Studies On Nitrilase Catalyzed Preparation Of Enantiopure Mandelic Acid And Rational Design Of Hydroxynitrile Lyase

Enantiomerically pure mandelic acid is a key intermediate for the synthesis of various pharmaceuticals, agrochemicals and fine chemicals. Nitrilase catalyzed enantioselective hydrolysis of racemic mandelonitrile represents one of the most interesting approaches to the biocatalytic preparation of enantiomerically pure mandelic acid. Hydroxynitrile lyase has attracted more and more attention from academia and industry as one of the few enzyme classes capable of catalyzing C-C bond coupling reactions. Besides cyanide, hydroxynitrile lyase can also catalyze the stereoselective addition of nitro alkanes to aldehydes or ketones to yield enantiopure nitroaldols, which are important building blocks in synthetic organic chemistry.This dissertation describes the cloning, expression and purification of a nitrilase from Alcaligenes sp., and biochemical properties of the recombinant nitrilase. Enzymatic production of enantiopure mandelic acid by enantioselective hydrolysis of racemic mandelonitrile using recombinant E. coli cells as catalyst was studied in detail. The hydroxynitrile lyase activity of a plant esterase (SABP2) derived hydroxynitrile lyase was significantly enhanced by rational design and the evolution route from an esterase to a hydroxynitrile lyase was proposed. Four ancestral enzymes of esterases and hydroxynitrile lyases were constructed and their capabilities for catalyzing novel and unnatural reactions were explored. This dissertation can be divided into four parts as follows:In the first part, a nitrilase from Alcaligenes sp. was cloned and over-expressed in E. coli, and the biochemical properties of the recombinant nitrilase were characterized. The expression level of nitrilase in E. coli was>160-folds higher than that in the wild type strain Alcaligenes sp. The optimal substrate is phenylacetonitrile and the optimal temperature and pH for the recombinant nitrilase are40-45℃and8.0, respectively. The thermostability and pH stability of the recombinant nitrilase are significantly better than other published nitrilases. Vmax and KM of the nitrilase measured were27.9μmol min-1mg-1and21.8mM, respectively.The second part, enzymatic preparation of (R)-(-)-mandelic acid by recombinant E. coli cells expressing nitrilase. The substrate tolerance of the wild type strain Alcaligenes sp. was merely20mM, while in the case of recombinant E. coli cells the substrate tolerance was200mM. The recombinant nitrilase was very stable and the residual activity after10batches of reaction was still40%of the initial activity. Mandelonitrile, benzaldehyde and acidic environment were found to be inhibitors of nitrilase activity. To relieve the substrate inhibition, water-toluene biphasic reaction system was adopted, and the substrate tolerance was further increased from200mM in monophasic system to500mM in biphasic system. In a fed-batch reaction in biphasic reaction system, the cumulative product concentration reached as high as93g/L. To stabilize the catalyst in the biphasic system and for the convenience of product and catalyst separation, the recombinant E. coli cells were entrapped in calcium alginate, the biocatalytic hydrolysis of mandelonitrile was successfully carried out in a stirred reactor (2-L scale) by repeated use of the calcium alginate entrapped cells for5batches, affording110.7g (R)-(-)-mandelic acid in98.0%ee and a specific production of13.8g (mandelic acid) g-1(cell), respectively.The third part, rational design of hydroxynitrile lyase. The hydroxynitrile lyase activity of a plant esterase (SABP2) derived hydroxynitrile lyase (G12T/M239K) was greatly increased up to300-fold via3additional amino acid substitutions (H80E/F151L/F155L). The best variant with five amino acid substitutions catalyzes the cleavage of mandelonitrile more efficiently than the natural HNLs (Kcat=12913min-1versus110min-1&1300min-1for natural HNLs). Most importantly, the best variant can also catalyze unnatural nitroaldol reaction with specific activity and catalytic efficiency of80U/mg and30000min-1, respectively. Finally, a possible evolution route from an esterase to a hydroxynitrile lyase was proposed.The fourth part, construction and rational design of ancestral enzymes. Four ancestral enzymes of esterases and hydroxynitrile lyases were constructed, and all of these four ancestral enzymes showed low to high hydroxynitrile lyase activity, of them, the best hydroxynitrile lyase (HNL2) catalyzes the cleavage of mandelonitrile with a specific activity of8U/mg. Two of the ancestral enzymes (HNL6&HNL8) were found to be good esterase with specific activity of0.46U/mg and0.23U/mg, respectively, while the other two ancestral enzymes (HNL2&HNL4) do not show any detectable esterase activity. Interestingly, all of these four ancestral enzymes also show nitroaldolase activity, and the specific activity of the best nitroaldolase (HNL2) was about2U/mg, which is almost200-fold that of natural hydroxynitrile lyase. At last, esterase activity was successfully introduced into HNL4without impairing the hydroxynitrile lyase activity by rational design.