PROTEIN

EXPRESSION

AND

PURIFICATION

2, So-93 (19%)

Overproduction of G lutamate Racemase of Pediococcus pentosaceus in Escherichia co/i Clone Cells and Its Purification Soo-Young Choi, Nobuyoshi Esaki, Tohru Yoshimura, and Kenji Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan

Soda1

Received March 12,199l

W e previously isolated a 6.0-kb DNA fragment that specifies glutamate racemase activity from the chromosomal DNA of Pediococcus pentosaceus by digestion with HindI (N. Nakajima, K. Tanizawa, H. Tanaka, and K. Soda, 1986), A&c. Biol. Chem. 50, 28232830). W e digested it further with EcoRI to obtain a fragment of 1.8 kb, which was blunt-ended and ligated into the SmaI site of vector plasmid pKK223-3. The recombinant plasmid showed a high glutamate racemase activity upon transformation of Escheriehia coli W 3 1 1 0 cells with it; the plasmid was named pICR223. Glutamate racemase was overproduced in the clone cells and occurred in inclusion bodies in the cells. The by dialenzyme was solubilized with 6 M urea, renatured ysis to remove urea, and purified to homogeneity with an overall yield of about 7 0 % after a single DEAE-cellulose column chromatography. The amount of enzyme produced by the clone cells corresponded to about 3 8 % 0 1991 Academic Press, inc. of the total insoluble protein.

Glutamate racemase (EC 5.1.1.3) catalyzes racemization of L- and D-glutamate and occurs in lactic acid bacteria. A product, D-glutamate, is an important component of the bacterial peptidoglycan (l-5). The enzyme was purified to homogeneity from Pediococcus pentosaceuS and shown to be independent of cofactors including pyridoxal5’-phosphate, which is usually required as the coenzyme for various amino acid racemases (6). The enzymatic mechanism by which a-amino acids such as glutamate are racemized with neither pyridoxal5’-phosphate nor any other cofactor has not been elucidated (7-9). The study has been hampered mainly by difficulties in preparing the homogeneous enzyme. W e have

1 To whom correspondence 90

should be addressed.

cloned the glutamate racemase gene from P. pentosaceuS into Escherichia coli and increased the bacterial productivity of the enzyme (10). However, it is not easy to purify the enzyme to homogeneity even from the clone cells. W e have subcloned the enzyme gene and then ligated it downstream of the tat promoter in expression vector pKK223-3; the enzyme was overexpressed in the clone cells. W e describe here the construction of the vector and a simple procedure for purification of the enzyme. MATERIALS

AND

METHODS

Materials Expression vector pKK223-3 was obtained from Pharmacia; restriction enzymes, pUC19, and pUC119 were from Takara Shuzo, Kyoto, Japan; dimethyl adipimidate. 2HC1, triethanolamine HCl, urea, and DEAEcellulose were from Nacalai Tesque, Kyoto, Japan. All other reagents were of analytical grade. Construction of Recombinant Plusmid pICR223 Carrying the Glutamate Racemase Gene W e previously cloned the DNA fragment (6.0 kb) that specifies glutamate racemase activity from the chromosomal DNA of P. pentosaceus (10). W e digested the recombinant plasmid with EcoRI and HindIII, isolated the insert DNA of 1.8 kb by agarose gel electrophoresis, and made both ends blunt. The DNA fragment was ligated into the SmaI site of pKK223-3 and was named pICR223. Cell Cultivation E. coli W3110 transformed with pICR223 was grown in a medium containing 16 g of Bacto tryptone, 10 g of yeast extract, and 10 g of NaCl per liter at 37°C with 1046~5928/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

PURIFICATION

OF GLUTAMATE

shaking. Large-scale cultivation was done in a New Brunswick Scientific fermenter (MF128S, vessel volume 28 liters) with 20 liters of the above medium at 37°C. Isopropyl-fi-D-thiogalactopyranoside (IPTG) was added to the medium at a final concentration of 0.1 mM 4 h after inoculation. After 5 h, the cells were harvested and then kept frozen at -80°C until use.

Enzyme

and Protein

Assay

Glutamate racemase activity was determined with Lglutamate dehydrogenase. The standard assay mixture contained 10 pmol of D-glutamate, 100 prnol of TrisHCl, 5 pmol of NAD+, and 5 unit of L-glutamate dehydrogenase from bovine liver (Sigma) in a total volume of 1 ml. The reaction was started by addition of glutamate racemase after preincubation at 37°C for 1 min and was followed by measurement of absorbance at 340 nm. One unit of enzyme was defined as the amount of enzyme that catalyzes the formation of 1 pmol of L-glutamate. The specific activity of the enzyme was expressed as units per milligram of protein. Protein was determined by the method of Bradford (11) with bovine serum albumin as a standard. Protein elution patterns were followed by measurement of absorbance at 280 nm.

Purification

of Glutamate

Racemase

The enzyme was purified from insoluble pellets of E. coli W3110 harboring pICR223. All procedures were done at 05°C unless otherwise stated. Twenty grams of frozen cells Step 1: Solubilization. was thawed in 100 ml of the standard buffer consisting of 50 mM Tris-HCl (pH 7.5), 1mM DL-glutamate, 10% glycerol, 0.1% 2-mercaptoethanol, and 0.1 mM phenylmethanesulfonyl fluoride and disrupted by sonic oscillation for 20 min. The cell debris and inclusion bodies were collected by centrifugation at 12,000g for 1 h and then washed twice with the standard buffer supplemented with 0.25% Triton X-100 and 10 mM EDTA. The precipitate containing the inclusion bodies was solubilized with 100 ml of the standard buffer supplemented with 6 M urea. After 12 h, the solution was centrifuged at 12,000g for 1 h. The supernatant solution was dialyzed against 2500 vol of the standard buffer for 12 h. Step 2: DEAE-cellulose column chromatography. The enzyme solution,was applied to a DEAE-cellulose column (3 x 60 cm) equilibrated with the standard buffer. The column was washed with 300 ml of the buffer. A linear gradient elution was performed with the buffer supplemented with NaCl by increasing the concentration from 0 to 0.3 M. The flow rate was kept at about 50 ml/h with a peristaltic pump. The active fractions were collected and concentrated by ultrafiltration with an Amicon PM-10 membrane.

91

RACEMASE

Determination

of Molecular

Weight

The molecular weight of the enzyme was measured by gel filtration. The enzyme solution (0.05 ml; 1 mg/ml) was applied to a YMC-pack Diol high-performance liquid chromatography column (5 X 80 cm) (Yamamura Chemical Laboratory, Kyoto, Japan): mobile phase, 100 mM potassium phosphate buffer (pH 7.0) containing 0.2 M NaCl; flow rate, 0.5 ml/min; detection, absorbance at 280 nm. Molecular weight markers were /3-galactosidase (M,, 116,000), albumin (66,000), carbonic anhydrase (29,000), and aprotinin (6500). The molecular weight of the subunit was measured by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The enzyme was cross-linked with a bifunctional crosslinking reagent, dimethyl adipimidate, in 0.2 ml of 0.2 M triethanolamine HCl (pH 8.5) at about 20°C for 3 h (12). The concentrations used were protein, 1 mg/ml; dimethyl adipimidate s 2HC1,12 mg/ml. The enzyme thus treated was subjected to SDS-PAGE without dialysis. RESULTS AND DISCUSSION Expression

of Glutamate

Racemase Gene in E. coli

The glutamate racemase gene of P. pentosaceus was placed downstream of the tat promoter in the recombinant plasmid pICR223. We found that the E. coli clone cells carrying pICR223 took on an abnormally long shape upon addition of IPTG to the medium. We showed the occurrence of inclusion bodies in the longshaped cells by phase-contrast microscopy. The cell extract showed little glutamate racemase activity, but significant activity was found in the cell debris. When the cell pellet was solubilized with 6 M urea followed by dialysis, the enzyme solution showed a specific activity of about 45. These results indicate that the enzyme occurs in inclusion bodies in the clone cells. The specific activity of the enzyme purified to homogeneity is about 121, as shown below. The E. coli W3110 host cells showed no glutamate racemase activity. Therefore, the enzyme gene is overproduced to about 38% of the total insoluble protein of the clone cells that is solubilized with 6 M urea. SDS-PAGE of the cell pellet solubilized with 6 M urea also showed the occurrence of the overexpressed enzyme gene (Fig. 1). The crude extract of P. pentosaceus showed a specific activity of about 0.006 (13), and the enzyme productivity was increased more than 7500-fold by the gene cloning. Moreover, P. pentosaceus cells are obtained only by complicated and tedious cultivation. In contrast, the E. coli clone cells are grown rapidly with a high cell yield. The previous E. coli clone cells carrying pICR221 derived from pBR322 produced glutamate racemase only 14 times more than P. pentosaceus. Thus, the enzyme productivity was extensively increased by means of the tat promoter.

92

CHOI

Purification of Glutamate W31 lo-pIcR223

Molecular

Weight, and Subunit

Structure

The purified enzyme can be stored in the standard buffer at -20°C for several months without loss of activity. The enzyme was found to be stable in the pH range

94,000 67,000 43,000

30,000

20,400

14,400 12

TABLE

Racemase from E. coli

We purified glutamate racemase from about 20 g wet cells of E. coli W3110-pICR223 and obtained about 40 mg of the homogeneous enzyme with an overall yield of 70% (Table 1). The purification was achieved using only a single DEAE-cellulose column chromatography. The specific activity of the enzyme was elevated 2.7fold from that of the cell debris solubilized with urea. It was about 5 times higher than that of the preparation from E. coli C600-pICR221. This is probably due to partial inactivation of the enzyme in the previous preparation: the homogeneous enzyme was obtained only after 130-fold purification with several column chromatography steps. The enzyme did not form inclusion bodies in the previous clone cells, probably due to the low enzyme productivity of the cells. The new purification method described here has several advantages over the previous method. The formation of inclusion bodies in the cells simplified the purification procedure. The urea solubilization and a single column chromatography step replaced the many tedious purification steps used previously. Thus, the time required for purification was reduced from several weeks to a few days. Moreover, the enzyme yield was increased by a simple and rapid purification. Stability,

ET AL.

3

4

FIG. 1. Polyacrylamide gel electrophoresis of glutamate racemase. Lane 1, molecular weight markers; lane 2, the debris of E. coli W3110pKK223-3 solubilized with 6 M urea; lane 3, the debris of E. coli W3110-pICR223 solubilized with 6 M urea; lane 4, the final preparation after DEAE-cellulose column chromatography.

1

Purification of Glutamate Racemase from 20 g of Wet E. coli W3110-pICR223 Cells Coding for the Glutamate Racemase Gene of Pediococcus pentosacew”

Step Urea solubilization DEAE-cellulose a About

Total protein (md

Total activity (units)

Specific activity

Yield (%I

150 40

6,750 4,840

45 121

100 70

20 g of wet cells was used.

between 6.5 and 8.5 when the enzyme solution (0.1 mg/ ml) was incubated in the standard buffer at 25°C for 15 min. Repeated thawing and melting of the enzyme (pH 7.2) caused inactivation of the enzyme. However, the activity was recovered by solubilization of the enzyme with 6 M urea followed by dialysis to remove urea in the presence of dithiothreitol. Thus, the inactivationisprobably due to oxidation of the sulfhydryl group(s) of the enzyme. The molecular weight of the enzyme in the absence of denaturants was estimated to be about 29,000 by gel filtration. SDS-PAGE gave a single band, which showed a molecular weight of about 29,000. These results suggest that the enzyme is composed of a single polypeptide chain. Attempts to detect oligomers crosslinked with dimethyl adipimidate were unsuccessful. This also showed the monomeric structure of the enzyme. Our previous preparation of the enzyme from E. coli C600-pICR221 was also shown to be composed of a single polypeptide chain (10). The nucleotide sequence of the enzyme gene coded by pICR223 indicates that the enzyme is composed of 265 amino acids with a calculated molecular weight of 29,143, as described elsewhere (14). This value is inconsistent with the molecular weight (about 40,000) of the previous preparation of the enzyme purified from E. coli C600-pICR221. The enzyme preparation previously obtained was possibly a fusion protein of glutamate racemase with a polypeptide derived from the inherent pBR322 sequence. The fact that the previous enzyme preparation was far less active than the present preparation suggests that an enzyme expressed in E. coli C600 acquired an extra peptide with a molecular weight of about 11,000, although the reason is unknown. We are planning to determine the covalent structure of the enzyme preparation by protein and DNA sequence determination. Supply of a sufficient amount of the enzyme obtained enables us to study the reaction mechanism of the enzyme by various spectroscopies. REFERENCES 1. Soda, K., and Esaki, N. (1985) Other microbial transaminases, in “Transaminase” (Christen, P., and Metzler, D. E., Eds.), pp. 463469, Wiley, New York.

PURIFICATION

OF GLUTAMATE

2. Ayengar, P., and Roberts, E. (1952) Utilization of D-glutamic acid by Lactobacillus arabinosus: Glutamic acid racemase. J. Biol. Chem. 197,453-460. 3. Narrod, S. A., and Wood, W. A. (1952) Evidence for a glutamic acid racemase in Lactobacillus arabinosus. Arch. Biochem. Biophys. 36,462-463. 4. Glaser, L. (1960) Glutamic acid racemase from Lactobacillus arabinosus. J. Biol. Ch.em. 235, 2095-2098. 5. Diven, W. F. (1969) Studies on the amino acid racemase. II. Purification and properties of the glutamate racemase from Lactobacil1~s fermenti. Biochim. Biophys. Acta 191, 702-706. 6. Adams, E. (1976) Catalytic aspects of enzymatic racemization, in “Advances in Enzymology” (Meister, A., Ed.), Vol. 44, pp. 69138, Academic Press, New York. 7. Rudnick, G., and Abeles, R. H. (1975) Reaction mechanism and structure of the active sites of proline racemase. Biochemistry 14, 4515-4522. 8. Ramaswamy, S. G. (1984) Hydroxyproline 2-epimerase of Pseudomonas. Subunit structure and active site studies. J. Biol. Chem. 259, 249-254.

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93

9. Wiseman, J. S., and Nichols, J. S. (1984) Purification and properties of diaminopimelic acid epimerase from Ercherichia coli. J. Biol. Chem. 259,8907-8914. 10. Nakajima, N., Tanizawa, K., Tanaka, H., and Soda, K. (1986) Cloning and expression in Escherichia coli of the glutamate racemase gene from Pediococcus pentosaceus. Agric. Biol. Chem. 50,

2823-2830. 11. Bradford, M. M. (1976) A rapid and sensitive method for the quantititation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248-254. 12. Davies, G. E., and Stark, G. R. (1970) Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric structure of oligermeric protein. Proc. Natl. Acad. Sci. USA 66,651-656. 13. Nakajima, N., Tanizawa, K., Tanaka, H., and Soda, K. (1988) Distribution of glutamate racemase in lactic acid bacteria and further characterization of the enzyme from Pediococcuspentosaceus. Agric. Biol. Chem. 52, 3099-3104. 14. Choi, S.-Y., Esaki, N., Yoshimura, T., and Soda, K., (1991) Manuscript submitted for publication.

Overproduction of glutamate racemase of Pediococcus pentosaceus in Escherichia coli clone cells and its purification.

We previously isolated a 6.0-kb DNA fragment that specifies glutamate racemase activity from the chromosomal DNA of Pediococcus pentosaceus by digesti...
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