Efficient Discovery Of Novel Nitrilase And Its Applications Research On Production Of Optically Pure α-hydroxylcarboxylic Acid

Optically pure a-hydroxylcarboxylic acids are versatile intermediates for the production of various pharmaceuticals and fine chemicals. Among them, optically pure mandelic acid and substituted derivatives are regarded as the most important representatives.(R)-(-)-mandelic acid is widely used for the production of semisynthetic cephalosporins, penicillins, antitumor agents and antiobesity agents. It is also used as an universal acidic chiral resolving agent for the resolution of racemic alcohols and amines.(R)-o-chloromandelic acid is the key precursor for the synthesis of Clopidogrel, a platelet aggregation inhibitor widely administered to atherosclerotic patients with the risk of a heart attack or stroke that are caused by the formation of a clot in the blood. Many chemical and enzymatic approaches to produce optically pure aromatic a-hydroxy carboxylic acid have been developed. Among them, nitrilase-mediated pathway becomes increasingly popular because of the absence of the cofactor involvement, cheap starting material in the form of mandelonitrile, high enantioselectivity and theoretically100%of the product.In this thesis, a novel arylacetonitrilase (BCJ2315) was efficiently discovered from Burkholderia cenocepacia J2315by phylogeny-based enzymatic substrate specificity prediction (PESSP) combination with traditional genome mining technology. BCJ2315demonstrated higher specificity, enantioselectivity and substrate tolarence based on the study of enzymatic properties. Thus BCJ2315showed great potential in the production of (R)-(-)-mandelic acid by hydrolysis of mandelonitrile. The optimization of the reaction conditions and reaction mode were succefully performed to achieve the production of (R)-(-)-mandelic acid and (R)-o-chloromandelic acid in a high concentration and enantioselectivity. Protein engineering of BCJ2315was also used to achieve the production (R)-o-chloromandelic acid in a high concentration and enantioselectivity. The obtained results laid a solid foundation for the industrial production of (R)-(-)-mandelic acid and (R)-o-chloromandelic acid. The results were as fllows:First, seven mandelonitrile hydrolases were discovered from GenBank by phylogeny-based enzymatic substrate specificity prediction (PESSP) combination with traditional genome mining technology. All of them showed relatively high enantioselectivity toward mandelonitrile. Among them, the nitrilase BCJ2315from Burkholderia cenocepacia J2315demonstrated the highest specificity (27.79U/mg) and the highest enantioselectivity (98.4%). No byproduct was observed in the biotransformation. The highest identity was71%compared with other enzymes in amino acid sequence to demonstrate that BCJ2315is a novel nitrilase. BCJ2315was chosen for further study because of its high specific activity and enantioselectivity.Second, We carried out a systematic study on the enzyme properties of the BCJ2315. BCJ2315showed one single band on the SDS-PAGE with a molecular weight of38kDa. The molecular weight of the purified native BCJ2315estimated by conducting a gel filtration chromatography was about450kDa. This result indicated that the native BCJ2315consisted of12subunits with identical size. The optimum temperature and pH of the purified BCJ2315were45℃and8.0, respectively. BCJ2315showed a very broad substrate spectrum, it could hydrolyze most of the24assayed nitriles. A clear preference of BCJ2315for arylacetonitriles as substrates indicated that this enzyme is an arylacetonitrilase. A lower activity was also observed with the aliphatic and heterocyclic nitriles. No detectable activities were observed with the aromatic nitriles. Different from other reported highly enantioselctive arylacetonitrilases which had phenylacetonitrile as their optimal substrate, BCJ2315showed the highest activity toward mandelonitrile (8.8times more than that of phenylacetonitrile), indicating that BCJ2315is a highly active mandelonitrile hydrolase. The low value of Km indicated that BCJ2315had high affinity toward mandelonitrile. The Km-values of other highly enantioselective mandelonitrile hydrolases were one to three orders of magnitude higher than that of BCJ2315. BCJ2315also had a high catalytic efficiency. The Kcat and Kcat/Km were15.4s-1and1.1×105M-1s-1, respectively. These results demonstrated great potential of BCJ2315in hydrolyzing mandelonitrile to produce optically pure (R)-(-)-mandelic acid.Third, the catalytic efficiency of the recombinant E. coli M15/BCJ2315was also tested in hydrolyzing mandelonitrile to produce (R)-(-)-mandelic acid to further investigate the potential of BCJ2315. M15/BCJ2315had a strong substrate tolerance and could completely hydrolyze mandelonitrile (100mM) with fewer amounts of wet cells (10mg/ml) within1h. The ee value of the product was97.6%. M15/BCJ2315could be reused up to14cycles without any loss of activity and thus was very stable. An ethyl acetate-water biphasic system was built to relieve the substrate inhibition and improve the yield of (R)-(-)-mandelic acid. A total of500mM (R)-(-)-mandelic acid was produced in ethyl acetate-water biphasic system (10%v/v) within4h with a high enantiomeric excess of97.1%, which efficiently relieved the substrate inhibition and improved the yield of (R)-(-)-mandelic acid. To achieve production of (R)-(-)-mandelic acid in high concentration, a fed-batch mode was also used. Mandelonitrile was fed into the reaction system to a final concentration of100mM when the mandelonitrile in the reaction was exhausted. After8batches, the cumulative concentration of744mM (R)-(-)-mandelic acid was produced in18h. In an modified fed-batch mode, we make the activity of BCJ2315in a high level by continuous substrate feeding and control of reaction pH. In300ml of reaction scale, the accumulation concentration of (R)-(-)-mandelic acid reached2.1M after18h of biotrasformation with97.6%ee value. When the reaction was scaled up to30L, the reaction time was shortened to18.5h when the concentration of product reached2.1M as the improved mix effect between enzyme and substrate, giving a ee value of97.6%. Through the optimization of the reaction conditions and reaction mode, the concentration of (R)-(-)-mandelic acid has been raised to2.1M, which is higer than that of other reports. The obtained results had laid a good foundation for further industry applications.Fourth, screening the existing enzyme library, mining new enzymes, solvent engineering and protein engineering were used to achieve high enantioselective production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile. Seven mandelonitrile hydrolases from the existing enzyme library and ten new enzymes were used to measure their enantioselectivity toward o-chloromandelonitrile. The nitrilase from Labrenzia aggregata showed the highest enantioselectivity (96.3%).Unfortunately, when our work was in progress, this nitrilase was reported. The nitrilase showed a relative low specific activity toward o-chloromandelonitrile. The rest of the nitrilases demonstated low enantioselectivity toward o-chloromandelonitrile with ee values no more than90%. The nitrilase BCJ2315still showed the highest specific activity in the hydrolysis of o-chloromandelonitrile to (R)-o-chloromandelic acid, thus it was chosen as the candidate catalyst. Solvent and protein engineering were used to improve the BCJ2315’s enantioselectivity toward o-chloromandelonitrile from the reaction conditions and amino acid level. Improving the reaction temperature and pH was conducive to improving the BCJ2315’s enantioselectivity. Seven kinds of water-soluble organic solvents were great helpful to improving the BCJ2315’s enantioselectivity and activity when added into the reaction mixture. When30%of ethanol as co-solvents was added into the reaction mixture, the ee value of product could be increased to98.2%. A continuous substrate feeding mode was used to produce high concentration of (R)-o-chloromandelic acid. The substrate o-chloromandelonitrile dissolved in ethanol was fed continuously to improve the concentration of ethanol in the reaction system in order to increase the enantioselectivity of the reaction. The cumulative concentration of product was415mM (R)-o-chloromandelic acid after8h of hydrolysis with ee value of96.7%in100mL reaction system. Through the random mutations technology combined with site-directed mutagenesis technology, we have successfully obtained the mutant of the nitrilase BCJ2315which had a higher relative activity and enantioselectivity toward o-chloromandelonitrile compared with those of the nitrilase BCJ2315. The mutant contained two mutations, I113M and Y199G. The relative activity of the mutant was three times more than that of wild type. The ee value of the product was increased to98.7%from89.2%of the wild-type. Using ethyl acetate/water two-phase system, the reaction time of fully hydrolyzing500mM o-chloromandelonitrile was shortened to3h, compated with7h of the wild-type.The ee value of the final product was97.2%. The mutant could produce (R)-(-)-chloromandelic acid in high enantioselectivity and concentration, therefore had great potential in the industrial production of (R)-(-)-chloromandelic acid. Four key’ hot spots’ had been identified that had great influence on the enantioselectivity of BCJ2315and saturated mutant library has been constructed at these four sites. They offered great opportunity to study the mechanism of the enantioselectivity of the nitrilase BCJ2315.