Acevedo Juan Pablo, Reetz Manfred T, Asenjo Juan A, Parra Loreto P
Facultad de Medicina y Facultad de Ingeniería y Ciencias Aplicadas, Universidad de los Andes, San Carlos de Apoquindo, 2200 Santiago, Chile.
Max-Planck-Institut für Kohlenforschung, 45070 Mülheim, Germany; Chemistry Department, Philipps-University, 35032 Marburg, Germany.
Enzyme Microb Technol. 2017 May;100:60-70. doi: 10.1016/j.enzmictec.2017.02.005. Epub 2017 Feb 13.
Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T. The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity.
由于低温下具有高效性且能源成本低，低温活性酶在工业生物过程中备受关注。自然进化的冷活性酶的一个特点是其在低温下酶活性增加，然而这类酶呈现出的低热稳定性仍是其在生物催化应用中的一个主要缺点。将冷适应酶定向进化为更具热稳定性的形式，似乎是满足工业稳定性和活性要求的一种有吸引力的策略。本文描述了一种来自GH家族10的新型高活性冷适应木聚糖酶（Xyl-L）的重组表达和表征，以及使用一种新颖的一步联合定向进化技术，该技术包括饱和诱变和聚焦易错PCR，作为提高热稳定性的可行半理性策略。Xyl-L酶从海洋南极细菌嗜冷杆菌属菌株2-17中克隆，在大肠杆菌BL21(DE3)菌株中重组表达并进行酶学表征。使用Xyl-L催化结构域的同源模型进行分子动力学模拟，以检测柔性区域和残基，这些被认为是定义该酶热不稳定性的可能结构元件。设计诱变文库，以便通过在一些已鉴定的柔性区域和残基中引入突变来稳定蛋白质。发现十二个阳性突变克隆提高了该酶的T值，在某些情况下不影响25°C时的活性。最佳突变体的T值提高了4.3°C。定向进化方法的效率也有望在立体选择性的蛋白质工程中发挥作用。
