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

大自然是一些难题的潜在解决方案的宝库。纽约理工大学的Richard Gross和Jin Montclare以及纽约大学的Richard Bonneau和Glenn Butterfoss组成的调查小组相信这一点。问题是确定分解塑料材料的方法，特别是PET塑料。潜在的解决方案是在酶催化剂中寻找攻击类似的天然聚合物。角质素是一种生物聚酯，由C16和C18 ω -羟基脂肪酸组成，具有保护植物表面免受病原生物入侵的功能。角质酶是一种自然平衡反应，是一种存在于各种病原体中的酶，它会攻击天然生物聚酯。然而，迄今为止，相对于其他酯水解酶家族，角质酶是一个受到关注的酶家族。这种情况正在改变，因为角质酶正在成为合成聚合物改性的主要基准水解酶之一，因为它们表现出非凡的能力，可以催化许多重要的聚合物生物转化，如聚乙二甲酸乙二醇酯（PET）、尼龙6,6、聚醋酸乙烯酯、聚丙烯腈等。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不同。这些pi已经将一个精心策划的项目放在一起，以提供一个彻底的研究，从而深入了解导致高热稳定性和增强角质酶活性的结构特征。该计划包括动力学和机制研究的角质酶催化水解PET和其他特定的聚合物材料。迄今为止，在已发表的文献中还缺乏对这一类型的全面研究。为了做到这一点，计划包括工程AoC变体（米曲霉表皮酶），将使用上述底物合成和测试，目标是实现高稳定性（温度，pH值）和催化活性。研究将包括建模工作和降解产物分析，以进一步建立理解。降解聚合物的角质酶活性只是这项工作的一个特点。许多聚合物应用需要定制表面特性，以增强生物相容性、耐化学性、疏水性、附着力和润湿性。目前改性聚合物表面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。这些方法表现出负面的特点，包括产生大量的溶剂废物，限制批量处理和安全隐患。此外，对表面具有自清洁功能、排斥和/或杀死微生物并具有先进生物特性的材料的需求不断增加。pi将能够考虑一种具有足够稳定性和活性的工程角质酶，可以固定在这些表面上，并具有修饰或降解材料表层的功能，从而设计出各种表面特性。这笔资金将为纽约理工大学的学生群体提供重要的研究机会。纽约理工大学的学生群体具有不同的人口结构、社会经济背景，其中包括许多第一代移民的子女，他们渴望通过教育达到经济阶梯的下一个台阶。pi参加了纽约理工学院制度化的UG暑期研究项目，有一个非常活跃的高中指导项目（每年6-10名学生），并与儿童科学挑战赛团队合作，为3至6年级的学生创建新的模块，例如，关于神奇微生物的教学。

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.