Overproduction ofd -Aminoacylase fromAlcaligenes xylosoxydanssubsp.xylosoxydansA-6 inEscherichia coliand Its Purification

PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.

7, 395–399 (1996)

0059

Overproduction of D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 in Escherichia coli and Its Purification Mamoru Wakayama, Shin-ichi Hayashi, Yukinori Yatsuda, Yutaka Katsuno, Kenji Sakai, and Mitsuaki Moriguchi1 Department of Applied Chemistry, Faculty of Engineering, Oita University, Oita 870-11, Japan

Received October 10, 1995, and in revised form January 21, 1996

We constructed the high-expression plasmid for Daminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. The appropriate Shine–Dalgarno sequence (AAGGAG) was introduced to the eight bases upstream of start codon (ATG) of D-aminoacylase structural gene by site-directed mutagenesis, and then the 1.75-kb DNA fragment including the open reading frame was inserted into the downstream of the tac promoter of plasmid vector pKK223-3. The resultant plasmid, which was named pKNSD2, showed a high D-aminoacylase activity in Escherichia coli JM109 cells transformed with it. The enzyme was purified to homogeneity in only two steps with a final yield of 24% (sp act, 2023 U/mg). q 1996 Academic Press, Inc.

D-Aminoacylase catalyzes the hydrolysis of N-acyl derivatives of various neutral D-amino acids to Damino acids and fatty acids and has been reported in Pseudomonas (1 – 3), Streptomyces (4,5), and Alcaligenes (6 – 10). The authors observed the enzyme in Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) (6) and Alcaligenes denitrificans subsp. xylosoxydans MI-4 (7 – 9). Alcaligenes A-6 additionally produces both N-acyl-D-aspartate (D-Asp) amidohydrolase and N-acyl-D-glutamate (D-Glu) amidohydrolase, which are highly specific for N-acylD-Asp and N-acyl-D-Glu, respectively (11 – 13). These enzymes, which have been found to possess strict substrate specificity, are highly useful for the optical resolution of DL-amino acids, but are being produced

Sequence data for D-aminoacylase have been assigned DDBJ Accession No. D45918. 1 To whom correspondence should be addressed. Fax: (0975) 547890.

only in small amounts in Alcaligenes A-6. To enhance the enzyme production and to elucidate the structure – function relationships of these enzymes, the genes of these enzymes from Alcaligenes A-6 have been cloned in Escherichia coli and their nucleotide sequences were determined (14 – 16). However, the expression level of these enzymes in E. coli was not adequate for obtaining sufficient pure preparation for the optical resolution of DL-amino acids and structural studies such as X-ray crystallography. First, in order to achieve higher expression of the D-aminoacylase gene in E. coli, modification of the gene and subcloning of the modified gene to downstream of the tac promoter in expression vector pKK223-3 have been performed. The enzyme was overexpressed in the cloned cells. The authors describe here the construction of the high-expression plasmid for D-aminoacylase and rapid purification of the enzyme. MATERIALS AND METHODS

Materials Expression vector pKK223-3 was purchased from Pharmacia. pUC118 and the site-directed mutagenesis kit (Mutan-K) were from Takara Shuzo. pUC18, restriction enzymes, and other DNA-modifying enzymes were from Nippon Gene. pET-23d(/), E. coli BL21(DE3), and BL21(DE3)pLysS were from Novagen. Oligonucleotide primers for site-directed mutagenesis were PCR grade from Funakoshi. The Taq DyeDeoxy Terminator Cycle Sequencing kit was from Applied Biosystems, Inc. DEAE–Toyopearl was from Toso. Butyl–Cellulofine was from Seikagakukogyo. All other chemicals were of analytical grade. Bacterial Strain and Cultivation E. coli JM109 was used as the recipient strain for transfomation experiments. E. coli CJ236 and BMH 395

1046-5928/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FIG. 1. Construction of high-expression plasmids for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. B, BamHI; E, EcoRI; H, HindIII.

71-18 mutS were used for site-directed mutagenesis. E. coli BL21(DE3) and BL21(DE3)pLysS were used as the hosts of pET-23d(/). There was no D-aminoacylase activity observed in the E. coli cells used in

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this study. The recombinant cells were grown in a Luria – Bertani medium containing 50 mg/ml of ampicillin with or without isopropyl-b-D-thiogalactopyranoside (0.1 mM).

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Site-Directed Mutagenesis We previously cloned the DNA fragment (5.8 kb) that specifies D-aminoacylase activity from the chromosomal DNA of Alcaligenes A-6 and determined its nucleoteide sequence (14). A BamHI – HindIII fragment (about 4 kb) excised from pAND1, which contains the entire coding region for Alcaligenes A-6 Daminoacylase, was subcloned into plasmid pUC118. Synthesis of mutant DNA was performed by the method of Kunkel et al. (17). The resultant plasmid pAND118 was introduced into E. coli CJ236 (dut0 ung0) cells, and the single-stranded DNA-containing uracil was purified from the culture supernatant. The three oligonucleotide primers designed to contain appropriate mismatched bases (indicated by underlines) for site-directed mutagenesis are shown in Fig. 1. A primer 1 was used for introduction of the canonical Shine – Dalgarno (SD) sequence (AAGGAG) to 8 bases upstream of the ATG initiation codon of D-aminoacylase structural gene. The AGGA sequence in this introduced SD sequence exists in those of both l phage (cro) and lacZ. The resultant plasmid was designated as pANSD1. The single-stranded DNA was prepared from pANSD1 in the same way as pAND118. A primer 2 was for introduction of EcoRI site to 33 bases upstream of ATG. A primer 3 was for introduction of a HindIII site to 264 bases downstream of the TGA stop codon. These positions of the new restriction sites introduced using primer 2 and primer 3 were selected on the basis of both their sequence homologies with the recoginition sites of EcoRI or HindIII and their positional relations with promoter or terminator. The resultant plasmid was designated pANSD1HE (Fig. 1). The sequences of nucleotides corresponding to the mutated regions were confirmed by the dideoxy chain termination method with an ABI 373A DNA sequencer.

Construction of a Series of High-Expression Plasmids The plasmid pANSD1 was composed of pUC118 and the 4-kb BamHI – HindIII fragment containing the introduced SD sequence. The 4-kb BamHI – HindIII fragment excised from pANSD1 was inserted into BamHI – HindIII sites of pKK223-3 and pET23d(/). The resultant plasmids were designated pKNSD1 and pETND1, respectively. The plasmid pANSD2 was constructed by subcloning the 1.75-kb EcoRI – HindIII fragment of pANSD1HE into the EcoRI – HindIII site of pUC118. The 1.75-kb EcoRI – HindIII fragment excised from pANSD1HE was inserted into the downstream of the tac promoter in the expression vector pKK223-3. The resultant plasmid was designated pKNSD2 (Fig. 1).

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Purification of the Enzyme Produced in E. coli JM109/pKNSD2 Recombinant E. coli JM109 cells transformed with pKNSD2 (about 18 g wet weight) cultured at 307C for 20 h in 2-l Sakaguchi flasks with vigorous shaking were used for enzyme purification. All operations were done at 0–47C and the buffer used was 10 mM potassium phosphate buffer (pH 7.0) unless otherwise stated. Cells in buffer (about 100 mg cell/ml) were disrupted by sonication. The cell debris was removed by centrifugation, and the supernatant was used as the cell extract. The cell extract was applied to a DEAE–Toyopearl column (4 1 28 cm) equilibrated with the buffer. After the column was washed with 4000 ml of the buffer containing 50 mM NaCl, the enzyme was eluted with the buffer containing 100 mM NaCl. The active fractions were collected and concentrated by ultrafiltration with an Amicon PM-10 membrane. After glycerol (10%), 2-mercaptoethanol (0.01%), and 0.01 mM ZnCl2 were added to the enzyme solution, solid (NH4)2SO4 was added to the enzyme solution to give 20% saturation. The precipitate formed was removed and the supernatant was applied to a Butyl–Cellulofine column (2 1 8 cm) previously equilibrated with the buffer containing 20% (NH4)2SO4 , 10% glycerol, 0.01% 2-mercaptoethanol, and 0.01 mM ZnCl2 . The column was washed with the buffer containing 15% (NH4)2SO4 and the enzyme was eluted with the buffer containing 10% (NH4)2SO4 . The enzyme collected was dialyzed against the buffer and then concentrated by ultrafiltration. Assays of Enzyme Activity and Protein The reaction mixture contained l00 mM Hepes buffer (pH 7.0), 20 mM N-acetyl-D-leucine, and enzyme in a final volume of 0.5 ml. After incubating at 307C for 10 to 30 min, the reaction was stopped by the addition of 0.25 ml of 0.25 M NaOH. D-Leucine formed was measured by the 2,4,6-trinitrobenzenesulfonic acid method (18). One unit of enzyme activity was defined as the amount of the enzyme that catalyzes the formation of 1 mmol of D-leucine per min. Specific activity of the enzyme was expressed as units per milligram of protein. Protein was estimated by the method of Lowry et al. (19), with crystalline egg albumin as a standard. Electrophoresis Polyacrylamide gel electrophoresis (PAGE) was done in vertical slabs (14 1 13.5 cm) with 12.5% (wt/vol) acrylamide in the presence of sodium dodecyl sulfate (SDS) by the method of Laemmli (20). Phosphorylase b (97 kDa, rabbit muscle), albumin (66 kDa, bovine serum), aldolase (42 kDa, rabbit muscle), carbonic anhydrase (30 kDa, bovine erythrocyte), and trypsin inhibitor (20 kDa, soybean) were used as marker proteins.

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enough pure enzyme for the optical resolution of DLamino acids and structural studies such as X-ray crystallography.

Purification of Recombinant D-Aminoacylase from E. coli JM109/pKNSD2 Cellsa

Step

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Yield (%)

Purification of D-Aminoacylase from E. coli JM109/ pKNSD2

Cell-free extract DEAE–Toyopearl Butyl–Cellulofine

2574 600.3 50

417,168 446,667 100,959

162.1 740.7 2023.2

100 107 24.2

D-Aminoacylase was purified from about 18 g wet cells of E. coli JM109/pKNSD2 and obtained about 50 mg of homogeneous enzyme with an overall yield of 24%. The purification was achieved using only two column chromatography steps, i.e., DEAE and Butyl–Cellulofine (Table 1). Eight percent of the total soluble intracellular proteins of recombinant E. coli exhibited D-aminoacylase activity on the basis of the specific activity of the purified enzyme. The new purification method described here is advantageous over the previous method in many aspects (14). The two column chromatography steps, which replaced the five purification steps used previously, reduced the time required for purification from several weeks to 1 week. The final preparation was homogeneous by SDS–PAGE (Fig. 2) and showed a specific activity of 2,023 U/mg, which is significantly higher than that of the enzyme purified previously from E. coli JM109/pAND1 (1000 U/mg). This could be attributed to the rapid purification which must have been made possible to avoid the aging of the enzyme. The enzyme yield (24%) was about threefold higher than that (8.4%) of E. coli JM109/pAND1. The overproduction of D-aminoacylase achieved in the E. coli JM109 transformant by introducing the canonical SD sequence, eliminating the extra regions of the gene, and inserting the gene into the downstream of the tac promoter of expression vector pKK223-3 would

a

About 18 g of wet cells was used.

RESULTS AND DISCUSSION

Expression of the Constructed Plasmids Encoding the D-Aminoacylase Gene in E. coli The introduction of canonical SD sequence was very effective for expression of D-aminoacylase gene in E. coli. pANSD1 showed about 7-fold higher enzyme productivity than pAND1, while pKNSD1 showed 1.1-fold higher activity than pANSD1. These results indicated that the introduction of canonical SD sequence caused more effective translation to D-aminoacylase, but the insertion of the 4-kb BamHI –HindIII fragment into the downstream of the tac promoter was not effective on the enzyme production. E. coli JM109 cells carrying pANSD2 showed 55-fold higher specific activity, 54.8 U/mg, than E. coli JM109 cells carrying pAND1. E. coli JM109 cells carrying pKNSD2 produced 23,000 U of the enzyme per gram of cells (wet weight). The cellfree extract of these recombinant E. coli cells exhibited a specific activity of 162 units/mg, which is over 150fold higher than that of pAND1. These facts suggested that the elimination of the extra region in the inserted DNA fragment was very effective for the high expression of the D-aminoacylase in E. coli. The E. coli BL21(DE3) carrying pETND1 showed almost the same specific activity as E. coli JM109 carrying pAND1, but E. coli BL21(DE3)pLysS carrying pETND1 produced a 20-fold higher amount of D-aminoacylase than that of pAND1. E. coli BL21(DE3) pLysS carrying the plasmid pETND2 obtained by subcloning the 1.75-kb EcoRI – HindIII fragment of pANSD1HE into the EcoRI –HindIII site of pET-23d(/) exhibited a small amount of enzyme activity in the cell-free extract. The pET system is known as a powerful system for the expression of recombinant proteins in E. coli. But the proteins expressed in this system were reported to be susceptible to the formation of an inclusion body (21,22). In this case also, it was presumed that the translated enzyme protein aggregated and formed the inclusion body. In conclusion, from the experiments described above, the expression level of D-aminoacylase in E. coli JM109 carrying pKNSD2 is adequate for obtaining

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FIG. 2. SDS–PAGE of recombinant D-aminoacylase. Lane 1, molecular weight markers; lane 2, the final preparation after Butyl–Cellulofine column chromatography; lane 3, cell-free extract; lane 4, the preparation after DEAE–Toyopearl column chromatography.

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make possible the extensive application of D-aminoacylase for the industrial D-amino acid production by optical resolution and the X-ray crystallographic analysis of this enzyme. REFERENCES 1. Kameda, Y., Toyoura, E., Yamazoe, H., Kimura, Y., and Yasuda, Y. (1952) Hydrolysis and metabolism by soil bacteria of benzoyl derivatives of D- and L-forms of some amino acids. Nature (London) 170, 888–889. 2. Kameda, Y., Hase, H., Kanamoto, S., and Kita, Y. (1978) Studies on acylase activity and microorganism. Purification and properties of D-aminoacylase (N-acyl-D-amino acid amidohydrolase) from AAA 6029 (Pseudomonas sp.). Chem. Pharm. Bull. (Tokyo) 26, 2698–2704. 3. Kubo, K., Ishikura, T., and Fukagawa, Y. (1980) Deacetylation of PS-5, a new b-lactam compound. II. Separation and purification of L- and D-amino acid acylases from Pseudomonas sp. 1158. J. Antibiot. 43, 550–555. 4. Sugie, M., and Suzuki, H. (1978) Purification and properties of D-aminoacylase of Streptomyces olivaceus. Agric. Biol. Chem. 42, 107–113. 5. Sugie, M., and Suzuki, H. (1980) Optical resolution of DL-amino acids with D-aminoacylase of Streptomyces. Agric. Biol. Chem. 44, 1089–1095. 6. Moriguchi, M., Sakai, K., Miyamoto, Y., and Wakayama, M. (1993) Production, purification, and characterization of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. Biosci. Biotechnol. Biochem. 57, 1149–1152. 7. Moriguchi, M., and Ideta, K. (1988) Production of D-aminoacylase from Alcaligenes denitrificans subsp. xylosoxydans MI4. Appl. Environ. Microbiol. 54, 2767–2770. 8. Sakai, S., Obata, T., Ideta, K., and Moriguchi, M. (1991) Purification and properties of D-aminoacylase from Alcaligenes denitrificans subsp. xylosoxydans MI-4. J. Ferment. Bioeng. 71, 79– 82. 9. Sakai, K., Obata, T., Takano, S., and Moriguchi, M. (1989) A novel inducer, g-methyl-D-leucine, of D-aminoacylase from Alcaligenes denitrificans subsp. xylosoxydans MI-4. Agric. Biol. Chem. 53, 2285–2286. 10. Tsai, Y. C., Tseng, C. P., Hsiao, K. M., and Chen, L. Y. (1988) Production and purification of D-aminoacylase from Alcaligenes denitrificans and taxonomic study of the strain. Appl. Environ. Microbiol. 54, 984–989.

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11. Sakai, K., Imamura, K., Goto, M., Hirashiki, I., and Moriguchi, M. (1990) Occurrence of novel enzyme, N-acetyl-D-glutamate deacylase and N-acetyl-D-aspartate deacylase, in Alcaligenes xylosoxydans subsp. xylosoxydans A-6. Agric. Biol. Chem. 54, 841– 844. 12. Sakai, K., Imamura, K., Sonoda, Y., Kido, H., and Moriguchi, M. (1991) Purification and characterization of N-acyl-D-glutamate deacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A6. FEBS Lett. 289, 44–46. 13. Moriguchi, M., Sakai, K., Katsuno, Y., Maki, T., and Wakayama, M. (1993) Purification and characterization of novel N-acyl-Daspartate amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. Biosci. Biotechnol. Biochem. 57, 1145–1148. 14. Wakayama, M., Katsuno, Y., Hayashi, S., Miyamoto, Y., Sakai, K., and Moriguchi, M. (1995) Cloning and sequencing of a gene encoding D- aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 and expression of the gene in Escherichia coli. Biosci. Biotechnol. Biochem. 59, 2115–2119. 15. Wakayama, M., Watanabe, E., Takenaka, Y., Miyamoto, Y., Tau, Y., Sakai, K., and Moriguchi, M. (1995) Cloning, expression, and nucleotide sequence of the gene of N-acyl-D-aspartate amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. J. Ferment. Bioeng. 80, 311–317. 16. Wakayama, M., Ashika, T., Miyamoto, Y., Yoshikawa, T., Sonoda, Y., Sakai, K., and Moriguchi, M. (1995) Primary structure of N-acyl-D-glutamate amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. J. Biochem. 118, 204–209. 17. Kunkel, T. A., Robert, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367–382. 18. Fields, R. (1972) The rapid determination of amino groups with TNBS. Methods Enzymol. 25, 464–468. 19. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. 20. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685. 21. Tabor, S., and Richardson, C. C. (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82, 1074–1078. 22. Arcone, R., Pucci, P., Zappacosta, F., Fontaine, V., Malorni, A., Marino, G., and Ciliberto, G. (1991) Single-step purification and characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector. Eur. J. Biochem. 198, 541–547.

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