| Statins are the most prescribed drugs in the treatment of hyperlipidemia so far, including atorvastatin, rosuvastatin, and L-carnitine is a class of indispensable functional moleculars for the metabolism in the body. As important intermediates for synthesis of side chains of statins and L-carnitine, preparation of (R)- and ethyl (S)-3-hydroxyglutrate is of greatly economic and social benefits. By now, owing to a single and inefficient synthetic route for synthesis of (R)- and ethyl (S)-3-hydroxyglutrate, and the correspondingly catalytic processes have not elucidated in details, so the yield and optical purity of these two chiral compounds fail to meet the criterion of the intermediate to side chain of stains. Therefore, there has been no breakthrough in study on synthesis of both chiral compounds. In this study, the microbial cell and enzyme were used as biocatalysts in their biosynthesis. Optically pure ethyl (S)-3-hydroxyglutrate was prepared after detailed studies on the reaction process and mechanism; and chiral ethyl (R)-3-hydroxyglutrate was also synthesized in one-step bioconversion by introducing a newly synthetic route catalyzed by a novel biocatalyst, and the screening, indentification, cultivation and application of this biocatalyst were investigated in details.By introducing ion-pair HPLC, we firstly established a novel HPLC analytic method for 3-hydroxyglutarate to solve an unresolved difficulty in the retention of separative components in C18 column, and the newly analytic method was also helping to optimize the reaction conditions and study catalytic process and mechanism by cell and enzyme in details. Optically pure ethyl (S)-3-hydroxyglutarate (ee > 95%) was prepared by the following processes including semi-preparative HPLC separation, ethyl acetate extraction and reduced pressure distillation of reaction mixture, after an enantioselective hydrolysis of prochiral substrate diethyl 3-hydroxyglutarate with Novozym 435 (CALB), the structure and configuration of ethyl (S)-3-hydroxyglutarate were confirmed by UV, IR、ESI-MS、NMR and measurement of specific rotation. Owing to no chiral column for separation of (R)- and ethyl (S)-3-hydroxyglutarate available, a novel analytical method for its optical purity was built up to solve the difficulty in accurate determination of ee value of ethyl (S)-3-hydroxyglutarate based on derivatization of (R)- and ethyl (S)-3-hydroxyglutarate to the corresponding diastereomers with (R)-(+)-phenylethylamine, and ee value of ethyl (S)-3-hydroxyglutarate could be determined by measuring the corresponding peak areas after silicagel column separation of these two diastereomers.The optimized conditions at pH 7.0, temperature 40 oC, substrate concentration 0.15 M, agitation speed 200 rpm were obtained through optimization of biocatalytic reaction conditions and process in the enantioselective hydrolysis with Novozym 435, and detailed studies on the effects of external mass transfer and intra-particle diffusion resistances on reaction process. Under this optimum conditions, optically pure ethyl (S)-3-hydroxyglutarate (ee > 95%) was synthesized. The inhibition of ethanol on reaction obeying an uncompetitive inhibition pattern was confirmed by study on inhibition kinetics, and the apparent kinetic constants were also obtained through non-linear regression, with values of Vmax 1.29 mmol/min·g, Km 0.06 mol/L, and Ki 0.37 mol/L, respectively.A high-throughput screening method for nitrilase strain which could catalyze enantioselective hydrolysis of ethyl 3-hydroxy-4-cyanobutyrate to ethyl (R)-3-hydroxyglutarate was established. It was based on a colorimetric reaction of Co2+ with the released NH3 from a nitrile hydrolytic reaction in Tris-HCl buffer (pH 7.0, 20 mM) to form a complex [Co2+(NH3)]2+, resulting in a notable color change in reaction mixture from pink to yellow. This assay overcomes the difficulty in strain screening by quick analysis of substrate consumption and product accumulation. A strain ZJB-0910 capable of transforming racemicβ-hydroxy aliphatic nitrile, displaying no hydrolase activity on the ester bond of ethyl 4-cyano-3-hydroxybutyate, was isolated by employing this high-throughput screening method. The structure and configuration of ethyl (R)-3-hydroxyglutarate were also determined by IR、ESI-MS、NMR and measurement of specific rotation. Strain ZJB-0910 was identified as Rhodococcus erythropolis based on morphology, physiological tests, and 16S rDNA sequence. This was the first report on R. erythropolis strain (R)-stereoselectively hydrolyzing racemicβ-hydroxy aliphatic nitrile, and such stereoselective hydrolysis of racemicβ-hydroxy aliphatic nitrile was previously considered very hard to be realized by nitrilase in general, because few enzymes show chiral recognition towardβ-hydroxy nitriles bearing a remote chiral center from the reacting cyano moiety in comparison withα-hydroxy nitriles.The medium composition for nitrilase from R. erythropolis ZJB-0910 was optimized by single factors and response surface methodology (RSM). The optimum medium composition was given as below (g/L): glucose 20.986, yeast extraction 8.468, MgSO4 0.22,K2HPO4 0.5,KH2PO4 0.5. Meanwhile, the optimum conditions for cell growth and nitrilase synthesis were as follows: temperature 30 oC, inoculum size 3%, medium volumetric ratio 20% (v/v), initial pH 7.0. Under the above conditions: 4.23 g/L biomass and 75.6 U/g DCW specific nitrilase activity were achieved after 45 h cultivation. Studies on the inducer influence on enzymatic production in R. erythropolis ZJB-0910 demonstrated that two enzymatic pathways of nitriles in the strain exist, therein, it exhibited constitutive nitrilase activity and inducible nitrile hydratase activity.The influences of reaction conditions on resting cells activity and enantioselectivity were also investigated, and the optimum reaction conditions were obtained as follows: Temperature 30 oC, pH 7.5, ethyl (R/S)-3-hydroxy-4-cyanobutyrate 20 mM, resting cells 1.6 g DCW/L. Under these conditions, ethyl (R)-3-hydroxyglutarate in ee>99% was synthesized after reaction for 4 h, and the conversion and E were 46.2% and >200, respectively. The half-life (t1/2) of resting cells at 30 and 40 oC were determined to be 93.3 and 37.6 h. Inhibitive kinetic study showed that no inhibition of ethyl (R/S)-3-hydroxy-4-cyanobutyrate on resting cells activity was observed; ethyl (R)-3-hydroxyglutarate, however, inhibited the resting cells activity following noncompetitive pattern. The kinetic constants including maximum reaction velocity (Vmax), Michaelis constant (Km) and dissociation constant (Ki) were calculated to be 85.6μmol/min·g, 0.01 M and 0.028 M, respectively. After eight reuse cycles, 50% of the resting cell activity was remained, suggesting it exhibited rather good batch stability. |
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