Structure-activity relationship analysis of the nitrilase form the cyanobacterium Synechocystis sp. PCC6803 through site-directed mutagenesis: improving catalytic properties for bioprocesses

聚球藻基因组中的一种新的氰化酶已被电子计算机检测到。PCC6803，并进行重组表达和鉴定。这种丁腈酶表现出对脂肪族腈的偏好，这在这些酶中是一种非常不寻常的性质；因此，它代表了一个独特的机会来研究“脂肪族”丁腈酶的结构-活性相关性。在拟议的项目中，将通过定点突变对酶进行合理的修饰，并分析突变体的酶性质。所获得的结果将有助于确定使酶优先转化脂肪族底物的结构特征，这将最终产生一个详细的模型，将某些氨基酸残基与酶的某些功能和能力联系起来。在α取代腈上活跃的丁腈酶在工业生物转化中具有重要意义。这些腈的对映体选择性水解产生手性α-取代羧酸，这是一种有商业价值的化学物质。聚合胞菌腈酶转化这些α-取代腈的能力将被分析并进行遗传改良。将尝试获得在底物专一性、对映体选择性和/或对这些底物的特定活性方面具有改进特性的点突变体。突变的酶将被纯化和固定化以保证操作的稳定性，并将从产量、稳定性和成本方面分析将该酶应用于更大规模生物转化的可行性。该项目将有助于了解腈酶的结构，并将提高这些酶在广泛潜在底物中的工业可获得性。

A novel nitrilase has been previously detected by an in silico approach in the genome of Synechocystis sp. PCC6803 and recombinantly expressed and characterized. This nitrilase demonstrated a preference for aliphatic nitriles, a quite unusual property among these enzymes; therefore, it represents a unique opportunity to study structure-activity correlations for an “aliphatic” nitrilase. In the proposed project the enzyme will be rationally modified by site-directed mutagenesis and the enzymatic properties of the mutants analyzed. The results obtained will allow to identify the structural features which enable the enzyme to preferentially convert aliphatic substrates and this will finally result in a detailed model that relates certain amino acid residues with certain functions and abilities of the enzyme.Nitrilases that are active on alpha-substituted nitriles are of great interest for industrial biotransformations. The enantioselective hydrolysis of these nitriles produces chiral alpha-substituted carboxylic acids, valuable chemicals of commercial interest. The ability of the Synechocystis nitrilase to transform these alpha-substituted nitriles will be analyzed and genetically improved. It will be attempted to obtain point mutants with improved characteristics in terms of substrate specificity, enantioselectivity and/or specific activity towards these substrates. The mutated enzyme will be purified and immobilized for operational stability and the feasibility of applying this nitrilase to larger scale biotransformations will be analyzed in terms of yield, stability and costs.This project will contribute to structural knowledge of nitrilases and will also improve the industrial availability of these enzymes for a wide range of potential substrates.