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

一种新的腈水解酶已被检测到的集胞藻PCC 6803的基因组中的电子方法和重组表达和表征。这种腈水解酶表现出对脂肪族腈的偏好，这是这些酶中非常不寻常的特性;因此，它代表了一个独特的机会来研究“脂肪族”腈水解酶的结构-活性相关性。在本研究中，我们将通过定点突变的方法对酶进行合理的修饰，并对突变体的酶学性质进行分析。所获得的结果将允许确定的结构特征，使酶优先转换脂肪族底物，这将最终导致在一个详细的模型，涉及某些氨基酸残基与某些功能和能力的酶。腈水解酶是活跃的α-取代腈是工业生物转化的极大兴趣。这些腈的对映选择性水解产生手性α-取代的羧酸，具有商业价值的有价值的化学品。集胞藻腈水解酶转化这些α-取代腈的能力将被分析和遗传改进。将尝试获得在底物特异性、对映体选择性和/或对这些底物的特异性活性方面具有改进的特征的点突变体。突变酶将被纯化和固定化，以确保操作稳定性，并将从产量、稳定性和成本方面分析将这种腈水解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水解酶的结构，并将提高这些酶在广泛潜在底物中的工业可用性。

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.