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A Novel Method For (R)-(−)-Mandelic Acid Production

With the development of medicine, the synthesis of various pharmaceuticals is often required. Pharmaceutical intermediates, a chemical medium, play an important role during the synthesis of pharmaceutical. Therefore, more and more attention should be paid to the synthesis of pharmaceutical intermediate.

Mandelic acid, especially (R)-(−)-mandelic acid, is a significant chiral pharmaceutical intermediate and is used as a chiral synthon for the synthesis of various pharmaceuticals. For example, (R)-(−)-mandelic acid is applied as a precursor for the synthesis of penicillin, cephalosporin, and anti-tumor and anti-obesity agents [1]. Many approaches have been used for enantioselective synthesis (R)-(−)-mandelic acid [2-5]. The most popular methods, biocatalysts, have been attracting more and more interest in the synthesis of chiral compounds since they carry out processes in mild ambient conditions and show specificity for a particular substrate [6].

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On the other hand, we have found that mandelonitrile was the medical compound which would produce mandelic acid by nitrilase. Free nitrilase is the nitrile degrading enzyme and possess enantio- and regioselectivity [7, 8], and may be used in pharmaceutical industries for the production of (R)-(−)- mandelic acid as an optical resolving agent. However, free nitrilas has some inevitable drawbacks, such as poor stability in aqueous solutions, difficulty in being separated from its product, and bad reusability, which limits this application [9, 10]. Fortunately, these problems can be overcome by forming immobilized enzymes with immobilization. Besides, enzyme immobilization could offer many advantages with increasing enzyme stability, reusing of the enzyme, and the possibility of continuous processing, which makes them more economical and efficient [11, 12].

Nitrilase has already been immobilized on various supports in the past decades, such as membranes [13], alginates [14], carrageenans [15], and polyvinyl alcohol [16], which suffer from diffusion limitations, deactivation during immobilization, and low loading capacity. D155 resin, as a cheap and non-harmful polymer, has hydrophobic hydrocarbon skeleton and carboxyl (-COOH) groups and possesses many advantages, such as high porosity, large adhesion area, easy regeneration, and convenient operation [17]. However, the hydrophobic hydrocarbon skeleton and the hydrophilic group of enzyme protein repel each other, leading to narrow slits during immobilization. This may be solved through grafting with L-lysine as a spacer arm. Acting as a spacer arm, L-lysine helps in reducing the effect of steric hindrance and improving the mass transfer [18].

Therefore, an immobilized nitrilase (I-N-L-D155) immobilized with D155 resin modified by L-lysine (L-D155) was synthesized and used in the production of (R)-(−)-mandelic acid. After the immobilization, the yield of (R)-(−)-mandelic acid was achieved 53.42±0.63% with the enantiomeric excess above 99%. Km value of I-N-L-D155 was 12.74±0.23 mmol/L, which increased by 22.74±2.08% compared to the free nitrilase. However, the enzyme activity of I-N-L-D155 decreased by 45.98±0.45%, this might be for reason that the steric hindrance between enzyme and L-D155, and the diffusion of substrate molecules to active site of the enzyme was difficult [19]. The thermostability of I-N-L-D155 was also increased by 5 oC compared to free nitrilase, and the enzyme activity of residual I-N-L-D155 was retained 92.48±0.32% of its initial activity after continuous 15th reuse in mandelonitrile hydrolysis. The high reuse stability could reduce operational cost. I believe that the immobilized enzyme mentioned above has great potential as a biocatalyst for the application in biotechnological fields, such as the production of (R)-(−)-mandelic acid and its derivatives.

These findings are described in the article entitled Production of (R)-(−)-mandelic acid with nitrilase immobilized on D155 resin modified by L-lysine, recently published in the Biochemical Engineering Journal. This work was conducted by Jiang Wen, Tao Renyou, Yang Yang, Xu Yang, Kang Hongkuan, Zhou Xiaohua, Zhou Zhiming from School of Chemistry and Chemical Engineering of Chongqing University, PR China.

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References:

  1. K. Tang, K. Huang, G. Zhang, Biphasic recognition chiral extraction: A novel method for separation of mandelic acid enantiomers. Chirality. 21 (2009) 390–395.
  2. G.D. Yadav, P. Sivakumar, Enzyme-catalysed optical resolution of mandelic acid via RS (∓)-methyl mandelate in non-aqueous media. Biochem. Eng. J. 19 (2004) 101–107.
  3. L. Xu, Y.Y. Yang, Y.Q. Wang, J.Z. Gao, Chiral salen Mn(III) complex-based enantioselective potentiometric sensor for L-mandelic acid. Anal. Chim. Acta. 63 (2009) 217–221.
  4. D.A. Evans, M.M. Morrissey, R.L. Dorow, ChemInform Abstract: ASYMMETRIC OXYGENATION OF CHIRAL IMIDE ENOLATES. A GENERAL APPROACH TO THE SYNTHESIS OF ENANTIOMERICALLY PURE α-HYDROXY CARBOXYLIC ACID SYNTHONS. J. Am. Chem. Soc. 107 (1985) 4346–4348.
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  7. S.C. Naik, P. Kaul, B. Barse, A. Banerjee, U.C. Banerjee, Studies on the production of enantioselective nitrilase in a stirred tank bioreactor by Pseudomonas putida MTCC 5110. Bioresour Technol. 99 (2008) 26-31.
  8. S.V. Sohoni, D. Nelapati, S. Sathe, V. Javadekar-Subhedar, R.P. Gaikaiwari, P.P. Wangikar, Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour Technol. 188 (2015) 202-208.
  9. N.B. Akacha, A. Zehlila, T. Mejri, 2008. Effect of gamma-ray on activity and stability of alcohol-dehydrogenase from Saccharomyces cerevisiae. Gargouri, Biochem. Eng. J. 40 (2008) 184–188.
  10. Y.P. Xue, Z.Q. Liu, M. Xu, Y.J. Wang, Y.G. Zheng, Y.C. Shen, Enhanced biotransformation of (RS)-mandelonitrile to (R)-(−)-mandelic acid with in situ production removal by addition of resin, Biochem. Eng. J. 53 (2010) 143–149.
  11. X.P. Jiang, T.T. Lu, C.H. Liu, X.M. Ling, M.Y. Zhuang, J.X. Zhang, Y.W. Zhang, Immobilization of dehydrogenase onto epoxy-functionalized nanoparticles for synthesis of (R)-mandelic acid. Int J Biol Macromol. 88 (2016) 9-17.
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  13. J.F. Zhang, Z.Q. Liu, X.H. Zhang, Y.G. Zheng, Biotransformation of iminodiacetonitrile to iminodiacetic acid by Alcaligenes faecalis cells immobilized in ACA-membrane liquid-core capsules. Chemical Papers. 68 (2014) 53-64.
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