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

米曲霉角质酶工程提高其在合成聚酯底物上的稳定性和活性

• 批准号: 1414309
• 负责人: Richard Gross
• 金额: $ 6.58万
• 依托单位: Rensselaer Polytechnic Institute
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-31 至 2014-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1414309&HistoricalAwards=false
• 关键词: Engineering; Aspergillus; oryzae; cutinase; improve

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.

大自然是解决一些困难问题的潜在解决方案的存储库。因此，请相信纽约理工大学的理查德·格罗斯（Richard Gross）和金·蒙克雷（Jin Montclare）的调查员团队，纽约大学理查德·邦诺（Richard Bonneau）和格伦·巴特福斯（Glenn Butterfoss）。问题是确定一种分解塑料材料的方法，特别是宠物塑料。潜在的溶液是在攻击类似天然聚合物的酶催化剂中寻求的。 Cutin是一种由复杂的C16和C18欧米茄羟基脂肪酸阵列建造的生物座酯，其功能可保护植物表面免受致病生物的侵袭。切丁蛋白酶是均衡反应的天性，并且是各种病原体中存在的酶，它将攻击自然生物生物生物植物。然而，切碎蛋白是一个酶家族，到目前为止，相对于其他酯水解酶家族而言，其关注很少。这是随着切割蛋白的出现而改变的，它是合成聚合物修饰的主要基准水解酶之一，因为它们具有非凡的能力，可以催化多种（乙基苯甲酸乙二醇酯）（PET），尼龙6,6，聚氯乙酸，聚乙酸，聚乙二醇烯烯丙基甲烯烯烃和其他。这是显着的，因为这些聚合物底物在结构上偏离了这些酶的自然底物。 PI制定了一个精心计划的程序，以提供一项彻底的研究，从而深入了解结构特征，从而导致热稳定性和增强切蛋白酶活性。该程序包括用于切割酶的PET和其他特定聚合物材料的切割酶催化水解的动力学和机械研究。到目前为止，对这类类型的全面研究缺乏已发表的文献。为了做到这一点，计划包括工程AOC变体（曲曲霉切蛋白酶），这些变体将使用上述底物合成和测试，以实现高稳定性（温度，pH）和催化活性。研究将包括建模工作和分析降解产品以进一步建立理解。聚合物降解的切割酶活性只是这项工作的一个特征。许多聚合物的应用需要量身定制表面性能，以增强生物相容性，耐疏水性，疏水性，粘附性和润湿性。当前修饰聚合物表面的方法包括湿化学修饰，血浆处理以及聚合物表面涂层的应用。这些方法表现出负面特征，包括产生大量溶剂废物，批处理处理的限制和安全危害。此外，对具有表面的材料的需求增加了，这些材料可以在自我清洁，排斥和/或杀死微生物并具有先进的生物学特性。 PI将能够考虑具有足够稳定性和活性的工程切割蛋白，以固定在此类表面和功能上，以修改或降解材料的表面层，从而在这些表面特性中进行工程。资金将为NYU-Poly学生团体提供重要的研究机会，该学生在人口统计学上，社会经济上都有多种多样的研究机会，其中包括许多第一代移民的孩子，渴望通过教育来达到经济阶梯的下一步。 PI参加Nyupolys制度化的UG夏季研究计划，拥有非常活跃的高中指导计划（每年6-10名学生），并与儿童科学挑战赛团队合作，创建了针对3至6年级的新模块，例如，教授魔术微生物。