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Enzyme and Microbial Technology 26 (2000) 165–170 The relative importance of intracellular proteolysis and transport on the yield of the periplasmic enzyme penicillin amidase in Escherichia coli夞 Z. Ignatovaa, S.-O. Enforsb, M. Hobbiea, S. Taruttisa, C. Vogta, V. Kaschea,* a Department of Biotechnology II, Technical University of Hamburg–Harburg, Denickestrasse 15, 21071 Hamburg, Germany b Department of Biotechnology, KTH, S-10044 Stockholm, Sweden Received 27 May 1999; received in revised form 4 August 1999; accepted 26 August 1999 Abstract Intracellular proteolysis is an important mechanism for regulating the level of the periplasmic enzyme penicillin amidase in Escherichia coli. Evidence is presented that the active enzyme is localized in the periplasmic space and maturation of pro-enzyme occurs during transport through the cytoplasmic membrane or rapidly after its entrance in the periplasm. The rate constants of the transport through cytoplasmic membrane and of the intracellular proteolysis were estimated to be 0.01 h and 0.5 h, respectively. This indicates that more than 90% of the synthesized pre-pro-enzyme is lost by intracellular proteolysis occurring in the cytoplasm. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Penicillin amidase; Proteolysis; E. coli; Membrane transport 1. Introduction Protein turnover is a normal process in cells to regulate the levels of specific proteins and eliminating damaged or abnormal proteins [1]. Proteins (enzymes) that are no longer required are hydrolyzed in energy– dependent reactions catalyzed by intracellular proteinases [2]. Cells contain a large number of endopeptidases that are localized in the cytoplasm, periplasm and cytoplasmic membrane [3,4]. In addition to endopeptidases, exopeptidases further degrade the peptides generated by endoproteolytic degradation of proteins [2]. Intracellular protein degradation is an active metabolic process and plays an important physiological function. For the biotechnical production of intra- and extracellular enzymes this process reduces the yield of these proteins [5]. The intracellular proteolysis has been studied in detail to minimize it. These includes the use of protease– deficient strains [6], growth of the host cells at low temperature [7], coexpression of molecular chaperones [8], optimization of the fermentation conditions [9,10], reducing the 夞 This work has been supported by DFG (Graduiertenkolleg, Biotechnologie, GK 95-3-98), DAAD (313/S-PPP) and Swedish National Research Council for Engineering Sciences (TFR). * Corresponding author. Tel.: ⫹49-40-42878-3018; fax: ⫹49-4042878-2127. E-mail address: [email protected] (V. Kasche) growth rate of the cells [11], and replacement of protease specific amino acid residues to eliminate proteinase cleavage sites [12,13]. The rate of intracellular proteolysis has until now been studied only for the intracellular proteins [4,5]. Penicillin amidase (PA) belongs to the group of bacterial enzymes that must be proteolytically processed to obtain enzymatic activity [14]. The enzyme is synthesized as a pre-pro-PA (ppPA) precursor form consisting of a secretory signal peptide and a pro-PA (pPA). The ppPA is transported to the periplasm (with removal of the signal sequence) and processed to a small A-subunit (24 kDa) and a larger Bsubunit (63 kDa) by proteolytic removal of a small spacer peptide [15,16]. The process starts with an intramolecular autoproteolytic step yielding a free N-terminal Ser of the B-chain [17,18]. In the further processing the small peptide (A-chain) is shortened from the C-end by intra- and intermolecular autoproteolysis [18,19]. The aim of this study was to determine the relative importance of the intracellular and periplasmic proteolysis, transport of ppPA and pPA maturation on the yield of active PA. 2. Materials and methods 2.1. Strain and plasmids Escherichia coli ATCC11105 (mutant strain derived from E. coli strain ATCC 9637; thr⫺, thi⫺) strain was 0141-0229/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 9 9 ) 0 0 1 3 0 - 1 166 Z. Ignatova et al. / Enzyme and Microbial Technology 26 (2000) 165–170 obtained from the German Culture Collection (DSMZ). E. ⫺ ⫺ ⫺ coli K5 (r⫺ k , mk , thr , thi ) was used as a host for a plasmid r pHM12 (tet ) carries a pac gene of a slowly processed (Gly263-Ser264) mutant PA precursor, where Thr263 has been replaced with Gly by site directed mutagenesis [20]. The expression of the mutant PA precursor is under the control of the tac promoter that was induced by IPTG. 2.2. Growth conditions The E. coli ATCC 11105 strain producing a wild type PA and E. coli K5 host strain expressing a mutant (Gly263Ser264) precursor, respectively, were grown at 28°C in 300 ml shake flasks containing 50 ml Luria–Bertani medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH7.5) supplemented with 10 g/ml tetracycline for cells harboring plasmid pHM12. The PA expression was induced at OD660 of approximately 1.0 with phenylacetic acid (PAA; 1g/liter) for cells expressing the wild type PA gene and IPTG (1 mM) for cells carrying a mutant (Gly263-Ser264) precursor gene. Growing cells of E. coli K5 host strain without plasmid served as a negative control. 2.3. Cell fractionation The cells harvested by centrifugation at 4000 ⫻ g for 20 min, were disrupted by sonication in phosphate buffer pH 7.5, I ⫽ 0.01 (Branson sonifier W 450 for 10 min with 50% duty cycle at 4°C) or by osmotic shock procedure according to Rodrigez et al. [21] with modification of the osmotic buffer (30 mM KH2PO4, pH 7.5, 1.4% EDTA, 40% saccharose). The periplasmatic fraction was separated from the spheroplasts suspension by centrifugation at 8000 ⫻ g for 40 min at 4°C. The cytoplasmic fraction (spheroplasts) were disrupted by sonication in distillated water and cell debris were harvested by centrifugation at 13 000 ⫻ g for 10 min. 2.4. Measurement of in vivo proteolysis To study the rate of intracellular proteolysis the E. coli ATCC 11105 (mutant strain derived from E. coli strain ATCC 9637; host strain producing a wild type PA) was grown under the same conditions described above. Three hours after induction of the PA synthesis with PAA (1g/ liter), chloramphenicol was added to the culture to a final concentration 100 g/ml to stop protein synthesis in accordance with the measurements of in vivo proteolysis of intracellular protein A [13]. Four-milliliter samples of this culture were removed at intervals. Each sample was analyzed by SDS-PAGE and Western blot hybridization methods after centrifugation and cell pellet sonication. Rate constants were calculated from the rate of disappearance of the scanned area of the full–length protein (ppPA). 2.5. Electrophoresis and immunoblotting SDS-PAGE of proteins using 12% gel was conducted according to the method of Laemmli [22]. Protein bands were detected by Coomasie blue staining and immunoblotting (Immun-blot Assay Kit, Bio–Rad). Proteins were transferred to the PVDF–membrane (Boehringer). Monoclonal antibody against the epitope in the B-chain of PA was purified as described by Kasche et al. [23]. The secondary antibody used was goat anti-mouse IgG (H⫹L) AP. Detection was accomplished by using 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium. PA with isoelectric point 7.0 (PA7.0) and pure mutant (Gly263-Ser264) precursor were purified as described in [20,23] and were used as standards. The calculations of relative optical density of the Western-blot were performed by using the program ONEDscan™ (1994; Scananalytics, A division of CSPI, Billerica, MA, USA). 2.6. Determination of enzyme activity A spectrophotometric assay by using the chromogenic substrate 6-nitro-3- phenylacetamido benzoic acid (NIPAB) was used [19]. The change in the absorbance at 380 nm per minute after mixing 950 l phosphate buffer pH 7.5 I ⫽ 0.2, 25 l NIPAB stock solution (5 mM in phosphate buffer, pH 7.5, I ⫽ 0.2) and 25 l sample with PA (solution or homogenized cells) was measured and converted in benzylpenicillin units. Under these conditions for pure PA (1 mg/ml) the change in the absorbance at 380 nm/min is 3.0, which converted in benzylpenicillin units corresponds to 42 U/ml. 2.7. Protein sequence analysis Automated Edman degradation was performed by using an Applied Biosystems pulsed liquid sequencer model 473A (Weiterstadt, Germany) with on-line analysis of the phenylthiohydantoin derivatives. After SDS-PAGE the protein bands were electroblotted to a polyvinylidene diflouride membrane (Boehringer Mannheim, Germany). The membrane was stained with Ponceau S. The bands of interest were cut out and activated by wetting them first with 100% methanol and then with 20% (v/v) methanol in distilled water. The bands were inserted in the slot of the sequencer’s cartridge and analyzed. 3. Results and discussion 3.1. Localization of active PA and the possible proteolysis products The whole cell lysate, periplasmic, and cytoplasmic fractions from E. coli expressing a wild-type PA and E. coli K5 strain harboring the plasmid pHM12 were separated on Z. Ignatova et al. / Enzyme and Microbial Technology 26 (2000) 165–170 167 Fig. 1. Localization of precursor, active PA, and proteolysis products of the penicillin amidase by SDS gel stained with Coomassie (A) and by Western blot of SDS gel (B). Lanes: 2, pure PA7.0; 3, sonicated homogenate from E. coli ATCC 11105 cells producing wild type PA; 4, supernatant after osmotic shock from E. coli ATCC 11105 cells producing wild type PA; 5, sonicated washed cells after osmotic shock from E. coli ATCC 11105 cells producing wild type PA; 6, sonicated homogenate from E. coli K5 host strain without plasmid-negative control; 7, pure mutant PA-precursor; 8, sonicated homogenate from E. coli K5 cells producing mutant (Gly263-Ser264) precursor; 9, supernatant after osmotic shock from E. coli K5 cells producing mutant (Gly263-Ser264) precursor; 10, sonicated washed cells after osmotic shock from E. coli K5 cells producing mutant (Gly263-Ser264) precursor; 1, 11, molecular weight markers. The E. coli ATCC 11105 strain expressing a wild type PA gene or E. coli K5 harboring the plasmid pHM12 were grown in LB medium for 6 h at 28°C after PAA (1g/liter) or IPTG (1 mM) induction respectively. In all experiments the same amount of cells was used. 12.5% polyacrylamide gels. The ppPA, pPA and mature enzyme were localized by immunoblotting analysis using a monoclonal antibody with epitope against B-chain of PA. The active PA was localized in the periplasmic space and practically all active enzyme could be obtained by osmotic shock procedure (Fig. 1, Lane 4). This was also verified by direct measurements of the enzyme activity in the periplasmic fraction (Fig. 1, Lane 4) and the cytoplasmic fraction (Fig. 1, Lane 5) after osmotic shock (data not shown). More than 95% of the PA-activity was found in the cell supernatant after the osmotic shock procedure. The pre-pro-PA (ppPA) with signal peptide has a MW about 3.5 kDa larger than pPA. This was, however, found in the cells but not in the periplasm (Fig. 1B, Lanes 3, 8). The unprocessed mutant (Gly263-Ser264) precursor was found mainly in the periplasm too (Fig. 1, Lane 9), but not in the homogenate of plasmid-free host strain (Fig. 1, Lane 6) serving as a negative control. Little or none of precursor (pPA) was observed in Western blot of the periplasmic proteins in cells producing the wild type PA (Fig. 1B, Lane 4). These observations allow us to conclude that the maturation of the active enzyme must occur during transport through the cell membrane or rapidly after its entrance into the periplasm. This is in agreement with previous studies [15,24]. In the Western blot of sonicated cells (Fig. 1B, Lanes 3, 8) and of sonicated spheroplasts (Fig. 1B, Lanes 5, 10), a large number of protein bands besides ppPA, pPA and B-chain of PA are specifically blotted. As unspecific blotting can be ruled out (Fig. 1, Lane 6), no bands in the sonicated host cells served as a negative control were blotted. All additional blotted bands also possess an epitope for anti-PA monoclonal antibody. Their molecular weights differ from the B-chain of PA or unprocessed mutant (Gly263Ser264) precursor and their intensity in the cytoplasm (sonicated spheroplasts) is much larger than in the periplasm (Fig. 1B, Lanes 4, 9). This demonstrates that all these products are derived from the PA-precursor or active PA and are produced in the cells by intracellular proteolysis. The band intensity is proportional to the strength of monoclonal antibody/antigen interactions and depends on the antigen-structure. For example the relative intensity of the band at 42 kDa is much larger than the intensity of the B-chain PA-band presumably due to the better accessibility of the epitope for the first antibody used. Thus the band intensities are not reliable for concentration comparison and the sum intensity of all proteolytic products is much greater than descendant protein (e.g. ppPA). 3.2. Proteolysis of ppPA in vivo The growth curve of E. coli ATCC 11105 strain showed a characteristic exponential growth during the first 5 h followed by stationary phase (Fig. 2B). The synthesis of PA was strongly correlated with cell growth. The maximum PA-activity was obtained after 6 h of induction with PAA (1g/liter) in the early stationary phase. In the Western blot of the samples from the cultivation of E. coli ATCC 11105 expressing a wild type PA the intensity of ppPA-band was constant in the first 6 h after induction with PAA (Fig. 2A) and disappeared progressively after entrance of the cells in the stationary phase (Fig. 1B, Lanes 8, 9). This indicates that the ppPA synthesis stops after approximately 6 h (Fig. 2B). A similar result were observed for large scale fed batch fermentation of the same strain (data not shown). The Western blot analysis during 0 to 9 h after induction of the PA-synthesis with PAA reveals as well bands with molecular mass corresponding to the full-length ppPA (97 kDa), pPA (92 kDa) and B-chain of PA (63 kDa) as well additional bands representing protein fragments with remained anti-PA binding properties. These results confirm the presence of proteolysis products but they provide no information on the rate of intracellular proteolysis. To mea- 168 Z. Ignatova et al. / Enzyme and Microbial Technology 26 (2000) 165–170 Fig. 2. (A) Western blot of SDS-PAGE of growing E. coli ATCC 11105 culture producing a wild type PA subsequent to the addition of inducer PAA. Lane 1, pure PA7.0; Lanes 2–9, a sonicated homogenate 0, 1, 2, 3, 4, 5, 7, 9 h after induction with PAA; Lane 10, pure mutant (Gly263-Ser264) precursor. (B) Change in the PA-activity of growing E. coli ATCC 11105, Total units (U per liter of culture medium, open circles) as a function of time after induction with PAA (1g/liter); Biomass dry weight (mg per liter of culture medium, closed circles). (C) Western blot of SDS-PAGE of the E. coli ATCC 11105 producing a wild type PA after inhibition of de novo protein synthesis. Lane 1, pure PA7.0; Lane 13, pure mutant (Gly263-Ser264) precursor; Lanes 2–12, a sonicated homogenate from E. coli strain incubated (3 h after induction with PAA) with chloramphenicol (100 g/ml) at 28°C for 0, 20, 40, 60, 80, 100, 120, 140, 160, 200, and 240 min, respectively. The arrows indicate the 97-kDa band of ppPA. (D) Change in the intensity of full-length protein ppPA-band (97 kDa) in Western blot (Fig. 2C) of samples taken 3 h after induction with PAA and after addition of chloramphenicol (100 g/ml). For each point the same number of cells were analyzed. sure the rate of degradation of ppPA in vivo, chloramphenicol (100 g/ml) was added to the induced culture of E. coli ATCC 11105 to stop de novo full–length protein synthesis while the culture was further incubated under the same growth conditions as described by Yang et al. [13]. The cells were removed at intervals after chloramphenicol addition and the protein pattern was analyzed by immunoblotting (Fig. 2C). A progressive linear decrease of the amounts of ppPA, pPA, and B-chain of PA and a contaminant increase of the bands of the degradation products was observed. The major degradation products have a molecular mass about 67 kDa, 60 kDa, 50 kDa, and 42 kDa. The N-terminal sequence analysis of this degradation product shows that the 60 kDa-band is a result of cleavage between amino acids Lys273-Ala274 (numbering is accordingly to the published primary protein structure of the pPA [15]. The 50 kDa and 42 kDa-degradation bands are results of the following cleavage occurring between Ser346-Ala347 and Thr396-Gln397, respectively. All these cleavage sites are localized in the B-chain of the mature PA. The proteolysis in vivo could be followed by analysis of the intensity of the 97-kDa band (ppPA) from the Western blot pattern (indicated by the arrow, Fig. 2C). The ratio of degradation products to full-length protein (ppPA) was significantly increased and this proteolysis continues until most of ppPA is degraded. To quantify the kinetics of the in vivo degradation shown in Fig. 2C, the relative amounts of ppPA (Fig. 2D) and degradation products were estimated from the gel scanning of the polypeptide peak areas. The origin of the ⬇ 100-kDa-protein band that appeared after chloramphenicol addition is not known (Fig. 2C). Moreover the intensities of this band increases parallel with the decreasing intensity of ppPA-band. It might be result of cross-reactions of proteolysis products with host proteins, as previously suggested by Keilmann et al. [7] The amino acids composition influences the proteolysis in proteins. The N-end rule [25] for eucaryotic proteins have also relevance in E. coli as observed for intracellular proteolysis of intracellular proteins [13]. Twenty-three from the first 100 amino acids from the N-terminus of B-chain have basic or aromatic side chains. The presence of aromatic or basic amino acid residues in the N-terminus destabilizes proteins and provokes proteolysis. 3.3. Estimation of the fraction of pre-pro-PA (ppPA) converted to the mature enzyme The yield of active PA is controlled by the following processes—intracellular proteolysis and inclusion body formation, membrane transport and maturation of ppPA, and proteolysis of PA in the periplasm (Fig. 3). The inclusion body formation by high-level expression of proteins in the wild type E. coli itself can result in the accumulation of aggregates from unprocessed and untranslocated ppPA only Z. Ignatova et al. / Enzyme and Microbial Technology 26 (2000) 165–170 169 Fig. 3. (A) Scheme of the conversion of ppPA to PA including the side reactions. kd is the first order rate constant for intracellular proteolysis of ppPA, kt is the first order rate constant for transport and maturation of ppPA. pPA is activated in a first order process by intramolecular proteolysis, kd,p is the first order rate constant for proteolysis of PA in the periplasm. (B) Scheme of the PA-processing. The subscript c, p denotes cytoplasm and periplasm, respectively. when the growing culture at 28°C was shifted to 42°C [26]. In our case the E. coli ATCC 11105 strain expressing the wild type PA was grown only at 28°C and the inclusion body formation could not influence the ppPA transport and can be neglected. Then the change in the concentration of ppPA in the cytoplasm per cell is given by the following relation: d[ppPA]c/dt ⫽ synthesis rate ⫺ 共k d ⫹ k t兲 䡠 [ppPA]c (1) where kd represents the rate constant of intracellular proteolysis and kt is transport and maturation rates constant. The change of active PA in the periplasm per cell assuming a steady state in the pro-enzyme content in the membrane is determined by the relation: d[PA]p/dt ⫽ k t [ppPA]c ⫺ k d,p [PA]p (2) where the last term on the right hand side (kd,p [PA]p) represents hydrolysis of PA in the periplasm by peptidases with the first order rate constant kd,p (Fig. 3A). From the curve of PA activity it follows that the synthesis of PA ends after about 6 h (Fig. 2B). The concentration of PA in the periplasm is practically constant for the following 7 h and then the rate of proteolysis in the periplasm (last term in the Eq. (2)) can be neglected. PA-activity usually remained constant up to 70 h in the cell lysate and in the separated periplasmic fraction by incubation at room temperature (data not shown). The measurements of the PA-activity in sonicated cells during the cultivation verified that the PA content per cell increases by a factor of approximately 2 during the synthesis phase after induction (data not shown). This was also observed for the direct measurement of the PA-content per cell from the intensities of the B-chain in Fig. 2A. They increased from 0.18 to 0.30 in the first three hours. In this time the intensity of the ppPA-band was practically constant (3.7 ⫾ 0.1). Assuming that the B-chain and ppPA have the same intensity for the same protein content kt can be determined from the integration of Eq. (2), using the above intensities. Then: [PA]p,t ⫺ [PA]p,0 ⫽ k t [ppPA]c 䡠 t (3) k t was determined to be 0.01 h. This value has a large experimental error. To determine (kd ⫹ kt ) for the degradation shown in Fig. 2C, the intensity of ppPA-band was determined from the Western blot (Fig. 2D). Chloramphenicol inhibit the protein, resp. de novo full–length ppPA synthesis. At this point the transport across cytoplasmic membrane is assumed to remain constant and in this case after integration Eq. (1) simplifies to: ln ([ppPA]c,t/[ppPA]c,0) ⫽ ⫺ (k d ⫹ k t) 䡠 t (4) where [ppPA]c,0 is the initial concentration of ppPA before addition of chloramphenicol. The value of kd may be estimated from the disappearance of the ppPA-band (97 kDa) and it was determined to be 0.5 h. This value is of the same order of magnitude as has been observed previously for 170 Z. Ignatova et al. / Enzyme and Microbial Technology 26 (2000) 165–170 ppPA [27] and other intracellular proteins [13]. From the values of k t and (kd ⫹kt ) it follows that more than 90% of ppPA is degraded by intracellular proteolysis. 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