Engineering of Aspergillus oryzae cutinase to improve its stability and activity on synthetic polyester substrates

自然界是一些难题的潜在解决方案的宝库。纽约理工大学的理查德·格罗斯和金·蒙克莱尔以及纽约大学的理查德·博诺和格伦·巴特福斯的调查团队也是这样认为的。问题是确定一种分解塑料材料的方法，特别是PET塑料。潜在的解决方案是在攻击类似天然聚合物的酶催化剂中寻找。角质是一种由C16和C18欧米伽-羟基脂肪酸组成的复杂的生物聚酯，其功能是保护植物表面免受病原体的入侵。瓜氨酸酶是一种天然的均衡反应，是存在于各种病原体中的一种酶，它会攻击天然的生物多聚体。然而，角质酶是一个酶家族，到目前为止，与其他酯水解酶家族相比，它受到的关注不成比例。这种情况正在改变，角质酶正在成为合成聚合物修饰的主要基准水解酶之一，因为它们表现出非凡的能力，可以催化聚对苯二甲酸乙二酯(PET)、尼龙6，6、聚乙酸乙烯酯、聚丙烯腈等许多重要的聚合物生物转化。这是值得注意的，因为这些聚合物底物在结构上与这些酶的天然底物有很大的偏离。PI已经制定了一个计划周密的计划，以提供全面的研究，从而深入了解导致高热稳定性和角质酶活性增强的结构特征。该计划包括角质酶催化的PET和其他特定聚合物材料的水解的动力学和机理研究。到目前为止，在已出版的文献中还缺乏对这一类型的全面研究。为了做到这一点，计划包括改造AOC变体(米曲霉角质酶)，将使用上述底物合成和测试AOC变体，以实现高稳定性(温度、pH)和催化活性。研究将包括对降解产物进行建模和分析，以进一步加深理解。降解聚合物的谷氨酸酶活性只是这项工作的一个特点。许多聚合物应用都需要对表面特性进行调整，以增强生物相容性、耐化学性、疏水性、附着力和润湿性。目前聚合物表面改性的方法包括湿化学改性、等离子体处理和聚合物表面涂层的应用。这些方法的缺点包括产生大量的溶剂废物，限制批处理，以及安全隐患。此外，对表面具有自清洁、排斥和/或杀灭微生物功能并具有先进生物性能的材料的需求也在增加。PI将能够考虑将具有足够稳定性和活性的工程化角质酶固定在这样的表面上，并具有修饰或降解材料表面层的功能，从而在这些表面特性中进行工程。资金将为纽约大学保利分校的学生群体提供重要的研究机会，该群体在人口结构、社会经济上都是多样化的，包括许多第一代移民的孩子，他们渴望通过教育达到经济阶梯的下一步。PI参与NYUPOLY制度化的UG暑期研究计划，拥有非常活跃的高中辅导计划(每年6-10名学生)，并与儿童科学挑战团队合作创建新的模块，针对3至6年级的学生教授例如神奇微生物的知识。

Nature is a repository of potential solutions to some difficult problems. So believe the team of Investigators of Richard Gross and Jin Montclare of the Polytechnic University of New York and Richard Bonneau and Glenn Butterfoss of New York University. The problem is to determine a method for decomposing plastic materials, in particular PET plastics. The potential solution is sought in enzyme catalysts that attack similar natural polymers. Cutin is a biopolyester built from a complex array of C16 and C18 omega-hydroxyfatty acids which functions to protect plant surfaces from invasion by pathogenic organisms. Cutinase is natures equalizing response and is an enzyme present in various pathogens which will attack the natural biopolyesters. However, cutinases are an enzyme family that, thus far, has received disproportionally little attention relative to other ester hydrolase enzyme families. This is changing as cutinases are emerging as one of the primary benchmark hydrolase enzymes for synthetic polymer modification as they exhibit the extraordinary ability to catalyze a number of important polymer biotransformations on poly(ethyleneterephthalate) (PET), Nylon 6,6, polyvinylacetate, polyacrylonitrile and others. This is remarkable as these polymer substrates deviate dramatically in structure from the natural substrate for these enzymes. The PIs have put together a well-planned program to provide a thorough study leading to a deep understanding of structural features that lead to high thermal stability and enhanced activity of cutinases. The program includes kinetics and mechanistic studies for cutinase-catalyzed hydrolysis of PET and other specific polymeric materials. A comprehensive study of this type is thus far lacking in published literature. In order to do this, plans include engineering AoC variants (Aspergillus oryzae cutinases) that will be synthesized and tested using the above substrates with the goal of achieving both high stability (temperature, pH) and catalytic activity. Studies will include modeling efforts and analysis of degradation products to further build understanding. Cutinase activity for polymer degradation is only one feature of this work. Numerous polymer applications require tailoring of surface properties to enhance biocompatibility, chemical resistance, hydrophobicity, adhesion and wettability. Current methodologies to modify polymer surfaces include wet chemical modification, plasma treatments, and application of polymeric surface coatings. These methodologies exhibit negative features including generation of large volumes of solvent waste, limitation to batch processing, and safety hazards. Furthermore, there is an increased demand for materials with surfaces that can function to self-clean, repel and/or kill microbes, and have advanced biological properties. The PIs will be able to consider an engineered cutinase with sufficient stability and activity to be immobilized on such surfaces and function to modify or degrade the surface layer of a material, thereby engineering in various of these surface properties. Funding will provide important research opportunities to the NYU-POLY student body which is diverse demographically, socio-economically and includes many children of first generation immigrants, eager to reach the next step of the economic ladder through education. The PIs participate in NYUPOLYs institutionalized UG summer research program, have a very active high school mentoring program (6-10 students per year) and work with the Kids Science Challenge team to create new modules aimed at 3rd to 6th graders to teach, for example, about magic microbes.