Enantioselective Enzymatic Hydrolysis of rac-Mandelonitrile to R

Article pubs.acs.org/IECR

Enantioselective Enzymatic Hydrolysis of rac-Mandelonitrile to R‑Mandelamide by Nitrile Hydratase Immobilized on Poly(vinyl alcohol)/Chitosan−Glutaraldehyde Support Sandip V. Pawar and Ganapati D. Yadav* Department of Chemical Engineering, Institute of Chemical Technology, Nathalal Parekh Marg Matunga, Mumbai - 400 019 India S Supporting Information *

ABSTRACT: Nitriles, amides and carboxylic acids have varied applications in active pharmaceutical intermediate (API), drug and chemical industry, and particularly in the synthesis of pure enantiomers of chiral compounds. The chemical hydrolysis of nitrile compounds is well studied, but it necessitates harsh conditions along with the protection of sensitive functional groups, which on deprotection leads to generation of a large amount of byproducts. Immobilization of nitrile hydratase using a novel support reduces the cost and allows green processes. In this work, enantioselective hydrolysis of rac-mandelonitrile was studied using poly(vinyl alcohol) (PVA)/chitosan−glutaraldehyde cross-linked Rhodococcus rhodochrous ATCC BAA-870 nitrile hydratase (NHase). Immobilized NHase converted rac-mandelonitrile to (R)-amide by dynamic kinetic resolution with enantiomeric excess (ee) up to 81%. The temperature of 40 °C and pH 8 were found to be optimum for the enantioselective nitrile hydrolysis. In the presence of various cosolvents, immobilized NHase showed higher retention of activity in methanol compared to other cosolvents. The kinetic constants for free and immobilized enzyme were obtained from the Lineweaver−Burk plot. The immobilized NHase was found to be reusable up to nine successive batch reactions.

1. INTRODUCTION Of late, enormous efforts have been directed toward synthesis of carboxylic acids and amides using nitrile hydrolyzing enzymes. Nitriles, amides and carboxylic acids have varied applications in active pharmaceutical intermediate (API), drug and chemical industry, particularly in pure enatiomers of chiral compounds.1 The chemical hydrolysis of nitrile compounds is well studied, but it necessitates harsh conditions along with protection of sensitive functional group, which on deprotection leads to generation of a large amount of byproducts.2 Microbial hydrolysis of nitriles involves three different groups of enzymes. Nitrilase enzymes (EC 3.5.5.1 and 3.5.5.7) catalyze the hydrolysis of nitriles to carboxylic acids, and it forms ammonia; nitrile hydratase (NHase, EC 4.2.1.84) is a class of enzyme that catalyzes the hydrolysis of a variety of nitrile compounds into high-value amides, the amides can be further hydrolyzed by amidases (EC 3.5.1.4). Nitrile hydratase (NHase; EC 4.2.1.84), originally found from microorganism and characterized by Asano et al. in 1980,3 is a heterodimeric enzyme with either a nonheme iron or a noncorrin cobalt as the prosthetic group.4 NHases are industrially important enzymes in nitrile metabolism in microorganisms and catalyze the hydrolysis of nitriles at ambient temperature and pH with high selectivity and high yield.5,6 Many processes catalyzing the hydrolysis of nitriles to the corresponding amides have been successfully adapted by the chemical industry for production of acrylamide, nicotinamide, and 5-cyanovaleramide.7−9 However, NHase still needs to be explored to increase its potential for production of valuable amides. NHases are usually labile and prone to substrate or product inhibition at high concentration and have limited substrate acceptability.9 The low enantioselectivity of nitrile hydrolyzing enzymes was a hurdle in developing efficient © 2014 American Chemical Society

approaches for resolution of racemic nitriles. Notwithstanding, numerous reports indicated that nitrile converting enzymes can mediate enantioselective kinetic resolution10−12 and dynamic kinetic resolution13−15 through hydrolysis of nitriles; however, enzymes are also found to be regioselective which prefer a single nitrile group of dinitrile compound.16,17 The industrial applications of NHases are limited due to their notable instability either in isolated or purified form, and hence the prerequisite for their effective use is to enhance the stability. Immobilization of enzyme is often regarded as method of choice to improve the stability for application in industry.18 Enzyme immobilization is beneficial in terms of improving the process economics and is mainly performed to enable enzyme reuse and enhance the overall productivity and robustness.19,20 There are various materials available for development of supports for immobilization of enzymes. The chitosan and PVA are widely used and possess many characteristic properties, including high affinity to proteins, good compatibility, improved resistance to chemical degradation, ease of preparation and availability of reactive functional groups for direct reaction with enzyme and chemical modification.21,22 Immobilization of enzyme on chitosan supports is readily performed by using the bifunctional cross-linker glutaraldehyde (GA); its functional group (−CHO) reacts with the binding site of chitosan (−NH2) and the amino terminal of the enzyme to form the imine bond.21,23 The present study reports immobilization of partially purified R. rhodochrous ATCC BAA870 NHase using poly(vinyl alcohol)/chitosan biocompatible Received: Revised: Accepted: Published: 7986

February 9, 2014 April 3, 2014 April 14, 2014 April 14, 2014 dx.doi.org/10.1021/ie500564b | Ind. Eng. Chem. Res. 2014, 53, 7986−7991

Industrial & Engineering Chemistry Research

Article

Scheme 1. Nitrile Hydratase Immobilized on Poly(vinyl alcohol)/Chitosan−Glutaraldehyde Support

complex for enantioselective nitrile hydrolysis. In the first step of immobilization the R. rhodochrous ATCC BAA-870 was cross-linked with chitosan and subsequently entrapped in poly(vinyl alcohol) which was further lyophilized and employed for nitrile hydrolysis. R. rhodochrous ATCC BAA-870 possesses a nitrile hydratase/amidase biocatalytic system which is capable of metabolizing a wide range of aliphatic and aromatic nitriles and was previously exploited in a number of nitrile biotransformations.1,24,25 The preparation and characterization of PVA/chitosan−GA cross-linked R. rhodochrous ATCC BAA870 NHase has been reported recently by our lab.26 The study indicated that the immobilized NHase exhibited greatly effective catalytic activity, thermal stability and increased tolerance to the varied pH conditions for nitrile hydrolysis.26 Not many nitrile hydratases have been examined for their activity toward mandelonitrile and other cynanohydrins. Herein, the immobilized R. rhodochrous ATCC BAA-870 nitrile hydratase was employed and evaluated for stereoselective nitrile hydrolysis of racemic mandelonitrile, and the effects of various parameters affecting reaction rate, activity and enantiomeric excess were studied.

subsequently 0.8 g R. rhodochrous ATCC BAA-870 NHase dissolved in 3 mL of deionized water was added to the chitosan solution. The cross-linking step was carried out by treating the resulting mixture of chitosan and NHase with 0.5% w/v of glutaraldehyde (GA) solution. It was kept for 6 h under constant magnetic stirring at 4 °C. Thereafter, the chitosan/ glutaraldehyde cross-linked NHase solution was slowly added to previously prepared PVA solution. The resulting gel was kept under constant magnetic stirring for another 3 h at 4 °C. It was thereafter poured in to a Petri dish and kept for freeze-drying in LABCONCO Freeze-Dryer (Kansas City, U.S.A.). The developed polymer blend of PVA/chitosan−GA cross-linked NHase was then cut off into several sections of 2−3 mm2 and was employed for the enantioselective nitrile hydrolysis of mandelonitrile (Scheme 1). 2.3. Enantioselective Hydrolysis of rac-Mandelonitrile Using Immobilized Nitrile Hydratase. The reaction mixture (up to 10 mL) consisted of immobilized 25 mg of R. rhodochrous ATCC BAA-870 NHase suspended in phosphate buffer (0.1 M, pH 7). To initiate the reaction, 30 mM mandelonitrile was added to the reaction mixture and kept in an incubator shaker (30 °C, 200 rpm). The reactions were carried out in triplicate. Samples were taken periodically and analyzed by HPLC. One unit of enzyme activity for nitrile hydratase was defined as the amount of enzyme that catalyzed the production of 1 μmol of mandelamide/min under standard assay conditions. 2.4. HPLC Analysis. RP-HPLC: The analyses were carried out on an Agilent 1260 infinity HPLC-system (pumps DEAB802848, autosampler DEAB305842, diode-array-detector DEAAX01774) using Zorbax Eclipse XDB-C18 column (0.46 mm × 250 mm; 5 μm) under the following conditions: mobile phase, water/acetonitrile/trifluoroacetic acid (60:40:0.1, v/v); flow rate, 1.0 mL/min; column temperature, 25 °C. The retention time of mandelamide and mandelonitrile were 2.5 and 5.6 min, respectively. Chiral HPLC: The analyses were carried out on an Agilent 1260 infinity HPLC system with Chiralpak IA column (0.46 mm × 250 mm; 5 μm) under the following conditions: mobile phase, n-hexane/2-propanol/TFA (95:5:0.1, v/v); flow rate, 1.0 mL/min. The retention time of (S)-mandelonitrile and (R)-mandelonitrile were 6.4 and 6.9 min, respectively, whereas the retention time of (R)mandelamide was 13.6 min. The RP-HPLC chromatogram (Figure S1) and CHIRAL HPLC chromatogram (Figure S2) are provided in the Supporting Information (SI).

2. EXPERIMENTAL SECTION 2.1. Materials. The R. rhodochrous ATCC BAA-870 nitrile hydratase (light brown powder, activity approximately 1000 U/ g partially purified enzyme) was obtained as gift sample from ZA Biotech (Pty) Ltd., South Africa. Chitosan (obtained from shrimp shells, degree of deacetylation ≥75%, Mol. Wt. ≳ 375,000 Da) was purchased from Himedia, Mumbai. Poly(vinyl alcohol) (Mol wt. ≈ 125,000) and glutaraldehyde (25% solution), with the highest purity available, were purchased from SD. Fine Chemicals Ltd., India. Buffer salts, solvents, and other chemicals with highest purity were obtained from firms of repute in Mumbai, India. 2.2. Preparation of PVA/Chitosan−GA Cross-Linked NHase. A facile method for preparation of cross-linked nitrile hydratase was developed using biocompatible complex of PVA and chitosan.26−28 At first, 4.0 g PVA polymer powder was dissolved in 40 mL of distilled water under magnetic stirring, at 80−90 °C to prepare PVA solution which was cooled down to ambient temperature after complete dissolution. Then 1 g chitosan flakes were dissolved in 80 mL of 0.5% acetic acid solution under constant magnetic stirring at room temperature. After complete dissolution of chitosan flakes, 1 M NaOH was added to adjust the pH of solution in the range of 5−5.5. The chitosan solution was then kept at 4 °C for stirring; 7987

dx.doi.org/10.1021/ie500564b | Ind. Eng. Chem. Res. 2014, 53, 7986−7991


Article

3. RESULTS AND DISCUSSION 3.1. Conversion of rac-Mandelonitrile. The hydration of nitriles has attracted substantial interest because conventional chemical methods for nitrile hydration involve the use of severe conditions such as the use of concentrated acid or base and high temperatures. In contrast, the reactions catalyzed by nitrile hydrolyzing enzymes proceed under mild conditions and produce a high yield of a stereospecific product. The immobilized NHase was employed for the stereoselective nitrile hydrolysis of rac-mandelonitrile (Scheme 2), Scheme 2. Reaction Scheme for Stereoselective Nitrile Hydrolysis of rac-Mandelonitrile by Immobilized Rhodococcus rhodochrous ATCC BAA-870

Figure 1. Conversion of rac-mandelonitrile and ee. The ee value of (R)-mandelamide was calculated by comparison of the peak areas of the respective (S)- and (R)-enantiomers obtained by chiral HPLC.

and reaction was monitored by HPLC. Mandelonitrile was almost stoichiometrically converted to mandelamide in 180 min. Due to the instability of mandelonitrile in phosphate buffer, it breaks down into benzaldehyde and cyanide followed by spontaneous racemization to form racemic mandelonitrile. The higher yield of enantiopure mandelamide is possible by dynamic kinetic resolution of mandelonitrile as shown in the reaction scheme. The NHase was shown to produce preferentially the (R)-enantiomer of the mandelamide. After 180 min of conversion time at 30 °C, the enantiomeric excess of mandelamide was 78% (Figure 1). In an investigation reported by Knife et al.21 incubation of whole cells of R. rhodochrous ATCC 870 with β-hydroxy nitrile was found to give selective production of (R)-amide by nitrile hydratase and subsequently selective access to (S)-acid due to the presence of amidase enzyme. Cohen et al.29 reported an immobilized whole cell system of Rhodococcus sp. SP 361 for hydrolysis of racemic 2-(4-isobutylphenyl)propionitrile which exhibited (R)-selectivity toward formation of product. Rhodococcus sp. AJ 270 and Moraxella sp. 3L-A-1-5-1a-1 were found to have selectivity toward the (R) product for 2-phenylbutyronitrile and 2-(4isobutylphenyl)propionitrile, respectively.30,7 The nitrile hydratase from Klebsiella oxytoca,12 R. equi A431 exhibited Sselectivity for various aromatic nitriles. 3.2. Effect of Temperature and pH. The biocatalytic system is sensitive to temperature. The immobilized R. rhodochrous ATCC BAA-870 NHase was employed for enantioselective nitrile hydrolysis of racemic mandelonitrile over a period of 3 h at six different temperatures ranging from 20 to 50 °C (Figure 2). The conversion increased from 72% at 20 °C to 92% at 40 °C, and there was a drastic reduction in conversion to 79% at 50 °C which could be due to instability of nitrile hydratase at temperatures above 40 °C. The ee increased marginally from 77.2 to 80.9% from 20 to 30 °C, and thereafter

Figure 2. Effect of temperature on the conversion rac-mandelonitrile and ee.

it was 79.9% at 40 °C, suggesting practically the same ee. It would mean that the activation energies of the two reactions are very close, and therefore temperature did not influence ee. The effect of pH on enantioselective nitrile hydrolysis of mandelonitrile was studied in the pH range of 6−9 (Figure 3). The enantioselective nitrile hydrolysis was favored at pH > 7 and the maximum conversion was achieved at pH 8, and the ee was above 80%. The R. rhodochrous ATCC BAA-870 NHase was found to be stable at alkaline pH, and there was a substantial reduction in conversion of rac-mandelontrile at pH > 8. In stereoselective nitrile hydrolysis of mandelonitrile, the unreacted mandelonitrile present in the reaction mixture undergoes spontaneous racemization via formation of benzal7988



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higher activity in comparison to the all organic solvents studied in the reaction. The ee of mandelamide was found to be below 50% in the presence of aprotic solvents, whereas it was 79.8% in methanol as cosolvent. The nature of a solvent regulates the microenvironment and thus the kinetics of the enantioselective path giving (R)-mandelamide. The enzyme undergoes inactivation in the presence of organic solvents due to denaturation and exhibits reduced activity compared to that of the control experiment. In a biphasic mixture of water− organic solvents the enzyme activity depends on its conformation flexibility as well the structural rigidity.32 3.4. Effect of Enzyme Concentration. The effect of catalyst loading was optimized by carrying out the immobilization of varying amounts of nitrile hydratase (0.04−0.24 g of NHase) per g of polymer support. The optimum enzyme loading with effective enzyme activity was found to be 0.16 g of NHase per g of polymer support. The nitrile hydrolysis of racmandelonitrile was studied by changing the concentration of immobilized R. rhodochrous ATCC BAA-870 NHase from 10 mg to 30 mg (containing 0.16 g of NHase per g of polymer support). The rate of reaction increased linearly with the increase in the concentration of nitrile hydratase (Figure 5).

Figure 3. Effect of pH on the conversion of rac-mandelonitrile and ee.

dehyde and hydrogen cyanide, and the racemization is favored at slightly alkaline pH.14 3.3. Effect of Cosolvents. The nonaqueous systems are highly desirable in biocatalysis to improve the rate of enzymatic reactions; the insolubility of nitrile substrates in an aqueous reaction mixture decreases the rate of enzymatic reactions.32 The presence of biocatalyst in a mixture of water−organic solvents affects its catalytic properties and stability, depending on exposure to the type of solvent. Various cosolvents were used to study their effect on the activity of immobilized nitrile hydratase. Nine different organic solvents were used as cosolvents (at 10%, v/v) (Figure 4). It was observed that the

Figure 5. Effect of enzyme concentration on conversion of racmandelonitrile.

There was an insignificant increase in conversion above an enzyme concentration of 25 mg; this suggests that the amount (active sites) of enzyme present in the reaction medium was greater than the desired concentration, and there was no more conversion. The enantiomeric excess of mandelamide was found to be 81.5% at optimum enzyme concentration. 3.5. Kinetic Constants for Immobilized NHase. The kinetic constants for the free and immobilized R. rhodochrous ATCC BAA-870 NHase were determined by studying the nitrile hydrolysis at different substrate concentrations (5−100 mM) of mandelonitrile. The initial rates were obtained from the time versus concentration profile, and the values of Michaelis constant (Km) and maximum reaction rate (Vmax) for immobilized NHase and free NHase were determined from the Lineweaver−Burk plot (Figure 6). The values for Michaelis constant (Km) and maximum velocity (Vmax) were 17.07 ± 1.04 mM, 10.74 ± 0.72 mM min−1 for immobilized enzyme, and

Figure 4. Effect of various cosolvents on the conversion racmandelonitrile.

extent of nitrile hydrolysis had significantly increased in cosolvents such as methanol, ethanol, and tert-butanol. These solvents were tolerated to a greater extent vis-á-vis other solvents used in the study. The enzyme activity was found to be on the lower side with the use of aprotic solvents such as DMSO and DMF and acetone, whereas the conversion decreased with increase in log P of organic solvents. The highest conversion was observed in methanol as cosolvent; however, the cosolvent-free control experiment exhibited 7989



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4. CONCLUSION The present study investigates the potential application of nitrile hydratase for enantioselective nitrile hydrolysis of racmandelonitrile. The immobilization of R. rhodochrous ATCC BAA-870 nitrile hydratase using a biocompatible polymer complex of PVA and chitosan is a prospective method for preparation of nitrile hydrolyzing biocatalyst. The immobilized R. rhodochrous ATCC BAA-870 NHase shows effective conversion of rac-mandelonitrile to (R)-amide by dynamic kinetic resolution. The kinetic parameters were determined from a Lineweaver−Burk plot, and catalyst reusability was studied up to nine cycles. Constantly improving methods for immobilization of nitrile hydrolyzing biocatalysts will add more applications of nitrile converting enzymes to the industry.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 6. Lineweaver−Burk plot.

13.6 ± 0.91, 11.02 ± 1.41 for free enzyme, respectively. The higher value of Km for immobilized enzyme compared to that of free enzyme indicates lower affinity of substrate for immobilized NHase. However, the reduced Vmax of immobilized NHase compared to free enzyme is attributed to mass transfer limitations imposed by polymer support on substrate or immobilization of enzyme molecule in inactive or substrateblocked configuration.33 3.6. Reusability Study. The reusability study of the immobilized NHase was carried out to determine stability of supported enzyme under optimized reaction conditions (40 °C, pH 8, cosolvent - methanol, reaction time - 120 min). The immobilized NHase was separated from the reaction mass after the experiment by filtration, dried at room temperature, and was reused for repeat reactions wherein no makeup quantity was added in the subsequent experiment. Table 1 shows the

*E-mail: [email protected]. Telefax: +91-22-33611001, Fax: +91-22-3361-1020. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.V.P. thanks the University Grant Commission for an award of fellowship. The authors are greatly thankful to ZA Biotech (Pty) Ltd, South Africa, for providing the gift sample of nitrile hydratase. G.D.Y. received support from R.T. Mody Distinguished Professor endowment and J. C. Bose National Fellowship of Department of Science and Technology, Government of India.

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Table 1. Reusability Study of Immobilized NHase number of reuses

conversion % after each reuse

1 2 3 4 5 6 7 8 9

96.2 84.9 75.2 62.8 53.4 42.5 31.6 22.4 10.3

AUTHOR INFORMATION

Corresponding Author

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NOMENCLATURE DMF = dimethylformamide DMSO = dimethyl sulfoxide GA = glutaraldehyde Km = Michaelis constant (mM) NHase = nitrile hydratase PVA = poly(vinyl alcohol) Vmax = maximum velocity (mM min−1) REFERENCES

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Enantioselective Enzymatic Hydrolysis of rac-Mandelonitrile to R

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