CN116064470A - 一种角质酶突变体及其对pet高效降解的应用 - Google Patents
一种角质酶突变体及其对pet高效降解的应用 Download PDFInfo
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Abstract
本发明公开了一种角质酶突变体及其对PET高效降解的应用。所述角质酶突变体的氨基酸序列如SEQ ID NO.2所示,是将SEQ ID NO.1所示的野生型角质酶的氨基酸序列第239位的苯丙氨酸替换为丙氨酸。本发明构建的角质酶突变体通过在大肠杆菌中表达,突变酶的活性与野生型酶相比,稳定性增加,催化效率提升,PET塑料(聚对苯二甲酸乙二醇酯,PET)降解产物的产量增加了42.6倍。改造后的角质酶突变体,提高PET降解效率,可降低生产成本,更适合工业应用。
Description
技术领域
本发明属于酶工程技术领域,具体涉及一种角质酶突变体及其对PET高效降解的应用。
背景技术
塑料工业的迅猛发展改变了人们传统的生产、生活方式,给人们的生活带来了极大的便利,但产生的大量塑料垃圾在自然环境中不断累积,给全球的生态环境造成了严重的负担。每年有大量的塑料垃圾进入海洋生态系统,给海洋的鸟类、鱼类等生物造成了严重的生存威胁。聚对苯二甲酸乙二醇酯(Polyethylene glycol terephthalate,PET)由石油衍生的对苯二甲酸和乙二醇为材料合成,具有烃链长、分子量高、气体渗透性低、难降解等特点。因生产成本低、耐用性好和使用简便等优点,PET成为使用量最多的聚酯材料,全球一半以上的合成纤维和塑料瓶由PET制成。由于PET分子的主链上存在芳香族化合物而难以自然降解,发展PET塑料的绿色降解处理技术对于全球生态环境保护等具有重要的意义。
角质酶(Cutinase)属于α/β折叠水解酶超家族,具有丝氨酸、组氨酸、天冬氨酸残基组成的催化三联体,是目前有效降解PET的重要酶类。在酶的作用下PET被降解为对苯二甲酸乙二醇酯(BHET)、对苯二甲酸单乙二醇酯(MHET)和对苯二甲酸(TPA),可被有效回收再利用。PET塑料的玻璃化温度为76℃,在高温下稳定的角质酶,是降解PET的重要类群。蛋白质工程改造角质酶,可明显的提高野生型酶的PET降解活力,热稳定性和环境适应潜力。海洋类诺卡氏菌Nocardioides sp.SCSIO 66511来源的角质酶ScCut,最适酶反应温度为70℃,但活性相对较低。因此,本专利通过结构分析构建了具备高活性的突变体,在环境中废PET塑料降解中表现出较好工业应用潜力。
发明内容
本发明的第一个目的是提供一种角质酶突变体,其氨基酸序列如SEQ ID NO.2所示。所述的角质酶突变体,是在SEQ ID NO.1所述的野生型角质酶基础上,将第239位苯丙氨酸替换为丙氨酸。
本发明的第二个目的是提供一种编码上述角质酶突变体的编码基因。优选,其核苷酸序列如SEQ ID NO.4所示。
本发明的第三个目的是提供一种含有上述编码基因的重组载体。
本发明的第四个目的是提供一种含有上述重组载体的重组工程菌。
优选,所述重组工程菌为大肠杆菌。
优选,所述大肠杆菌为BL21(DE3)。
本发明的第五个目的是提供上述角质酶突变体、上述重组载体或上述重组工程菌在PET降解中的应用。
本发明的第六个目的是提供一种降解PET的方法,是用上述角质酶突变体与PET进行反应。
优选,所述角质酶突变体的浓度为1mg/mL。
本发明通过分子对接模拟确定了角质酶与底物的结合口袋,发现了影响活性的关键氨基酸残基(239位),通过定点突变提升了PET的催化效率,提高了降解产物的释放量。与野生型菌株相比,角质酶突变体催化产物提升了42.6倍,降解活性超过了世界最好降解活性酶之一的ICCG,在降低生产成本,提高生产效率等方面显示出更好的工业应用潜力。
诺卡氏菌Nocardioides sp.SCSIO 66511保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号CGMCC No.26044,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,邮编:100101,保藏日期为:2022年12月4号。
附图说明
图1是角质酶ScCut与分子MHET和BHET对接示意图;(A,与分子MHET对接图;B,与分子BHET对接图)。
图2是计算模拟野生酶与突变体结构对比示意图;(绿色为野生酶结构,蓝色为突变体结构)。
图3是PET降解产物液相检测吸收峰(野生型酶ScCut;突变酶ScCut-Phe239Ala;对照酶ICCG,Control是HPLC的检测基线)。
图4是降解产物芳香族化合物对比(野生型酶ScCut;突变酶ScCut-Phe239Ala;对照酶ICCG)。
具体实施方式
以下实施例中未作具体的分子生物学实验方法,均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行。
在本发明中采用如下定义:
1.氨基酸和DNA核苷酸序列的命名法
使用氨基酸残基的公认IUPAC命名法,用三字字母代码形式。DNA核苷酸序列采用公认IUPAC命名法。
2.角质酶突变体的标识,采用“原始氨基酸位置替换的氨基酸”来表示ScCut突变体中突变的氨基酸。如Phe239Ala,表示位置239的氨基酸由野生型的Phe替换成Ala,位置的编号对应于SEQ ID No.1中野生型ScCut的氨基酸序列编号。
以下将结合附图和实施例对本发明作进一步地解释说明。
实施例1:野生型角质酶与降解产物MHET和BHET分子对接
以角质酶ScCut基因为模板,在Alpha fold 2进行蛋白结构预测,采用AutoDock4.2软件进行对接,根据结合模式发现底物结合口袋的上方有类似“盖子”的结构(图1),阻碍了反应底物的进入。因此为了提高活性,选择对该“盖子”结构进行突变,将239位置的苯丙氨酸替换为丙氨酸后底物结合口袋明显变大(图2)。
实施例2:定点突变株的获得
以海洋类诺卡氏菌Nocardioides sp.SCSIO 66511的基因组作为模板,以SC-F1(5’-gga gatatacatatgGATGCACCGTGCGCGCCGCT-3’),SC-R1(5’-ggtgctcgagGCGTGCCGCGTCGC GCAGGC-3’)为引物,扩增编码ScCut酶的基因(SEQ ID NO.3的第91-951位核苷酸,1-90位为信号肽序列);PCR反应体系见表1,PCR反应程序为:预变性95℃10min;32个循环(变性95℃30s;退火55℃30s;延伸72℃1min;)延伸72℃10min;保存:4℃。将扩增后的PCR产物使用PCR纯化试剂盒进行纯化;将纯化后的产物与线性载体pET-22b(+)(NdeI和XhoI双酶切)进行同源重组,50℃水浴反应5min;将5μL重组产物加入到100μL感受态细胞中,冰浴30min,然后42℃热激30s,冰浴反应2min,向离心管在加入500μL LB培养基(不含抗生素),37℃,120r/min下培养1h后,将大肠杆菌细胞涂布于含有氨苄青霉素(100μg/mL)的LB培养皿上,37℃培养12h,挑取单克隆进行测序验证,验证成功的E.coli DH5a(pET-22b-ScCut)扩大培养,提取质粒备用。
表1PCR反应体系
基于一步法定点突变的方法将核苷酸序列由SEQ ID NO.3突变为SEQ ID NO.4,即SEQ ID NO.3的第715-717的TTC突变为SEQ ID NO.4中的GCA,即将氨基酸由野生型的Phe替换成Ala,获得突变体。以环形质粒pET-22b-ScCut为模板,使用引物239M-F1(5′-GACACGTCGATCGCACAGCAGGCCCTGAAGATG-3′)与239M-R1(5′-TGCGATCGACGTGTCTGCGAGTGCGGACTTGCC-3′)进行扩增;PCR反应体系见表1,PCR反应程序为:预变性95℃10min;32个循环(变性95℃30s;退火55℃30s;延伸72℃4min;)延伸72℃10min;保存:4℃。将扩增后的PCR产物使用PCR纯化试剂盒进行纯化,得到线性化的质粒;将5μL纯化后的产物(线性化的质粒)加入到100μL大肠杆菌XL1-Blue感受态细胞中,冰浴30min,然后42℃热激30s,冰浴反应2min,向离心管在加入500μL LB培养基(不含抗生素),37℃,120r/min下培养1h后,将大肠杆菌细胞涂布于含有氨苄青霉素(100μg/mL)的LB培养皿上,37℃培养12h,挑取单克隆进行测序验证(扩增引物为SC-F1/R1),确认第715-717位核苷酸为GCA。验证成功的E.coli XL1-Blue(pET-22b-ScCu t-Phe239Ala)扩大培养,提取质粒备用。将提取的质粒pET-22b-ScCut-Phe239Ala转化至大肠杆菌BL21(DE3)感受态细胞,在37℃,120r/min下培养1h后,将大肠杆菌细胞涂布于含有氨苄青霉素(100μg/mL)的LB培养皿上,37℃培养12h,挑取单克隆进行测序验证,验证成功的为含有角质酶的基因工程菌BL21(DE3)/ScCut-Phe239Ala。
按相同的方法将pET-22b-ScCut转入大肠杆菌BL21(DE3)中获得含有野生角质酶的基因工程菌BL21(DE3)/ScCut。
实施例3:新型角质酶ScCut-Phe239Ala蛋白的制备
将基因工程菌BL21(DE3)/ScCut-Phe239Ala(或基因工程菌BL21(DE3)/ScCut)在含100mL LB液体培养基的500mL锥形瓶中37℃,180rpm/min震荡培养4h,按体积比2%的接种量接种至含有氨苄青霉素(100μg/mL)的LB液体培养基中,在37℃,180rpm/min震荡培养至OD600为0.6-0.8;然后加入终浓度为0.1mM的IPTG,在16℃下震荡培养20h。在4℃条件下8000r/min离心10min收集菌体,用Tris-HCl缓冲液(50mM、pH8.0)洗涤菌体3次,重悬菌体并超声破碎,然后4℃,10000r/min离心去除沉淀,得到的上清为粗酶液。粗酶液用Ni2+亲和层析柱进行蛋白纯化,用咪唑梯度洗脱,收集洗脱液,然后用10kDa的超滤管置换为50mMpH8.0的Tris-HCl缓冲液,并进行浓缩。利用SDS-PAGE检验蛋白的纯度,用超微量蛋白检测仪检测浓缩蛋白的浓度。由此获得角质酶ScCut和角质酶ScCut-Phe239Ala。
实施例4:角质酶定点突变酶对PET塑料降解效率的影响
角质酶ScCut和角质酶ScCut-Phe239Ala对PET塑料膜的降解实验按照以下步骤进行:
将PET薄膜裁剪成1cm2大小,然后用75%的乙醇浸泡灭菌1h,取出塑料膜放置于无菌培养皿中,在无菌操作台中挥发掉塑料表面乙醇。在10mL玻璃瓶中2mL 20mM Tris-HCl(pH8.5),然后加入纯化后的角质酶,终浓度为1mg/mL,放入3片无菌处理后的PET塑料膜;对照组为加入2mL 20mM Tris-HCl(pH8.5),放入3片无菌处理后的PET塑料膜。每组处理设置三个平行,将锥形瓶置于恒温摇床中,70℃,180r/min培养48h,检测PET降解产物。以ICCG酶作为对照。
PET降解后产物分析流程:取1mL降解溶液,8000rpm/min离心10min,上清液使用0.22μm水性滤膜过滤,备用。使用安捷伦1260高效液相色谱检测TPA,MHET和BHET的组成。色谱柱;Zorbax SB-C18ODS(4.6*150mm,5μm),检测波长240nm。流动相:A是含有0.1%(v/v)甲酸的去离子水;B是含有0.1%(v/v)甲酸的甲醇。梯度洗脱条件:0-5min 10%B;5-20min10%-100%B溶液;流速1.0mL/min,柱温保持30℃。
经测定,突变后的角质酶PET降解活性显著提升,在70℃,反应48h,突变酶可将PET降解,并释放407.5μM的芳香族化合物,是野生酶释放产物的42.6倍(图4),说明突变效果很明显。液相检测的降解产物主要为TPA,降解效果优于被大量研究的ICCG酶(图3)。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。SEQ ID NO.1(野生型酶ScCut氨基酸序列)
MKVRALLASAAMVAGGSMLAVGSTTAPASADAPCAPLQVVGVPGTGYELIFDGQGMPNGLDLLKPGVLIKDVAAKLQPERDAGRVTYQQVPYPADIGITMSYRKSVRVGTAATKTYIKAKSIQCPGSRFALIGYSQGARVAGNVLHSIGKGNGPIAPDKLAVGGLWSDPGRSTSDQLIGPGVPGVGIDRLRKGGFGAVNGRTFSVCAPGDIVCSTDDTTLLRPLVRKFGKSALADTSIFQQALKMLRRNGFDLQKWSREFGEQDPMSLLPKAFKTGFEIDRYVREGSHGHYLVGNTISVDGQSSIDWVTDRLRDAARSEQ ID NO.2(突变酶ScCut-Phe239Ala氨基酸序列)
MKVRALLASAAMVAGGSMLAVGSTTAPASADAPCAPLQVVGVPGTGYELIFDGQGMPNGLDLLKPGVLIKDVAAKLQPERDAGRVTYQQVPYPADIGITMSYRKSVRVGTAATKTYIKAKSIQCPGSRFALIGYSQGARVAGNVLHSIGKGNGPIAPDKLAVGGLWSDPGRSTSDQLIGPGVPGVGIDRLRKGGFGAVNGRTFSVCAPGDIVCSTDDTTLLRPLVRKFGKSALADTSIAQQALKMLRRNGFDLQKWSREFGEQDPMSLLPKAFKTGFEIDRYVREGSHGHYLVGNTISVDGQSSIDWVTDRLRDAARSEQ ID NO.3(编码野生酶ScCut的核苷酸序列)
ATGAAGGTACGAGCACTACTGGCCTCCGCAGCAATGGTCGCGGGTGGCTCGATGTTGGCTGTCGGGTCCACTACTGCGCCGGCAAGCGCAGATGCACCGTGCGCGCCGCTGCAGGTCGTCGGCGTTCCAGGCACCGGGTACGAGCTCATCTTCGACGGGCAAGGTATGCCGAACGGCCTCGATCTGCTCAAGCCAGGCGTGTTGATCAAGGATGTGGCCGCCAAGTTGCAGCCTGAGCGCGACGCAGGCAGGGTCACCTACCAGCAGGTCCCCTACCCCGCGGACATCGGCATCACCATGTCGTACCGCAAGTCGGTCCGGGTCGGAACGGCCGCTACGAAGACCTACATCAAGGCAAAGTCGATCCAGTGCCCGGGCAGCCGGTTCGCGCTCATCGGGTACTCGCAGGGTGCGAGGGTCGCCGGAAACGTGCTGCATTCCATCGGCAAGGGCAACGGGCCGATCGCGCCCGATAAGCTCGCGGTCGGCGGCTTGTGGTCCGATCCCGGCCGATCGACGTCCGACCAGCTGATCGGACCGGGAGTACCGGGTGTTGGCATCGACCGGCTACGCAAGGGAGGCTTCGGCGCGGTCAACGGCCGTACCTTCTCGGTCTGCGCTCCCGGCGACATCGTCTGCTCGACGGACGATACGACGCTGCTGCGTCCGCTCGTGCGCAAGTTCGGCAAGTCCGCACTCGCAGACACGTCGATCTTCCAGCAGGCCCTGAAGATGCTCCGCCGCAACGGATTCGACCTACAGAAGTGGTCGCGCGAGTTCGGTGAGCAGGATCCGATGTCGCTGCTGCCGAAGGCGTTCAAGACCGGGTTCGAGATCGACCGGTACGTCCGCGAGGGCAGCCACGGCCATTACCTCGTCGGCAACACCATCAGTGTCGACGGCCAGTCGTCGATCGACTGGGTGACGGATCGCCTGCGCGACGCGGCACGCSEQ ID NO.4(编码突变酶ScCut-Phe239Ala的核苷酸序列)
ATGAAGGTACGAGCACTACTGGCCTCCGCAGCAATGGTCGCGGGTGGCTCGATGTTGGCTGTCGGGTCCACTACTGCGCCGGCAAGCGCAGATGCACCGTGCGCGCCGCTGCAGGTCGTCGGCGTTCCAGGCACCGGGTACGAGCTCATCTTCGACGGGCAAGGTATGCCGAACGGCCTCGATCTGCTCAAGCCAGGCGTGTTGATCAAGGATGTGGCCGCCAAGTTGCAGCCTGAGCGCGACGCAGGCAGGGTCACCTACCAGCAGGTCCCCTACCCCGCGGACATCGGCATCACCATGTCGTACCGCAAGTCGGTCCGGGTCGGAACGGCCGCTACGAAGACCTACATCAAGGCAAAGTCGATCCAGTGCCCGGGCAGCCGGTTCGCGCTCATCGGGTACTCGCAGGGTGCGAGGGTCGCCGGAAACGTGCTGCATTCCATCGGCAAGGGCAACGGGCCGATCGCGCCCGATAAGCTCGCGGTCGGCGGCTTGTGGTCCGATCCCGGCCGATCGACGTCCGACCAGCTGATCGGACCGGGAGTACCGGGTGTTGGCATCGACCGGCTACGCAAGGGAGGCTTCGGCGCGGTCAACGGCCGTACCTTCTCGGTCTGCGCTCCCGGCGACATCGTCTGCTCGACGGACGATACGACGCTGCTGCGTCCGCTCGTGCGCAAGTTCGGCAAGTCCGCACTCGCAGACACGTCGATCGCACAGCAGGCCCTGAAGATGCTCCGCCGCAACGGATTCGACCTACAGAAGTGGTCGCGCGAGTTCGGTGAGCAGGATCCGATGTCGCTGCTGCCGAAGGCGTTCAAGACCGGGTTCGAGATCGACCGGTACGTCCGCGAGGGCAGCCACGGCCATTACCTCGTCGGCAACACCATCAGTGTCGACGGCCAGTCGTCGATCGACTGGGTGACGGATCGCCTGCGCGACGCGGCACGC。
Claims (10)
1.一种角质酶突变体,其特征在于,其氨基酸序列如SEQ ID NO.2所示。
2.一种编码权利要求1所述的角质酶突变体的编码基因。
3.根据权利要求2所述的编码基因,其特征在于,其核苷酸序列如SEQ ID NO.4所示。
4.一种含有权利要求2所述的编码基因的重组载体。
5.一种含有权利要求4所述的重组载体的重组工程菌。
6.根据权利要求5所述的重组工程菌,其特征在于,所述重组工程菌为大肠杆菌。
7.根据权利要求6所述的重组工程菌,其特征在于,所述大肠杆菌为BL21(DE3)。
8.权利要求1所述的角质酶突变体、权利要求4所述的重组载体或权利要求5所述的重组工程菌在PET降解中的应用。
9.一种降解PET的方法,其特征在于,是用权利要求1所述的角质酶突变体与PET进行反应。
10.根据权利要求9所述的方法,其特征在于,所述角质酶突变体的浓度为1mg/mL。
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