Mol Biotechnol DOI 10.1007/s12033-008-9094-z
RESEARCH
Enantioselective Nitrilase from Pseudomonas putida: Cloning, Heterologous Expression, and Bioreactor Studies Anirban Banerjee Æ Sachin Dubey Æ Praveen Kaul Æ Brajesh Barse Æ Markus Piotrowski Æ U. C. Banerjee
Ó Humana Press 2008
Abstract Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(?) and over-expressed as histidinetagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTGinduced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(-)-mandelic acid. Keywords Nitrilase cloning Heterologous expression Pseudomonas putida (R)-(-)-mandelic acid Bioreactor studies
A. Banerjee S. Dubey P. Kaul B. Barse U. C. Banerjee (&) Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, Punjab 160 062, India e-mail:
[email protected] M. Piotrowski Department of Plant Physiology, Ruhr-Universita¨t, Universita¨tsstr. 150, 44801 Bochum, Germany
Introduction Nitrilases constitute an important class of hydrolases that can hydrolyze the cyanide functionality to the corresponding carboxylic acid under ambient conditions [1]. This has resulted in the increased recognition of their potential owing to the possibility of conducting such conversion under mild condition that would not alter other labile reactive groups. The added attribute of -chemo, -regio, and -enantio selectivity exhibited by this class of enzymes offers synthetic possibilities that are difficult to achieve by conventional catalytic approaches [2]. In spite of their tremendous potential, a bio-option will only come in question if the chemical arsenal cannot achieve the synthesis of the target molecule. This is so, because the appropriate strain and enzymes are often missing. The big gap between their potential and application is largely filled in by the lack of the enzyme availability in a cheap and reusable form. The required activities may be accessed using screening methods or the biocatalyst may be procured from various culture collection centers [3, 4]. Although a few commercial preparations have come up recently, but they are too expensive for synthetic applications and this has prompted the use of immobilized enzymes [5, 6]. There are very few reports on the fermentative production of nitrile hydrolases, particularly on nitrilases [7]. Pseudomonas putida MTCC 5110 has earlier been shown to harbor a highly enantioselective nitrilase for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid [4], a versatile chiral building block, and a resolving agent [8–10]. Therefore, the objective of the present investigation was to clone and over-express the nitrilase gene from P. putida in a suitable host and to perform studies regarding the effect of various process parameters on nitrilase production in a stirred-tank reactor. P. putida nitrilase gene has been deposited in Genbank
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Accession No is EF467660. The recombinant enzyme was also used to produce (R)-mandelic acid from mandelonitrile.
Materials and Methods Chemicals Mandelonitrile was obtained from Aldrich Chemicals (Milawukee, USA). All media components were supplied by Hi Media laboratories Ltd (Mumbai, India). All inorganic salts were supplied by Qualigens Fine Chemicals (Mumbai, India). Bradford reagent and different inhibitors were obtained from Sigma Chemical Company (St. Louis, USA). Different restriction enzymes, alkaline phosphatase, and DNA-ligase were procured from Takara Bio Inc. (Shiga, Japan). All other reagents or chemicals used were of analytical grade and obtained from standard companies. Microorganisms, Cultivation Conditions, and Vectors P. putida MTCC 5110 was earlier screened and isolated as a nitrilase producer for enantioselective hydrolysis of mandelonitrile to (R)-mandelic acid. Escherichia coli XL1-Blue [recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac (F0 , proAB, lacIqZ, M15, Tn10)] (Stratagene, SanDiego, USA) was used for routine sub-cloning experiments and E. coli BL21 CodonPlus (DE3) [F-, ompT, hsdSB, (rB-, mB) gal dcm (DE3)] (Novagen, Madison, USA) was used for the expression of the gene of interest, respectively. pGEMÒ-T (Promega, Madison, USA) and pBluescript II SK (±) (Stratagene, LaJolla, USA) were used as sequencing vectors and pET 21b(?) (Novagen, Madison, USA) was used as expression vector, respectively. The stock culture was maintained on Luria Bertani plates containing ampicillin. The microorganism was initially grown at 37°C for 16 h in 29 YT medium of the following composition—yeast extract (10 g/l), tryptone (16 g/l), and sodium chloride (5 g/l). After 16 h, 10% (v/v) of the culture was transferred to 6.6 l Bioflo 3000Ò bioreactor (New Brunswick Scientific, Edison, NJ, USA) containing the same media. The cells were grown for 24 h at 37°C, with an aeration and agitation rates of 1 vvm and 200 rpm, respectively. Standard Molecular Biology Techniques Standard molecular biology techniques were used as described by Ausubel et al. [11]. Sequencing of DNA was done by MWG Biotech (Ebersberg, Germany). Sequence analysis, database searches, and comparisons were carried out with DNAMAN (Lynon Corporation, Quebec, Canada) program with default parameters and BLAST search facilities at NCBI [12].
Polymerase Chain Reaction (PCR) and Cloning of Nitrilase Gene Genomic DNA from P. putida was isolated according to the procedure of Ausubel et al. [11]. Sense (50 -CATATGCAGACAAGAAAAATCGTCCGG-30 ) and antisense primers (50 -CTCGAGGACGGTTCTTGCACCAGTAGC-30 ) were used to amplify the nitrilase gene (GeneBank Accession No. EF467660) from genomic DNA of P. putida. The sense primer contained an Nde I site while the antisense primer contained an Xho I site (restriction sites are indicated by underlined bases) to facilitate cloning of the PCR product. Each PCR reaction mixture contained 5 ll PCR reaction buffer (109) with 1.5 mM MgCl2, each dideoxynucleotide triphosphate (dNTP) at a concentration of 0.2 mM, sense and antisense primers at a concentration of 1 lM, 1 unit of DNA polymerase, and 0.1 lg genomic DNA of P. putida as template, in a final volume of 50 ll. The following PCR program was used: initial denaturation step (94°C, 5 min), followed by 40 cycles consisting of a denaturation step at 94°C for 30 s, an annealing step at 64°C for 45 s and an elongation step at 72°C for 1 min. The polymerization step lasted for 1 h. The PCR product was cloned in to the pGEMÒ-T vector using TA cloning and positive clones were verified by restriction digestion and DNA sequencing. Finally, the PCR product was digested with Nde I and Xho I and cloned in pET 21b(?) vector under control of T7 promoter to generate expression plasmids pET 21b(?)-PspNit. Intactness of the nitrilase gene in the recombinant plasmid was verified by DNA sequencing, and E. coli BL21 (DE3) cells were transformed by pET 21b(?)-PspNit plasmid by electroporation. Purification of Nitrilase E. coli BL21 (DE3) (pET 21b(?)-PspNit) cells were grown overnight in 29 YT medium with ampicillin (100 lg/ml). The cells were subsequently diluted (1:100 v/v) in 600-ml fresh medium. IPTG (1 mM) was added to induce the nitrilase gene when the optical density (OD) of the bacterial culture at 600 nm reached about 0.25. The cells were harvested after 5 h by centrifugation (10,000g, 10 min, 4°C) washed twice with 20 ml Na-phosphate buffer (10 mM, pH 7.5) and resuspended in 10 ml of the same buffer. The cells were disrupted using a French Pressure Cell (16,000 psi) and the homogenate was centrifuged (50,000g, 45 min, 4°C) to obtain the crude cell extract. The supernatant (soluble protein) was used for further experiments. Since the recombinant nitrilase carried a C-terminal affinity tag of six consecutive histidine residues, it was purified using the QIAexpress nickel-nitrilotriaceticacid (Ni-NTA) protein purification system (Qiagen, Hilden, Germany). Spin columns were packed with 1 ml Ni-NTA agarose matrix. The protein purification was done with 30–40 mg protein/
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ml Ni-NTA agarose. The column was pre-equilibrated with phosphate buffer (0.05 M, pH 8.0) containing 300 mM NaCl and 10 mM imidazole. Flow through was collected and the column was washed with the same buffer containing 40 mM imidazole to wash out loosely bound proteins. The C-terminal His-tagged nitrilase was finally eluted from the column using a linear gradient of imidazole (40–400 mM) in phosphate buffer (0.05 mM, pH 8.0) containing 300 mM NaCl. The eluted protein was buffer exchanged with phosphate buffer (0.01 M, pH 7.5) containing 1 mM DTT using PD-10 columns (Amersham Biosciences, Uppasala, Sweden), to remove imidazole. Enzyme Assay The standard reaction mixture (2 ml) consisted of wet cell paste (10 mg) suspended in phosphate buffer (100 mM, pH 7.5). Mandelonitrile (5 mM) was added to initiate the reaction and the mixture was incubated at 37°C for 20 min. Amount of mandelic acid formed was determined by analytical high-performance liquid chromatography (model 10AD VP) (Shimadzu, Japan) equipped with a LiChroCART-RP-18 column (250 9 4 mm, 5 lm) (Merck, Germany) at a flow rate of 0.8 ml/min with a solvent system (65:35, v/v) of phosphate buffer (0.01 M, pH 4.8) and methanol. The retention times for mandelic acid and mandelonitrile were 2.6 and 18.3 min, respectively. A254 nm was measured. The optical purity of mandelic acid was determined by the analysis of the enantiomers on CHIRALCELOD-H column (250 9 0.46 mm, 5 lm) (Daicel Chemical Industries, USA) at a flow rate of 0.5 ml/min with a mobile phase containing hexane, isopropanol, and trifluoro acetic acid (90:10:0.2, v/v). The retention times for (S)-(?) and (R)-(-)-isomers were 15.5 and 17.5 min, respectively. A254 nm was measured.
Inc., NJ, USA) having a working volume of 4.5 l. Fermentation was carried out at 37°C for 24 h with aeration (1 vvm) and agitation (200 rpm) unless otherwise stated. pH and other parameters were measured online by specific probes.
Results Cloning of P. putida Nitrilase in E. coli P. putida nitrilase gene was PCR-amplified from P. putida genomic DNA. The amplified gene was flanked by restriction sites NdeI and XhoI. The NdeI and XhoI digested nitrilase gene and pET-21b vector were ligated and transformed in BL-21 (DE-3) cells. The colonies were screened for the insert by digesting the plasmids isolated from respective colonies with NdeI and XhoI and positive clone was selected. Expression of Nitrilase in E. coli
The growth of the organism in the fermenter was estimated by taking 1 ml aliquot periodically and measuring the OD at 600 nm by UV-visible spectrophotometer (Beckman DU 7400, USA).
Recombinant E. coli BL21 (DE3) cells containing pET 21b(?)-PspNit were cultured aerobically in 29 YT medium containing 100 lg/ml ampicillin and 30 lg/ml chloramphenicol at 37°C for overnight. This inoculum (10%, v/v) was then transferred to fresh 29 YT medium containing only ampicillin (100 lg/ml). After 1 h, IPTG was added to the culture broth at a final concentration of 1 mM to induce the nitrilase production. Four hours after IPTG addition, cells were harvested by centrifugation (10,000g, 10 min, 4°C) and suspended in phosphate buffer (0.05 M, pH 8.0) containing 300 mM NaCl and 1 mg/ml lysozyme (lysis buffer). The cell suspension was sonicated for 1 min and centrifuged at 12,000g for 20 min at 4°C to obtain clear supernatant which was designated as cell-free extract (CFE). As a control, E. coli pET 21b(?) without the nitrilase insert was cultivated under identical condition and both the cell extracts were compared using an SDS–PAGE (Fig. 1). In E. coli pET 21b(?)-PspNit, an additional protein of the predicted size was observed, however, the control also showed a very faint band of nitrilase. The nitrilase activity in the supernatant of the recombinant E. coli cells was found to be 0.44 U/mg, which was approximately fourfold higher than that of P. putida. Time course of enzyme production by the recombinant E. coli cells was examined and it was observed that the nitrilase activity was highest after 6 h of cultivation. On quantitative analysis of the gel tracks by QuantityOneÒ software, the nitrilase formed corresponded to about 27% of total soluble protein (data not shown) of the E. coli cells.
Bioreactor Studies
Purification of the Recombinant Nitrilase
All the bioreactor studies were carried out in a 6.6 l benchtop fermenter (BIOFLO 3000, New Brunswick Scientific Co.,
Nitrilase was purified from cell extracts prepared from IPTG-induced cells of E. coli pET 21b(?)-PspNit (Fig. 2).
Estimation of Total Reducing Sugar The total reducing sugar in the medium was measured by the standard procedure [13]. A standard curve with glucose was made under the same condition. Estimation of Growth
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His-tagged protein was 28-fold higher than the activity of the wild-type nitrilase (data not shown). No significant differences in the kinetic constants were observed between the wild-type and the His-tagged nitrilase, its indicating that the addition of the six histidine residues at the carboxy terminus did not affect the overall catalytic properties of the enzyme (data not shown). Bioreactor Studies
Fig. 1 Expression analysis of nitrilase using SDS–PAGE in cell extracts from uninduced (lanes 2 and 3) cells of control vector pET 21b(?) and IPTG induced (lanes 4 and 5) cells containing pET 21b(?)-PspNit construct (10 lg crude extract per lane). Lane 1 represents molecular weight markers
One of the key issues with the cultivation of recombinant organisms is the requirement of a selection pressure of antibiotic to maintain plasmid stability and in most cases, need for a costly inducer such as IPTG. This directly affects the economics and results in a compromised process. Our preliminary experiments suggest that the replacement of IPTG with lactose (0.8%) resulted in comparable growth and nitrilase activity (Table 1). Further studies in the bioreactor level were therefore carried out using lactose as an inducer instead of IPTG. Effect pH
Fig. 2 Purification of recombinant nitrilase by Ni-NTA agarose chromatography (lane 1, Mol. wt. marker; lane 2, CFE; lane 3, flow through from Ni-NTA column; lane 4, Ni-NTA fraction)
Due to the C-terminal (His)6 affinity tag, the recombinant nitrilase could be easily purified using the Ni-NTA purification system. The protein was purified to homogeneity by a factor of 3.21% with a yield of 83.49% from the CFE of E. coli using mandelonitrile as substrate. This resulted in enzyme preparation that converted mandelonitrile with a specific activity of 1.26 U/mg. The activity of the purified
The effect of initial pH on the growth and nitrilase production by E. coli was examined by carrying out the fermentation at different initial pH (6.5–7.5) in the bioreactor (Table 2). pH of the medium was adjusted initially to the desired value after which it was monitored but not controlled. Nitrilase activity as well as growth was found to be highest when the initial pH of the medium was set at pH 7.5. The effect of controlled pH (6, 8, and 9) on the growth and nitrilase production was also observed by recombinant E. coli cells in a stirred tank reactor (Table 2). The pH of the medium was maintained by the addition of 1 N H2SO4 and 1 N NaOH as and when required. Choice of the pH controlled study was based on the effects observed during the cultivation at different initial pH condition. Maximum specific activity and growth was obtained when the pH was controlled at 7.5. Controlling of pH only resulted in very slight increase in specific growth rate (l) and no change in specific product formation rate (qP). However, the yield coefficient for enzyme production as a function of cell mass (YP/X) resulted in a slight enhancement under controlled pH condition. Effect of Agitation Proper growth of a microorganism in a bioreactor requires efficient mixing; mass and heat transfer and these have a tremendous effect on the physiology and metabolic activity of the microorganism. These also affect cell morphology,
Mol Biotechnol Table 1 Effect of inducer replacement on the growth and enzyme production by E. coli in a stirred tank reactor Inducer
Cell mass (g/l)
l (h-1)
qp (h-1)
Y(P/X)
Enzyme activity (U/mg)
Time for maximum nitrilase expression (h)
Lactose
3.6
0.31
0.78
2.45
22.8
5
IPTG
2.59
0.34
0.77
2.24
21.7
5
Table 2 Effect of initial and controlled pH on the growth and enzyme production by E. coli in a stirred tank reactor
Initial pH
Controlled pH
pH
Cell mass (g/l)
l (h-1)
qp (h-1)
Y(P/X)
Enzyme activity (U/mg)
Time for maximum nitrilase expression (h)
6.5
4.9
0.10
1.32
12.78
15.39
5
7.5
7.19
0.11
1.41
13.22
18.92
7
8.5
6.34
0.094
1.21
21.88
15.14
7
6.5
6.31
0.11
1.32
11.8
16.03
7
7.5
7.3
0.18
1.4
18.05
7
8.5
6.54
0.12
1.3
16.27
7
7.66 10.0
Fig. 3 Effect of agitation on the (a) specific activity, (b) total reducing sugar concentration, (c) growth, and (d) DO during the cultivation of E. coli in a 2.5 l reactor (d, 200 rpm; m, 300 rpm; and j, 400 rpm)
growth, and product formation kinetics. Often the shear forces generated in the vessel are enough to cause cell rupture and may also lead to excessive foaming. Hence effect of mixing (200, 300, and 400 rpm) on the production of nitrilase by E. coli was studied (Fig. 3). Maximum enzyme activity was observed at 200 rpm, while cell mass generated at 200 or 300 rpm were similar, but lower to that obtained at 400 rpm. The reason for the less cell mass growth may be attributed to the depletion of dissolved oxygen experienced by the organisms due to insufficient mixing (low agitation rate).
As the agitation was increased, the organism showed faster growth rate and required less time for maximal nitrilase expression. However, specific activity showed a reverse trend, that is, the activity decreased as the agitation was increased. Higher amount of cell disruption was also visualized at higher agitation rates (observed by optical microscope). Thus, it could be concluded that although the organism grew faster and better at the higher agitation, the enzyme production is adversely affected at this agitation, probably due to the cell disintegration as a result of higher stress.
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Effect of Aeration Aeration is regarded as one of the essential tools for maintaining metabolic activities of aerobic organisms and also proper mixing of the bioreactor contents. The most important step in oxygen transfer is its path from air bubbles, through the liquid medium, to the microbial cells. The effect of increasing aeration rate was checked by sparging the bioreactor at different aeration rates (0.5, 1, and 1.5 vvm) (Fig. 4). Growth and enzyme production showed a relative reverse relationship with aeration rate. As the aeration rate was increased, the cell mass increased in quantity (growth) but decreased in quality (enzyme production). Increasing the aeration rate brought a decline in the specific activity of the enzyme with practically no activity being obtained at higher aeration rate (1.5 vvm). This may be due to the oxidation of the sulfhydryl residue near the active site of nitrilase involved in nitrile hydrolysis. Hence, a low aeration rate can be said to be optimal for obtaining higher enzyme activity. However, the cell mass was found to increase at a faster rate with higher aeration rate. Production of (R)-(-)-mandelic Acid Using Recombinant Nitrilase Recombinant E. coli cells with superior nitrilase activity were subsequently used for the enantioselective hydrolysis of racemic mandelonitrile to (R)-(-)-mandelic acid. The reactions were carried out for 6 h at 35°C with 50 mM
Fig. 4 Effect of aeration on the (a) specific activity, (b) total reducing sugar concentration, (c) growth and, and (d) DO during the cultivation of E. coli in a 2.5 l reactor (d, 0.50 vvm; m, 1 vvm; and j, 1.5 vvm)
Fig. 5 Enantioselective hydrolysis of mandelonitrile by genetically engineered E. coli cells (Mandelonitrile hydrolysis was carried out using recombinant E. coli cells. The reaction conditions were as follows: temperature, 35°C; agitation, 200 rpm; reaction volume, 100 ml; cell mass, 400 mg; substrate concentration, 50 mM. The reaction was carried out for 6 h in phosphate buffer (0.1 M, pH 7.5) and after harvesting the biocatalyst, the products were extracted using the standard protocols. The amount of mandelic acid formed and the residual mandelonitrile were determined by RP-HPLC. The ee (%) of the mandelic acid formed was determined by chiral HPLC)
mandelonitrile (Fig. 5). Due to the auto-degradation of mandelonitrile to benzaldehyde and hydrogen cyanide, the concentration of mandelonitrile in the reaction mixture decreased rapidly with the concomitant formation of ammonia and mandelic acid. Finally, after a single batch reaction, (R)-(-)-mandelic acid was obtained with 87% yield and 99.99% enantiomeric excess (ee). The volumetric
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also prove handful to circumvent these problems. Studies are in progress in our laboratory in these directions.
productivity (g mandelic acid/l/h) and the catalyst productivity (g mandelic acid/g cell mass) were found to be 1.14 and 1.36, respectively. These values were significantly higher than those obtained employing non-engineered P. putida (volumetric productivity was 0.76 g mandelic acid/l/h and catalyst productivity was 0.90 g mandelic acid/g cell mass).
Acknowledgments Anirban Banerjee gratefully acknowledges financial assistance provided by CSIR Govt. of India, and DAAD Fellowship, Sachin Dubey and Praveen Kaul gratefully acknowledge financial assistance provided by DBT and CSIR, Govt. of India. This is NIPER communication number 413.
Discussion
References
P. putida nitrilase is an arylacetonitrilase having substrate specificity toward arylacetonitriles. Rhodococcus rhodochrous K22 nitrilase is an aliphatic nitrilase having substrate specificity for aliphatic nitriles. Nagasawa et al. [14] purified an arylacetonitrilase from Alcaligenes faecalis JM3. The P. putida nitrilase gene sequence was found to be highly homologous with the sequence from A. faecalis. Though the amino acid sequence of the A. faecalis JM3 and P. putida nitrilases are very similar ([95%), the kinetic constants of the two enzymes are not identical (Km values of 0.010 and 3.61 mM for A. faecalis JM3 and P. putida with phenylacetonitrile as substrate, respectively), revealing probably a different pattern of folding of the two proteins in different environment, exposing different amino acid residues actively involved in catalysis. The nitrilase gene of P. putida was cloned between Nde I and Xho I sites in pET 21b (?) vector under control of T7 promoter. This T7 promoter was in-turn under the control of a lac promoter in BL21 (DE3) cells of E. coli and therefore the nitrilase production was inducible by IPTG. The overall process economics was severely affected because of the high cost of IPTG. We could successfully replace IPTG by readily available lactose as inducer for enzyme production and, therefore, the cost of the overall process was lowered by several folds. No significant differences in the kinetic constants were observed between the wild-type and the His-tagged variant of the nitrilase, implying that the catalytic properties of the protein were not changed because of the attachment of histidine residues. Bioreactor studies allowed optimization of different physicochemical parameters for nitrilase production in a stirred tank reactor. Finally, the genetically modified E. coli cells were utilized for the production of (R)-(-)-mandelic acid with higher yield and ee. Diffusion of the hydrophobic substrate and product into and out of the cells offers certain challenges because of the presence of cell membrane as barrier and lowering this diffusional resistance would certainly increase the overall product yield. Engineering the reaction medium may prove one alternative that would reduce the diffusional limitations. Preparations of cross-linked enzyme crystals or cross-linked enzyme aggretates may
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