CN110241097B - Pet降解酶突变体及其应用 - Google Patents
Pet降解酶突变体及其应用 Download PDFInfo
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Abstract
本发明属于酶工程技术领域，涉及一种酶的突变体及其应用。一种PET降解酶突变体，所述的突变体是PET降解酶7029PETase上第61位氨基酸由亮氨酸突变为苏氨酸形成的，其氨基酸序列如SEQ ID NO.2所示，其核苷酸序列如SEQ ID NO.10所示；PET降解酶7029PETase的氨基酸序列如SEQ ID NO.1所示，基因核苷酸序列如SEQ ID NO.9所示。本发明的PET降解酶突变体，能够在常温下降解PET酶，与现有的PET降解酶相比，对商业化PET塑料及其单体BHET的降解速率有了一定的提高，具有良好的工业前景。
Description
技术领域
本发明属于酶工程技术领域，涉及一种酶的突变体及其应用。
背景技术
聚对苯二甲酸乙二醇酯（polyethylene terephthalate，PET）是塑料的一种，因其具有高机械强度、低透气性、重量轻、成本价格低等优点，目前已在全世界范围内得到了广泛的应用。同时由于其化学惰性，自然状态下较难被降解，导致其在环境中大量累积，造成严重的环境污染。目前对于PET废弃物的处理方法中，最常用的为机械回收方法，虽然部分废弃塑料得到了回收利用，但这些工艺不能用于回收有色或者不透明的塑料，而这些塑料通常最终会进入垃圾填埋场或焚烧炉，造成很大的资源浪费与环境污染。
微生物代谢的多样性使它们在环境修复方面具有极大的潜力，许多细菌已被证明可以产生某种水解酶，可以在不同程度上降解PET，但到目前为止发现的这些水解酶大部分需要较高的温度，或者在常温下降解效率不高。因此，如果能找到常温条件下的高效PET降解酶，或者得到常温PET降解酶的突变体，便有可能实现常温下降解PET塑料，从而为节能条件下PET塑料废弃物的处理提供一种工具。
发明内容
鉴于现有技术中存在的上述问题，本发明目的在于提供一种能够在常温下有效降解PET塑料的降解酶及其突变体和应用。
本发明解决其技术问题采用的第一种技术方案，是提供一种PET降解酶：一种PET降解酶，编码该酶的基因命名为7029PETase，其氨基酸序列如SEQ ID NO.1 所示，基因核苷酸序列如SEQ ID NO.9 所示。
本发明解决其技术问题采用的第二种技术方案，是提供PET降解酶突变体，所述的突变体是在7029PETase的底物结合位点发生单点或多点突变。
进一步的，所述的突变体是7029PETase上第61位氨基酸由亮氨酸突变为苏氨酸形成的，其氨基酸序列如SEQ ID NO.2 所示，其核苷酸序列如SEQ ID NO.10 所示。
进一步的，所述的突变体是7029PETase上第132位氨基酸由色氨酸突变为组氨酸形成的，其氨基酸序列如SEQ ID NO.3 所示，其核苷酸序列如SEQ ID NO.11 所示。
进一步的，所述的突变体是7029PETase上第259位氨基酸由精氨酸突变为丙氨酸形成的，其氨基酸序列如SEQ ID NO.4 所示，其核苷酸序列如SEQ ID NO.12 所示。
进一步的，所述的突变体是7029PETase上第61位氨基酸由亮氨酸突变为苏氨酸，同时第132位氨基酸由色氨酸突变为组氨酸形成的，其氨基酸序列如SEQ ID NO.5 所示，其核苷酸序列如SEQ ID NO.13 所示。
进一步的，所述的突变体是7029PETase上第61位氨基酸由亮氨酸突变为苏氨酸，同时第259位氨基酸由精氨酸突变为丙氨酸形成的，其氨基酸序列如SEQ ID NO.6 所示，其核苷酸序列如SEQ ID NO.14 所示。
进一步的，所述的突变体是7029PETase上第132位氨基酸由色氨酸突变为组氨酸，同时第259位氨基酸由精氨酸突变为丙氨酸形成的，其氨基酸序列如SEQ ID NO.7 所示，其核苷酸序列如SEQ ID NO.15 所示。
进一步的，所述的突变体是7029PETase上第61位氨基酸由亮氨酸突变为苏氨酸，第132位氨基酸由色氨酸突变为组氨酸，同时第259位氨基酸由精氨酸突变为丙氨酸形成的，其氨基酸序列如SEQ ID NO.8 所示，其核苷酸序列如SEQ ID NO.16 所示。
本发明还提供一种重组载体，所述的载体包含所述的7029PETase突变体。
本发明还提供一种重组菌，所述的重组菌包含所述的重组载体。
本发明还提供PET降解酶突变体在降解PET塑料中的应用。
本发明的PET降解酶及其突变体，能够在常温下降解PET酶，与现有的PET降解酶相比，对商业化PET塑料及其单体BHET的降解速率有了一定的提高，具有良好的工业前景。
附图说明
图1是 PET降解酶7029PETase在常温下降解商业化PET的降解流程预测图（底物无论是PET塑料或者其单体BHET，反应②为主要反应过程，MHET为主要反应产物）；
图2为野生型7029PETase降解商业化PET塑料降解液的高效液相色谱图；
图3为7029PETase最适体外反应条件的探索示意图；
图4为7029PETase的蛋白结构预测图；
图5A为各突变体以BHET为底物，降解产物中MHET的量的对比；
图5B为各突变体以BHET为底物，降解产物中TPA的量的对比；
图5C为各突变体以PET为底物，降解产物中MHET的量的对比；
图5D为各突变体以PET为底物，降解产物中BHET与TPA的量的对比。
具体实施方式
下面结合附图和实施例对本发明的PET降解酶及其突变体和应用作详细的说明和阐述。
本发明实施例中，实验所用菌株DSM7029购自DSMZ，大肠杆菌BL21(DE3)购自北京鼎国昌盛生物技术有限责任公司，表达载体pET22b购自优宝生物
所用试剂：
DNA扩增所用的高保真DNA聚合酶primeSTAR MAX、琼脂糖凝胶电泳所用的DNAmarker购自北京宝日医生物技术有限公司；DNA内切酶和Gibson组装预混液购自NewEngland Biolabs公司；琼脂糖凝胶回收试剂盒购自北京天根生化科技有限公司。
所用仪器：
PCR扩增仪（Eppendorf）、高速冷冻离心机（Eppendorf）、琼脂糖凝胶成像系统（biolab）、高效液相色谱仪（岛津公司）。
本发明使用了基因工程和分子生物学领域常规的技术和方法。本领域的技术人员可以在本发明提供的实施方式的基础上采用本领域其它常规技术、方法和试剂，而不限于本发明具体实施例的限定。
实施例1 PET降解酶7029PETase的来源及其在E.coli BL21(DE3)中异源表达载体构建
通过分析DSM7029（ATCC 53080）中的基因组数据，在其基因组中发现了一个沉默表达的基因，将其氨基酸序列及核酸序列与已经报道过的PET降解酶IsPETase的序列对比发现它们存在一定的同源性，氨基酸序列相似度为64.3%，核酸序列相似度为72.6%，因此预测该基因所编码的酶为PET降解酶，将该基因命名为7029PETase，其氨基酸序列如SEQ IDNO.1 所示，其核苷酸序列如SEQ ID NO.9 所示。经异源表达与体外活性检测，该PET降解酶7029PETase具有在常温下降解商业化PET的能力，降解流程如图1所示。
使用DSM7029的基因组DNA为模板，使用带有同源臂的引物7029-22b-1和7029-22b-2扩增7029PETase，使用琼脂糖凝胶回收试剂盒对目标片段进行切胶回收；使用Nco1与Xho1双酶切载体pET22b，使用琼脂糖凝胶回收试剂盒对载体片段进行切胶回收；将载体与片段进行Gibson组装，获得野生型7029PETase的重组表达载体pET22b-7029PETase。
上述扩增过程使用的引物序列为：
7029-22b-1：TCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGAT
CAGACCAACCCCTACCAGCG，SEQ ID NO.17；
7029-22b-2：CTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTG
GTACGGGCAGCTCTCGCGGTACTC，SEQ ID NO.18。
其中，下划线序列表示同源臂部分。
（1）片段扩增的PCR程序为：
预变性95℃1 min，每个循环包括98℃ 10 s、55℃ 15 s、72℃ 20 s，共30个循环，最终延伸72℃ 10 min。
（2）Nco1与Xho1双酶切的体系为：
Nco1: 4μl，Xho1: 4μl，cutsmart buffer: 20μl，ddH2O: 92μl，质粒：80μl，37℃，3 hours。
（3）Gibson组装、转化与鉴定：
150ng PCR扩增7029PETase产物片段，150ng载体pET22b双酶切片段，加入ddH2O补足5微升，加入5微升Gibson预混液，50℃反应40min。将反应完成的组装液通过化学转化的方法转入大肠杆菌DH5α中，37℃过夜培养，挑取单克隆酶切、测序鉴定。经鉴定正确的重组载体用化学转化的方法转入大肠杆菌BL21(DE3)中，并将该重组菌株命名为BL21-PETase以备进行后续实验。
实施例2：PET降解酶7029PETase突变体的构建
利用overlap PCR的方法对野生型7029PETase进行定点突变，以野生型7029PETase的重组载体pET22b-7029PETase为模板，使用引物7029-L61T-1和7029-22b-1扩增7029PETase的前端编码序列，使用引物7029-L61T-2和7029-22b-2扩增7029PETase的后端编码序列及终止子序列，并引入使第61位亮氨酸突变为苏氨酸的核酸序列；最终使用重叠延伸PCR的产物为模板，使用7029-22b-1和7029-22b-2两条引物扩增得到突变体的编码序列及终止子序列。片段扩增使用的PCR程序与实施例1中相同，所得到的突变体命名为7029PETaseL61T，是由7029PETase的第61位氨基酸由亮氨酸突变为苏氨酸形成的，其氨基酸序列如SEQ ID NO.2 所示，核苷酸序列如SEQ ID NO.10所示。
然后使用实施例1中的方法构建7029PETaseL61T的重组载体，经鉴定正确的重组载体用化学转化的方法转入大肠杆菌BL21(DE3)中，并将该重组菌株命名为BL21-L61T以备进行后续实验。
其他突变体的突变方法与7029PETaseL61T相同，所得到的突变体分别命名为7029PETaseW132H、7029PETaseR259A、7029PETaseL61T/W132H、7029PETaseW132H/R259A、7029PETaseL61T/R259A、7029PETaseL61T/W132H/R259A；所构建的重组菌株分别命名为BL21-W132H、BL21-R259A、BL21-L61T/W132H、BL21-L61T/R259A、BL21-W132H/R259A、BL21-L61T/W132H/R259A。
突变过程中使用的引物序列如下：
7029-L61T-1：tgctggactggcgggcggtgtagccgggca，SEQ ID NO.19；
7029-L61T-2：accgcccgccagtccagcat，SEQ ID NO.20；
7029-W132H-1：ccgcctccgcccatcgaatggcccatcaccgc，SEQ ID NO.21；
7029-W132H-2：cattcgatgggcggaggcggca，SEQ ID NO.22；
7029-R259A-1：gccctctcggagtaccgcga，SEQ ID NO.23；
7029-R259A-2：cgcggtactccgagagggcgctgctgcgca，SEQ ID NO.24。
其中，下划线部分为突变区域。
实施例3：野生型7029PETase及其突变体蛋白的表达纯化
野生型7029PETase的表达纯化过程如下：
1) 取重组菌株BL21-PETase于LB固体培养基（含有氨苄抗生素100 μg/ml）上划线，37℃过夜培养；
2) 挑取单克隆菌体于LB液体培养基（含有氨苄抗生素50μg/ml）中，37℃，200 rpm过夜培养；
3) 以1％的接种量将过夜培养所得菌液转接至2L LB液体培养基（含有氨苄抗生素50μg/ml）中，37℃，200 rpm培养至OD600约为0.8时，加入0.1 mM IPTG进行诱导，然后18℃，200 rpm继续培养16~18 h；
4) 离心菌液（4500 rpm，15 min，4℃），弃去上清，将菌体用80 ml Buffer A（50mM Tris-HCl， 300 mM NaCl， 10 mM imidazole， pH 7.6）重悬；
5) 将菌体重悬液在冰浴上进行超声破碎，然后将裂解液离心（20000 rpm，25min，4℃），弃去沉淀，将上清液加到用Buffer A 平衡好的Ni柱上，用Buffer A冲洗未结合的蛋白后，用Buffer B（50 mM Tris-HCl，300 mM NaCl，250 mM imidazole， pH 7.6）洗脱目的蛋白。之后使用PD-10凝胶柱（GE Healthcare）去除咪唑，蛋白最终保存在Buffer C(50 mM Tris-HCl，300 mM NaCl，10%甘油，pH 7.6)中。
7029PETase突变体蛋白的表达纯化方法步骤与野生型相同。
实施例4：7029PETase最适反应条件
为确定7029PETase的最适反应条件，我们测定了其在不同pH（6.0~10.0），以及温度（25℃~42℃）条件下的酶活。在500 μl含有4 mM BHET的反应缓冲液（pH 6.0-8.0时，50mM Na2HPO4-HCl；pH 9.0-10.0时，50mM glycine-NaOH）中加入一定浓度（200 nM）纯化的酶，一定温度下孵育18 h。之后通过热失活（80℃，10 min）终止反应，反应液通过0.22 μm滤膜过滤后，使用HPLC分析产物。在底物分别为BHET与商业化PET塑料膜时，7029PETase的最适反应pH是不同的，实验结果表明，7029PETase的最适反应pH分别是7.0（底物为BHET，图3A）和8.0（底物为PET，图 3B），最适反应温度是30℃（图3C）。
实施例5：7029PETase各个突变体的活性与野生型7029PETase及其突变体的比较
当底物是PET的单体形式BHET时，比较酶对底物的活性方法如下。纯化的野生型7029PETase及其突变体分别加入到500 μl含有4 mM BHET的反应缓冲液（50 mM Na2HPO4-HCl，pH 7.0）中，酶的终浓度为100 nM，在30℃下孵育18 h，每组做三个平行。
当底物是商业化的PET塑料时，比较酶对底物的活性方法如下。纯化的野生型7029PETase及其突变体分别加入到500 μl反应缓冲液（50 mM Na2HPO4-HCl, pH 8.0）中，酶的终浓度为100 nM，在缓冲液中加入直径为5 mm的塑料片。在30℃下孵育18 h，每组做三个平行。
反应完成后，热处理（80℃，10 min）终止反应，将反应液通过0.22 μm滤膜之后，使用反相HPLC分析终产物，结果如下：
(A)各突变体以BHET为底物，降解产物中MHET的量的对比（野生型7029PETase降解液中MHET的量定义为1，MHET为主要产物），见图5A；
(B)各突变体以BHET为底物，降解产物中TPA的量的对比（单位：mM），见图5B；
(C)各突变体以PET为底物，降解产物中MHET的量的对比（野生型7029PETase降解液中MHET的量定义为1，MHET为主要产物），见图5C；
(D)各突变体以PET为底物，降解产物中BHET与TPA的量的对比（单位：μM），见图5D。
结合图5A与图5B可知，当以PET的单体BHET作为底物时，7029PETase的单点突变体与多点突变体的活性与野生型活性相比均有不同程度的提高。而当底物变成商业化PET塑料时，由图5C与图5D可知，各个突变体酶解产生的次要产物BHET与TPA的量相差不大，但产生的主要产物MHET有较大差距，其中突变体7029PETaseW132H、7029PETaseR259A、7029PETaseW132H/R259A和7029PETaseL61T/W132H/R259A比野生型酶活性分别提高了1.04倍、1.28倍、1.12倍和1.52倍。
序列表
<110> 山东大学
<120> PET降解酶及其突变体和应用
<141> 2019-05-22
<160> 24
<170> SIPOSequenceListing 1.0
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<213> 野生型7029PETase的氨基酸序列(Artificial Sequence)
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Gln Thr Asn Pro Tyr Gln Arg Gly Pro Asp Pro Thr Thr Arg Asp Leu
1 5 10 15
Glu Asp Ser Arg Gly Pro Phe Arg Tyr Ala Ser Thr Asn Val Arg Ser
20 25 30
Pro Ser Gly Tyr Gly Ala Gly Thr Ile Tyr Tyr Pro Thr Asp Val Ser
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Gly Ser Val Gly Ala Val Ala Val Val Pro Gly Tyr Leu Ala Arg Gln
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Ser Ser Ile Arg Trp Trp Gly Pro Arg Leu Ala Ser His Gly Phe Val
65 70 75 80
Val Ile Thr Leu Asp Thr Arg Ser Thr Ser Asp Gln Pro Ala Ser Arg
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Ser Ala Gln Gln Met Ala Ala Leu Arg Gln Val Val Ala Leu Ser Glu
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Thr Arg Ser Ser Pro Ile Tyr Gly Lys Val Asp Pro Asn Arg Leu Ala
115 120 125
Val Met Gly Trp Ser Met Gly Gly Gly Gly Thr Leu Ile Ser Ala Arg
130 135 140
Asp Asn Pro Ser Leu Lys Ala Ala Val Pro Phe Ala Pro Trp His Asn
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Thr Ala Asn Phe Ser Gly Val Gln Val Pro Thr Leu Val Ile Ala Cys
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Glu Asn Asp Thr Val Ala Pro Ile Ser Arg His Ala Ser Ser Phe Tyr
180 185 190
Asn Ser Phe Ser Ser Ser Leu Ala Lys Ala Tyr Leu Glu Ile Asn Asn
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Gly Ser His Thr Cys Ala Asn Thr Gly Asn Ser Asn Gln Ala Leu Ile
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Gly Lys Tyr Gly Val Ala Trp Ile Lys Arg Phe Val Asp Asn Asp Thr
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<211> 269
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<213> 7029PETase突变体L61T氨基酸序列(Artificial Sequence)
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Gln Thr Asn Pro Tyr Gln Arg Gly Pro Asp Pro Thr Thr Arg Asp Leu
1 5 10 15
Glu Asp Ser Arg Gly Pro Phe Arg Tyr Ala Ser Thr Asn Val Arg Ser
20 25 30
Pro Ser Gly Tyr Gly Ala Gly Thr Ile Tyr Tyr Pro Thr Asp Val Ser
35 40 45
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50 55 60
Ser Ser Ile Arg Trp Trp Gly Pro Arg Leu Ala Ser His Gly Phe Val
65 70 75 80
Val Ile Thr Leu Asp Thr Arg Ser Thr Ser Asp Gln Pro Ala Ser Arg
85 90 95
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100 105 110
Thr Arg Ser Ser Pro Ile Tyr Gly Lys Val Asp Pro Asn Arg Leu Ala
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130 135 140
Asp Asn Pro Ser Leu Lys Ala Ala Val Pro Phe Ala Pro Trp His Asn
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165 170 175
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180 185 190
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<213> 7029PETase突变体W132H氨基酸序列(Artificial Sequence)
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Gln Thr Asn Pro Tyr Gln Arg Gly Pro Asp Pro Thr Thr Arg Asp Leu
1 5 10 15
Glu Asp Ser Arg Gly Pro Phe Arg Tyr Ala Ser Thr Asn Val Arg Ser
20 25 30
Pro Ser Gly Tyr Gly Ala Gly Thr Ile Tyr Tyr Pro Thr Asp Val Ser
35 40 45
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50 55 60
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85 90 95
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100 105 110
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115 120 125
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130 135 140
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145 150 155 160
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165 170 175
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180 185 190
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245 250 255
Ser Ser Arg Leu Ser Glu Tyr Arg Glu Ser Cys Pro Tyr
260 265
<210> 4
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<212> PRT
<213> 7029PETase突变体R259A氨基酸序列(Artificial Sequence)
<400> 4
Gln Thr Asn Pro Tyr Gln Arg Gly Pro Asp Pro Thr Thr Arg Asp Leu
1 5 10 15
Glu Asp Ser Arg Gly Pro Phe Arg Tyr Ala Ser Thr Asn Val Arg Ser
20 25 30
Pro Ser Gly Tyr Gly Ala Gly Thr Ile Tyr Tyr Pro Thr Asp Val Ser
35 40 45
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Ser Ser Ile Arg Trp Trp Gly Pro Arg Leu Ala Ser His Gly Phe Val
65 70 75 80
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85 90 95
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100 105 110
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130 135 140
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145 150 155 160
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165 170 175
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180 185 190
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195 200 205
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260 265
<210> 5
<211> 269
<212> PRT
<213> 29PETase双突变体L61T/W132H氨基酸序列(Artificial Sequence)
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Gln Thr Asn Pro Tyr Gln Arg Gly Pro Asp Pro Thr Thr Arg Asp Leu
1 5 10 15
Glu Asp Ser Arg Gly Pro Phe Arg Tyr Ala Ser Thr Asn Val Arg Ser
20 25 30
Pro Ser Gly Tyr Gly Ala Gly Thr Ile Tyr Tyr Pro Thr Asp Val Ser
35 40 45
Gly Ser Val Gly Ala Val Ala Val Val Pro Gly Tyr Thr Ala Arg Gln
50 55 60
Ser Ser Ile Arg Trp Trp Gly Pro Arg Leu Ala Ser His Gly Phe Val
65 70 75 80
Val Ile Thr Leu Asp Thr Arg Ser Thr Ser Asp Gln Pro Ala Ser Arg
85 90 95
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100 105 110
Thr Arg Ser Ser Pro Ile Tyr Gly Lys Val Asp Pro Asn Arg Leu Ala
115 120 125
Val Met Gly His Ser Met Gly Gly Gly Gly Thr Leu Ile Ser Ala Arg
130 135 140
Asp Asn Pro Ser Leu Lys Ala Ala Val Pro Phe Ala Pro Trp His Asn
145 150 155 160
Thr Ala Asn Phe Ser Gly Val Gln Val Pro Thr Leu Val Ile Ala Cys
165 170 175
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180 185 190
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195 200 205
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245 250 255
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260 265
<210> 6
<211> 269
<212> PRT
<213> 7029PETase双突变体L61T/R259A氨基酸序列(Artificial Sequence)
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Gln Thr Asn Pro Tyr Gln Arg Gly Pro Asp Pro Thr Thr Arg Asp Leu
1 5 10 15
Glu Asp Ser Arg Gly Pro Phe Arg Tyr Ala Ser Thr Asn Val Arg Ser
20 25 30
Pro Ser Gly Tyr Gly Ala Gly Thr Ile Tyr Tyr Pro Thr Asp Val Ser
35 40 45
Gly Ser Val Gly Ala Val Ala Val Val Pro Gly Tyr Thr Ala Arg Gln
50 55 60
Ser Ser Ile Arg Trp Trp Gly Pro Arg Leu Ala Ser His Gly Phe Val
65 70 75 80
Val Ile Thr Leu Asp Thr Arg Ser Thr Ser Asp Gln Pro Ala Ser Arg
85 90 95
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100 105 110
Thr Arg Ser Ser Pro Ile Tyr Gly Lys Val Asp Pro Asn Arg Leu Ala
115 120 125
Val Met Gly Trp Ser Met Gly Gly Gly Gly Thr Leu Ile Ser Ala Arg
130 135 140
Asp Asn Pro Ser Leu Lys Ala Ala Val Pro Phe Ala Pro Trp His Asn
145 150 155 160
Thr Ala Asn Phe Ser Gly Val Gln Val Pro Thr Leu Val Ile Ala Cys
165 170 175
Glu Asn Asp Thr Val Ala Pro Ile Ser Arg His Ala Ser Ser Phe Tyr
180 185 190
Asn Ser Phe Ser Ser Ser Leu Ala Lys Ala Tyr Leu Glu Ile Asn Asn
195 200 205
Gly Ser His Thr Cys Ala Asn Thr Gly Asn Ser Asn Gln Ala Leu Ile
210 215 220
Gly Lys Tyr Gly Val Ala Trp Ile Lys Arg Phe Val Asp Asn Asp Thr
225 230 235 240
Arg Tyr Ser Pro Phe Leu Cys Gly Ala Pro His Gln Ala Asp Leu Arg
245 250 255
Ser Ser Ala Leu Ser Glu Tyr Arg Glu Ser Cys Pro Tyr
260 265
<210> 7
<211> 269
<212> PRT
<213> 7029PETase双突变体W132H/R259A氨基酸序列(Artificial Sequence)
<400> 7
Gln Thr Asn Pro Tyr Gln Arg Gly Pro Asp Pro Thr Thr Arg Asp Leu
1 5 10 15
Glu Asp Ser Arg Gly Pro Phe Arg Tyr Ala Ser Thr Asn Val Arg Ser
20 25 30
Pro Ser Gly Tyr Gly Ala Gly Thr Ile Tyr Tyr Pro Thr Asp Val Ser
35 40 45
Gly Ser Val Gly Ala Val Ala Val Val Pro Gly Tyr Leu Ala Arg Gln
50 55 60
Ser Ser Ile Arg Trp Trp Gly Pro Arg Leu Ala Ser His Gly Phe Val
65 70 75 80
Val Ile Thr Leu Asp Thr Arg Ser Thr Ser Asp Gln Pro Ala Ser Arg
85 90 95
Ser Ala Gln Gln Met Ala Ala Leu Arg Gln Val Val Ala Leu Ser Glu
100 105 110
Thr Arg Ser Ser Pro Ile Tyr Gly Lys Val Asp Pro Asn Arg Leu Ala
115 120 125
Val Met Gly His Ser Met Gly Gly Gly Gly Thr Leu Ile Ser Ala Arg
130 135 140
Asp Asn Pro Ser Leu Lys Ala Ala Val Pro Phe Ala Pro Trp His Asn
145 150 155 160
Thr Ala Asn Phe Ser Gly Val Gln Val Pro Thr Leu Val Ile Ala Cys
165 170 175
Glu Asn Asp Thr Val Ala Pro Ile Ser Arg His Ala Ser Ser Phe Tyr
180 185 190
Asn Ser Phe Ser Ser Ser Leu Ala Lys Ala Tyr Leu Glu Ile Asn Asn
195 200 205
Gly Ser His Thr Cys Ala Asn Thr Gly Asn Ser Asn Gln Ala Leu Ile
210 215 220
Gly Lys Tyr Gly Val Ala Trp Ile Lys Arg Phe Val Asp Asn Asp Thr
225 230 235 240
Arg Tyr Ser Pro Phe Leu Cys Gly Ala Pro His Gln Ala Asp Leu Arg
245 250 255
Ser Ser Ala Leu Ser Glu Tyr Arg Glu Ser Cys Pro Tyr
260 265
<210> 8
<211> 269
<212> PRT
<213> 7029PETase三突变体L61T/W132H/R259A氨基酸序列(ArtificialSequence)
<400> 8
Gln Thr Asn Pro Tyr Gln Arg Gly Pro Asp Pro Thr Thr Arg Asp Leu
1 5 10 15
Glu Asp Ser Arg Gly Pro Phe Arg Tyr Ala Ser Thr Asn Val Arg Ser
20 25 30
Pro Ser Gly Tyr Gly Ala Gly Thr Ile Tyr Tyr Pro Thr Asp Val Ser
35 40 45
Gly Ser Val Gly Ala Val Ala Val Val Pro Gly Tyr Thr Ala Arg Gln
50 55 60
Ser Ser Ile Arg Trp Trp Gly Pro Arg Leu Ala Ser His Gly Phe Val
65 70 75 80
Val Ile Thr Leu Asp Thr Arg Ser Thr Ser Asp Gln Pro Ala Ser Arg
85 90 95
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100 105 110
Thr Arg Ser Ser Pro Ile Tyr Gly Lys Val Asp Pro Asn Arg Leu Ala
115 120 125
Val Met Gly His Ser Met Gly Gly Gly Gly Thr Leu Ile Ser Ala Arg
130 135 140
Asp Asn Pro Ser Leu Lys Ala Ala Val Pro Phe Ala Pro Trp His Asn
145 150 155 160
Thr Ala Asn Phe Ser Gly Val Gln Val Pro Thr Leu Val Ile Ala Cys
165 170 175
Glu Asn Asp Thr Val Ala Pro Ile Ser Arg His Ala Ser Ser Phe Tyr
180 185 190
Asn Ser Phe Ser Ser Ser Leu Ala Lys Ala Tyr Leu Glu Ile Asn Asn
195 200 205
Gly Ser His Thr Cys Ala Asn Thr Gly Asn Ser Asn Gln Ala Leu Ile
210 215 220
Gly Lys Tyr Gly Val Ala Trp Ile Lys Arg Phe Val Asp Asn Asp Thr
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Arg Tyr Ser Pro Phe Leu Cys Gly Ala Pro His Gln Ala Asp Leu Arg
245 250 255
Ser Ser Ala Leu Ser Glu Tyr Arg Glu Ser Cys Pro Tyr
260 265
<210> 9
<211> 807
<212> DNA
<213> 野生型7029PETase的核苷酸序列(Artificial Sequence)
<400> 9
cagaccaacc cctaccagcg aggcccggac ccgacgaccc gcgacctgga agacagccga 60
gggccgttcc gctacgccag caccaacgtg cgctcgccca gcggttatgg cgcgggcacg 120
atctactacc ccaccgacgt cagcggcagc gtcggcgcgg tggcggtggt gcccggctac 180
ctcgcccgcc agtccagcat ccgctggtgg gggccgcgcc tggcctcgca cgggtttgtc 240
gtcatcaccc tcgacacccg ctcgacctcc gaccagcccg ccagccgttc cgctcaacag 300
atggcggcgc tgcggcaggt ggtggcgctc agcgagacgc gcagcagccc gatctacggc 360
aaggtcgatc ccaaccgtct cgcggtgatg ggctggtcga tgggcggagg cggcacgctg 420
atctctgcgc gcgacaaccc cagcttgaag gccgccgtgc cattcgcccc gtggcacaac 480
accgccaact tctcgggcgt gcaagtgccg acgctcgtga tcgcttgcga aaacgacacc 540
gtcgccccga tctcgcgaca tgcctcgtcc ttctacaaca gcttttcgag ctcgctcgcc 600
aaggcctatc tcgagatcaa caacggctcg cacacctgcg ccaacaccgg caacagcaac 660
caggccctga tcggcaaata cggtgtggcg tggatcaagc gcttcgtcga caacgacaca 720
cgctacagcc ccttcctctg cggcgcacca catcaggccg acctgcgcag cagccgcctc 780
tcggagtacc gcgagagctg cccgtac 807
<210> 10
<211> 807
<212> DNA
<213> 7029PETase突变体L61T核苷酸序列(Artificial Sequence)
<400> 10
cagaccaacc cctaccagcg aggcccggac ccgacgaccc gcgacctgga agacagccga 60
gggccgttcc gctacgccag caccaacgtg cgctcgccca gcggttatgg cgcgggcacg 120
atctactacc ccaccgacgt cagcggcagc gtcggcgcgg tggcggtggt gcccggctac 180
accgcccgcc agtccagcat ccgctggtgg gggccgcgcc tggcctcgca cgggtttgtc 240
gtcatcaccc tcgacacccg ctcgacctcc gaccagcccg ccagccgttc cgctcaacag 300
atggcggcgc tgcggcaggt ggtggcgctc agcgagacgc gcagcagccc gatctacggc 360
aaggtcgatc ccaaccgtct cgcggtgatg ggctggtcga tgggcggagg cggcacgctg 420
atctctgcgc gcgacaaccc cagcttgaag gccgccgtgc cattcgcccc gtggcacaac 480
accgccaact tctcgggcgt gcaagtgccg acgctcgtga tcgcttgcga aaacgacacc 540
gtcgccccga tctcgcgaca tgcctcgtcc ttctacaaca gcttttcgag ctcgctcgcc 600
aaggcctatc tcgagatcaa caacggctcg cacacctgcg ccaacaccgg caacagcaac 660
caggccctga tcggcaaata cggtgtggcg tggatcaagc gcttcgtcga caacgacaca 720
cgctacagcc ccttcctctg cggcgcacca catcaggccg acctgcgcag cagccgcctc 780
tcggagtacc gcgagagctg cccgtac 807
<210> 11
<211> 807
<212> DNA
<213> 7029PETase突变体W132H核苷酸序列(Artificial Sequence)
<400> 11
cagaccaacc cctaccagcg aggcccggac ccgacgaccc gcgacctgga agacagccga 60
gggccgttcc gctacgccag caccaacgtg cgctcgccca gcggttatgg cgcgggcacg 120
atctactacc ccaccgacgt cagcggcagc gtcggcgcgg tggcggtggt gcccggctac 180
ctcgcccgcc agtccagcat ccgctggtgg gggccgcgcc tggcctcgca cgggtttgtc 240
gtcatcaccc tcgacacccg ctcgacctcc gaccagcccg ccagccgttc cgctcaacag 300
atggcggcgc tgcggcaggt ggtggcgctc agcgagacgc gcagcagccc gatctacggc 360
aaggtcgatc ccaaccgtct cgcggtgatg ggccattcga tgggcggagg cggcacgctg 420
atctctgcgc gcgacaaccc cagcttgaag gccgccgtgc cattcgcccc gtggcacaac 480
accgccaact tctcgggcgt gcaagtgccg acgctcgtga tcgcttgcga aaacgacacc 540
gtcgccccga tctcgcgaca tgcctcgtcc ttctacaaca gcttttcgag ctcgctcgcc 600
aaggcctatc tcgagatcaa caacggctcg cacacctgcg ccaacaccgg caacagcaac 660
caggccctga tcggcaaata cggtgtggcg tggatcaagc gcttcgtcga caacgacaca 720
cgctacagcc ccttcctctg cggcgcacca catcaggccg acctgcgcag cagccgcctc 780
tcggagtacc gcgagagctg cccgtac 807
<210> 12
<211> 807
<212> DNA
<213> 7029PETase突变体R259A核苷酸序列(Artificial Sequence)
<400> 12
cagaccaacc cctaccagcg aggcccggac ccgacgaccc gcgacctgga agacagccga 60
gggccgttcc gctacgccag caccaacgtg cgctcgccca gcggttatgg cgcgggcacg 120
atctactacc ccaccgacgt cagcggcagc gtcggcgcgg tggcggtggt gcccggctac 180
ctcgcccgcc agtccagcat ccgctggtgg gggccgcgcc tggcctcgca cgggtttgtc 240
gtcatcaccc tcgacacccg ctcgacctcc gaccagcccg ccagccgttc cgctcaacag 300
atggcggcgc tgcggcaggt ggtggcgctc agcgagacgc gcagcagccc gatctacggc 360
aaggtcgatc ccaaccgtct cgcggtgatg ggctggtcga tgggcggagg cggcacgctg 420
atctctgcgc gcgacaaccc cagcttgaag gccgccgtgc cattcgcccc gtggcacaac 480
accgccaact tctcgggcgt gcaagtgccg acgctcgtga tcgcttgcga aaacgacacc 540
gtcgccccga tctcgcgaca tgcctcgtcc ttctacaaca gcttttcgag ctcgctcgcc 600
aaggcctatc tcgagatcaa caacggctcg cacacctgcg ccaacaccgg caacagcaac 660
caggccctga tcggcaaata cggtgtggcg tggatcaagc gcttcgtcga caacgacaca 720
cgctacagcc ccttcctctg cggcgcacca catcaggccg acctgcgcag cagcgccctc 780
tcggagtacc gcgagagctg cccgtac 807
<210> 13
<211> 807
<212> DNA
<213> 7029PETase双突变体L61T/W132H核苷酸序列(Artificial Sequence)
<400> 13
cagaccaacc cctaccagcg aggcccggac ccgacgaccc gcgacctgga agacagccga 60
gggccgttcc gctacgccag caccaacgtg cgctcgccca gcggttatgg cgcgggcacg 120
atctactacc ccaccgacgt cagcggcagc gtcggcgcgg tggcggtggt gcccggctac 180
accgcccgcc agtccagcat ccgctggtgg gggccgcgcc tggcctcgca cgggtttgtc 240
gtcatcaccc tcgacacccg ctcgacctcc gaccagcccg ccagccgttc cgctcaacag 300
atggcggcgc tgcggcaggt ggtggcgctc agcgagacgc gcagcagccc gatctacggc 360
aaggtcgatc ccaaccgtct cgcggtgatg ggccattcga tgggcggagg cggcacgctg 420
atctctgcgc gcgacaaccc cagcttgaag gccgccgtgc cattcgcccc gtggcacaac 480
accgccaact tctcgggcgt gcaagtgccg acgctcgtga tcgcttgcga aaacgacacc 540
gtcgccccga tctcgcgaca tgcctcgtcc ttctacaaca gcttttcgag ctcgctcgcc 600
aaggcctatc tcgagatcaa caacggctcg cacacctgcg ccaacaccgg caacagcaac 660
caggccctga tcggcaaata cggtgtggcg tggatcaagc gcttcgtcga caacgacaca 720
cgctacagcc ccttcctctg cggcgcacca catcaggccg acctgcgcag cagccgcctc 780
tcggagtacc gcgagagctg cccgtac 807
<210> 14
<211> 807
<212> DNA
<213> 7029PETase双突变体L61T/R259A核苷酸序列(Artificial Sequence)
<400> 14
cagaccaacc cctaccagcg aggcccggac ccgacgaccc gcgacctgga agacagccga 60
gggccgttcc gctacgccag caccaacgtg cgctcgccca gcggttatgg cgcgggcacg 120
atctactacc ccaccgacgt cagcggcagc gtcggcgcgg tggcggtggt gcccggctac 180
accgcccgcc agtccagcat ccgctggtgg gggccgcgcc tggcctcgca cgggtttgtc 240
gtcatcaccc tcgacacccg ctcgacctcc gaccagcccg ccagccgttc cgctcaacag 300
atggcggcgc tgcggcaggt ggtggcgctc agcgagacgc gcagcagccc gatctacggc 360
aaggtcgatc ccaaccgtct cgcggtgatg ggctggtcga tgggcggagg cggcacgctg 420
atctctgcgc gcgacaaccc cagcttgaag gccgccgtgc cattcgcccc gtggcacaac 480
accgccaact tctcgggcgt gcaagtgccg acgctcgtga tcgcttgcga aaacgacacc 540
gtcgccccga tctcgcgaca tgcctcgtcc ttctacaaca gcttttcgag ctcgctcgcc 600
aaggcctatc tcgagatcaa caacggctcg cacacctgcg ccaacaccgg caacagcaac 660
caggccctga tcggcaaata cggtgtggcg tggatcaagc gcttcgtcga caacgacaca 720
cgctacagcc ccttcctctg cggcgcacca catcaggccg acctgcgcag cagcgccctc 780
tcggagtacc gcgagagctg cccgtac 807
<210> 15
<211> 807
<212> DNA
<213> 7029PETase双突变体W132H/R259A核苷酸序列(Artificial Sequence)
<400> 15
cagaccaacc cctaccagcg aggcccggac ccgacgaccc gcgacctgga agacagccga 60
gggccgttcc gctacgccag caccaacgtg cgctcgccca gcggttatgg cgcgggcacg 120
atctactacc ccaccgacgt cagcggcagc gtcggcgcgg tggcggtggt gcccggctac 180
ctcgcccgcc agtccagcat ccgctggtgg gggccgcgcc tggcctcgca cgggtttgtc 240
gtcatcaccc tcgacacccg ctcgacctcc gaccagcccg ccagccgttc cgctcaacag 300
atggcggcgc tgcggcaggt ggtggcgctc agcgagacgc gcagcagccc gatctacggc 360
aaggtcgatc ccaaccgtct cgcggtgatg ggccattcga tgggcggagg cggcacgctg 420
atctctgcgc gcgacaaccc cagcttgaag gccgccgtgc cattcgcccc gtggcacaac 480
accgccaact tctcgggcgt gcaagtgccg acgctcgtga tcgcttgcga aaacgacacc 540
gtcgccccga tctcgcgaca tgcctcgtcc ttctacaaca gcttttcgag ctcgctcgcc 600
aaggcctatc tcgagatcaa caacggctcg cacacctgcg ccaacaccgg caacagcaac 660
caggccctga tcggcaaata cggtgtggcg tggatcaagc gcttcgtcga caacgacaca 720
cgctacagcc ccttcctctg cggcgcacca catcaggccg acctgcgcag cagcgccctc 780
tcggagtacc gcgagagctg cccgtac 807
<210> 16
<211> 807
<212> DNA
<213> 7029PETase三突变体L61T/W132H/R259A核苷酸序列(ArtificialSequence)
<400> 16
cagaccaacc cctaccagcg aggcccggac ccgacgaccc gcgacctgga agacagccga 60
gggccgttcc gctacgccag caccaacgtg cgctcgccca gcggttatgg cgcgggcacg 120
atctactacc ccaccgacgt cagcggcagc gtcggcgcgg tggcggtggt gcccggctac 180
accgcccgcc agtccagcat ccgctggtgg gggccgcgcc tggcctcgca cgggtttgtc 240
gtcatcaccc tcgacacccg ctcgacctcc gaccagcccg ccagccgttc cgctcaacag 300
atggcggcgc tgcggcaggt ggtggcgctc agcgagacgc gcagcagccc gatctacggc 360
aaggtcgatc ccaaccgtct cgcggtgatg ggccattcga tgggcggagg cggcacgctg 420
atctctgcgc gcgacaaccc cagcttgaag gccgccgtgc cattcgcccc gtggcacaac 480
accgccaact tctcgggcgt gcaagtgccg acgctcgtga tcgcttgcga aaacgacacc 540
gtcgccccga tctcgcgaca tgcctcgtcc ttctacaaca gcttttcgag ctcgctcgcc 600
aaggcctatc tcgagatcaa caacggctcg cacacctgcg ccaacaccgg caacagcaac 660
caggccctga tcggcaaata cggtgtggcg tggatcaagc gcttcgtcga caacgacaca 720
cgctacagcc ccttcctctg cggcgcacca catcaggccg acctgcgcag cagcgccctc 780
tcggagtacc gcgagagctg cccgtac 807
<210> 17
<211> 60
<212> DNA
<213> 引物7029-22b-1(Artificial Sequence)
<400> 17
tctgctgctc ctcgctgccc agccggcgat ggccatggat cagaccaacc cctaccagcg 60
<210> 18
<211> 64
<212> DNA
<213> 引物7029-22b-2(Artificial Sequence)
<400> 18
ctttgttagc agccggatct cagtggtggt ggtggtggtg gtacgggcag ctctcgcggt 60
actc 64
<210> 19
<211> 30
<212> DNA
<213> 引物7029-L61T-1(Artificial Sequence)
<400> 19
tgctggactg gcgggcggtg tagccgggca 30
<210> 20
<211> 20
<212> DNA
<213> 引物7029-L61T-2(Artificial Sequence)
<400> 20
accgcccgcc agtccagcat 20
<210> 21
<211> 32
<212> DNA
<213> 引物7029-W132H-1(Artificial Sequence)
<400> 21
ccgcctccgc ccatcgaatg gcccatcacc gc 32
<210> 22
<211> 22
<212> DNA
<213> 引物7029-W132H-2(Artificial Sequence)
<400> 22
cattcgatgg gcggaggcgg ca 22
<210> 23
<211> 20
<212> DNA
<213> 引物7029-R259A-1(Artificial Sequence)
<400> 23
gccctctcgg agtaccgcga 20
<210> 24
<211> 30
<212> DNA
<213> 引物7029-R259A-2(Artificial Sequence)
<400> 24
cgcggtactc cgagagggcg ctgctgcgca 30
Claims (7)
1.一种PET降解酶突变体，其特征在于：所述的突变体是PET降解酶7029PETase上第61位氨基酸由亮氨酸突变为苏氨酸形成的，其氨基酸序列如SEQ ID NO.2 所示，其核苷酸序列如SEQ ID NO.10 所示；PET降解酶7029PETase的氨基酸序列如SEQ ID NO.1 所示，核苷酸序列如SEQ ID NO.9 所示。
2.一种PET降解酶突变体，其特征在于：所述的突变体是PET降解酶7029PETase上第61位氨基酸由亮氨酸突变为苏氨酸，同时第132位氨基酸由色氨酸突变为组氨酸形成的，其氨基酸序列如SEQ ID NO.5 所示，其核苷酸序列如SEQ ID NO.13 所示；PET降解酶7029PETase的氨基酸序列如SEQ ID NO.1 所示，核苷酸序列如SEQ ID NO.9 所示。
3.一种PET降解酶突变体，其特征在于：所述的突变体是PET降解酶7029PETase上第61位氨基酸由亮氨酸突变为苏氨酸，同时第259位氨基酸由精氨酸突变为丙氨酸形成的，其氨基酸序列如SEQ ID NO.6 所示，其核苷酸序列如SEQ ID NO.14 所示；PET降解酶7029PETase的氨基酸序列如SEQ ID NO.1 所示，核苷酸序列如SEQ ID NO.9 所示。
4.一种PET降解酶突变体，其特征在于：所述的突变体是PET降解酶7029PETase上第61位氨基酸由亮氨酸突变为苏氨酸，同时第132位氨基酸由色氨酸突变为组氨酸，第259位氨基酸由精氨酸突变为丙氨酸形成的，其氨基酸序列如SEQ ID NO.8 所示，其核苷酸序列如SEQ ID NO.16 所示；PET降解酶7029PETase的氨基酸序列如SEQ ID NO.1 所示，核苷酸序列如SEQ ID NO.9 所示。
5.一种重组载体，其特征在于：所述的载体包含权利要求1-4任一项所述的PET降解酶突变体。
6.一种重组菌，其特征在于：所述的重组菌包含权利要求5所述的重组载体。
7.如权利要求1-4任一项所述的PET降解酶突变体的用途，其特征在于：PET降解酶突变体在降解PET塑料中的应用。
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| CN113684195B (zh) * | 2020-05-19 | 2022-07-22 | 中国海洋大学 | 一种甾醇酯酶和其编码基因及其突变体 |
| CN114480343B (zh) * | 2020-10-27 | 2023-07-04 | 湖北大学 | 具提升活性的pet水解酶 |
| CN114438140B (zh) * | 2020-11-02 | 2024-05-17 | 华南理工大学 | Pet的降解方法 |
| CN115125225B (zh) * | 2021-03-25 | 2023-07-04 | 湖北大学 | 具有提升的热稳定性的pet降解酶 |
| CN113774041B (zh) * | 2021-08-06 | 2022-09-02 | 天津大学 | PET水解酶IsPETase突变酶及编码基因及工程菌 |
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| CN114591933A (zh) | 2022-06-07 |
| CN114591933B (zh) | 2023-09-22 |
| CN110241097A (zh) | 2019-09-17 |
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