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Xue Y.-P.,Zhejiang University of Technology | Liu Z.-Q.,Zhejiang University of Technology | Xu M.,Zhejiang Laiyi Biotechnology Co. | Wang Y.-J.,Zhejiang University of Technology | Zheng Y.-G.,Zhejiang University of Technology
Journal of Chemical Technology and Biotechnology

BACKGROUND: (R)-(-)-Mandelic acid (R-MA) is an important intermediate and chiral regent with broad uses. An efficient method for the separation of R-MA from the bioreaction mixture with high yield is of great importance, thus, the main objective of this work is to investigate the recovery of R-MA using an ion-exchange process. RESULTS: The equilibrium isotherms for the separation of R-MA by resin HZ202 were obtained in the pH range 5.0-9.0 and temperature range 25-35 °C. The equilibrium data are well fitted by the Langmuir isotherm. Batch kinetic experiments showed that the mobility of R-MA- in solution was rapid and the R-MA-/OH- ion-exchange process reached equilibrium after about 60 min. Adsorption kinetics were analyzed by a linear driving force mass-transfer model, yielding good prediction of the kinetic behavior. In fixed bed column experiments, the breakthrough curves of R-MA from the solution on resin HZ202 were determined at different flow rates and R-MA was eluted with different concentrations of HCl. A favorable breakthrough curve and optimal eluant concentration were obtained. The results were used for the separation of R-MA biosynthesized from (R,S)-mandelonitrile with nitrilase, and separation was successfully achieved with above 90% recovery yield. CONCLUSION: Resin HZ202 presents favorable behavior for the recovery of R-MA, in terms of capacity, kinetics, affinity, and susceptibility to regeneration. The results of this study provide an efficient method for R-MA recovery from bioreaction mixture and could potentially be used in industry. © 2010 Society of Chemical Industry. Source

Xue Y.-P.,Zhejiang University of Technology | Liu Z.-Q.,Zhejiang University of Technology | Xu M.,Zhejiang Laiyi Biotechnology Co. | Wang Y.-J.,Zhejiang University of Technology | And 2 more authors.
Biochemical Engineering Journal

Bioconversion of (R,. S)-mandelonitrile (R,. S-MN) to prepare (R)-(-)-mandelic acid (R-MA) with nitrilase is an attractive method in industrial application. However, during this bioconversion by whole cells of Alcaligenes faecalis CCTCC M 208168, R-MA was found to inhibit its own production. To improve R-MA productivity, a new biocatalytic process of in situ product removal (ISPR) has been developed utilizing anion-exchange resin. To optimize the bioconversion of R-MA from R,. S-MN, several anion-exchange resins were examined: HZ202, demonstrated several exciting features including high R-MA and low R,. S-MN adsorbance. In batch biotransformation, ISPR by addition of HZ202 increased R-MA volumetric productivity to 0.285. mmol/l/min. The kinetic models for enantioselective hydrolysis of R,. S-MN by A. faecalis CCTCC M 208168 with ISPR were established, and they were well fitted to the experimental data of the reaction kinetics. In fed-batch biotransformation with ISPR was also performed. As compared to the conventional fed-batch mode, this approach allowed R-MA volumetric productivity and biocatalyst productivity to be increased from 0.083. mmol/l/min and 55.1. mmol/g to 0.281. mmol/l/min and 185.5. mmol/g, respectively. © 2010 Elsevier B.V. Source

Liu Z.-Q.,Zhejiang University of Technology | Zhang X.-H.,Zhejiang University of Technology | Xue Y.-P.,Zhejiang University of Technology | Xu M.,Zhejiang Laiyi Biotechnology Co. | Zheng Y.-G.,Zhejiang University of Technology
Journal of Agricultural and Food Chemistry

Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(-)-mandelic acid after 7.5 h of conversion reached 693 mM with an enantiomeric excess of 99%, and the space-time productivity of Mut3 was 21.50-fold higher than that of wild-type nitrilase. The Km, Vmax, and kcat of wild-type and Mut3 for mandelonitrile were 20.64 mM, 33.74 μmol mg-1 min-1, 24.45 s-1, and 9.24 mM, 47.68 μmol mg -1 min-1, and 34.55 s-1, respectively. A homology modeling and molecular docking study showed that the diameter of the catalytic tunnel of Mut3 became longer and that the tunnel volume was smaller. These structural changes are proposed to improve the hydrolytic activity and pH stability of Mut3. Mut3 has the potential for industrial applications in the upscale production of (R)-(-)-mandelic acid. © 2014 American Chemical Society. Source

Xue Y.-P.,Zhejiang University of Technology | Xu M.,Zhejiang Laiyi Biotechnology Co. | Chen H.-S.,Zhejiang University of Technology | Liu Z.-Q.,Zhejiang University of Technology | And 2 more authors.
Organic Process Research and Development

An integrated bioprocess for the enantioselective hydrolysis of mandelonitrile to (R)-(-)-mandelic acid (R-MA) with immobilized Alcaligenes faecalis ZJUTB10 cells was constructed. Production of A. faecalis ZJUTB10 nitrilase in a pilot-scale fermenter (700 L) with high activity was achieved after optimizing cultivation conditions. A. faecalis ZJUTB10 cells were then immobilized in Ca-alginate. Efficient reusability of the biocatalyst up to 9 batches was obtained by immobilization, and treatment with polyethyleneimine (PEI) and glutaraldehyde (GA) further extended the longevity to 19 batches. The immobilized cells showed maximum activity at 40 C and pH 8.0. A method for in situ product recovery (ISPR) based on an external extraction loop was established to overcome product inhibition. Anion-exchange column containing resin HZ202 was coupled to the packed bed bioreactor and enabled product recovery by continuously recirculating reaction mixture through the ISPR unit. This integrated bioprocess led to a high productivity of 8.87 mM/h after 16 h of reaction. The productivity of R-MA did not drop significantly even after 80 h of reaction, and the accumulative R-MA amount reached a final value of 550 mmol with excellent enantiomeric excess (>99%). The present studies demonstrated the potential of using the integrated bioprocess for continuous production of R-MA on an industrial scale. © 2013 American Chemical Society. Source

Liu Z.-Q.,Zhejiang University of Technology | Zhou L.-M.,Zhejiang University of Technology | Liu P.,Zhejiang University of Technology | Baker P.J.,Zhejiang University of Technology | And 4 more authors.
Applied Microbiology and Biotechnology

A new two-step chemo-enzymatic approach for highly efficient synthesis of all-trans-retinyl palmitate is constructed in this study. In the first step, retinyl acetate as starting material was fully hydrolyzed to retinol by potassium hydroxide. In the hydrolysis system, anhydrous ethanol was the best co-solvent to increase the solubility of retinyl acetate. The addition amounts of 5 M potassium hydroxide and anhydrous ethanol were 8 and 10 mL against 10 g retinyl acetate, respectively, and 100 % hydrolysis rate was obtained. In the second step, esterification was catalyzed by immobilized lipase on macroporous acrylic resin AB-8 using the extracted retinol and palmitic acid as substrates in non-aqueous system. After optimization, the parameters of esterification reaction were confirmed as follows: non-aqueous solvent was selected as n-hexane, washing times of extraction solution was four times, retinol concentration was 300 g/L, substrate molar ratio of retinol to palmitic acid was 1:1.1, the amount of immobilized enzyme was 10 g/L, and the esterification temperature was 30 °C. Under the optimal conditions, this protocol resulted in a 97.5 % yield of all-trans-retinyl palmitate in 700-L reactor. After purification, all-trans-retinyl palmitate was obtained with above 99 % of purity and 88 % of total recovery rate. This methodology provides a promising strategy for the large-scale production of all-trans-retinyl palmitate. © 2015, Springer-Verlag Berlin Heidelberg. Source

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