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Cloning and co-expression of d -amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli
Enzyme and Microbial Technology 35 (2004) 514–518
Cloning and co-expression of d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli Hui Luo, Huimin Yu, Qiang Li, Zhongyao Shen∗ Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, Beijing 100084, PR China
Abstract To convert cephalosporin C to 7-aminocephalosporin (7-ACA), a d-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21(DE3)/pET-DAAO, a high DAAO activity of 250 U ml−1 was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E. coli BL21(DE3)/pET-ACY with a high expression level of 3000 U l−1 . A novel recombinant strain, BL21(DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml−1 and GL-7-ACA acylase activity of 950 U l−1 were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly. © 2004 Elsevier Inc. All rights reserved. Keywords: d-Amino acid oxidase; GL-7-ACA acylase; Co-expression; Recombinant E. coli; Induction and expression
1. Introduction d-Amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) are industrially important enzymes for the production of 7-aminocephalosporanic acid (7-ACA) from cephalosporin C (CPC) in a process involving two steps [1,2]. The first one involves the conversion by DAAO of cephalosporin C to 7--(5-carboxy-5-oxopentanamido)-cephalosporanic acid (keto-AD-7-ACA), which reacts spontaneously with the hydrogen peroxide produced in this reaction to render 7--(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA). In the second step, GL-7-ACA is further hydrolyzed to 7-ACA by a GL-7-ACA acylase. Therefore, the development of efficient enzymatic procedures is an important goal for pharmaceutical companies. Traditionally, these two enzymes are separately cultured and expressed with the recombinant strains with individual DAAO or GL-7-ACA acylase activity, which results in the ∗
Corresponding author. Tel.: +86 10 62788568; fax: +86 10 62770304. E-mail address: [email protected] (Z. Shen).
0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2004.08.036
complexity of operations and relatively high cost of enzymes production [3,4]. Several DAAOs have been characterized in a wide variety of organisms, and some DAAO genes have been cloned and sequenced. Among them, the yeast Trigonopsis variabilis has been reported to be a potent producer of DAAO [5]. In our previous work, a gene of d-amino acid oxidase was obtained by reverse transcription (RT)-PCR from T. variabilis, and the DAAO gene was expressed in recombinant Escherichia coli BL21(DE3)/pET-DAAO with a high DAAO activity level of 175 U l−1 [6]. In our previous work on GL-7-ACA acylase, a PCR-based method was developed to rapidly obtain the GL-7-ACA acylase gene of Pseudomonas sp. from soil samples and the obtained gene was successfully expressed in a recombinant E. coli [7]. In the present paper, the growth conditions for overproduction of DAAO and GL-7-ACA acylase were optimized with recombinant strains BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY. To simplify the culture of enzymes and directly catalyze CPC to 7-ACA with a single strain, an engineered bacterium possessing the activity of both d-amino
H. Luo et al. / Enzyme and Microbial Technology 35 (2004) 514–518
acid oxidase and GL-7-ACA acylase were also constructed and expressed in E. coli. 2. Materials and methods
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cell growth and then changed to 28 ◦ C when 0.3 mmol l−1 IPTG was added to induce the recombinant protein. The pH value was controlled at 7.0 with 4 mol l−1 NaOH solution. Agitation and aeration were set to 600 rev min−1 and 2.0 l min−1 .
2.1. Chemicals, enzymes and strains Restriction endonuclease, T4 DNA ligase and Pfu DNA polymerase were obtained from Takara Biotechnology Co. Ltd (Dalian, China). GL-7-ACA, which was used as a substrate in the GL-7-ACA acylase reaction, was synthesized according to reference [8]. E. coli TOP-10F (Invitrogen, The Netherlands) and BL21 (DE3) (Promega, USA) were used as host strains for genetic cloning and expression, respectively. The recombinant plasmids pET-DAAO and pET-ACY were constructed as previously described [6,7]. All other chemicals were of reagent grade and obtained from commercial sources. 2.2. Construction of a plasmid containing two co-expressed genes The plasmid containing two co-expressed genes was constructed by subcloning of pET-ACY and pET-DAAO. The plasmid pET-ACY was digested by Bgl II and Xho I, and the 2.2 kb DNA fragment was recovered. The plasmid pETDAAO was digested by BamH I and Xho I, and the 6.3 kb DNA fragment was recovered. After linkage of these two fragments and transforming the E. coli TOP-10F , a recombinant plasmid pET-DA harboring both DAAO and GL-7-ACA acylase genes was confirmed to be correct by digestion with suitable restriction endonucleases. The recombinant E. coli BL21 (DE3)/pET-DA was finally constructed by transformation of pET-DA into the host strain BL21 (DE3).
2.4. Assay of d-amino acid oxidase and GL-7-ACA acylase The DAAO activity was assayed by measuring the productivity of keto acid according to the method described previously [6]. GL-7-ACA acylase was assayed by the calorimetric method [9]. Whole cell protein SDS–PAGE was performed with 4% stacking gel and 12.5% running gel as described [10]. 2.5. Bioconversion of cephalosporin C Action of recombinant strain BL21 (DE3)/pET-DA on cephalosporin C was monitored by high performance liquid chromatography (HPLC). The cell pellets were collected by centrifugation at 10,000 rpm for 5 min and resuspended in 100 mmol l−1 sodium phosphate buffer (pH 8.0). The reaction mixture contained 1 ml of cell pellet-resuspending solution from culture, and 9 ml of cephalosporin C (0.05%, w/v). After incubation at 28 ◦ C for 60 min, 10 ml of HCl (0.2 M) was added to stop the reaction. The products were analyzed by HPLC with a Shimadzu C18 column with the UV detector set at 260 nm. 3. Results and discussion 3.1. Construction of recombinant plasmids of pET-DAAO, pET-ACY and pET-DA
2.3. Overproduction of DAAO and GL-7-ACA acylase Expression of recombinant DAAO and GL-7-ACA acylase in a flask was carried out as described previously [5]. Briefly, each bacteria clone was grown in LB medium supplemented with kanamycin (50 g ml−1 ) at 37 ◦ C with shaking (200–250 rev min−1 ). After incubation to an optical density at 600 nm of 1.0, 0.5 mmol l−1 isopropyl-lthiogalactopyranoside (IPTG) was added to the medium to induce the synthesis of desired proteins. The cultures were further cultivated at 28 ◦ C for another 20 h, and then collected by centrifugation. The fed-batch culture for overproduction of recombinant proteins was performed in a 5-l fermenter (B. Braun Company, Gemeny) containing 2 l semi-defined medium: glucose (5 g l−1 ), (NH4 )2 SO4 (2.0 g l−1 ), yeast extract (5 g l−1 ), peptone (10 g l−1 ), Na2 HPO4 ·12H2 O (6.0 g l−1 ), KH2 PO4 (1.5 g l−1 ), CaCl2 (20 mg l−1 ) and MgSO4 ·7H2 O (0.5 g l−1 ) with a pH of 7.0. The feed solution used for the fed-batch culture contained, per liter: 300 g glucose, 100 g yeast extract and 200 g peptone. The temperature was maintained at 37 ◦ C for
In traditional studies, the DAAO and GL-7-ACA acylase used to catalyze CPC are separately cultured and expressed. However, the production cost of 7-ACA is rather high at present with this method. In this work, a novel plasmid with co-expressed DAAO and GL-7-ACA acylase genes was further constructed to reduce the expense of these two enzymes. A gene of DAAO was obtained by RT-PCR from T. variabilis, and the DAAO gene was inserted into a prokaryotic expression vector pET-28a as described previously [6]. The DAAO recombinant plasmid pET-DAAO is shown in Fig. 1A. In our previous work on GL-7-ACA acylase [7], a PCRbased strategy was developed to rapidly obtain GL-7ACA acylase genes from soil samples, and a GL-7-ACA acylase recombinant plasmid pET-ACY was constructed as shown in Fig. 1B. Based on the techniques of genetic engineering and bioinformatics, the co-expressed plasmid pET-DA was constructed by subcloning of pET-ACY and pET-DAAO. A 2.2 kb DNA fragment containing the GL-7-ACA acylase gene was yielded
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Fig. 1. Physical map of expression plasmids pET-DAAO, pET-ACY and pET-DA. Symbols denote: PT7, T7 promoter; pBR322 ori, origin of replication; lacI, the mutant repressor gene of lac operon; kan, kanamycin-resistant marker; DAAO, d-amino acid oxidase gene; Acylase, GL-7-ACA acylase gene. The transcriptional direction of the DAAO and GL-7-ACA acylase genes is indicated with arrows.
by digestion of pET-ACY with Bgl II and Xho I. This fragment was then inserted into the pET-DAAO cleaved with BamH I and Xho I (Fig. 1C). In pET-DA, the transcription of the GL7-ACA acylase gene followed the DAAO gene and possessed its own T7 promoter. 3.2. Expression of DAAO It is interesting that the wild strain T. variabilis could not detect the d-amino acid oxidase activity by normal cultivation. But the gene from this strain could show high-level activity when expressed in E. coli. As described previously, the influences of induction and expression conditions on the expression of the recombinant protein were investigated. After the induction conditions in the flask were optimized, a high DAAO activity of 175 U ml−1 was obtained by a fed-batch culture with lactose induction in a 5-l fermenter [6]. Since the DAAO was intracellularly expressed, the DAAO activity could reach 250 U ml−1 after the cell was disrupted by sonication.
3.4. Expression of the co-expressed DAAO and GL-7-ACA acylase in plasmid pET-DA To simplify the operation of separate cultivation of these two individually expressed recombinant strains, the BL21(DE3)/pET-DA harboring both the DAAO gene and the GL-7-ACA acylase gene was constructed. The co-expression of these two genes was investigated in a flask with IPTG induction. Fig. 3 shows the SDS–PAGE analysis of the whole cell of E. coli BL21(DE3) harboring pET-DA, pET-DAAO and pET-ACY. The data in Fig. 3 revealed that DAAO and GL-7-ACA acylase could be overproduced by BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY, respectively. These two genes could also successfully be expressed in the co-expressed strain, BL21(DE3)/pET-DA, and the overall induced protein in BL21(DE3)/pET-DA is comparable to that in BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY. But in BL21(DE3)/pET-DA, a large amount of GL-7-ACA acylase was expressed as the precursor, a kind of immature protein without activity [12].
3.3. Expression of GL-7-ACA acylase As described in a previous work, the GL-7-ACA acylase gene of Pseudomonas sp. in soil was rapidly cloned with a novel PCR-based method [7]. To facilitate the expression of this gene in E. coli, the sequence encoding of a signal peptide of Pseudomonas GL-7-ACA acylase was deleted by a PCR method. The modified GL-7-ACA acylase gene was inserted into the prokaryotic expression vector pET-28a and the recombinant strain BL21(DE3)/pET-ACY was constructed. Although the recombinant DAAO was overproduced with lactose induction [6], the GL-7-ACA activity induced with lactose was only 30% of that with IPTG induction, indicating that the mechanisms of lactose inducing recombinant protein in E.coli might be complex [11]. Based on the optimal conditions of a flask level of 300 ml, a high expression level of 1200 U l−1 was obtained in a 5-l fermenter by fed-batch culture with 0.3 mmol l−1 IPTG induction (Fig. 2). The activity of GL-7-ACA acylase could reach 3000 U l−1 after the cell was disrupted by sonication.
Fig. 2. Cell growth and GL-7-ACA acylase activity accumulation of BL21(DE3)/pET-ACY. In a fed-batch culture, cell concentrations of BL21(DE3)/pET-ACY () and GL-7-ACA acylase activity () were measured and plotted. 0.3 mmol l−1 IPTG was added at 6 h, which is indicated with an arrow.
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Fig. 5. HPLC chromatography of cephalosporin C catalyzed by BL21(DE3)/pET-DA. Three major peaks in the HPLC chromatogram at retention times of 2.1, 3.1 and 11.3 min corresponded with 7-ACA, CPC and GL-7-ACA, respectively.
Fig. 3. SDS–PAGE analysis of the whole cell of E. coli BL21 (DE3) harboring pET-DA, pET-DAAO and pET-ACY. Lane 1, protein size marker with molecular weights of 94, 67, 43, 31 and 14 kDa, respectively. Lane 2–4, E. coli BL21 (DE3) harboring pET-DA, pET-DAAO and pET-ACY, respectively, grown at 28 ◦ C, with 0.5 mM IPTG induction. The proteins corresponding to DAAO, GL-7-ACA acylase precursor,␣-subunit and -subunit are indicated with arrows.
By a fed-batch culture with the same condition as BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY, the DAAO and GL-7-ACA acylase were expressed in BL21(DE3)/pET-DA with a similar trend as shown in Fig. 4. The highest values of DAAO activity and GL-7-ACA acylase activity of intact BL21(DE3)/pET-DA cells could reach
95 and 390 U l−1 , respectively. After disruption by sonication, DAAO activity and GL-7-ACA acylase activity were 140 and 950 U l−1 , which represented 56 and 32% of the solely expressed strains, BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY, respectively. Despite lower values than solely expressed strains in this work, the expression of the strain with two co-expressed genes is still a rather high level [3,13]. 3.5. Bioconversion of cephalosporin C by the co-expressed DAAO and GL-7-ACA acylase Intact cells of E. coli BL21(DE3)/pET-DA were used for the bioconversion of cephalosporin C, and the product of 60minutes’ reaction was analyzed by HPLC (Fig. 5). Three
Fig. 4. Cell growth, DAAO activity and GL-7-ACA acylase activity accumulation of BL21(DE3)/pET-DA. In a fed-batch culture, cell concentrations of BL21(DE3)/pET-DA (), DAAO activity () and GL-7-ACA acylase activity () were measured and plotted. 0.3 mmol l−1 IPTG was added at 10 h, which is indicated with an arrow.
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major peaks at retention times of 2.1, 3.1 and 11.3 min corresponded to 7-ACA, CPC and GL-7-ACA, respectively. The results showed that CPC can be hydrolyzed directly to 7-ACA by a single strain BL21(DE3)/pET-DA possessing the activity of both DAAO and GL-7-ACA acylase. However, there was still GL-7-ACA found in the reaction sample, indicating that the DAAO activity is redundant to the GL-7-ACA acylase in the co-expressed strain. Since the product (7-ACA) can be obtained directly with high productivity by intact BL21(DE3)/pET-DA cells, the culture and catalyzing process of this strain were much more simple. Moreover, the multiple, costly steps including purification and immobilization of the two enzymes were avoided, which would then reduce the cost of 7-ACA production possibly on a large scale. Thus, the strain and the catalyzing process in this work show great potential in the pharmaceutical industry. Acknowledgment This work was supported by Grant No. 200345 of the Foundation for the Author of National Excellent Doctoral Dissertation of PR China. References [1] Parmar A, Kumar H, Marwaha SS, Kennedy JF. Recent trends in enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid (7-ACA). Crit Rev Biotechnol 1998;18:1–12. [2] Luo H, Li Q, Tong YZ, Si YT, Shen ZY. Progress in enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid. Modern Chem Ind 2002;22:18–22.
[3] Hwang TS, Fu HM, Lin LL, Hsu WH. High-level expression of Trigonopsis variabilis d-amino acid oxidase in Escherichia coli using lactose as inducer. Biotechnol Lett 2000;22:655– 8. [4] Zheng YG, Li Y, Chen JF, Jiang WH, Zhao GP, Wang ED. Two novel engineered bacteria for secretory expression of glutaryl 7amino-cephalosporanic acid acylase. Biotechnol Lett 2001;23:1781– 7. [5] Pilone MS. d-Amino acid oxidase: new findings. Cell Mol Life Sci 2000;57:1732–47 [review]. [6] Luo H, Tong YZ, Li Q, Yu HM, Shen ZY. Cloning and expression of Trigonopsis variabilis d-amino acid oxidase gene in E. coli. Acta Microbiol Sinica 2004;44:336–9. [7] Shen ZY, Luo H, Li Q, Yu HM. A rapid procedure to obtain glutaryl7-aminocephalosporanic acid acylase genes in soil. Chinese Patent, 2003; Application No. 03143198.4. [8] Shibuya Y, Matsumoto K, Fujii T. Isolation and properties of 7(4-carboxybutanamido) cephalosporanic acid acylase-producing bacteria. Agric Biol Chem 1981;45:1561–7. [9] Kim DW, Yoon KH. Cloning and high expression of glutaryl 7aminocephalosporanic acid acylase gene from Pseudomonas diminuta. Biotechnol Lett 2001;23:1067–71. [10] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989. p. 880–6. [11] Hansen LH, Knudsen S, Sorensen SJ. The effect of the lacY gene on the induction of IPTG inducible promoters studied in Escherichia coli and Pseudomonas fluorescens. Curr Microbiol 1998;36:341– 7. [12] Li Y, Chen JF, Jiang WH, Mao X, Zhao GP, Wang D. In vivo posttranslational processing and subunit reconstitution of cephalosporin acylase from pseudomonas sp. 130. Eur Biochem 1999;262: 713–9. [13] Zheng YG, Li Y, Chen JF, Jiang WH, Zhao GP, Wang D. Two novel engineered bacteria for secretory expression of glutaryl 7-amino-cephalosporanic acid acylase. Biotechnol Lett 2001;23: 1781–7.
Cloning and co-expression of d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli Hui Luo, Huimin Yu, Qiang Li, Zhongyao Shen∗ Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, Beijing 100084, PR China
Abstract To convert cephalosporin C to 7-aminocephalosporin (7-ACA), a d-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21(DE3)/pET-DAAO, a high DAAO activity of 250 U ml−1 was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E. coli BL21(DE3)/pET-ACY with a high expression level of 3000 U l−1 . A novel recombinant strain, BL21(DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml−1 and GL-7-ACA acylase activity of 950 U l−1 were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly. © 2004 Elsevier Inc. All rights reserved. Keywords: d-Amino acid oxidase; GL-7-ACA acylase; Co-expression; Recombinant E. coli; Induction and expression
1. Introduction d-Amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) are industrially important enzymes for the production of 7-aminocephalosporanic acid (7-ACA) from cephalosporin C (CPC) in a process involving two steps [1,2]. The first one involves the conversion by DAAO of cephalosporin C to 7--(5-carboxy-5-oxopentanamido)-cephalosporanic acid (keto-AD-7-ACA), which reacts spontaneously with the hydrogen peroxide produced in this reaction to render 7--(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA). In the second step, GL-7-ACA is further hydrolyzed to 7-ACA by a GL-7-ACA acylase. Therefore, the development of efficient enzymatic procedures is an important goal for pharmaceutical companies. Traditionally, these two enzymes are separately cultured and expressed with the recombinant strains with individual DAAO or GL-7-ACA acylase activity, which results in the ∗
Corresponding author. Tel.: +86 10 62788568; fax: +86 10 62770304. E-mail address: [email protected] (Z. Shen).
0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2004.08.036
complexity of operations and relatively high cost of enzymes production [3,4]. Several DAAOs have been characterized in a wide variety of organisms, and some DAAO genes have been cloned and sequenced. Among them, the yeast Trigonopsis variabilis has been reported to be a potent producer of DAAO [5]. In our previous work, a gene of d-amino acid oxidase was obtained by reverse transcription (RT)-PCR from T. variabilis, and the DAAO gene was expressed in recombinant Escherichia coli BL21(DE3)/pET-DAAO with a high DAAO activity level of 175 U l−1 [6]. In our previous work on GL-7-ACA acylase, a PCR-based method was developed to rapidly obtain the GL-7-ACA acylase gene of Pseudomonas sp. from soil samples and the obtained gene was successfully expressed in a recombinant E. coli [7]. In the present paper, the growth conditions for overproduction of DAAO and GL-7-ACA acylase were optimized with recombinant strains BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY. To simplify the culture of enzymes and directly catalyze CPC to 7-ACA with a single strain, an engineered bacterium possessing the activity of both d-amino
H. Luo et al. / Enzyme and Microbial Technology 35 (2004) 514–518
acid oxidase and GL-7-ACA acylase were also constructed and expressed in E. coli. 2. Materials and methods
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cell growth and then changed to 28 ◦ C when 0.3 mmol l−1 IPTG was added to induce the recombinant protein. The pH value was controlled at 7.0 with 4 mol l−1 NaOH solution. Agitation and aeration were set to 600 rev min−1 and 2.0 l min−1 .
2.1. Chemicals, enzymes and strains Restriction endonuclease, T4 DNA ligase and Pfu DNA polymerase were obtained from Takara Biotechnology Co. Ltd (Dalian, China). GL-7-ACA, which was used as a substrate in the GL-7-ACA acylase reaction, was synthesized according to reference [8]. E. coli TOP-10F (Invitrogen, The Netherlands) and BL21 (DE3) (Promega, USA) were used as host strains for genetic cloning and expression, respectively. The recombinant plasmids pET-DAAO and pET-ACY were constructed as previously described [6,7]. All other chemicals were of reagent grade and obtained from commercial sources. 2.2. Construction of a plasmid containing two co-expressed genes The plasmid containing two co-expressed genes was constructed by subcloning of pET-ACY and pET-DAAO. The plasmid pET-ACY was digested by Bgl II and Xho I, and the 2.2 kb DNA fragment was recovered. The plasmid pETDAAO was digested by BamH I and Xho I, and the 6.3 kb DNA fragment was recovered. After linkage of these two fragments and transforming the E. coli TOP-10F , a recombinant plasmid pET-DA harboring both DAAO and GL-7-ACA acylase genes was confirmed to be correct by digestion with suitable restriction endonucleases. The recombinant E. coli BL21 (DE3)/pET-DA was finally constructed by transformation of pET-DA into the host strain BL21 (DE3).
2.4. Assay of d-amino acid oxidase and GL-7-ACA acylase The DAAO activity was assayed by measuring the productivity of keto acid according to the method described previously [6]. GL-7-ACA acylase was assayed by the calorimetric method [9]. Whole cell protein SDS–PAGE was performed with 4% stacking gel and 12.5% running gel as described [10]. 2.5. Bioconversion of cephalosporin C Action of recombinant strain BL21 (DE3)/pET-DA on cephalosporin C was monitored by high performance liquid chromatography (HPLC). The cell pellets were collected by centrifugation at 10,000 rpm for 5 min and resuspended in 100 mmol l−1 sodium phosphate buffer (pH 8.0). The reaction mixture contained 1 ml of cell pellet-resuspending solution from culture, and 9 ml of cephalosporin C (0.05%, w/v). After incubation at 28 ◦ C for 60 min, 10 ml of HCl (0.2 M) was added to stop the reaction. The products were analyzed by HPLC with a Shimadzu C18 column with the UV detector set at 260 nm. 3. Results and discussion 3.1. Construction of recombinant plasmids of pET-DAAO, pET-ACY and pET-DA
2.3. Overproduction of DAAO and GL-7-ACA acylase Expression of recombinant DAAO and GL-7-ACA acylase in a flask was carried out as described previously [5]. Briefly, each bacteria clone was grown in LB medium supplemented with kanamycin (50 g ml−1 ) at 37 ◦ C with shaking (200–250 rev min−1 ). After incubation to an optical density at 600 nm of 1.0, 0.5 mmol l−1 isopropyl-lthiogalactopyranoside (IPTG) was added to the medium to induce the synthesis of desired proteins. The cultures were further cultivated at 28 ◦ C for another 20 h, and then collected by centrifugation. The fed-batch culture for overproduction of recombinant proteins was performed in a 5-l fermenter (B. Braun Company, Gemeny) containing 2 l semi-defined medium: glucose (5 g l−1 ), (NH4 )2 SO4 (2.0 g l−1 ), yeast extract (5 g l−1 ), peptone (10 g l−1 ), Na2 HPO4 ·12H2 O (6.0 g l−1 ), KH2 PO4 (1.5 g l−1 ), CaCl2 (20 mg l−1 ) and MgSO4 ·7H2 O (0.5 g l−1 ) with a pH of 7.0. The feed solution used for the fed-batch culture contained, per liter: 300 g glucose, 100 g yeast extract and 200 g peptone. The temperature was maintained at 37 ◦ C for
In traditional studies, the DAAO and GL-7-ACA acylase used to catalyze CPC are separately cultured and expressed. However, the production cost of 7-ACA is rather high at present with this method. In this work, a novel plasmid with co-expressed DAAO and GL-7-ACA acylase genes was further constructed to reduce the expense of these two enzymes. A gene of DAAO was obtained by RT-PCR from T. variabilis, and the DAAO gene was inserted into a prokaryotic expression vector pET-28a as described previously [6]. The DAAO recombinant plasmid pET-DAAO is shown in Fig. 1A. In our previous work on GL-7-ACA acylase [7], a PCRbased strategy was developed to rapidly obtain GL-7ACA acylase genes from soil samples, and a GL-7-ACA acylase recombinant plasmid pET-ACY was constructed as shown in Fig. 1B. Based on the techniques of genetic engineering and bioinformatics, the co-expressed plasmid pET-DA was constructed by subcloning of pET-ACY and pET-DAAO. A 2.2 kb DNA fragment containing the GL-7-ACA acylase gene was yielded
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Fig. 1. Physical map of expression plasmids pET-DAAO, pET-ACY and pET-DA. Symbols denote: PT7, T7 promoter; pBR322 ori, origin of replication; lacI, the mutant repressor gene of lac operon; kan, kanamycin-resistant marker; DAAO, d-amino acid oxidase gene; Acylase, GL-7-ACA acylase gene. The transcriptional direction of the DAAO and GL-7-ACA acylase genes is indicated with arrows.
by digestion of pET-ACY with Bgl II and Xho I. This fragment was then inserted into the pET-DAAO cleaved with BamH I and Xho I (Fig. 1C). In pET-DA, the transcription of the GL7-ACA acylase gene followed the DAAO gene and possessed its own T7 promoter. 3.2. Expression of DAAO It is interesting that the wild strain T. variabilis could not detect the d-amino acid oxidase activity by normal cultivation. But the gene from this strain could show high-level activity when expressed in E. coli. As described previously, the influences of induction and expression conditions on the expression of the recombinant protein were investigated. After the induction conditions in the flask were optimized, a high DAAO activity of 175 U ml−1 was obtained by a fed-batch culture with lactose induction in a 5-l fermenter [6]. Since the DAAO was intracellularly expressed, the DAAO activity could reach 250 U ml−1 after the cell was disrupted by sonication.
3.4. Expression of the co-expressed DAAO and GL-7-ACA acylase in plasmid pET-DA To simplify the operation of separate cultivation of these two individually expressed recombinant strains, the BL21(DE3)/pET-DA harboring both the DAAO gene and the GL-7-ACA acylase gene was constructed. The co-expression of these two genes was investigated in a flask with IPTG induction. Fig. 3 shows the SDS–PAGE analysis of the whole cell of E. coli BL21(DE3) harboring pET-DA, pET-DAAO and pET-ACY. The data in Fig. 3 revealed that DAAO and GL-7-ACA acylase could be overproduced by BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY, respectively. These two genes could also successfully be expressed in the co-expressed strain, BL21(DE3)/pET-DA, and the overall induced protein in BL21(DE3)/pET-DA is comparable to that in BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY. But in BL21(DE3)/pET-DA, a large amount of GL-7-ACA acylase was expressed as the precursor, a kind of immature protein without activity [12].
3.3. Expression of GL-7-ACA acylase As described in a previous work, the GL-7-ACA acylase gene of Pseudomonas sp. in soil was rapidly cloned with a novel PCR-based method [7]. To facilitate the expression of this gene in E. coli, the sequence encoding of a signal peptide of Pseudomonas GL-7-ACA acylase was deleted by a PCR method. The modified GL-7-ACA acylase gene was inserted into the prokaryotic expression vector pET-28a and the recombinant strain BL21(DE3)/pET-ACY was constructed. Although the recombinant DAAO was overproduced with lactose induction [6], the GL-7-ACA activity induced with lactose was only 30% of that with IPTG induction, indicating that the mechanisms of lactose inducing recombinant protein in E.coli might be complex [11]. Based on the optimal conditions of a flask level of 300 ml, a high expression level of 1200 U l−1 was obtained in a 5-l fermenter by fed-batch culture with 0.3 mmol l−1 IPTG induction (Fig. 2). The activity of GL-7-ACA acylase could reach 3000 U l−1 after the cell was disrupted by sonication.
Fig. 2. Cell growth and GL-7-ACA acylase activity accumulation of BL21(DE3)/pET-ACY. In a fed-batch culture, cell concentrations of BL21(DE3)/pET-ACY () and GL-7-ACA acylase activity () were measured and plotted. 0.3 mmol l−1 IPTG was added at 6 h, which is indicated with an arrow.
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Fig. 5. HPLC chromatography of cephalosporin C catalyzed by BL21(DE3)/pET-DA. Three major peaks in the HPLC chromatogram at retention times of 2.1, 3.1 and 11.3 min corresponded with 7-ACA, CPC and GL-7-ACA, respectively.
Fig. 3. SDS–PAGE analysis of the whole cell of E. coli BL21 (DE3) harboring pET-DA, pET-DAAO and pET-ACY. Lane 1, protein size marker with molecular weights of 94, 67, 43, 31 and 14 kDa, respectively. Lane 2–4, E. coli BL21 (DE3) harboring pET-DA, pET-DAAO and pET-ACY, respectively, grown at 28 ◦ C, with 0.5 mM IPTG induction. The proteins corresponding to DAAO, GL-7-ACA acylase precursor,␣-subunit and -subunit are indicated with arrows.
By a fed-batch culture with the same condition as BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY, the DAAO and GL-7-ACA acylase were expressed in BL21(DE3)/pET-DA with a similar trend as shown in Fig. 4. The highest values of DAAO activity and GL-7-ACA acylase activity of intact BL21(DE3)/pET-DA cells could reach
95 and 390 U l−1 , respectively. After disruption by sonication, DAAO activity and GL-7-ACA acylase activity were 140 and 950 U l−1 , which represented 56 and 32% of the solely expressed strains, BL21(DE3)/pET-DAAO and BL21(DE3)/pET-ACY, respectively. Despite lower values than solely expressed strains in this work, the expression of the strain with two co-expressed genes is still a rather high level [3,13]. 3.5. Bioconversion of cephalosporin C by the co-expressed DAAO and GL-7-ACA acylase Intact cells of E. coli BL21(DE3)/pET-DA were used for the bioconversion of cephalosporin C, and the product of 60minutes’ reaction was analyzed by HPLC (Fig. 5). Three
Fig. 4. Cell growth, DAAO activity and GL-7-ACA acylase activity accumulation of BL21(DE3)/pET-DA. In a fed-batch culture, cell concentrations of BL21(DE3)/pET-DA (), DAAO activity () and GL-7-ACA acylase activity () were measured and plotted. 0.3 mmol l−1 IPTG was added at 10 h, which is indicated with an arrow.
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major peaks at retention times of 2.1, 3.1 and 11.3 min corresponded to 7-ACA, CPC and GL-7-ACA, respectively. The results showed that CPC can be hydrolyzed directly to 7-ACA by a single strain BL21(DE3)/pET-DA possessing the activity of both DAAO and GL-7-ACA acylase. However, there was still GL-7-ACA found in the reaction sample, indicating that the DAAO activity is redundant to the GL-7-ACA acylase in the co-expressed strain. Since the product (7-ACA) can be obtained directly with high productivity by intact BL21(DE3)/pET-DA cells, the culture and catalyzing process of this strain were much more simple. Moreover, the multiple, costly steps including purification and immobilization of the two enzymes were avoided, which would then reduce the cost of 7-ACA production possibly on a large scale. Thus, the strain and the catalyzing process in this work show great potential in the pharmaceutical industry. Acknowledgment This work was supported by Grant No. 200345 of the Foundation for the Author of National Excellent Doctoral Dissertation of PR China. References [1] Parmar A, Kumar H, Marwaha SS, Kennedy JF. Recent trends in enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid (7-ACA). Crit Rev Biotechnol 1998;18:1–12. [2] Luo H, Li Q, Tong YZ, Si YT, Shen ZY. Progress in enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid. Modern Chem Ind 2002;22:18–22.
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