca tggggtctcg gtatgccaat ggtcgcaaaa 180
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aattttctat tcactgcacc ttattcacct tcaatagctg gaaatacgtt aactcatact 300
gcttcaattc caacttattc gcacaacctc gcgcgattgt tcgctagtac gcatgggctc 360
gcggtgcgcg ctatcgcgat tgaagtccag gacgcggagt tggcgtataa tataagtgtc 420
gcaaatggcg cgaaaccttc gtcctctccg attaaactcg atgaaggcgt agttctttcg 480
gagattcagc tatacggaga cgttgttctt cgttatctca gcttcaaaaa cacaaaccag 540
agttgtccat ttttacctgg gtttgaagaa gtgggtgaag tttcgtcatc tcgaggacta 600
gattttggta ttagaaggtt agaccatgca gtaggaaatg taccaaattt ggcagaagca 660
ataggatatt tgaaagagtt taccgggttt catgagtttg ctgagttcac agcagaagat 720
gtaggaacca ccgaaagcgg gttgaactcc atagttttag ctagtaatga tgaaatggta 780
ttgttaccaa tgaatgagcc tgtttatgga acaaagagaa agagccagat acaaacttat 840
ttggaacata atgaaggtgc tggtgttcag catttggctt tggttagtga agatatattt 900
actactttga gagaaatgag gagaagaagt ggtgttggtg gttttgagtt catgccttca 960
ccaccaccaa cttattataa gaatctgaag aatagagctg gtgatgtttt gagtgatgag 1020
caaattaaag agtgtgagga attgggcatt ttggtggata gagatgcaca gggtactttg 1080
cttcagattt tcaccaagcc tgttggggat aggccaacta tatttgttga gataattcag 1140
aggcttggat gcatgttaaa ggatgaagaa gggaaaacat atcagaaggc aggatgtgga 1200
ggatttggga agggtaactt ctctgagctc ttcaaatcta ttgaagaata tgagaagact 1260
ttggaggcca aagctaatgt tgttgctgca tag 1293
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Met Val Pro Ser Thr Ala Ser Asn Leu Lys Leu Val Gly His Thr Asn
1 5 10 15
Phe Val His Asn Asn Pro Lys Ser Asp Lys Phe His Val Lys Lys Phe
20 25 30
His His Ile Glu Phe Trp Ser Thr Asp Ala Thr Asn Thr Ala Arg Arg
35 40 45
Phe Ser Trp Gly Leu Gly Met Pro Met Val Ala Lys Ser Asp Leu Ser
50 55 60
Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Arg Ser Gly Glu Leu
65 70 75 80
Asn Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Ile Ala Gly Asn Thr
85 90 95
Leu Thr His Thr Ala Ser Ile Pro Thr Tyr Ser His Asn Leu Ala Arg
100 105 110
Leu Phe Ala Ser Thr His Gly Leu Ala Val Arg Ala Ile Ala Ile Glu
115 120 125
Val Gln Asp Ala Glu Leu Ala Tyr Asn Ile Ser Val Ala Asn Gly Ala
130 135 140
Lys Pro Ser Ser Ser Pro Ile Lys Leu Asp Glu Gly Val Val Leu Ser
145 150 155 160
Glu Ile Gln Leu Tyr Gly Asp Val Val Leu Arg Tyr Leu Ser Phe Lys
165 170 175
Asn Thr Asn Gln Ser Cys Pro Phe Leu Pro Gly Phe Glu Glu Val Gly
180 185 190
Glu Val Ser Ser Ser Arg Gly Leu Asp Phe Gly Ile Arg Arg Leu Asp
195 200 205
His Ala Val Gly Asn Val Pro Asn Leu Ala Glu Ala Ile Gly Tyr Leu
210 215 220
Lys Glu Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala Glu Asp
225 230 235 240
Val Gly Thr Thr Glu Ser Gly Leu Asn Ser Ile Val Leu Ala Ser Asn
245 250 255
Asp Glu Met Val Leu Leu Pro Met Asn Glu Pro Val Tyr Gly Thr Lys
260 265 270
Arg Lys Ser Gln Ile Gln Thr Tyr Leu Glu His Asn Glu Gly Ala Gly
275 280 285
Val Gln His Leu Ala Leu Val Ser Glu Asp Ile Phe Thr Thr Leu Arg
290 295 300
Glu Met Arg Arg Arg Ser Gly Val Gly Gly Phe Glu Phe Met Pro Ser
305 310 315 320
Pro Pro Pro Thr Tyr Tyr Lys Asn Leu Lys Asn Arg Ala Gly Asp Val
325 330 335
Leu Ser Asp Glu Gln Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val
340 345 350
Asp Arg Asp Ala Gln Gly Thr Leu Leu Gln Ile Phe Thr Lys Pro Val
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Gly Asp Arg Pro Thr Ile Phe Val Glu Ile Ile Gln Arg Leu Gly Cys
370 375 380
Met Leu Lys Asp Glu Glu Gly Lys Thr Tyr Gln Lys Ala Gly Cys Gly
385 390 395 400
Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu
405 410 415
Tyr Glu Lys Thr Leu Glu Ala Lys Ala Asn Val Val Ala Ala
420 425 430
Claims (6)
1.一种提高黑色素产率的基因，其特征在于，该基因是将基因Cjhppd的编码蛋白的氨基酸序列的第280位的Tyr突变成Phe，命名为Cjhppd Y280F，基因Cjhppd Y280F的核苷酸序列如SEQ ID NO : 1所示。
2.一种权利要求1所述的提高黑色素产率的基因所编码的蛋白，其特征在于，其氨基酸序列如SEQ ID NO : 2所示。
3.一种含有如权利要求1所述的提高黑色素产率的基因的质粒。
4.一种含有如权利要求1所述的提高黑色素产率的基因的工程菌株。
5.一种权利要求1所述的提高黑色素产率的基因在生产黑色素中的应用。
6.根据权利要求5所述的提高黑色素产率的基因在生产黑色素中的应用，其特征在于，将基因Cjhppd Y280F连接到质粒载体上，导入大肠杆菌中，获得能够产生黑色素的重组菌株，活化培养，诱导表达，产生色素，分离提取，获得黑色素。
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