Purification and Characterization of Limonoate Dehydrogenase from ...

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limonin formation or to eliminate this limonoid from citrus juices (3, 6, 12, 20). Limonoate dehydrogenase (LDase), an enzyme detected in different species of ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1997, p. 3385–3389 0099-2240/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 63, No. 9

Purification and Characterization of Limonoate Dehydrogenase from Rhodococcus fascians ´ PEZ-RUIZ, MARI´A T. MERINO, JOSE ´ MANUEL ROLDA ´ N, LOURDES HUMANES, ANTONIO LO ´ S DIEZ* AND JESU Departamento de Bioquı´mica y Biologı´a Molecular, Centro de Experimentacio ´n Biolo ´gica, Facultad de Veterinaria, Universidad de Co ´rdoba, 14071 Co ´rdoba, Spain Received 11 April 1997/Accepted 1 July 1997

Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD1. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect. electrophoretic homogeneity and the sequencing of LDase as the first step in the process. The enzyme has also been characterized, particularly with respect to substrate specificity and overexpression by limonoate.

Bitterness in citrus juices is a problem of great economic importance (7). Citrus tissues possess limonoate A-ring lactone (LARL), a nonbitter limonoid and a natural precursor of limonin, which is the compound responsible for bitter taste (18, 19). After juice extraction, acidic pH conditions facilitate the conversion of LARL to limonin, a process known as delayed bitterness (6). Different approaches have been used to avoid limonin formation or to eliminate this limonoid from citrus juices (3, 6, 12, 20). Limonoate dehydrogenase (LDase), an enzyme detected in different species of microorganisms, can prevent limonin production by catalyzing the oxidation of LARL to the corresponding 17-dehydrolimonoate form, a nonbitter derivative which cannot be converted into limonin (Fig. 1). The enzyme has been isolated from Arthrobacter globiformis (2), Pseudomonas sp. strain 321-18 (8), and Rhodococcus fascians (5). In these studies a partial purification and characterization of LDase was described. However, the optimal pH of LDase (8.0 to 9.0) hampered the use of the isolated enzyme in debittering processes due to the acidic pH of the juices. Consequently, complete microorganisms were immobilized and used in debittering attempts (9, 10, 12, 23). Recently, the use of genetic engineering techniques has been proposed to solve the problem of bitterness. Thus, LDase from A. globiformis has been purified and its amino terminal sequence has been determined with the aim of isolating and inserting the LDase gene in citrus fruits (22). The aim of our research is to sequence LDase from R. fascians in order to clone and overexpress the protein in GRAS (“generally recognized as safe”) organisms for their use in debittering reactors. In addition, the enzyme could eventually be used to develop biosensors for the determination of limonin levels in juices. In this paper, we report the purification to

MATERIALS AND METHODS Organism and growth conditions. R. fascians NRRL-B-15096, from the Agricultural Research Service culture collection, was grown in liquid cultures in the medium described by Martı´nez-Madrid et al. (20). The medium was adjusted to pH 7.0 with 3 M NaOH and inoculated with 1% of a 48- to 72-h-old culture. Incubation was carried out at 25°C on an orbital shaker (120 oscillations/min). For culture assays, fructose was replaced by other carbon sources at 0.4% (wt/vol). Growth was monitored by measuring the increase in absorbance of the culture at 600 nm. After 72 h, cells were collected by centrifugation at 15,000 3 g for 10 min, washed with 0.1 M phosphate buffer at pH 7.0, and stored frozen at 220°C until used. Preparation of crude extracts. Cells were broken in a vibration homogenizer (Vibrogen Vi 4; Edmund Bu ¨hler, Tu ¨bingen, Germany) with glass beads (0.1 mm in diameter), and the broken material was extracted with 50 mM phosphate buffer, pH 7.0, containing 2 mM dithioerythritol, 1 mM EDTA, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 5 mM ε-NH2 caproic acid, and 30% glycerol (10 ml of buffer/g of wet cells). The homogenate was filtered through a porous disk filter (G1). To remove nucleic acids a solution of 100 mM streptomycin sulfate, pH 7.0, was added to the filtrate obtained previously (1 ml of streptomycin sulfate/10 ml of filtrate). After 15 min at 4°C with gentle stirring, the nucleic acids were precipitated by centrifugation at 70,000 3 g for 50 min. The resulting supernatant was used as the starting material for purification of the enzyme. Purification procedure. LDase was purified by a procedure consisting of the following steps, performed at 4°C except as otherwise indicated. (i) DEAE-Sepharose fast flow chromatography. The crude extract was passed through a DEAE-Sepharose column (2.6 by 18 cm) equilibrated with 50 mM phosphate buffer, pH 7.0, containing 1 mM EDTA, 2 mM dithioerythritol, and 30% glycerol (standard buffer). The chromatography was run at a flow rate of 330 ml/h. The column was washed with 0.05 M NaCl in standard buffer until absorbance at 280 nm decreased to 0. LDase was eluted with a linear gradient from 0.05 to 0.30 M NaCl. The fractions containing high LDase activity were brought to 1.0 M ammonium sulfate. (ii) Phenyl-Sepharose high-performance (HP) chromatography. The pool of fractions from the previous step was applied to a phenyl-Sepharose HP column (1.6 by 5 cm) equilibrated with standard buffer containing 1 M ammonium sulfate. The flow rate was 115 ml/h, and the column was washed with the same buffer until the absorbance at 280 nm decreased to 0. The enzyme activity was eluted with a step gradient by using standard buffer containing 0.5 and 0.1 M ammonium sulfate.

* Corresponding author. Mailing address: Departamento de Bioquı´mica y Biologı´a Molecular, Centro de Experimentacio ´n Biologı´a, Facultad de Veterinaria, Universidad de Co ´rdoba, Avda. Medina Azahara s/n, 14071 Co ´rdoba, Spain. Phone: 34 57 21 10 75. Fax: 34 57 21 86 88. E-mail: [email protected]. 3385

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FIG. 1. Production of 17-dehydrolimonoate A-ring lactone by LDase.

(iii) Cibacron blue 3GA agarose chromatography. LDase-containing fractions from the previous step were mixed, dialyzed for 4 h against standard buffer, and applied to a Cibacron blue column (1.6 by 10.5 cm) equilibrated with standard buffer and run at a flow rate of 60 ml/h. The activity was eluted after applying 30 ml of standard buffer containing 10 mM NAD1 followed by 1 M NaCl in standard buffer. (iv) Mono Q HR 5/5 chromatography. The pool from the Cibacron blue column was desalted with a PD-10 (Sephadex G-25) gel filtration column (Pharmacia). Then two anion-exchange chromatography procedures were performed on a Mono Q HR 5/5 column at room temperature with a fast-performance liquid chromatography (FPLC) system. In the first step the column was loaded with the enzyme in standard buffer at a flow rate of 1 ml/min, washed with the same buffer supplemented with 0.1 M NaCl until the absorbance at 280 nm became 0, and then eluted with a linear gradient from 0.1 to 0.3 M NaCl. The pool of high-LDase-activity fractions was diluted to achieve the electrical conductivity of the standard buffer and applied again to the Mono Q column under the same conditions. The column was washed with standard buffer for 15 min and then with the same buffer containing 0.15 M NaCl for 5 min. Enzyme activity was eluted with a linear gradient from 0.15 to 0.3 M NaCl. Enzyme assay and protein determination. LDase activity was measured spectrophotometrically at 25°C by monitoring the production of NADH at 340 nm. Activity was assayed in 1 ml of reaction mixture containing 100 mM Tris-HCl (pH 8.5), 0.5 mM NAD1, 4 mM disodium limonoate, and an aliquot of the enzyme (5). One unit of activity was the amount of enzyme that catalyzed the formation of 1 mmol of product per minute. The protein content of soluble fractions was determined according to the method of Lowry et al. (16), with bovine serum albumin as the standard. Limonoate and LARL production. Limonin was obtained from Citrus seeds as described by Barton et al. (1). Disodium limonoate was prepared by adding 48 ml of 0.1 M NaOH to 1 g of limonin; the mixture was then refluxed for 20 min, until most of the limonin was transformed into limonoate, and neutralized to pH 7.5 with 2 N HCl (17). For production of LARL, purified limonoate D-ring lactone (LDRL) hydrolase immobilized on a Q-Sepharose column (6.5 by 1.6 cm) was used (21). The column was equilibrated with 100 mM Tris-HCl, pH 8.0. Then, a solution of 100 mM triethanolamine, pH 8.0, containing 30% acetonitrile and 0.48 mM limonin, was passed through at a flow rate of 0.1 ml/min. Once the lactone was produced, acetonitrile was evaporated by vacuum concentration of the resulting solution. PAGE and molecular weight determinations. Native electrophoresis was performed in slab gels containing 10% acrylamide for the running gel and 5% for the stacking gel. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was carried out in gels containing 12% acrylamide for the running gel and 5% for the stacking gel (13). For molecular weight determinations of enzyme subunits by SDS-PAGE, the

following molecular weight standards were used: ovotransferrin (Mr, 78,000), bovine serum albumin (Mr, 66,250), ovalbumin (Mr, 45,000), carbonic anhydrase (Mr, 30,000), myoglobin (Mr, 16,949), and cytochrome c (Mr, 12,400). LDase was detected in native electrophoresis gels by the fluorescence of NADH. Following electrophoresis, the gels were incubated in 20 ml of a modified assay mixture containing 375 mM Tris-HCl (pH 8.8), 0.5 mM NAD1, and 2 mM disodium limonoate. Then, the gels were placed on a UV light box. After a few minutes a band of yellow fluorescence was observed. The gels were later stained for protein detection with Coomassie blue. The molecular weight of the native enzyme was determined by gel filtration chromatography at room temperature on a Superdex 200 column by using an FPLC system. The column was equilibrated and eluted with standard buffer containing 0.2 M NaCl at a flow rate of 0.4 ml/min. Tyroglobulin (Mr, 669,000), ferritin (Mr, 440,000), aldolase (Mr, 161,000), isocitrate dehydrogenase (Mr, 61,000), cytochrome c (Mr, 12,400), and vitamin B12 (Mr, 1,355) were used as standards and subjected to chromatography under the same conditions. Immunochemical techniques. Anti-LDase monospecific antibodies were raised in rabbits and characterized according to Lo ´pez-Ruiz et al. (15). For immunoblot analysis, proteins were first separated by SDS gel electrophoresis and transferred to polyvinylidene membranes as previously described (11). Determination of amino acid sequence. The NH2-terminal amino acid sequence of the protein was determined in samples of purified enzyme after running them in an SDS–15% polyacrylamide gel. The protein was transferred to Immobilon membranes and stained (14). The band, corresponding to LDase, was excised, and the sequence was determined in a Beckman LF 300 sequencer equipped with a PTH amino acid analyzer (System Gold; Beckman).

RESULTS R. fascians cells grown on fructose (4 g/liter) showed a basal level of activity (specific activity, 5.5 mU/mg), while LDase activity was five times higher (28.0 mU/mg) in limonoate (1 g/liter)-containing cultures. Cells previously grown on lowfructose medium (1g/liter) which was supplemented 72 h later with 0.1 g of limonoate/liter also showed a marked increase in enzyme activity levels, to 18.0 mU/mg, similar to those previously reported by Hasegawa and King (5) with cells grown on medium containing 4 g of limonoate/liter. On the other hand, when cells were transferred to fresh fructose medium (4 g/liter) after the limonoate was exhausted, LDase activity decreased to the basal level (5.0 mU/mg). Purification of LDase from R. fascians was a complex and laborious process, probably due to the low intracellular levels of the enzyme and its poor stability, particularly when protein concentration decreases during the purification process. The enzyme lability problem was partially solved by using glycerol in buffers throughout the purification procedure, which consisted of the steps summarized in Table 1. It is worth mentioning that the two applications of Mono Q FPLC were highly resolving (Fig. 2) and produced marked increases in specific activity. Electrophoresis of purified enzyme samples under nondenaturing conditions showed a single band of protein that corresponded to the band due to NADH fluorescence in the specific in situ assay for LDase activity (Fig. 3A). SDS-PAGE of purified enzyme also yielded a single band with an approximate molecular weight of 30,000 (Fig. 3B). Since the native enzyme appears to exhibit a molecular weight of around 128,000, according to the results obtained by size exclusion Superdex 200

TABLE 1. Purification of LDase from R. fasciansa Chromatographic step

Protein (mg)

Activity (U)

Sp act (U/mg)

Yield (%)

Purification (fold)

None (crude extract) DEAE-Sepharose Phenyl-Sepharose Cibacron blue 1st Mono Q 2nd Mono Q

1,872 179.82 39.05 7.65 0.31 0.07

18.80 14.34 10.33 6.03 4.05 3.25

0.01 0.08 0.26 0.79 13.06 46.42

100 76 55 32 22 17

1 8 26 79 1,301 4,624

a

Experimental details are given in Materials and Methods.

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FIG. 2. Elution profile of LDase from R. fascians by Mono Q HR 5/5 FPLC. Fractions with high LDase activity eluted from the Cibacron blue 3GA agarose column were mixed and applied to the Mono Q column. Other experimental details are described in Materials and Methods. ---, LDase activity; O, absorbance at 280 nm.

FPLC (data not shown), LDase from R. fascians seems to be composed of four similar or identical subunits. On the other hand, the purified enzyme was subjected to isoelectric focusing, yielding a pI value of 4.9. We studied the possible differences in the properties of LDase obtained from fructose- versus limonoate-grown cells. Partially purified enzymes from both sources showed similar optimum pHs, around 9.0. Similarly, heat stability studies showed that both preparations were completely inactivated after 10 min of incubation at 60°C, but they were not affected at 50°C even after 2 h of incubation. Although significant activity stimulation by Zn ions on enzymes from Arthrobacter and Pseudomonas species (2, 8) has been reported, no effect of this cation has been observed in our case. Mn and Mg partially inactivated LDase from R. fascians, while the enzyme was completely inactivated by Cu ions (not shown). Comparison of Km values for purified enzymes from fructose- and limonoate-grown cells is presented in Table 2. The affinity for NAD1 of LDases from both sources was quite

similar. In contrast, the enzyme from limonoate extracts exhibited a greater affinity for disodium limonoate, as is indicated by the lower Km value. Moreover, LDase obtained from fructose and limonoate cultures had Km values for LARL which were eight and five times lower than for disodium limonoate, respectively (Table 2). In addition, affinity for LARL was slightly higher in the case of LDase purified from limonoate-grown cells. On the other hand, the catalytic efficiency of LDase was clearly higher for LARL, which has been described as the physiological substrate for the enzyme (7), than for disodium limonoate. The ratio of Vmax to Km, obtained from the data shown in Table 2 and Fig. 4, was around 30 times higher for LARL. Homogeneous LDase samples were used to determine the NH2-terminal amino acid sequence by automatic Edman degradation. The sequence of the 20 analyzed residues was G-LG-P-Y-D-R-L-K-G-E-V-A-I-V-V-G-A-G-T. Immunoblotting experiments were performed in order to determine if the increase in enzyme activity found in limonoate-grown cells was due to new synthesis of the enzyme or to activation of preexisting LDase. However, no clear conclusions were obtained so a different approach was used. The addition of chloramphenicol to cell cultures before adding limonoate prevented such an increase in LDase activity (Fig. 5). On the other hand, in vitro incubation with limonoate of LDase obtained from fructose-grown cells did not promote any significant enzyme activation.

TABLE 2. Km values for LDase from cells grown in fructose or limonoatea Substrate 1

NAD Limonoate LARL FIG. 3. Gel electrophoresis of LDase from R. fascians. (A) Native gel electrophoresis of homogeneous LDase. Lanes: 1, gel stained for protein detection (2 mg); 2, in situ activity assay. (B) SDS-PAGE of homogeneous LDase (1.2 mg).

a

Km (mM)b for LDase from: Fructose-grown cells

Limonoate-grown cells

0.17 6 0.012 0.78 6 0.086 0.10 6 0.011

0.12 6 0.012 0.27 6 0.035 0.05 6 0.008

LDase purified as described in Materials and Methods. Values are means 6 standard errors for triplicate measurements from four separate experiments. b

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raphy (2, 5, 8). Recently, however, LDase from A. globiformis has been purified to homogeneity by using affinity and ionexchange chromatography. The purification protocol presented here for R. fascians is more complex, due probably to the extremely low intracellular levels and the instability of LDase from Rhodococcus species. The molecular weights of both native enzyme and subunits of LDase from R. fascians appear to be similar to those previously reported for the enzyme of A. globiformis (22), with values around 120,000 and 30,000, respectively. A comparison of NH2-terminal sequences of R. fascians and A. globiformis (22) reveals a significant degree of homology (indicated below by boldface type) for LDase from both organisms, with complete coincidence in a series of eight amino acids: 1

GLGPYDRLKGEVAIVVGAGT (R. fascians) 1 MPFNRLENEVAIVVGA (A. globiformis)

FIG. 4. LDase activity with disodium limonoate or LARL as substrates. Purified enzyme samples from limonoate-grown cells were assayed in the presence of the specified concentrations of disodium limonoate (■) or LARL (F).

DISCUSSION LDase from Arthrobacter (2, 22), Pseudomonas (8), and Acinetobacter (23) species is inducible by limonoate, whereas the enzyme from R. fascians is constitutively expressed. However, overexpression of LDase has been obtained with R. fascians cells grown in disodium limonoate-containing media (5). Since this limonoid is not commercially available, it has to be obtained from pure limonin, a costly chemical. Consequently, we have tried to optimize the induction of the enzyme by limonoate in this species. Under our experimental conditions, R. fascians cells showed levels of LDase activity similar to those previously reported (5) but with a concentration of limonoate 40 times lower in the growth media. LDase from Arthrobacter, Pseudomonas, and R. fascians has been partially purified by using a procedure which includes ammonium sulfate precipitation and ion-exchange chromatog-

FIG. 5. Effect of chloramphenicol on LDase induction. R. fascians cells growing in low concentration of fructose (1g/l) for 72 h were collected, divided into four aliquots, and transferred to standard growth medium containing 4 g of fructose/liter (■), 0.1 g of limonoate/liter (F), 4 g of fructose/liter plus 25 mg of chloramphenicol/ml (h), or 0.1 g of limonoate/liter plus 25 mg of chloramphenicol/ml (E). The antibiotic was added to culture aliquots 45 min before the addition of the carbon source. At the indicated times, crude extracts were obtained and LDase activity was assayed as described in Materials and Methods.

Previous studies on LDase substrate specificity and the electron acceptor of the enzymatic reaction indicate that both NAD1 and NADP1 can act as cofactors of the enzyme from Pseudomonas but not from Arthrobacter and Rhodococcus, for which NAD1 is the specific electron acceptor (2, 5, 8). Although LARL has been proposed as the physiological electron donor for LDase (7), disodium limonoate is the limonoid generally used for in vitro assays (2, 4, 5, 8). Chemical lactonization of disodium limonoate at acidic pH, or limonin hydrolysis under alkaline conditions, primarily produces a mixture of both lactones, A and D, of limonoate. However, LARL is much more unstable than LDRL and, consequently, previous efforts to obtain homogeneous LARL solutions were unsuccessful (19). To circumvent that problem we have used immobilized LDRL hydrolase. This approach has allowed us to obtain practically homogeneous LARL solutions, with limonin contaminations lower than 2% (21). In any case, the presence of limonin does not affect the LDase assay, since the enzyme is inactive towards that limonoid due to its closed D ring (2). Comparison of enzyme activity on disodium limonoate (both A and D rings open) and other related limonoids also indicated the need for an epoxide group and a furane ring in the molecule in order for it to be enzymatically attacked. On the other hand, removal of carboxy or hydroxyl groups in the open A ring promoted a slight increase in enzyme activity (2). In our case, the catalytic efficiency of the R. fascians enzyme greatly increased when the A ring was closed in the substrate. This result suggests that the A ring could participate in enzyme-substrate binding, probably through a nonpolar area of the protein. Finally, the proposal of Hasegawa and Maier (7) that LARL could be the physiological substrate of LDase is further corroborated. Cells grown in limonoate possess LDase with higher affinity for LARL and disodium limonoate than the enzyme obtained from fructose cultures. It is difficult to explain this difference in affinity, particularly considering that LDase from both sources showed identical molecular weight, heat stability, and optimum pH. Since in vitro incubation of the enzyme with limonoate did not activate LDase, the higher activity detected in limonoategrown cells (see Results) may be the consequence of de novo enzyme synthesis, which is corroborated by the prevention of such an increase of LDase activity in the presence of chloramphenicol (Fig. 5). Consequently, we suggest that the higher levels of the enzyme in limonoate-grown cells are due to synthesis of new LDase protein. We are currently obtaining the carboxy-terminal sequence of LDase. This information, together with that described in this

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paper for the amino end, would allow the use of PCR for amplification of the LDase gene. Molecular engineering could then be further used to clone the LDase gene and overexpress that protein in GRAS organisms.

10.

11.

ACKNOWLEDGMENTS This work was supported by CICYT (Spain) grant Bio 94-0378 and Junta de Andalucia grant 1240. L. Humanes and M. T. Merino were recipients of fellowships from the Universidad de Co ´rdoba and OTRI, Spain, respectively. We are indebted to A. Padilla, J. F. Martı´nez, and G. Dorado for helpful discussions.

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