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EFFICIENT PRODUCTION OF STAPHYLOCOCCUS SIMULANS LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT ESCHERICHIA COLI a
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Piotr Szweda , Grzegorz Gorczyca , Pawel Filipkowski , Magdalena Zalewska & Slawomir Milewski
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Department of Pharmaceutical Technology and Biochemistry , Gdansk University of Technology , Gdansk , Poland b
Department of Food Chemistry , Technology and Biotechnology, Gdansk University of Technology , Gdansk , Poland Accepted author version posted online: 06 Aug 2013.Published online: 06 Aug 2013.
To cite this article: Preparative Biochemistry and Biotechnology (2013): EFFICIENT PRODUCTION OF STAPHYLOCOCCUS SIMULANS LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT ESCHERICHIA COLI , Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2013.829499 To link to this article: http://dx.doi.org/10.1080/10826068.2013.829499
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ACCEPTED MANUSCRIPT Efficient production of Staphylococcus simulans lysostaphin in a benchtop bioreactor by recombinant Escherichia coli Piotr Szweda1, Grzegorz Gorczyca1, Pawel Filipkowski2, Magdalena Zalewska1, Slawomir Milewski1 1
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Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Gdansk, Poland 2 Department of Food Chemistry, Technology and Biotechnology, Gdansk University of Technology, Gdansk, Poland Corresponding author: Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, ul. G. Narutowicza 11/12, 80233, Gdańsk, Poland. Tel.: 0048583471693. Fax: 0048583471144. E-mail: [email protected] Abstract Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high costs of its production. In the present study we have determined the optimal conditions for lysostaphin production in 5 l batch bioreactor. The enzyme production was based on, constructed earlier in our laboratory, heterologous, E. coli expression system assigned as pBAD2Lys. An evident influence of both physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. The efficiency of production of about 13000 U/L have been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH = 6 and temperature of 37 ºC in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally over 80 000 units of lysostaphin
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ACCEPTED MANUSCRIPT were obtained from one (batch) bioreactor – 3L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. From our best knowledge, it is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.
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KEYWORDS: lysostaphin, Staphylococcus aureus, bioreactor, Escherichia coli expression system
INTRODUCTION Lysostaphin was first isolated from Staphylococcus simulans biovar staphylolyticus by Schindler and Schuhardt in 1964[1]. The enzyme is a zinc metalloproteinase, which degrades the cell wall of almost all known staphylococcal species. The target of the lysostaphin activity are the pentaglycine interpeptide bridges of the unique staphylococcal peptidoglycan[2,3]. Other Gram-positive and Gram-negative bacteria are not susceptible to this enzyme [1]. The unique biological activity of lysostaphin presents numerous possibilities for applications of this enzyme as an antistaphylococcal agent in medicine, veterinary and food industry. The high therapeutic potential of the enzyme has been confirmed in many animal models of staphylococcal infections, including endocarditis[4] and ocular infections[5]. Several reports have shown that lysostaphin is a promising agent also in the eradication of biofilm of staphylococci from biotic and abiotic surfaces including medical devices e.g. catheters[6–9]. The latest proposed clinical application of lysostaphin is its immobilization on the surface of wound dressing materials or meshes which are used in herniorrhaphy[10,11]. The enzyme has been also
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ACCEPTED MANUSCRIPT successfully used in the treatment of staphylococcal bovine mastitis, a disease of major economic importance in the veterinary and dairy industry[12]. In 2005, the Wall’s group constructed transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 µg/mL of their milk. Protection against S. aureus mastitis appeared to be achievable with as little as 3 µg/mL of lysostaphin in milk. The antistaphylocoocal activity of the
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lysostaphin-containing milk was confirmed by in vitro assays[13]. Szweda and coworkers demonstrated potential application of lysostaphin as a food preservative. The enzyme effectively reduced the number of staphylococcal cells in milk and pork, however was less active in eradication of staphylococci from mayonnaise salads[14].
In spite of so many advantages and promising results of preliminary research, the enzyme still has not been widely used in medical or veterinary practice or in food industry. This is in part a consequence of still limited data on possible toxicity or other side effects of lysostaphin for humans but also results from the high costs of lysostaphin production. In this paper, we have presented a novel approach for production of recombinant lysostaphin, using a heterologous, E. coli expression system, constructed earlier in our laboratory[14]. Conditions of the fermentation process in the 5 l batch bioreactor have been optimized to maximize the yield of the active protein, with a relatively low cost of its production.
MATERIALS AND METHODS Bacterial Strains, Plasmid And Reagents
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ACCEPTED MANUSCRIPT The recombinant lysostaphin was produced in the cells of E. coli TOP10F’ strain (Invitrogen, USA) transformed with the plasmid pBAD2Lys, constructed in our laboratory[14]. The strain of Staphylococcus aureus ATCC29213 was used for analysis of bactericidal – lytic activity of recombinant enzyme. All media and buffers’ components
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were purchased from Sigma.
Growth Media E. coli TOP10F’ transformed with the pBAD2Lys plasmid and S. aureus ATCC29213 were grown and stored on the petri dishes with LA agar (Peptone 10 g/L, Yeast extract 5 g/L, NaCl 10 g/L, agar 20 g/L). Three different media were used for optimization of production of recombinant lysostaphin in E. coli cells: LB medium (Peptone 10 g/L, Yeast extract 5 g/L, NaCl 10 g/L), enriched LB medium (Peptone 16 g/L, Yeast extract 10 g/L, NaCl 5 g/L, K2HPO4 4 g/L, glycerol 3 g/L) and medium assigned as HCDC (high cell density cultivation, composed of glucose 10 g/L, Yeast extract 2 g/L, KH2PO4 10 g/L, (NH4)2HPO4 4 g/L, MgSO4 x 7H2O 1.4 g/L and sea salts 3.5 g/L). After induction of expression of the enzyme the enriched LB medium was continuously supplemented with glycerol (at feeding rate of 3 or 9 g/Lxh) and HCDC medium was supplemented with “second stage medium” (glucose 700 g/L, MgSO4 x 7H2O 20 g/L and Yeast extract 40 g/L) at feeding rate of 3 mL/Lxmin.
Optimization Of Fermentation Conditions The preliminary optimization of production of recombinant lysostaphin was performed using LB medium. A single colony of E. coli TOP10F’ transformed with pBAD2Lys was
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ACCEPTED MANUSCRIPT inoculated into 300 mL the medium containing ampicillin (100 mg/L). The bacteria were cultivated overnight aerobically on a rotator shaker (200 rpm), at 37 °C. The obtained cell suspension was transferred to the bioreactor containing 2.7-L of a sterile LB medium supplemented with ampicillin (100 mg/L). All fermentations were carried out in a 5-L bioreactor (Biostat C, Braun Co., Germany) with control modules for pH, temperature,
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agitation, dissolved oxygen (DO) and air flow. The aeration rate was maintained at 1.0 vvm (volume per volume per minute - gas volume flow per unit of liquid volume per minute) and then increased to 1.2 vvm when required, as a result the concentration of dissolved oxygen was kept above 5 % saturation. Agitation was controlled by changing the Motor speed ratio in the 400-800 rpm range. The pH of the medium was controlled at the designed level (range 5 to 8.5) by adding 25 % (w/w) ammonium hydroxide or 1M sulfuric acid solutions. Temperature was maintained constant at 25 °C, 30 °C or 37 °C. Protein expression was induced by addition of a filter sterilized solution of arabinose to the final concentration of 0.1 %, when the OD600 reached 0.2 – 0.5. Samples of the cell suspension were collected at 0, 2, 4, 6, 8 h after induction to measure the following parameters: biomass wet weight (WCW) (g/L), protein production yield (mg/L), lysostaphin specific activity (U/mg) and specific yield (U/L).
After completion of desired time of production, cells were harvested by centrifugation (9000 rpm for 20 min at 4 °C) and obtained cell pellet was stored at − 20 °C.
In order to obtain higher yields of production of the enzyme two alternative media were tested. In comparison to LB both of them contained more carbon and nitrogen
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ACCEPTED MANUSCRIPT components: Peptone, Yeast extract and glycerol - enriched LB medium and Yeast extract and glucose – HCDC medium. Important components of alternative media were also mineral salts: KH2PO4 – enriched LB medium and KH2PO4, (NH4)2HPO4, MgSO4 x 7H2O and sea salts – HCDC. In the case of enriched LB medium expression of the enzyme was induced exactly in the same conditions as in the case of LB medium, whilst
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in the case of HCDC medium the inducer was introduced after 12 hours of growing bacteria (OD600 was not determined). After induction three different strategies of glycerol feeding of enriched LB medium were tested: 1) feeding with glycerol at range of 3 g/Lxh, 2) feeding with glycerol at range of 9 g/Lxh, 3) feeding with glycerol at range of 9 g/Lxh, and with additional portion of inducer, which was solubilized in glycerol. The HCDC was supplemented with the “second stage medium” at range of 3 mL/min. For all tested media cultivation of bacteria was carried out at the time of eight hours after induction.
Protein Purification The purification of the enzyme was carried out according to the method proposed by Szweda and coworkers[15], which was optimized for two other expression systems of production of recombinant lysostaphin constructed in our laboratory. The results of expression and purification were analyzed with SDS PAGE and Western blotting, using anti-His6 antibodies conjugated with the horseradish peroxidase. Protein concentration was determined according to Bradford[16]. The purified enzyme was dialyzed against the buffer 20 mM Tris-HCl, pH 7.5, 200 mM NaCl. The obtained final solution of the protein was frozen using liquid nitrogen and kept in – 80 °C for further analysis.
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ACCEPTED MANUSCRIPT Measurement Of Lysostaphin Specific Activity The bacteriolytic activity of lysostaphin was determined by the spectrophotometric assay according to Marova and Kovar[17], with slight modifications. The reaction mixture, containing 6 mL suspension of S. aureus ATCC 29213 cells diluted in 0.1 M phosphate buffer (pH 7.5) to OD600 = 0.25, was preincubated at 37 ºC for 10 min, and then different
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volumes of lysostaphin solutions were added. After 10 min of incubation at 37ºC, the changes in turbidity of the reaction mixture were determined. One unit (U) of the lysostaphin activity was defined as an amount of preparation causing a 50 % reduction in turbidity of the 6 mL cell suspension within 10 min at 37 ºC.
Other Methods SDS-PAGE was performed in 12 % (w/v) gels by the method of Laemmli[18], the proteins were detected by staining with Coomassie brilliant blue. In the case of western blot analysis the proteins were electroblotted onto nitrocellulose membranes and incubated for 1 h with anti-His6 antibodies conjugated to horseradish peroxidase (a dilution of 1:1000). Blots were visualized with 3,3’,5,5’-tetramethylbnzidine solution. A PageRulerTMPlus purchased from Thermo Scientific was used as a protein molecular mass standard (250, 130, 95, 72, 55, 36, 28, 17 and 10 kDa) for both SDS-PAGE and western blot analysis.
RESULTS AND DISCUSSION Lysostaphin is one of the most promising non-antibiotic antistaphylococcal agent[19]. Within last 15 years, there have been numerous reports, which definitely confirm therapeutic potential of this enzyme and possibilities of its application as a food
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ACCEPTED MANUSCRIPT preservative. Unfortunately, despite the successful results of research carried in many laboratories, lysostaphin is still not used in clinical practice, nor in the food industry. In fact, it is widely used only in the research field as a staphylolytic agent for the liberation of intracellular enzymes, nucleic acids, and cell membrane and surface components of these bacteria. In order to use substance of interest as a pharmaceutical agent or food
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preservative, the major limitations are high cost and great amount of time required for carry out all preclinical and clinical research. At present, lysostaphin is at the second stage of clinical trials. After approval, next crucial issue is the enzyme large-scale production. Effective production process and its low cost should be taken here into consideration. Although several efficient heterologous systems of expression of lysostaphin has been developed[14,15,21–24], its’ optimization usually was limited to laboratory-scale production. To date the most economical technology of lysostaphin production has been developed by Mierau and coworkers[21,22]. Using a Lactococcus lactis nisin-controlled system, the authors optimized conditions of recombinant lysostaphin production at the 3000 L-scale and obtained final yield of lysostaphin after purification about 120 g per batch (40 mg/L). The same group of researchers revealed that in the small scale (1L fermentor) the efficiency of the expression system can be increased to the level of 300 mg/L[22]. Obvious requirement of solution proposed Mierau and coworkers is the use of mentioned Lactococcus lactis nisin- controlled system, which is less known and more laborious than the most popular E. coli systems applied for production of heterologous proteins.
Optimization Of Fermentation Conditions
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ACCEPTED MANUSCRIPT The carried out investigation, revealed a strong influence of most important parameters of fermentation process on the performance of production of the recombinant protein defined as a number of specific units of the enzyme produced in 1 L of culture (U/L) Tables 1–3. The conditions of growing bacteria affected both: amount of produced protein (mg/L) and its specific activity (U/mg), which in combination give specific
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activity yield (U/L). The amount of produced protein was the most dependent on the mechanical stirrer speed (aeration) and also, but less temperature. The most effective production of the enzyme (16.8 mg/L) was obtained in the experiment with the lowest stirrer’s speed (400 rpm). Twofold increase in the speed of the stirrer (800 rpm) resulted in almost five-fold decrease in the amount of produced protein (3.62 mg/L), and because aerationdid not affect specific activity of produced lysostaphin it was equivalent with about five-fold decrease of the specific activity yield of the enzyme (from 6200 U/L to 1194 U/L respectively) (Table 1). In contrast to temperature and aeration, the pH of growing media, affected mainly specific activity (U/mg), which is probably a consequence of influence of pH of the environment on the folding of the produced molecules of the protein. The enzyme characterized with the best specific activity (695 U/mg, when expressed in 37 ºC) was obtained, when bacteria were grown in pH 6.0, in comparison the enzyme produced in pH 5.0 and 7.0 characterized with activity 573 and 396 U/mg respectively (when grown in 37 ºC) (Table 3). Growing bacteria with optimum conditions of aeration (400 rpm) and pH (6.0) but in 25 or 30 ºC resulted in production of enzyme with higher specific activity 863 and 914 U/mg respectively, however with lower amount yield 7.9 and 13.1 mg/L respectively in comparison to 19.3 mg/L obtained in 37 ºC. The carried out investigation revealed that the maximum level of production of
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ACCEPTED MANUSCRIPT recombinant lysostaphin in bioreactor can be achieved in the following conditions: pH 6.0, temperature 37 °C and mechanical stirrer speed 400 rpm. We also found that the optimum time of growing of bacteria after adding the inducer was 8 hours. Extension of time of expression of the protein resulted in decrease in its specific activity (Table 5). This observation is in agreement with results of our previous investigations of production
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of recombinant lysostaphin in arabinose induced systems using a classical flask approach[15]. Under these conditions, in the case of the most effective of three carried out experiments (Table 3 and 4), the following culture parameters values were obtained: wet cell weight 6.43 g/l; lysostaphin mass produced per liter of culture medium 19.32 mg/l; lysostaphin specific activity 695 U/mg; number of lysostaphin units obtained from 1 liter of culture medium 13427.
Optimization Of Media Composition Further improvement in the production efficiency of the enzyme was achieved as a result of modification of the growth medium composition (Table 6). However four different media were tested (including three different glycerol feeding strategies of enriched LB medium), the positive effect was observed only in the case of enriched LB media supplemented with glycerol at the rate of 3 g/Lxh, started at the time of induction. Additional glycerol administration has led to almost twofold increase in the specific activity yield of the enzyme (25923 U/L), which in fact was mainly a consequence of increase in the amount of produced biomass (66 %) in comparison to LB medium. Neither evident increase of WCW parameter (mg of enzyme/g wet biomass) nor specific activity (U/mg) was observed in comparison to process carried out in classical LB
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ACCEPTED MANUSCRIPT medium. Finally over 80 000 units of lysostaphin were obtained from one (batch) bioreactor – 3L of culture of E. coli TOP10F’ transformed with pBAD2lys plasmid.
The tested HCDC medium was earlier successfully used by Liu and coworkers for production of recombinant nitrilase[25]. Because of high content of carbon sources
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(especially glucose) using this medium led to evident increase in the amount of biomass of cells of grown bacteria, which was observed by Liu and coworkers and in presented investigation. On the other hand large amount of glucose, which is an inhibitor of arabionose operon was a reason of low level of expression of gene coding for lysostaphin. In fact this result was rather expected, and the medium was only used to determine whether it is possible to improve production efficiency of biomass of cells of E. coli transformed with pBAD2Lys plasmid using a rich carbon source media in comparison to LB medium. Surprisingly the performance of production of grown E. coli cells in enriched LB medium supplemented with glycerol at range of 3 and 9 g/Lxh was nearly the same, and production of the enzyme was much better in the case of media supplemented with less amount of glycerol (Table 6).
CONCLUSIONS The production efficiency of recombinant lysostaphin achieved in the presented research is comparable with these obtained in the industrial scale technology developed by Mierau and coworkers, 35.6 and 40 mg/L respectively, and is much better then in described to date other methods of production of this protein using E. coli expression systems. From our best knowledge, the most efficient methods of recombinant lysostaphin production
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ACCEPTED MANUSCRIPT using E. coli as a host have been developed and described in our group[14,15]. The efficiency of investigated previously systems assigned as pBADLys and pETLys were 3125 and 13687 U/L, respectively. Optimization of the pBAD2Lys system developed in 2007, with fermentation performed in the small scale flask culture resulted in obtaining 10 mg of the protein with specific activity 498 U/mg from 1 L of culture. Thus the
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technology described in this paper led to five-fold increase in specific activity yield in comparison to the flask culture method in the case of pBAD2Lys system and two-fold increase in comparison to the pETLys system. Our previous research revealed that production of recombinant lysostaphin using pBAD system is definitely easier in comparison to pET system, which is much more sensitive to many parameters of protein production process, such us: strain of the host bacteria, moment of induction and temperature of the culture after induction, which strongly affects the amount of produced enzyme. These observations were the most important reason of selection of pBAD2Lys expression system for developing a bioreactor technology of production of recombinant lysostaphin. There are also no doubts that using pBAD or any other E. coli systems is easier and more often used in most laboratories in comparison to the Lactococcus lactis nisin – controlled system.
The enzyme is commercially available, but is still very expensive. At the moment the price of 1 and 15 mg of lysostaphin (specific activity 500 U/mg) produced by leading supplier is 100 and 800 €, respectively, which can be a problem even for its application in research field or as therapeutic agent and is completely unacceptable for its using as
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ACCEPTED MANUSCRIPT food preservative. The calculated costs of production of 1000 units of the enzyme with proposed technology is only about 3.5 Euros (Fig. 1).
The novel method of recombinant lysostaphin production presented in this paper is convenient and low-cost. Enhanced availability of the enzyme is crucial not only for its
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application in medicine, veterinary and food technology, but also for further tests of its properties during pre-clinical and clinical research. Thus we think that presented results can be interesting for many research groups, or small biotechnology companies. The described technology can be easily adapted in any laboratory for production of large amount of active lysostaphin for their own research or as a commercial product, which could be rather difficult in the case of technology developed by the group of Mierau[21,22]. Moreover we declare that all plasmids constructed in our laboratory, which can be used for production of recombinant lysostaphin in E. coli as a host are freely available, when used for research, not commercial purposes.
ACKNOWLEDGMENTS This research was supported by the grant no. N N405 420739 from the Polish Ministry of Science and Foundation for Polish Science program VENTURES (VENTURES/20105/2).
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ACCEPTED MANUSCRIPT 25.
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ACCEPTED MANUSCRIPT TABLE 1. Influence of agitation intensity on parameters of lysostaphin production in batch fermentation using the pBAD2Lys expression system; T= 37 oC, pH=7
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Rpm 400
600
800
WCW [g/l]
4,20
8,90
6,95
yield [mg/l]
16,81
5,47
3,62
yield on WCW [mg/g]
4,00
0,61
0,52
specific activity [U/mg]
369
326
330
6203
1782
1194
1477,0
200,2
171,8
9
9
9
689,28
197,97
132,64
1,87
0,61
0,40
specific yield [U/l] Lys specific yield on WCW [U/g] process time [h] specific productivity [U/L·h] space time yield [mg/L·h]
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ACCEPTED MANUSCRIPT TABLE 2. Influence of pH on parameters of lysostaphin production in batch fermentation using the pBAD2Lysexpression system; T=37 oC, rpm 400
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pH 5
5,5
6
7
WCW [g/l]
4,47
5,65
6,43
4,20
yield [mg/l]
17,49
28,33
19,32
16,81
yield on WCW [mg/g]
3,92
5,01
3,00
4,00
specific activity [U/mg]
573,4
429
695
369
specific yield [U/l]
10030
12155
13427
6203
Lys specific yield on WCW
2245,5
2149,5
2086,6
1477,10
9
9
9
9
1114,44
1350,55
1491,84
689,28
1,94
3,15
2,15
1,87
[U/g] process time [h] specific productivity [U/L·h] space time yield [mg/L·h]
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ACCEPTED MANUSCRIPT TABLE 3. Influence of temperature on parameters of lysostaphin production in batch fermentation using the pBAD2Lysexpression system; rpm=400, pH=6
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T [oC] 25
30
37
WCW [g/l]
7,61
4,49
6,41
yield [mg/l]
7,90
13,14
19,71
yield on WCW [mg/g]
1,04
2,93
3,07
specific activity [U/mg]
862,9
914
641,00
specific yield [U/l]
6813
12014
12632
Lys specific yield on WCW
895,5
2677,6
2245,5
9
9
9
756,97
1334,85
1403,54
0,88
1,46
2,19
[U/g] process time [h] specific productivity [U/L·h] space time yield [mg/L·h]
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ACCEPTED MANUSCRIPT TABLE 4. Result of two additional batch fermentations in optimal conditions: T=37 oC, pH=6, rpm=400, using pBAD2Lysexpression system
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Repetition 1
2
WCW [g/l]
6,43
4,47
yield [mg/l]
19,32
17,49
yield on WCW [mg/g]
3,00
3,92
specific activity [U/mg]
695
573,4
specific yield [U/l]
13427
10030
Lys specific yield on WCW
5279,0
5726,1
9
9
1491,84
1114,44
2,15
1,94
[U/g] process time [h] specific productivity [U/L·h] space time yield [mg/L·h]
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ACCEPTED MANUSCRIPT TABLE 5. Influence of the time of bacteria growth after adding the inducer on parameters of enzme using the pBAD2Lys expression system; rpm=400; pH=6, T=37 oC
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Time of growing bacteria from the moment of induction [h] 0
2
4
6
8
OD600
0,30
0,86
0,95
0,96
0,99
pO2 [%]
77,50
59,00
69,00
68,20
80,60
WCW [g/l]
---
5,52
6,10
6,22
6,41
yield [mg/l]
---
8,13
12,41
16,23
19,71
yield on WCW [mg/g]
---
1,47
2,03
2,61
3,07
specific activity [U/mg]
---
590
630
680
641
specific yield [U/l]
---
4797
7818
11036
12634
Lys specific yield on WCW
---
869,0
1281,7
1774,3
1971,0
---
3
5
7
9
specific productivity [U/L·h] ---
1598,90
1563,66
1576,63
1403,79
space time yield [mg/L·h]
2,71
2,48
2,32
2,19
[U/g] process time [h]
---
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ACCEPTED MANUSCRIPT TABLE 6. Influence of growing media composition on the efficiency production of recombinant lysostaphin, using the pBAD2Lysexpression system; T=37 oC, pH=6, rpm=400 Medium modifications:
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LB
Glycerol
Glycerol
Glycerol
9g/Lxh
9g/Lxh
3g/Lxh
HCDC
2x inductor WCW [g/l]
6,43
10,47
8,59
10,70
29,12
yield [mg/l]
19,32
13,26
4,22
35,61
1,89
yield on WCW [mg/g]
3,00
1,27
0,49
3,33
0,06
specific activity [U/mg]
695
379
741
728
357,7
specific yield [U/l]
13427
5026
3129
25923
676
Lys specific yield on
2086,6
480,0
364,1
2422,9
676
process time [h]
9
9
9
9
22
specific productivity
1491,84
558,45
122,70
2880,33
30,72
2,15
1,47
0,17
3,96
0,09
WCW [U/g]
[U/L·h] space time yield [mg/L·h]
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ACCEPTED MANUSCRIPT FIGURE 1. Detailed cost analysis for a single experiment carried out in the optimal conditions and medium. The analysis included all stages of enzyme production (inoculum preparation, bioreactor process, purification, enzyme parameters estimation). The salary for qualified laboratory personnel was assumed at 1200 EUR/month (160 h/month; single experiment time - 25h). Electricity consumption needed for power and control unit of
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bioreactor was adopted at a safe level of 5kWh. Because of its minimal effect, energy cost for other laboratory works was ignored. Prices of reagents and media components were based on Sigma-Aldrich catalogue. Under these conditions the cost of the single experiment was set at 267 EUR. Cost involved with production of 1000 units of lysostaphin is then at a level of 3.34 EUR.
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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
EFFICIENT PRODUCTION OF STAPHYLOCOCCUS SIMULANS LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT ESCHERICHIA COLI a
a
b
a
Piotr Szweda , Grzegorz Gorczyca , Pawel Filipkowski , Magdalena Zalewska & Slawomir Milewski
a
a
Department of Pharmaceutical Technology and Biochemistry , Gdansk University of Technology , Gdansk , Poland b
Department of Food Chemistry , Technology and Biotechnology, Gdansk University of Technology , Gdansk , Poland Accepted author version posted online: 06 Aug 2013.Published online: 06 Aug 2013.
To cite this article: Preparative Biochemistry and Biotechnology (2013): EFFICIENT PRODUCTION OF STAPHYLOCOCCUS SIMULANS LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT ESCHERICHIA COLI , Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2013.829499 To link to this article: http://dx.doi.org/10.1080/10826068.2013.829499
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ACCEPTED MANUSCRIPT Efficient production of Staphylococcus simulans lysostaphin in a benchtop bioreactor by recombinant Escherichia coli Piotr Szweda1, Grzegorz Gorczyca1, Pawel Filipkowski2, Magdalena Zalewska1, Slawomir Milewski1 1
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Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Gdansk, Poland 2 Department of Food Chemistry, Technology and Biotechnology, Gdansk University of Technology, Gdansk, Poland Corresponding author: Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, ul. G. Narutowicza 11/12, 80233, Gdańsk, Poland. Tel.: 0048583471693. Fax: 0048583471144. E-mail: [email protected] Abstract Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high costs of its production. In the present study we have determined the optimal conditions for lysostaphin production in 5 l batch bioreactor. The enzyme production was based on, constructed earlier in our laboratory, heterologous, E. coli expression system assigned as pBAD2Lys. An evident influence of both physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. The efficiency of production of about 13000 U/L have been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH = 6 and temperature of 37 ºC in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally over 80 000 units of lysostaphin
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ACCEPTED MANUSCRIPT were obtained from one (batch) bioreactor – 3L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. From our best knowledge, it is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.
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KEYWORDS: lysostaphin, Staphylococcus aureus, bioreactor, Escherichia coli expression system
INTRODUCTION Lysostaphin was first isolated from Staphylococcus simulans biovar staphylolyticus by Schindler and Schuhardt in 1964[1]. The enzyme is a zinc metalloproteinase, which degrades the cell wall of almost all known staphylococcal species. The target of the lysostaphin activity are the pentaglycine interpeptide bridges of the unique staphylococcal peptidoglycan[2,3]. Other Gram-positive and Gram-negative bacteria are not susceptible to this enzyme [1]. The unique biological activity of lysostaphin presents numerous possibilities for applications of this enzyme as an antistaphylococcal agent in medicine, veterinary and food industry. The high therapeutic potential of the enzyme has been confirmed in many animal models of staphylococcal infections, including endocarditis[4] and ocular infections[5]. Several reports have shown that lysostaphin is a promising agent also in the eradication of biofilm of staphylococci from biotic and abiotic surfaces including medical devices e.g. catheters[6–9]. The latest proposed clinical application of lysostaphin is its immobilization on the surface of wound dressing materials or meshes which are used in herniorrhaphy[10,11]. The enzyme has been also
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ACCEPTED MANUSCRIPT successfully used in the treatment of staphylococcal bovine mastitis, a disease of major economic importance in the veterinary and dairy industry[12]. In 2005, the Wall’s group constructed transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 µg/mL of their milk. Protection against S. aureus mastitis appeared to be achievable with as little as 3 µg/mL of lysostaphin in milk. The antistaphylocoocal activity of the
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lysostaphin-containing milk was confirmed by in vitro assays[13]. Szweda and coworkers demonstrated potential application of lysostaphin as a food preservative. The enzyme effectively reduced the number of staphylococcal cells in milk and pork, however was less active in eradication of staphylococci from mayonnaise salads[14].
In spite of so many advantages and promising results of preliminary research, the enzyme still has not been widely used in medical or veterinary practice or in food industry. This is in part a consequence of still limited data on possible toxicity or other side effects of lysostaphin for humans but also results from the high costs of lysostaphin production. In this paper, we have presented a novel approach for production of recombinant lysostaphin, using a heterologous, E. coli expression system, constructed earlier in our laboratory[14]. Conditions of the fermentation process in the 5 l batch bioreactor have been optimized to maximize the yield of the active protein, with a relatively low cost of its production.
MATERIALS AND METHODS Bacterial Strains, Plasmid And Reagents
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ACCEPTED MANUSCRIPT The recombinant lysostaphin was produced in the cells of E. coli TOP10F’ strain (Invitrogen, USA) transformed with the plasmid pBAD2Lys, constructed in our laboratory[14]. The strain of Staphylococcus aureus ATCC29213 was used for analysis of bactericidal – lytic activity of recombinant enzyme. All media and buffers’ components
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were purchased from Sigma.
Growth Media E. coli TOP10F’ transformed with the pBAD2Lys plasmid and S. aureus ATCC29213 were grown and stored on the petri dishes with LA agar (Peptone 10 g/L, Yeast extract 5 g/L, NaCl 10 g/L, agar 20 g/L). Three different media were used for optimization of production of recombinant lysostaphin in E. coli cells: LB medium (Peptone 10 g/L, Yeast extract 5 g/L, NaCl 10 g/L), enriched LB medium (Peptone 16 g/L, Yeast extract 10 g/L, NaCl 5 g/L, K2HPO4 4 g/L, glycerol 3 g/L) and medium assigned as HCDC (high cell density cultivation, composed of glucose 10 g/L, Yeast extract 2 g/L, KH2PO4 10 g/L, (NH4)2HPO4 4 g/L, MgSO4 x 7H2O 1.4 g/L and sea salts 3.5 g/L). After induction of expression of the enzyme the enriched LB medium was continuously supplemented with glycerol (at feeding rate of 3 or 9 g/Lxh) and HCDC medium was supplemented with “second stage medium” (glucose 700 g/L, MgSO4 x 7H2O 20 g/L and Yeast extract 40 g/L) at feeding rate of 3 mL/Lxmin.
Optimization Of Fermentation Conditions The preliminary optimization of production of recombinant lysostaphin was performed using LB medium. A single colony of E. coli TOP10F’ transformed with pBAD2Lys was
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ACCEPTED MANUSCRIPT inoculated into 300 mL the medium containing ampicillin (100 mg/L). The bacteria were cultivated overnight aerobically on a rotator shaker (200 rpm), at 37 °C. The obtained cell suspension was transferred to the bioreactor containing 2.7-L of a sterile LB medium supplemented with ampicillin (100 mg/L). All fermentations were carried out in a 5-L bioreactor (Biostat C, Braun Co., Germany) with control modules for pH, temperature,
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agitation, dissolved oxygen (DO) and air flow. The aeration rate was maintained at 1.0 vvm (volume per volume per minute - gas volume flow per unit of liquid volume per minute) and then increased to 1.2 vvm when required, as a result the concentration of dissolved oxygen was kept above 5 % saturation. Agitation was controlled by changing the Motor speed ratio in the 400-800 rpm range. The pH of the medium was controlled at the designed level (range 5 to 8.5) by adding 25 % (w/w) ammonium hydroxide or 1M sulfuric acid solutions. Temperature was maintained constant at 25 °C, 30 °C or 37 °C. Protein expression was induced by addition of a filter sterilized solution of arabinose to the final concentration of 0.1 %, when the OD600 reached 0.2 – 0.5. Samples of the cell suspension were collected at 0, 2, 4, 6, 8 h after induction to measure the following parameters: biomass wet weight (WCW) (g/L), protein production yield (mg/L), lysostaphin specific activity (U/mg) and specific yield (U/L).
After completion of desired time of production, cells were harvested by centrifugation (9000 rpm for 20 min at 4 °C) and obtained cell pellet was stored at − 20 °C.
In order to obtain higher yields of production of the enzyme two alternative media were tested. In comparison to LB both of them contained more carbon and nitrogen
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ACCEPTED MANUSCRIPT components: Peptone, Yeast extract and glycerol - enriched LB medium and Yeast extract and glucose – HCDC medium. Important components of alternative media were also mineral salts: KH2PO4 – enriched LB medium and KH2PO4, (NH4)2HPO4, MgSO4 x 7H2O and sea salts – HCDC. In the case of enriched LB medium expression of the enzyme was induced exactly in the same conditions as in the case of LB medium, whilst
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in the case of HCDC medium the inducer was introduced after 12 hours of growing bacteria (OD600 was not determined). After induction three different strategies of glycerol feeding of enriched LB medium were tested: 1) feeding with glycerol at range of 3 g/Lxh, 2) feeding with glycerol at range of 9 g/Lxh, 3) feeding with glycerol at range of 9 g/Lxh, and with additional portion of inducer, which was solubilized in glycerol. The HCDC was supplemented with the “second stage medium” at range of 3 mL/min. For all tested media cultivation of bacteria was carried out at the time of eight hours after induction.
Protein Purification The purification of the enzyme was carried out according to the method proposed by Szweda and coworkers[15], which was optimized for two other expression systems of production of recombinant lysostaphin constructed in our laboratory. The results of expression and purification were analyzed with SDS PAGE and Western blotting, using anti-His6 antibodies conjugated with the horseradish peroxidase. Protein concentration was determined according to Bradford[16]. The purified enzyme was dialyzed against the buffer 20 mM Tris-HCl, pH 7.5, 200 mM NaCl. The obtained final solution of the protein was frozen using liquid nitrogen and kept in – 80 °C for further analysis.
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ACCEPTED MANUSCRIPT Measurement Of Lysostaphin Specific Activity The bacteriolytic activity of lysostaphin was determined by the spectrophotometric assay according to Marova and Kovar[17], with slight modifications. The reaction mixture, containing 6 mL suspension of S. aureus ATCC 29213 cells diluted in 0.1 M phosphate buffer (pH 7.5) to OD600 = 0.25, was preincubated at 37 ºC for 10 min, and then different
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volumes of lysostaphin solutions were added. After 10 min of incubation at 37ºC, the changes in turbidity of the reaction mixture were determined. One unit (U) of the lysostaphin activity was defined as an amount of preparation causing a 50 % reduction in turbidity of the 6 mL cell suspension within 10 min at 37 ºC.
Other Methods SDS-PAGE was performed in 12 % (w/v) gels by the method of Laemmli[18], the proteins were detected by staining with Coomassie brilliant blue. In the case of western blot analysis the proteins were electroblotted onto nitrocellulose membranes and incubated for 1 h with anti-His6 antibodies conjugated to horseradish peroxidase (a dilution of 1:1000). Blots were visualized with 3,3’,5,5’-tetramethylbnzidine solution. A PageRulerTMPlus purchased from Thermo Scientific was used as a protein molecular mass standard (250, 130, 95, 72, 55, 36, 28, 17 and 10 kDa) for both SDS-PAGE and western blot analysis.
RESULTS AND DISCUSSION Lysostaphin is one of the most promising non-antibiotic antistaphylococcal agent[19]. Within last 15 years, there have been numerous reports, which definitely confirm therapeutic potential of this enzyme and possibilities of its application as a food
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ACCEPTED MANUSCRIPT preservative. Unfortunately, despite the successful results of research carried in many laboratories, lysostaphin is still not used in clinical practice, nor in the food industry. In fact, it is widely used only in the research field as a staphylolytic agent for the liberation of intracellular enzymes, nucleic acids, and cell membrane and surface components of these bacteria. In order to use substance of interest as a pharmaceutical agent or food
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preservative, the major limitations are high cost and great amount of time required for carry out all preclinical and clinical research. At present, lysostaphin is at the second stage of clinical trials. After approval, next crucial issue is the enzyme large-scale production. Effective production process and its low cost should be taken here into consideration. Although several efficient heterologous systems of expression of lysostaphin has been developed[14,15,21–24], its’ optimization usually was limited to laboratory-scale production. To date the most economical technology of lysostaphin production has been developed by Mierau and coworkers[21,22]. Using a Lactococcus lactis nisin-controlled system, the authors optimized conditions of recombinant lysostaphin production at the 3000 L-scale and obtained final yield of lysostaphin after purification about 120 g per batch (40 mg/L). The same group of researchers revealed that in the small scale (1L fermentor) the efficiency of the expression system can be increased to the level of 300 mg/L[22]. Obvious requirement of solution proposed Mierau and coworkers is the use of mentioned Lactococcus lactis nisin- controlled system, which is less known and more laborious than the most popular E. coli systems applied for production of heterologous proteins.
Optimization Of Fermentation Conditions
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ACCEPTED MANUSCRIPT The carried out investigation, revealed a strong influence of most important parameters of fermentation process on the performance of production of the recombinant protein defined as a number of specific units of the enzyme produced in 1 L of culture (U/L) Tables 1–3. The conditions of growing bacteria affected both: amount of produced protein (mg/L) and its specific activity (U/mg), which in combination give specific
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activity yield (U/L). The amount of produced protein was the most dependent on the mechanical stirrer speed (aeration) and also, but less temperature. The most effective production of the enzyme (16.8 mg/L) was obtained in the experiment with the lowest stirrer’s speed (400 rpm). Twofold increase in the speed of the stirrer (800 rpm) resulted in almost five-fold decrease in the amount of produced protein (3.62 mg/L), and because aerationdid not affect specific activity of produced lysostaphin it was equivalent with about five-fold decrease of the specific activity yield of the enzyme (from 6200 U/L to 1194 U/L respectively) (Table 1). In contrast to temperature and aeration, the pH of growing media, affected mainly specific activity (U/mg), which is probably a consequence of influence of pH of the environment on the folding of the produced molecules of the protein. The enzyme characterized with the best specific activity (695 U/mg, when expressed in 37 ºC) was obtained, when bacteria were grown in pH 6.0, in comparison the enzyme produced in pH 5.0 and 7.0 characterized with activity 573 and 396 U/mg respectively (when grown in 37 ºC) (Table 3). Growing bacteria with optimum conditions of aeration (400 rpm) and pH (6.0) but in 25 or 30 ºC resulted in production of enzyme with higher specific activity 863 and 914 U/mg respectively, however with lower amount yield 7.9 and 13.1 mg/L respectively in comparison to 19.3 mg/L obtained in 37 ºC. The carried out investigation revealed that the maximum level of production of
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ACCEPTED MANUSCRIPT recombinant lysostaphin in bioreactor can be achieved in the following conditions: pH 6.0, temperature 37 °C and mechanical stirrer speed 400 rpm. We also found that the optimum time of growing of bacteria after adding the inducer was 8 hours. Extension of time of expression of the protein resulted in decrease in its specific activity (Table 5). This observation is in agreement with results of our previous investigations of production
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of recombinant lysostaphin in arabinose induced systems using a classical flask approach[15]. Under these conditions, in the case of the most effective of three carried out experiments (Table 3 and 4), the following culture parameters values were obtained: wet cell weight 6.43 g/l; lysostaphin mass produced per liter of culture medium 19.32 mg/l; lysostaphin specific activity 695 U/mg; number of lysostaphin units obtained from 1 liter of culture medium 13427.
Optimization Of Media Composition Further improvement in the production efficiency of the enzyme was achieved as a result of modification of the growth medium composition (Table 6). However four different media were tested (including three different glycerol feeding strategies of enriched LB medium), the positive effect was observed only in the case of enriched LB media supplemented with glycerol at the rate of 3 g/Lxh, started at the time of induction. Additional glycerol administration has led to almost twofold increase in the specific activity yield of the enzyme (25923 U/L), which in fact was mainly a consequence of increase in the amount of produced biomass (66 %) in comparison to LB medium. Neither evident increase of WCW parameter (mg of enzyme/g wet biomass) nor specific activity (U/mg) was observed in comparison to process carried out in classical LB
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ACCEPTED MANUSCRIPT medium. Finally over 80 000 units of lysostaphin were obtained from one (batch) bioreactor – 3L of culture of E. coli TOP10F’ transformed with pBAD2lys plasmid.
The tested HCDC medium was earlier successfully used by Liu and coworkers for production of recombinant nitrilase[25]. Because of high content of carbon sources
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(especially glucose) using this medium led to evident increase in the amount of biomass of cells of grown bacteria, which was observed by Liu and coworkers and in presented investigation. On the other hand large amount of glucose, which is an inhibitor of arabionose operon was a reason of low level of expression of gene coding for lysostaphin. In fact this result was rather expected, and the medium was only used to determine whether it is possible to improve production efficiency of biomass of cells of E. coli transformed with pBAD2Lys plasmid using a rich carbon source media in comparison to LB medium. Surprisingly the performance of production of grown E. coli cells in enriched LB medium supplemented with glycerol at range of 3 and 9 g/Lxh was nearly the same, and production of the enzyme was much better in the case of media supplemented with less amount of glycerol (Table 6).
CONCLUSIONS The production efficiency of recombinant lysostaphin achieved in the presented research is comparable with these obtained in the industrial scale technology developed by Mierau and coworkers, 35.6 and 40 mg/L respectively, and is much better then in described to date other methods of production of this protein using E. coli expression systems. From our best knowledge, the most efficient methods of recombinant lysostaphin production
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ACCEPTED MANUSCRIPT using E. coli as a host have been developed and described in our group[14,15]. The efficiency of investigated previously systems assigned as pBADLys and pETLys were 3125 and 13687 U/L, respectively. Optimization of the pBAD2Lys system developed in 2007, with fermentation performed in the small scale flask culture resulted in obtaining 10 mg of the protein with specific activity 498 U/mg from 1 L of culture. Thus the
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technology described in this paper led to five-fold increase in specific activity yield in comparison to the flask culture method in the case of pBAD2Lys system and two-fold increase in comparison to the pETLys system. Our previous research revealed that production of recombinant lysostaphin using pBAD system is definitely easier in comparison to pET system, which is much more sensitive to many parameters of protein production process, such us: strain of the host bacteria, moment of induction and temperature of the culture after induction, which strongly affects the amount of produced enzyme. These observations were the most important reason of selection of pBAD2Lys expression system for developing a bioreactor technology of production of recombinant lysostaphin. There are also no doubts that using pBAD or any other E. coli systems is easier and more often used in most laboratories in comparison to the Lactococcus lactis nisin – controlled system.
The enzyme is commercially available, but is still very expensive. At the moment the price of 1 and 15 mg of lysostaphin (specific activity 500 U/mg) produced by leading supplier is 100 and 800 €, respectively, which can be a problem even for its application in research field or as therapeutic agent and is completely unacceptable for its using as
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ACCEPTED MANUSCRIPT food preservative. The calculated costs of production of 1000 units of the enzyme with proposed technology is only about 3.5 Euros (Fig. 1).
The novel method of recombinant lysostaphin production presented in this paper is convenient and low-cost. Enhanced availability of the enzyme is crucial not only for its
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application in medicine, veterinary and food technology, but also for further tests of its properties during pre-clinical and clinical research. Thus we think that presented results can be interesting for many research groups, or small biotechnology companies. The described technology can be easily adapted in any laboratory for production of large amount of active lysostaphin for their own research or as a commercial product, which could be rather difficult in the case of technology developed by the group of Mierau[21,22]. Moreover we declare that all plasmids constructed in our laboratory, which can be used for production of recombinant lysostaphin in E. coli as a host are freely available, when used for research, not commercial purposes.
ACKNOWLEDGMENTS This research was supported by the grant no. N N405 420739 from the Polish Ministry of Science and Foundation for Polish Science program VENTURES (VENTURES/20105/2).
REFERENCES 1.
Schindler, C.A.; Schuhardt, V.T. Lysostaphin: a new bacteriolytic agent for the
Staphylococcus. Proc. Natl. Acad. Sci. USA. 1964, 51, 414–421.
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ACCEPTED MANUSCRIPT 2.
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ACCEPTED MANUSCRIPT TABLE 1. Influence of agitation intensity on parameters of lysostaphin production in batch fermentation using the pBAD2Lys expression system; T= 37 oC, pH=7
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Rpm 400
600
800
WCW [g/l]
4,20
8,90
6,95
yield [mg/l]
16,81
5,47
3,62
yield on WCW [mg/g]
4,00
0,61
0,52
specific activity [U/mg]
369
326
330
6203
1782
1194
1477,0
200,2
171,8
9
9
9
689,28
197,97
132,64
1,87
0,61
0,40
specific yield [U/l] Lys specific yield on WCW [U/g] process time [h] specific productivity [U/L·h] space time yield [mg/L·h]
ACCEPTED MANUSCRIPT 18
ACCEPTED MANUSCRIPT TABLE 2. Influence of pH on parameters of lysostaphin production in batch fermentation using the pBAD2Lysexpression system; T=37 oC, rpm 400
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pH 5
5,5
6
7
WCW [g/l]
4,47
5,65
6,43
4,20
yield [mg/l]
17,49
28,33
19,32
16,81
yield on WCW [mg/g]
3,92
5,01
3,00
4,00
specific activity [U/mg]
573,4
429
695
369
specific yield [U/l]
10030
12155
13427
6203
Lys specific yield on WCW
2245,5
2149,5
2086,6
1477,10
9
9
9
9
1114,44
1350,55
1491,84
689,28
1,94
3,15
2,15
1,87
[U/g] process time [h] specific productivity [U/L·h] space time yield [mg/L·h]
ACCEPTED MANUSCRIPT 19
ACCEPTED MANUSCRIPT TABLE 3. Influence of temperature on parameters of lysostaphin production in batch fermentation using the pBAD2Lysexpression system; rpm=400, pH=6
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T [oC] 25
30
37
WCW [g/l]
7,61
4,49
6,41
yield [mg/l]
7,90
13,14
19,71
yield on WCW [mg/g]
1,04
2,93
3,07
specific activity [U/mg]
862,9
914
641,00
specific yield [U/l]
6813
12014
12632
Lys specific yield on WCW
895,5
2677,6
2245,5
9
9
9
756,97
1334,85
1403,54
0,88
1,46
2,19
[U/g] process time [h] specific productivity [U/L·h] space time yield [mg/L·h]
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ACCEPTED MANUSCRIPT TABLE 4. Result of two additional batch fermentations in optimal conditions: T=37 oC, pH=6, rpm=400, using pBAD2Lysexpression system
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Repetition 1
2
WCW [g/l]
6,43
4,47
yield [mg/l]
19,32
17,49
yield on WCW [mg/g]
3,00
3,92
specific activity [U/mg]
695
573,4
specific yield [U/l]
13427
10030
Lys specific yield on WCW
5279,0
5726,1
9
9
1491,84
1114,44
2,15
1,94
[U/g] process time [h] specific productivity [U/L·h] space time yield [mg/L·h]
ACCEPTED MANUSCRIPT 21
ACCEPTED MANUSCRIPT TABLE 5. Influence of the time of bacteria growth after adding the inducer on parameters of enzme using the pBAD2Lys expression system; rpm=400; pH=6, T=37 oC
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Time of growing bacteria from the moment of induction [h] 0
2
4
6
8
OD600
0,30
0,86
0,95
0,96
0,99
pO2 [%]
77,50
59,00
69,00
68,20
80,60
WCW [g/l]
---
5,52
6,10
6,22
6,41
yield [mg/l]
---
8,13
12,41
16,23
19,71
yield on WCW [mg/g]
---
1,47
2,03
2,61
3,07
specific activity [U/mg]
---
590
630
680
641
specific yield [U/l]
---
4797
7818
11036
12634
Lys specific yield on WCW
---
869,0
1281,7
1774,3
1971,0
---
3
5
7
9
specific productivity [U/L·h] ---
1598,90
1563,66
1576,63
1403,79
space time yield [mg/L·h]
2,71
2,48
2,32
2,19
[U/g] process time [h]
---
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ACCEPTED MANUSCRIPT TABLE 6. Influence of growing media composition on the efficiency production of recombinant lysostaphin, using the pBAD2Lysexpression system; T=37 oC, pH=6, rpm=400 Medium modifications:
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LB
Glycerol
Glycerol
Glycerol
9g/Lxh
9g/Lxh
3g/Lxh
HCDC
2x inductor WCW [g/l]
6,43
10,47
8,59
10,70
29,12
yield [mg/l]
19,32
13,26
4,22
35,61
1,89
yield on WCW [mg/g]
3,00
1,27
0,49
3,33
0,06
specific activity [U/mg]
695
379
741
728
357,7
specific yield [U/l]
13427
5026
3129
25923
676
Lys specific yield on
2086,6
480,0
364,1
2422,9
676
process time [h]
9
9
9
9
22
specific productivity
1491,84
558,45
122,70
2880,33
30,72
2,15
1,47
0,17
3,96
0,09
WCW [U/g]
[U/L·h] space time yield [mg/L·h]
ACCEPTED MANUSCRIPT 23
ACCEPTED MANUSCRIPT FIGURE 1. Detailed cost analysis for a single experiment carried out in the optimal conditions and medium. The analysis included all stages of enzyme production (inoculum preparation, bioreactor process, purification, enzyme parameters estimation). The salary for qualified laboratory personnel was assumed at 1200 EUR/month (160 h/month; single experiment time - 25h). Electricity consumption needed for power and control unit of
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bioreactor was adopted at a safe level of 5kWh. Because of its minimal effect, energy cost for other laboratory works was ignored. Prices of reagents and media components were based on Sigma-Aldrich catalogue. Under these conditions the cost of the single experiment was set at 267 EUR. Cost involved with production of 1000 units of lysostaphin is then at a level of 3.34 EUR.
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