Journal of Biotechnology, 17 (1991) 99-108 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0168-1656/91/$03.5091 ADONIS 016816569100051E
99
BIOTEC 00556
Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis Joo H y u n Kang
1, Young
H w a n g 2 and O o k Joon Y o o 1
1 Department of Biological Science and Engineering, The Korea Advanced Institute of Science and Technology, Chongyang, Seoul, Korea and 2 Department of Biotechnology, Korea Explosives Group, Research and Engineering Center, Yousung-Ku, Daejeon, Korea (Received 14 February 1990; revision accepted 4 May 1990)
Summary Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated. Recombinant Escherichia coli clones were screened for clear halo forming activity on the lawn of Staphylococcus aureus ATCC 6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-(a)phenylglycine methylester. The gene was contained within a 2.8 kb DNA fragment and expressed efficiently "when transferred from E. coli to Bacillus subtilis. A twenty times greater amount of enzyme was produced in B. subtilis transformant than that in B. megaterium. The purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000. The enzyme was able to react on various cephalosporins, i.e., cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin and cephradine. Penicillin G acylase; Cloning; B. megaterium; Expression; Substrate specificity
Introduction
Penicillin G acylases (penicillin amidase or benzyl penicillin amidohydrolase EC 3.5.1.11) which catalyze the deacylation of penicillin G are used for commercial Correspondence to: O.J. Yoo, Dept. of Biological Science and Engineering, The Korea Advanced Institute of Science and Technology, P.O. Box 150, Chongyang, Seoul 130-650, Korea.
100 production of 6-aminopenicillanic acid (6-APA), an important precursor for the synthesis of many semisynthetic penicillins (Vandamme and Voets, 1974). Because of the high industrial value of penicillin G acylase, much effort has been made toward the search for microorganisms producing the enzyme (Mahajam, 1984) and for overproducing strains expressing a higher level using conventional mutagenesis. With recently developed recombinant DNA technology, research has been focused on the cloning of penicillin G acylase genes. Consequently, penicillin G acylase genes from Arthrobacter viscosus 8895GU (Ohashi et al., 1988), Bacillus sphaericus ATCC 14577 (Olsson et al., 1985), Escherichia coli ATCC 11105 (Mayer et al., 1979), Kluyvera citrophila ATCC 21285 (Garcia and Buesa, 1986), Proteus rettgeri ATCC 31052 (Daumy et al., 1986), and Pseudomonas sp. strain GK16 (Matsuda and Komatsu, 1985) were isolated. In addition, with the thorough characterization of the penicillin G acylases from several bacterial strains, it has been found that some of the enzymes have broad substrate specificity so they can be introduced to produce commercially more valuable semisynthetic/3-1actam antibiotics using their reverse reaction activity (Shimizu et al., 1975; Fujii et al., 1976). This report details the isolation of the gene encoding penicillin G acylase exhibiting broad substrate specificity from Bacillus megateriurn and the expression of the cloned gene in E. coli and B. subtilis and the characterization of the purified enzyme from the culture fluid of the B. subtilis transformant.
Materials and Methods
Bacterial strains and plasmids
The bacterial strains and plasmids used in this work are listed in Table 1. Media and reagents
NY (Maniatis et al., 1982) and LB (Maniatis et al., 1982) medium were prepared as required. The clones resistant against antibiotics were selected on the media containing 50/~g m1-1 of ampicillin, 12.5/~g m1-1 of tetracycline or 10/~g m1-1 of kanamycin depending on the plasmid DNA used. All the antibiotics, their related chemicals and p-dimethylaminobenzaldehyde were purchased from Sigma Chemical Company, MO, U.S.A. 2,4-Pentanedione and D-(a)-phenylglycine methylester were from Wako Pure Chemicals, Japan. Restriction endonucleases, Bal31 exonuclease and T4 DNA ligase were purchased from New England Biolabs, U.S.A. and Bethesda Research Laboratories, U.S.A. and used under the conditions described by the suppliers. Preparation of DNA
Chromosomal DNA from B. megaterium ATCC 14945 was prepared following the method described by Marmur (1961). Plasmid DNAs from E. coli and B.
101 TABLE 1 Bacterial strains and plasmids Strains and plasmids
Relevant characteristics
Source or reference
Escherichia coli HBIO1
Host for gene constructions
Escherichia coli JM83
Host for subcloning the gene into pUC19 Penicillin G acylase producer Indicator strain of cephalexin Host for subcloning the gene into pUBll0 Apr, Tc r Apr Apr Kmr Tc r, penicillin G acylase positive TC, penicillin G acylase positive Tc r, penicillin G acylase positive Apr, penicillin G acylase positive Apr, penicillin G acylase positive Apr, overproduction of penicillin G acylase Krnr, Apr, penicillin G acylase positive (replicated in E. coli and B. subtilis) Km~, penicillin acylase positive (replicated only in B. subtilis)
Boyer and Roulland-Dussoix (1969) Vieira and Messing (1982)
Bacillus megaterium Staphylococcus aureus Bacillus subtilis ~BR322 ~UC19 ~TTQ19 ~UBll0 ~CSE220 ~CSE130 ~CSE94 ~UCSE70 9UCSE59 ~TCSE76 pUBC103 pUBC73
ATCC 14945 ATCC 6538P Bolivar et al. (1977) Norrander et al. (1983) Stark (1987) Keggins et al. (1978) This paper This paper This paper This paper This paper This paper This paper This paper
subtilis were prepared according to the methods published by Birnboim and D o l y (1979) and H a r d y (1985). Transformation of cells The transformations of E. coli and B. subtilis were performed as described by H a n a h a n (1983) and D u b n a u and Davidoff-Abelson (1971).
Screening for penicillin G acylase-positive clones E. coli clones carrying B. megaterium D N A were replicated to L B - A g a r (LA) plates and incubated overnight. They were then overlaid with 3 ml of soft agar containing overnight-grown Staphylococcus aureus A T C C 6538P, 7 - A D C A (25 m M ) and D-(a)-phenylglycine methylester (50 mM). The plates were incubated for additional 6 - 7 h at 37°C. Positive clones showed transparent halo on the lawn of S. aureus colony due to the cephalexin p r o d u c e d b y enzymatic acylation. Assay for penicillin G acylase activity One ml of assay mixture contained 1% (w v -1) penicillin G (potassium salt) in a 0.1 M sodium phosphate buffer ( p H 6.8). The reaction was started by adding
102 enzyme (suspended cells or enzyme solution) and the mixture was incubated at 37 °C for 1 h. The reaction was stopped by heating in a boiling water bath for 3 rain. The amount of produced 6-APA was determined as described by Kornfeld (1978). One unit of activity is defined as the amount of enzyme required to hberate 1 ~tmol of 6-APA per 1 h at 37°C. To study the hydrolyzing activities of the penicillin G acylase on various cephalosporins, reactivities were determined according to the method developed by Balasingham et al. (1972). Acylating activities of 7-ADCA and 7-aminocephalosporanic acid (7-ACA) for D-(a)-phenylglycine methylester were determined according to the method described by Fujii et al. (1976).
Isolation of penicillin G acylase from culture fluid of B. subtilis transformant B. subtilis harboring the recombinant plasmid, pUBC73, was grown in a 8 1 LB medium containing 10/~g m1-1 kanamycin at 37°C with stirring at 700 rpm. After the O1)560 reached 9.0, cells were harvested at 9000 rpm for 15 min. The cell-free culture fluid was acidified to pH 6.4 with diluted acetic acid and mixed with acid-washed Celite (No. 545 Jhones-Manville Co., New York) at a ratio of 12 g 1-1. The Cehte was collected by filtration and washed with 1 1 of cold 0.01 M Tris-C1 buffer (pH 8.4). This immobilized enzyme was extracted with 350 ml of 24% (w v -1) ammonium sulfate in 0.1 M Tris-C1 (pH 8.4). The 350 ml of extract solution was dialyzed against 0.02 M sodium phosphate buffer (pH 6.8). The enzyme was further purified by fractionation through diethylaminoethyl (DEAE) Sephadex A-50 column chromatography (1.7 x 20 cm) with a linear salt gradient (0-1.0 M NaC1 in 0.02 M sodium phosphate buffer, pH 6.8) and gel filtration column chromatography (2.2 x 69 cm) using Sephacryl $200 (Pharmacia) in a 0.1 M sodium phosphate buffer (pH 6.8). Protein assay and SDS-polyacrylamide gel electrophoresis Protein concentration was measured by the method originated by Bradford (1976), with bovine serum albumin as the standard. 10% sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis was carried out as described by Laemmli (1970).
Results
Cloning and expression of penicillin G acylase gene in E. coli and B. subtilis Chromosomal DNA from B. megaterium ATCC 14945 was partially digested with PstI and ligated with PstI-cleaved pBR322. The resulting recombinant plasrnids were used to transform E. coli HB101. About 18,500 tetracycline-resistant clones were transferred to LA plates, grown overnight and screened for cephalexinsemisynthesizing activity using the procedure described in Materials and Methods. Only two clones formed clear halo on the lawn of S. aureus. The plasmids isolated
103
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c
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Fig. 1. Scheme showing construction of various recombinant plasmids. D N A plasmid vectors are represented by empty boxes (pBR322), dotted boxes (pUC19) and hatched boxes (pUBll0). Solid thick lines represent the regions carrying the penicillin G acylase gene. B, BamHI; Bg, BgllI; C, ClaI; H, HindlII; P, PstI; Pv, PvulI; R, EcoRI; S, ScaI; S*, partial ScaI; Ss, SstI; X, XbaI; Xh, XhoI.
from both clones were shown to have identical restriction patterns. The size of the plasmid was 22.0 kb including 17.6 kb foreign DNA. The plasmid, designated pCSE220, was subjected to subcloning as illustrated in Fig. 1. Two D N A fragments, 8.6 kb and 9.0 kb, were obtained by treating pCSE220 with the enzyme mixture of XhoI and PstI. These fragments were treated with Klenow enzyme to make blunt ended terminals and ligated into ScaI-cleaved pBR322. After transforming the subcloned plasmids, tetracycline-resistant colonies were screened for the activity. The resulting recombinant plasmid isolated from the positive clones, designated pCSE130, contained 8.6 kb foreign D N A fragment, pCSE130 was digested with ClaI and the larger fragment (9.4 kb) was isolated and self ligated. The resulting plasmid, designated pCSE94, was composed of a 5.6 kb insert containing the gene and a 3.8 kb D N A fragment driven from pBR322. The plasmid, pCSE94, was subjected to further subcloning into pUC19 since it had been found that fl-lactamase produced from pUC19 could not interfere in the detection of cephalexin
104 TABLE 2 Penicillin G acylase activity in various strains and subclones a Strains and subclones
Cultured supernatant (unit)
Cell pellet (unit)
B. B. B. E. E. E. E. E.
0.6 ND 13.8 ND ND ND ND ND
ND b ND 0.3 ND 0.17 0.16 0.16 0.15
megaterium subtilis (pUB110) subtilis (pUBC73) coli (pBR322) coli (pCSE220) coli (pCSE130) coli (pCSE94) coli (pUCSE59)
a Assays were performed using the 10 ml culture fluid or the collected cells as enzyme sources. b N D , none detected.
produced by acylating reaction of 7-ADCA and D-(a)-phenylglycine methylester. pCSE94 was partially digested with Sau3AI and randomly ligated into BamHIcleaved pUC19. Among the transformants possessing cephalexin semisynthesizing activity, the clone which contained the shortest insert in the plasmid was isolated. This recombinant plasmid, consisting of 3.8 kb insert DNA, 0.4 kb fragment from pBR322 and 2.7 kb of pUC19 DNA, was designated as pUCSE70. A smaller recombinant plasmid pUCSE59, containing 2.8 kb foreign DNA, was constructed in such a way that pUCSE70 was treated with ClaI followed by Bal31 and then self ligated. B. megaterium secretes the penicillin G acylase into the culture medium; therefore we intended, by means of gene manipulation, to construct B. subtilis that can secrete the enzyme extracellularly. A shuttle vector plasmid for E. coli and B. subtilis, designated pUBC103, was constructed by inserting EcoRI-cleaved pUBll0 in the EcoRI site of pUCSE59, pUBC103, however, was not stably replicated in B. subtilis. The problem has been solved by constructing a smaller recombinant plasmid, pUBC73, in such a way that the 7.3 kb fragment generated from XbaIcleaved pUBC103 was self ligated. In order to produce the enzyme in a greater amount, we transferred the gene from pUCSE59 to an expression vector, pTTQ19 (Stark, 1987). The resulting recombinant plasmid was designated as pTCSE76. Penicillin G acylase activities expressed by various recombinant clones were determined and the results were shown in Table 2. The activities in the E. coli clones were lower than those in B. megaterium. On the other hand, a twenty times greater amount of the enzyme was produced and secreted extracellularly by B. subtilis (harboring pUBC73) than that produced by B. megaterium. Purification and characterization of penicillin G acylase from culture fluid of B. subtilis transformant From 8 1 of the culture fluid of B. subtilis (pUBC73), penicillin G acylase was purified by a series of procedures using Celite, DEAE-Sephadex A-50 column
105 TABLE 3 Summary of purification of penicillin G acylase from B. subtilis transformant Purification step
Vol (ml)
Total activity (U)
Total protein (mg)
Specific activity (U mg- 1)
Yield (%)
Crude reaction mixture Celite DEAE-Sephadex A-50 Sephacryl $200
8,000 350 35 15
12,700 9,890 7,810 6,900
7,050 122 77 62
1.8 81.1 101.4 111.3
100 78 61 54
chromatography and gel filtration on Sephacryl $200. The detailed purification steps were described in Materials and Methods and summarized in Table 3. From the DEAE-Sephadex column, the enzyme eluted between 0.35-0.45 M NaCI. The molecular weight of the native enzyme estimated by the Sephacryl $200 column was about 115,000 Da. The enzyme proteins obtained from the culture fluid of B. megaterium and purified from B. subtilis transformants were analyzed by a 10% SDS-polyacrylamide gel electrophoresis. The subunit molecular weight of penicillin G acylase isolated from B. megaterium and B. subtilis transformant was estimated to be 57,000 as illustrated in Fig. 2a, lane B and C, respectively. A thick protein band appeared from the total cell proteins of E. coli harboring pTCSE76 (lane E) when compared with the control (lane D). The band, however, turned out to be larger than the protein secreted from B. subtilis by about 3000 Da as judged by a prolonged SDS-polyacrylamide gel electrophoresis (Fig. 2b). The results from SDSpolyacrylamide gel electrophoresis and gel filtration indicate that the native enzyme consists of two identical subunits.
TABLE 4 Hydrolyzingactivities of penicillin G acylase Substrate
Rate of hydrolysis a (U mg-1)
Relative activity (%)
Penicillin G Cephaiothin Cefamandole Cephaioridine Cephaloglycin Cephalexin Cephradine Ampicillin Cephapirin Cefotaxime Cephalosporin C Penicillin N
111.3 95.0 81.7 7413 40.2 33.4 13.5 11.4 ND b ND ND ND
100 85.4 73.4 66.8 36.1 30.0 12.1 10.2 ND ND ND ND
a Rates of hydrolysiswere expressed as specific activities when each substrate was used. b ND, none detected.
106 Substrate specificity of purified penicillin G acylase
To investigate the industrial value, hydrolyzing activities of the enzyme were examined with various fl-lactam antibiotics. The enzyme effectively deacylated penicillin G, cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin, cephradine and ampicillin. The rate of hydrolysis of these substrates by the enzyme was different from each other and the relative rates to penicillin G were listed in the order of their rates in Table 4. This enzyme, however, could not hydrolyze penicillin N, cephapirin, cefotaxime or cephalosporin C.
Discussion
In this paper, we have described the cloning of the gene encoding penicillin G acylase from B. megaterium and the production of the enzyme in E. coli and B. subtilis. Attempts to clone the gene have been hampered by the poor expression in E. coli. The method employed in this study used S. aureus which is one of the most sensitive strains against cephalexin for the screening of E. coli transformants. As a consequence, colonies forming clear haloes were easily isolated on the lawn of S. aureus . Table 2 shows that B. subtilis harboring pUBC73 produced a more significantly increased level of the active enzyme than that produced by the original strain. When the gene was transferred to an expression vector, pTTQ19, penicillin G acylase was produced at the level of more than 30% of the total cell protein as illustrated in Fig. 2a, lane E. The enzyme protein, however, was mostly inactive and existed as inclusion bodies which were visible under the phase contrast microscope. Efforts for renaturing the enzyme are under progress in our laboratory. The expressed protein in E. coli was larger than the secreted protein from B. subtilis (Fig. 2b) by about 3000 Da. Apparently the signal sequence of the enzyme is being processed in B. subtilis. It has been reported that penicillin acylases produced by A. oiscosus (Ohashi et al., 1988), E. coli ATCC 11105 (Bock et al., 1983a, b), K. citrophila (Barbero et al., 1986), P. rettgeri ATCC 31052 (Daumy et al., 1985) and Pseudomonas sp. strain GK16 (Ichikawa et al., 1981) consist of two distinct subunits with respective molecular weights of 24,000 and 60,000, 20,500 and 69,000, 23,000 and 62,000, 24,500 and 65,000 and 16,000 and 54,000. On the other hand, penicillin V amidase from B. sphaericus consists of four identical subunits, each with a molecular weight of 36,500 (Olsson et al., 1985). In the case of B. megaterium, the enzyme consists of two identical subunits and its relative subunit size was 57,000 Da. This result was supported by the size of the subunit that appeared on a 10% SDS-polyacrylamide gel (Fig. 2) and the molecular weight of the native protein based on Sephacryl $200. It is likely that the enzyme prefers the aromatic ring structure as substitutes at a-carbon atom rather than alkyl groups. This enzyme tends to recognize and react on all of 6-APA, 7-ADCA and 7-ACA. The enzyme has also been tested for the acylating reactions between D-(a)-phenylglycine methylester and 7-ADCA and
107
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Fig. 2. Identification of penicillin G acylase by SDS-polyacrylamide gel electrophoresis. Samples were analyzed by 10% SDS-polyacrylamide gel electrophoresis. The protein bands were visualized with Coomassie Brilliant Blue R-250. (a) Lane A, contained marker proteins of known molecular weight. Lane B, concentrated culture fluid of B. megaterium using Celite. Lane C, purified proteins from culture fluid of B. subtilis harboring pUBC73 (for details see Materials and Methods). Small amount of other protein band appeared at the lower part of this lane. Lane D, total cell proteins of E. coli harboring pTTQ19. Lane E, total cell proteins of E. eoli harboring pTCSE76. The arrows show the bands corresponding to the penicillin G acylase protein. BPB, bromophenol blue dye. (b) Lane A, secreted proteins from B. subtilis harboring pUBC73. Lane B, mixed proteins applied on lane A and C. Lane C, one tenth of the amount of the proteins used for lane E of Fig. 2a.
D - ( a ) - p h e n y l g l y c i n e m e t h y l e s t e r a n d 7 - A C A to p r o d u c e c e p h a l e x i n a n d c e p h a l o g l y c i n , r e s p e c t i v e l y . I n b o t h cases, t h e rates of a c y l a t i n g r e a c t i o n s w e r e c l o s e l y p a r a l l e l e d to the rates o f d e a c y l a t i n g r e a c t i o n s . T h e s e facts s u g g e s t p o s s i b i l i t i e s o f s y n t h e s i z i n g p e n i c i l l i n s a n d c e p h a l o s p o r i n s in l a r g e scale u s i n g t h e r e v e r s e r e a c t i o n o f this e n z y m e .
References
Barbero, J.L., Buesa, J.M., Buitrago, G.G., Mendez, E., Perez-Aranda, A. and Garcia, J.L. (1986) Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila. Gene 49, 69-80. Balasingham, K., Warburton, D., Dunnill, P. and Lilly, M.D. (1972) The isolation and kinetic of penicillin amidase from Es.cheriehia coli. Biochim. Biophys. Acta 276, 250-256. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523. Bock, A., Wirth, R., Schmid, G., Schumacher, G., Lang, G. and Buckel, P. (1983a) The penicillin acylase from Eseherichia coil ATCC 11105 consists of two dissimilar subunits. FEMS Microbiol. Lett. 20, 135-139. Bock, A., Wirth, R., Schmid, G., Schumacher, G., Lang, G. and Buckel, P. (1983b) The two subunits of penicillin acylase are processed from a common precursor. FEMS Microbiol. Lett. 20, 141-144. Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., Crosa, J.H. and Falkow, S. (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95-113.
108 Boyer, H.W. and Roulland-Dussoix, D. (1969) A complementation analysis of the restriction and modification of DNA in Escherichm coll. J. Mol. Biol. 41,459-472. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 75, 248-254. Daumy, G.O., Danley, D. and McColl, A.S. (1985) Role of protein subunits in Proteus rettgeri penicillin G acylase. J. Bacteriol. 163, 1279-1281. Daumy, G.O., Williams, J.A., McColl, A.S., Zuzel, T.J. and Danley, D. (1986) Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coll. J. Bacteriol. 168, 431-433. Dubnau, D. and Davidoff-Abelson, R. (1971) Fate of transforming DNA following uptake by competent Bacillus subtilis. J. Mol. Biol. 56, 209-221. Fujii, T., Matsumoto, K. and Watanabe, T. (1976) Enzymatic synthesis of cephalexin. Process Biochem. 21-25. Garcia, J.L. and Buesa, J.M. (1986) An improved method to clone penicillin acylase gene: cloning and expression in Escherichia coli of penicillin G acylase from Kluyoera citrophila. J. Biotechnol. 3, 187-195. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557-580. Hardy, K.G. (1985) Bacillus cloning methods. In: Glover, D.M. (Ed.), DNA Cloning, Vol. II, IRL Press, Oxford. pp. 1-17. Ichikawa, S., Shibuya, Y., Matsumoto, K., Fujii, T., Komatsu, K. and Kodaira, R. (1981) Purification and properties of 7-beta-(4-carboxybutanamido)-cephalosporanic acid acylase produced by mutants derived from Pseudomonas. Agr. Biol. Chem. 45, 2231-2236. Keggins, K.M., Lovett, P.S. and Duvall, E.J. (1978) Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUBll0. Proc. Natl. Acad. Sci. 75, 1423-1427. Kornfeld, J.M. (1978) A new colorimetric method for the determination of 6-aminopenicillanic acid. Anal. Biochem. 86, 118-126. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. Mahajam, P.B. (1984) Penicillin acylases, An update. Appl. Biochem. Biotechnol. 9, 537-554. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Marmur, J. (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3, 208-218. Matsuda, A. and Komatsu, K. (1985) Molecular cloning and structure of the gene for 7-beta-(4-carboxybutanamido)-cephalosporanic acid acylase from a Pseudomonas strain. J. Bacteriol. 163, 1222-1228. Mayer, H., Collins, J. and Wagner, F. (1979) Cloning of the penicillin G acylase gene of Escherichia coli ATCC 11105 on multicopy plasmids. In: Timmis, K.N. and Puhler, A. (Eds.), Plasmids of Medical, Environmental and Commercial Importance, Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 459-470. Norrander, J., Kempe, T. and Messing, J. (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26, 101-106. Ohashi, H., Katsuta, Y., Hashizume, T., Abe, S., Kajiura, H., Hattori, H., Kamei, T. and Yano, M. (1988) Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus. Appl. Environ. Microbiol. 54, 2603-2607. Olsson, A., Hagstrom, T., Nilsson, B., Uhlen, M. and Gatenbeck, S. (1985) Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis. Appl. Environ. Microbiol. 49, 1084-1089. Shimizu, M., Okachi, R., Kimura, K. and Nara, T. (1975) Purification and properties of penicillin acylase from Kluyvera citrophila. Agr. Biol. Chem. 39, 1655-1661. Stark, M.J.R. (1987) Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of gene in Escherichia coli. Gene 51, 255-267. Vandamme, E.J. and Voets, J.P. (1974) Microbial penicillin acylases. Adv. Appl. Microbiol. 17, 311-369. Vieira, J. and Messing, J. (1982) The pUC plasmids, an M13 mp7-derived system for insertion mutagenesis and sequencing with synthetic primers. Gene 19, 259-268.
99
BIOTEC 00556
Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis Joo H y u n Kang
1, Young
H w a n g 2 and O o k Joon Y o o 1
1 Department of Biological Science and Engineering, The Korea Advanced Institute of Science and Technology, Chongyang, Seoul, Korea and 2 Department of Biotechnology, Korea Explosives Group, Research and Engineering Center, Yousung-Ku, Daejeon, Korea (Received 14 February 1990; revision accepted 4 May 1990)
Summary Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated. Recombinant Escherichia coli clones were screened for clear halo forming activity on the lawn of Staphylococcus aureus ATCC 6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-(a)phenylglycine methylester. The gene was contained within a 2.8 kb DNA fragment and expressed efficiently "when transferred from E. coli to Bacillus subtilis. A twenty times greater amount of enzyme was produced in B. subtilis transformant than that in B. megaterium. The purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000. The enzyme was able to react on various cephalosporins, i.e., cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin and cephradine. Penicillin G acylase; Cloning; B. megaterium; Expression; Substrate specificity
Introduction
Penicillin G acylases (penicillin amidase or benzyl penicillin amidohydrolase EC 3.5.1.11) which catalyze the deacylation of penicillin G are used for commercial Correspondence to: O.J. Yoo, Dept. of Biological Science and Engineering, The Korea Advanced Institute of Science and Technology, P.O. Box 150, Chongyang, Seoul 130-650, Korea.
100 production of 6-aminopenicillanic acid (6-APA), an important precursor for the synthesis of many semisynthetic penicillins (Vandamme and Voets, 1974). Because of the high industrial value of penicillin G acylase, much effort has been made toward the search for microorganisms producing the enzyme (Mahajam, 1984) and for overproducing strains expressing a higher level using conventional mutagenesis. With recently developed recombinant DNA technology, research has been focused on the cloning of penicillin G acylase genes. Consequently, penicillin G acylase genes from Arthrobacter viscosus 8895GU (Ohashi et al., 1988), Bacillus sphaericus ATCC 14577 (Olsson et al., 1985), Escherichia coli ATCC 11105 (Mayer et al., 1979), Kluyvera citrophila ATCC 21285 (Garcia and Buesa, 1986), Proteus rettgeri ATCC 31052 (Daumy et al., 1986), and Pseudomonas sp. strain GK16 (Matsuda and Komatsu, 1985) were isolated. In addition, with the thorough characterization of the penicillin G acylases from several bacterial strains, it has been found that some of the enzymes have broad substrate specificity so they can be introduced to produce commercially more valuable semisynthetic/3-1actam antibiotics using their reverse reaction activity (Shimizu et al., 1975; Fujii et al., 1976). This report details the isolation of the gene encoding penicillin G acylase exhibiting broad substrate specificity from Bacillus megateriurn and the expression of the cloned gene in E. coli and B. subtilis and the characterization of the purified enzyme from the culture fluid of the B. subtilis transformant.
Materials and Methods
Bacterial strains and plasmids
The bacterial strains and plasmids used in this work are listed in Table 1. Media and reagents
NY (Maniatis et al., 1982) and LB (Maniatis et al., 1982) medium were prepared as required. The clones resistant against antibiotics were selected on the media containing 50/~g m1-1 of ampicillin, 12.5/~g m1-1 of tetracycline or 10/~g m1-1 of kanamycin depending on the plasmid DNA used. All the antibiotics, their related chemicals and p-dimethylaminobenzaldehyde were purchased from Sigma Chemical Company, MO, U.S.A. 2,4-Pentanedione and D-(a)-phenylglycine methylester were from Wako Pure Chemicals, Japan. Restriction endonucleases, Bal31 exonuclease and T4 DNA ligase were purchased from New England Biolabs, U.S.A. and Bethesda Research Laboratories, U.S.A. and used under the conditions described by the suppliers. Preparation of DNA
Chromosomal DNA from B. megaterium ATCC 14945 was prepared following the method described by Marmur (1961). Plasmid DNAs from E. coli and B.
101 TABLE 1 Bacterial strains and plasmids Strains and plasmids
Relevant characteristics
Source or reference
Escherichia coli HBIO1
Host for gene constructions
Escherichia coli JM83
Host for subcloning the gene into pUC19 Penicillin G acylase producer Indicator strain of cephalexin Host for subcloning the gene into pUBll0 Apr, Tc r Apr Apr Kmr Tc r, penicillin G acylase positive TC, penicillin G acylase positive Tc r, penicillin G acylase positive Apr, penicillin G acylase positive Apr, penicillin G acylase positive Apr, overproduction of penicillin G acylase Krnr, Apr, penicillin G acylase positive (replicated in E. coli and B. subtilis) Km~, penicillin acylase positive (replicated only in B. subtilis)
Boyer and Roulland-Dussoix (1969) Vieira and Messing (1982)
Bacillus megaterium Staphylococcus aureus Bacillus subtilis ~BR322 ~UC19 ~TTQ19 ~UBll0 ~CSE220 ~CSE130 ~CSE94 ~UCSE70 9UCSE59 ~TCSE76 pUBC103 pUBC73
ATCC 14945 ATCC 6538P Bolivar et al. (1977) Norrander et al. (1983) Stark (1987) Keggins et al. (1978) This paper This paper This paper This paper This paper This paper This paper This paper
subtilis were prepared according to the methods published by Birnboim and D o l y (1979) and H a r d y (1985). Transformation of cells The transformations of E. coli and B. subtilis were performed as described by H a n a h a n (1983) and D u b n a u and Davidoff-Abelson (1971).
Screening for penicillin G acylase-positive clones E. coli clones carrying B. megaterium D N A were replicated to L B - A g a r (LA) plates and incubated overnight. They were then overlaid with 3 ml of soft agar containing overnight-grown Staphylococcus aureus A T C C 6538P, 7 - A D C A (25 m M ) and D-(a)-phenylglycine methylester (50 mM). The plates were incubated for additional 6 - 7 h at 37°C. Positive clones showed transparent halo on the lawn of S. aureus colony due to the cephalexin p r o d u c e d b y enzymatic acylation. Assay for penicillin G acylase activity One ml of assay mixture contained 1% (w v -1) penicillin G (potassium salt) in a 0.1 M sodium phosphate buffer ( p H 6.8). The reaction was started by adding
102 enzyme (suspended cells or enzyme solution) and the mixture was incubated at 37 °C for 1 h. The reaction was stopped by heating in a boiling water bath for 3 rain. The amount of produced 6-APA was determined as described by Kornfeld (1978). One unit of activity is defined as the amount of enzyme required to hberate 1 ~tmol of 6-APA per 1 h at 37°C. To study the hydrolyzing activities of the penicillin G acylase on various cephalosporins, reactivities were determined according to the method developed by Balasingham et al. (1972). Acylating activities of 7-ADCA and 7-aminocephalosporanic acid (7-ACA) for D-(a)-phenylglycine methylester were determined according to the method described by Fujii et al. (1976).
Isolation of penicillin G acylase from culture fluid of B. subtilis transformant B. subtilis harboring the recombinant plasmid, pUBC73, was grown in a 8 1 LB medium containing 10/~g m1-1 kanamycin at 37°C with stirring at 700 rpm. After the O1)560 reached 9.0, cells were harvested at 9000 rpm for 15 min. The cell-free culture fluid was acidified to pH 6.4 with diluted acetic acid and mixed with acid-washed Celite (No. 545 Jhones-Manville Co., New York) at a ratio of 12 g 1-1. The Cehte was collected by filtration and washed with 1 1 of cold 0.01 M Tris-C1 buffer (pH 8.4). This immobilized enzyme was extracted with 350 ml of 24% (w v -1) ammonium sulfate in 0.1 M Tris-C1 (pH 8.4). The 350 ml of extract solution was dialyzed against 0.02 M sodium phosphate buffer (pH 6.8). The enzyme was further purified by fractionation through diethylaminoethyl (DEAE) Sephadex A-50 column chromatography (1.7 x 20 cm) with a linear salt gradient (0-1.0 M NaC1 in 0.02 M sodium phosphate buffer, pH 6.8) and gel filtration column chromatography (2.2 x 69 cm) using Sephacryl $200 (Pharmacia) in a 0.1 M sodium phosphate buffer (pH 6.8). Protein assay and SDS-polyacrylamide gel electrophoresis Protein concentration was measured by the method originated by Bradford (1976), with bovine serum albumin as the standard. 10% sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis was carried out as described by Laemmli (1970).
Results
Cloning and expression of penicillin G acylase gene in E. coli and B. subtilis Chromosomal DNA from B. megaterium ATCC 14945 was partially digested with PstI and ligated with PstI-cleaved pBR322. The resulting recombinant plasrnids were used to transform E. coli HB101. About 18,500 tetracycline-resistant clones were transferred to LA plates, grown overnight and screened for cephalexinsemisynthesizing activity using the procedure described in Materials and Methods. Only two clones formed clear halo on the lawn of S. aureus. The plasmids isolated
103
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Fig. 1. Scheme showing construction of various recombinant plasmids. D N A plasmid vectors are represented by empty boxes (pBR322), dotted boxes (pUC19) and hatched boxes (pUBll0). Solid thick lines represent the regions carrying the penicillin G acylase gene. B, BamHI; Bg, BgllI; C, ClaI; H, HindlII; P, PstI; Pv, PvulI; R, EcoRI; S, ScaI; S*, partial ScaI; Ss, SstI; X, XbaI; Xh, XhoI.
from both clones were shown to have identical restriction patterns. The size of the plasmid was 22.0 kb including 17.6 kb foreign DNA. The plasmid, designated pCSE220, was subjected to subcloning as illustrated in Fig. 1. Two D N A fragments, 8.6 kb and 9.0 kb, were obtained by treating pCSE220 with the enzyme mixture of XhoI and PstI. These fragments were treated with Klenow enzyme to make blunt ended terminals and ligated into ScaI-cleaved pBR322. After transforming the subcloned plasmids, tetracycline-resistant colonies were screened for the activity. The resulting recombinant plasmid isolated from the positive clones, designated pCSE130, contained 8.6 kb foreign D N A fragment, pCSE130 was digested with ClaI and the larger fragment (9.4 kb) was isolated and self ligated. The resulting plasmid, designated pCSE94, was composed of a 5.6 kb insert containing the gene and a 3.8 kb D N A fragment driven from pBR322. The plasmid, pCSE94, was subjected to further subcloning into pUC19 since it had been found that fl-lactamase produced from pUC19 could not interfere in the detection of cephalexin
104 TABLE 2 Penicillin G acylase activity in various strains and subclones a Strains and subclones
Cultured supernatant (unit)
Cell pellet (unit)
B. B. B. E. E. E. E. E.
0.6 ND 13.8 ND ND ND ND ND
ND b ND 0.3 ND 0.17 0.16 0.16 0.15
megaterium subtilis (pUB110) subtilis (pUBC73) coli (pBR322) coli (pCSE220) coli (pCSE130) coli (pCSE94) coli (pUCSE59)
a Assays were performed using the 10 ml culture fluid or the collected cells as enzyme sources. b N D , none detected.
produced by acylating reaction of 7-ADCA and D-(a)-phenylglycine methylester. pCSE94 was partially digested with Sau3AI and randomly ligated into BamHIcleaved pUC19. Among the transformants possessing cephalexin semisynthesizing activity, the clone which contained the shortest insert in the plasmid was isolated. This recombinant plasmid, consisting of 3.8 kb insert DNA, 0.4 kb fragment from pBR322 and 2.7 kb of pUC19 DNA, was designated as pUCSE70. A smaller recombinant plasmid pUCSE59, containing 2.8 kb foreign DNA, was constructed in such a way that pUCSE70 was treated with ClaI followed by Bal31 and then self ligated. B. megaterium secretes the penicillin G acylase into the culture medium; therefore we intended, by means of gene manipulation, to construct B. subtilis that can secrete the enzyme extracellularly. A shuttle vector plasmid for E. coli and B. subtilis, designated pUBC103, was constructed by inserting EcoRI-cleaved pUBll0 in the EcoRI site of pUCSE59, pUBC103, however, was not stably replicated in B. subtilis. The problem has been solved by constructing a smaller recombinant plasmid, pUBC73, in such a way that the 7.3 kb fragment generated from XbaIcleaved pUBC103 was self ligated. In order to produce the enzyme in a greater amount, we transferred the gene from pUCSE59 to an expression vector, pTTQ19 (Stark, 1987). The resulting recombinant plasmid was designated as pTCSE76. Penicillin G acylase activities expressed by various recombinant clones were determined and the results were shown in Table 2. The activities in the E. coli clones were lower than those in B. megaterium. On the other hand, a twenty times greater amount of the enzyme was produced and secreted extracellularly by B. subtilis (harboring pUBC73) than that produced by B. megaterium. Purification and characterization of penicillin G acylase from culture fluid of B. subtilis transformant From 8 1 of the culture fluid of B. subtilis (pUBC73), penicillin G acylase was purified by a series of procedures using Celite, DEAE-Sephadex A-50 column
105 TABLE 3 Summary of purification of penicillin G acylase from B. subtilis transformant Purification step
Vol (ml)
Total activity (U)
Total protein (mg)
Specific activity (U mg- 1)
Yield (%)
Crude reaction mixture Celite DEAE-Sephadex A-50 Sephacryl $200
8,000 350 35 15
12,700 9,890 7,810 6,900
7,050 122 77 62
1.8 81.1 101.4 111.3
100 78 61 54
chromatography and gel filtration on Sephacryl $200. The detailed purification steps were described in Materials and Methods and summarized in Table 3. From the DEAE-Sephadex column, the enzyme eluted between 0.35-0.45 M NaCI. The molecular weight of the native enzyme estimated by the Sephacryl $200 column was about 115,000 Da. The enzyme proteins obtained from the culture fluid of B. megaterium and purified from B. subtilis transformants were analyzed by a 10% SDS-polyacrylamide gel electrophoresis. The subunit molecular weight of penicillin G acylase isolated from B. megaterium and B. subtilis transformant was estimated to be 57,000 as illustrated in Fig. 2a, lane B and C, respectively. A thick protein band appeared from the total cell proteins of E. coli harboring pTCSE76 (lane E) when compared with the control (lane D). The band, however, turned out to be larger than the protein secreted from B. subtilis by about 3000 Da as judged by a prolonged SDS-polyacrylamide gel electrophoresis (Fig. 2b). The results from SDSpolyacrylamide gel electrophoresis and gel filtration indicate that the native enzyme consists of two identical subunits.
TABLE 4 Hydrolyzingactivities of penicillin G acylase Substrate
Rate of hydrolysis a (U mg-1)
Relative activity (%)
Penicillin G Cephaiothin Cefamandole Cephaioridine Cephaloglycin Cephalexin Cephradine Ampicillin Cephapirin Cefotaxime Cephalosporin C Penicillin N
111.3 95.0 81.7 7413 40.2 33.4 13.5 11.4 ND b ND ND ND
100 85.4 73.4 66.8 36.1 30.0 12.1 10.2 ND ND ND ND
a Rates of hydrolysiswere expressed as specific activities when each substrate was used. b ND, none detected.
106 Substrate specificity of purified penicillin G acylase
To investigate the industrial value, hydrolyzing activities of the enzyme were examined with various fl-lactam antibiotics. The enzyme effectively deacylated penicillin G, cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin, cephradine and ampicillin. The rate of hydrolysis of these substrates by the enzyme was different from each other and the relative rates to penicillin G were listed in the order of their rates in Table 4. This enzyme, however, could not hydrolyze penicillin N, cephapirin, cefotaxime or cephalosporin C.
Discussion
In this paper, we have described the cloning of the gene encoding penicillin G acylase from B. megaterium and the production of the enzyme in E. coli and B. subtilis. Attempts to clone the gene have been hampered by the poor expression in E. coli. The method employed in this study used S. aureus which is one of the most sensitive strains against cephalexin for the screening of E. coli transformants. As a consequence, colonies forming clear haloes were easily isolated on the lawn of S. aureus . Table 2 shows that B. subtilis harboring pUBC73 produced a more significantly increased level of the active enzyme than that produced by the original strain. When the gene was transferred to an expression vector, pTTQ19, penicillin G acylase was produced at the level of more than 30% of the total cell protein as illustrated in Fig. 2a, lane E. The enzyme protein, however, was mostly inactive and existed as inclusion bodies which were visible under the phase contrast microscope. Efforts for renaturing the enzyme are under progress in our laboratory. The expressed protein in E. coli was larger than the secreted protein from B. subtilis (Fig. 2b) by about 3000 Da. Apparently the signal sequence of the enzyme is being processed in B. subtilis. It has been reported that penicillin acylases produced by A. oiscosus (Ohashi et al., 1988), E. coli ATCC 11105 (Bock et al., 1983a, b), K. citrophila (Barbero et al., 1986), P. rettgeri ATCC 31052 (Daumy et al., 1985) and Pseudomonas sp. strain GK16 (Ichikawa et al., 1981) consist of two distinct subunits with respective molecular weights of 24,000 and 60,000, 20,500 and 69,000, 23,000 and 62,000, 24,500 and 65,000 and 16,000 and 54,000. On the other hand, penicillin V amidase from B. sphaericus consists of four identical subunits, each with a molecular weight of 36,500 (Olsson et al., 1985). In the case of B. megaterium, the enzyme consists of two identical subunits and its relative subunit size was 57,000 Da. This result was supported by the size of the subunit that appeared on a 10% SDS-polyacrylamide gel (Fig. 2) and the molecular weight of the native protein based on Sephacryl $200. It is likely that the enzyme prefers the aromatic ring structure as substitutes at a-carbon atom rather than alkyl groups. This enzyme tends to recognize and react on all of 6-APA, 7-ADCA and 7-ACA. The enzyme has also been tested for the acylating reactions between D-(a)-phenylglycine methylester and 7-ADCA and
107
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Fig. 2. Identification of penicillin G acylase by SDS-polyacrylamide gel electrophoresis. Samples were analyzed by 10% SDS-polyacrylamide gel electrophoresis. The protein bands were visualized with Coomassie Brilliant Blue R-250. (a) Lane A, contained marker proteins of known molecular weight. Lane B, concentrated culture fluid of B. megaterium using Celite. Lane C, purified proteins from culture fluid of B. subtilis harboring pUBC73 (for details see Materials and Methods). Small amount of other protein band appeared at the lower part of this lane. Lane D, total cell proteins of E. coli harboring pTTQ19. Lane E, total cell proteins of E. eoli harboring pTCSE76. The arrows show the bands corresponding to the penicillin G acylase protein. BPB, bromophenol blue dye. (b) Lane A, secreted proteins from B. subtilis harboring pUBC73. Lane B, mixed proteins applied on lane A and C. Lane C, one tenth of the amount of the proteins used for lane E of Fig. 2a.
D - ( a ) - p h e n y l g l y c i n e m e t h y l e s t e r a n d 7 - A C A to p r o d u c e c e p h a l e x i n a n d c e p h a l o g l y c i n , r e s p e c t i v e l y . I n b o t h cases, t h e rates of a c y l a t i n g r e a c t i o n s w e r e c l o s e l y p a r a l l e l e d to the rates o f d e a c y l a t i n g r e a c t i o n s . T h e s e facts s u g g e s t p o s s i b i l i t i e s o f s y n t h e s i z i n g p e n i c i l l i n s a n d c e p h a l o s p o r i n s in l a r g e scale u s i n g t h e r e v e r s e r e a c t i o n o f this e n z y m e .
References
Barbero, J.L., Buesa, J.M., Buitrago, G.G., Mendez, E., Perez-Aranda, A. and Garcia, J.L. (1986) Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila. Gene 49, 69-80. Balasingham, K., Warburton, D., Dunnill, P. and Lilly, M.D. (1972) The isolation and kinetic of penicillin amidase from Es.cheriehia coli. Biochim. Biophys. Acta 276, 250-256. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523. Bock, A., Wirth, R., Schmid, G., Schumacher, G., Lang, G. and Buckel, P. (1983a) The penicillin acylase from Eseherichia coil ATCC 11105 consists of two dissimilar subunits. FEMS Microbiol. Lett. 20, 135-139. Bock, A., Wirth, R., Schmid, G., Schumacher, G., Lang, G. and Buckel, P. (1983b) The two subunits of penicillin acylase are processed from a common precursor. FEMS Microbiol. Lett. 20, 141-144. Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., Crosa, J.H. and Falkow, S. (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95-113.
108 Boyer, H.W. and Roulland-Dussoix, D. (1969) A complementation analysis of the restriction and modification of DNA in Escherichm coll. J. Mol. Biol. 41,459-472. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 75, 248-254. Daumy, G.O., Danley, D. and McColl, A.S. (1985) Role of protein subunits in Proteus rettgeri penicillin G acylase. J. Bacteriol. 163, 1279-1281. Daumy, G.O., Williams, J.A., McColl, A.S., Zuzel, T.J. and Danley, D. (1986) Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coll. J. Bacteriol. 168, 431-433. Dubnau, D. and Davidoff-Abelson, R. (1971) Fate of transforming DNA following uptake by competent Bacillus subtilis. J. Mol. Biol. 56, 209-221. Fujii, T., Matsumoto, K. and Watanabe, T. (1976) Enzymatic synthesis of cephalexin. Process Biochem. 21-25. Garcia, J.L. and Buesa, J.M. (1986) An improved method to clone penicillin acylase gene: cloning and expression in Escherichia coli of penicillin G acylase from Kluyoera citrophila. J. Biotechnol. 3, 187-195. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557-580. Hardy, K.G. (1985) Bacillus cloning methods. In: Glover, D.M. (Ed.), DNA Cloning, Vol. II, IRL Press, Oxford. pp. 1-17. Ichikawa, S., Shibuya, Y., Matsumoto, K., Fujii, T., Komatsu, K. and Kodaira, R. (1981) Purification and properties of 7-beta-(4-carboxybutanamido)-cephalosporanic acid acylase produced by mutants derived from Pseudomonas. Agr. Biol. Chem. 45, 2231-2236. Keggins, K.M., Lovett, P.S. and Duvall, E.J. (1978) Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUBll0. Proc. Natl. Acad. Sci. 75, 1423-1427. Kornfeld, J.M. (1978) A new colorimetric method for the determination of 6-aminopenicillanic acid. Anal. Biochem. 86, 118-126. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. Mahajam, P.B. (1984) Penicillin acylases, An update. Appl. Biochem. Biotechnol. 9, 537-554. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Marmur, J. (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3, 208-218. Matsuda, A. and Komatsu, K. (1985) Molecular cloning and structure of the gene for 7-beta-(4-carboxybutanamido)-cephalosporanic acid acylase from a Pseudomonas strain. J. Bacteriol. 163, 1222-1228. Mayer, H., Collins, J. and Wagner, F. (1979) Cloning of the penicillin G acylase gene of Escherichia coli ATCC 11105 on multicopy plasmids. In: Timmis, K.N. and Puhler, A. (Eds.), Plasmids of Medical, Environmental and Commercial Importance, Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 459-470. Norrander, J., Kempe, T. and Messing, J. (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26, 101-106. Ohashi, H., Katsuta, Y., Hashizume, T., Abe, S., Kajiura, H., Hattori, H., Kamei, T. and Yano, M. (1988) Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus. Appl. Environ. Microbiol. 54, 2603-2607. Olsson, A., Hagstrom, T., Nilsson, B., Uhlen, M. and Gatenbeck, S. (1985) Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis. Appl. Environ. Microbiol. 49, 1084-1089. Shimizu, M., Okachi, R., Kimura, K. and Nara, T. (1975) Purification and properties of penicillin acylase from Kluyvera citrophila. Agr. Biol. Chem. 39, 1655-1661. Stark, M.J.R. (1987) Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of gene in Escherichia coli. Gene 51, 255-267. Vandamme, E.J. and Voets, J.P. (1974) Microbial penicillin acylases. Adv. Appl. Microbiol. 17, 311-369. Vieira, J. and Messing, J. (1982) The pUC plasmids, an M13 mp7-derived system for insertion mutagenesis and sequencing with synthetic primers. Gene 19, 259-268.
