Stereo-Selective Conversion of Mandelonitrile to (R)-(2)-Mandelic Acid Using Immobilized Cells of Recombinant Escherichia Coli
Total Page：16
File Type：pdf, Size：1020Kb
3 Biotech (2012) 2:319–326 DOI 10.1007/s13205-012-0058-4 ORIGINAL ARTICLE Stereo-selective conversion of mandelonitrile to (R)-(2)-mandelic acid using immobilized cells of recombinant Escherichia coli Sandip V. Pawar • Vachan Singh Meena • Shubhangi Kaushik • Ashwini Kamble • Sandeep Kumar • Yusuf Chisti • U. C. Banerjee Received: 22 January 2012 / Accepted: 14 March 2012 / Published online: 29 March 2012 Ó The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Immobilized cells of a recombinant Esche- Introduction richia coli expressing nitrilase from Pseudomonas putida were used to catalyze the hydrolysis of mandelonitrile (2- The conventional harsh chemical methods are extensively hydroxy-2-phenylacetonitrile) to (R)-(-)-mandelic acid. substituted by environment-friendly biocatalysts for the syn- The cells had been immobilized by entrapment in an thesis of pharmaceutically important intermediates (Kumar alginate matrix. Conditions for the hydrolysis reaction were et al. 2010). The interest towards use of nitrilase has been optimized in shake flasks and in a packed bed reactor. In tremendously increasing since past decade leading to the easy shake flasks the best conditions for the reaction were a hydrolysis of nitriles (Martinkova and Mylerova 2003;Chen temperature of 40 °C, pH 8, biocatalyst bead diameter of et al. 2008;Kaplanetal.2006; Rustler et al. 2007); it offers 4.3 mm, sodium alginate concentration in the gel matrix of significant advantages over other routes as cheap starting 2 % (w/v, g/100 mL), a cell dry mass concentration in the material, absence of cofactor involvement and the possibility bead matrix of 20 mg/mL, an initial substrate concentra- of carrying reactions under mild condition (Banerjee et al. tion of 50 mM and a reaction time of 60 min. Under these 2002;Heetal.2010;Heetal.2007). Additionally, the exis- conditions, the conversion of mandelonitrile was nearly tence of nitrile-hydrolyzing enzymes that show enatio-and 95 %. In the packed bed reactor, a feed flow rate of 20 mL/ regio-selectivity offers synthetic possibilities that are difficult h at a substrate concentration of 200 mM proved to be the to achieve by conventional catalytic approaches (Naik et al. best at 40 °C, pH 8, using 4.3 mm beads (2 % w/v sodium 2008; Kiziak et al. 2005). Immobilized whole cells of alginate in the gel matrix, 20 mg dry cell concentration per microorganisms are widely used as catalysts for various bio- mL of gel matrix). This feed flow rate corresponded to a transformation processes (Bucko et al. 2005; Niladevi and residence time of 0.975 h in the packed bed. Prema 2008). When applicable, whole cell biocatalysis avoids the need to extract and purify intracellular enzymes and is Keywords Nitrilase Á Mandelic acid Á Mandelonitrile Á therefore less expensive compared with immobilized enzyme Packed bed reactor Á Immobilized cells catalysts (Givry et al. 2008;Takeruetal.2005). (R)-(-)-mandelic acid is an important pharmaceutical intermediate for the production of semi-synthetic cephalo- sporins, penicillins (Terreni et al. 2001; Tang et al. 2009) antitumor agents (Surivet and Vatele 1998) and antiobesity S. V. Pawar Á V. S. Meena Á S. Kaushik Á A. Kamble Á drugs (Mills et al. 1983). Its derivatives are used also as chiral & S. Kumar Á U. C. Banerjee ( ) resolving agents (Kinbara et al. 1996; Xue et al. 2011). Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Polymers of mandelic acid have been developed into anti- Sector-67, SAS Nagar, 160 062 Punjab, India microbial contraceptives for vaginal prophylaxis (Zaneveld e-mail: [email protected] et al. 2002). Derivatives of mandelic acid have been shown to have antifungal activity (Kope et al. 1991), broad spectrum b- Y. Chisti School of Engineering, Massey University, Private Bag 11 222, lactamase inhibitor activity (Mollard et al. 2001) and anti- Palmerston North, New Zealand oxidative properties (Ley and Bertram 2001). 123 320 3 Biotech (2012) 2:319–326 Mandelic acid is produced by chemical as well as Aldrich Chemical Co. (Milwaukee, USA). Growth media enzymatic methods; several approaches to obtain enatio- components were obtained from Hi-Media Inc. (Mumbai, merically pure mandelic acid have been reported in the India). Solvents, buffer salts, inorganic salts and other literature (Yadav and Sivakumar 2004; Xiao et al. 2005, chemicals used were of analytical grade. 2008; Oda et al. 1992; Patterson et al. 1981; Miyamoto and Ohta 1992). Nitrilase-mediated production of (R)-(-)- Microorganism and cultivation conditions mandelic acid has significant advantage over chemical methods of production; it offers excellent enatioselectivity Recombinant E. coli BL21 (DE3) was used for the present and above all possibility of carrying out dynamic kinetic study. The bacterium harbored the nitrilase gene of P. resolution which provides 100 % theoretical yield of the putida MTCC 5110 and had been cloned in our laboratory product (Yamamoto et al. 1991; Banerjee et al. 2006; Singh (Genbank accession no. EF467660) (Banerjee et al. 2009). et al. 2005; Xue et al. 2011) (Fig. 1). In recent years, the The stock culture was maintained on plates of Luria–Ber- hydrolysis of mandelonitrile has been reported using tani agar supplemented with ampicillin. The microorgan- recombinant nitrilase from Pseudomonas fluorescens ism was initially grown in shake flasks (37 °C, 200 rpm) EBC191 (Bucko et al. 2005), Alcaligenes faecalis for 16 h in a medium of the following composition: yeast ATCC8750 (Rey et al. 2004), newly isolated nitrilase extract (10 g/L), tryptone (16 g/L) and sodium chloride producer Alcaligenes sp. ECU0401 (He et al. 2007;He (5 g/L). After 16 h, 10 % (v/v) of the culture was inocu- et al. 2010) and A. faecalis ZJUTB10 (Xue et al. 2011). lated into fresh flasks containing the aforementioned This study reports the use of immobilized whole cells of specified medium. The inducer isopropyl-b-D-1-thiogalac- the recombinant Escherichia coli BL21 (DE3) for pro- topyranoside (IPTG) was added to each flask after 1 h of ducing (R)-(-)-mandelic acid from mandelonitrile in shake inoculation and the final IPTG concentration in the flask flask and packed bed reactor. The recombinant bacterium was 1 mM. Cells were harvested after 4 h by centrifugation used harbored the nitrilase gene from P. putida MTCC at 10,000g (4 °C) and washed with Tris–HCl buffer 5110 in the IPTG-inducible plasmid pET 21 b (?) (100 mM, pH 7) before use. expression vector. P. putida was reported as an ideal can- didate for stereo-selective hydrolysis of mandelonitrile, Immobilization of whole cells considering its higher growth rate, higher reaction rate and higher stability as compared with other nitrilase-producing Washed cells harvested as specified above, were immobi- microorganisms. P. putida nitrilase attains comparable lized by entrapment in a sodium alginate matrix (Varma specific activity within a shorter period of time and the and Gaikwad 2010; Givry et al. 2008). For the entrapment, fermentation time was also reported to be lower for bio- 200 mg (dry weight basis) of cells was resuspended in catalyst generation (Kaul et al. 2004). 10 mL of Tris–HCl buffer (100 mM, pH 7). This slurry was added to a 2 % (w/v, g/100 mL) solution of sodium alginate (prepared in Tris–HCl buffer, 100 mM, pH 7) with Materials and methods vigorous stirring to ensure complete mixing. The resulting suspension was added dropwise via a syringe to an aqueous Chemicals solution of CaCl2 (0.2 M). Ca-alginate beads thus formed were allowed to stand in CaCl2 solution for 1 h for hard- Mandelonitrile, sodium alginate, glutaraldehyde (GA) and ening. The hardened beads were washed with distilled polyethyleneimine (PEI) were purchased from Sigma- water and used for the biotransformation reaction. Fig. 1 Reaction scheme for OH OH OH enantioselective hydrolysis of Nitrilase (R,S)-mandelonitrile for CN CN synthesis of (R)-mandelic acid COOH + 2H2O + (R,S)-Mandelonitrile (S)-(+)-Mandelonitrile (R)-(-)-Mandelic acid Chemical equilibrium CHO + HCN Benzaldehyde 123 3 Biotech (2012) 2:319–326 321 Biotransformation conditions sodium alginate were satisfactory in every respect, but were not as good at converting the substrate as the Immobilized cells (200 mg dry weight basis) were sus- aforementioned beads. This was likely because the harder pended in 10 mL of Tris–HCl buffer (100 mM, pH 7). The gels formed at sodium alginate concentrations of [2% final concentration of immobilized cells was 20 mg/mL. reduced the diffusion of the substrate in the beads. Due to Mandelonitrile is not soluble in aqueous solvent (sparingly the liable nature of nitrilase, milder methods are generally soluble); hence 10 % (v/v) methanol was used as a co- used for the immobilization of whole cells/enzymes. The solvent to make it soluble (Kaul and Banerjee 2008). It was potential adverse effects from the matrix or reagents used added to an initial concentration of 30 mM in reaction are less in the entrapment method of the cell immobili- mixture. The mixture was incubated in shake flasks held at zation. It has been reported in the literature (Panova et al. 40 °C, 200 rpm, for 3 h. Bead-free samples were taken at 2007; Ben-Bassat et al. 2008) that by encapsulation of regular intervals and analyzed by HPLC. whole cells, there were effective recovery and recycle of the enzyme in the case of glycolic acid production. In Analytical methods view of this, cells immobilized at an alginate concentra- tion of 2 % were used in all subsequent experiments, The concentration of mandelic acid formed in the reaction unless stated otherwise. mixture was measured by high-performance liquid chro- matography (Shimadzu 10AD VP, Kyoto, Japan). Man- Effect of bead size delic acid, mandelamide and mandelonitrile were assayed Ò on a LiChroCART C18 column (250 9 4 mm, 5 lm) Immobilized biocatalysts commonly experience mass (Merck, Darmstadt, Germany).