CN103820401B - 一种碱性细菌漆酶在酵母中的高通量表达方法 - Google Patents
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Abstract
本发明公开了一种碱性细菌漆酶在酵母中的高通量表达方法,包括以下步骤:A、带有碱性细菌漆酶基因的毕赤酵母表达载体构建;B、阳性转化子的筛选;C、酵母表达载体的线性化;D、酵母电转感受态的制备E、重组子的表达鉴定;F、融合蛋白的纯化;G、酶动力学参数测定。本发明通过基于语义的模型发现来帮助建模者,更加高效、合理的完成模型的组合建模,从而解决了碱性细菌漆酶在原核表达系统中易形成包涵体及产量、酶活性低的问题。
Description
技术领域
本发明属于生物工程技术领域,涉及一种碱性细菌漆酶在酵母中的高通量表达方法。
背景技术
漆酶(Laccase EC1.10.3.2)是一种含铜的多酚氧化酶,能催化酚类和芳香类化合物的氧化,使之生成相应的苯醌,同时伴随电子的转移,将分子氧还原成水。由于漆酶能够氧化芳香类化合物和其它一些非芳香类有机物,具有广泛的底物特异性,因此在纸浆漂白、纺织品染料脱色、有毒废弃物的去除、生物修复、医疗诊断或作伪抗癌药物制备的催化剂以及生物传感器等方面具有巨大的应用潜力。漆酶不仅存在于植物中,还广泛存在于真菌中。而最新研究表明,某些昆虫甚至细菌也可产生具有漆酶活性的物质。近年来随着研究的深入,真菌漆酶的工业用途日益受到人们的关注,但真菌漆酶只在酸性范围有活性,严重地限制了该酶的工业应用。而细菌漆酶则在某些方面如高温、高压、高pH的条件下比植物漆酶和真菌漆酶更具有优势。但目前细菌漆酶主要在原核表达系统中表达,其表达量及活性仍然较低,需要进一步提高才能满足工业上的需要。相比较而言,酵母表达系统具有分泌表达的优势,可进行高密度发酵,重组蛋白表达量高,因此可以利用酵母表达系统的优势来实现细菌漆酶的高通量表达。
发明内容
为了克服现有技术中存在的缺陷,本发明提供一种碱性细菌漆酶在酵母中的高通量表达方法,通过基于语义的模型发现来帮助建模者,更加高效、合理的完成模型的组合建模,从而解决了碱性细菌漆酶在原核表达系统中易形成包涵体及产量、酶活性低的问题。其技术方案如下:
一种碱性细菌漆酶在酵母中的高通量表达方法,包括以下步骤:
A、碱性细菌漆酶基因的毕赤酵母表达载体构建
(1)在50μl的体系中,加入5μl10×限制性内切酶反应缓冲液,并按1μg DNA:3-5U限制性内切酶的比例加入DNA和限制性内切酶CpoI和NotI,混匀后37℃保温2-12h,使用该方法分别用相同的限制性内切酶消化切割ppic9K质粒DNA和已扩增的细菌漆酶基因DNA,为了方便纯化,在基因终止密码子后设计一段组氨酸标签;
(2)用1%琼脂糖凝胶电泳分离限制性内切酶消化样品,在紫外灯下用刀片切出目的DNA片段,然后按DNA片段回收试剂盒的方法回收目的DNA片段;
(3)在25μl的体系中,加入2.5μl10×T4DNA连接酶反应缓冲液,并按1∶5的比例加入限制性内切酶消化过的pPic9k质粒DNA和细菌漆酶基因DNA,再加入1μlT4DNA连接酶,16℃水浴4-12h将细菌漆酶基因插入ppic9K质粒上的多克隆位点形成一个重组表达质粒;
B、阳性转化子的筛选
利用常规的CaCl2法制备E.coli DHlOB感受态细胞,然后将5-10μl连接反应混合物与100μlE.coli DHlOB感受态细胞混合,冰浴30min,42℃热休克1.5min后立即置冰上5min,加入900μl新鲜LB培养基后在37℃摇床上培养45min,摇床转速为150rpm,然后将细菌涂布在含100μg/ml氨苄青霉素(Amp)的LB平板上,37℃培养12h,平板上长出的单个菌落即为阳性转化子;
C、酵母表达载体的线性化
SalI分别酶切原始载体重组载体ppic9K-laccase,1%琼脂糖凝胶回收含目的基因的重组线性化载体,15ulddH2O-20℃保存备用;
D、酵母电转感受态的制备
挑取单克隆GS115接种于10mlYPD液体培养基中,200rpm/min,30℃摇床培养过夜,以1%接种量转接至100mlYPD液体培养基,30℃摇床过夜OD=1.1-1.3,在4℃离心5000rpm,5min,弃上清,用100ml冰预冷无菌水重悬菌体,重复一次,在4℃离心5000rpm,5min,弃上清,用20ml冰预冷1mol/L的山梨醇重悬菌体,重复1次,山梨醇的量减为5ml,最后在4℃6000rpm,8min离心弃上清,用400ul冰预冷1mol/L的山梨醇重悬均匀,按100ul分装于1.5ml预冷无菌EP管中,零度备用。
E、重组子的表达鉴定
挑取MD平板上的转化子接于5ml BMGY:0.1mol/L PB pH6.0,CuSO40.4mmol/L中。,标记菌株后置于30度摇床200rpm/min过夜培养,直到OD600约2.5左右时,8000rpm/min离心5分钟,去上清,每管以5ml BMMY:0.1mol/L PB pH6.0,0.5%甲醇,CuSO40.4mmol/L重悬,200rpm/min转速下30度摇床诱导培养,每隔24小时补加5%的甲醇,从第36小时开始以ABTS检测漆酶活性,每隔12小时取100ul菌液,12000rpm/min离心3分钟后取其上清用于检测漆酶活性。以选择漆酶表达量高的酵母重组菌株,漆酶反应体系为:0.02mol/L醋酸-醋酸钠缓冲液pH3.6,0.5mmol/L ABTS,20ul酵母表达上清,0.2mmol/L CuSO4,补充双蒸水至1ml,以转化的负对照为本底,在420nm处测定其在3分钟内催化底物ABTS的umol数;
F、融合蛋白的纯化
将50ml上清粗酶液与10mlNi2+离子亲和层析树脂混合,4℃缓慢摇动4-12h,然后将混合物装柱,柱直径2mM、柱高10mM,用10倍柱床体积的PBS*(pH7.4)缓冲溶液平衡柱子,流速控制在1mL/min至1.2mL/min,当柱中平衡溶液快接近凝胶柱界面时,封住下端出口,将结合后的填料再次上柱分别用5倍柱体积的I-4040mM Imidazole in PBS Buffer和2倍柱体积的I-45Buffer45mM Imidazole in PBS Buffer洗脱,流速为自然流出,收集洗脱液,每次洗脱时,用1.5ml Eppendorf管收集,每管收集1.5ml;这一步用来除去与镍非特异性结合的杂蛋白。分别用用I-250Buffer250mM Imidazole in PBS Buffer洗脱,流速为自然流出,收集洗脱液。收集方法与上相同。目的蛋白在这一步从柱上被洗脱下来,最后用I-1000Buffer1M Imidazole in PBS Buffer洗脱,流速为自然流出,不必收集洗脱液;因为这一部与蛋白的纯化无关,通常是为了清洁镍柱。
收集洗脱峰,目的蛋白经10%SDS-PAGE电泳鉴定,纯化后的蛋白经65%硫酸铵沉淀浓缩后再用50mM磷酸缓冲液(pH7.2)透析去除残余的硫酸铵,在实验室摇瓶发酵条件下,从1L培养物中可获得50-80mg纯蛋白;
G、酶动力学参数测定
使用漆酶底物ABTS(2,2’-联氮基-双(3-乙基苯丙噻唑啉-6-磺酸))和DMP(2,6-二甲基苯)检测,麦芽糖结合蛋白-细菌漆酶融合蛋白具有与未融合的细菌漆酶相似的酶动力学参数,以DMP为底物时,反应体系为50mM TrisHCl(pH8.0)、0.2mM CuSO4和不同浓度的DMP0.2、0.3、0.4、0.6和1mM,以ABTS为底物时,反应体系为50mM醋酸缓冲液(pH3.0)、0.2mMCuSO4和不同浓度的ABTS0.2、0.4、0.6、0.8、1.0和2mM,在反应体系中加入1-5μl适量稀释的纯酶后启动反应,用MV-2550型紫外分光光度计在37℃条件下测定477nm,用于DMP,或420nm,用于ABTS的吸收值1-3min,再根据吸收值的改变(ΔA)值和消光系数(ε)计算出单位时间内产物的生成量即酶反应速度(υ),DMP在477nm处的消光系数为14.8mM-1cm-1,ABTS在420nm处的消光系数为36mM-1cm-1,酶活力定义为在37℃条件下每分钟氧化1μg底物为1个酶活力单位,然后再根据Lineweaver-Burk双倒数作图法1/v=(Km/Vmax)*1/[S]+1/Vmax求出酶动力学参数Km、Vmax和Km/Vmax值。
与现有技术相比,本发明的有益效果:
本发明实现了细菌漆酶在酵母中的稳定高效表达,利用组氨酸标签简化了纯化步骤,易于操作,一步纯化后就可得到教纯的蛋白;充分利用毕赤酵母分泌表达优势的同时还保留了细菌漆酶优良的热稳定性。由于毕赤酵母是目前公认的食品级安全酵母,故通过该系统发酵产生的漆酶可直接用于食品中的一些应用,如果汁澄清、食品保鲜中。
附图说明
图1是经镍柱纯化后的漆酶,如图中的箭头所示;
图2是511漆酶pH的稳定性;
图3是DMP为底物时Lineweaver-Burk双倒数法作图;
图4是ABTS为底物时Lineweaver-Burk双倒数法作图。
具体实施方式
下面结合实施例进一步说明本发明的技术方案。
利用本发明表达的一株苍白杆菌511漆酶pH稳定性的研究表明,511漆酶pH在6.5的稳定性最好,20h以后,依然具有90%的活性。pH7.5、pH8.5有很大的稳定性,处理16h依然有超过80%的相对酶活力。在弱酸和弱碱pH环境中也具有一定的稳定性,pH3.0和pH9.5环境中,处理12h仍有75%的活力,处理16h以后有约60%的活力。其中pH9.5处理20小时还有40%左右的相对酶活力。
以上所述,仅为本发明最佳实施方式,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (1)
1.一种碱性细菌漆酶在酵母中的高通量表达方法,其特征在于,包括以下步骤:
A、带有碱性细菌漆酶基因的毕赤酵母表达载体构建
(1)从一株苍白杆菌基因组扩增得到漆酶编码基因,命名为511lac经测序鉴定如:SEQID NO:1所示;
(2)在50μl的体系中,加入5μl 10×限制性内切酶反应缓冲液,并按1μg DNA∶3-5U限制性内切酶的比例加入DNA和限制性内切酶Cpo I和Not I,混匀后37℃保温2-12h,使用该方法分别用相同的限制性内切酶消化切割pPic9K质粒DNA和已扩增的细菌漆酶基因DNA,在基因终止密码子后设计一段组氨酸标签;
(3)用1%琼脂糖凝胶电泳分离限制性内切酶消化样品,在紫外灯下用刀片切出目的DNA片段,然后按DNA片段回收试剂盒的方法回收目的DNA片段;
(4)在25μl的体系中,加入2.5μl 10×T4DNA连接酶反应缓冲液,并按1∶5的比例加入限制性内切酶消化过的pPic9k质粒DNA和细菌漆酶基因DNA,再加入1μl T4DNA连接酶,16℃水浴4-12h将细菌漆酶基因插入pPic9K质粒上的多克隆位点形成一个重组表达质粒;
B、阳性转化子的筛选
利用常规的CaCl2法制备E.coli DH10B感受态细胞,然后将5-10μl连接反应混合物与100ul E.coli DH10B感受态细胞混合,冰浴30min,42℃热休克1.5min后立即置冰上5min,加入900μl新鲜LB培养基后在37℃摇床上培养45min,摇床转速为150rpm,然后将细菌涂布在含100μg/ml氨苄青霉素Amp的LB平板上,37℃培养12h,平板上长出的单个菌落即为阳性转化子;
C、酵母表达载体的线性化
Sal I分别酶切原始载体重组载体pPic9K-laccase,1%琼脂糖凝胶回收含目的基因的重组线性化载体,15ul ddH2O-20℃保存备用;
D、酵母电转感受态的制备
挑取单克隆GS115接种于10ml YPD液体培养基中,200rpm/min,30℃摇床培养过夜,以1%接种量转接至100ml YPD液体培养基,30℃摇床过夜OD=1.1-1.3,在4℃离心5000rpm,5min,弃上清,用100ml冰预冷无菌水重悬菌体,重复1次,在4℃离心5000rpm,5min,弃上清,用20ml冰预冷1mol/L的山梨醇重悬菌体,重复1次,山梨醇的量减为5ml,最后在4℃6000rpm,8min离心弃上清,用400ul冰预冷1mol/L的山梨醇重悬均匀,按100ul分装于1.5ml预冷无菌EP管中,零度备用;
E、重组子的表达鉴定
挑取MD平板上的转化子接于5ml BMGY:0.1mol/L PB pH6.0,CuSO40.4mmol/L中,标记菌株后置于30度摇床200rpm/min过夜培养,直到OD600为2.5时,8000rpm/min离心5分钟,去上清,每管以5ml BMMY:0.1mol/L PB pH6.0,0.5%甲醇,CuSO40.4mmol/L重悬,200rpm/min转速下30度摇床诱导培养,每隔24小时补加5%的甲醇,从第36小时开始以ABTS检测漆酶活性,每隔12小时取100ul菌液,12000rpm/min离心3分钟后取其上清用于检测漆酶活性,以选择漆酶表达量高的酵母重组菌株,漆酶反应体系如下:0.02mol/L醋酸-醋酸钠缓冲液pH3.6,0.5mmol/L ABTS,20ul酵母表达上清,0.2mmol/L CuSO4,补充双蒸水至1ml,以转化的负对照为本底,在420nm处测定其在3分钟内催化底物ABTS的μmol数;
F、融合蛋白的纯化
将50ml上清粗酶液与10ml Ni2+离子亲和层析树脂混合,4℃缓慢摇动4-12h,然后将混合物装柱,柱直径2mM、柱高10mM,用10倍柱床体积的PBS*(pH7.4)缓冲溶液平衡柱子,流速控制在1mL/min至1.2mL/min,当柱中平衡溶液快接近凝胶柱界面时,封住下端出口,将结合后的填料再次上柱分别用5倍柱体积的I-40:40mM Imidazole in PBS Buffer和2倍柱体积的I-45Buffer:45mM Imidazole in PBS Buffer洗脱,流速为自然流出,收集洗脱液,每次洗脱时,用1.5ml Eppendorf管收集,每管收集1.5ml,这一步用来除去与镍非特异性结合的杂蛋白,分别用用I-250Buffer:250mM Imidazole in PBS Buffer洗脱,流速为自然流出,收集洗脱液,收集方法与上相同,目的蛋白在这一步从柱上被洗脱下来,最后用I-1000Buffer:1M Imidazole in PBS Buffer洗脱,流速为自然流出,不必收集洗脱液,收集洗脱峰,目的蛋白经10%SDS-PAGE电泳鉴定,纯化后的蛋白经65%硫酸铵沉淀浓缩后再用50mM磷酸缓冲液pH7.2透析去除残余的硫酸铵,在实验室摇瓶发酵条件下,从1L培养物中可获得50-80mg纯蛋白;
G、酶动力学参数测定
使用漆酶底物ABTS(2,2’-联氮基-双(3-乙基苯丙噻唑啉-6-磺酸))和DMP(2,6-二甲基苯)检测,麦芽糖结合蛋白-细菌漆酶融合蛋白具有与未融合的细菌漆酶相似的酶动力学参数,以DMP为底物时,反应体系为50mM TrisHCl pH8.0、0.2rnM CuSO4和不同浓度的DMP0.2、0.3、0.4、0.6和1mM,在反应体系中加入1-5μl适量稀释的纯酶后启动反应,用MV-2550型紫外分光光度计在37℃条件下测定477nm的吸收光度值;以ABTS为底物时,反应体系为50mM醋酸缓冲液pH3.0、0.2mM CuSO4和不同浓度的ABTS0.2、0.4、0.6、0.8、1.0和2mM,在反应体系中加入1-5μl适量稀释的纯酶后启动反应,用MV-2550型紫外分光光度计在37℃条件下测定420nm的吸收光度值;再根据吸收值的改变ΔA值和消光系数ε计算出单位时间内产物的生成量即酶反应速度υ,DMP在477nm处的消光系数为14.8mM-1cm-1,ABTS在420nm处的消光系数为36mM-1cm-1,酶活力定义为在37℃条件下每分钟氧化1μg底物为1个酶活力单位,然后再根据Lineweaver-Burk双倒数作图法1/v=(Km/Vmax)*1/[S]+1/Vmax求出酶动力学参数Km、Vmax和Km/Vmax值。
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