Dror Adi, Fishman Ayelet
Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, 32000, Israel.
Comput Struct Biotechnol J. 2012 Oct 23;2:e201209011. doi: 10.5936/csbj.201209011. eCollection 2012.
Oxygenases are ubiquitous enzymes that catalyze the introduction of one or two oxygen atoms to unreactive chemical compounds. They require reduction equivalents from NADH or NADPH and comprise metal ions, metal ion complexes, or coenzymes in their active site. Thus, for industrial purposes, oxygenases are most commonly employed using whole cell catalysis, to alleviate the need for co-factor regeneration. Biotechnological applications include bioremediation, chiral synthesis, biosensors, fine chemicals, biofuels, pharmaceuticals, food ingredients and polymers. Controlling activity and selectivity of oxygenases is therefore of great importance and of growing interest to the scientific community. This review focuses on protein engineering of non-heme monooxygenases and dioxygenases for generating improved or novel functionalities. Rational mutagenesis based on x-ray structures and sequence alignment, as well as random methods such as directed evolution, have been utilized. It is concluded that knowledge-based protein engineering accompanied with targeted libraries, is most efficient for the design and tuning of biocatalysts towards novel substrates and enhanced catalytic activity while minimizing the screening efforts.
加氧酶是一种普遍存在的酶，可催化将一个或两个氧原子引入到不活泼的化合物中。它们需要来自NADH或NADPH的还原当量，并且在其活性位点包含金属离子、金属离子络合物或辅酶。因此，出于工业目的，加氧酶最常用于全细胞催化，以减轻对辅因子再生的需求。生物技术应用包括生物修复、手性合成、生物传感器、精细化学品、生物燃料、药物、食品成分和聚合物。因此，控制加氧酶的活性和选择性非常重要，并且越来越受到科学界的关注。本综述重点关注非血红素单加氧酶和双加氧酶的蛋白质工程，以产生改进的或新的功能。基于X射线结构和序列比对的理性诱变以及诸如定向进化等随机方法已被采用。得出的结论是，基于知识的蛋白质工程与靶向文库相结合，对于设计和调整生物催化剂以适应新底物并提高催化活性同时最小化筛选工作最为有效。
