中国科学院微生物研究所、中国微生物学会主办
文章信息
- 赵夷培, 王浩, 武攀, 李志帅, 刘夫锋, 顾群, 刘卫东, 高健, 韩旭
- ZHAO Yipei, WANG Hao, WU Pan, LI Zhishuai, LIU Fufeng, GU Qun, LIU Weidong, GAO Jian, HAN Xu
- 来源于海洋宏基因组塑料降解酶Ple629的耐热性提升改造
- Engineering the plastic degradation enzyme Ple629 from marine consortium to improve its thermal stability
- 生物工程学报, 2023, 39(5): 2040-2052
- Chinese Journal of Biotechnology, 2023, 39(5): 2040-2052
- 10.13345/j.cjb.221045
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文章历史
- Received: December 29, 2022
- Accepted: March 9, 2023
2. 中国科学院天津工业生物技术研究所 工业酶国家工程研究中心, 天津 300308;
3. 国家合成生物技术创新中心, 天津 300308;
4. 中国科学院大学, 北京 100049
2. National Engineering Center for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;
3. National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China;
4. University of Chinese Academy of Sciences, Beijing 100049, China
塑料是一类以不同石化来源单体为原料通过加聚或缩合形成的高分子聚合物,它们具有绝缘性、耐热性、可塑性及足够的强度,被广泛地应用于日常生活和其他领域[1]。聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)由对苯二甲酸(terephthalic acid, TPA)和乙二醇(ethylene glycol, EG)通过酯键聚合而成,被广泛用作饮料、食品包装材料以及纺织纤维等,是重要的大规模生产的石化塑料之一[2],然而,塑料的广泛使用带来了大量的环境污染及白色污染,因为其降解速度极慢,并且已经严重威胁到全球的生态系统。聚己二酸/对苯二甲酸丁二醇酯(polybutylene adipate terephthalate, PBAT)是一种可生物降解的脂肪族芳香族共聚酯,通过将1, 4-丁二醇与芳香族二羧酸酯化,然后与琥珀酸缩聚合成[3],它既保留了聚酯塑料的一些优良特性,又可以较快地被生物降解[4],所以人们认为它可以作为聚乙烯(polyethylene, PE)等塑料的环保替代品,具有较广泛的应用潜力[5]。PBAT可以单独使用,也可与聚乳酸(polylactic acid, PLA)、聚-β-羟基丁酸-β-羟基戊酸酯(polyhydroxybutyrate-co-valerate, PHBV)和纤维素等其他材料混合使用[6-12],并且已经作为商业产品用于工业规模生产,如地膜、有机垃圾袋或包装材料等[13]。PBAT在土壤和堆肥条件下可被微生物降解[14-16],在高温堆肥条件下,可在数月完全分解[17]。对聚酯类塑料如PET和PBAT,利用生物酶法进行降解并对降解产物进行高值化利用,是一个重要的研究方向[18-19]。
近年来筛选和鉴定出大量聚酯类塑料降解酶,如酯酶(esterase)、脂肪酶(lipase)和角质酶(cutinase)等,一些以降解PET塑料为主的酶,对PBAT也有降解能力[20],随着聚酯类塑料降解酶催化机制和定向改造的突破性进展,已经获得了一些具有优良性质的突变体酶[21-23]。较高的反应温度有利于塑料的降解,因而高温酶在塑料的生物降解中有明显的优势[24],提升酶的热稳定性将有助于酶法生物循环的应用[25]。近年来,许多研究者通过酶工程对塑料降解酶如来自大阪伊德氏杆菌(Ideonella sakaiensis) 201-F6的IsPETase改造来提高其热稳定性[26]。在蛋白结构中增添二硫键是提升酶的热稳定性的有效方法之一[27-29],该方法被成功用在塑料降解酶上,来自于热裂菌(Thermobifida fusca) KW3聚酯水解酶TfCut2通过在蛋白结构中引入二硫键(D204C-E253C),其热稳定性Tm提升了25 ℃,酶的催化效率也提高了1倍,并通过蛋白质晶体学证明了此二硫键的形成[30-31]。除了上述基于酶结构的改造,其他技术如人工智能、机器学习,以及FoldX虚拟突变能量计算等也被用来进行辅助研究[32],基于贪婪积累策略方法获得的DuraPETase,比野生型IsPETase的Tm增加了31 ℃ [33],进一步结合卷积神经网络训练后,得到了目前构建IsPETase突变体库中Tm最高的突变体DuraPETase+ N233K,Tm约为83.5 ℃ [34]。
以PBAT为唯一碳源的海洋微生物基因组中发现的塑料降解酶Ple629[35],对PBAT和PET纳米颗粒都具有水解活性,有较好的应用潜能。近期本团队解析了其空间结构[36]和底物复合体的结构[37],并参照PET降解酶的改造[38],对该酶进行了活性提升改造,获得了活性提升的突变体。由于该酶是来源于海洋环境的中温酶,本研究拟通过设计提升其热稳定性,以增强其后续应用的潜能。参照已报道的PET水解酶利用生成二硫键提升热稳定性的结果,本研究在Ple629中相应位置引入了一对关键二硫键,并结合虚拟计算设计找到了一些可能的相关位点,对这些突变蛋白进行表达制备,验证了突变体热稳定性的变化情况,获得了一些热稳定性提升的突变体,并对突变的潜在影响进行了分析。本研究为后续聚酯类塑料的降解研究提供了更多的理论基础和实验依据,对推进相应塑料的绿色降解具有积极意义。
1 材料与方法 1.1 材料大肠杆菌(Escherichia coli) BL21(DE3)感受态细胞购于北京全式金生物技术有限公司;异丙基-β-d-硫代半乳糖苷(isopropyl-β- d-thiogalactoside, IPTG)购于北京索莱宝科技有限公司。PET纳米颗粒由非晶态PET薄膜(购自Goodfellow公司)制备而成,对苯二甲酸购自Sigma公司。质粒提取试剂盒和DNA纯化试剂盒购于天根生化科技(北京)有限公司(Tiangen)。
1.2 Ple629重组质粒的合成和定点突变海洋微生物来源的Ple629 (GenBank登录号:OK558825) [28]基因经密码子优化后,由上海捷瑞生物技术公司合成,并连接至pET32a载体。经过定点突变PCR,在PCR产物中加入Dpn I酶进行消化,去除甲基化模板后,用DNA纯化试剂盒纯化,纯化后的产物转入E. coli DH5α感受态细胞中,测序正确的克隆按照质粒小提试剂盒上的步骤进行质粒抽提,存放于–20 ℃,用于后续实验,突变体引物设计如表 1所示。
Mutants | Primer sequence (5′→3′) |
N65D | TATCCGACCGANACCACCGGCACGATGGCG |
V80C | CCGGGCTTTTGNAGCCCGGAAAGCAGCA |
V80M | CCGGGCTTTATGAGCCCGGAAAGCAGC |
V80C/G108C | CCGGGCTTTATGAGCCCGGAAAGCAGC ACCAACAGCTGNTTTGATCAGCCGGCG |
G108C | ACCAACAGCTGNTTTGATCAGCCGGCG |
Q111D | GGCTTTGATGANCCGGCGAGCCGTGCGA |
D226C/S281C | CCGCCACACAGTTCCACAAACGCTTTCGCG ATTCACAAATGCTGCGATCGCTTTCATGGTT |
S237F | AATGGTAGCTTNGGTTTTGGCGGTAGCTATA |
S237W | AATGGTAGCTGGGGTTTTGGCGGTAGCTAT |
T231C | CGGCCATTGTTGCGCGAATGGTAGCTC |
T231D | CGGCCATGACTGCGCGAATGGTAGCTC |
将重组质粒pET32a-Ple629以及相应突变体质粒分别转化至E. coli BL21(DE3),挑取单菌落于含有100 μg/L氨苄青霉素(ampicillin, Amp)的5 mL Luria-Bertani (LB)小试管中,220 r/min培养约14–16 h,逐级扩大培养至1 L含有相同抗性的LB培养基中,在37 ℃摇床中以220 r/min的转速培养至OD600值为0.6–0.8左右,加入终浓度为0.4 mmol/L的诱导剂IPTG,降温至16 ℃继续培养18–22 h,5 000 r/min离心20 min后,收集菌体。将菌体重悬于25 mmol /L Tris-HCl,150 mmol /L NaCl,20 mmol /L咪唑,pH 7.5的缓冲液中,混合均匀后利用低温高压均质破碎后,将破碎后的菌液在4 ℃、15 500 r/min条件下低温高速离心1 h,上清液用于后续纯化。上清液载于经上述缓冲液平衡后的Ni-NTA亲和柱上,用含有25 mmol/L Tris-HCl,150 mmol/L NaCl,250 mmol/L咪唑,pH 7.5的缓冲液梯度洗脱蛋白,目的蛋白大约在含有100 mmol/L咪唑的条件中洗脱出来,收集目的蛋白,在4 ℃冰箱中,用25 mmol/L Tris-HCl、150 mmol/L NaCl、pH 7.5的缓冲液过夜透析,同时加入适量TEV酶切掉其载体标签。将去除标签的目的蛋白重新经过Ni-NTA亲和柱进一步纯化,最后获得较纯蛋白,透析于25 mmol/L Tris-HCl、150 mmol/L NaCl、pH 7.5的缓冲液中,利用聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate- polyacrylamide gel electrophoresis, SDS-PAGE)确定纯化蛋白的情况,测定蛋白质浓度后,保存于−80 ℃冰箱中。
1.4 蛋白结构分析前期已经解析了Ple629的蛋白质晶体结构(PDB ID: 7VPA),它和已知的PET水解酶家族类似,属于α/β水解酶超家族[39],Ple629结构上有9个β折叠,外围被8个α螺旋包围,具有保守的丝氨酸水解酶序列Gly-x1-Ser-x2-Gly (G150-W151-S152-M153-G154)和催化三联体S152-D198-H230。选取和Ple629结构及序列较为相似的PET水解酶TfCut2、枝叶堆肥的宏基因组来源的嗜热PET水解酶LCC和IsPETase等为研究对象,利用多种软件分析比较其蛋白结构、序列和这些高同源蛋白的关系。
1.5 计算设计分析在有稳定可靠的蛋白质结构信息时,基于使用能量函数对蛋白能量进行计算,可以得到理论上自由能更小、更有利于蛋白质稳定的突变设计[40],相应方法有成功应用的实例。本文使用FoldX软件包[41]中的PositionScan功能,利用已经解析的Ple629蛋白质晶体为模板,对其所有氨基酸残基进行虚拟饱和突变,计算得到所有突变的DDG (DDG=DGMut–DGWt),保留DDG < –0.45 kcal/mol的突变,选择能量最低的突变进行后续实验。
1.6 热稳定性Tm值以及活性测定Ple629野生型蛋白和突变体蛋白分别通过蛋白质稳定分析仪(unchained labs, model: UNcle)测定其样品的熔点温度(melting temperature, Tm)。将纯化后的蛋白稀释至1.0 mg/mL (PBS缓冲液,pH 7.4)后放入样品池中,分析仪升温参数的起始温度设置为15 ℃、终点温度为95 ℃,升温速率为0.25 ℃/min。实验结束后用分析软件UNcle Analysis 5.03自动计算Tm和开始聚集温度(Tagg),默认BCM (Barycentric mean)方法分析Tm。
检测Ple629野生型和最优热稳定性突变体D226C/S281C的活性,将5 mg PET-NP溶解在PBS (pH 7.4)缓冲液中,每个实验的总反应体积为1 mL,加入0.32 µmol/L蛋白,在30 ℃、300 r/min条件下反应24 h,用相同体积的预冷甲醇稀释终止反应。反应液经过12 000 r/min离心10 min后,取上清通过0.22 µm滤膜,使用Welch Ultimate XB-C18色谱柱(4.6 mm× 250 mm, 5 µm)进行液相检测,流动相为19%的乙腈和81%的含有0.1%甲酸的水溶液,流速为0.8 mL/min,进样量为10 µL,柱温为30 ℃,检测254 nm处产物对苯二甲酸单(2-羟乙基) [mono(2-hydroxyethyl)terephthalate, MHET]和对苯二甲酸(TPA)的吸收峰。
1.7 分子动力学模拟以Ple629野生型晶体结构为模板,使用COOT[42]软件(版本号0.9.8)对相应氨基酸进行原位突变,并进行整体链优化以去除潜在冲突。使用Gromacs[43]软件包(版本号2022.3)进行模型准备和分子动力学模拟,将蛋白分子放置在正方体的水盒子中,用TIP3P模型[44]的水分子填充溶剂及离子使系统呈中性,用最速下降法[45]进行能量最小化,保持体系温度恒定为40 ℃,并且使用弱耦合方法控制压力为1 bar[46]进行100 ns的分子模拟,模拟的时间步长设置为2 fs,使用稳定后的结果进行均方根偏差(root mean square deviation, RMSD)以及均方根波动(root mean square fluctuation, RMSF)等计算分析。
2 结果与分析 2.1 Ple629野生型和突变体蛋白表达在大肠杆菌BL21(DE3)中对Ple629野生型蛋白和N65D、V80C、V80M、V80C/G108C、G108C、Q111D、D226C/S281C、S237F、S237W、T231C、T231D突变体蛋白进行诱导表达,成功表达的蛋白经过Ni-NTA亲和层析洗脱,透析切除载体标签,再次经过Ni-NTA亲和层析纯化后,蛋白分子量大小约为30 kDa,与目的蛋白的分子量理论值(30.2 kDa)一致,SDS- PAGE分析结果如图 1所示。
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图 1 纯化蛋白SDS-PAGE分析 Fig. 1 SDS-PAGE analysis of purified proteins. M: Standard molecular weight protein; 1: WT; 2: N65D; 3: V80V; 4: V80M; 5: V80C/G108C; 6: G108C; 7: Q111D; 8: D226C/S281C; 9: S237F; 10: S237W; 11: T231C; 12: T231D. |
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利用FoldX软件对Ple629蛋白进行单位点逐个虚拟饱和突变后,共获得了5 691个不同的突变体及相关能量参数,其中有215虚拟突变的位点DDG < –1,进一步筛选了9个潜在突变体,相应点突变的能量(DDG)计算结果如下:Q111D=−5.08 kcal/mol,V80C=−2.73 kcal/mol,G108C=−2.26 kcal/mol,V80M=−1.58 kcal/mol,S237F=−1.41 kcal/mol,S237W=−1.32 kcal/mol,T231D=−1.05 kcal/mol,N65D=−0.83 kcal/mol,T231C=−1.06 kcal/mol。
2.3 结构比较分析及二硫键位置确定比较蛋白质结构数据库中和Ple629 (PDB: 7VPA)同源性较近的蛋白结构,选取蛋白序列一致性40%以上的65个结构,分析了它们的序列主链一致性以及二硫键的情况,结果如表 2所示。从表 2中可以看出,Ple629和上述同源结构蛋白都很类似,主链均方根偏差RMSD均小于1 Å;其中大部分蛋白结构中仅有一个二硫键,少数蛋白结构中有2个二硫键,Ple629蛋白结构中含有3个二硫键。
The type of enzyme |
Source | PDB[47] | Main chain RMSD (Å) |
Numbers and location of disulfide bonds |
References |
Lipase EC 3.1.1.3 |
Streptemyces efoliatus | 1JFR | 0.559 | 1, (CYS 242-CYS 258) | [48] |
Metagenome library in gelatin degradation reactor | 7EC8 | 0.475 | 2, (CYS 218-CYS 255, CYS 289 -CYS 306) |
[49] | |
Cutinase EC 3.1.1.74 |
Thermobifida alba AHK119 | 3VIS | 0.701 | 1, (CYS 280-CYS 298) | [50] |
Thermobifida fusca | 4CG1 | 0.675 | 1, (CYS 241-CYS 259) x | [51] | |
Metagenome library in leaf-branch compost | 4EB0 | 0.834 | 1, (CYS 275-CYS 292) | [52] | |
Saccharomonospora viridis AHK190 | 4WFI | 0.602 | 1, (CYS 287-CYS 302) | [53] | |
Thermobifida cellulosilytica | 5LUI | 0.712 | 1, (CYS 242-CYS 260) | [54] | |
Hydrolase EC 3.1.1.101 |
Ideonella sakaiensis 201-F6 | 5XFY | 0.565 | 2, (CYS 174-CYS 210, CYS 244-CYS 260) |
[55] |
Burkhoderiales bacterium | 7CWQ | 0.533 | 2, (CYS 333-CYS 370, CYS 404-CYS 424) |
[56] | |
Pseudomonas aestusnigri | 6SCD | 0.382 | 2, (CYS 214-CYS 251, CYS 285-CYS 302) |
[57] | |
Rhizobacter gummiphilus | 7DZT | 0.552 | 2, (CYS 200-CYS 236, CYS 270-CYS 290) |
[58] | |
Compost metagenome library | 7CUV | 0.653 | 1, (CYS 242-CYS 257) | [59] | |
Marine microbial consortium | 7VPA | 0.000 | 3, (CYS 17-CYS 21, CYS 195- CYS 232, CYS 270- CYS 287) |
[37] |
目前已知聚酯类塑料降解酶的结构都比较类似[38],有研究表明可以在蛋白结构的相应位点引入二硫键以提高其性能,如PET塑料降解酶TfCut2、PET2[49]、LCC[60-61]以及IsPETase[62]等。Ple629和这几个蛋白的结构主链很相似(主链RMSD见表 2),而且上述引入二硫键区域的主链走势都很类似(图 2),参照上述聚酯类塑料降解酶引入二硫键的位点,本研究在Ple629对应位置上尝试引入二硫键(D226C/S281C)。
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图 2 Ple629和同源蛋白比对以及二硫键位置的确定 Fig. 2 Structural comparison with homolog proteins and determination of disulfide bond positions. Green: Ple629; Cyan: Tfcut2; Yellow: LCC; Pink: IsPETase; Stick: Disulfide bond. |
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Ple629野生型和突变体蛋白分别用Uncle测定其Tm值,结果如表 3所示。野生型蛋白的Tm为38.6 ℃,与文献报道[36]的38.1 ℃较为接近,突变体中Tm提升较多的是V80C和D226C/ S281C,分别提升了5.2 ℃和6.9 ℃。其中D226C/S281C的活性比野生型的活性提高了1.5倍(野生型=0.77 mmol/L,D226C/S281C= 1.925 mmol/L)。
Items | Tm (ºC) |
WT | 38.6 |
N65D | 35.7 |
V80C | 43.8 |
V80M | 38.1 |
G108C | 34.1 |
Q111D | 38.5 |
D226C/S281C | 45.5 |
S237F | 35.3 |
S237W | 35.2 |
T231C | 30.5 |
T231D | 36.3 |
为了进一步研究相关突变对蛋白质整体结构的潜在影响,对野生型Ple629及热稳定性提升以及能量计算应该稳定但实际没有提升的突变体进行了分子动力学模拟,温度为40 ℃,模拟时长为100 ns。本研究利用整体蛋白主链的均方根偏差RMSD表征系统考量的稳定性(图 3A),从图中可以看出,所有突变稳定的体系其RMSD都比较小,从RMSD的变化趋势可以看出,野生型及所有突变体均在60 ns后达到了比较稳定的平衡状态,表明模拟是稳定的。选择模拟达到平衡后即100 ns的主链Calpha的均方根涨落RMSF来表征单个残基波动对蛋白质整体动力学的影响(图 3B–3C)。除了少数特殊区域(红色虚线方框)外,野生型蛋白和突变体的大多数残基波动大小相似。热稳定性提升的突变体的整体RMSF都小于野生型(图 3B),表明突变之后蛋白质的整体结构更加趋于稳定,因而有更好的耐温能力;而在活性降低的突变体中(图 3C),大部分区域的波动情况和野生型类似,但是235–242区域(图 3C红色虚线框)的波动明显比野生型及活性提升的突变体大(最高差别0.3 Å左右)。从晶体结构上看,该235–242区域的B值相对整体结构较高(图 3D中红色loop),这些活性降低的突变位点虽然有些离该区域较远,但实际突变中通过间接离子键等作用的影响,增加了这个区域构象的波动性,从整体结构上导致更加不稳定,因而热稳定性下降。本文中所选择的突变位点均位于结构的表面(图 3D)。
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图 3 Ple629野生型和突变分子模拟结果比较 Fig. 3 Comparison of molecular simulations between Ple629 wild-type and mutants. |
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对塑料降解酶的性能改造是提升塑料降解效率重要因素之一,本研究针对来源于海洋的中温塑料降解酶Ple629进行了研究,在前期获得蛋白质结构的基础上,通过结构比较以及相应能量分析,设计了一些可能对热稳定性有提升的突变体,并对它们进行了表达纯化和性质鉴定。研究者通过在蛋白结构增添二硫键来提升酶的热稳定性,通过在聚酯水解酶TfCut2蛋白结构中引入二硫键(D204C/E253C),其熔点温度Tm提升了25 ℃,酶的催化效率提高了1倍[30-31];LCC在相同位点引入二硫键(D238C/S283C),熔点温度Tm提升了9.8 ℃[60-61];IsPETase在其相同位点引入一对二硫键N233C/S282C,突变体TS-PETase (ThermoPETase+N233C/S282C)比ThermoPETase (Tm=57.6 ℃)的Tm提高了11.8 ℃[25, 62],D1 (DuraPETase+N233C/S282C)比DuraPETase (Tm=77.0 ℃)的Tm提高了4.1 ℃[26]。根据上述同源酶的结构分析比对,在Ple629结构中相应位点引入二硫键,其突变体D226C/ S281C热稳定性明显提升,Tm比野生型提高6.9 ℃,活性也提高了1.5倍。通过虚拟突变能量计算设计出来的突变体中,虽然也获得了性能提升的突变体,V80C的Tm提升了5.2 ℃,但其他突变体的相关性能并没有显著提升,这也与文献报道的一致[63]。分子动力学模拟的结果表明,热稳定性提升的突变体整体结构更加趋于稳定,而虚拟计算能量较低的许多突变体的235–242区域loop明显柔性更强,无法保证稳定的热稳定性,因而导致实际Tm并没有比理论值升高。本研究将通过对Ple629的进一步研究探索其在工业上的应用价值,为酶法绿色降解聚酯类塑料及回收再利用提供理论基础。
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