CN110295182A - 一种源于集胞藻的漆酶基因slr1573及在染料脱色中的应用 - Google Patents
一种源于集胞藻的漆酶基因slr1573及在染料脱色中的应用 Download PDFInfo
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- CN110295182A CN110295182A CN201910599343.7A CN201910599343A CN110295182A CN 110295182 A CN110295182 A CN 110295182A CN 201910599343 A CN201910599343 A CN 201910599343A CN 110295182 A CN110295182 A CN 110295182A
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- slr1573
- laccase
- cytoalgae
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- laccase gene
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Abstract
本发明涉及一种源于集胞藻Synechocystis 6803的漆酶基因slr1573及在染料脱色中的应用，属于生物技术领域。该漆酶基因的DNA序列如SEQ ID NO.1所示，编码蛋白质氨基酸序列如SEQ ID NO.2所示。本发明构建了集胞藻漆酶基因slr1573的原核表达载体，并转化大肠杆菌表达菌株BL21(DE3)，成功获得表达重组Slr1573的菌株，对重组漆酶粗酶液酶学性质进行了检测，本发明获得的slr1573漆酶基因获得高效表达，重组漆酶可高效催化孔雀绿等染料脱色。
Description
技术领域
本发明涉及一种源于集胞藻Synechocystis 6803的漆酶基因slr1573及在染料脱色中的应用，属于生物技术领域。
背景技术
漆酶是一种含铜多酚氧化酶，能催化氧化酚类和非酚类芳香化合物成水，属于环保型酶。漆酶催化底物十分广泛，通过形成漆酶－ 介体系统可进一步扩大底物的作用范围。
漆酶资源丰富，按照不同的来源可划分为三类：植物漆酶、动物漆酶和微生物漆酶（包括真菌漆酶和细菌漆酶），最早发现的漆酶是漆树漆酶，来源于动物的漆酶目前报道较少，真菌漆酶是漆酶的主要来源，如白腐菌、 子囊菌亚门(Ascomycotina) 及半知菌亚门(Deuteromycotina)。随着人们对漆酶的研究，发现细菌中也存在大量的漆酶。
目前植物漆酶、真菌漆酶和细菌漆酶已经有大量的研究报道，不同来源的漆酶性质和功能也有所不同，酸碱耐受性、温度稳定性等区别很大，在生产应用中的潜力也有所限制和不同。来源不同的漆酶染料降解能力和偏好性也大不相同，在添加中间介质后，漆酶催化底物范围扩大，催化效果增强。重组表达Kurthia 属菌株漆酶Lacl K属碱性酶，在中性偏碱环境、添加ABTS等介质条件下能够降解90%以上的对维多利亚蓝和乙基紫，但对孔雀绿的降解率不高（郭翔，2016）。芽孢杆菌B. subtilis 168 CotA漆酶，没有中间介质参与，对结晶紫、溴酚蓝、孔雀绿和刚果红的降解绿分别为95%、81%、62%和59%，但对甲基红和亮黄降解率较低，仅为32%和19%（Samak et al.,2017）。因此需要不断发掘新型来源、具有广谱耐受性和酶活更稳定的漆酶。
藻类漆酶是一种新型来源的漆酶，目前报道较少。现有技术中，关于集胞藻漆酶slr1573基因的酶学性质及其应用尚未有相关报道。
发明内容
针对现有技术中藻类漆酶来源较少等问题，本发明提供了一种源于集胞藻漆酶基因。
本发明还提供了一种源于集胞藻漆酶基因在染料脱色中的应用。
本发明为了实现上述目的所采用的技术方案为：
本发明提供了一种源于集胞藻漆酶基因，该漆酶基因的DNA序列如SEQ ID NO.1所示，编码蛋白质氨基酸序列如SEQ ID NO.2所示。
本发明提供的漆酶基因的克隆方法为：提取集胞藻Synechocystis 6803基因组DAN作为模板，设计引物slr1573-NheI-F/slr1573-XhoI-R，以集胞藻6803基因组为模板扩增得到slr1573漆酶基因开放阅读框795 bp，扩增引物slr1573-NheI-F序列如SEQ ID NO.3所示，slr1573-XhoI-R序列如SEQ ID NO.4所示。
本发明还提供了一种集胞藻漆酶基因的重组漆酶原核表达载体的构建方法，包括以下步骤：
(1)提取集胞藻Synechocystis 6803基因组DAN；
（2）以集胞藻6803基因组DNA为模板，slr1573-NheI-F/slr1573-XhoI-R为上、下游引物，扩增slr1573基因；
（3）回收纯化目标PCR产物。
（4）将回收纯化的slr1573 PCR产物与原核表达载体pET-28a（+）连接得到重组载体pET-28a（+）-slr1573。
本发明还提供了一种源于集胞藻漆酶基因的制备方法，其特征在于，包括以下步骤：
(1)提取集胞藻Synechocystis 6803基因组DAN；
（2）以集胞藻6803基因组DNA为模板，slr1573-NheI-F/slr1573-XhoI-R为上、下游引物，扩增slr1573基因；
（3）回收纯化目标PCR产物。
（4）将回收纯化的slr1573 PCR产物与原核表达载体pET-28a（+）连接得到重组载体pET-28a（+）-slr1573；
（5）将构建好的pET-28a（+）-slr1573转化大肠杆菌BL2(DE3)，得到能够大量表达Slr1573重组漆酶的BL21(DE3)/pET-28a（+）-slr1573菌株，发酵后得重组漆酶。
本发明的再一目的为提供了一种上述源于集胞藻漆酶基因在染料脱色中的应用。
进一步的，所述染料为孔雀绿、活性蓝、溴酚蓝、品红、结晶紫、甲基橙。最优化的染料为孔雀绿。
本发明获得的漆酶酶学性质的测定，步骤如下：
（1）pH对重组酶酶活及稳定性影响；
（2）温度对重组酶酶活及稳定性影响；
（3）金属离子及抑制剂对重组酶酶活的影响；
（4）卤离子对重组酶酶活的影响。
本发明针对集胞藻6803中新型漆酶Slr1573进行研究，通过大肠杆菌原核表达，对其酶学性质进行研究，明确了该酶的最适温度、pH、温度稳定性、pH稳定性、离子稳定性等酶学特性。我们用粗酶液对染料进行降解实验，获得了Slr1573染料对孔雀蓝的降解功能，为集胞藻6803漆酶Slr1573的应用提供基础。
本发明的有益效果为：本发明构建了集胞藻漆酶基因slr1573的原核表达载体，并转化大肠杆菌表达菌株BL21(DE3)，成功获得表达重组Slr1573的菌株，对重组漆酶粗酶液酶学性质进行了检测，本发明获得的slr1573漆酶基因获得高效表达，重组漆酶可高效催化孔雀绿等染料脱色。
附图说明
图1 PCR扩增slr1573基因全长;
图2 重组质粒验证电泳图；
其中，M为Trans 2K Plus DNA Marker，1泳道为pET-28a（+）-slr1573质粒，2泳道为pET-28a（+）-slr1573质粒经NheI和XhoI限制性内切酶双酶切产物。
图3 pET-28a（+）-slr1573转大肠杆菌BL21(DE3)菌液PCR鉴定；
其中，M是Trans2K DNA Marker ; 1-7是pET-28a（+）-slr1573菌液PCR条带。
图4 最佳诱导剂浓度筛选；
其中，M是蛋白预染marker；空载为BL21/PET28a（+）诱导表达产物；0h、2h、4h和6h为BL21/pET28a（+）-slr1573不同浓度IPTG诱导表达产物；上清和沉淀为BL21/pET28a（+）-slr1573不同浓度IPTG诱导表达6h产物超声破碎的上清和沉淀样品。
图5 Slr1573融合蛋白不同诱导温度表达；
其中，M是蛋白预染marker；空载为BL21/PET28a（+）诱导表达产物；0h、2h、4h和6h为BL21/PET28a（+）-slr1573不同浓度IPTG诱导表达产物；上清和沉淀为BL21/PET28a（+）-slr1573不同浓度IPTG诱导表达6h产物超声破碎的上清和沉淀样品。
图6 Slr1573融合蛋白Western blot检测；
其中，1-2都为BL21/PET28a（+）-slr1573表达融合蛋白。
图7 不同pH对Slr1573漆酶催化活力的影响；
图8 以ABTS为底物，不同pH对于Slr1573漆酶稳定性影响；
图9 不同温度对Slr1573漆酶催化活力的影响；
图10 以ABTS为底物，Slr1573漆酶温度稳定性；
图11 卤素对Slr1573漆酶酶活的影响；
具体实施方式
下面结合实施例对本发明的技术方案做进一步说明，但本发明所保护范围不限于此。
生物材料来源
集胞藻（Synechocystis sp.）PCC6803基因组DNA为本实验室提取保藏；
大肠杆菌DH5α和BL21 (DE3)感受态购自北京全式金生物技术有限公司；
DNA凝胶回收试剂盒、质粒回收试剂盒购自Omega公司；
限制性内切酶和T4 DNA连接酶购自TAKARA公司；
PCR扩增高保真酶和PCR检测用酶购自青岛擎科梓熙生物技术有限公司；
his tag兔多克隆抗体、辣根过氧化物酶（HRP）标记的羊抗兔二抗和ECL高灵敏度化学发光检测试剂盒购自康为世纪生物科技有限公司；
愈创木酚、ABTS、SGZ和2,6-DMP购自sigma公司；
溴酚蓝、结晶紫、活性蓝、孔雀绿、甲基橙购自生工生物工程(上海)股份有限公司；
其他所用酶、试剂和试剂盒均购自市售产品。
实施例1集胞藻漆酶slr1573基因PCR扩增
根据Genebank数据库中已登录的slr1573基因，设计引物slr1573-NheI-F/slr1573-XhoI-R，以集胞藻6803基因组DNA为模板扩增得到slr1573漆酶基因开放阅读框789 bp（PCR产物电泳图谱见图1），核苷酸序列如SEQ ID NO.1所示，编码蛋白序列如SEQ ID NO.2所示，扩增引物如下：
slr1573-NheI-F：5′-ACTC GCTAGC ATGGGGGACG ATCTCTGGGG -3′（内含NheI酶切位点），（SEQ ID NO.3）
slr1573-XhoI-R：5′-ACTC CTCGAG TCAGTAACTGACAATGCCAG-3′（内含XhoI酶切位点）（SEQID NO.4）
PCR反应条件：98℃预变性2min；98℃变性10s，55℃复性15s，72℃延伸15s，30个循环后；72℃延伸5min，4℃保存。本实验PCR扩增片段所用DNA聚合酶为I-5™ 2×High-Fidelity Master Mix（购自北京擎科新业生物技术有限公司）。
PCR反应结束后电泳切胶回收，然后分别连接到pEASY-Blunt Simple克隆载体（购自北京全式金生物技术有限公司）后转化大肠杆菌DH5α，筛选阳性克隆（阳性质粒鉴定图谱见图2）。
实施例2 slr1573表达载体构建
将实施1中扩增获得的阳性克隆（含slr1573片段）用NheI和XhoI双酶切，回收slr1573片段；pET28a（+）质粒同样用NheI和XhoI限制性内切酶进行处理，回收pET28a（+）载体片段。用回收的slr1573片段与pET28a（+）载体片段进行连接，获得pET28a（+）- slr1573质粒。
实施例3 重组集胞藻漆酶基因slr1573在大肠杆菌的表达
实施2获得的pET28a（+）- slr1573质粒转化大肠杆菌BL21（DE3）（PCR鉴定图谱见图3），获得BL21/PET28a（+）-slr1573重组表达菌株，优化培养条件，获得高效表达重组漆酶的培养条件，并对Slr1573重组蛋白进行Western blot检测。
1.最佳IPTG诱导浓度筛选：
液体LB活化菌种过夜培养，按菌种和培养基1:50比例接种于50ml LB液体培养基中，置于37℃，180rpm培养至OD600~0.6时，分别加入终浓度为0.1mM、0.5mM和1mM不同浓度诱导剂IPTG诱导表达，筛选最佳IPTG诱导浓度（诱导结果见图4）。
2.可溶性蛋白最佳诱导温度筛选：
液体LB活化菌种过夜培养后，按菌种和培养基1:50比例接种于50ml LB液体培养基中，置于37℃，180rpm培养至OD600~0.6时，加入上述最佳IPTG诱导浓度，然后置于20℃、28℃和37℃诱导表达，6 h时筛选可溶性蛋白表达最佳温度（诱导结果见图5）。
上述LB液体培养基，每升组分如下：
胰蛋白胨(tryptone) 10g，酵母提取物(yeast extract) 5g，氯化钠(NaCl) 10g，水定容至1L。
3.Western blot检测
将诱导表达的Slr1573重组蛋白经SDS-PAGE电泳后，用转膜仪将该蛋白转至PVDF膜上。转印好的PVDF膜用含有5%脱脂奶粉的TBST溶液室温下封闭2 h（或4℃过夜封闭），随后用TBST溶液漂洗膜3次，每次5 min；漂洗后用his标签兔多克隆抗体室温摇床孵育1 h，后用TBST溶液漂洗3次，每次5 min；漂洗后加入辣根过氧化物酶（HRP）标记的羊抗兔抗体温室摇床孵育1 h，最后用ECL增强型发光液在Bio-Rad伯乐凝胶成像系统上拍照观察（结果见图6）。
上述 TBST溶液配方，每升组分如下：
1M Tris-HCl（pH=7.5）10ml,NaCl 5.8g,加ddH2O定容至1L后加500ul Tween-20，磁力搅拌器混匀。
实施例4 Slr1573重组漆酶酶学性质的测定
本发明以ABTS为底物进行酶活测定，Slr1573重组漆酶酶学性质测定，包括pH对酶活的影响、温度对酶活的影响、不同离子对酶活的影响、卤素对酶活的影响。具体酶活测定反应体系及测定方法步骤如下：
以ABTS（ε420 =36,000M-1cm-1）为底物，反应体系和测定方法为：1ml反应体系中，含有100ul 终浓度为1mmol/L的ABTS，100ul粗酶液，800ul 0.1mol/L Britton-Robison缓冲溶液（pH=4）（预实验酶活最高pH为4）,70℃（预实验最高酶活温度）水浴保温3min后，立即冰水浴冷却终止反应，在420nm处测定吸光度。酶学性质测定过程根据要求调整pH和温度。
酶活单位定义：在1min中催化氧化1umolABTS的漆酶的酶量为1个酶活单位（IU）。
已知ABTS在420nm处的摩尔消光系数为ε420=36,000M-1cm-1。
计算公式：酶活（IU/L）= n×Vt×OD420/(3.6×104×Ve×3/106)(n为酶液稀释倍数、Ve为酶液体积、Vt为反应体系总体积)。
上述Britton-Robison缓冲液的配制方法如下：
在100ml三酸混合液（磷酸、乙酸、硼酸，浓度均为0.04mol/L）中，加入0.2mol/LNaOH，在酸度计测量下调至所需pH即得表中相应PH值的缓冲溶液。
1.pH对酶活的影响
（1）最适pH测定：pH选用3、4、5、6、7、8、9 Britton-Robison缓冲液，配好的反应体系置于温度为70℃的水浴锅反应3min后立即放入冰水浴中终止反应，待温度降低后，用分光光度计测量420nm处吸光度。每个pH都设空白对照，对照组中底物和缓冲液不变，用去离子水代替酶液，每个pH做3个平行，取其平均值；以酶活最高的设为100，计算出其它pH的相对酶活，最终以pH为横坐标，以相对酶活为纵坐标作图（结果见图7）。
（2）pH稳定性测定。选择pH为3、4、5、6、7、8和9的Britton-Robison缓冲液，未添加底物ABTS的反应体系置于4℃保温4h后，按反应体系量添加底物ABTS测定其剩余酶活，以未经处理的酶液做为对照，设其酶活为100，计算其它pH的相对酶活，以不同pH为横坐标，以相对酶活为纵坐标作图（结果见图8）。
上述pH测定反应体系为1ml：800ul Britton-Robison缓冲液、100ul酶液、100ulABTS底物。每个pH都设空白对照，用去离子水替代替酶液。
2.温度对漆酶酶活性的影响
（1）重组漆酶的最适反应温度测定。反应体系选择pH为4（预实验测定该漆酶最适反应pH=4）的Britton-Robison缓冲液，分别选取30℃、40℃、50℃、60℃、70℃、80℃和90℃，反应3min后立即放置到冰水浴中结束反应，待温度降低后，用分光光度计测量420nm处吸光度。在每个温度都设空白对照，用去离子水代替酶液，每个梯度做3个平行组，取其平均值；以最高酶活设为100，计算其它温度相对酶活，以温度范围为横坐标，以相对酶活为纵坐标作图（结果见图9）。
（2）重组漆酶温度稳定性：设定反应温度分别为50℃、60℃和70℃（预实验结果显示该酶属于高温酶），反应均在最适pH=4的Britton-Robison缓冲液中进行；将粗酶液分别在50℃、60℃和70℃中保温，在0min、5min、10min、15min、20min、25min、30min、35min、40min进行剩余酶活测定，每个保温时间设3个平行，同时空白对照组；以0min的酶活为100，计算不同保温时间的相对酶活，以保温时间为横坐标，以相对剩余酶活为纵坐标作图（结果见图10）。
上述温度对酶活影响测定反应体系为1ml：800ul的Britton-Robison缓冲液、100ul酶液、100ulABTS底物。每个梯度都要求有对照组，对照用去离子水替代酶液即可。
3.不同金属离子和抑制剂对酶活的影响
先配制该漆酶最适pH=4的磷酸氢二钾-磷酸二氢钾缓冲液（pH=4），在1ml的反应体系中分别加入Na+、K+、Mn2+、Ni+、Pb2+、Mg+、Li+、Zn+、Ca+、Fe2+、Fe3+、Co2+以及SDS、EDTA、二甲基亚砜和尿素溶液，其终浓度分别为1mmol/L和10mmol/L，加入缓冲液至800ul，再向溶液中加入100ul酶液，混匀后放置与4℃下10min后，向各组反应溶液中加入100ul的底物ABTS（终浓度为1mmol/L），置于最适温度70℃反应3min后立即放入冰水浴中冷却降温，终止反应，测定其酶活；以未加金属离子和抑制剂的酶液组作为最高酶活，设定其酶活为100，空白对照以去离子水代替酶液，每种离子组做3个平行，计算各组相对酶活(结果见表1)。
表1
4.卤素对漆酶活性的影响
在1mL反应体系中分别加入终浓度为0、100、200、300、400、500mM的NaCl、NaBr和NaF，再加入Britton-Robison缓冲液（pH4.0）补足至800ul，加入100ul的粗酶液，将反应体系置于4℃ 10min后加入100ul底物ABTS（终浓度为1mM)，70℃反应3min后立即冰水浴终止反应，测定残余酶活，每组设置3个平行。以不加卤化物的酶反应体系设为活力100,计算其他组相对酶活，以卤素浓度为横坐标，相对酶活为纵坐标作图（结果见图11）。
实施例5重组漆酶对染料降解作用
探索重组漆酶Slr1573在室温（25℃）对5种合成染料脱色效果。本发明测定重组漆酶Slr1573对合成染料的降解作用，合成染料包括：溴酚蓝（λmax=592nm，Bromophenol blue，100mg/L）、结晶紫（λmax=594nm，Crystal violet，100mg/L）、活性蓝（λmax=595nm，Reactiveblue 4，800mg/L）、孔雀绿（λmax=618nm,Malachite green，100mg/L）、甲基橙（λmax=464nm，Methyl orange，100mg/L）。
按上述浓度配制染料溶液，染料使用去离子水溶解，脱色反应体系为5mL，5mL的染色液中加入300uL的漆酶Slr1573粗酶液，每种染料设置一个对照组，对照组以水代替酶液液。室温放置48h后观察脱色效果，测定脱色前后染料在其最高吸收波长下的吸光度值变化，计算脱色率（结果见图12，表2）。公式为：脱色率=（A0-A）/A0×100%，其中A0为染料溶液的初始吸光值，A为染料溶液经脱色反应后的吸光值。
表2
结果分析
本发明成功克隆集胞藻Synechocytis sp. PCC6803漆酶基因slr1573，构建了pET-28a(+)-slr1573原核表达载体，转化大肠杆菌BL21(DE3)，成功表达。IPTG诱导浓度为0.5mM/L、最适诱导温度为20℃时，为重组蛋白Slr1573最佳诱导表达条件；以ABTS为底物该酶的最适反应温度为70℃，最适pH为4.0；Slr1573耐高温，在50℃保温40 min后酶活仅降低10%，在pH5.0-7.0保存4 h后酶活仅降低20%；金属离子Pb2+、Zn2+、Ca2+等对酶活抑制作用明显，微量K+、Na+对酶活有促进作用；对孔雀绿降解明显，室温（～25 ℃）条件下处理48 h时，降解率达到56.8%，对溴酚蓝和结晶紫的降解率分别为44.4%和30.5%，但对活性蓝、甲基橙和刚果红降解率很低，降解率仅为4.5%、3.0%和1.4%。结果表明，Slr1573属高温、酸性酶，在不加任何中间介体时能够有效降解孔雀绿，在该染料的降解应用中具有很大潜力。
核苷酸序列表
<110> 山东省农业科学院生物技术研究中心
<120> 一种源于集胞藻的漆酶基因slr1573及在染料脱色中的应用
<160> 4
<210>1
<211>789
<212>DNA
<213>集胞藻（synechocystis）
<220>
<223>
<400>1
atgggggacg atctctgggg atggcagaac atcaatggtt caccctatct cacctgtgct 60
ttgttggctc cctggcccca tgcctttttc accagagcct tctatcccca attgcccgaa 120
caactgatca cctatcttga tccccaagga aaagctttcc gggtcaaaca ggttcatggg 180
gacgtaaccc taacggcaac ggaaattggc caaactccct tggccccaga cagtacccat 240
cccccagcgg atggcatcat tagcgacgct ccccaccaag gggtctgggt ggccagtgcg 300
gactgtaccc cggtattgat tggcgatttg attggtaaac gtgtggcggc gattcatgca 360
ggctggcggg ggaccaaagc cagaattgtg cccaaaacca tcgataaatt tctggccctg 420
ggtagtgagt taaaggattt acgggttgcc ctaggcccgg cgatcgccgg agaggtgtat 480
caagtcgatc cctgggtggc cttggaagtg gggcaaagtg tgcaagcagt acaaaaatta 540
gcgacagaag aacagcaatg ggaccattta tccaccatgg tgaacccgcc cgtgttacct 600
gatgcagaac cggaaaaata tcgccttgat gtgcgtcgca tcaatcaatt gcaactgtta 660
gagttaggtt ttgcccagga acagattgct gtggcccccc actgtacttt ccaaatggaa 720
gaactttttt tctcctatcg ccgtacccac accaaggagg tgcaatggtc tggcattgtc 780
agttactga 789
<210>2
<211>20
<212>DNA
<220>
<223>
<400>2
Met Gly Asp Asp Leu Trp Gly Trp Gln Asn Ile Asn Gly Ser
1 5 10 15
Pro Tyr Leu Thr Cys Ala Leu Leu Ala Pro Trp Pro His Ala Phe Phe
20 25 30
Thr Arg Ala Phe Tyr Pro Gln Leu Pro Glu Gln Leu Ile Thr Tyr Leu
35 40 45
Asp Pro Gln Gly Lys Ala Phe Arg Val Lys Gln Val His Gly Asp Val
50 55 60
Thr Leu Thr Ala Thr Glu Ile Gly Gln Thr Pro Leu Ala Pro Asp Ser
65 70 75 80
Thr His Pro Pro Ala Asp Gly Ile Ile Ser Asp Ala Pro His Gln Gly
85 90 95
Val Trp Val Ala Ser Ala Asp Cys Thr Pro Val Leu Ile Gly Asp Leu
100 105 110
Ile Gly Lys Arg Val Ala Ala Ile His Ala Gly Trp Arg Gly Thr Lys
115 120 125
Ala Arg Ile Val Pro Lys Thr Ile Asp Lys Phe Leu Ala Leu Gly Ser
130 135 140
Glu Leu Lys Asp Leu Arg Val Ala Leu Gly Pro Ala Ile Ala Gly Glu
145 150 155 160
Val Tyr Gln Val Asp Pro Trp Val Ala Leu Glu Val Gly Gln Ser Val
165 170 175
Gln Ala Val Gln Lys Leu Ala Thr Glu Glu Gln Gln Trp Asp His Leu
180 185 190
Ser Thr Met Val Asn Pro Pro Val Leu Pro Asp Ala Glu Pro Glu Lys
195 200 205
Tyr Arg Leu Asp Val Arg Arg Ile Asn Gln Leu Gln Leu Leu Glu Leu
210 215 220
Gly Phe Ala Gln Glu Gln Ile Ala Val Ala Pro His Cys Thr Phe Gln
210 215 220
Met Glu Glu Leu Phe Phe Ser Tyr Arg Arg Thr His Thr Lys Glu Val
225 230 235 240
Gln Trp Ser Gly Ile Val Ser Tyr
260
<210>3
<211>30
<212>DNA
<220>
<223>
<400>3
ACTCGCTAGC ATGGGGGACG ATCTCTGGGG 30
<210>4
<211>30
<212>DNA
<220>
<223>
<400>4
ACTCCTCGAG TCAGTAACTG ACAATGCCAG 30
Claims (7)
1.一种源于集胞藻漆酶基因，其特征在于，该漆酶基因的DNA序列如SEQ ID NO.1所示，编码蛋白质氨基酸序列如SEQ ID NO.2所示。
2. 根据权利要求1所述的集胞藻漆酶基因，其特征在于，该漆酶基因的克隆方法为：提取集胞藻Synechocystis 6803基因组DAN作为模板，设计引物slr1573-NheI-F/slr1573-XhoI-R，以集胞藻6803基因组为模板扩增得到slr1573漆酶基因开放阅读框789 bp，扩增引物slr1573-NheI-F序列如SEQ ID NO.3所示，slr1573-XhoI-R序列如SEQ ID NO.4所示。
3.一种如权利要求1或2所述的集胞藻漆酶基因的重组漆酶原核表达载体的构建方法，其特征在于，包括以下步骤：
(1)提取集胞藻Synechocystis 6803基因组DAN；
（2）以集胞藻6803基因组DNA为模板，slr1573-NheI-F/slr1573-XhoI-R为上、下游引物，扩增slr1573基因；
（3）回收纯化目标PCR产物。
（4）将回收纯化的slr1573 PCR产物与原核表达载体pET-28a（+）连接得到重组载体pET-28a（+）-slr1573。
4.一种如权利要求1所述的源于集胞藻漆酶基因的制备方法，其特征在于，包括以下步骤：
(1)提取集胞藻Synechocystis 6803基因组DAN；
（2）以集胞藻6803基因组DNA为模板，slr1573-NheI-F/slr1573-XhoI-R为上、下游引物，扩增slr1573基因；
（3）回收纯化目标PCR产物。
（4）将回收纯化的slr1573 PCR产物与原核表达载体pET-28a（+）连接得到重组载体pET-28a（+）-slr1573；
（5）将构建好的pET-28a（+）-slr1573转化大肠杆菌BL2(DE3)，得到能够大量表达Slr1573重组漆酶的BL21(DE3)/pET-28a（+）-slr1573菌株，发酵后得重组漆酶。
5.一种如权利要求1或2所述的源于集胞藻漆酶基因在染料脱色中的应用。
6.根据权利要求5所述的应用，其特征在于，所述染料为孔雀绿、活性蓝、溴酚蓝、品红、结晶紫、甲基橙。
7.根据权利要求5所述的应用，其特征在于，所述染料为孔雀绿。
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