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ACS Catal. | 基于GRAPE策略的计算设计实现温和条件下塑料生物降解

发布: 2022-04-01 381阅读 107评论

2021年1月，中国科学院微生物研究所的吴边团队在ACS Catalysis上发表文章提出了一种新型蛋白质稳定性计算设计策略——GRAPE（greedy accumulated strategy for protein engineering）（图1），利用该策略研究人员对PET塑料降解酶IsPETase进行了稳定性改造，使得突变体DuraPETase的熔融温度升高31℃，并且在温和温度下对PET膜的降解增强30%（图3A）。还实现了在温和条件下将2 g/L微塑料完全生物降解为水溶性产品（图3E）。

图1. GRAPE 策略示意图

GRAPE策略主要分为3个部分

1、基于四种不同的单点突变预测算法ABACUS、FoldX、Rosetta –ddg、 Consensus Analysis预测到253个潜在位点，经过人工检查筛选（破坏氢键、疏水残基暴露等）和实验验证最终获得了21个有益单点突变；

2、通过K-means聚类算法，将21个有益单点突变分为3个Cluster；

3、组合阶段根据热稳定性和降解性能来决定是有益突变还是有害突变，根据贪婪算法（图2）对每个聚类中的突变进行累积，以识别出稳定性和酶活性提高的多点组合突变。值得注意的是，贪婪算法不从整体最优上加以考虑，算法得到的是某种意义上的局部最优解。

图2.贪婪算法示意图

经过10轮迭代叠加得到突变体IsPETase-M10(S214H-I168R-W159H-S188Q-R280A-A180I-G165A-Q119Y-L117F-T140D, M10)，研究人员将其命名为DuraPETase，并对其进行了活性检测、扫描电镜分析和蛋白晶体结构解析。结果显示，DuraPETase对30%结晶度PET薄膜的降解效率相较于野生型提升了300倍（图3B），对甲醇和乙二醇的耐受性显著提高（图3C）；经DuraPETase处理后的PET薄膜内部结构发生了显著的腐蚀变化（图3D）；在动力学模拟中观察到W159H突变与Ser160形成一个新的氢键（图4a），I168R和D186之间形成新的盐桥相互作用（图4b），L117F和Q119Y与Tyr87形成了“T形” π-π 相互作用（图4c）。

图3（A）DuraPETase相较于野生型 Is PETase在不同温度下对30%结晶度PET薄膜的降解性能；（B）DuraPETase及野生型 I s PETase在37°C条件下对30%结晶度PET薄膜的降解曲线；（C）DuraPETase及野生型 Is PETase在有机溶剂中的降解性能；（D）PET薄膜在不同溶剂中孵育10天的扫描电镜图；（E）DuraPETase及野生型 Is PETase对纳米塑料及微塑料的降解曲线。

图4  W159H，I168R-S188Q， S214H-L117F-Q119Y突变的结构分析

GRAPE策略的提出是酶设计方法的重大进步，其优势在于结合了聚类算法和贪婪算法，通过最少的实验筛选，在较短时间内最大限度的探索序列空间叠加路径。该研究为计算机辅助蛋白质改造提供了新思路，并为减少环境微塑料积聚的研究开辟了道路。近期，该团队已经开发了基于此策略的网站服务器，地址https://nmdc.cn/grape-web/，更方便广大研究人员的应用。

论文链接：https://pubs.acs.org/doi/full/10.1021/acscatal.0c05126

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