Dec 9, 2005 - Abstract The highly enantioselective arylacetonitrilase of. Pseudomonas putida was purified to homogeneity using a combination of ...
Arch Microbiol (2006) 184: 407–418 DOI 10.1007/s00203-005-0061-9
O R I GI N A L P A P E R
Anirban Banerjee Æ Praveen Kaul Æ U. C. Banerjee
Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida
Received: 10 July 2005 / Revised: 26 October 2005 / Accepted: 9 November 2005 / Published online: 9 December 2005 Springer-Verlag 2005
Abstract The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH4)2SO4 fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (kcat/Km) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (3-phenyl propionitrile>4-phenyl butyronitrile). There appeared to be a limit on the bulk being tolerated by the enzyme at 2 position in the side chain, as substitution of the hydrogen atom (phenylacetonitrile) in that position by –OH group (mandelonitrile), –NH2 group (phenylglycinenitrile) or –CH3 group (2-phenyl propionitrile) dramatically lowered the activity. Molecular activity, kinetic constants and enzyme kinetics The specific activity of the purified nitrilase against mandelonitrile was 3.26 lmol/min/mg of protein under standard assay condition. Assuming the molecular
weight of the enzyme to be 412 kDa, its molecular activity is calculated to be 1.34 Kmol/min/mol of nitrilase at 35C under standard assay condition. The molecular activity of R. rhodochrous K22 nitrilase was reported to be 0.479 Kmol/min/mol of nitrilase with crotononitrile as substrate (Kobayashi et al. 1990), whereas molecular activities of Brevibacterium sp. and Pseudomonas chlororaphis B23 nitrile hydratases (NHase) were found to be 161 and 184 kmol/min/mol of NHase, respectively, with propionitrile as the substrate (Nagasawa et al. 1986, 1987). The marked difference in molecular activities between nitrilase and NHase reflected differences with respect to reaction mechanism and the presence of co-factors in these enzymes. To study the affinity of the nitrilase from P. puida towards mandelonitrile, the kinetic parameters of the nitrilase were estimated over a range of mandelonitrile concentration (5–30 mM) at 35C in phosphate buffer (0.01 M, pH 7.5). The maximal hydrolysis rate (Vmax) and apparent Michaelis–Menten constant (Km) of nitrilase for mandelonitrile were calculated from Lineweaver–Burk plot. Km and Vmax for mandelonitrile were found to be 13.39 mM and 16.50 lmol/min/mg of protein, respectively. Kinetic constants for other preferred substrates (Phenylacetonitrile, Indole 3-acetonitrile) were also determined in order to obtain a better estimation of the catalytic properties of the nitrilase (Table 3). A comparison of the kinetic constants revealed that the specificity constant (kcat/Km) for phenylacetonitrile and indole 3-acetonitrile was approximately three- and sixfold higher than mandelonitrile. This was essentially related to the substrate binding as the Km value for phenylacetonitrile and indole 3-acetonitrile was fourfold and 2.5-fold lower than the same for mandelonitrile, while kcat values were of the same order. The high activity of the nitrilase towards phenylacetonitrile was not surprising as P. putida was isolated from soil using phenylacetonitrile as a nitrile substrate of choice (Kaul et al. 2004). It is assumed that selection during acclimation culture gives access to enzyme systems that are highly adapted to the target substrate. The stoichiometry of nitrile consumption and acid formation during the hydrolysis of mandelonitrile was examined in a reaction mixture containing 20 lmol phosphate buffer (pH 7.5), 2 lmol DTT, 10 lmol mandelonitrile and the enzyme in a final volume of 2 ml. The reactions were carried out at 35C in airtight tubes.
412 Table 2 Substrate specificity of P. putida nitrilase
At different time intervals samples (100 ll) were withdrawn and amount of mandelic acid, ammonia, cyanide ion and residual mandelonitrile were determined.
Throughout the reaction mandelonitrile was stoichiometrically hydrolyzed with the concomitant formation of mandelic acid and ammonia (Fig. 2). No formation of
413 Table 2 (Contd.)
(Nitrilase activity against various nitriles (5 mM) was estimated by measuring the liberated ammonia in the reaction mixture using standard assay protocol. The activity for mandelonitrile that corresponded to 3.26 U/mg of protein was taken as 100% and relative activities with respect to mandelonitrile were represented)
mandelamide was detected at any stage in the reaction unlike other nitrilases where amide product is usually 20%) also, the enzyme activity dropped down significantly, revealing the occurrence of protein denaturation in the presence of higher concentration of organic solvent. The results obtained signify the incorporation of small volume of organic solvent in the enzymatic reaction mixture to avoid the diffusional barrier and thus increase the availability of such hydrophobic substrate for efficient hydrolysis. Synechocystis sp. nitrilase also required 10– 20% (v/v) methanol for its increased activity towards long chain aliphatic nitriles and napthalenecarbonitrile (Heinemann et al. 2003). Nitrilase from Pseudomonas sp. DSM 11387 retained considerable amount of activity in the presence of buffer-saturated alcohol and an inverse relationship was observed between the retained enzyme activity and increasing chain length of the alcohol (Layh and Willetts 1998).
Conclusion Biocatalysts are inherently labile and therefore their operational stability is of paramount importance for any bioprocess. Poor biocatalyst stability would result in longer process operations (resulting from decreased catalytic activity); increased frequency of catalyst replacement and reduced product yield. Since thermostability is regarded as an index of overall biocatalyst stability, higher stability of P. putida nitrilase under operational condition (30C) implied that the enzyme could be used repeatedly for long-term biocatalytic reaction under these conditions, allowing a more flexible process viability study. Moreover, cyanohydrins (e.g. mandelonitrile) are known to undergo in-situ racemization by spontaneous degradation to corresponding aldehyde and hydrogen cyanide at slightly alkaline pH (Brady et al. 2004). This can constitute an
417 Fig. 5 Hydrolysis of mandelonitrile by purified nitrilase in the presence of organic solvent (The hydrolytic reaction mixture consisted of 0.01 M phosphate buffer and specified concentrations of organic solvents. After incubation for 10 min with the organic solvents at 35C, mandelonitrile, 5 mM, was added. The reaction was stopped after 20 min with 10%, v/v, 1 M HCl. Precipitated protein was removed by centrifugation and the amount of mandelic acid formed was determined by RP-HPLC. DMSO, dimethyl sulfoxide; DMF, Dimethyl formamide; THF, Tetrahydrofuran; IPA, Isopropyl alcohol)
DMF DMSO EtOH MeOH IPA Acetone 180 160
THF Dioxan
140 120 100 80
Relative activity (%)
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40
5 10 Volume (%) 20 30
20
efficient dynamic–kinetic resolution process to obtain the optically pure acid, provided the pH is maintained alkaline and the enzyme retains its stability under these conditions for efficient resolution. The greater stability of the enzyme at slightly alkaline pH could therefore be exploited to achieve an efficient in-situ racemization of the unreacted (S)-mandelonitrile and attain yield of (R)-( )-mandelic acid close to 100%. Further cloning and over-expression of the nitrilase gene in a suitable host will allow an intensive look at the molecular mechanisms responsible for such strict substrate specificity and high enantiomeric excess exhibited by the enzyme. Works in these aspects are currently in progress in our laboratory. Acknowledgement A. Banerjee and P. Kaul gratefully acknowledge the fellowship provided by CSIR, Govt. of India. This is NIPER communication number 347.
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