CN115044565B - 一种胆绿素还原酶突变体及其编码基因和应用 - Google Patents
一种胆绿素还原酶突变体及其编码基因和应用 Download PDFInfo
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- CN115044565B CN115044565B CN202210638772.2A CN202210638772A CN115044565B CN 115044565 B CN115044565 B CN 115044565B CN 202210638772 A CN202210638772 A CN 202210638772A CN 115044565 B CN115044565 B CN 115044565B
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Abstract
本发明公开了一种胆绿素还原酶突变体及其编码基因和应用。所述的胆绿素还原酶突变体的氨基酸序列与SEQ ID NO:2所示的氨基酸序列相比，在SEQ ID NO:2所示的氨基酸序列的第151位、第226位、第228位进行单点突变。所述的胆绿素还原酶突变体可用于胆红素的合成制备，将其作为生物催化剂转化底物胆绿素生成胆红素，反应后产物经验证，反应转化率>95％。本发明构建的胆绿素还原酶突变体与野生型酶相比，催化活性显著提高，能显著降低酶的使用量，有利于实现生产成本下降及利润提高，进而有效提高胆绿素还原酶在工业上的应用价值。
Description
技术领域
本发明涉及生物酶工程技术领域，具体涉及一种胆绿素还原酶突变体及其编码基因和应用。
背景技术
胆红素(bilirubin，CAS号635-65-4)是一种胆色素，主要由血红素(heme，CAS号14875-96-8)降解产生。胆红素是牛黄的主要成分之一，牛黄是珍稀名贵中药材，具有极高的药用价值，在我国药用已有二千多年历史，是世界上公认的珍贵稀有药料。以牛黄配制的中成药方有180多个品种，如日本的救心丹、我国的安宫牛黄丸等。但是天然牛黄来源稀少，为了缓解天然牛黄药源短缺，我国药学工作者在上个世纪70年代根据天然牛黄的成分，成功地研制出人工牛黄。但是，人工牛黄粉胆红素含量太低(仅0.7％)，远远低于天然牛黄35％的含量标准，效果自然也存在差异，因此提高胆红素的含量和纯度有利于提高人工牛黄的品质。此外，胆红素也是药典记载的100多种中成药制剂的主要成份，具有镇静、镇惊、解热、降压等作用。
目前，胆红素基本都是从动物胆汁中提取，但胆汁中天然胆红素含量相当低，如猪胆汁中的胆红素含量仅为0.05％左右，由于原料来源受到限制，所以国内开发活猪造瘘引流胆汁的技术，该技术虽然解决了胆汁来源的问题，但是涉及的动物伦理问题引起很大的社会争议，因此该应用受到限制，开发非动物胆汁来源的胆红素生产方法具有重要经济价值和社会意义。
胆绿素还原酶(biliverdin reductase，简称BvdR，EC 1.3.1.24)是在血红素代谢中，催化胆绿素还原生成胆红素的酶，因此胆绿素还原酶可用于生物转化胆绿素制备胆红素。胆绿素还原酶可通过从动物肝脏直接提取的方法获得，但这种方法制备的胆绿素还原酶成本高，且活性不够理想，限制了其在胆红素工业化生产中的应用。因此，采用基因工程的方法制备胆绿素还原酶是当前经济有效的办法之一。
例如中国专利CN113186235A公开了一种生物转化法合成胆红素的方法，是以表达胆绿素还原酶的基因重组大肠杆菌细胞为生物催化剂，以胆绿素为底物生物转化合成胆红素。富含胆红素的大肠杆菌菌体作为胆红素提取的原料，克服了从动物胆汁中提取胆红素原料来源不足的问题。
蛋白质三维结构模拟和蛋白定向进化技术，是近年来发展起来的对原始基因序列进行人工改造、以满足工业化应用需求的高科技技术，其中蛋白定向进化技术更是获得了2018年诺贝尔化学奖。因此结合蛋白质三维结构模拟和蛋白定向进化技术，进一步寻找和开发新的具有更高酶活性、适用于工业大规模生产的胆绿素还原酶是目前研究的方向之一。
发明内容
针对现有技术存在的不足，本发明要解决的技术问题是提供一种胆绿素还原酶突变体，以解决现有胆绿素还原酶活性不理想，难以实现工业化生产的问题。本发明利用生物信息分析胆绿素还原酶的三维结构，模拟分子间对接相互作用，找出酶蛋白的活性或特性具有关键性的氨基酸，并针对这些特定氨基酸进行定向突变来提高酶活性，将有利于实现生产成本下降及利润提高，进而有效提高胆绿素还原酶在工业上的应用价值。
为解决上述技术问题，本发明提供以下技术方案：
本发明提供的胆绿素还原酶突变体来源于Rattus norvegicus(Norway rat)的野生型胆绿素还原酶bvr。bvr的编码基因核苷酸序列通过常州基宇生物技术有限公司全基因合成所得，依据NCBI上提供的bvr基因序列经大肠杆菌偏好密码子优化，并在编码区两端分别添加NdeI和HindIII限制性内切酶位点，优化后的核苷酸序列如SEQ ID NO:1所示。该基因编码的蛋白质的氨基酸序列如SEQ ID NO:2所示。合成的DNA片段通过限制性内切酶NdeI和HindIII酶切后，与经过同样双酶切的pET28a(+)载体(Novagen公司)进行连接、转化和筛选，筛选得到的阳性质粒bvr-pET28a(+)转入BL21(DE3)宿主菌中，从而构建bvr的体外异源表达体系。
bvr的突变体的构建，是通过定向进化的技术手段得到的。具体地，通过分析bvr的三维蛋白质结构，利用能量最低原理和分子对接技术预测出可能的与催化相关的一个或多个位点，然后对这些位点进行饱和突变，从中筛选出活性有显著提高的突变体。
更为具体的过程如下：本发明通过自由能计算预测出的可能与催化和底物结合相关的位点为151、226和228。分别对这三个位点进行bvr饱和突变，利用紫外分光光度计来进行突变体的筛选。更为具体的为：当位点151的苯丙氨酸(F)突变为色氨酸(W)时，突变体酶活得到了提高；当位点226的精氨酸(R)突变为色氨酸(W)时，突变体酶活相对野生型酶来说得到了提高；当位点228的缬氨酸(V)突变为色氨酸(W)时，突变体酶活得到了显著提高。
因此，一方面，本发明请求保护一种胆绿素还原酶突变体，其氨基酸序列与SEQ IDNO:2所示的氨基酸序列相比，在SEQ ID NO:2所示的氨基酸序列的第151位、第226位、第228位进行单点突变。
具体地，所述的单点突变为：
当SEQ ID NO:2所示的氨基酸序列的第151位由苯丙氨酸突变为色氨酸，所述的胆绿素还原酶突变体的氨基酸序列如SEQ ID NO:4所示；
或，当SEQ ID NO:2所示的氨基酸序列的第226位由精氨酸突变为色氨酸，所述的胆绿素还原酶突变体的氨基酸序列如SEQ ID NO:6所示；
或，当SEQ ID NO:2所示的氨基酸序列的第228位由缬氨酸突变为色氨酸，所述的胆绿素还原酶突变体的氨基酸序列如SEQ ID NO:8所示。
另一方面，本发明还请求保护上述胆绿素还原酶突变体的编码基因。
具体地，氨基酸序列如SEQ ID NO:4所示的胆绿素还原酶突变体的编码基因的核苷酸序列如SEQ ID NO:3所示；
或，氨基酸序列如SEQ ID NO:6所示的胆绿素还原酶突变体的编码基因的核苷酸序列如SEQ ID NO:5所示；
或，氨基酸序列如SEQ ID NO:8所示的胆绿素还原酶突变体的编码基因的核苷酸序列如SEQ ID NO:7所示。
根据现有公共知识，任何基因经操作或者改造后连入各类表达载体，转化至合适宿主细胞，经适当条件诱导均能过量表达目的蛋白。
因此，又一方面，本发明还请求保护含有上述编码基因的载体。
具体地，所述的载体可以为各种表达载体，包括但不限于pET表达载体、pCW表达载体、pUC表达载体或pPIC9k表达载体中的任意一种表达载体。
又一方面，本发明还请求保护含有上述编码基因的宿主细胞。
具体地，所述的宿主细胞可以为任一种合适的宿主细胞，包括但不限于大肠杆菌、毕赤酵母、链霉菌或枯草芽孢杆菌中的任意一种宿主细胞。
又一方面，本发明还请求保护上述胆绿素还原酶突变体、编码基因、载体、宿主细胞在制备胆红素中的应用。
再一方面，本发明还提供了一种制备胆红素的方法，包括以下步骤：
S1.配置反应体系，包含：1-5g/L上述的胆绿素还原酶突变体，50mM pH8.5磷酸钠缓冲液，1-1.5g/LNADPH，0.5-1g/L胆绿素；控制反应体系温度为30℃，进行搅拌反应；
S2.反应2h后进行紫外分光光度法检测，即得胆红素。
反应产物经紫外分光光度法检测，反应转化率＞95％，从而可证明该酶突变体可作为生物催化剂，转化底物胆绿素生成胆红素。此外，可进行上述生物催化反应的酶包括纯酶、相应的重组菌休止细胞、粗酶液或者粗酶粉等其他存在形态。
相对于现有技术，本发明具有以下有益效果：
本发明构建的胆绿素还原酶突变体与野生型酶相比，突变体的单位酶活得到大大提高，从而可以显著加快反应速率、降低酶的使用量，降低反应时间和生产成本，有效提高胆绿素还原酶在工业上的应用价值。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例中，未注明具体条件的实验方法，通常按常规条件，如《分子克隆实验指南》(J.萨姆布鲁克，D.W.拉塞尔著，黄培堂，汪嘉玺，朱厚础等译，第三版，北京：科学出版社，2002)中所述的方法进行，或按照试剂盒生产商建议方法操作。
实施例一原核表达体系的构建
bvr基因片段由常州基宇生物技术有限公司合成，并重组到PUC57载体上。经限制性内切酶NdeI和HindIII(购自New England Biolabs公司，NEB)在37℃双酶切4h后，酶切产物经1％琼脂糖凝胶电泳分离并进行切胶回收(胶回收试剂盒购自天根生化科技(北京)有限公司)。随后与经过同样双酶切的表达载体pET28a(+)(Novagen公司)，在T4 DNA连接酶(购自Takara公司)作用下于16℃连接过夜。连接液转化Top10感受态细胞(购自天根生化科技(北京)有限公司)，并进行菌落PCR筛选和测序验证，从而得到阳性重组质粒bvr-pET28a(+)。
将阳性重组质粒bvr-pET28a(+)转化表达宿主菌BL21(DE3)(购自天根生化科技(北京)有限公司)，得到原核表达菌株bvr-pET28a(+)/BL21(DE3)，作为后续定向进化和发酵的原代菌株。
实施例二酶的摇瓶发酵制备酶冻干粉
上述构建的表达菌株bvr-pET28a(+)/BL21(DE3)在加有终浓度为30μg/mL硫酸卡那霉素的5mL LB液体培养基【10g/L胰蛋白胨(OXIOD)，5g/L酵母粉(OXIOD)，10g/L氯化钠(国药试剂)】中，于37℃、200rpm振荡培养过夜后，按1％(V/V)比例接种于含有终浓度为30μg/mL硫酸卡那霉素的500mL LB液体培养基中，于37℃、200rpm振荡培养。待OD600在0.8-1.0之间时，加入终浓度为0.1mM的诱导剂IPTG(异丙基-β-D-硫代半乳糖苷，IPTG)，并在30℃诱导过夜。菌体在4℃、8000rpm条件下离心收集，然后悬浮于50mM pH8.5磷酸钠缓冲液中，超声破碎(200W，3s/5s，20min)，4℃、12000rpm离心20min，取上清进行冷冻干燥，即得粗酶粉。
实施例三突变体的构建
突变体的构建：以RatBVR晶体结构(PDB：1GCU)为出发点，利用分子对接技术预测出可能与催化和底物结合有关的位点，初步选择151、226、228三个位点。随后以bvr-pET28a(+)重组质粒为模板，对这三个位点分别进行了饱和突变(具体突变操作参照stratagene公司的
Site-Directed Mutagenesis Kit操作说明)。
其中：
151位点苯丙氨酸突变为色氨酸
正向引物(SEQ ID NO:9)：
5'CAGCTTGCGTNNKACCGCTTCTCCGTT 3'，
反向引物(SEQ ID NO:10)：
5'AACGGAGAAGCGGTMNNACGCAAGCTG 3'；
226位点精氨酸突变为色氨酸
正向引物(SEQ ID NO:11)：
5'GGTCCGGGCTTGAAACGCAATNNKTATGTTAATTTTCAATT 3'，
反向引物(SEQ ID NO:12)：
5'AATTGAAAATTAACATAMNNATTGCGTTTCAAGCCCGGACC 3'；
228位点缬氨酸突变为色氨酸
正向引物(SEQ ID NO:13)：
5'CGCAATCGCTATNNKAATTTTCAATTCACCTCTGGC 3'，
反向引物(SEQ ID NO:14)：
5'GCCAGAGGTGAATTGAAAATTMNNATAGCGATTGCG 3'。
实施例四突变体的培养
突变体培养：将上述突变得到的质粒转化BL21(DE3)宿主菌后，涂布于含30μg/mL卡那霉素的LB固体培养基上，37℃倒置培养过夜，随后从平板上挑取单克隆置于96孔板中进行培养。过夜培养的菌液再转接于含有新鲜LB培养基的96孔板中，于37℃、200rpm振荡培养4h后加入终浓度为0.1mM的IPTG进行诱导，30℃培养过夜。于4℃、8000rpm离心10min收集菌体，用50mM pH8.5磷酸钠缓冲液悬浮后加入溶菌酶破碎，离心后取上清进行单位酶活测定。
实施例五突变体的筛选
突变体的筛选：底物胆绿素浓度40mg/L，NADPH浓度0.2g/L，50mM pH8.5磷酸钠缓冲液，按1％比例加入上述制备的破碎酶液，混匀后放于96孔板中反应，使用酶标仪在450nm处扫描5min，记录5min前后吸光值的变化，通过产物胆红素的生成量来计算单位酶活。
将单位酶活有显著提高的克隆进行扩大培养后测序验证突变情况。测序结果显示，突变体酶活得到显著提高的克隆中含有的突变位点如下：位点151的苯丙氨酸(F)突变为色氨酸(W)，位点226的精氨酸(R)突变为色氨酸(W)，位点228的缬氨酸(V)突变为色氨酸(W)。具体酶活数值如下表1所示。
表1野生型与不同突变体的酶活
| 氨基酸编号 | 野生型/突变体名称 | 单位酶活(U/mg) | 提高倍数 |
| SEQ ID NO:2 | 野生型bvr | 0.04 | -- |
| SEQ ID NO:4 | F151W | 0.12 | 3.0 |
| SEQ ID NO:6 | R226W | 0.11 | 2.8 |
| SEQ ID NO:8 | V228W | 0.052 | 1.3 |
*1U定义为单位时间内(1min)生产1μmol产物所需的酶量。
因此，当SEQ ID NO:2所示的氨基酸序列的第151位由苯丙氨酸突变为色氨酸，胆绿素还原酶突变体的氨基酸序列如SEQ ID NO:4所示，相应地，其编码基因的核苷酸序列如SEQ ID NO:3所示。
或，当SEQ ID NO:2所示的氨基酸序列的第226位由精氨酸突变为色氨酸，胆绿素还原酶突变体的氨基酸序列如SEQ ID NO:6所示，相应地，其编码基因的核苷酸序列如SEQID NO:5所示。
或，当SEQ ID NO:2所示的氨基酸序列的第228位由缬氨酸突变为色氨酸，胆绿素还原酶突变体的氨基酸序列如SEQ ID NO:8所示，相应地，其编码基因的核苷酸序列如SEQID NO:7所示。
实施例六突变体的生物催化
将10mg底物胆绿素盐酸盐溶解于10mL 50mM pH8.5磷酸钠缓冲液中，待底物完全溶解后加入15mg NADPH、10mg胆绿素还原酶突变体，在30℃恒温磁力搅拌器下搅拌反应，反应2h后分别使用HPLC和紫外分光光度法检测底物的消耗量和产物的生产量。不同突变体的底物转化率和产物生成率见下表2。
表2野生型与不同突变体的底物转化率和产物生成率
最后应当说明的是，以上内容仅用以说明本发明的技术方案，而非对本发明保护范围的限制，本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换，均不脱离本发明技术方案的实质和范围。
序列表
<110> 中山俊凯生物技术开发有限公司
<120> 一种胆绿素还原酶突变体及其编码基因和应用
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Asn Arg Tyr Val Asn Phe Gln Phe Thr Ser Gly Ser Leu Glu Glu Val
225 230 235 240
Pro Ser Val Gly Val Asn Lys Asn Ile Phe Leu Lys Asp Gln Asp Ile
245 250 255
Phe Val Gln Lys Leu Leu Asp Gln Val Ser Ala Glu Asp Leu Ala Ala
260 265 270
Glu Lys Lys Arg Ile Met His Cys Leu Gly Leu Ala Ser Asp Ile Gln
275 280 285
Lys Leu Cys His Gln Lys Lys
290 295
<210> 3
<211> 888
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggatgccg aaccgaaacg caaatttggt gttgttgttg ttggtgttgg tcgtgctggc 60
tctgttcgtt tgcgcgattt gaaagatcct cgtagcgctg cttttcttaa tttgattggc 120
tttgtttctc gccgcgaatt gggtagcttg gatgaagttc gtcaaatttc tttggaagat 180
gctttgcgca gccaagaaat tgatgttgcc tatatttgca gcgaaagcag ctctcatgaa 240
gattatattc gtcagttttt gcaagccggc aaacatgttt tggttgaata tcctatgacc 300
ttgtcttttg ccgctgctca ggaattgtgg gaattggccg cccagaaagg ccgtgttttg 360
catgaagaac atgttgaatt gttgatggaa gaatttgaat ttctgcgccg tgaagttttg 420
ggcaaagaat tgttgaaagg cagctggcgt tttaccgctt ctccgttgga agaagaacgt 480
tttggctttc cggcctttag cggcatttct cgcttgacct ggttggttag cttgtttggt 540
gaattgagct tgatttctgc taccttggaa gaacgcaaag aagatcaata tatgaaaatg 600
accgtccagt tggaaaccca aaataagggt ttgttgtctt ggattgaaga aaaaggtccg 660
ggcttgaaac gcaatcgcta tgttaatttt caattcacct ctggctcttt ggaagaagtt 720
ccgtctgttg gcgttaataa gaatattttt ctgaaggatc aggatatctt cgttcaaaaa 780
ttgttggatc aggtttctgc cgaagatttg gctgctgaaa aaaaacgtat tatgcattgt 840
ttgggtttgg ccagcgatat tcagaaattg tgccatcaaa aaaagtaa 888
<210> 4
<211> 295
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Asp Ala Glu Pro Lys Arg Lys Phe Gly Val Val Val Val Gly Val
1 5 10 15
Gly Arg Ala Gly Ser Val Arg Leu Arg Asp Leu Lys Asp Pro Arg Ser
20 25 30
Ala Ala Phe Leu Asn Leu Ile Gly Phe Val Ser Arg Arg Glu Leu Gly
35 40 45
Ser Leu Asp Glu Val Arg Gln Ile Ser Leu Glu Asp Ala Leu Arg Ser
50 55 60
Gln Glu Ile Asp Val Ala Tyr Ile Cys Ser Glu Ser Ser Ser His Glu
65 70 75 80
Asp Tyr Ile Arg Gln Phe Leu Gln Ala Gly Lys His Val Leu Val Glu
85 90 95
Tyr Pro Met Thr Leu Ser Phe Ala Ala Ala Gln Glu Leu Trp Glu Leu
100 105 110
Ala Ala Gln Lys Gly Arg Val Leu His Glu Glu His Val Glu Leu Leu
115 120 125
Met Glu Glu Phe Glu Phe Leu Arg Arg Glu Val Leu Gly Lys Glu Leu
130 135 140
Leu Lys Gly Ser Trp Arg Phe Thr Ala Ser Pro Leu Glu Glu Glu Arg
145 150 155 160
Phe Gly Phe Pro Ala Phe Ser Gly Ile Ser Arg Leu Thr Trp Leu Val
165 170 175
Ser Leu Phe Gly Glu Leu Ser Leu Ile Ser Ala Thr Leu Glu Glu Arg
180 185 190
Lys Glu Asp Gln Tyr Met Lys Met Thr Val Gln Leu Glu Thr Gln Asn
195 200 205
Lys Gly Leu Leu Ser Trp Ile Glu Glu Lys Gly Pro Gly Leu Lys Arg
210 215 220
Asn Arg Tyr Val Asn Phe Gln Phe Thr Ser Gly Ser Leu Glu Glu Val
225 230 235 240
Pro Ser Val Gly Val Asn Lys Asn Ile Phe Leu Lys Asp Gln Asp Ile
245 250 255
Phe Val Gln Lys Leu Leu Asp Gln Val Ser Ala Glu Asp Leu Ala Ala
260 265 270
Glu Lys Lys Arg Ile Met His Cys Leu Gly Leu Ala Ser Asp Ile Gln
275 280 285
Lys Leu Cys His Gln Lys Lys
290 295
<210> 5
<211> 888
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggatgccg aaccgaaacg caaatttggt gttgttgttg ttggtgttgg tcgtgctggc 60
tctgttcgtt tgcgcgattt gaaagatcct cgtagcgctg cttttcttaa tttgattggc 120
tttgtttctc gccgcgaatt gggtagcttg gatgaagttc gtcaaatttc tttggaagat 180
gctttgcgca gccaagaaat tgatgttgcc tatatttgca gcgaaagcag ctctcatgaa 240
gattatattc gtcagttttt gcaagccggc aaacatgttt tggttgaata tcctatgacc 300
ttgtcttttg ccgctgctca ggaattgtgg gaattggccg cccagaaagg ccgtgttttg 360
catgaagaac atgttgaatt gttgatggaa gaatttgaat ttctgcgccg tgaagttttg 420
ggcaaagaat tgttgaaagg cagcttgcgt tttaccgctt ctccgttgga agaagaacgt 480
tttggctttc cggcctttag cggcatttct cgcttgacct ggttggttag cttgtttggt 540
gaattgagct tgatttctgc taccttggaa gaacgcaaag aagatcaata tatgaaaatg 600
accgtccagt tggaaaccca aaataagggt ttgttgtctt ggattgaaga aaaaggtccg 660
ggcttgaaac gcaattggta tgttaatttt caattcacct ctggctcttt ggaagaagtt 720
ccgtctgttg gcgttaataa gaatattttt ctgaaggatc aggatatctt cgttcaaaaa 780
ttgttggatc aggtttctgc cgaagatttg gctgctgaaa aaaaacgtat tatgcattgt 840
ttgggtttgg ccagcgatat tcagaaattg tgccatcaaa aaaagtaa 888
<210> 6
<211> 295
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Asp Ala Glu Pro Lys Arg Lys Phe Gly Val Val Val Val Gly Val
1 5 10 15
Gly Arg Ala Gly Ser Val Arg Leu Arg Asp Leu Lys Asp Pro Arg Ser
20 25 30
Ala Ala Phe Leu Asn Leu Ile Gly Phe Val Ser Arg Arg Glu Leu Gly
35 40 45
Ser Leu Asp Glu Val Arg Gln Ile Ser Leu Glu Asp Ala Leu Arg Ser
50 55 60
Gln Glu Ile Asp Val Ala Tyr Ile Cys Ser Glu Ser Ser Ser His Glu
65 70 75 80
Asp Tyr Ile Arg Gln Phe Leu Gln Ala Gly Lys His Val Leu Val Glu
85 90 95
Tyr Pro Met Thr Leu Ser Phe Ala Ala Ala Gln Glu Leu Trp Glu Leu
100 105 110
Ala Ala Gln Lys Gly Arg Val Leu His Glu Glu His Val Glu Leu Leu
115 120 125
Met Glu Glu Phe Glu Phe Leu Arg Arg Glu Val Leu Gly Lys Glu Leu
130 135 140
Leu Lys Gly Ser Leu Arg Phe Thr Ala Ser Pro Leu Glu Glu Glu Arg
145 150 155 160
Phe Gly Phe Pro Ala Phe Ser Gly Ile Ser Arg Leu Thr Trp Leu Val
165 170 175
Ser Leu Phe Gly Glu Leu Ser Leu Ile Ser Ala Thr Leu Glu Glu Arg
180 185 190
Lys Glu Asp Gln Tyr Met Lys Met Thr Val Gln Leu Glu Thr Gln Asn
195 200 205
Lys Gly Leu Leu Ser Trp Ile Glu Glu Lys Gly Pro Gly Leu Lys Arg
210 215 220
Asn Trp Tyr Val Asn Phe Gln Phe Thr Ser Gly Ser Leu Glu Glu Val
225 230 235 240
Pro Ser Val Gly Val Asn Lys Asn Ile Phe Leu Lys Asp Gln Asp Ile
245 250 255
Phe Val Gln Lys Leu Leu Asp Gln Val Ser Ala Glu Asp Leu Ala Ala
260 265 270
Glu Lys Lys Arg Ile Met His Cys Leu Gly Leu Ala Ser Asp Ile Gln
275 280 285
Lys Leu Cys His Gln Lys Lys
290 295
<210> 7
<211> 888
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggatgccg aaccgaaacg caaatttggt gttgttgttg ttggtgttgg tcgtgctggc 60
tctgttcgtt tgcgcgattt gaaagatcct cgtagcgctg cttttcttaa tttgattggc 120
tttgtttctc gccgcgaatt gggtagcttg gatgaagttc gtcaaatttc tttggaagat 180
gctttgcgca gccaagaaat tgatgttgcc tatatttgca gcgaaagcag ctctcatgaa 240
gattatattc gtcagttttt gcaagccggc aaacatgttt tggttgaata tcctatgacc 300
ttgtcttttg ccgctgctca ggaattgtgg gaattggccg cccagaaagg ccgtgttttg 360
catgaagaac atgttgaatt gttgatggaa gaatttgaat ttctgcgccg tgaagttttg 420
ggcaaagaat tgttgaaagg cagcttgcgt tttaccgctt ctccgttgga agaagaacgt 480
tttggctttc cggcctttag cggcatttct cgcttgacct ggttggttag cttgtttggt 540
gaattgagct tgatttctgc taccttggaa gaacgcaaag aagatcaata tatgaaaatg 600
accgtccagt tggaaaccca aaataagggt ttgttgtctt ggattgaaga aaaaggtccg 660
ggcttgaaac gcaatcgcta ttggaatttt caattcacct ctggctcttt ggaagaagtt 720
ccgtctgttg gcgttaataa gaatattttt ctgaaggatc aggatatctt cgttcaaaaa 780
ttgttggatc aggtttctgc cgaagatttg gctgctgaaa aaaaacgtat tatgcattgt 840
ttgggtttgg ccagcgatat tcagaaattg tgccatcaaa aaaagtaa 888
<210> 8
<211> 295
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Asp Ala Glu Pro Lys Arg Lys Phe Gly Val Val Val Val Gly Val
1 5 10 15
Gly Arg Ala Gly Ser Val Arg Leu Arg Asp Leu Lys Asp Pro Arg Ser
20 25 30
Ala Ala Phe Leu Asn Leu Ile Gly Phe Val Ser Arg Arg Glu Leu Gly
35 40 45
Ser Leu Asp Glu Val Arg Gln Ile Ser Leu Glu Asp Ala Leu Arg Ser
50 55 60
Gln Glu Ile Asp Val Ala Tyr Ile Cys Ser Glu Ser Ser Ser His Glu
65 70 75 80
Asp Tyr Ile Arg Gln Phe Leu Gln Ala Gly Lys His Val Leu Val Glu
85 90 95
Tyr Pro Met Thr Leu Ser Phe Ala Ala Ala Gln Glu Leu Trp Glu Leu
100 105 110
Ala Ala Gln Lys Gly Arg Val Leu His Glu Glu His Val Glu Leu Leu
115 120 125
Met Glu Glu Phe Glu Phe Leu Arg Arg Glu Val Leu Gly Lys Glu Leu
130 135 140
Leu Lys Gly Ser Leu Arg Phe Thr Ala Ser Pro Leu Glu Glu Glu Arg
145 150 155 160
Phe Gly Phe Pro Ala Phe Ser Gly Ile Ser Arg Leu Thr Trp Leu Val
165 170 175
Ser Leu Phe Gly Glu Leu Ser Leu Ile Ser Ala Thr Leu Glu Glu Arg
180 185 190
Lys Glu Asp Gln Tyr Met Lys Met Thr Val Gln Leu Glu Thr Gln Asn
195 200 205
Lys Gly Leu Leu Ser Trp Ile Glu Glu Lys Gly Pro Gly Leu Lys Arg
210 215 220
Asn Arg Tyr Trp Asn Phe Gln Phe Thr Ser Gly Ser Leu Glu Glu Val
225 230 235 240
Pro Ser Val Gly Val Asn Lys Asn Ile Phe Leu Lys Asp Gln Asp Ile
245 250 255
Phe Val Gln Lys Leu Leu Asp Gln Val Ser Ala Glu Asp Leu Ala Ala
260 265 270
Glu Lys Lys Arg Ile Met His Cys Leu Gly Leu Ala Ser Asp Ile Gln
275 280 285
Lys Leu Cys His Gln Lys Lys
290 295
<210> 9
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cagcttgcgt nnkaccgctt ctccgtt 27
<210> 10
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aacggagaag cggtmnnacg caagctg 27
<210> 11
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggtccgggct tgaaacgcaa tnnktatgtt aattttcaat t 41
<210> 12
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aattgaaaat taacatamnn attgcgtttc aagcccggac c 41
<210> 13
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cgcaatcgct atnnkaattt tcaattcacc tctggc 36
<210> 14
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gccagaggtg aattgaaaat tmnnatagcg attgcg 36
Claims (7)
1.一种胆绿素还原酶突变体，其特征在于，所述的胆绿素还原酶突变体的氨基酸序列与SEQ ID NO:2所示的氨基酸序列相比，在SEQ ID NO:2所示的氨基酸序列的第151位、第226位、第228位进行单点突变；
所述的单点突变为：
当SEQ ID NO:2所示的氨基酸序列的第151位由苯丙氨酸突变为色氨酸，所述的胆绿素还原酶突变体的氨基酸序列如SEQ ID NO:4所示；
或，当SEQ ID NO:2所示的氨基酸序列的第226位由精氨酸突变为色氨酸，所述的胆绿素还原酶突变体的氨基酸序列如SEQ ID NO:6所示；
或，当SEQ ID NO:2所示的氨基酸序列的第228位由缬氨酸突变为色氨酸，所述的胆绿素还原酶突变体的氨基酸序列如SEQ ID NO:8所示。
2.权利要求1所述的胆绿素还原酶突变体的编码基因，其特征在于，氨基酸序列如SEQID NO:4所示的胆绿素还原酶突变体的编码基因的核苷酸序列如SEQ ID NO:3所示；
或，氨基酸序列如SEQ ID NO:6所示的胆绿素还原酶突变体的编码基因的核苷酸序列如SEQ ID NO:5所示；
或，氨基酸序列如SEQ ID NO:8所示的胆绿素还原酶突变体的编码基因的核苷酸序列如SEQ ID NO:7所示。
3.含有权利要求2所述的编码基因的载体。
4.根据权利要求3所述的载体，其特征在于，所述的载体为pET表达载体、pCW表达载体、pUC表达载体或pPIC9k表达载体。
5.含有权利要求2所述的编码基因的宿主细胞，其特征在于，所述的宿主细胞为大肠杆菌、毕赤酵母、链霉菌或枯草芽孢杆菌。
6.权利要求1所述的胆绿素还原酶突变体、权利要求2所述的编码基因、权利要求3或4所述的载体、权利要求5所述的宿主细胞在制备胆红素中的应用。
7.一种制备胆红素的方法，其特征在于，所述的方法包括以下步骤：
S1.配置反应体系，包含：1-5g/L权利要求1所述的胆绿素还原酶突变体，50mM pH8.5磷酸钠缓冲液，1-1.5g/L NADPH，0.5-1g/L胆绿素；控制反应体系温度为30℃，进行搅拌反应；
S2.反应2h后进行紫外分光光度法检测，即得胆红素。
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