CN102080069A - 一种新的细胞色素p450基因、表达蛋白及其用途 - Google Patents
一种新的细胞色素p450基因、表达蛋白及其用途 Download PDFInfo
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Abstract
本发明“一种新的细胞色素P450基因、表达蛋白及其用途”，属于生物技术领域。P450基因，编码CYP107213酶，其核苷酸序列如SEQ ID NO 1所示。本发明P450基因，具有位点特异性氧化阿维菌素4’-羟基成为4’-羰基的功能，为生物转化阿维菌素提供了新的功能基因资源，对进一步改造和重组该基因，提高阿维菌素生物转化效率、降低甲氨基阿维菌素苯甲酸盐的生产成本奠定了基础。含有该基因的野生型链霉菌菌株Streptomyces sp.ZB01，具有位点特异性转化阿维菌素生成4’-羰基阿维菌素的功能，转化效率为36.8％，明显高于现有的野生型链霉菌的转化效率以及大多数ema异源表达蛋白的转化效率。
Description
技术领域
本发明属于生物技术领域，具体涉及一种新的细胞色素P450基因、蛋白及其应用。
背景技术
细胞色素P450(cytochrome P450，CYP)是广泛存在于生物体内的含血红素和硫羟基的蛋白质，是一类以还原态与CO结合后在波长450nm处有吸收峰的含血红素的单链蛋白质。CYP酶系能够在生物体内催化多种内源性物质的生物合成，还参与许多外源性难降解有机物的生物氧化和降解等，被认为可能是自然界中最具催化作用的生物催化剂之一。细胞色素P450酶系构成了一个超基因家族，氨基酸序列同一性超过40％的细胞色素P450属于同一家族，而同一性超过55％的细胞色素P45O则属于相同亚家族。链霉菌中含有数量众多不同家族的CYP，在链霉菌次生代谢产物的生物合成和外来物质代谢过程中发挥了重要作用。
阿维菌素(avermectins)是由阿维链霉菌(Streptomyces.avermitilis)发酵产生的一种重要的农用抗生素，已在世界范围内被推广使用，并受到广泛重视。阿维菌素应用至今，某些不足已逐渐显露出来。试验证明：它对许多害虫的卵没有活性，无内吸性，并已对某些害虫产生抗性。甲氨基阿维菌素苯甲酸盐(简称甲维盐)是从阿维菌素B1合成的一种新型高效的杀虫、杀螨剂。甲维盐与母体阿维菌素相比，药效比阿维菌素提高1-3个数量级，对鳞翅目害虫杀虫活性比阿维菌素高160多倍，对哺乳动物、节肢动物、水产动物低毒，对任何农作物没有药害，被确认为高毒农药的替代品种。
甲维盐的化学合成主要包括以下5个步骤：①将阿维菌素B1的C5-位活性羟基保护起来；②4”-位羟基氧化成羰基；③4”-羰基的还原甲氨化；④除去C5上的保护基；⑤与苯甲酸盐反应成盐。其中，对C5-位活性羟基的保护-去保护过程是甲维盐化学合成过程中操作复杂，成本提高的关键步骤。因此寻找合适的催化剂位点特异性氧化4”-位羟基，对简化甲维盐生产步骤、降低生产成本具有重要的意义。
Volker Jungmann等人(Volker J，Istva′n M，Philip E H，et al.Biocatalytic conversion ofavermectin to4-Oxo-Avermectin：Characterization of Biocatalytically Active Bacterial Strains andof cytochromeP450 monooxygenase enzymes and their genes.Applied and environmentalmicrobiology，2005，71(11)：6968-6976)从3334株微生物中筛选到17株能够催化4”-位羟基生成4”-羰基阿维菌素的链霉菌菌株。其中以S.tubercidicusI-1529的转化效率最高，在96h内，能够使初始浓度为0.75g/L的阿维菌素转化率达到16％。经过研究，上述17株链霉菌中催化该反应的酶具有很高的同源性，有的是同一种酶，共计12种ema蛋白，均为细胞色素P450，组成了一个新的CYP107Z亚家族。
I Molnar等人(Molna′r I，Jungmann V，Stege J，et al.Biocatalytic conversion of avermectininto 4”-oxo-avermectin：discovery，characterization，heterologous expression and specificityimprovement of the cytochrome P450enzyme.Biochem Soc Trans，2006.34：1236-1240)将这些P450基因在E.coli，S.lividans，以及溶剂耐受性强的Pseudomonas putida菌株中成功表达。鉴于体外分析P450表达产物需要FD(铁氧还蛋白)和FRE(FD:NADP+还原酶)，并以NADPH提供电子，研究者还克隆了S.coelicolor和其他具有催化的链霉菌菌株中的FDs和FREs，构建与P450的共表达体系。通过使用不同的载体和启动子，截短基因和碱基替换等手段，优化电子转移条件。使P450异源表达产物的转化效率有不同程度的提高，但转化效率均低于25％[I Molnar，et al.2006，同前]。Axel Trefzer等在前人研究成果的基础上，采用定向进化技术改进P450基因的功能，构建了4个基因重组突变库，筛选了总计达13.7万个克隆，得到1个重组ema-V5b基因，表达ema-V5b的S.tubercidicus R922重组菌位点特异性转化阿维菌素为4”-羰基阿维菌素的转化效率为73％，比野生型Ema1基因的转化效率提高5倍[Axel T，Volker J，Molna′r I，et al.Improvement of Cytochrome P450 Monooxygenase specificityby directed evolution.Appl Environ Microbiol，2007，73(13)：4317-4325]。
发明内容
本发明提供了一种新的P450基因，具有位点特异性氧化阿维菌素4’-羟基成为4’-羰基，为生物转化阿维菌素提供了新的功能基因资源，对进一步改造和重组该基因，提高阿维菌素生物转化效率、降低甲氨基阿维菌素苯甲酸盐的生产成本奠定了基础。
CYP107Z13酶，其氨基酸序列如SEQ ID NO 2所示。
一种P450基因，其编码CYP107Z13酶，其核苷酸序列如SEQ ID NO 1所示。
含有P450基因的重组表达载体。
所述重组表达载体是pET28a-ema。
含有P450基因的重组菌株。
所述重组菌株的宿主菌为E.coli BL21。
所述重组菌株在特异性氧化阿维菌素4’-羟基成为4’-羰基中的应用。
CYP107Z13酶在特异性氧化阿维菌素4’-羟基成为4’-羰基中的应用。
含有该P450的野生菌株Streptomyces sp.ZB01，其保藏编号为：CGMCC NO.2804。
含有该P450的野生菌株Streptomyces sp.ZB01在特异性氧化阿维菌素4’-羟基成为4’-羰基中的应用。
本发明通过大量筛选，获得了一株链霉菌，编号为Streptomyces sp.ZB01(保藏编号CGMCC NO.2804)。该菌株具有位点特异性转化阿维菌素生成4’-羰基阿维菌素的功能，转化效率为36.8％，明显高于现有的17株野生型链霉菌的转化效率以及大多数ema异源表达蛋白的转化效率。
以该菌株的基因组DNA为模板，采用PCR技术，克隆获得P450部分基因序列，再采用染色体步移技术，分别获得已知部分序列的上、下游序列，将上述DNA序列拼接后得到完整的P450基因序列，验证拼接序列的正确性，进行序列同源性比对和蛋白保守序列分析，将其命名为CYP107Z13。将CYP107Z13基因序列的上下游分别引入XbaI和XhoI酶切位点，与pET28a连接，构建pET28a-ema重组质粒，转化E.coli BL21(DE3)感受态细胞。筛选获得转化子。IPTG诱导表达目的蛋白。重组蛋白经纯化、脱盐和浓缩后，采用HPLC检测其催化阿维菌素生成4”-羰基阿维菌素的功能。
本发明为生物转化阿维菌素提供了新的功能基因资源，对进一步改造和重组该基因，提高阿维菌素生物转化效率、降低甲氨基阿维菌素苯甲酸盐的生产成本奠定了基础。
链霉菌菌株Streptomyces sp.ZB01，分类命名：链霉菌Streptomyces sp.
保藏号：CGMCC No.2804，
保藏日期：2008年12月15日
保藏单位：中国微生物菌种保藏管理委员会普通微生物中心
保藏地址：北京市朝阳区大屯路中国科学院微生物研究所。
附图说明
图1阿维菌素纯品的HPLC图
其中1：B1a；2：4’-羰基阿维菌素
图2Streptomyces sp.ZB01代谢阿维菌素的代谢产物
其中1：B1a；2：4’-羰基阿维菌素；3：B1b；4：代谢副产物
图3Streptomyces sp.ZB01基因组DNA电泳图
其中1：DNAMarker；2：基因组DNA
图4拼接产物电泳图
图5：CYP107Z亚家族蛋白系统发生树
图6诱导前后菌体蛋白的SDS-PAGE电泳图
其中1：未诱导菌体，2：诱导后菌体，3：蛋白条带Marker，自上至下分别为97.2Kd、66.4Kd、44.3Kd、29.0Kd、20.1Kd、14.3Kd
图7结合不同样品酶促显色吸光值随时间变化曲线
图8SDS-PAGE检测蛋白的表达
其中1：诱导后菌体破碎后的上清液，2：未诱导菌体总蛋白对照，3：诱导后的菌体总蛋白，4：平衡液(含20mM咪唑)洗脱杂蛋白，5：含50mM咪唑的洗脱缓冲液洗脱，6：含100mM咪唑的洗脱缓冲液洗脱，7：蛋白Marker自上至下大小分别为97.2kD、66.4kD、44.3kD、29.0kD、20.1kD、14.3kD，8：含200mM咪唑的洗脱缓冲液洗脱(注：黑色箭头显示为目的蛋白)
图9蛋白浓度-吸光值标准曲线
图10阿维菌素纯品的HPLC图
其中1：B1a；2：4’-羰基阿维菌素
图11表达蛋白代谢阿维菌素的代谢产物
其中1：B1a；2：4’-羰基阿维菌素；3：B1b；4：代谢副产物
具体实施方式
实施例1链霉菌生物催化转化阿维菌素生成4’-羰基阿维菌素的活性检测
1-1.链霉菌菌种培养和样品制备
分离培养不同来源的链霉菌菌株，在YEME培养基中保存。菌种活化后，将链霉菌孢子接种于ISP2液体培养基中(ISP2：酵母膏4.0g；麦芽汁10.0g；葡萄糖4.0g；琼脂20.0g；蒸馏水1000ml。pH 7.0)，28℃120rpm培养3d后，补充1g/L阿维菌素(贮存液浓度20g/LDMSO∶Tween-40＝1∶1，阿维菌素纯品浓度为97％，其中B1a含量84％)。继续培养24h。取30ml培养物用30ml甲基叔丁基醚(简称MTBE)抽提，收集醚相并真空干燥。残留物重新溶解在1.2ml乙腈中，HPLC检测阿维菌素衍生物的生成。
2.HPLC检测阿维菌素生物转化为4’-羰基阿维菌素的活性
采用HPLC色谱法分析阿维菌素及其衍生物，高效液相色谱仪(HP1100-VWD)，层析柱使用SBC18柱(安捷伦)。30min内流速为1.2ml/min的线性梯度为50％-100％的CH3CN，在243nm下检测化合物。阿维菌素在这些条件下的保留时间：阿维菌素B1b 19.4min；阿维菌素B1a 22.4min；4’-羰基阿维菌素B1a 26.7min。阿维菌素纯品的HPLC检测结果见图1，链霉菌Streptomyces sp.ZB01代谢阿维菌素的代谢产物见图2。检测结果显示，在试验条件下阿维菌素B1a转化为4’-羰基阿维菌素的转化率为36.8％。
实施例2.CYP107Z13基因的克隆与序列分析
1.链霉菌Streptomyces sp.ZB01基因组DNA的提取
YEME培养基(酵母膏3g；蛋白胨5g；麦芽膏3g；葡萄糖10g；蔗糖340g；补蒸馏水至1000ml，灭菌，使用前加入5mM灭过菌的MgCl2·6H2O)30℃200rpm液体摇床培养链霉菌ZB0148h。离心收集菌体(50mg)，用STE缓冲液洗涤，离心，去上清；重复上述步骤。将菌体用500ul 2-3mg/ml溶菌酶溶液悬浮，37℃水浴作用60min。加入1％SDS，37℃水浴2h。加入等体积中性酚/氯仿，小心混匀约10min，离心取上清。加入1/10体积的3MNaAc(pH4.8)，加等体积的异丙醇，摇匀，弃上清，吸出沉淀块。加70％乙醇清洗沉淀。用500ul TE溶解沉淀，加RNAse至终浓度50ug/ml，37℃作用30min。加SDS至终浓度0.5％，加pH8.0的EDTA至终浓度50ug/ml，加蛋白酶K至终浓度150ug/ml，50℃作用1h。加等体积中性酚/氯仿，混匀，离心取上清。加氯仿，混匀，离心，取上清。加1/10体积3M NaAc(pH4.8)，加2倍体积的无水乙醇，混匀，吸出沉淀块。70％乙醇洗，将沉淀移至另一管。将管室温开盖放置至可见的痕量乙醇挥发，加入TE溶解沉淀。取3ul进行琼脂糖凝胶电泳检验，结果见图3。
2.CYP107Z13基因的获得
(1)CYP107Z13基因片段的克隆
根据Genebank中CYP107Z家族基因的同源序列设计引物
P1：5′-CGCGGCCGGTTCATGGACGAC-3′
P2：5-ACCGCACCAGCGCATCAGCTC-3
以链霉菌ZB01基因组DNA为模板，建立50ul PCR反应体系：其中含有模板DNA 1ul，dNTP Mixture 8ul，2×GC Buffer 25ul，TaKaRa LA Taq 0.5ul，引物p1和p2各1ul，ddH2O13.5ul。PCR反应条件：94℃预变性3min，94℃变性1min，62℃1min，72℃1min，30个循环，72℃延伸10min。反应结束后，1％琼脂糖凝胶电泳，切胶回收PCR产物，连接入pMD18-T载体，转化入E.coli DH5a感受态细胞。提取转化子质粒，检验后测序。测序结果为SEQ ID No1 P1和P2之间部分。
(2)CYP107Z13全基因的克隆
根据上述已知DNA序列，采用染色体步移技术(genome walking)获取上述已知107Zema基因片断的上、下游未知序列。在上、下游分别各设计3条同向且退火温度较高的特异性引物。如下：
F1 GACCTCATTCCGCACTTCGCCTAT
F2 ATCACCGTCATCTGCGAACTGG
F3 AGCTGCAACTGCTCAAGTCCGA
R1 ATGTGGTCGATCATCTCCGGGAAC
R2 AATCCCCACCAGTTCGCAGATGAC
R3 GATAGGCGAAGTGCGGAATGA
使用TAKARA公司Genome Walking Kit，以链霉菌基因组为模板，进行3次巢式PCR反应。
①第1次PCR反应
以链霉菌基因组DNA为模板，以Genome Walking Kit提供的AP1作为上游引物，分别以F1和R1作为下游引物，建立2个50ul PCR反应体系：其中含有模板DNA 1ul，dNTP Mixture 8ul，10×LA PCR Buffer II 5ul，TaKaRa LA Taq 0.5ul，引物AP1 1ul，F1或R1 1ul，ddH2O 33.5ul反应条件如下：
②第2次PCR反应
将第1次PCR反应液稀释100倍后，取1ml作为第2次PCR反应的模板，以AP1作为上游引物，分别以F2和R2作为下游引物，建立2个50ul PCR反应体系：其中含有模板DNA 1ul，dNTP Mixture 8ul，10×LA PCR Buffer II 5ul，TaKaRa LA Taq 0.5ul，引物AP1 1ul，F2或R2 1ul，ddH2O 33.5ul
反应条件如下：
③第3次PCR反应
将第2次PCR反应液稀释100倍后，取1ml作为第3次PCR反应的模板，以AP1作为上游引物，分别以F3和R3作为下游引物，建立2个50ul PCR反应体系：其中含有模板DNA 1ul，dNTP Mixture 8ul，10×LA PCR Buffer II 5ul，TaKaRa LATaq 0.5ul，引物AP1 1ul，F3或R31ul，ddH2O 33.5ul。反应条件同第2次PCR反应。
④序列片段拼接
通过3次PCR反应，分别获得已知序列的上、下游序列。拼接后得到SEQ ID No1。
⑤拼接DNA序列的鉴定
根据拼接序列设计引物，Jd1：5′-CTGGTCCGGGGATCCTCTAGAGATTC-3′；Jd2：5′-CTCAGTGAGATTCGGTGCGGTCG-3′。以不吸水链霉菌基因组DNA为模板，建立50ul PCR反应体系：模板DNA 1ul，dNTP Mixture 8ul，2×GC BufferI 25ul，TaKaRa LA Taq 0.5ul，引物Jd1 1ul，Jd2 1ul，ddH20 13.5ul。PCR反应条件：94℃预变性3min，94℃变性1min，60℃1min，72℃1min，30个循环，72℃延伸10min。反应结束后，1％琼脂糖凝胶电泳(图4)，PCR产物切胶回收，连接入pMD18-T载体，转化E.coli DH5a感受态细胞。提取转化子质粒进行测序鉴定，结果同SEQ ID No1。证明拼接获得的DNA序列是正确的。
⑥CYP107Z13序列分析
序列SEQ ID No.l全长1386bps，开放阅读框的位置是70-1358，GC含量为70％，编码429个氨基酸，通过http://us.expasy.org/tools预测蛋白的分子量为47.89kD，等电点pI为5.74。其氨基酸序列为SEQ ID No.2所示。在蛋白质序列的370-379、56-60、262-266、301-304处分别有细胞色素P450的特征性保守序列-血红素结合区、螺旋C、螺旋I和螺旋K。在http://www.ncbi.nlm.nih.gov/BLAST/BLAST/Bl ast.cgi上分别用Blastn和Blastp软件进行同源性分析。显示该基因的碱基序列和氮基酸序列同链霉菌来源的CYP107Z亚家族基因和蛋白有高度同源性。用DNAman软件将全部CYP107Z亚家族编码蛋白绘制系统发生树。见图5，CYP107Z13与CYP107Z家族其他蛋白(ema)的同源性为66％-83％。将该基因提交到国际P450命名委员会，被命名为CYP107Z13。
实施例3CYP107Z13基因在大肠杆菌中的表达、纯化和鉴定
1.CYP107Z13原核表达载体的构建
根据CYP107Z13全长编码序列，设计扩增出编码阅读框的引物(分别对应于编码序列5’和3’端约20个以上核苷酸)，并在正反引物上分别引入XbaI和XhoI酶切位点。引物序列如下：
上游：5-TCTAGACACTGCACGCCACAGGAGA-3
下游：5-CTCGAGGTTCAACCGCAGCGGCAG-3
以链霉菌ZB01基因组DNA为模板，PCR扩增获得目的片段，PCR反应条件：94℃预变性3min后，94℃预变性1min，65℃1min，72℃1min，30个循环，72℃延伸10min。PCR产物切胶回收，连接入pMD18-T-simple载体，转化E.coliDH5a感受态细胞，筛选转化子，提取质粒，经XbaI和XhoI双酶切，与同样经过双酶切的pET28a连接，获得重组质粒pET28a-ema，转化E.coli BL21(DE3)感受态细胞，获得转化子。
2.重组菌株的培养和诱导表达
重组菌株E.coli BL21(DE3)(pET28a-ema)在28℃下200rpm摇床培养过夜，按1％接种量转接于50ml含50mg/L卡那霉素的LB培养基中，继续震荡培养3h，加0.2mM IPTG诱导，并继续培养6h，8000r/min离心10min收集菌体，菌体沉淀悬于PBS缓冲液中，超声破碎，分别取诱导前、后菌体破碎液，进行SDS-PAGE电泳。结果见图6，诱导后菌体蛋白表达条带中含有预期大小的蛋白表达条带。
3.ELISA鉴定目的蛋白的表达
原理：带有6-his标签的表达蛋白，与特异识别His标签的小鼠抗体结合，带有辣根过氧化物酶的二抗(羊抗鼠)再特异性的识别小鼠抗体并与之结合，当加入辣根过氧化物酶的底物TMB时，酶便催化底物反应产生蓝色物质，用酶标仪在405nm下便可通过检测吸光值从而估测出各种样品中目的蛋白的相对含量。
操作步骤：
IPTG诱导前、后重组菌株E.coli BL21(DE3)(pET28a-ema)的菌体，分别超声破碎并离心，取上清与沉淀，进行ELISA鉴定。以带有6-His标签的PI蛋白作为阳性对照，无菌水作为阴性对照。样品加入酶标板中，每孔100ul，4℃吸附过夜。将酶标板中的样品倒去。每孔中加入100ul PBS，静置1min后倒去，重复清洗3次。加入100ul 2％牛血清蛋白封闭，37℃30min(室温1～2h)。将2％牛血清蛋白封闭液倒去，用PBS清洗3次，加入100ul一抗1∶1000稀释液，37℃ 30min(室温1～2h)，倒去一抗稀释液。用PBS清洗3次，加入100ul二抗1∶1000稀释液，37℃ 30min(室温1～2h)，倒去二抗稀释液。用PBS清洗3次。加入100ul现配的底物溶液，0～40min内，于酶标仪上以最适波长450nm及不敏感波长630nm双波长检测样品吸光度。结果见图7：
根据图7显示，诱导前菌体上清和沉淀中也含有少量的表达蛋白，诱导后菌体破碎物上清中含有较高浓度的6-His标记的表达蛋白，沉淀中含有少量的表达蛋白，说明诱导后重组菌大量表达6-His标记的目的蛋白，并多为可溶性蛋白。此外，表达蛋白的吸光值随时间变化呈现出一条类似于竞争性酶促反应的S型曲线，可能参与了辣根过氧化物酶与底物TMB的酶促反应。
4.表达蛋白的纯化和脱盐浓缩
采用亲和层析的方法纯化目的蛋白，具体操作如下(在4℃条件下进行)：将1ml预装填料加到空柱子(容量5ml)中，待其沉降后加入冰预冷的平衡缓冲液(50mM磷酸缓冲液，0.5M NaCl，20mM咪唑，PH7.4)15ml平衡柱子。将50ml菌体破碎、离心后的上清用0.45nm的微孔滤膜过滤，滤液加到层析柱中，挂柱。用15ml冰预冷的平衡缓冲液洗脱非特异性结合的杂蛋白。再用冰预冷的3-10ml含50-200mM咪唑的洗脱缓冲液(PH7.4)洗脱目的蛋白，经2次纯化，收集40-100mM洗脱液，合并，加入10kD超滤管中，4℃ 4500g/min离心30min，再加入50mM磷酸缓冲液5ml，离心，重复上述步骤共5次循环。最终获得浓缩并脱盐后的蛋白溶液。分别加入甘油至终浓度50％保存于-20℃。
以SDS-PAGE电泳检测上述步骤收集的样品中是否含有的目的蛋白，同时以未纯化的粗蛋白提取液及诱导和未诱导菌悬液作为对照。结果见图8：在诱导后菌体、诱导后菌体破碎物上清中均含有目的蛋白，50mM和100mM咪唑的洗脱缓冲液的洗脱液中含有纯化的目的蛋白，而在未诱导菌体、平衡液中没有或有极少量目的蛋白，200mM咪唑的洗脱缓冲液的洗脱液中含有微量的目的蛋白，显示层析柱中的目的蛋白已大部分被洗脱出来。
5、蛋白浓度测定
以BCA蛋白浓度检测试剂盒检测纯化浓缩后的蛋白溶液中的蛋白浓度。以BSA为标准品，设置0、12.5、62.5、125、250、500、1000、2000ug/ml共8个浓度梯度。于酶标板上每孔中加入标准品溶液，每个浓度3个重复。每孔中加入25ul待测样品溶液，每个样品3个重复。每孔加入组分A(50∶1)组分B的混合物200ul。震荡30s混匀后，室温静置2h，用酶标仪于492nm处检测各样品吸光值，绘制标准曲线，如下：
检测浓缩蛋白样品的吸光值，根据蛋白-吸光值标准曲线回归方程y＝0.0005x+0.048，得到浓缩、脱盐并加入甘油保存的纯化蛋白浓度为347.9ug/ml。
6.表达蛋白的功能验证
在铁氧还蛋白、铁氧还蛋白-NADP-还原酶体系反应中，利用HPLC检测重组蛋白催化阿维菌素反应的产物。具体操作如下：400ul浓缩蛋白溶液(含蛋白100～200ug)中加入终浓度3.5uM的铁氧还蛋白、40mU/ml的铁氧还蛋白-NADP-还原酶和30mg/L阿维菌素，混合物在30℃预培养5min，加入终浓度为0.2mM的NADPH启动反应。孵育30min后，加入500ulMTBE抽提终止反应，离心后取上清于通风橱中吹干，加入500ul乙腈重溶，HPLC检测。结果见图10，11。显示在反应体系中，有4”-羰基阿维菌素生成，验证了重组蛋白具有氧化阿维菌素4”-羟基生成4”-羰基阿维菌素的功能。
附录：
SEQUENCE LISTING
<110>中国农业科学院植物保护研究所
<120>一种新的细胞色素P450基因、表达蛋白及其用途
<130>P09476/ZWB
<160>2
<170>PatentIn version 3.3
<210>1
<211>1386
<212>DNA
<213>P450基因
<400>1
ctggtccggg gatcctctag agattccttc gagcccagct catttcactg cacgccacag 60
gagagcgaca tgaccgaact aacggactcc ccgttcagcg agttcgtcgg aaagcacccc 120
ggcgaaccga acgtgatgga accggccctg ctcacggacc cgttcgccgg ctacggcgcg 180
ctgcgcgagc agggcccggt ggtccgcggc cggttcgtgg acgacacgcc ggtgtggttc 240
atcacccgct tcgaggaggc ccgggaggta ctgcgcgacc agcggttcgc caacagtccc 300
gcgcactcgg cgggcggcgg gagcgcggac acccccatcg accggctgct ggagatcatg 360
ggactgcccg agcactaccg ggcgtacctc tccgggacca tcctcaacat ggacgccccc 420
gaccacaccc ggctccggcg gttggtctcc cgggccttca ccgcccgcaa gatcaccgat 480
ctgcgtcccc gggtggcgga catcgcggaa gacgcgctgc gccggctgcc cgaacacgcc 540
gtggacggcg tcgtcgacct cattccgcac ttcgcctatc cgctgcccat caccgtcatc 600
tgcgaactgg tggggattcc ggaggccgac cggccgcagt ggcgggaatg gagcacgcac 660
ctggtctccc tgcggccgga gctgcaccct gagacgttcc cggagatgat cgaccacatc 720
cacgcgctga tccgggaacg gcggacggcg ctcaccgacg atctgctcag cgagctgatc 780
cgggtgcacg acgacgacgg cagccgcctc agcgacgtcg agatggtgac gttggtcctg 840
accctcgtcc tggccggtca cgaaaccacc gcccacctga tcaccaacgg cgtcgccgcc 900
ctgctcaccc atcccgacca gctgcaactg ctcaagtccg agccggcgtt gctgccccgt 960
gcggtgcacg agctgatgcg ctggtgcggt ccggtgcacc tgacccagat gcggtacgcc 1020
accgaggacg tcgagctggc cggcgtgcgg atcaagaagg gcgaggccgt cacgcccgtc 1080
ctggtcgcgg cgaaccacga tccgcgccac ttcgccgatc ccgaccggct cgacctcacc 1140
cgccagccgg cgggccgggc cgagaaccat gtcggcttcg ggcacggcat gcactactgc 1200
ctgggcgcca ccctggcccg tcaggaggcc gaggtcgcct tcggaaagct gctcgcgcac 1260
tatccggacg tggcgctggc ggtggcgccg gaggacctcc agcgggtccc gttgccgggc 1320
agttggcggc tggcctcgct gccgctgcgg ttgaactgaa aggcgaccgc accgaatctc 1380
actgag 1386
<210>2
<211>429
<212>PRT
<213>CYP107Z13酶的氨基酸序列
<400>2
Met Thr Glu Leu Thr Asp Ser Pro Phe Ser Glu Phe Val Gly Lys His
1 5 10 15
Pro Gly Glu Pro Asn Val Met Glu Pro Ala Leu Leu Thr Asp Pro Phe
20 25 30
Ala Gly Tyr Gly Ala Leu Arg Glu Gln Gly Pro Val Val Arg Gly Arg
35 40 45
Phe Val Asp Asp Thr Pro Val Trp Phe Ile Thr Arg Phe Glu Glu Ala
50 55 60
Arg Glu Val Leu Arg Asp Gln Arg Phe Ala Asn Ser Pro Ala His Ser
65 70 75 80
Ala Gly Gly Gly Arg Ala Asp Thr Pro Ile Asp Arg Leu Leu Glu Ile
85 90 95
Met Gly Leu Pro Glu His Tyr Arg Ala Tyr Leu Ser Gly Thr Ile Leu
100 105 110
Asn Met Asp Ala Pro Asp His Thr Arg Leu Arg Arg Leu Val Ser Arg
115 120 125
Ala Phe Thr Ala Arg Lys Ile Thr Asp Leu Arg Pro Arg Val Ala Asp
130 135 140
Ile Ala Glu Asp Ala Leu Arg Arg Leu Pro Glu His Ala Val Asp Gly
145 150 155 160
Val Val Asp Leu Ile Pro His Phe Ala Tyr Pro Leu Pro Ile Thr Val
165 170 175
Ile Cys Glu Leu Val Gly Ile Pro Glu Ala Asp Arg Pro Gln Trp Arg
180 185 190
Glu Trp Ser Thr His Leu Val Ser Leu Arg Pro Glu Leu His Pro Glu
195 200 205
Thr Phe Pro Glu Met Ile Asp His Ile His Ala Leu Ile Arg Glu Arg
210 215 220
Arg Thr Ala Leu Thr Asp Asp Leu Leu Ser Glu Leu Ile Arg Val His
225 230 235 240
Asp Asp Asp Gly Ser Arg Leu Ser Asp Val Glu Met Val Thr Leu Val
245 250 255
Leu Thr Leu Val Leu Ala Gly His Glu Thr Thr Ala His Leu Ile Thr
260 265 270
Asn Gly Val Ala Ala Leu Leu Thr His Pro Asp Gln Leu Gln Leu Leu
275 280 285
Lys Ser Glu Pro Ala Leu Leu Pro Arg Ala Val His Glu Leu Met Arg
290 295 300
Trp Cys Gly Pro Val His Leu Thr Gln Met Arg Tyr Ala Thr Glu Asp
305 310 315 320
Val Glu Leu Ala Gly Val Arg Ile Lys Lys Gly Glu Ala Val Thr Pro
325 330 335
Val Leu Val Ala Ala Asn His Asp Pro Arg His Phe Ala Asp Pro Asp
340 345 350
Arg Leu Asp Leu Thr Arg Gln Pro Ala Gly Arg Ala Glu Asn His Val
355 360 365
Gly Phe Gly His Gly Met His Tyr Cys Leu Gly Ala Thr Leu Ala Arg
370 375 380
Gln Glu Ala Glu Val Ala Phe Gly Lys Leu Leu Ala His Tyr Pro Asp
385 390 395 400
Val Ala Leu Ala Val Ala Pro Glu Asp Leu Gln Arg Val Pro Leu Pro
405 410 415
Gly Ser Trp Arg Leu Ala Ser Leu Pro Leu Arg Leu Asn
420 425
Claims (10)
1.CYP107Z13酶，其氨基酸序列如SEQ ID NO 2所示。
2.一种P450基因，其编码权利要求1所述的CYP107Z13酶，其核苷酸序列如SEQ ID NO 1所示。
3.含有权利要求2所述的P450基因的重组表达载体。
4.权利要求3所述的重组表达载体是pET28a-ema。
5.含有权利要求2所述的P450基因的重组菌株。
6.权利要求5所述的重组菌株，其宿主菌为E.coli BL21。
7.权利要求5或6所述的重组菌株在特异性氧化阿维菌素4’-羟基成为4’-羰基中的应用。
8.权利要求1所述的CYP107Z13酶在特异性氧化阿维菌素4’-羟基成为4’-羰基中的应用。
9.含有权利要求2所述的P450基因的野生菌株Streptomyces sp.ZB01，其保藏编号为：CGMCC NO.2804。
10.权利要求9所述的野生菌株Streptomyces sp.ZB01在特异性氧化阿维菌素4’-羟基成为4’-羰基中的应用。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399796A (zh) * | 2011-11-09 | 2012-04-04 | 南京林业大学 | 巨大芽孢杆菌ala2细胞色素p450酶基因及其重组质粒的构建及酶的纯化方法 |
| CN104745615A (zh) * | 2013-12-30 | 2015-07-01 | 泸州医学院 | Cyp119酶表达载体及其纯化方法 |
| CN106268694A (zh) * | 2016-09-23 | 2017-01-04 | 中南大学 | 一种一维纳米尺度聚间苯二胺/链霉菌复合材料及其制备和应用 |
| CN106497956A (zh) * | 2016-10-25 | 2017-03-15 | 上海交通大学 | Cyp101酶重组载体及构建方法、cyp101酶高效表达纯化方法 |
| CN111198185A (zh) * | 2020-02-20 | 2020-05-26 | 迈科若(苏州)医疗科技有限公司 | 一种硒单糖的酶催化检出方法 |
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|---|---|---|---|---|
| CN1472323A (zh) * | 2003-05-16 | 2004-02-04 | 上海交通大学 | 梅岭霉素细胞色素p450羟化酶基因 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399796A (zh) * | 2011-11-09 | 2012-04-04 | 南京林业大学 | 巨大芽孢杆菌ala2细胞色素p450酶基因及其重组质粒的构建及酶的纯化方法 |
| CN104745615A (zh) * | 2013-12-30 | 2015-07-01 | 泸州医学院 | Cyp119酶表达载体及其纯化方法 |
| CN106268694A (zh) * | 2016-09-23 | 2017-01-04 | 中南大学 | 一种一维纳米尺度聚间苯二胺/链霉菌复合材料及其制备和应用 |
| CN106497956A (zh) * | 2016-10-25 | 2017-03-15 | 上海交通大学 | Cyp101酶重组载体及构建方法、cyp101酶高效表达纯化方法 |
| CN111198185A (zh) * | 2020-02-20 | 2020-05-26 | 迈科若(苏州)医疗科技有限公司 | 一种硒单糖的酶催化检出方法 |
| CN111198185B (zh) * | 2020-02-20 | 2023-02-21 | 迈科若(苏州)医疗科技有限公司 | 一种硒单糖的酶催化检出方法 |
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