| The nitrilases are an important class of industrial enzymes belonging to the nitrilase superfamily, which can hydrolyze the cheap nitriles to the high value-added carboxylic acids in one step. Nitrilases have exhibited great potential in the synthesis of a-hydroxy acids, such as (R)-(-)-mandelic acid. However, due to the impact of high steric effects, the performance of nitrilase in the aspects of preparation of (R)-(-)-chloromandelic acid is not satisfactory, and thus limited its industrial applications. In addition, though there have been many reports about nitrilase at home and abroad, the nitrilase which could produce (S)-(-)-mandelic acid was rare, especially with high S enantioselectivity. In order to solve the above problems, this study committed to the development of new nitrilase resources and a nitrilase library was constructed by genome mining. The recombinant nitrilase GPnor51 from Luminiphilus syltensisNOR5\-B with high activity and high enantioselectivity toward o-chloromandelonitrile and nitrilase PpL19 from Pseudomonas psychrotolerans L19 with S enantioselectivity were screened. Specific research contents and the results obtained are as follows:First, we applied the traditional method of data mining to search the database, and took the nitrilase from Burkholderia cenocepacia J2315 as template, which showed great potential in the production of (R)-mandelic acid. A small nitrilase library containing 43 novel nitrilases was constructed, and laid the foundation of the subsequent screening for different substrates.(1) Screening the nitrilase with high activity and high enantioselectivity toward o-chloromandelonitrile and its enzyme properties of the GPnor51 The first screening was performed by taking o-chloromandelonitrile as substrate, the recombinant nitrilase from Luminiphilus syltensis NOR51-B which showed the highest activity (14.2 U/mg) and enantioselectivity (98.2%) was selected. Then the enzyme properties of the GPnor51 was studied, and GPnor51 showed good temperature and pH stability. The nitrilase GPnor51 exhibited a broad substrate spectrum, and it can be able to hydrolyze a large number of nitriles with structural diversity, such as heterocyclic nitriles, dinitriles and some aliphatic nitriles. The Lineweaver-Burk plot obtained for the conversion of o-chloromandelonitrile under standard assay conditions showed that the Km was 0.38 mM. The low value of Km indicated that GPnor51 had high affinity toward o-chloromandelonitrile. The nitrilase GPnor51 also displayed high activity and enantioselectivity toward mandelonitrile derivatives (substitutions with halogen at ortho, meta and para-position), and showed great potential in producing optically pure a-hydroxyl carboxylic acids.(2) The study of recombinant nitrilase GPnor51 in the production of (R)-(-)-chloromandelic acid from o-chloromandelonitrile In order to produce (R)-(-)-chloromandelic acid with high concentration and high enantioselectivity, the effects of seven miscible organic solvents on the activity and enantioselectivity of nitrilase GPnor51 were investigated firstly. The results showed that the addition of the organic solvents resulted in significant loss of activity. The effects of solvents on the enantioselectivity of GPnor51 were more significant, and the enantioselectivity was reduced harshly and even reversed. There was no ideal results obtained in the constructed two-phase system, Immobilization technology was applied to the nitrilase GPnor51, and the immobilized cells were used to hydrolyze o-chloromandelonitrile. After immobization, the nitrilase GPnor51 was more stable and could hydrolyze the reaction with 10 cycles and with no evident activity loss. The concentration of the (R)-(-)-chloromandelic acid was 458 mM (85 g/L), with an ee value of 97.9%, which achieved the production of (R)-(-)-chloromandelic acid with high concentration.(3) The screening and directed evolution of nitrilase with S-selectivityTaken (S)-(+)-mandelic acid as the product, a novel S-selective nitrilase from Pseudomonas psychrotolerans L19, which produced (S)-mandelic acid with an enantiomeric excess (ee) of 52.1% was screened. To enhance the enzyme’s S-selectivity, directed evolution was performed on it through random mutagenesis, site-directed mutagenesis, and site-saturation mutagenesis, which yielded several variants that harbored mutations in corresponding residues and showed distinct selectivity. Four "hot spots" (M113, R128, A136 and 1168) responsible for enantioselectivity toward mandelonitrile were identified and characterized. By combining beneficial mutations, variants PpL19-LH was evolved which exhibited S-selectivity with an ee value of 91.1%, which is the first reported nitrilase that is highly S-selective toward mandelonitrile, and it holds considerable potential for use in one-step synthesis of (S)-mandelic acid from racemic mandelonitrile. Notably, we inverted the selectivity of nitrilase PpL19 to generate a highly R-selective mutant PpL19-GYY more than 90% ee, which demonstrates an innovative approach for developing novel biocatalysts.(4) The homology modeling and molecular docking analysis of the nitrilase PpL19 and its mutantsIn order to understand how the introduced mutations enhanced and inverted the enantioselectivity in the hydrolysis of mandelonitrile and to guide the engineering of the nitrilase further, The homology modeling of nitrilase PpL19 was modeled. The homology model obtained was assessed by Ramachandran plot and Profile-3D to ensure that the model of the nitrilase PpL19 was with high confidence level, which laid the basis of the molecular docking analysis. The refined models were docked with the substrates (R)-and (S)-mandelonitrile. The docking models was analyzed from two aspects which were the distances between the substrate and the catalytic sites and the formation of hydrogen bonds, and the explaination was given for the change of the enantioselectivity of nitrilase PpL19. These analysis results may play a guidance role in reshaping the enantioselectivity of other arylacetonitrilases. |
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